cyclin-d1 and Disease-Models--Animal

The D-type and E-type cyclins control the G(1) to S phase transition during normal cell cycle progression and are critical components of steroid- and growth factor-induced mitogenesis in breast epithelial cells. Mammary epithelial cell-specific overexpression of these genes leads to mammary carcinoma, while in cyclin D1-deficient mice mammary gland development is arrested prior to lobuloalveolar development. Cyclin D1 null mice are resistant to mammary carcinoma induced by the neu and ras oncogenes, indicating an essential role for cyclin D1 in the development of some mammary cancers. Cyclin D1 and E1 are commonly overexpressed in primary breast cancer, with some evidence of an association with an adverse patient outcome. This observation may result in part from their ability to confer resistance to endocrine therapies. The functional consequences of cyclin E overexpression in breast cancer are likely related to its role in cell cycle progression, whereas that of cyclin D1 may also be a consequence of a more recently defined role in transcriptional regulation.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Mice; Oncogene Proteins

Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer, with 45-50% of primary ductal carcinomas overexpressing this oncoprotein. Targeted deletion of the gene encoding cyclin D1 demonstrates an essential role in normal mammary gland development while transgenic studies provide evidence that cyclin D1 is a weak oncogene in mammary epithelium. In a recent exciting development, Yu et al. demonstrate that cyclin D1-deficient mice are resistant to mammary carcinomas induced by c-neu and v-Ha-ras, but not those induced by c-myc or Wnt-1. These findings define a pivotal role for cyclin D1 in a subset of mammary cancers in mice and imply a functional role for cyclin D1 overexpression in human breast cancer.

Topics: Animals; Carcinoma, Ductal, Breast; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Mice; Mice, Transgenic

Rodent mammary tumors induced by chemical carcinogens have proven to be very useful in the genetic analysis of initiation, promotion and progression of mammary carcinogenesis. We are studying rat mammary carcinomas induced by the chemical carcinogen, N-nitroso-N-methylurea. The earliest genetic event observed in the mammary gland is the activation of Ha-ras oncogenes, which is followed by promotion of the initiated cells by hormones involved in puberty. Preferential amplification of the mutated Ha-ras allele, of PRAD-1 and IGF2, loss of expression of the mitogenic growth factor gene, MK, and mutation in the tumor suppressor gene, p53, are seen in the mammary tumors during tumor progression.

Topics: Animals; Cyclin D1; Cyclins; Disease Models, Animal; Female; Gene Amplification; Genes, p53; Genes, ras; Mammary Neoplasms, Experimental; Methylnitrosourea; Oncogene Proteins; Rats

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cisplatin; Cyclin D1; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; MicroRNAs

The main strategy of cancer cells for survival is uncontrolled cell division and escape from apoptosis. The use of anticancer agents inducing the production of reactive oxygen species (ROS) and controlling cell division might be a therapeutic approach to eradicate cancer cells. Herein, we examined the therapeutic effects of Auraptene on CT26 cells as well as on a mouse model of colorectal cancer (CRC). The spheroid assay was also conducted to analyze the anti-proliferative activity of Auraptene. We also assessed the in vitro analysis of ROS generation. The impact of Auraptene on oxidant/antioxidant markers, as well as the mRNA expression of Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin genes, was evaluated by qPCR in tumor samples. As a result, Auraptene significantly reduced the size of CT26 spheroids at a dose of 200 µM. After 12 h, ROS levels were significantly elevated in CT26 cells. The administration of Auraptene induced apoptosis and the cell cycle arrest by modulating Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin mRNA levels. Furthermore, our results demonstrated that Auraptene suppressed CAT, GSH (reduced Glutathione), and FRAP while increasing MDA in tissue homogenates which in turn could raise oxidative stress and stimulate apoptosis. Therefore, Auraptene may act as a powerful adjuvant therapy in CRC since it triggers apoptosis and cell cycle.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Colorectal Neoplasms; Coumarins; Cyclin D1; Disease Models, Animal; Mice; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Survivin

Hastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients.. This study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms.. C57BL/6 J mice with carbon tetrachloride (CCl. These findings suggest that hastatoside can bind to GSK-3β and promote its activity, while inhibiting the GSK-3β downstream effector expression of β-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.

Topics: Animals; beta Catenin; Chemical and Drug Induced Liver Injury; Cyclin D1; Disease Models, Animal; Glycogen Synthase Kinase 3 beta; Hepatic Stellate Cells; Humans; Iridoid Glycosides; Liver; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Quality of Life; Signal Transduction

Although Huntington's disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

Topics: Animals; Corpus Striatum; Cyclin D1; Disease Models, Animal; Huntingtin Protein; Huntington Disease; Interneurons; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons

Penile cancer is a rare malignancy with a poor prognosis, even with various treatment options. Considering the little progress in the study of the pathogenesis and treatment of penile cancer because of the lack of models that mimic the biological properties of the tumor, we have developed a patient-derived xenograft (PDX) model and paired hydrogel-embedded histoculture drug sensitivity test (HDST) to screen for drugs that can inhibit tumors. The increased expression of XPO1, as a key nuclear export protein involved in the transport of various tumor suppressors and cell cycle regulatory proteins, is associated with the prognosis of a variety of tumors [World J Uroly 27(2):141-150, 2009]. Selinexor is an inhibitor of XPO1, which can treat cancers, such as multiple myeloma, gastric cancer, triple-negative breast cancer, and non-small cell carcinoma [Transl Androl Urol 6(5):785-790, 2017; OncoTargets Therapy 13:6405-6416, 2020]. However, whether XPO1 inhibition has a role in penile cancer remains unknown. Therefore, this article used the PDX and HDST models to investigate whether the inhibition of XPO1 has an effect on penile cancer and its underlying mechanism.. We used penile cancer tumor tissues to construct a PDX model of penile cancer and paired PDXE model and confirmed the consistency of PDX tumor tissues in source patients. Then, we assessed the ability of Selinexor to inhibit penile cancer tissues in vivo using a PDX model and in vitro by HDST. We also examined the potential mechanism of XPO1 action on penile cancer by IHC and TUNEL. Finally, we assessed the safety of the drug treatment by H&E and biochemical blood analysis.. Result showed that the penile cancer PDX model and patient penile cancer tissues were clinically consistent in morphological characteristics and protein expression. In addition, Selinexor could inhibit tumor growth in PDX models and HDST. We found that P53, P21 expression was upregulated; Cyclin D1 expression was downregulated, and apoptosis of tumor cells was increased in the Selinexor-treated PDX model. Moreover, it had no significant effect on liver, kidney, and cardiac function.. The PDX model of penile cancer was a powerful tool for penile cancer research and new drug development. It showed that Selinexor can effectively inhibit penile cancer in vitro and in vivo. In addition, XPO1 may affect P53, P21, and Cyclin D1 expression to regulate the growth and apoptosis of penile carcinoma.

Topics: Active Transport, Cell Nucleus; Animals; Carcinoma; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Heterografts; Humans; Hydrazines; Hydrogels; Karyopherins; Male; Penile Neoplasms; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

The high prevalence of colorectal cancer (CRC) and its leading death causing rate have placed a considerable burden on patients and healthcare providers. There is a need for a therapy that has fewer adverse effects and greater efficiency. Zearalenone (ZEA), an estrogenic mycotoxin, has been demonstrated to exert apoptotic properties when administrated in higher doses. However, it is unclear whether such apoptotic effect remains valid in an in vivo setting. The current study aimed to investigate the effect of ZEA on CRC and its underlying mechanisms in the azoxymethane/ dextran sodium sulfate (AOM/DSS) model. Our results revealed that ZEA significantly lowered the total number of tumours, colon weight, colonic crypt depth, collagen fibrosis and spleen weight. ZEA suppressed Ras/Raf/ERK/cyclin D1 pathway, increasing the expression of apoptosis parker, cleaved caspase 3, while decreasing the expression of proliferative marker, Ki67 and cyclin D1. The gut microbiota composition in ZEA group showed higher stability and lower vulnerability in the microbial community when compared to AOM/DSS group. ZEA increased the abundance of short chain fatty acids (SCFAs) producing bacteria unidentified Ruminococcaceae, Parabacteroidies and Blautia, as well as the faecal acetate content. Notably, unidentified Ruminococcaceae and Parabacteroidies were substantially correlated with the decrease in tumour count. Overall, ZEA demonstrated a promising inhibitory effect on colorectal tumorigenesis and exhibited the potential for further development as a CRC treatment.

Topics: Animals; Azoxymethane; Bacteria; Carcinogenesis; Cell Transformation, Neoplastic; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Zearalenone

The mechanisms underlying the transition from colitis-associated inflammation to carcinogenesis and the cell origin of cancer formation are still unclear. The azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model reproduces human colitis-associated colorectal cancer. To elucidate the mechanisms of cancer development and dynamics of the linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr)-positive cells, we explored the early stages of colitis-associated colorectal cancer in AOM/DSS mice. The AOM/DSS mice were sacrificed at 4 to 6 weeks following AOM administration. To analyze the initial lesions, immunofluorescence staining for the following markers was performed: β-catenin, Ki67, CDK4, Sox9, Bmi1, cyclin D1, and pSmad2/3L-Thr. Micro-neoplastic lesions were flat and unrecognizable, and the uni-cryptal ones were either open to the surfaces or hidden within the mucosae. These neoplastic cells overexpressed β-catenin, Sox9, Ki67, and Cyclin D1 and had large basophilic nuclei in the immature atypical cells. In both the lesions, pSmad2/3L-Thr-positive cells were scattered and showed immunohistochemical co-localization with β-catenin, CDK4, and Bmi1 but never with Ki67. More β-catenin-positive neoplastic cells of both lesions were detected at the top compared to the base or center of the mucosae. We confirmed initial lesions in the colitis-associated colorectal cancer model mice and observed results that suggest that pSmad2/3L-Thr is a biomarker for tissue stem cells and cancer stem cells.

Topics: Animals; Azoxymethane; beta Catenin; Colitis; Colitis-Associated Neoplasms; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Humans; Ki-67 Antigen; Mice; Mice, Inbred C57BL; Neoplastic Stem Cells

Topics: Animals; Azoxymethane; Chemokines, CC; Colonic Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred ICR; Proto-Oncogene Proteins c-akt; Receptors, CCR1; Smad2 Protein; Xanthophylls

Osteoarthritis (OA) is the most prevalent degenerative joint disease. In vitro experiments are an intuitive method used to investigate its early pathogenesis. Chondrocyte inflammation models in rats and mice are often used as in vitro models of OA. However, similarities and differences between them in the early stages of inflammation have not been reported.. This paper seeks to compare the chondrocyte phenotype of rats and mice in the early inflammatory state and identify chondrocytes suitable for the study of early OA.. Under similar conditions, chondrocytes from rats and mice were stimulated using the same IL-1β concentration for a short period of time. The phenotypic changes of chondrocytes were observed under a microscope. The treated chondrocytes were subjected to RNA-seq to identify similarities and differences in gene expression. Chondrocytes were labelled with EdU for proliferation analysis. Cell proliferation-associated proteins, including minichromosome maintenance 2 (MCM2), minichromosome maintenance 5 (MCM5), Lamin B1, proliferating cell nuclear antigen (PCNA), and Cyclin D1, were analysed by immunocytochemical staining, cell immunofluorescence, and Western blots to verify the RNA-seq results.. RNA-seq revealed that the expression patterns of cytokines, chemokines, matrix metalloproteinases, and collagen were similar between the rat and mouse chondrocyte inflammation models. Nonetheless, the expression of proliferation-related genes showed the opposite pattern. The RNA-seq results were further verified by subsequent experiments. The expression levels of MCM2, MCM5, Lamin B1, PCNA, and Cyclin D1 were significantly upregulated in rat chondrocytes (P < 0.05) and mouse chondrocytes (P < 0.05).. Based on the findings, the rat chondrocyte inflammation model may help in the study of the early pathological mechanism of OA.

Topics: Animals; Cell Proliferation; Chondrocytes; Cyclin D1; Disease Models, Animal; Gene Expression; Immunoblotting; Immunohistochemistry; Inflammation; Interleukin-1beta; Mice; Osteoarthritis; Proliferating Cell Nuclear Antigen; Rats; RNA-Seq

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic.

Topics: Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Caspase 3; CD36 Antigens; Choroid; Choroidal Neovascularization; Cyclin D1; Cyclin D2; Disease Models, Animal; Endothelial Cells; Interleukin-17; Peptides; Proto-Oncogene Proteins c-bcl-2; Rats; Thrombospondin 1; Vascular Endothelial Growth Factor A

Space radiation-induced gastrointestinal (GI) cancer risk models for future interplanetary astronauts are being developed that primarily rely on quantitative animal model studies to assess radiation-quality effects of heavy-ion space radiation exposure in relation to γ-rays. While current GI-cancer risk estimation efforts are focused on sporadic GI-cancer mouse models, emerging in-vivo data on heavy-ion radiation-induced long-term GI-inflammation are indicative of a higher but undetermined risk of GI-inflammation associated cancers, such as colitis-associated cancer (CAC). Therefore, we aimed to assess radiation quality effects on colonic inflammation, colon cancer incidence, and associated signaling events using an in-vivo CAC model i.e., Il10-/- mice. Male Il10-/- mice (8-10 weeks, n = 12/group) were irradiated with either sham, γ-rays or heavy-ions (28Si or 56Fe), and histopathological assessments for colitis and CAC were conducted at 2.5 months post-exposure. qPCR analysis for inflammation associated gene transcripts (Ptges and Tgfb1), and in-situ staining for markers of cell-proliferation (phospho-histone H3), oncogenesis (active-β-catenin, and cyclin D1), and inflammation (phospho-p65NF-κB, iNOS, and COX2) were performed. Significantly higher colitis and CAC frequency were noted after heavy-ion exposure, relative to γ and control mice. Higher CAC incidence after heavy-ion exposure was associated with greater activation of β-catenin and NF-κB signaling marked by induced expression of common downstream inflammatory (iNOS and COX2) and pro-proliferative (Cyclin D1) targets. In summary, IR-induced colitis and CAC incidence in Il10-/- mice depends on radiation quality and display co-activation of β-catenin and NF-κB signaling.

Topics: Animals; beta Catenin; Carcinogenesis; Colitis; Colonic Neoplasms; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Gastritis; Inflammation; Male; Mice; Mice, Inbred C57BL; Neoplasms, Radiation-Induced; NF-kappa B

Liver ischemia-reperfusion injury (IRI) is an inevitable process during liver transplantation, hemorrhagic shock, resection, and other liver surgeries. It is an important cause of postoperative liver dysfunction and increased medical costs. The protective effects of the vagus nerve on hepatic IRI have been reported, but the underlying mechanism has not been fully understood. We established a hepatic vagotomy (Hv) mouse model to study the effect of the vagus on liver IRI and to explore the underlying mechanism. Liver IRI was more serious in mice with Hv, which showed higher serum ALT and AST activities and histopathological changes. Further experiments confirmed that Hv significantly downregulated the expression of IL-22 protein and mRNA in the liver, blocking the activation of the STAT3 pathway. The STAT3 pathway in the livers of Hv mice was significantly activated, and liver injury was clearly alleviated after treatment with exogenous IL-22 recombinant protein. In conclusion, Hv can aggravate hepatic IRI, and its mechanism may be related to inhibition of IL-22 expression and downregulation of the STAT3 pathway in the liver.

Topics: Animals; Cyclin D1; Disease Models, Animal; Disease Progression; Disease Susceptibility; Gene Expression; Immunohistochemistry; Interleukin-22; Interleukins; Liver Diseases; Male; Mice; Nerve Block; Phosphorylation; Reperfusion Injury; STAT3 Transcription Factor; Vagus Nerve

Therapeutic discovery for mantle cell lymphoma (MCL) has been hindered by a lack of preclinical mouse models that recapitulate human disease. In this issue, Pieters and colleagues (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202280) establish a novel mouse model of MCL driven by overexpression of cyclin D2 and identify fetal-derived B1a cells as putative cell of origin for MCL.

Topics: Animals; Cyclin D1; Disease Models, Animal; Lymphoma, Mantle-Cell; Mice

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3'-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Base Sequence; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Disease Models, Animal; Disease Progression; DNA Breaks, Double-Stranded; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Gene Ontology; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes; Mice, Inbred C57BL; MicroRNAs; RNA, Messenger; Up-Regulation

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Dyrk Kinases; Female; Glioblastoma; Humans; Mice; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction

Vascular remodeling and contraction contribute to the development of hypertension. We investigated the role of miR-212-5p and its downstream target in vascular smooth muscle cell (VSMC) proliferation, migration, and contraction. MicroRNA microarray and PCR analyses showed that miR-212-5p expression was increased with angiotensin II treatment in vivo and in vitro. Moreover, miR-212-5p mimic treatment attenuated and miR-212-5p inhibitor treatment increased VSMC proliferation and migration. Additionally, miR-212-5p mimic treatment suppressed VSMC contraction and related gene expression [Ras homolog gene family member A (RhoA) and Rho-associated protein kinase 2], while miR-212-5p inhibitor treatment exerted opposite effects. Bioinformatics analysis revealed that platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is a target of miR-212-5p. miR-212-5p mimic treatment significantly reduced and miR-212-5p inhibitor treatment increased PAFAH1B2 expression. Furthermore, PAFAH1B2 expression was decreased in angiotensin II-treated aortic tissues and VSMCs. PAFAH1B2 was ubiquitously expressed in most adult rat tissues. In the vasculature, PAFAH1B2 was only distributed in the cytoplasm. PAFAH1B2 overexpression decreased A10 cell proliferation, while PAFAH1B2 knockdown increased A10 cell proliferation and cyclin D1 mRNA levels. PAFAH1B2 knockdown stimulated VSMC contraction and RhoA expression. These results suggest that miR-212-5p and PAFAH1B2 are novel negative regulators of VSMC proliferation, migration, and contraction in hypertension.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Angiotensin II; Animals; Antagomirs; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; Down-Regulation; Hypertension; MicroRNAs; Muscle Contraction; Muscle, Smooth, Vascular; Rats; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Vascular Remodeling

A xanthophyll of fucoxanthin (Fx) is a potential chemopreventive agent. Familial adenomatous polyposis (FAP) is an inherited disease that is associated with a high risk of developing colorectal cancer. However, it remains unclear whether Fx can modify colorectal tumorigenesis in Apc. We investigated the chemopreventive effect of Fx in dextran sodium sulfate (DSS)-treated Apc. Administration of Fx in the diet for 5 weeks significantly suppressed the number of colorectal adenocarcinomas in DSS-treated male Apc. Fx possesses chemopreventive potential against progression of colorectal carcinogenesis in Apc

Topics: Adenomatous Polyposis Coli; Animals; Anticarcinogenic Agents; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Male; Mice; Xanthophylls

Glomerular mesangial cell (GMC) proliferation is a histopathological alteration in human mesangioproliferative glomerulonephritis (MsPGN) or in animal models of MsPGN, e.g., the rat Thy-1 nephritis (Thy-1N) model. Although sublytic C5b-9 assembly on the GMC membrane can trigger cell proliferation, the mechanisms are still undefined. We found that sublytic C5b-9-induced rat GMC proliferation was driven by extracellular signal-regulated kinase 1/2 (ERK1/2), sry-related HMG-box 9 (SOX9), and Cyclin D1. Here, ERK1/2 phosphorylation was a result of the calcium influx-PKC-α-Raf-MEK1/2 axis activated by sublytic C5b-9, and Cyclin D1 gene transcription was enhanced by ERK1/2-dependent SOX9 binding to the Cyclin D1 promoter (-582 to -238 nt). In addition, ERK1/2 not only interacted with SOX9 in the cell nucleus to mediate its phosphorylation at serine residues 64 (a new site identified by mass spectrometry) and 181 (a known site), but also indirectly induced SOX9 acetylation by elevating the expression of general control non-repressed protein 5 (GCN5), which together resulted in Cyclin D1 synthesis and GMC proliferation. Moreover, our in vivo experiments confirmed that silencing these genes ameliorated the lesions of Thy-1N rats and reduced SOX9 phosphorylation, acetylation and Cyclin D1 expression. Furthermore, the renal tissue sections of MsPGN patients also showed higher phosphorylation or expression of ERK1/2, SOX9, and Cyclin D1. In summary, these findings suggest that sublytic C5b-9-induced GMC proliferation in rat Thy-1N requires SOX9 phosphorylation and acetylation via enhanced Cyclin D1 gene transcription, which may provide a new insight into human MsPGN pathogenesis.

Topics: Acetylation; Animals; Biomarkers; Calcium; Calcium Signaling; Cell Proliferation; Complement Membrane Attack Complex; Cyclin D1; Disease Models, Animal; Disease Susceptibility; Gene Expression Regulation; Gene Knockdown Techniques; Glomerulonephritis; Male; MAP Kinase Signaling System; Mesangial Cells; Models, Biological; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Rats; SOX9 Transcription Factor

Remifentanil is a potent, short-acting opioid analgesic drug that can protect tissues from ischemia and reperfusion injury though anti-inflammatory effects. However, the utility of remifentanil in liver regeneration after hepatectomy is not known. Using a 70% hepatectomy mouse model (PHx), we found that preconditioning animals with 4 μg/kg remifentanil enhanced liver regeneration through supporting hepatocyte proliferation but not through anti-inflammatory effects. These effects were also phenocopied in vitro where 40 mM remifentanil promoted the proliferation of primary mouse hepatocyte cultures. We further identified that remifentanil treatment increased the expression of β-arrestin 2 in vivo and in vitro. Demonstrating specificity, remifentanil preconditioning failed to promote liver regeneration in liver-specific β-arrestin 2 knockout (CKO) mice subjected to PHx. While remifentanil increased the expression of activated (phosphorylated)-ERK and cyclin D1 in PHx livers, their levels were not significantly changed in remifentanil-treated CKO mice nor in WT mice pretreated with the ERK inhibitor U0126. Our findings suggest that remifentanil promotes liver regeneration via upregulation of a β-arrestin 2/ERK/cyclin D1 axis, with implications for improving regeneration process after hepatectomy.

Topics: Analgesics, Opioid; Animals; beta-Arrestin 2; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; Hepatectomy; Hepatocytes; Liver Regeneration; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Remifentanil; Reperfusion Injury; Up-Regulation

Icariside II (ICA II), an active flavonoid monomer, has been proven to restore post-prostatectomy erectile dysfunction in rats; however, the high cost of extraction from natural plants limits the application of ICA II.. To investigate the therapeutic effect and possible mechanism of action of YS-10, a new flavonoid compound, which was designed and synthesized based on the structure of ICA II in a rat model in of cavernous nerve injury.. Eight of 32 adult male Sprague-Dawley rats were selected as the normal control (NC) group and received vehicle treatment. The remaining rats were subjected to bilateral cavernous nerve injury (BCNI) and randomized into three groups: BCNI group, BCNI + ICA II group (2.5 mg/kg/day), and BCNI + YS-10 group (2.5 mg/kg/day). The total procedure lasted for 21 days, followed by a washout period of 3 days. All animals were evaluated for erectile function, and tissues were harvested for histopathological analyses.. It was observed that in YS-10 group, the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP) and the area under the ICP/MAP curve were effectively enhanced. The maximum ICP/MAP increased by 30% in the YS-10 group (0.86 ± 0.085) compared with the BCNI group (0.66 ± 0.058), which is close to 82% of the NC group (1.05 ± 0.033). Histopathological changes demonstrated significant reduction of smooth muscle atrophy, collagen deposition, and endothelial and neural dysfunction after YS-10 treatment, which have no statistical differences compared with ICA II group. Additionally, high-protein expression levels of β-Catenin and cyclin D1 were observed in the treatment groups.. YS-10, a novel synthesized flavonoid compound, could effectively improve erectile dysfunction in rats after BCNI by alleviating pathological impairments; this effect may associate with the upregulation of β-Catenin and cyclin D1 in Wnt signaling pathway.

Topics: Animals; beta Catenin; Cyclin D1; Disease Models, Animal; Erectile Dysfunction; Flavonoids; Male; Penile Erection; Penis; Peripheral Nerve Injuries; Rats; Rats, Sprague-Dawley; Up-Regulation; Wnt Signaling Pathway

Imbalances in bone formation and resorption cause osteoporosis. Mounting evidence supports that brain-derived neurotrophic factor (BDNF) implicates in this process. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived small molecular TrkB agonist, mimics the functions of BDNF. We show that both BDNF and 7,8-DHF promoted the proliferation, osteogenic differentiation, and mineralization of MC3T3-E1 cells. These effects might be attributed to the activation of the Wnt/β-catenin signaling pathway as the expression of cyclin D1, phosphorylated-glycogen synthase kinase-3β (p-GSK3β), β-catenin, Runx2, Osterix, and osteoprotegerin (OPG) was all significantly up-regulated. Knockdown of β-catenin restrained the up-regulation of Runx2 and Osterix stimulated by 7,8-DHF. In particular, blocking TrkB by its specific inhibitor K252a suppressed 7,8-DHF-induced osteoblastic proliferation, differentiation, and expression of osteoblastogenic genes. Moreover, BDNF and 7,8-DHF repressed osteoclastic differentiation of RAW264.7 cells. The transcription factor c-fos and osteoclastic genes such as tartrate-resistant acid phosphatase (TRAP), matrix metalloprotein-9 (MMP-9), Adamts5 were inhibited by 7,8-DHF. More importantly, 7,8-DHF attenuated bone loss, improved trabecular microarchitecture, tibial biomechanical properties, and bone biochemical indexes in an ovariectomy (OVX) rat model. The current work highlights the dual regulatory effects that 7,8-DHF exerts on bone remodeling.

Topics: Animals; beta Catenin; Bone and Bones; Bone Remodeling; Cell Differentiation; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Cyclin D1; Disease Models, Animal; Female; Flavones; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Osteoprotegerin; Ovariectomy; Rats; Sp7 Transcription Factor; Wnt Signaling Pathway

Cell proliferation and cell death abnormalities are strongly linked to the development of cancer, including lung cancer. The purpose of this study was to investigate the effect of pterostilbene on cell proliferation and cell death via cell cycle arrest during the transition from G1 to S phase and the p53 pathway. A total of 24 female Balb/C mice were randomly categorized into four groups (n = 6): N-nitroso-tris-chloroethyl urea (NTCU) induced SCC of the lungs, vehicle control, low dose of 10 mg/kg PS + NTCU (PS10), and high dose of 50 mg/kg PS + NTCU (PS50). At week 26, all lungs were harvested for immunohistochemistry and Western blotting analysis. Ki-67 expression is significantly lower, while caspase-3 expression is significantly higher in PS10 and PS50 as compared to the NTCU (p < 0.05). There was a significant decrease in cyclin D1 and cyclin E2 protein expression in PS10 and PS50 when compared to the NTCU (p < 0.05). PS50 significantly increased p53, p21, and p27 protein expression when compared to NTCU (p < 0.05). Pterostilbene is a potential chemoprevention agent for lung SCC as it has the ability to upregulate the p53/p21 pathway, causing cell cycle arrest.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclins; Disease Models, Animal; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice, Inbred BALB C; Stilbenes; Tumor Suppressor Protein p53; Up-Regulation

Early differential diagnosis between malignant and benign tumors and their underlying intrinsic differences are the most critical issues for life-threatening cancers. To study whether human acral melanomas, deadly cancers that occur on non-hair-bearing skin, have distinct origins that underlie their invasive capability, we develop fate-tracing technologies of melanocyte stem cells in sweat glands (glandular McSCs) and in melanoma models in mice and compare the cellular dynamics with human melanoma. Herein, we report that glandular McSCs self-renew to expand their migratory progeny in response to genotoxic stress and trauma to generate invasive melanomas in mice that mimic human acral melanomas. The analysis of melanocytic lesions in human volar skin reveals that genetically unstable McSCs expand in sweat glands and in the surrounding epidermis in melanomas but not in nevi. The detection of such cell spreading dynamics provides an innovative method for an early differential diagnosis of acral melanomas from nevi.

Topics: Animals; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epidermis; Gene Amplification; Genomic Instability; Melanocytes; Melanoma; Mice, Inbred C57BL; Nevus; Risk Factors; Skin; Skin Pigmentation; Stem Cells; Sweat Glands; Ultraviolet Rays

Benign prostatic hyperplasia (BPH) is a common disorder in men aged over 60 years and significantly contributes to the distressing lower urinary tract symptoms. Cucurbitacins are triterpene derivatives with diverse medicinal uses including prostate diseases. Cucurbitacin E glucoside was evaluated against testosterone-induced prostatic hyperplasia in mice. Our data indicate that it significantly inhibited the increase in prostate weight and prostate index. The compound ameliorated histopathological changes in prostatic architecture and inhibited the increase in glandular epithelial length induced by testosterone. These results were confirmed by decreased expression of cyclin D1 in prostatic tissues compared to those obtained from the testosterone-alone group. Also, it showed significant antioxidant activity as evidenced by inhibiting lipid peroxides accumulation, glutathione depletion and superoxide exhaustion. Further, it exhibited anti-inflammatory activity as it decreased cyclooxygenase-2 and interleukin-1β protein expression in prostatic tissues. Masson's trichrome staining of prostate sections indicated obvious antifibrotic activity that was supported by decreased α-smooth muscle actin expression. In conclusion, Cucurbitacin E glucoside inhibits testosterone-induced experimental BPH in mice due to, at least partly, its antiproliferative, antioxidant, anti-inflammatory, and antifibrotic effects.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Citrullus colocynthis; Cyclin D1; Disease Models, Animal; Glucosides; Male; Mice; Prostatic Hyperplasia; Testosterone; Triterpenes

Rapamycin (RAPA) is a potent angiogenic inhibitor and the aim of this study is to identify the inhibitory effect of RAPA on retinal neovascularization (RNV) in experimental oxygen-induced retinopathy (OIR).. Forty-two 7-day-old C57BL/6 J mice were randomly divided into normoxia control group (14 mice), OIR group (14 mice), and rapamycin (RAPA) group. OIR model was induced in OIR and RAPA group. Vehicle and RAPA (2 mg/kg/d) was injected intraperitoneally daily from postnatal day 12 (P12) in OIR and RAPA groups, respectively. RNV was evaluated using fluorescence angiography and histopathology on P17. Non-perfused areas of retina were analyzed by Image-Pro plus 6.0 software. Retinal expression of cyclin D1 was detected both at mRNA and protein levels.. RAPA treatment significantly decreased RNV, non-perfused areas and number of endothelial cell nuclei breaking through the internal limiting membrane (ILM) in OIR mice. Moreover, RAPA decreased activation of cyclin D1 in retina caused by OIR.. RAPA can inhibit RNV by downregulating the expression of cyclin D1, which indicates its therapeutic potential in treating RNV-related diseases.

Topics: Animals; Animals, Newborn; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Oxygen; Retinal Neovascularization; RNA, Messenger; Sirolimus

Resveratrol has shown benefit for pulmonary hypertension improvement. Our previous reports showed NR4A3/cyclin D1 pathway promoted pulmonary arterial smooth muscle cells (PASMCs) proliferation. This study tried to explore the mechanism underlying this process, focusing on the role of resveratrol in regulation of miRNA and NR4A3.. Rats were injected with monocrotaline (MCT) to establish pulmonary hypertension (PH) models. Resveratrol was used to prevent pulmonary vascular remodeling. Primary rat PASMCs were cultured in vitro and stimulated by platelet-derived growth factor (PDGF) with or without resveratrol. Cells proliferation and expression of miR-638 as well as NR4A3 were evaluated.. MCT resulted in significant pulmonary vascular remodeling and down-regulation of miR-638, which could be suppressed by resveratrol. Moreover, PDGF-induced PASMC proliferation and miR-638 down-regulation were both significantly prevented by resveratrol treatment in vitro. MiR-638 mimics markedly inhibited PASMC proliferation and percentage of PCNA-positive cells in vitro. But anti-miR-638 could markedly promote cells proliferation and percentage of PCNA-positive cells. The luciferase reporter assay showed that NR4A3 was a direct target of miR-638. The loss-of-function and gain-of-function experiments indicated that NR4A3 promoted proliferation via cyclin D1 pathway.. Our data indicated that resveratrol prevented MCT-induced pulmonary vascular remodeling via miR-638 regulating NR4A3/cyclin D1 pathway.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; DNA-Binding Proteins; Hypertension, Pulmonary; Male; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nerve Tissue Proteins; Pulmonary Artery; Rats, Wistar; Resveratrol; Signal Transduction; Vascular Remodeling

Topics: Animals; Azoxymethane; Carcinogenesis; Caspase 3; Colon; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Cytokines; Dextran Sulfate; Dietary Carbohydrates; Disease Models, Animal; Edible Grain; Interleukin-1beta; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; RNA, Messenger; Tumor Necrosis Factors; Tumor Suppressor Protein p53

YKL-40 is a chitinase-like protein which was significantly elevated in asthma patients and related closely to asthma severity and airway remodeling. Airway remodeling in asthma involves complicated physical and pathological processes, including increased airway smooth muscle mass due to proliferation, migration of airway smooth muscle cells, epithelial-mesenchymal transition (EMT) and sub-epithelial fibrosis. However, the precise effect and underlying mechanism of YKL-40 in this pathological alteration remained unelucidated. In this study, we demonstrated that YKL-40 could promote asthma airway remodeling by increasing airway smooth muscle mass, inducing EMT and sub-epithelial fibrosis. Furthermore, we identified that FAK and MAPK signaling pathways are activated in the process. Inhibiting FAK or MAPK pathway could significantly ameliorate airway remodeling induced by excessive secretion of YKL-40

Topics: Airway Remodeling; Animals; Asthma; Cell Movement; Cell Proliferation; Chitinase-3-Like Protein 1; Cyclin D1; Cyclin-Dependent Kinases; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Focal Adhesion Protein-Tyrosine Kinases; Humans; MAP Kinase Signaling System; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; src-Family Kinases

Salvia officinalis L. (Lamiaceae) is known to have antibacterial properties possibly conducive to the healing process of infected wounds.. The present study aimed to evaluate the effects of an ointment containing Salvia officinalis essential oil (SOO) on an infected wound model.. Essential oil hydrodistillated from the dried leaves of the plant was analyzed by GC-FID and GC-MS. After creating two full-thickness cutaneous wounds, mice were classified into four groups, control, and animals treated with 2 % mupirocin® (standard positive drug), and 2 % and 4 % (w/w) of SOO. In order to evaluate the effects of SOO on the wound healing phases, the expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), cyclin-D1, Bcl-2, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factors (VEGF) were analyzed using qRT-PCR. Immunohistochemistry analysis, tissue total antioxidant capacity (TAC) and malondialdehyde (MDA) were further assessed in all groups.. Concerning essential oil, the main compounds were found to be cis-thujone (26.8 %), camphor (16.4 %), trans-thujone (14.1 %) and 1,8-cineole (10.8 %). Our findings showed that the topical application of SOO was able to shorten the inflammatory phase and accelerate the cellular proliferation, re-vascularization, collagen deposition and re-epithelialization in comparison to the control group (p < 0.05). Moreover, increased mRNA levels of FGF-2 and VEGF, and up-regulation of cyclin-D1 and Bcl-2 were observed following the topical application of SOO compared to the control group (p < 0.05). The expression levels of IL-6, IL-1β and TNF-α were reduced in animals treated with SOO on days 3, 7 and 14 (p < 0.05).. Administration of SOO increased the TAC level and reduced the MDA content and levels of IL-1β and TNF-α. It is concluded that SOO is able to accelerate the wound healing process by regulating the expression of pro-inflammatory cytokines, growth factors, and antioxidant properties.

Topics: Administration, Cutaneous; Animals; Anti-Bacterial Agents; Cyclin D1; Cytokines; Disease Models, Animal; Fibroblast Growth Factor 2; Inflammation Mediators; Male; Mice, Inbred BALB C; Oils, Volatile; Plant Oils; Proto-Oncogene Proteins c-bcl-2; Pseudomonas Infections; Salvia officinalis; Staphylococcal Infections; Time Factors; Vascular Endothelial Growth Factor A; Wound Healing; Wound Infection

To better understand the influence of ultraviolet (UV) irradiation on the initial steps of skin carcinogenesis, we examine patches of labeled keratinocytes as a proxy for clones in the interfollicular epidermis (IFE) and measure their size variation upon UVB irradiation. Multicolor lineage tracing reveals that in chronically irradiated skin, patches near hair follicles (HFs) increase in size, whereas those far from follicles do not change. This is explained by proliferation of basal epidermal cells within 60 μm of HF openings. Upon interruption of UVB, patch size near HFs regresses significantly. These anatomical differences in proliferative behavior have significant consequences for the cell of origin of basal cell carcinomas (BCCs). Indeed, a UV-inducible murine BCC model shows that BCC patches are more frequent, larger, and more invasive near HFs. These findings have major implications for the prevention of field cancerization in the epidermis.

Topics: Animals; Carcinoma, Basal Cell; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epidermis; Hair Follicle; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms, Radiation-Induced; Skin Neoplasms; Stem Cells; Tumor Suppressor Protein p53; Ultraviolet Rays

Osteosarcoma is one of the most common malignant bone tumors with the annual global incidence of approximately four per million. Upregulated gene 4 (URG4) expression in the osteosarcoma tissue is closely associated with recurrence, metastasis, and poor prognosis of osteosarcoma. However, the biological function and underlying mechanisms of URG4 in osteosarcoma have not been elucidated. This study aimed to explore the expression and underlying mechanism of URG4 in osteosarcoma.. The expression level of URG4 in osteosarcoma and normal tissues was compared using immunohistochemistry (IHC). PCR and western blotting (WB) techniques are used to detect URG4 mRNA and protein levels. Wound healing and Transwell analysis to assess the effect of URG4 on osteosarcoma cell migration and invasion. Cell Counting Kit-8 assay and colony proliferation assay were performed to evaluate the effects of silencing URG4 on the inhibition of cell proliferation. The cell cycle distribution was detected by flow cytometry, and a xenograft mouse model was used to verify the function of URG4 in vivo.. URG4 was found to be highly expressed in osteosarcoma tissues and cells, and its high expression was correlated with advanced Enneking stage, large tumor size, and tumor metastasis in osteosarcoma patients. The proliferation in osteosarcoma cell lines and cell cycle in the S phase was suppressed when siRNA was used to downregulate URG4. URG4 promoted cell proliferation and tumorigenesis in vitro and in vivo. WB verified that URG4 promotes cell proliferation in osteosarcoma via pGSK3β/β-catenin/cyclinD1 signaling.. URG4, which is high-expressed in osteosarcoma, promotes cell cycle progression via GSK3β/β-catenin/cyclin D1 signaling pathway and may be a novel biomarker and potential target for the treatment of osteosarcoma.

Topics: Adolescent; Adult; Animals; beta Catenin; Bone Neoplasms; Cell Culture Techniques; Cell Cycle; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Osteosarcoma; Signal Transduction; Young Adult

The influence of cilostazol on learning and memory, and cyclin D1 expression in the cerebral cortex of rats with chronic cerebral ischemia were investigated. A chronic cerebral ischemia model was established using the permanent bilateral common carotid artery occlusion method (2VO), learning and memory capacity was detected using the Morris water maze, and expression changes in apoptosis regulating gene cyclin D1 were tested by RT-PCR. Results of the Morris water maze indicated that significant extensions were found in the escape latent period and swimming path of rats in the ischemia group (2VO group), learning and memory results in the cilostazol group was obviously superior compared to the 2VO group (P<0.05), and the expression of cyclin D1 was observed to increase in both the ischemia and cilostazol intervention groups at the 9th week of ischemia. A significant difference was observed, compared with the sham operation group (P<0.05), the expression level decreased in the ischemia group compared with the cilostazol group, and a significant difference was identified compared with the ischemia group (P<0.05). Cilostazol can reduce nerve function impairment and improve learning and memory functions by affecting changes in apoptosis regulating genes.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Chronic Disease; Cilostazol; Cyclin D1; Disease Models, Animal; Male; Maze Learning; Memory; Neuroprotective Agents; Rats; Rats, Wistar

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Gene Knockdown Techniques; Hepatocyte Nuclear Factor 4; Hepatocytes; Liver; Liver Regeneration; Male; Mice, Inbred BALB C; Mice, Knockout

Cardiovascular diseases are associated with proliferation and phenotypic switch. Platelet-derived growth factor-BB (PDGF-BB) is a major initiating factor for proliferative vascular diseases, such as neointimal lesion formation, restenosis after angioplasty, and atherosclerosis. Ruxolitinib, a potent Janus kinase (JAK) 1 and 2 inhibitor, has been reported to significantly block the proliferation-related signaling pathway of JAK2/signal transducers and activators of transcription 3 (STAT3) and harbor a broad spectrum of anti-cancer activities, including proliferation inhibition, apoptosis induction, and anti-inflammation. However, the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation remains to be elucidated. Thus, this study investigates the role of ruxolitinib in regulating PDGF-BB-induced VSMC proliferation and its underlying mechanisms.. In vivo, the medial thickness of the carotid artery was evaluated using a mouse carotid ligation model, ruxolitinib was administered orally to the mice every other day, and the mice were euthanized on day 28 to evaluate the therapeutic effects of ruxolitinib. Cell proliferation markers were measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. In vitro, VSMCs were treated with ruxolitinib with or without PDGF-BB at an indicated time and concentration. Cell proliferation and apoptosis were measured using Cell Counting Kit-8 assay, MTS assays and flow cytometry. The JAK2/STAT3 signaling pathway involved in the effects of ruxolitinib on VSMCs was detected by western blotting with the specific pathway inhibitor AG490.. In vivo, ruxolitinib significantly decreased the ratio-of-intima ratio (I/M ratio) by inhibiting the expression of PCNA and cyclinD1 (p <0.05). In vitro, ruxolitinib inhibited PDGF-BB-induced VSMC proliferation compared with the PDGF-BB treatment group (p <0.05). In addition, ruxolitinib inhibited the PDGF-BB-induced activation of the JAK2/STAT3 signaling pathway and decreased the expression of proliferation related-proteins cyclinD1 and PCNA in VSMCs (p <0.05).. Our findings suggest that ruxolitinib inhibits VSMC proliferation in vivo and in vitro by suppressing the activation of the JAK2/STAT3 signaling pathway. Therefore, ruxolitinib has a therapeutic potential for proliferative vascular diseases.

Topics: Animals; Becaplermin; Carotid Artery, Common; Carotid Stenosis; Cells, Cultured; Cyclin D1; Disease Models, Animal; Hyperplasia; Janus Kinase 2; Janus Kinase Inhibitors; Male; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Nitriles; Proliferating Cell Nuclear Antigen; Pyrazoles; Pyrimidines; Signal Transduction; STAT3 Transcription Factor

Epithelial to mesenchymal transition (EMT) is associated with the pathophysiology of chronic rhinosinusitis with nasal polyp (CRSwNP). Wnt signaling is causative for EMT, whereas the mechanism in CRSwNP is not fully understood.. We sought to evaluate the role of Wnt signaling in EMT of CRSwNP using a murine nasal polyp (NP) model and human tissues.. Inflammatory markers and EMT-related molecules were evaluated in NP models using adenomatosis polyposis coli (Apc). Apc. Wnt signaling may contribute to the pathogenesis of NPs through EMT. Therefore, inhibition of Wnt signaling may be a potential therapeutic strategy for patients with CRSwNP.

Topics: Actins; Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Biomarkers; Cadherins; Cyclin D1; Disease Models, Animal; Epithelial-Mesenchymal Transition; Humans; Indocyanine Green; Mice; Nasal Polyps; Rhinitis; Sinusitis; Twist-Related Protein 1; Up-Regulation; Wnt Signaling Pathway

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

Topics: Animals; Biopsy; Cell Transdifferentiation; Cell Transformation, Neoplastic; Chick Embryo; Child; Cyclin D1; Datasets as Topic; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; N-Myc Proto-Oncogene Protein; Neoplasm Invasiveness; Neural Stem Cells; Neural Tube; Oncogene Proteins, Fusion; Paired Box Transcription Factors; PAX3 Transcription Factor; PAX7 Transcription Factor; Rhabdomyosarcoma, Alveolar; S Phase

Transgenic adenocarcinoma mouse prostate (TRAMP) model is established to mimic human prostate cancer progression, where seminal vesicle lesions often occur and has been described as phyllodes-like epithelial-stromal tumors. However, the molecular mechanism regulating tumorigenesis and progression in seminal vesicles of TRAMP mice remains largely unknown. In this study, C57BL/6 TRAMP mice were found to have a significantly shorter lifespan than wild-type (WT) mice and all of the seminal vesicles were markedly increased in size and weight with age from 24 weeks exhibiting a clearly papillary-phyllode pattern, though no obvious difference was observed in multiple organs including heart, liver, spleen, lungs, kidneys, testicles and bone between TRAMP and WT mice, and less than 10% of TRAMP mice developed prostate tumors. Western blotting showed Cyclin (CCN) B1 and CCND1 were remarkably overexpressed in seminal vesicle tumors of TRAMP mice at 24 weeks of age and increased with age till the end of trial, which was confirmed by Immunohistochemistry (IHC). P21 and P27 were also significantly augmented, whereas P53 and phosphorylated P53 (p-P53) were constantly expressed in normal controls and P53 did not appear to be mutated. Not only cyclin-dependent kinase (CDK) 1 and phosphorylated forkhead box protein (FOX) O1 but also CDK4, CDK6 and phosphorylated retinoblastoma-associated protein (RB) had similar increase trends, so did epidermal growth factor receptor (EGFR), AKT serine/threonine kinase (AKT), and their respective phosphorylation levels. Signal transducer and activator of transcription (STAT) 3, p-STAT3, enhancer of zeste homolog 2 (EZH2) and EZH2 mediated trimethylation of histone H3 lysine 27 (H3K27me3) were considerably elevated, too. Taken together, this finding suggests P21 and P27 promote carcinogenesis and development in seminal vesicles of TRAMP mice via accelerating cell cycle progression, in which oncogenic transformation of P21 and P27 might be through regulation of EGFR-AKT signaling.

Topics: Animals; Carcinogenesis; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; ErbB Receptors; Immunohistochemistry; Male; Mice; Prostate; Prostatic Neoplasms; Receptors, Tumor Necrosis Factor, Member 25; Seminal Vesicles; Signal Transduction

Over proliferation of glomerular mesangial cells (MCs) disturbs mesangial homeostasis and leads to renal damage in mesangioproliferative glomerulonephritis. It is documented that transcriptional factors may be involved in the proliferation of MCs. This study aims to identify the key transcriptional factor that prevents the MCs from over proliferation and to clarify its regulatory mechanism. Microarray analysis of glomeruli isolated from Sprague-Dawley rats (SD rats) with or without anti-Thy1 nephritis (anti-Thy1N) showed that the cell cycle pathway was the most enriched pathway in anti-Thy1N model, and the D-site binding protein (DBP) ranked first in the cluster of transcription factors. Compare with normal rats, DBP is markedly decreased accompanied by an over proliferation of MCs in rats with anti-Thy1N. The cell proliferative capacity was measured by 5-Ethynyl-2'-deoxyuridine (EdU) assay in primary rat MCs with DBP knockdown or overexpression, respectively. The results showed that the knockdown of DBP significantly promoted the proliferation of MCs, whereas the overexpression of DBP inhibited the MCs' proliferation, compared to that of the control cells. Further study indicated that DBP arrested G1/S-phase transition by inhibiting the expression of p21, p27 and inducing the Cyclin D1 expression in MCs. The current data suggest that DBP effectively inhibits the proliferation of MCs through G1 phase arrest, and the decrease of DBP may induce mesangial over proliferation in rats with anti-Thy1N.

Topics: Animals; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA-Binding Proteins; Down-Regulation; Isoantibodies; Male; Mesangial Cells; Nephritis; Rats, Sprague-Dawley; RNA, Messenger; Transcription Factors

Cancer is a hyper-proliferative disease. Whether the proliferative state originates from the cell-of-origin or emerges later remains difficult to resolve. By tracking de novo transformation from normal hematopoietic progenitors expressing an acute myeloid leukemia (AML) oncogene MLL-AF9, we reveal that the cell cycle rate heterogeneity among granulocyte-macrophage progenitors (GMPs) determines their probability of transformation. A fast cell cycle intrinsic to these progenitors provide permissiveness for transformation, with the fastest cycling 3% GMPs acquiring malignancy with near certainty. Molecularly, we propose that MLL-AF9 preserves gene expression of the cellular states in which it is expressed. As such, when expressed in the naturally-existing, rapidly-cycling immature myeloid progenitors, this cell state becomes perpetuated, yielding malignancy. In humans, high CCND1 expression predicts worse prognosis for MLL fusion AMLs. Our work elucidates one of the earliest steps toward malignancy and suggests that modifying the cycling state of the cell-of-origin could be a preventative approach against malignancy.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Leukemic; Gene Knock-In Techniques; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Male; Mice, Transgenic; Myeloid Progenitor Cells; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Piperazines; Primary Cell Culture; Prognosis; Pyridines

Hepatic ischemia/reperfusion injury (IRI) is a common cause of morbidity and mortality in liver transplantation settings and involves severe cell death and inflammatory responses. MicroRNA-191 has recently been reported to be abnormally expressed in hepatocellular carcinoma and other liver diseases in the regulation of important cellular processes. However, little is known about its function and molecular mechanism in IRI. Here, we demonstrate that miR-191 is significantly upregulated in a cultured cell line during hypoxia/reperfusion (H/R) and in liver tissue during IRI in mice. The activation of miR-191 under hypoxic conditions is mediated by hypoxia-inducible factor-1α (HIF1α) binding to its promoter region. Global miR-191 KO mice were constructed by CRISPR/Cas9 system, and we found that miR-191 deficiency markedly reduces liver tissue damage, cell inflammatory responses and cell death in a mouse hepatic IRI model. Under the H/R condition, miR-191 overexpression promotes G0/G1 cell cycle arrest and cell apoptosis, but inhibition of miR-191 facilitates cell cycle progression and decreases cell death. Mechanistically, upon induction by hypoxia or ischemia, miR-191 suppresses expression of ZO-1-associated Y-box factor (ZONAB) and its downstream factor Cyclin D1, consequently resulting in cell death and tissue injury. Moreover, the effects of miR-191 on cell cycle arrest and cell apoptosis are abrogated by ZONAB overexpression, and vice versa. Taken together, our results indicate an important role of the HIF1α/miR-191/ZONAB signaling pathway in hepatic IRI and suggest miR-191 as a novel therapeutic target for the treatment of liver IRI.

Topics: Animals; Apoptosis; CCAAT-Enhancer-Binding Proteins; Cell Cycle Checkpoints; Cell Hypoxia; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; DNA-Binding Proteins; Gene Knockout Techniques; Heat-Shock Proteins; HEK293 Cells; Hepatocytes; Humans; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Reperfusion Injury; Transcription Factors; Transfection

The associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure promotes the proliferation of the future liver remnant, but evidence to support the feasibility of ALPPS in livers with fibrosis is needed. Therefore the aim of this study was to establish a fibrotic ALPPS model in the rat to compare the capacity of regeneration in the remnant liver with or without fibrosis.. In our study we first established a thioacetamide-induced fibrotic ALPPS model in rats. Then the ALPPS-induced regenerative capacities of normal and fibrotic liver were compared in this animal model. In addition, markers of regeneration, including the proliferative index and cyclin D1 and proliferating cell nuclear antigen levels, as well as various indicators of liver function were determined to evaluate the quality of the hepatic regeneration.. Compared with that of the sham group (opening of the peritoneal cavity with no further operative manipulation), the proliferation of the future liver remnant in fibrotic rat liver after the ALPPS procedure was increased on postoperative days 1, 2, and 5 (P < .039 each). In addition, the proliferative response was greater in the ALPPS group than in the ligation group subjected only to portal vein ligation of the left lateral, left middle, right, and caudate lobes (P = .099, P = .006, and P = .020 on postoperative days 1, 2, and 5, respectively). In contrast, the ALPPS-induced regenerative capacity in the fibrotic rat livers was attenuated compared with that in the normal liver on postoperative days 1, 2, and 5 (P < .031 for each) after stage I and on postoperative day 5 after stage II of the ALPPS procedure (P < .005). This attenuated the recovery of liver function, and the greater mortality rate indicated that functional proliferation was either delayed or not as extensive in the fibrotic rat livers.. Through establishing a rat model of thioacetamide-induced liver fibrosis, we found that ALPPS-derived liver regeneration was present and feasible in fibrotic livers, but this effect was attenuated compared with that in normal liver.

Topics: Animals; Biomarkers; Cyclin D1; Disease Models, Animal; Feasibility Studies; Hepatectomy; Ki-67 Antigen; Ligation; Liver; Liver Cirrhosis; Liver Regeneration; Portal Vein; Proliferating Cell Nuclear Antigen; Random Allocation; Rats, Sprague-Dawley; Thioacetamide

Housefly (Musca domestica) Larvae powder (HL) is rich in antioxidants. As oxidative stress is considered as one of the main pathogenesis in Alzheimer's Disease (AD), this study was designed to explore the protective effects of HL as an antioxidant on APP/PS1 mice. 2-Month-old APP/PS1 mice were divided into a model control (MC) group, a Donepezil group and a HL group, and C57BL/6 mice were used as the normal control (NC) group. After 180 days of treatment, the memory ability was measured by Morris Water Maze (MWM). The presence of Aβ and the expression of Uncoupling Protein 4 (UCP4) and CyclinD1 were detected by immunohistochemistry. The expressions of Superoxide Dismutase 1 (SOD1), Catalase (CAT) and Mitogen-activated Protein Kinase (MAPK) signal pathways were measured by western blotting. Compared with untreated APP/PS1 mice, the memory abilities of the HL-treated mice were significantly improved. Furthermore, the HL treatment not only down-regulated the deposition of Aβ and the expression of CylinD1, but also increased both the mRNA and protein levels of SOD, CAT, and UCP4, and enhanced the phosphorylation of JNK and P38 MAPK activation. In conclusion, these results suggest that HL may have a protective effect against memory impairment and prevent oxidative stress-induced injury via the regulation of UCP4 and CyclinD1 and the modulation of JNK and P38 MAPK signaling in AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cyclin D1; Disease Models, Animal; Houseflies; Humans; Larva; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Mitochondrial Uncoupling Proteins; Neuroprotective Agents; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction

The purpose of this study was to explore the role of microRNA-451a (miR-451a) in diabetic retinopathy through activating transcription factor 2 (ATF2). The epiretinal membrane samples from patients with proliferative diabetic retinopathy (PDR) were immunolabeled with an antibody for Ki-67 to identify the proliferative cells. The expression of miR-451a was measured by qRT-PCR in the retina of Akita mice and in RPE cells under diabetic conditions. The potential downstream targets of miR-451a were predicted by bioinformatics and confirmed by dual luciferase assay, qRT-PCR, and Western blotting. Mitochondrial function, cell proliferation, and migration assays were used to detect the functional change after transfection of miR-451a mimic and inhibitor. Proliferative RPE cells were identified in the epiretinal membrane from PDR patients. The expression of miR-451a was downregulated both in the retina of Akita mice and 4-hydroxynonenal (4-HNE)-treated RPE cells. Bioinformatic analysis and luciferase assay identified ATF2 as a potential target of miR-451a. miR-451a inhibited proliferation and migration of RPE cells. The mitochondrial function was enhanced by miR-451a mimic, but suppressed by miR-451a inhibitor. In diabetic conditions, miR-451a showed a protective effect on mitochondrial function. The results of qRT-PCR and Western blotting revealed that overexpression of miR-451a downregulated the expression of ATF2 and its downstream target genes CyclinA1, CyclinD1, and MMP2. In conclusion, miR-451a/ATF2 plays a vital role in the regulation of proliferation and migration in RPE cells through regulation of mitochondrial function, which may provide new perspectives for developing effective therapies for PDR.

Topics: Activating Transcription Factor 2; Adult; Aged; Animals; Cell Movement; Cell Proliferation; Cyclin A1; Cyclin D1; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Disease Models, Animal; Female; Humans; Male; Matrix Metalloproteinase 2; Mice; MicroRNAs; Middle Aged; Mitochondria; Retinal Pigment Epithelium

Adult neurogenesis is involved in cognitive performance but studies that manipulated this process to improve brain function are scarce. Here, we characterized a genetic mouse model in which neural stem cells (NSC) of the subventricular zone (SVZ) were temporarily expanded by conditional expression of the cell cycle regulators Cdk4/cyclinD1, thus increasing neurogenesis. We found that supernumerary neurons matured and integrated in the olfactory bulb similarly to physiologically generated newborn neurons displaying a correct expression of molecular markers, morphology and electrophysiological activity. Olfactory performance upon increased neurogenesis was unchanged when mice were tested on relatively easy tasks using distinct odor stimuli. In contrast, intriguingly, increasing neurogenesis improved the discrimination ability of mice when challenged with a difficult task using mixtures of highly similar odorants. Together, our study provides a mammalian model to control the expansion of somatic stem cells that can in principle be applied to any tissue for basic research and models of therapy. By applying this to NSC of the SVZ, we highlighted the importance of adult neurogenesis to specifically improve performance in a challenging olfactory task.

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase 4; Discrimination Learning; Disease Models, Animal; Male; Mice; Mice, Transgenic; Neural Stem Cells; Neurogenesis; Odorants; Olfactory Bulb

Primary and secondary traumatic brain injury (TBI) can cause tissue damage by inducing cell death pathways including apoptosis, necroptosis, and autophagy. However, similar pathways can also lead to senescence. Senescent cells secrete senescence-associated secretory phenotype proteins following persistent DNA damage response signaling, leading to cell disorders. TBI initially activates the cell cycle followed by the subsequent triggering of senescence. This study aims to clarify how the mRNA and protein expression of different markers of cell cycle and senescence are modulated and switched over time after TBI. We performed senescence-associated-β-galactosidase (SA-β-gal) staining, immunohistochemical analysis, and real-time PCR to examine the time-dependent changes in expression levels of proteins and mRNA, related to cell cycle and cellular senescence markers, in the cerebrum during the initial 14 days after TBI using a mouse model of controlled cortical impact (CCI). Within the area adjacent to the cerebral contusion after TBI, the protein and/or mRNA expression levels of cell cycle markers were increased significantly until 4 days after injury and senescence markers were significantly increased at 4, 7, and 14 days after injury. Our findings suggested that TBI initially activated the cell cycle in neurons, astrocytes, and microglia within the area adjacent to the hemicerebrum contusion in TBI, whereas after 4 days, such cells could undergo senescence in a cell-type-dependent manner.

Topics: Animals; Apoptosis; Autophagy; beta-Galactosidase; Brain Injuries, Traumatic; Cellular Senescence; Cerebrum; Cyclin D1; Disease Models, Animal; Gene Expression Profiling; Male; Mice; Mice, Inbred C57BL; Neurons; Proliferating Cell Nuclear Antigen; Signal Transduction

Around 80% of nonmelanoma skin cancers (NMSCs) are basal cell carcinoma (BCC), still studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of ultraviolet B (UVB) radiation-induced BCC formation, we excised skin samples from UVB exposed Ptch+/- and Ptch+/+ mice before tumor formation to study the promotion/progression of BCC and to determine the efficacy and target/s of silibinin, a well-known skin cancer chemopreventive agent. UVB exposure for 1 month increased the number of mast cells in Ptch+/- mice by ~48% (P < 0.05), which was completely inhibited by silibinin. Polymerase chain reaction profiler array analysis of skin samples showed strong molecular differences between Ptch+/+ and Ptch+/- mice which were either unexposed or UVB irradiated+/- silibinin treatment. Most notably, silibinin treatment significant decreased the expression of BMP-2, Bbc3, PUMA, and Ccnd1 in Ptch+/- mice irradiated with silibinin + UVB. Additional studies showed that silibinin targets UVB-induced expression of bone morphogenetic protein 2 (BMP-2) in Ptch+/- mouse skin. Last, our studies found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo only after single UVB exposure. Together, our results suggest a possible role of mast cell recruitment and BMP-2 activation in the early stages of BCC development; these are strongly inhibited by silibinin suggesting its possible chemopreventive efficacy against BCC formation in long-term UVB exposure regimen.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis Regulatory Proteins; Bone Morphogenetic Protein 2; Carcinoma, Basal Cell; Chemoprevention; Cyclin D1; Disease Models, Animal; DNA Damage; Mast Cells; Mice; Mice, Transgenic; Patched-1 Receptor; Signal Transduction; Silybin; Skin; Skin Neoplasms; Tumor Suppressor Proteins; Ultraviolet Rays

Prostate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa.. MTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1.. The current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide.. This study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment.

Topics: Animals; Apoptosis; Autophagy; Benzamides; Benzylamines; Biomarkers, Tumor; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glucose; Glucose Transporter Type 1; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Models, Biological; Molecular Targeted Therapy; Nitriles; Phenylthiohydantoin; Prostatic Neoplasms; Quinazolines; Signal Transduction; Xenograft Model Antitumor Assays

Transplanting donor livers with severe macrosteatosis is associated with increased risk of primary non-function (PNF). The purpose of this study was to identify steatosis-driven biomarkers as a predisposition to severe liver damage and delayed recovery following ischemia reperfusion injury. Wistar rats were fed a methionine- and choline-deficient (MCD) diet for up to three weeks to achieve severe macrosteatosis (>90%). Animals underwent diet withdrawal to control chow and/or underwent ischemia reperfusion and partial hepatectomy injury (I/R-PHx) and reperfused out to 7 days on control chow. For animals with severe macrosteatosis, hepatic levels of IL-33 decreased while Cyclin D1 levels increased in the absence of NF-κB p65 phosphorylation. Animals with high levels of nuclear Cyclin D1 prior to I/R-PHx either did not survive or had persistent macrosteatosis after 7 days on control chow. Survival 7 days after I/R-PHx fell to 57% which correlated with increased Cyclin D1 and decreased liver IL-33 levels. In the absence of I/R-PHx, withdrawing the MCD diet normalized IL-33, Cyclin D1 levels, and I/R-PHx survival back to baseline. In transplanted grafts with macrosteatosis, higher Cyclin D1 mRNA expression was observed. Shifts in Cyclin D1 and IL-33 expression may identify severely macrosteatotic livers with increased failure risk if subjected to I/R injury. Clinical validation of the panel in donor grafts with macrosteatosis revealed increased Cyclin D1 expression corresponding to delayed graft function. This pre-surgical biomarker panel may identify the subset of livers with increased susceptibility to PNF.

Topics: Adult; Animals; Biomarkers; Cyclin D1; Diet; Disease Models, Animal; Disease Susceptibility; Fatty Liver; Humans; Interleukin-33; Liver; Liver Failure; Liver Transplantation; Male; Middle Aged; Rats, Wistar; Reperfusion Injury; Survival Analysis

Proton radiation is a major component of the radiation field in outer space and is used clinically in radiation therapy of resistant cancers. Although epidemiologic studies in atom bomb survivors and radiologic workers have established radiation as a risk factor for colorectal cancer (CRC), we have yet to determine the risk of CRC posed by proton radiation owing to a lack of sufficient human or animal data. The purpose of the current study was to quantitatively and qualitatively characterize differential effects of acute and fractionated high-energy protons on colorectal carcinogenesis.. Significantly higher intestinal tumor number and grade, along with decreased differentiation, were observed after acute radiation relative to fractionated radiation. Acute protons induced upregulation of β-catenin and Akt pathways with increased proliferative marker phospho-histone H3. Increased DNA damage along with decreased DNA repair factors involved in mismatch repair and nonhomologous end joining were also observed after exposure to acute protons.. We show increased γH2AX, 53BP1, and 8-oxo-dG, suggesting that increased ongoing DNA damage along with decreased DNA repair factors and increased proliferative responses could be triggering a higher number of intestinal tumors after acute relative to fractionated proton exposures in Apc

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; beta Catenin; Carcinogenesis; Cell Differentiation; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Mismatch Repair; Dose Fractionation, Radiation; Female; Gene Expression; Genes, APC; Histones; Immunoblotting; Intestinal Neoplasms; Intestine, Small; Mice; Mice, Inbred C57BL; Neoplasms, Radiation-Induced; Proto-Oncogene Proteins c-akt; Protons; Radiation Dosage; Radiation Exposure; Space Flight; Tumor Suppressor p53-Binding Protein 1; Up-Regulation

This study investigated the effects of cyclin D1 gene silencing on cell proliferation and apoptosis of interleukin-1β (IL-1β)-induced osteoarthritis (OA) chondrocytes. Chondrocytes from healthy sprague-dawley rats were divided into blank, OA model (chondrocytes underwent IL-1β inducement), OA trial (chondrocytes underwent IL-1β inducement with cyclin D1-shRNA treatment), and negative control (NC; chondrocytes underwent IL-1β inducement and control-shRNA treatment) groups. Cell proliferation was assessed by CCK-8 assay, and cell cycle and apoptosis by flow cytometry. qRT-PCR and Western blotting were performed to detect cyclin D1 and apoptosis-related factors expression levels. Chondrocyte proliferation increased after 72-96 h after incubation. The OA trial group exhibited reduced cell proliferation at 48, 72, and 96 h after treatment. The OA model, OA trial, and NC groups all contained more cells arrested in G1 phase and had higher apoptosis rates than the blank group. Additionally, the OA trial group contained more cells arrested in G1 phase, with increased apoptosis rates compared to the OA model and NC groups. The OA model group had lowest expression of cyclin D1 whereas the blank group contained the highest among the four groups. qRT-PCR also showed that the OA model, OA trial, and NC groups all had increased expression levels of Bax and reduced expression levels of Bcl-2 and P53 compared to the blank group, whereby by the OA group had the most significant change. The combined evidence in our study shows that cyclin D1 gene silencing suppresses proliferation and induces apoptosis of rat chondrocytes in IL-1β-induced OA. J. Cell. Biochem. 119: 290-299, 2018. © 2017 Wiley Periodicals, Inc.

Topics: Animals; Apoptosis; Chondrocytes; Cyclin D1; Disease Models, Animal; Gene Silencing; Interleukin-1beta; Osteoarthritis; Rats; Rats, Sprague-Dawley

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.

Topics: Acrylamides; Animals; Antibodies, Monoclonal; Click Chemistry; Cyclin D1; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Humans; Immunoconjugates; Immunoglobulin Fragments; Leukemia; Mice; Molecular Targeted Therapy; Peptides; Polymers

Depression caused by genetic and environmental factors is acomplicated disease. Here, it is demonstrated that glycogen synthase kinase-3β is highly expressed and phosphorylated in the brain of a chronic stress mouse. Inhibition of glycogen synthase kinase-3βleads to decreased depression-like symptoms which manifest in open-field test, tail-suspension test, forced swim test, and a novelty suppressed feeding test. It was also found that β-catenin is attenuated, and its target genes Cyclin D1 and c-Myc are down-regulated. Glycogen synthase kinase-3β was also found to inhibit Erk-Creb-BDNF signaling. These results show that glycogen synthase kinase-3β may promote the progression of depression. Therefore, targeting glycogen synthase kinase-3β may be an effective therapeutic strategy.

Topics: Animals; Antidepressive Agents; beta Catenin; Brain; Brain-Derived Neurotrophic Factor; Chronic Disease; Cyclin D1; Depressive Disorder; Disease Models, Animal; Feeding Behavior; Glycogen Synthase Kinase 3 beta; Male; Mice, Inbred C57BL; Motor Activity; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-myc; Random Allocation; RNA, Messenger; Stress, Psychological; Thiadiazoles

Signals from growth factors or mechanical stimuli converge to promote vascular smooth muscle cell (VSMC) migration and proliferation, key events in the pathogenesis of intimal hyperplasia upon vascular injury. Spry1, a regulator of receptor tyrosine kinases (RTK), plays a role in maintaining the contractile phenotype of VSMC. The aim of the current study was to determine the role of Spry1 in VSMC proliferation in vitro and injury induced neointimal hyperplasia in vivo. VSMC proliferation and neointima formation were evaluated in cultured human aortic SMC (hAoSMC) and ligation-induced injury of mouse carotid arteries from Spry1 gene targeted mice, and their corresponding wild type littermates. Human Spry1 or non-targeting control lentiviral shRNAs were used to knock down Spry1 in hAoSMC. Time course cell cycle analysis showed a reduced fraction of S-phase cells at 12 and 24 h after growth medium stimulation in Spry1 shRNA transduced hAoSMC. Consistent with reduced S-phase entry, the induction of cyclinD1 and the levels of pRbS807/S811, pH3Ser10, and pCdc2 were also reduced, while the cell cycle inhibitor p27

Topics: Adaptor Proteins, Signal Transducing; Animals; Carotid Artery Injuries; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Gene Knockdown Techniques; Humans; Membrane Proteins; Mice; Muscle, Smooth, Vascular; Neointima; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.

Topics: Animals; Bone Marrow Cells; Cyclin D1; Disease Models, Animal; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Gene Silencing; Glial Fibrillary Acidic Protein; Humans; Locomotion; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats; Receptor, Nerve Growth Factor; Recovery of Function; RNA, Small Interfering; Spinal Cord; Spinal Cord Injuries

Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exhibits a diverse range of neuroprotective mechanisms against a variety of neurological disorders including ischemic stroke. We investigated the anti-apoptotic mechanisms of G-CSF against endoplasmic reticulum (ER) stress induced apoptosis. Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90 min. Rats were injected with G-CSF (n = 15; 50 μg/kg body weight s.c.) for 4 days, starting 24 h post-MCAO and brains were harvested after 4 days reperfusion (n = 16). Key proteins in ER stress apoptosis were analyzed by immunoblotting. G-CSF reduced infarct volume to 53% and improved neurological deficits. G-CSF treatment significantly (P < .05) attenuated the expression of proteins involved in ER stress apoptosis pathway; ATF4, ATF6, p-p38MAPK, pJNK and CHOP. G-CSF treatment also re-established ER homeostasis evident by the reduction of the intraluminal ER stress sensor, GRP78 as well as reducing the overall cellular stress level protein, HSP27. G-CSF also up-regulated anti-apoptotic proteins pAKT and Bcl-2 while down-regulated the pro-apoptotic protein Bax. G-CSF exerts neuroprotection from cerebral ischemia through the preservation of the ER, resulting in the attenuation of pro-apoptotic proteins and the potentiation of anti-apoptotic proteins.

Topics: Activating Transcription Factor 6; Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Brain Infarction; Cyclin D1; Disease Models, Animal; Endoplasmic Reticulum Stress; Granulocyte Colony-Stimulating Factor; Infarction, Middle Cerebral Artery; Male; Neurologic Examination; Neuroprotective Agents; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Receptors, Granulocyte Colony-Stimulating Factor; Signal Transduction

Rhodiola algida var. tangutica (Maxim.) S.H. Fu is a perennial plant of the Crassulaceae family that grows in the mountainous regions of Asia. The rhizome and roots of this plant have been long used as Tibetan folk medicine for preventing high latitude sickness.. The aim of this study was to determine the effect of bioactive fraction from R. algida (ACRT) on chronic hypoxia-induced pulmonary arterial hypertension (HPAH) and to understand the possible mechanism of its pharmacodynamic actions.. Male Sprague-Dawley rats were separated into five groups: control group, hypoxia group, and hypoxia+ACRT groups (62.5, 125, and 250mg/kg/day of ACRT). The chronic hypoxic environment was created in a hypobaric chamber by adjusting the inner pressure and oxygen content for 4 weeks. After 4 weeks, major physiological parameters of pulmonary arterial hypertension such as mPAP, right ventricle index (RV/LV+S, RVHI), hematocrit (Hct) levels and the medial vessel thickness (wt%) were measured. Protein and mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, p27Kip1 and cyclin-dependent kinase 4 (CDK4)) were detected by western blotting and real time PCR respectively. Chemical profile of ACRT was revealed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS).. The results showed that a successful HPAH rat model was established in a hypobaric chamber for 4 weeks, as indicated by the significant increase in mPAP, RV/LV+S, RV/BW and wt%. Compared with the normal group, administration of ACRT reduced mPAP, right ventricular hypertrophy, pulmonary small artery wall thickness, and damage in ultrastructure induced by hypoxia in rats. PCNA, cyclin D1, and CDK4 expression was reduced (p<0.05), and p27Kip1 expression increased (p<0.05) in hypoxia+ACRT groups compared to hypoxia. 38 constituents in bioactive fraction were identified by UHPLC-Q-TOF-MS/MS.. Our results suggest that ACRT could alleviate chronic hypoxia-induced pulmonary arterial hypertension. And its anti-proliferation mechanism in rats based on decreasing PCNA, cyclin D1, CDK4 expression level and inhibiting p27Kip1 degradation.

Topics: Animals; Arterial Pressure; Cell Proliferation; Chronic Disease; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Phytotherapy; Plant Extracts; Plants, Medicinal; Proliferating Cell Nuclear Antigen; Proteolysis; Pulmonary Artery; Rats, Sprague-Dawley; Rhodiola; Signal Transduction; Vascular Remodeling; Ventricular Function, Right; Ventricular Remodeling

Patients with a deep burn injury are characterized by losing the function of perspiration and being unable to regenerate the sweat glands. Because of their easy accession, multipotency, and lower immunogenicity, bone marrow-derived mesenchymal stem cells (BM-MSCs) represent as an ideal biological source for cell therapy. The aim of this study was to identify whether targeting the promotor of ectodysplasin (EDA) by CRISPR/dCas9-effector (dCas9-E) could induce the BM-MSCs to differentiate into sweat gland-like cells (SGCs).. Activation of EDA transcription in BM-MSCs was attained by transfection of naive BM-MSCs with the lenti-CRISPR/dCas9-effector and single-guide RNAs (sgRNAs). The impact of dCas9-E BM-MSCs on the formation of SGCs and repair of burn injury was identified and evaluated both in vitro and in a mouse model.. After transfection with sgRNA-guided dCas9-E, the BM-MSCs acquired significantly higher transcription and expression of EDA by doxycycline (Dox) induction. Intriguingly, the specific markers (CEA, CK7, CK14, and CK19) of sweat glands were also positive in the transfected BM-MSCs, suggesting that EDA plays a critical role in promoting BM-MSC differentiation into sweat glands. Furthermore, when the dCas9-E BM-MSCs with Dox induction were implanted into a wound in a laboratory animal model, iodine-starch perspiration tests revealed that the treated paws were positive for perspiration, while the paws treated with saline showed a negative manifestation. For the regulatory mechanism, the expression of downstream genes of NF-κB (Shh and cyclin D1) was also enhanced accordingly.. These results suggest that EDA is a pivotal factor for sweat gland regeneration from BM-MSCs and may also offer a new approach for destroyed sweat glands and extensive deep burns.

Topics: Animals; Bone Marrow Cells; Burns; Cell Differentiation; Cell Line; Cell- and Tissue-Based Therapy; Cellular Reprogramming Techniques; CRISPR-Cas Systems; Cyclin D1; Disease Models, Animal; Doxycycline; Ectodysplasins; Gene Editing; Hedgehog Proteins; HEK293 Cells; Humans; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Promoter Regions, Genetic; RNA, Guide, Kinetoplastida; Sweat Glands

The imbalance between cell proliferation and apoptosis can lead to tumor progression, causing oncogenic transformation, abnormal cell proliferation and cell apoptosis suppression. Tea polysaccharide (TPS) is the major bioactive component in green tea, it has showed antioxidant, antitumor and anti-inflammatory bioactivities. In this study, the chemoprophylaxis effects of TPS on colitis-associated colon carcinogenesis, especially the cell apoptosis activation and inhibition effects on cell proliferation and invasion were analyzed. The azoxymethane/dextran sulfate sodium (AOM/DSS) was used to induce the colorectal carcinogenesis in mice. Results showed that the tumor incidence was reduced in TPS-treated AOM/DSS mice compared to AOM/DSS mice. TUNEL staining and Ki-67 immunohistochemistry staining showed that the TPS treatment increased significantly the cell apoptosis and decreased cell proliferation among AOM/DSS mice. Furthermore, TPS reduced the expression levels of the cell cycle protein cyclin D1, matrix metalloproteinase (MMP)-2, and MMP-9. In addition, in vitro studies showed that TPS, suppressed the proliferation and invasion of the mouse colon cancer cells. Overall, our findings demonstrated that TPS could be a potential agent in the treatment and/or prevention of colon tumor, which promoted the apoptosis and suppressed the proliferation and invasion of the mouse colon cancer cells via arresting cell cycle progression.

Topics: Animals; Apoptosis; Biomarkers; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colitis; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Disease Progression; Gene Expression; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Plant Extracts; Polysaccharides; Tea

The current study was designed and performed to investigate the effect of mefloquine on the proliferation and tumor formation potential of liver cancer stem cells. CD133 + HepG2 cells were identified using MACS and showed markedly higher tumor formation potential compared to the parental cells. The secondary tumors formed by CD133 + cells were markedly large in size and more in number compared to the parental cells. Mefloquine treatment of CD133 + HepG2 cells inhibited the proliferation selectively in concentration based manner. The rate of proliferation was inhibited to 82 and 12% in parental and CD133 + sphere forming cells, respectively on treatment with 10 μM concentration of mefloquine. The number of secondary tumors formed by primary tumors was decreased significantly on treatment with 10 μM mefloquine concentration. Treatment of the liver cancer stem cells with mefloquine markedly decreased the potential to undergo self-renewal at 10 μM concentration after 48 h. The results from western blot analysis showed significantly higher expression of cancer stem cell molecules β-catenin and cyclin D1 in LCSCs. Treatment of the LCSCs with various concentrations of mefloquine reduced the expression levels of β-catenin and cyclin D1. Administration of the CD133 + cell tumor xenografts in the mice led to the formation of large sized tumors in the control group. However, the tumor growth was inhibited significantly in the mice on treatment with 10 mg/kg doses of mefloquine after day 21. The tumor weight was significantly lower in the animals of mefloquine treatment group compared to the control group. Thus, mefloquine treatment inhibits self-renewal and proliferation potential of cells through targeting β-catenin pathway.

Topics: AC133 Antigen; Animals; beta Catenin; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cyclin D1; Disease Models, Animal; Drug Combinations; Hep G2 Cells; Humans; Lithium Chloride; Liver Neoplasms; Male; Mefloquine; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Transplantation, Heterologous

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Animals, Newborn; bcl-2-Associated X Protein; Caspases; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Hypoxia; Membrane Potential, Mitochondrial; Mental Disorders; Mitochondrial Diseases; NAD; Oxidative Stress; Oxidoreductases; Pregnancy; Rats; Succinate Dehydrogenase

The present study, attempts to validate the molecular mechanism(s) of Poly-l-lysine (PLL) induced apoptosis, anti-proliferative and anti-tumorigenic properties in in-vitro HUVECs cells and Dalton's Ascitic Lymphoma (DAL) and in in-vivo DAL cell bearing BALB/c mice model.. The cell proliferation assay and morphological assay was carried out using the MTT assay and Giemsa staining method. The antitumor activity of PLL was evaluated in BALB/c mice at 20 and 40 mg/kg/b.w doses for 21 days for DAL solid tumor model. Several tumor evaluation endpoints, hematological and biochemical parameters were estimated. Additionally, the tumor apoptosis, anti-proliferative and anti-tumor angiogenesis effects were assessed using western blots and immunohistochemistry.. PLL significantly decreased cell proliferation in in-vitro HUVECs and DAL cells without significant effects on normal cell growth. PLL also induced alteration in cellular morphology in DAL cells. Therafter, in the BALB/c mouse model, PLL had noticeable inhibition in DAL-induced tumorigenesis. This inhibition was evident through reduced solid tumor volume and weight versus the control group. However, PLL promoted tumor apoptosis and suppressed cell-proliferation and tumor-angiogenesis. PLL also increased hematological markers significantly compared to 5-flurouracil (5-FU). The amount of TdT in the nuclei of DAL cells in mice treated with PLL was significantly increased while in contrast decreases of anti-apoptotic protein Bcl-2 expression were observed. PLL also significantly upregulated the pro-apoptotic protein Bax and activated caspase-3. Measurable decreases of cyclin-D. The present study offers opportunities and hopes for possible anti-tumortherapies with PLL in the near future and warrants further formulation developments.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Ascites; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cyclin D1; Disease Models, Animal; Down-Regulation; Female; Human Umbilical Vein Endothelial Cells; Humans; Kaplan-Meier Estimate; Ki-67 Antigen; Lymphoma; Mice, Inbred BALB C; Neovascularization, Pathologic; Polylysine; Survival Analysis

Tumor necrosis factor ligand-related molecule 1 A (TLlA) is closely related to the occurrence and development of inflammatory bowel disease.. We aimed to explore whether TLlA was involved in the occurrence of colitis-associated colorectal cancer (CAC).. Firstly, azoxymethane (AOM) and dextran sulfate sodium (DSS) were used to construct the CAC mice model in wild-type (WT) and TL1A transgenic (Tg) mice with TL1A high expression. The histopathological analysis was used for the evaluation of inflammation level, and the immunohistochemistry staining analysis was used to test the expression and location of proliferating cell nuclear antigen (PCNA) and β-catenin. Secondly, the HCT116 and HT29 cell lines were used for knockdown of TL1A gene for further assay including cell viability, cell clone, cell apoptosis and matrigel invasion. Western blot were used for quantitative protein expression of β-catenin and downstream oncogenes including c-myc and Cyclin D1 after knockdown of TL1A gene.. The evaluation of inflammation level showed that the disease activity index score and tumor formation rate were significantly higher in AOM + DSS/Tg group than that in AOM + DSS/WT group. The expression of PCNA, β-catenin, c-myc, and Cyclin D1 in AOM + DSS/Tg group was significantly higher than that in AOM + DSS/WT group. The cell experiment showed that TL1A knockdown inhibited the cell proliferation, invasion, and migration. Moreover, the expression of c-myc and Cyclin D1 was significantly decreased after TL1A knockdown.. TL1A can induce tumor cell proliferation and promote the occurrence of CAC by activating Wnt/β-catenin pathway.

Topics: Animals; Azoxymethane; beta Catenin; Cell Movement; Cell Proliferation; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Invasiveness; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Time Factors; Tumor Necrosis Factor Ligand Superfamily Member 15; Wnt Signaling Pathway

Taraxasterol has potent anti-inflammatory and anti-tumor activity. However, the effect and potential mechanisms of Taraxasterol on the growth of human liver cancer have not been clarified. Histidine triad nucleotide-binding protein 1 (Hint1) is a tumor suppressor and its downregulated expression is associated with the development of cancer. Here, we report that Taraxasterol treatment significantly suppressed cell proliferation and induced cell cycle arrest at G0/G1 phase and apoptosis in liver cancer cells, but not in non-tumor hepatocytes. Furthermore, Taraxasterol upregulated Hint1 and Bax, but downregulated Bcl2 and cyclin D1 expression, accompanied by promoting the demethylation in the Hint1 promoter region in liver cancer cells. The effects of Taraxasterol were abrogated by Hint1 silencing and partially mitigated by Bax silencing, Bcl2 or cyclin D1 over-expression in HepG2 cells. Moreover, oral administration with Taraxasterol did not affect body weight, urinary protein levels, and the heart, liver, and kidney morphology in BALB/c mice but effectively inhibited the growth of implanted SK-Hep1 tumor in vivo. Collectively, we demonstrate that Taraxasterol inhibits the growth of liver cancer at least partially by enhancing Hint1 expression to regulate Bax, Bcl2, and cyclin D1 expression. Taraxasterol may be a drug candidate for the treatment of human liver cancer.. Taraxasterol inhibits growth and induces apoptosis in human liver cancer cells. Taraxasterol enhances Hint1 expression by promoting demethylation in Hint1 promoter. Taraxasterol increases Hint1 levels to regulate Bax, Bcl2, and cyclinD1 expression. The effects of Taraxasterol are abrogated by Hint1 silencing in liver cancer cells. Taraxasterol inhibits the growth of subcutaneously implanted liver cancers in mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Nerve Tissue Proteins; Proto-Oncogene Proteins c-bcl-2; Sterols; Triterpenes; Xenograft Model Antitumor Assays

Atherosclerosis (AS) is the key cause of many cardiovascular and cerebrovascular diseases. The inflammatory response and lipid metabolism disorders contribute to the development and progression of AS. This work aims to study the anti-inflammatory effect and mechanism of low molecular weight fucoidan (LMWF) obtained from Saccharina japonica on atherosclerosis in apoE-knockout mice. The experimental results showed that LMWF statistically decreased the levels of triglyceride (TRIG) and oxidative low-density lipoproteins (ox-LDL) and stabilized established atherosclerotic lesions. LMWF ameliorated the inflammatory response by down regulating IL-6 and by up regulating IL-10 transcriptional levels, and LMWF returned p-JNK and cyclin D1 to normal levels. Moreover, LMWF increased the mRNA level of CD11b in the aorta and suppressed the expression of CD11b in the intimal layer of the aorta. Therefore, LMWF prevented macrophages from developing into foam cells and prevented SMCs from migrating into the intimal layer of the aorta, which inhibited the formation of atherosclerotic plaques; and ameliorated the occurrence and development of AS.

Topics: Animals; Anti-Inflammatory Agents; Apolipoproteins E; Atherosclerosis; Cyclin D1; Disease Models, Animal; Gene Expression Regulation; Humans; Interleukin-10; Interleukin-6; Lipid Metabolism; Lipid Peroxidation; MAP Kinase Kinase 4; Mice; Mice, Knockout; Molecular Weight; Phaeophyceae; Polysaccharides

Our aim was to investigate the role of interleukin-6 (IL-6) in myocardial regeneration from mice after cardiac injury. The newborn mice were divided into the following 4 groups (16 in each group): sham group, model group, IL-6-/- group (IL-6 knockout) and IL-6 group (IL-6 overexpression). Electrocardiography was performed on all mice and found higher LVEDD, LVESD and IVST and lower LVEF and LVFS in the IL-6 group compared with the sham group. Using HE staining, severe myocardial injury combined with infarction and fibrosis were observed in the IL-6-/- group, while the damaged myocardial tissue was repaired to some extent in the IL-6 group. The expression of IL-6 in the IL-6 group were significantly up-regulated. BrdU immunofluorescence found that the IL-6-/- group had the least number of BrdU positive cells, while the IL-6 group had more BrdU positive cells than the model group and the IL-6-/- group. Expressions of IL-6, cyclinD1 and Bcl-2 in the IL-6 group were up-regulated compared with other groups. In conclusion, IL-6 overexpression could enhance cardiomyocyte proliferation and relevant protein expression in mice myocardium, thus promoting cardiac regeneration.

Topics: Animals; Animals, Newborn; Cell Proliferation; Cyclin D1; Disease Models, Animal; Heart Diseases; Interleukin-6; Mice, Knockout; Myocytes, Cardiac; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Regeneration; Signal Transduction; STAT3 Transcription Factor; Stroke Volume; Vascular Endothelial Growth Factor A; Ventricular Function, Left

Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The. Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the. Abemaciclib induced quiescence in ES cell lines via a G. Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

Topics: Aminopyridines; Animals; Benzimidazoles; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Humans; Interferons; Mice; Sarcoma, Ewing; Signal Transduction; Xenograft Model Antitumor Assays

Epigallocatechin‑3‑gallate (EGCG) is a polyphenolic compound extracted and isolated from green tea, which has a variety of important biological activities in vitro and in vivo, including anti‑tumor, anti‑oxidation, anti‑inflammation and lowering blood pressure. The aim of the present study was to investigate the protective effect of EGCG against secondary osteoporosis in a mouse model via the Wnt/β‑catenin signaling pathway. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting were used to analyze runt‑related transcription factor 2 and osterix mRNA expression, and the protein expression of cyclin D1, Wnt and β‑catenin, and suppressed peroxisome proliferator‑activated receptor γ protein expression. The protective effect of EGCG against secondary osteoporosis was examined and its potential mechanism was analyzed. Treatment with EGCG significantly decreased serum calcium, urinary calcium, body weight and body fat, and increased leptin levels in mice with secondary osteoporosis. In addition, EGCG treatment significantly inhibited the structure score of articular cartilage and cancellous bone in proximal tibia metaphysis in mice with secondary osteoporosis. Treatment also significantly decreased alkaline phosphatase activity, runt‑related transcription factor 2 and osterix mRNA expression. EGCG also significantly induced the protein expression of cyclin D1, Wnt and β‑catenin, and suppressed peroxisome proliferator‑activated receptor γ protein expression in mice with secondary osteoporosis. Taken together, these results suggest that EGCG may be a possible new drug in clinical settings.

Topics: Animals; beta Catenin; Calcium; Cartilage; Catechin; Cell Proliferation; Cyclin D1; Disease Models, Animal; Humans; Mice; Osteoporosis; PPAR gamma; Tea; Wnt Signaling Pathway

A feasible large animal model to evaluate regenerative medicine techniques is vital for developing clinical applications. One such appropriate model could be to use retrorsine (RS) together with partial hepatectomy (PH). Here, we have developed the first porcine model using RS and PH. RS or saline control was administered intraperitoneally to Göttingen miniature pigs twice, two weeks apart. Four weeks after the second dose, animals underwent PH. Initially, we tested different doses of RS and resection of different amounts of liver, and selected 50 mg/kg RS with 60% hepatectomy as our model for further testing. Treated animals were sacrificed 3, 10, 17 or 28 days after PH. Blood samples and resected liver were collected. Serum and liver RS content was determined by Liquid Chromatograph-tandem Mass Spectrometer. Blood analyses demonstrated liver dysfunction after PH. Liver regeneration was significantly inhibited 10 and 17 days after PH in RS-treated animals, to the extent of 20%. Histological examination indicated hepatic injury and regenerative responses after PH. Immunohistochemical staining demonstrated accumulation of Cyclin D1 and suppression of Ki-67 and PCNA in RS-treated animals. We report the development of the first large animal model of sustained liver injury with suppression of hepatic regeneration.

Topics: Animals; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatectomy; Hepatocytes; Ki-67 Antigen; Liver; Liver Regeneration; Pyrrolizidine Alkaloids; Regenerative Medicine; Swine; Swine, Miniature

Cholestasis impairs liver regeneration following partial liver resection (PHx). Bile acid receptor farnesoid X-receptor (FXR) is a key mediator of liver regeneration. The effects of FXR agonist obeticholic acid (OCA) on liver (re)growth were therefore studied in cholestatic rats. Animals underwent sham surgery or reversible bile duct ligation (rBDL). PHx with concurrent internal biliary drainage was performed 7 days after rBDL. Animals were untreated or received OCA (10 mg/kg/day) per oral gavage from rBDL until sacrifice. After 7 days of OCA treatment, dry liver weight increased in the rBDL + OCA group, indicating OCA-mediated liver growth. Enhanced proliferation in the rBDL + OCA group prior to PHx concurred with a rise in Ki67-positive hepatocytes, elevated hepatic Ccnd1 and Cdc25b expression, and an induction of intestinal fibroblast growth factor 15 expression. Liver regrowth after PHx was initially stagnant in the rBDL + OCA group, possibly due to hepatomegaly prior to PHx. OCA increased hepatobiliary injury markers during BDL, which was accompanied by upregulation of the bile salt export pump. There were no differences in histological liver injury. In conclusion, OCA induces liver growth in cholestatic rats prior to PHx but exacerbates biliary injury during cholestasis, likely by forced pumping of bile acids into an obstructed biliary tree.

Topics: Administration, Oral; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; cdc25 Phosphatases; Chenodeoxycholic Acid; Cholestasis; Cyclin D1; Disease Models, Animal; Fibroblast Growth Factors; Gene Expression Regulation; Liver Regeneration; Male; Organ Size; Rats

Histones chaperones have been found to play critical roles in tumor development and progression. However, the role of histone chaperone CHAF1A in gastric carcinogenesis and its underlying mechanisms remain elusive.. CHAF1A expression in gastric cancer (GC) was analyzed in GEO datasets and clinical specimens. CHAF1A knockdown and overexpression were used to explore its functions in gastric cancer cells. The regulation and potential molecular mechanism of CHAF1A expression in gastric cancer cells were studied by using cell and molecular biological methods.. CHAF1A was upregulated in GC tissues and its high expression predicted poor prognosis in GC patients. Overexpression of CHAF1A promoted gastric cancer cell proliferation both in vitro and in vivo, whereas CHAF1A suppression exhibited the opposite effects. Mechanistically, CHAF1A acted as a co-activator in the Wnt pathway. CHAF1A directly interacted with TCF4 to enhance the expression of c-MYC and CCND1 through binding to their promoter regions. In addition, the overexpression of CHAF1A was modulated by specificity protein 1 (Sp1) in GC. Sp1 transcriptionally enhanced the expression of CHAF1A in GC. Furthermore, CHAF1A expression induced by Helicobacter pylori was Sp1 dependent.. CHAF1A is a potential oncogene in GC, and may serve as a novel therapeutic target for GC treatment.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chromatin Assembly Factor-1; Cyclin D1; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Mice; Models, Biological; Protein Binding; Proto-Oncogene Proteins c-myc; Sp1 Transcription Factor; Stomach Neoplasms; Transcription Factor 4

Some traditional Oriental herbal medicines, such as Acorus tatarinowii and Acorus gramineus, produce beneficial effects for cognition enhancement. An active compound in rhizomes and the bark of these plants is α-asarone.. This study investigated the effects of α-asarone on the proliferation and differentiation of neural progenitor cells (NPCs) in a primary culture and a murine model of ischemic stroke.. NPCs were isolated from mouse fetal cerebral cortices on embryonic day 15, and all experiments were performed using passage 3 NPCs. We utilized a cell counting kit-8 assay, flow cytometry, western blot, and immunohistochemical analysis to assess proliferation and differentiation of NPCs and employed α-asarone in NPC transplanted ischemic stroke mice to evaluate stroke-related functional recovery using behavioral and immunohistochemical analysis.. Treatment with 1 µM, 3 µM, or 10 μM α-asarone induced significant NPC proliferation compared to vehicle treatment. Induced NPCs expressed the neuronal marker neuronal nuclei (NeuN) or the astrocyte marker S100 calcium-binding protein B (S100β). Both immunohistochemistry and flow cytometry revealed that treatment with α-asarone increased the number of NeuN-immunoreactive cells and decreased the number of S100β-immunoreactive cells. Treatment with α-asarone also increased the expression of β-catenin, cyclin D1, and phosphorylated extracellular signal-regulated kinase (ERK) compared to vehicle treatment. In a murine model of ischemic stroke, treatment with α-asarone and transplanted NPCs alleviated stroke-related functional impairments. The corner and rotarod test results revealed that treatment with α-asarone in the NPC transplanted group had greater-than-additive effects on sensorimotor function and motor balance. Moreover, α-asarone treatment promoted the differentiation of transplanted NPCs into NeuN-, glial fibrillary acidic protein (GFAP)-, platelet-derived growth factor-α (PDGFR-α)-, and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase)-immunoreactive cells.. α-asarone may promote NPC proliferation and differentiation into neuron-lineage cells by activating β-catenin, cyclin D1, and ERK. Moreover, α-asarone treatment facilitated neurofunctional recovery after NPC transplantation in a murine model of ischemic stroke. Therefore, α-asarone is a potential adjunct treatment to NPC therapy for functional restoration after brain injuries such as ischemic stroke.

Topics: Acorus; Allylbenzene Derivatives; Animals; Anisoles; Astrocytes; beta Catenin; Brain Ischemia; Cell Differentiation; Cyclin D1; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Mice; Mice, Inbred C57BL; Neural Stem Cells; Neurons; Stroke

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Topics: Animals; Antineoplastic Agents; Carcinogenesis; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Disease Models, Animal; Endometrial Neoplasms; Female; Humans; Mice; Mice, Knockout; Piperazines; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Pyridines; Tamoxifen; Transplantation, Heterologous

The telomerase/telomere interacting protein PinX1 has been suggested as a tumor suppressor. However, the clinical and biological significance of PinX1 in human non-small cell lung cancer (NSCLC) is unclear.. PinX1 gene/expression pattern and its association with NSCLC patient survival were analyzed in cBioportal Web resource and two cohorts of NSCLC samples. A series of in vivo and in vitro assays were performed to elucidate the function of PinX1 on NSCLC cells proliferation and underlying mechanisms.. More frequency of gene PinX1 homozygous deletion and heterozygote deficiency was first retrieved from cBioportal Web resource. Low expression of PinX1 correlated with smoking condition, histological type, T stage, N stage, M stage and TNM stage, and was an independent predictor for overall survival in a learning cohort (n = 93) and a validation cohort (n = 51) of NSCLC patients. Furthermore, knockdown of PinX1 dramatically accelerated NSCLC cell proliferation and G1/S transition, whereas ectopic overexpression of PinX1 substantially inhibited cell viability and cell cycle transition in vitro and in vivo. p15/cyclin D1 pathway and BMP5 might contribute to PinX1-associated cell proliferation and cell cycle transition.. The cost-effective expression of PinX1 could constitute a novel molecular predictor/marker for NSCLC management.

Topics: Adult; Aged; Animals; Biomarkers, Tumor; Bone Morphogenetic Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Databases, Nucleic Acid; Disease Models, Animal; Female; Gene Deletion; Gene Silencing; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; Signal Transduction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

To resolve the controversy as to whether periostin plays a role in myocardial regeneration after myocardial infarction (MI), we created a neonatal mouse model of MI to investigate the influence of periostin ablation on myocardial regeneration and clarify the underlying mechanisms.. Neonatal periostin-knockout mice and their wildtype littermates were subjected to MI or sham surgery. In the wildtype mice after MI, fibrosis was detectable at 3 days and fibrotic tissue was completely replaced by regenerated myocardium at 21 days. In contrast, in the knockout mice, significant fibrosis in the infarcted area was present at even 3 weeks after MI. Levels of phosphorylated-histone 3 and aurora B in the myocardium, detected by immunofluorescence and western blotting, were significantly lower in knockout than in wildtype mice at 7 days after MI. Similarly, angiogenesis was decreased in the knockout mice after MI. Expression of both the endothelial marker CD-31 and α-smooth muscle actin was markedly lower in the knockout than in wildtype mice at 7 days after MI. The knockout MI group had elevated levels of glycogen synthase kinase (GSK) 3β and decreased phosphatidylinositol 3-kinase (PI3K), phosphorylated serine/threonine protein kinase B (p-Akt), and cyclin D1, compared with the wildtype MI group. Similar effects were observed in experiments using cultured cardiomyocytes from neonatal wildtype or periostin knockout mice. Administration of SB216763, a GSK3β inhibitor, to knockout neonatal mice decreased myocardial fibrosis and increased angiogenesis in the infarcted area after MI.. Ablation of periostin suppresses post-infarction myocardial regeneration by inhibiting the PI3K/GSK3β/cyclin D1 signalling pathway, indicating that periostin is essential for myocardial regeneration.

Topics: Animals; Animals, Newborn; Cell Adhesion Molecules; Cells, Cultured; Cyclin D1; Disease Models, Animal; Fibrosis; Mice, Knockout; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Regeneration; Repressor Proteins; Signal Transduction; Time Factors

Scrib is a membrane protein that is involved in the maintenance of apical-basal cell polarity of the epithelial tissues. However, Scrib has also been shown to be mislocalized to the cytoplasm in breast and prostate cancer. Here, for the first time, we report that Scrib not only translocates to the cytoplasm but also to the nucleus in hepatocellular carcinoma (HCC) cells, and in mouse and human liver tumor samples. We demonstrate that Scrib overexpression suppresses the growth of HCC cells in vitro, and Scrib deficiency enhances liver tumor growth in vivo. At the molecular level, we have identified the existence of a positive feed-back loop between Yap1 and c-Myc in HCC cells, which Scrib disrupts by simultaneously regulating the MAPK/ERK and Hippo signaling pathways. Overall, Scrib inhibits liver cancer cell proliferation by suppressing the expression of three oncogenes, Yap1, c-Myc and cyclin D1, thereby functioning as a tumor suppressor in liver cancer.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Gene Expression; Heterografts; Hippo Signaling Pathway; Humans; Liver Neoplasms; MAP Kinase Signaling System; Membrane Proteins; Mice; Phosphoproteins; Protein Binding; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins c-myc; Signal Transduction; Transcription Factors; Tumor Suppressor Proteins; YAP-Signaling Proteins

Topics: Activating Transcription Factor 4; Acute Kidney Injury; Animals; Antigens, Differentiation, B-Lymphocyte; Apoptosis; Autophagy; Cell Hypoxia; Cell Line; Cell Proliferation; Cyclin D1; Disease Models, Animal; Eukaryotic Initiation Factor-2; Female; Genetic Predisposition to Disease; Histocompatibility Antigens Class II; Intramolecular Oxidoreductases; Kidney Tubules, Proximal; Macrophage Migration-Inhibitory Factors; Male; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Regeneration; Reperfusion Injury; Secretory Leukocyte Peptidase Inhibitor; Signal Transduction; Time Factors; Transfection

Epigenetic mechanisms play a key role in gastrointestinal cancer (GIC) development and progression, and most studies have been focused on aberrant DNA methylation and histone modifying enzymes. However, the histone H3-H4 chaperone ASF1A is an important factor regulating chromatin assembling and gene transcription, while it is currently unclear whether ASF1A is involved in cancer pathogenesis. The present study is thus designed to address this issue. Here we showed that ASF1A expression was widespread in GIC-derived cell lines and up-regulated in primary GIC. Higher levels of ASF1A expression predicted significantly shorter patient overall survival in colorectal cancer (P=0.0012). The further analyses of the GEO dataset validate higher ASF1A expression as a prognostic factor for CRC patients. Mechanistically, ASF1A interacted with β-catenin and promoted the transcription of β-catenin target genes including c-MYC, cyclin D1, ZEB1 and LGR5, thereby stimulating proliferation, stemness and migration/invasion of GIC cells. β-Catenin inhibition abolished these effects of ASF1A. Moreover, the ASF1A-β-catenin-ZEB1 axis down-regulated E-Cadherin expression, thereby contributing to enhanced migration/invasion of GIC cells. ASF1A over-expression and depletion facilitated and inhibited in vivo tumor growth and/or metastasis in mouse xenograft models, respectively. Taken together, ASF1A is aberrantly over-expressed in GIC tumors and plays key roles in GIC development and progression by stimulating the transcription of β-catenin target genes. ASF1A may thus be a novel target for GIC therapy and a potential prognostic marker.

Topics: Animals; beta Catenin; Biomarkers, Tumor; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cyclin D1; Disease Models, Animal; Disease Progression; Gastrointestinal Neoplasms; Gene Expression; Genes, myc; Heterografts; Histone Chaperones; Humans; Immunohistochemistry; Molecular Chaperones; Prognosis; Promoter Regions, Genetic; Survival Analysis

Oesophageal cancer is one of the most common malignancies worldwide,and oesophageal squamous cell carcinoma (ESCC) is the predominant histological type both globally and in China. Collagen triple helix repeat containing 1 (CTHRC1) has been found to be upregulated in ESCC. However, its role in tumourigenesis and progression of ESCC remains unclear.. Using our previous ESCC mRNA profiling data, we screened upregulated genes to identify those required for proliferation. Immunohistochemistry was performed to determine the level of CTHRC1 protein expression in 204 ESCC patients. Correlations between CTHRC1 expression and clinicopathological characteristics were assessed. In addition, pyrosequencing and 5-aza-dC treatment were performed to evaluate methylation status of CTHRC1 promoter. In vitro and in vivo analyses were also conducted to determine the role of CTHRC1 in ESCC cell proliferation, migration and invasion, and RNA sequencing and molecular experiments were performed to study the underlying mechanisms.. Based on mRNA profiling data, CTHRC1 was identified as one of the most significantly upregulated genes in ESCC tissues (n = 119, fold change = 20.5, P = 2.12E-66). RNA interference screening also showed that CTHRC1 was required for cell proliferation. Immunohistochemistry confirmed markedly high CTHRC1 protein expression in tumour tissues, and high CTHRC1 expression was positively correlated with advanced T stage (P = 0.043), lymph node metastasis (P = 0.023), TNM stage (P = 0.024) and poor overall survival (P = 0.020). Promoter hypomethylation at cg07757887 may contribute to increased CTHRC1 expression in ESCC cells and tumours. Forced overexpression of CTHRC1 significantly enhanced cell proliferation, migration and invasion, whereas depletion of CTHRC1 suppressed these cellular functions in three ESCC cell lines and xenografts. CTHRC1 was found to activate FRA-1 (Fos-related antigen 1, also known as FOSL1) through the MAPK/MEK/ERK cascade, which led to upregulation of cyclin D1 and thus promoted cell proliferation. FRA-1 also induced snail1-mediated MMP14 (matrix metallopeptidase 14, also known as MT1-MMP) expression to facilitate ESCC cell invasion, migration, and metastasis.. Our data suggest that CTHRC1 may act as an oncogenic driver in progression and metastasis of ESCC, and may serve as a potential biomarker for prognosis and personalized therapy.

Topics: Adult; Aged; Animals; Biomarkers; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Disease Progression; DNA Methylation; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Extracellular Matrix Proteins; Female; Gene Expression; Heterografts; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 14; Middle Aged; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Signal Transduction; Snail Family Transcription Factors; Tumor Burden

Our previous study showed that WNT5A, a member of the noncanonical WNT pathway, is involved in interleukin-1beta induced matrix metalloproteinase expression in temporomandibular joint (TMJ) condylar chondrocytes. The purpose of this study is to further explore the roles of WNT5A in cartilage biology of the TMJ.. An early TMJ osteoarthritis-like rat model was constructed by a mechanical method (steady mouth-opening). The gene and protein levels of WNT5A during the condylar cartilage changes were measured. Effects of WNT5A on chondrocyte proliferation, hypertrophy and migration were analyzed after WNT5A gain or loss of function in vitro. A c-Jun N-terminal kinase (JNK) inhibitor SP600125 was used to evaluate the involvement of JNK pathway in these effects of WNT5A. The expression and transcription activity of cell cycle regulators c-MYC and Cyclin D1 were examined to determine the mechanism behind WNT5A regulation of chondrocyte proliferation.. WNT5A was significantly upregulated in the condylar cartilage of rats in the early TMJ osteoarthritis-like model. Activating WNT5A facilitated condylar chondrocyte proliferation, hypertrophy and migration. Conversely, inhibiting WNT5A activity in chondrocytes decreased their proliferation, hypertrophy and migration. Blockage of the JNK pathway by its inhibitor, SP600125, impaired these effects of WNT5A on chondrocytes. WNT5A regulated both the expression and transcriptional activity of c-MYC and Cyclin D1 in chondrocytes, both of which were upregulated in condylar cartilage of the rat early TMJ osteoarthritis.. WNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration. These findings provide new insights into the role of WNT5A signaling in TMJ cartilage biology and its potential in future therapy for TMJ degenerative diseases.

Topics: Animals; Cell Movement; Cell Proliferation; Chondrocytes; Cyclin D1; Disease Models, Animal; Hypertrophy; Mandibular Condyle; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Signal Transduction; Temporomandibular Joint Disorders; Up-Regulation; Wnt-5a Protein

Topics: Animals; Antigens, Ly; Bacteremia; Binge Drinking; Bone Marrow Cells; CCAAT-Enhancer-Binding Protein-beta; Cyclin D1; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Ethanol; Gene Expression Regulation; Granulocytes; Hematopoiesis; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nucleotidyltransferases; Proto-Oncogene Proteins c-kit; Signal Transduction; Toll-Like Receptor 4

Repair of large bone defects remains a challenge for surgeons, tissue engineering represents a promising approach. However, the use of this technique is limited by delayed vascularization in central regions of the scaffold. Growth differentiation factor 15(GDF15) has recently been reported to be a potential angiogenic cytokine and has an ability to promote the proliferation of human umbilical vein endothelial cells(HUVECs). Whether it can be applied for promoting vascularized bone regeneration is still unknown. In this study, we demonstrated that GDF15 augmented the expression of cyclins D1 and E, induced Rb phosphorylation and E2F-1 nuclear translocation, as well as increased HUVECs proliferation. Furthermore, we also observed that GDF15 promoted the formation of functional vessels at an artificially-induced angiogenic site, and remarkably improved the healing in the repair of critical-sized calvarial defects. Our results confirm the essential role of GDF15 in angiogenesis and suggest its potential beneficial use in regenerative medicine.

Topics: Animals; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin E; Disease Models, Animal; E2F1 Transcription Factor; Growth Differentiation Factor 15; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Neovascularization, Physiologic; Phosphorylation; Retinoblastoma Protein; Signal Transduction; Skull

The current study was designed to elucidate the mechanism of retinol binding protein 4 (RBP4) in cleft palate induced by all‑trans retinoic acid (atRA). To establish a cleft palate model in C57BL/6J mice, pregnant mice were administered atRA (100 mg/kg) by gavage at the tenth embryonic stage (E10.0). Control groups were given the equivalent volume of corn oil. Pregnant mice were dissected at E12.5, E13.5 and E14.5 to obtain the embryonic palates. The expression levels of RBP4 in the embryonic palatal mesenchyme (EPM) were determined by immunohistochemistry, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Human embryonic palatal mesenchymal cells were exposed to atRA to detect the variation in RBP4 induced by atRA in vitro. Small interfering RNA was used to suppress the expression of RBP4, and a plasmid overexpressing RBP4 was used to examine upregulated expression. The cell counting kit‑8 assay was used to evaluate the effect of RBP4 on cell proliferation. The expression levels of p27 and cyclin D1 were determined by RT‑qPCR and western blotting, while the expression levels of extracellular signal‑related kinase (ERK) 1/2 and protein kinase B (AKT) were assessed by western blotting. At E14.5, RBP4 was strongly expressed in the EPM, while it was downregulated following atRA treatment, which induced cleft palate in vivo. In vitro experiments indicated that atRA suppressed the expression of RBP4 and altered the expression of p27 and cyclin D1 to cause growth inhibition. Knockdown of RBP4 resulted in decreased expression of cyclin D1 and increased p27, and suppressed proliferation. Overexpression of RBP4 reversed the inhibitory effect of atRA and promoted proliferation via the ERK1/2 and AKT signaling pathways. These results suggested that RBP4 was involved in cleft palate induced by atRA and it can be suppressed by atRA to cause growth inhibition in the embryonic palate.

Topics: Animals; Cell Line; Cell Proliferation; Cleft Palate; Corn Oil; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Embryo, Mammalian; Excipients; Female; Gene Expression Regulation, Developmental; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Pregnancy; Proto-Oncogene Proteins c-akt; Retinol-Binding Proteins, Plasma; RNA, Small Interfering; Signal Transduction; Tretinoin

Abnormal proliferation is significantly associated with the promotion of malignant tumor. Growing evidence suggest that the signal pathways of p21. We evaluated PAK5 and p65 staining in breast cancer tissues (BCTs) and paired non-cancerous tissues (NTs) using tissue microarray (TMA) technology. The functions of PAK5 were studied in vitro and in vivo. Cell Counting Kit-8 (CCK-8) and flow cytometry were performed to determine proliferation of breast cancer cells. Phosphorylation assay and co-immunoprecipitation (co-IP) were employed to identify the regulation mechanism of p65 by PAK5. The activation of Cyclin D1 promoter was measured with luciferase reporter assay. Xenograft models in nude mice were established to explore the roles of PAK5 in breast cancer growth.. In this study, we show that PAK5 is highly expressed in breast cancer tissues and the increased PAK5 is significantly associated with breast cancer progression. Overexpression of PAK5 promotes the proliferation and cell-cycle progression by increasing the expression of Cyclin D1 in vitro and in vivo. Mechanistic studies demonstrated that PAK5 can promote the phosphorylation and the nuclear translocation of p65 subunit of nuclear factor-kappaB (NF-κB). Furthermore, p65 can directly bind to the promoter of Cyclin D1 and mediate an increase in its protein expression.. Taken together, our findings suggest that PAK5 may serve as a potential prognosis marker and therapeutic target for human breast cancer.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Follow-Up Studies; Gene Expression; Genes, Reporter; Heterografts; Humans; Immunohistochemistry; Mice; Neoplasm Staging; p21-Activated Kinases; Phosphorylation; Prognosis; Promoter Regions, Genetic; Protein Transport; Signal Transduction; Transcription Factor RelA; Transcriptional Activation; Tumor Burden

Experimental and clinical evidence points to a critical role of Upregulator of cell proliferation (URGCP/URG4) in controlling the progression of multiple tumors. However, the oncogenic role of URGCP in glioma still remains elusive. In this study we tried to investigate the oncogenic roles and molecular mechanisms of URGCP in glioma. We found that the levels of URGCP were upregulated in glioma, and that the high-levels of URGCP indicated a worse prognosis in glioma patients. URGCP and miR-16 are critical for glioma growth: silencing URGCP (shURGCP) inhibited glioma growth, while, the shURGCP-mediated proliferative inhibition could be recovered by antagonizing miR-16 (anta-miR-16) in vivo and in vitro. Mechanically, URGCP repressed miR-16 expression via activating NF-κB/c-myc pathway in glioma; Cyclins D1 and Cyclin E1 were identified as the direct targets of miR-16, thus, URGCP-mediated miR-16 downregulation accelerated cell proliferation by upregulating Cyclin D1 and Cyclin E1 expression. All these results suggested that URGCP accelerates glioma growth through the NF-κB/c-myc/miR-16/Cyclin D1/E1 pathway, and both URGCP and miR-16 function as a novel cell cycle regulators in glioma and could be considered as potential targets for glioma therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Disease Models, Animal; Flow Cytometry; Gene Expression; Gene Expression Regulation, Neoplastic; Glioma; Heterografts; Humans; Immunohistochemistry; Male; Mice; MicroRNAs; Neoplasm Proteins; NF-kappa B; Oncogene Proteins; Polymerase Chain Reaction; Signal Transduction

Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis with massive neuronal loss and severe gliosis. Aberrant neurogenesis has been shown in the epileptogenesis process of temporal lobe epilepsy. However, the molecular mechanisms underlying aberrant neurogenesis remain unclear. The roles of Wnt signalling cascade have been well established in neurogenesis during multiple aspects. Here, we used kainic acid-induced rat epilepsy model to investigate whether Wnt/β-catenin signalling pathway is involved in the aberrant neurogenesis in temporal lobe epilepsy. Immunostaining and western blotting results showed that the expression levels of β-catenin, Wnt3a, and cyclin D1, the key regulators in Wnt signalling pathway, were up-regulated during acute epilepsy induced by the injection of kainic acids, indicating that Wnt signalling pathway was activated in kainic acid-induced temporal lobe epilepsy. Moreover, BrdU labelling results showed that blockade of the Wnt signalling by knocking down β-catenin attenuated aberrant neurogenesis induced by kainic acids injection. Altogether, Wnt/β-catenin signalling pathway mediated hippocampal neurogenesis during epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy. Temporal lobe epilepsy is a chronic disorder of nerve system, mainly characterized by hippocampal sclerosis. Aberrant neurogenesis has been shown to involve in the epileptogenesis process of temporal lobe epilepsy. In the present study, we discovered that Wnt3a/β-catenin signalling pathway serves as a link between aberrant neurogenesis and underlying remodelling in the hippocampus, leading to temporal lobe epilepsy, which might provide new strategies for clinical treatment of temporal lobe epilepsy.

Topics: Animals; beta Catenin; Cyclin D1; Disease Models, Animal; Epilepsy, Temporal Lobe; Hippocampus; Kainic Acid; Male; Rats; Rats, Wistar; RNA Interference; RNA, Small Interfering; Up-Regulation; Wnt Signaling Pathway; Wnt3A Protein

Niemann-Pick disease type C (NPC) is a fatal, neurovisceral lipid storage disease, neuropathologically characterized by cytoplasmic sequestration of glycolipids in neurons, progressive neuronal loss, neurofibrillary tangles (NFTs) formation, and axonal spheroids (AS). Cytoskeletal pathology including accumulation of hyperphosphorylated cytoskeletal proteins is a neuropathological hallmark of the mouse model of NPC (npc mice). With a goal of elucidating the mechanisms underlying the lesion formation, we investigated the temporal and spatial characteristics of cytoskeletal lesions and the roles of cdc2, cdk4, and cdk5 in lesion formation in young npc mice. Cytoskeletal lesions were detectable in npc mice at three weeks of age. Importantly, concomitant activation of cdc2/cyclin B1 kinase and accumulation of a subsequently generated cohort of phospho-epitopes were detected. The activation of cdk4/cyclin D1 and cdk5/p25 kinases was observed during the fourth week of life in npc mice, and this activation contributed to the lesion formation. We concluded that the progression of cytoskeletal pathology in npc mice older than four weeks is accelerated by the cumulative effect of cdc2, cdk4, and cdk5 activation. Furthermore, cdc2/cyclin B1 may act as a key initial player one week earlier. Targeting cell cycle activation may be beneficial to slow down the NPC pathogenesis.

Topics: Animals; CDC2 Protein Kinase; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 5; Cytoskeleton; Disease Models, Animal; Humans; Mice; Niemann-Pick Disease, Type C; Phosphorylation; Phosphotransferases; Transcriptional Activation

The study aimed to investigate the protective effect of curcumin against oxidative stress-induced injury of Parkinson's disease (PD) through the Wnt/β-catenin signaling pathway in rats.. The successfully established PD rat models and normal healthy rats were randomly assigned into the 6-hydroxydopamine (6-OHDA), the curcumin (Cur) and the control groups. Immunohistochemistry was used to detect the positive expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and glial fibrillary acidic protein (GFAP). Deutocerebrum primary cells were extracted and classified into the control, 6-OHDA, Cur (5, 10, 15 µmol/L), Dickkopf-1 (DKK-1) and Cur + DKK-1 groups. MTT assays, adhesion tests and TUNEL staining were used to assess cell viability, adhesion and apoptosis, respectively. Western blotting and qRT-PCR were used to examine the protein and mRNA expressions of Wnt3a and β-catenin and the c-myc and cyclinD1 mRNA expressions.. TH and DAT expressions in the Cur group were elevated and GFAP was reduced compared with the 6-OHDA group. Curcumin enhanced viability, survival and adhesion and attenuated apoptosis of deutocerebrum primary cells by activating the Wnt/β-catenin signaling pathway. Higher Wnt3a and β-catenin mRNA and protein expressions and c-myc and cyclinD1 mRNA expressions, enhanced superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) contents, decreased malondialdehyde (MDA) content and elevated mitochondrial membrane potential (∆ψm) were found in the 10 and 15 µmol/L Cur groups compared with the 6-OHDA group. However, opposite tendencies were found in the Cur + DKK-1 group compared to the 10 µmol/L Cur group.. This study suggests that curcumin could protect against oxidative stress-induced injury in PD rats via the Wnt/β-catenin signaling pathway.

Topics: Animals; Apoptosis; Astrocytes; Behavior, Animal; beta Catenin; Cell Adhesion; Cells, Cultured; Curcumin; Cyclin D1; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Glial Fibrillary Acidic Protein; Glutathione Peroxidase; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Oxidative Stress; Oxidopamine; Parkinson Disease; Protective Agents; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine 3-Monooxygenase; Wnt Signaling Pathway; Wnt3 Protein

The utmost aim of this present study was to investigate the anti-inflammatory, antiproliferative and proapoptotic potential of Asiatic acid (AA) on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in experimental rats. Rats were divided into six groups and received modified pellet diet for 32 weeks. Group 1 served as control rats. Group 2 received AA (4 mg/kg b.w. p.o.). Group 3-6 rats received 15 DMH (20 mg/kg b.w., s.c.) injections once a week starting from the 4th week. Besides DMH, rats received AA (4 mg/kg b.w. p.o.) in group 4 starting 2 weeks before carcinogen treatment till the end of the last DMH; group 5 starting 2 days after last DMH till the end of the experiment; and group 6 throughout the experiment. Pre-neoplastic lesions, xenobiotic metabolizing enzymes, inflammation, cell proliferation and apoptotic markers were analysed in our study. Our results ascertained AA supplementation to DMH-exposed rats significantly decreased the incidence of aberrant crypt foci (ACF) and phase I xenobiotic enzymes; and increased the phase II xenobiotic enzymes and mucin content as compared to DMH-alone-exposed rats. Moreover the increased expressions of mast cells, argyrophilic nucleolar organizer regions (AgNORs), proliferating cell nuclear antigen (PCNA) and cyclin D1 observed in the DMH-alone-exposed rats were reverted and were comparable with those of the control rats, when treated with AA. Concordantly AA also induced apoptosis by downregulating the expression of Bcl-2 and upregulating Bax, cytochrome c, caspase-3 and -9 in the DMH-alone-exposed rats. Thus AA was able to inhibit DMH-induced colon carcinogenesis by detoxifying the carcinogen, decreasing the preneoplastic lesions by virtue of its anti-inflammatory, antiproliferative and proapoptotic effects. Therefore our findings suggest that AA could be used as an effective chemopreventive agent against DMH induced colon carcinogenesis.

Topics: 1,2-Dimethylhydrazine; Aberrant Crypt Foci; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Cytochrome P-450 Enzyme System; Disease Models, Animal; Down-Regulation; Liver; Male; Pentacyclic Triterpenes; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Up-Regulation

Although sorafenib was approved as antiangiogenic agent in case of hepatocellular carcinoma (HCC), the pathways mediating its antitumorigenic effects were not fully examined in vivo. This study was conducted to elucidate the molecular mechanisms underlying the antineoplastic effect of sorafenib in livers of rats exposed to the hepatocarcinogen diethyl nitrosamine (DENA) regarding oxidative stress, proliferation, and apoptotic pathways. Male albino rats were divided into three groups: normal control, DENA group, and sorafenib group. Sorafenib (10 mg/kg) was given daily to rats orally for 2 weeks, started 6 weeks after DENA (200 mg/kg, single i.p. dose). The histopathological results proved that sorafenib corrected neoplastic changes in the liver as evidenced by a decrease in size of hepatocellular foci. The liver index, glutathione, as well as Bcl-2 were significantly decreased in sorafenib group compared with DENA group. Sorafenib also exhibited antiproliferative effect through suppression of gene expression of cyclin D1 and β-catenin. Thus, the apoptotic and proliferative pathways in HCC could be interrupted by sorafenib, supporting the role of sorafenib as antineoplastic agent and nominating it as a candidate drug for other neoplasms.

Topics: Administration, Oral; Animals; Antineoplastic Agents; beta Catenin; Cell Proliferation; Cyclin D1; Disease Models, Animal; Glutathione; Histocytochemistry; Liver; Liver Neoplasms; Male; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Sorafenib; Treatment Outcome

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.

Topics: 4-Nitroquinoline-1-oxide; Adult; Animals; Areca; Carcinogenesis; Carcinoma, Squamous Cell; Cortactin; Cyclin D1; Disease Models, Animal; Disease-Free Survival; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Leukoplakia; Male; Mice; Mice, Inbred BALB C; Middle Aged; Mouth Neoplasms; Plant Extracts; Polymerase Chain Reaction; Prognosis; Proportional Hazards Models; Tongue Neoplasms; Up-Regulation

Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Disease Models, Animal; DNA Damage; DNA Replication; Heterografts; Humans; Lymphoma, Mantle-Cell; Mice; Proto-Oncogene Mas; RNA Interference; RNA, Small Interfering

The antibacterial agent helvolic acid, which was isolated from the active antitumor fraction of Cordyceps taii, showed potent cytotoxicity against different human cancer cells. In the present study, the in vivo antitumor effect of helvolic acid was investigated in murine sarcoma S180 tumor-bearing mice. Doses of 10 and 20 mg/kg/day helvolic acid did not exert significant antitumor activity. Interestingly, co-administration of 10 mg/kg/day helvolic acid and 20 mg/kg/day cyclophosphamide (CTX) - a well-known chemotherapy drug - showed promising antitumor activity with a growth inhibitory rate of 70.90%, which was much higher than that of CTX alone (19.5%). Furthermore, the combination markedly prolonged the survival of tumor-bearing mice. In addition, helvolic acid enhanced the immune organ index. The protein expression levels of β-catenin, cyclin D1, and proliferating cell nuclear antigen were significantly suppressed in mice treated with 20 mg/kg/day helvolic acid and in those receiving combination therapy. Taken together, these results indicated that helvolic acid in combination with CTX showed potent in vivo synergistic antitumor efficacy, and its mechanism of action may involve the Wnt/ β-catenin signaling pathway.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Cell Line, Tumor; Cordyceps; Cyclin D1; Cyclophosphamide; Disease Models, Animal; Drug Synergism; Fusidic Acid; Male; Mice; Proliferating Cell Nuclear Antigen; Sarcoma; Wnt Proteins; Wnt Signaling Pathway

Traumatic brain injury (TBI) is induced by complex primary and secondary mechanisms that give rise to cell death, inflammation, and neurological dysfunction. Understanding the mechanisms that drive neurological damage as well as those that promote repair can guide the development of therapeutic drugs for TBI. Kruppel-like factor 4 (KLF4) has been reported to negatively regulate axon regeneration of injured retinal ganglion cells (RGCs) through inhibition of JAK-STAT3 signaling. However, the role of KLF4 in TBI remains unreported. Reactive oxygen species (ROS)-induced neuronal death is a pathophysiological hallmark of TBI.. In this study, we used H. The results show that H. These findings provide evidence that KLF4 plays an important role in the pathophysiology of TBI. Blocking KLF4 may be a potential therapeutic strategy for the treatment of TBI, either alone or in combination with agents that target complementary mechanisms.

Topics: Animals; Apoptosis; Brain Injuries, Traumatic; Cerebral Cortex; Cyclin D1; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Hydrogen Peroxide; Janus Kinases; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Nerve Regeneration; Optic Nerve Injuries; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Signal Transduction; STAT3 Transcription Factor; Tumor Suppressor Protein p53; Tyrphostins

Pulmonary vascular remodelling is a common feature among the heterogeneous disorders that cause pulmonary arterial hypertension (PAH), and pulmonary arterial smooth muscle cells (PASMCs) proliferation impact the long-term prognosis of the patient. Isoquercitrin (IQC) is a flavonoid with anti-oxidative, anti-inflammatory and anti-proliferative activations. This study aimed to investigate whether IQC could prevent PASMCs proliferation and vascular remodelling in monocrotaline (MCT) induced PAH. Male Wistar rats were administered with Vehicle or 0.1% IQC maintain feed after MCT (40 mg/kg) injection. Haemodynamic changes, right ventricular hypertrophy and lung morphological features were assessed 3 weeks later. MCT-induced PAH, pulmonary vascular remodelling and PASMCs proliferation in Vehicle-treated rats. IQC reduced the right ventricle systolic pressure (RVSP), the ratio of RV/LV+S and the RV hypertrophy. IQC significantly alleviated the expression of proliferating cell nuclear antigen (PCNA), smooth muscle α-actin (α-SMA), and the percentage of fully muscularized small arterioles. In vitro studies, PASMCs were pretreated with IQC and stimulated with platelet-derived growth factor (PDGF)-BB (20 ng/mL). IQC suppressed PDGF-BB-induced PASMCs proliferation and caused G0/G1 phase cell cycle arrest. IQC downregulated the expression of Cyclin D1 and CDK4 as well as inhibited p27Kip1 degradation. Meanwhile, IQC negatively modulated PDGF-BB-induced phosphorylation of PDGF-Rβ, Akt/GSK3β and ERK1/2. IQC ameliorated MCT-induced pulmonary vascular remodelling via suppressing PASMCs proliferation and blocking PDGF-Rβ signalling pathway.

Topics: Animals; Antioxidants; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Flow Cytometry; Hypertension, Pulmonary; Male; Myocytes, Smooth Muscle; Pulmonary Artery; Quercetin; Rats, Wistar; Vascular Remodeling

Many victims of blast-induced traumatic brain injury are occupants of military vehicles targeted by land mines. Recently improved vehicle designs protect these individuals against blast overpressure, leaving acceleration as the main force potentially responsible for brain injury. We recently developed a unique rat model of under-vehicle blast-induced hyperacceleration where exposure to acceleration as low as 50G force results in histopathological evidence of diffuse axonal injury and astrocyte activation, with no evidence of neuronal cell death. This study investigated the effects of much higher blast-induced accelerations (1200 to 2800G) on neuronal cell death, neuro-inflammation, behavioral deficits and mortality. Adult male rats were subjected to this range of accelerations, in the absence of exposure to blast overpressure, and evaluated over 28days for working memory (Y maze) and anxiety (elevated plus maze). In addition, brains obtained from rats at one and seven days post-injury were used for neuropathology and neurochemical assays. Sixty seven percent of rats died soon after being subjected to blasts resulting in 2800G acceleration. All rats exposed to 2400G acceleration survived and exhibited transient deficits in working memory and long-term anxiety like behaviors, while those exposed to 1200 acceleration G force only demonstrated increased anxiety. Behavioral deficits were associated with acute microglia/macrophage activation, increased hippocampal neuronal death, and reduced levels of tight junction- and synapse- associated proteins. Taken together, these results suggest that exposure of rats to high underbody blast-induced G forces results in neurologic injury accompanied by neuronal apoptosis, neuroinflammation and evidence for neurosynaptic alterations.

Topics: Acceleration; Animals; Antigens, Differentiation; Blast Injuries; Brain; Brain Injuries, Traumatic; Caspase 3; Cyclin D1; Disease Models, Animal; Disks Large Homolog 4 Protein; Gene Expression Regulation; HSP70 Heat-Shock Proteins; Intracellular Signaling Peptides and Proteins; Male; Maze Learning; Membrane Proteins; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Time Factors; von Willebrand Factor; Zonula Occludens-1 Protein

Autosomal dominant polycystic kidney disease (ADPKD) leads to renal failure. The hallmark of ADPKD is increased epithelial proliferation, which has been proposed to be due to atypical signaling including abnormal JAK-STAT activity. However, the relative contribution of JAK-STAT family members in promoting proliferation in ADPKD is unknown. Here, we present siRNA JAK-STAT-focused screens discovering a previously unknown proliferative role for multiple JAK-STAT components (including STAT1, STAT2, STAT4, STAT5a, and STAT5b). Amongst these, we selected to study the growth hormone/growth hormone receptor/STAT5-axis because of its known role as a regulator of growth in nonrenal tissues. Loss of STAT5 function, facilitated by pharmacological inhibition or siRNAs, significantly reduced proliferation with an associated reduction in cyst growth in vitro. To study whether STAT5 is abnormally activated in vivo, we analyzed its expression using two independent mouse models of ADPKD. STAT5 was nuclear, thus activated, in renal epithelial cyst lining cells in both models. To test whether forced activation of STAT5 can modulate proliferation of renal cells in vivo, irrespective of the Pkd1 status, we overexpressed growth hormone. These mice showed increased STAT5 activity in renal epithelial cells, which correlated with de novo expression of cyclin D1, a STAT5 target gene. Chromatin immunoprecipitation experiments revealed that STAT5 transcriptionally activated cyclin D1 in a growth hormone-dependent fashion, thus providing a mechanism into how STAT5 enhances proliferation. Finally, we provide evidence of elevated serum growth hormone in Pkd1 mutant mice. Thus, the growth hormone/STAT5 signaling axis is a novel therapeutic target in ADPKD.

Topics: Animals; Carrier Proteins; Cell Line; Cell Nucleus; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epithelial Cells; Genotype; Growth Hormone; Humans; Janus Kinases; Kidney; Mice, Transgenic; Phenotype; Polycystic Kidney, Autosomal Dominant; Protein Kinase Inhibitors; RNA Interference; Signal Transduction; STAT5 Transcription Factor; Time Factors; Transfection; TRPP Cation Channels; Tumor Suppressor Proteins

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Differentiation; Cell Proliferation; Chemistry, Pharmaceutical; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Epidermis; Female; Inflammation; Mice; Phytic Acid; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured

Malignant melanoma is the deadliest form of all skin cancers. Itraconazole, a commonly used systemic antifungal drug, has been tested for its anti-tumor effects on basal cell carcinoma, prostate cancer, and non-small cell lung cancer. Whether itraconazole has any specific anti-tumor effect on melanoma remains unknown. However, the goal of this study is to investigate the effect of itraconazole on melanoma and to reveal some details of its underlying mechanism. In the in vivo xenograft mouse model, we find that itraconazole can inhibit melanoma growth and extend the survival of melanoma xenograft mice, compared to non-itraconazole-treated mice. Also, itraconazole can significantly inhibit cell proliferation, as demonstrated by Ki-67 staining in itraconazole-treated tumor tissues. In in vitro, we show that itraconazole inhibits the proliferation and colony formation of both SK-MEL-28 and A375 human melanoma cells. Moreover, we demonstrate that itraconazole significantly down-regulates Gli-1, Gli-2, Wnt3A, β-catenin and cyclin D1, while it up-regulates Gli-3 and Axin-1, indicating potent inhibitory effects of itraconazole on Hedgehog (Hh) and Wnt signaling pathways. Furthermore, itraconazole significantly suppresses the PI3K/mTOR signaling pathway - indicated by the down-regulated phosphorylation of p70S6K, 4E-BP1 and AKT - but has no effect on the phosphorylation of MEK or ERK. Our data suggest that itraconazole inhibits melanoma growth through an interacting regulatory network that includes Hh, Wnt, and PI3K/mTOR signaling pathways. These results suggest that this agent has several potent anti-melanoma features and may be useful in the synergesis of other anti-cancer drugs via blockage of the Hh, Wnt and PI3K/mTOR signaling pathways.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Itraconazole; Melanoma; Mice; Phosphatidylinositol 3-Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay; Wnt Proteins; Xenograft Model Antitumor Assays

IgA nephropathy (IgAN) has been considered to be the most frequent form of primary glomerulonephritis that occurs worldwide with a variety of factors involved in its occurrence and development. The impact of autophagy in IgAN, however, remains partially unclear. This study was designed to investigate the effects of rapamycin in an IgAN model.. After establishing an IgAN rat model, SD rats were divided into 4 groups: control, control + rapamycin, IgAN, IgAN + rapamycin. Proteinuria and the pathological changes and the level of autophagy of kidney were texted. Identify the expression of phosphorylation and total mammalian target of rapamycin (mTOR) and s6k1 as well as cyclin D1 in the kidney of rats through Western blot and immunohistochemistry.. With rapamycin treatment, we observed a significant reduction in the progression of proteinuria as well as alleviation of pathological lesions in IgAN rats. Besides, autophagy was inhibited, while the mTOR/S6k1 pathway was activated and expression of cyclin D1 was increased in IgAN. Rapamycin treatment increased autophagy and decreased the expression of cyclin D1.. These results may suggest that mTOR-mediated autophagy inhibition may result in mesangial cell proliferation in IgAN.

Topics: Animals; Autophagy; Cell Proliferation; Cyclin D1; Disease Models, Animal; Glomerulonephritis, IGA; Humans; Immunohistochemistry; Immunosuppressive Agents; Mesangial Cells; Microscopy, Electron; Proteinuria; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Quercetin, a natural polyphenolic flavonoid compound, can inhibit the growth of several malignant cancers. However, the mechanism still remains unclear. Our previous findings have suggested that quercetin can significantly inhibit HepG2 cell proliferation and induce cell apoptosis in vitro. It can also affect cell cycle distribution and significantly decrease cyclin D1 expression. In this study, we investigated the anti-cancer effect of quercetin on HepG2 tumor-bearing nude mice and its effect on cyclin D1 expression in the tumor tissue. First, the nude murine tumor model was established by subcutaneous inoculation of HepG2 cells, then quercetin was administered intraperitoneally, and the mice injected with saline solution were used as controls. The daily behavior of the tumor-bearing mice was observed and differences in tumor growth and survival rate were monitored. The expression of cyclin D1 in isolated tumor sections was evaluated by immunohistochemistry. We found that HepG2 tumor became palpable in the mice one-week post-inoculation. Tumors in the control group grew rapidly and the daily behavior of the mice changed significantly, including listlessness, poor feeding and ataxia. The mice in quercetin-treated group showed delayed tumor growth, no significant changes in daily behavior, and the survival rate was significantly improved. Finally, we observed increased tumor necrosis and a lighter cyclin D1 staining with reduced staining areas. Our findings thus suggest that quercetin can significantly inhibit HepG2 cell proliferation, and this effect may be achieved through the regulation of cyclin D1 expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Biomarkers; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Hep G2 Cells; Humans; Mice; Mice, Inbred BALB C; Quercetin; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

o-Nitroanisole is an intermediate in the manufacture of azo dyes. In a National Toxicology Program stop-exposure study,o-nitroanisole induced hyperplasia, papillomas, and papillary carcinomas in the urinary bladder of Fischer 344/N rats.o-Nitroanisole was investigated since occupational or environmental exposure to aniline and azo dyes is a risk factor for urinary bladder cancer in humans. The current study describes the morphology of urinary bladder neoplasms seen in rats with respect to those observed in humans. This study also evaluated immunohistochemical expression of the cell cycle-related proteins cyclin D1 and p53 and the differentiation markers cytokeratin 20 and uroplakin III in hyperplastic (n= 11) and neoplastic (n= 6 papillomas,n= 11 carcinomas) lesions of the urinary bladder epithelium from rats treated with o-nitroanisole and in normal (n= 6) urinary bladders from untreated rats. The tumors observed were more similar to the papillary type rather than the muscle-invasive type of urinary bladder cancer in humans. The preneoplastic and neoplastic lesions observed suggest progression from hyperplasia to papilloma to papillary carcinoma. With neoplastic progression (hyperplasia to papilloma to carcinoma), cyclin D1 immunoreactivity progressively increased in intensity, percentage of cells staining, and distribution. Overexpression of p53 was not found. Cytokeratin 20 staining decreased in superficial cells, while uroplakin III staining increased in intermediate and basal cells with progression from hyperplasia to carcinoma. The results are consistent with increased cell cycle dysregulation or proliferation (cyclin D1), decreased differentiation (cytokeratin 20), and abnormal differentiation (uroplakin III) as lesions progress toward malignancy.

Topics: Animals; Anisoles; Biomarkers, Tumor; Carcinoma, Papillary; Cyclin D1; Disease Models, Animal; Female; Humans; Hyperplasia; Immunohistochemistry; Keratin-20; Male; Papilloma; Precancerous Conditions; Rats; Rats, Inbred F344; Tumor Suppressor Protein p53; Urinary Bladder; Urinary Bladder Neoplasms; Uroplakin III

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerate muscle regeneration in normal mice, and it ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multiparameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the MEK1-ERK1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight the profound functions of a small metabolite as signaling molecule in mammalian cells.

Topics: Acetoacetates; Animals; Cell Proliferation; Cyclin D1; Disease Models, Animal; Gene Expression Regulation; Ketone Bodies; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Animal; Regeneration; Satellite Cells, Skeletal Muscle; Signal Transduction

Previous studies by this group have shown that Helicobacter pylori with high thioredoxin-1 (Trx1) expression might be involved in stomach carcinogenesis in vitro. To study histopathological changes of the stomach mucosa in vivo, a Mongolian gerbil model infected with H. pylori with high Trx1 expression was established.. Healthy, male Mongolian gerbils (n=75) were randomly divided into 3 groups: controls (n=15), which were not infected with H. pylori, high Trx1 (n=30) which were infected with H. pylori with high Trx1 expression and low Trx1 (n=30) which were infected with low Trx1 expression H. pylori. The animals were sacrificed at 4, 20, 34, 48, 70 and 90 weeks after inoculation.. The Mongolian gerbil model of H. pylori infection was successfully established. Three animals died during the study, leaving 72 animals (controls, n=14; low Trx1, n=29; high Trx1, n=29) examined on schedule. Histopathological analysis of the stomach mucosa showed gradually increased aggravation over time in the high and low Trx1 groups. Compared with control and low Trx1, the histopathological changes were more serious in the high Trx1 group. At 90 weeks, no abnormal changes were found in the controls, but 62.5% of the high Trx1 group and 33.3% of the low Trx1 showed adenocarcinomas. The H. pylori Trx1 level in gastric cancer tissue was significantly higher than that from gastritis tissue. Within gastric cancer cells, high Trx1 expression in H. pylori significantly upregulated cyclin D1.. High Trx1 expression in H. pylori promoted stomach carcinogenesis. More studies are needed to confirm this finding.

Topics: Adenocarcinoma; Animals; Carcinogenesis; Cyclin D1; Disease Models, Animal; Gastric Mucosa; Gerbillinae; Helicobacter Infections; Helicobacter pylori; Male; Stomach Neoplasms; Thioredoxins; Up-Regulation

Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body's immune system.. The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3+, CD19+, and CD11b+, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12β cytokines, but suppressed IL-10 level. At the tumor sites, CD11b+ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the genes controlling cell survival and function. Phosphorylation of STAT1 was absent in WEHI-3. Instead, similar treatments significantly decreased pSTAT3 signaling that regulates transcription of genes controlling tumor growth and proliferation.. Rice bran gramisterol possesses a promising anti-cancer effect against a tumor of white blood cells and induces the production of anti-cancer immune-related cytokines. Gramisterol induces cell cycle arrest and apoptosis via suppression of pSTAT3 signaling control of tumor cells' growth and progression. Gramisterol increased IFN-γ production and prevented the dysfunctional immune system of leukemic mice by enhancing pSTAT1 transcription signal controlling proliferation and functions of hematopoietic cells in the spleen. Together with IFN-γ, gramisterol efficiently facilitates leukemic mice immune system modulation leading to improvement of the AML condition. Administration of RBDS containing gramisterol potentiates immune recovery of leukemic mice and extends their survival. This finding encourages the medicinal application of rice bran gramisterol as a palliative treatment or an alternative agent for future drug development against AML.

Topics: Animals; Antigens, CD19; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; CD11b Antigen; CD3 Complex; Cell Proliferation; Cholestadienols; Cyclin D1; Cyclin E; Disease Models, Animal; Drug Screening Assays, Antitumor; G1 Phase; Gene Expression Regulation, Leukemic; Immune System; Leukemia, Myeloid, Acute; Mice; Mice, Inbred BALB C; Oryza; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sterols; Tumor Suppressor Protein p53

All serious liver injuries alter metabolism and initiate hepatic regeneration. Recent studies using partial hepatectomy (PH) and other experimental models of liver regeneration implicate the metabolic response to hepatic insufficiency as an important source of signals that promote regeneration. Based on these considerations, the analyses reported here were undertaken to assess the impact of interrupting the hypoglycemic response to PH on liver regeneration in mice. A regimen of parenteral dextrose infusion that delays PH-induced hypoglycemia for 14 hours after surgery was identified, and the hepatic regenerative response to PH was compared between dextrose-treated and control mice. The results showed that regenerative recovery of the liver was postponed in dextrose-infused mice (versus vehicle control) by an interval of time comparable to the delay in onset of PH-induced hypoglycemia. The regulation of specific liver regeneration-promoting signals, including hepatic induction of cyclin D1 and S-phase kinase-associated protein 2 expression and suppression of peroxisome proliferator-activated receptor γ and p27 expression, was also disrupted by dextrose infusion. These data support the hypothesis that alterations in metabolism that occur in response to hepatic insufficiency promote liver regeneration, and they define specific pro- and antiregenerative molecular targets whose regenerative regulation is postponed when PH-induced hypoglycemia is delayed.

Topics: Animals; Cyclin D1; Disease Models, Animal; Gene Expression Regulation; Glucose; Hepatectomy; Hepatic Insufficiency; Hydrogen-Ion Concentration; Hypoglycemia; Liver; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Models, Biological; Phosphorylation; PPAR gamma; S-Phase Kinase-Associated Proteins

Mouse gene inactivation has shown that the transcription factor Sox11 is required for mouse palatogenesis. However, whether Sox11 is primarily involved in the regulation of palatogenesis still remains elusive. In this study, we explored the role ofSox11in palatogenesis by analyzing the developmental mechanism in cleft palate formation in mutants deficient in Sox11. Sox11 is expressed both in the developing palatal shelf and in the surrounding structures, including the mandible. We found that cleft palate occurs only in the mutant in which Sox11is directly deleted. As in the wild type, the palatal shelves in the Sox11 mutant undergo outgrowth in a downward direction and exhibit potential for fusion and elevation. However, mutant palatal shelves encounter clefting, which is associated with a malpositioned tongue that results in physical obstruction of palatal shelf elevation at embryonic day 14.5 (E14.5). We found that loss of Sox11led to reduced cell proliferation in the developing mandibular mesenchyme via Cyclin D1, leading to mandibular hypoplasia, which blocks tongue descent. Extensive analyses of gene expression inSox11 deficiency identified FGF9 as a potential candidate target of Sox11 in the modulation of cell proliferation both in the mandible and the palatal shelf between E12.5 and E13.5. Finally we show, using in vitro assays, that Sox11 directly regulates the expression of Fgf9 and that application of FGF9 protein to Sox11-deficient palatal shelves restores the rate of BrdU incorporation. Taken together, the palate defects presented in the Sox11 loss mutant mimic the clefting in the Pierre Robin sequence in humans.

Topics: Animals; Binding Sites; Bromodeoxyuridine; Cell Proliferation; Cleft Palate; Cyclin D1; Disease Models, Animal; Embryo, Mammalian; Fibroblast Growth Factor 9; Gene Expression Regulation, Developmental; Humans; Mandible; Mesenchymal Stem Cells; Mice; Mice, Transgenic; Mutation; Palate; Pierre Robin Syndrome; Protein Binding; Signal Transduction; SOXC Transcription Factors; Tongue

Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Isoxazoles; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Organophosphonates; Prostatic Neoplasms; Tetrazoles; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

Topics: Animals; Apoptosis; Bacterial Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Hydrogen-Ion Concentration; Inhibitor of Apoptosis Proteins; Lacticaseibacillus casei; Mice; Probiotics; Survivin; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

The typical progression of oral cancer is from hyperplastic epithelial lesions through dysplasia to invasive carcinoma. It is important to investigate malignant oral cancer progression and development in order to determine useful approaches of prevention of dysplastic lesions. The present study aimed to gain insights into the underlying molecular mechanism of oral carcinogenesis by establishing a rat model of oral carcinogenesis using 4‑nitroquinoline 1‑oxide. Subsequently, transcription profile analysis using an integrating microarray was performed. The dynamic gene expression changes of the six stages of rat oral carcinogenesis (normal, mild epithelial dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and oral squamous cell carcinomas) were analyzed using component plane presentations (CPP)‑self‑organizing map (SOM). Six genes were verified by quantitative polymerase chain reaction, immunohistochemistry and succinate dehydrogenase (SDH) activity assay kit. Numerous differentially expressed genes (DEGs) were identified during rat oral carcinogenesis. CPP‑SOM determined that these DEGs were primarily enriched during cell cycle, apoptosis, inflammatory response and tricarboxylic acid cycle, indicating the coordinated regulation of molecular networks. In addition, the expression of specific DEGs, such as janus kinase 3, cyclin‑dependent kinase A‑1, B‑cell chronic lymphocytic leukaemia/lymphoma 2‑like 2, nuclear factor‑κB, tumor necrosis factor receptor superfamily member 1A, cyclin D1 and SDH were identified to have high concordance with the results from microarray data. The current study demonstrated that oral carcinogenesis is a multi‑step and multi‑gene process, with a distinct pattern alteration along a continuum of malignant transformation. In addition, this comprehensive investigation provided a theoretical basis for the understanding of the molecular alterations associated with oral carcinogenesis.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinogenesis; Cyclin D1; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Mouth Mucosa; Mouth Neoplasms; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Succinate Dehydrogenase; Tongue Neoplasms; Transcription Factor RelA; Transcriptome

To assess the risk of colorectal cancer in humans with inactivation of NRF2, Nrf2-proficient (Nrf2(+/+) ) and -deficient (Nrf2(-/-) ) mice were exposed to potassium bromate (KBrO3 ) at concentrations of 750 or 1500 ppm for 52 weeks. Neoplastic proliferative lesions were observed in the small intestine and exhibited accumulations of β-catenin and cyclin D1. The lesions had characteristics similar to those in experimental models of human hereditary colorectal cancer. An additional 13-week study was performed to examine the role of Nrf2 in the effects of oxidative stress. Significant increase in combined incidences of preneoplastic and neoplastic lesions in Nrf2(-/-) mice administered high-dose KBrO3 . In the short-term study, although 8-hydroxydeoxyguanosine (8-OHdG) levels in the epithelial DNA of Nrf2(-/-) mice at the high dose were significantly lower than those of the corresponding Nrf2(+/+) mice, the difference was very small. mRNA levels of Nrf2-regulated genes were increased in Nrf2(+/+) mice. Overexpression of cyclooxygenase 2 (COX2) and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in the jejunal crypts were observed in Nrf2(-/-) mice administered high-dose KBrO3 . Overall, these data suggested that individuals having single-nucleotide polymorphisms in NRF2 may have a risk of colorectal cancer to some extent.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; beta Catenin; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cytokines; Deoxyguanosine; Disease Models, Animal; Female; Gene Expression; Gene Silencing; Humans; Intestinal Mucosa; Intestine, Small; Mice; Mice, Knockout; NF-E2-Related Factor 2; Oxidative Stress; Proliferating Cell Nuclear Antigen

Müller glia, the principal glial cell type in the retina, have the potential to proliferate and regenerate neurons after retinal damage. However, unlike the situation in fish and birds, this capacity of Müller glia is extremely limited in mammals. To gain new insights into the mechanisms that hamper retinal regeneration in mammals, we examined the cell cycle progression and DNA damage response in Müller glia after retinal damage.. Expression of cell cycle-related proteins and DNA damage response were analyzed in adult rat and mouse retinas after N-methyl-N-nitrosourea (MNU)- or N-methyl-D-aspartate (NMDA)-induced retinal damage. Zebrafish and postnatal rat retinas were also investigated for comparison. Analysis was conducted by using immunofluorescence, Western blotting, and quantitative real-time polymerase chain reaction.. In the rat retina, most Müller glia reentered the cell cycle after MNU-induced photoreceptor damage while no proliferative response was observed in the mouse model. Cell cycle reentry of rat Müller glia was accompanied by DNA damage response including the phosphorylation of the histone variant H2AX and upregulation of p53 and p21. The DNA damage response was also observed in rat Müller glia after NMDA-induced loss of inner retinal neurons, but not in zebrafish Müller glia or rat retinal progenitor cells.. Our findings suggest that the DNA damage response induced by unscheduled cell cycle reentry may be one of the mechanisms that limit the proliferative and regenerative capacity of Müller glia in the mammalian retina.

Topics: Animals; Blotting, Western; Cell Count; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; DNA Damage; Ependymoglial Cells; Gene Expression Regulation; Immunohistochemistry; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Nerve Regeneration; Photoreceptor Cells; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Retina; Retinal Diseases; Retinal Neurons; RNA

Novel, effective and safe agents are needed for the chemoprevention of colorectal cancer (CRC). This study investigated the effects of chitosan oligosaccharides (COS) on CRC progression and their underlying mechanisms and safety profiles in mice. Using a mouse model of colitis-associated CRC, we found that oral administration of COS (500mg/kg/day) resulted in a ∼60% reduction of tumor size and tumor numbers/sectioning. In addition, COS treatment increased AMPK activity, suppressed the NF-κB-mediated inflammatory response and reduced the expressions of cyclin D1, phosphorylated ribosomal protein S6, and MMP-9 in the colon tissues of these mice. Importantly, administration of COS (500mg/kg/day; 50 days) had no adverse effects on renal or liver functions. Our results indicate that COS suppressed CRC progression via AMPK activation and the suppression of NF-κB and mTOR signaling. COS may be of potential utility in the chemoprevention of CRC.

Topics: AMP-Activated Protein Kinases; Animals; Anticarcinogenic Agents; Chitosan; Colitis; Colon; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Male; Matrix Metalloproteinase 9; Mice, Inbred C57BL; NF-kappa B; Oligosaccharides; TOR Serine-Threonine Kinases; Tumor Burden

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC).. The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies.. We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3β and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo.. Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

Topics: Animals; Antibodies, Monoclonal; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Computational Biology; Cyclin D1; Databases, Genetic; Disease Models, Animal; DNA Methylation; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Kaplan-Meier Estimate; Mice; Prognosis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Triple Negative Breast Neoplasms; Tumor Burden; Xenograft Model Antitumor Assays

To study the inhibitory effect and mechanism of Ganoderma lipsiense extract (GLE) on the growth of triple-negative breast cancer (TNBC) cell line MDA-MB-231-HM in a mouse model.. The mouse model of TNBC was established by subcutaneous injection of 1.5 x 10(6) of MDA-MB-231-HM cells into BALB/c-nu mouse. Twenty successfully modeled mice were divided into the GLE group and the negative control group according to random digit table, 10 in each group. GLE (0.2 mL 100 mg/mL) was peritoneally injected to mice in the GLE group, while equal dose of normal saline was peritoneally injected to mice in the negative control group. The medication was administered once per 3 days and discontinued after 45 days. The CD34 expression was detected using immunohistochemical assay for counting microvessels. Meanwhile, expressions of thrombospondin 1 (TSP-1) and cyclin D1 were detected using immunohistochemical assay.. The average weight was obviously lower in the GLE group than in the negative control group [(0.33 ± 0.16) g vs (0.68 ± 0.37)g, P < 0.05]. The tumor inhibition rate was 51.4% in the GLE group. The volume of transplanted tumor was obviously lesser in the GLE group than in the negative control group (P < 0.05). Results of immunohistochemical staining showed, the microvessel density (MVD) under every field was (20.7 ± 2.1), TSP-1 positive cell count was (66.2 ± 9.2), cyclin D1 positive cell count was (33.8 ± 16.4) in the GLE group, and they were 34.0 ± 2.0, 24.0 ± 6.6, and 168.2 ± 32.6, respectively in the negative control group. There was statistical difference in all indices between the two groups (P < 0.05).. GLE could inhibit malignant proliferation of tumor cells by suppressing angiogenesis of blood vessels in tumor tissues and regulating cell cycles, thereby inhibiting TNBC.

Topics: Animals; Biological Products; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Ganoderma; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microvessels; Neoplasm Transplantation; Neovascularization, Pathologic; Random Allocation; Thrombospondin 1; Triple Negative Breast Neoplasms

Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degron(ODC) (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-Kras(G12D) model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.

Topics: Animals; Carcinogenesis; Ceruletide; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; eIF-2 Kinase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Integrases; Mice; Mice, Transgenic; NF-kappa B; Pancreas; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins p21(ras); Signal Transduction; Trans-Activators

Alzheimer's disease (AD) is the most common type of dementia frequently responsible for cognitive decline in the elderly. The etiology and molecular mechanism of AD pathogenesis remain inconclusive. Aging and vascular factors are important independent causes and contributors to sporadic AD. Clinical imaging studies showed that cerebral blood flow decreases before cognitive impairment in patients with AD. To investigate the effect of chronic cerebral hypoperfusion (CCH) on cognitive impairment and morphological features, we developed a new manner of CCH mouse model by narrowing bilateral common carotid arteries. Mice started to manifest spatial memory deficits 1month after the surgery and exhibited behavioral changes in a time-dependent manner. Mice also presented memory deficits accompanied with morphological changes at the neuronal and synaptic levels. CCH damaged the normal neuronal morphology and significantly reduced the expression level of PSD95. CCH activated astrocytes, increased the co-expression of GFAP and AQP4, and destroyed the blood-brain barrier (BBB). Furthermore, CCH facilitated intracellular and extracellular Aβ deposition by up-regulating γ-secretase and β-secretase levels. Our results showed good reproducibility of post-CCH pathological processes, which are characterized by neuronal apoptosis, axonal abnormalities, glial activation, BBB damage, amyloid deposition, and cognitive dysfunction; these processes may be used to decipher the complex interplay and pathological process between CCH and AD. This study provides laboratory evidence for the prevention and treatment of cognitive malfunction and AD.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aquaporin 4; Aspartic Acid Endopeptidases; Brain Ischemia; Cyclin D1; Disease Models, Animal; Disks Large Homolog 4 Protein; Enzyme-Linked Immunosorbent Assay; GAP-43 Protein; Guanylate Kinases; Maze Learning; Membrane Proteins; Memory Disorders; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Nerve Tissue Proteins; Synapses; Time Factors; Tubulin

Limitless replicative potential is one of the hallmarks of cancer that is mainly due to the activity of telomerase. This holoenzyme maintains telomere length, adding TTAGGG repetitions at the end of chromosomes in each cell division. In addition to this function, there are extratelomeric roles of telomerase that are involved in cancer promoting events. It has been demonstrated that TERT, the catalytic component of telomerase, acts as a transcriptional modulator in many signaling pathways. Taking into account this evidence and our experience on the study of azidothymidine (AZT) as an inhibitor of telomerase activity, the present study analyzes the effect of AZT on some telomeric and extratelomeric activities. To carry out the present study, we evaluated the transcription of genes that are modulated by the Wnt/β-catenin pathway, such as c-Myc and cyclin-D1 (Cyc-D1) and cell processes related with their expression, such as, proliferation, modifications of the actin cytoskeleton, cell migration and cell cycle in a mammary carcinoma cell line (F3II). Results obtained after treatment with AZT (600 µM) for 15 passages confirmed the inhibitory effect on telomerase. Regarding extratelomeric activities, our results showed a decrease of 64, 38 and 25% in the transcription of c-Myc, Cyc-D1 and TERT, respectively (p<0.05) after AZT treatment. Furthermore, we found an effect on cell migration, reaching an inhibition of 48% (p<0.05) and a significant passage-dependent increase on cell doubling time during treatment. Finally, we evaluated the effect on cell cycle, obtaining a decline in G0/G1 in AZT-treated cells. These results allow us to postulate that AZT is not only an inhibitor of telomerase activity, but also a potential modulator of extratelomeric processes involved in cancer promotion.

Topics: Actin Cytoskeleton; Adenocarcinoma; Animals; Apoptosis; beta Catenin; Breast Neoplasms; Cell Division; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Humans; Proto-Oncogene Proteins c-myc; Telomerase; Telomere Homeostasis; Wnt Signaling Pathway; Zidovudine

Neurons that reenter the cell cycle die rather than divide, a phenomenon that is associated with neurodegeneration in Alzheimer's disease (AD). Reexpression of cell-cycle related genes in differentiated neurons in AD might be rooted in aberrant mitogenic signaling. Because microglia and astroglia proliferate in the vicinity of amyloid plaques, it is likely that plaque components or factors secreted from plaque-activated glia induce neuronal mitogenic signaling. Advanced glycation end products (AGEs), protein-bound oxidation products of sugar, might be one of those mitogenic compounds. Cyclin D1 positive neurons are colocalized with AGEs or directly surrounded by extracellular AGE deposits in AD brain. However, a direct proof of DNA replication in these cells has been missing. Here, we report by using fluorescent in situ hybridization that consistent with the expression of cell cycle proteins, hyperploid neuronal cells are in colocalization with AGE staining in AD brains but not in nondemented controls. To complement human data, we used apolipoprotein E-deficient mice as model of neurodegeneration and showed that increased oxidative stress caused an intensified neuronal deposition of AGEs, being accompanied by an activation of the MAPK cascade via RAGE. This cascade, in turn, induced the expression of cyclin D1 and DNA replication. In addition, reduction of oxidative stress by application of α-lipoic acid decreased AGE accumulations, and this decrease was accompanied by a reduction in cell cycle reentry and a more euploid neuronal genome.

Topics: Alzheimer Disease; Animals; Astrocytes; Brain; Cell Cycle; Cells, Cultured; Cyclin D1; Disease Models, Animal; DNA Replication; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Glycation End Products, Advanced; Humans; In Situ Hybridization, Fluorescence; Male; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Mitogen-Activated Protein Kinases; Nerve Degeneration; Neurons; Oxidative Stress; Signal Transduction; Thioctic Acid

Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non-coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte and based on Ang-II-induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR-16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR-16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR-16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR-16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post-transcriptionally by miR-16. In addition, the signal transducer and activator of transcription-3 and c-Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR-16 attenuation. Therefore, attenuation of miR-16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR-16 might be a target to manage cardiac hypertrophy.

Topics: Animals; Aorta, Abdominal; Cardiomegaly; Cell Line; Cyclin D1; Cyclin D2; Cyclins; Disease Models, Animal; Enzyme Activation; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Myocytes, Cardiac; Phenylephrine; Phosphorylation; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; STAT3 Transcription Factor

Multiple sclerosis (MS) has been associated with a history of sub-optimal exposure to ultraviolet light, implicating vitamin D3 as a possible protective agent. We evaluated whether 1,25(OH)2D3 attenuates the progression of experimental autoimmune encephalomyelitis (EAE), and explored its potential mechanisms. EAE was induced in C57BL/6 mice via immunization with MOG35-55, and some mice received 1,25(OH)2D3. 1,25(OH)2D3 inhibited EAE progression. Additionally, 1,25(OH)2D3 reduced inflammation, demyelination, and neuron loss in the spinal cord. The protective effect of 1,25(OH)2D3 was associated with significantly elevated expression of Beclin1, increased Bcl-2/Bax ratio, and decreased LC3-II accumulation. Thus, 1,25(OH)2D3 may represent a promising new MS treatment.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Beclin-1; Calcitriol; Calcium Channel Agonists; Cyclin D1; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Extremities; Female; Gene Expression Regulation; Mice; Microtubule-Associated Proteins; Myelin-Oligodendrocyte Glycoprotein; Neurodegenerative Diseases; Peptide Fragments; Phosphopyruvate Hydratase; Spinal Cord

Products of the Pim (the proviral integration site for the Moloney murine leukemia virus) family of proto—oncogenes possess serine/threonine kinase activity and belong to the Ca2+/calmodulin—dependent protein kinase group. Pim—3, a member of the Pim family is closely linked to the development of a variety of tumors. However, the role of Pim—3 in human glioblastoma remains unknown. In this study, we elucidated the role of Pim—3 in the growth and apoptosis of glioblastoma cells. Western blotting was used for determination of protein levels, and shRNA was used for Pim—3 knockdown. The MTT assay was used to evaluate cell proliferation and flow cytometry was used to determine cell cycle status and the number of apoptotic cells. A mouse xenograft model was established by injecting nude mice with Pim—3—depleted glioblastoma cells in order to determine tumor growth in vivo. We demonstrated that Pim—3 was highly expressed in human glioblastoma cell lines. We also found that knockdown of Pim—3 by specific shRNA slowed decreased proliferation, induced cell cycle arrest in the G0/G1 phase, and increased apoptosis in glioblastoma cells. Pim—3 knockdown potently inhibited the growth of subcutaneously implanted glioblastoma cells in vivo. We further revealed that Pim—3 knockdown induced growth inhibition by reducing the levels of the anti—apoptotic protein Bcl—xl and cell cycle regulatory proteins, including cyclin D1 and Cdc25C, and increasing the levels of the pro—apoptotic protein Bax.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinogenesis; cdc25 Phosphatases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Heterografts; Humans; Mice, Nude; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering

Previous studies by our group showed that Qianliening capsules (QC), a clinically proven effective traditional Chinese formulation that has long been used in the treatment of benign prostatic hyperplasia (BPH), is capable of inhibiting BPH in vivo and in vitro via the promotion of apoptosis, suppression of the EGFR/STAT3 signaling pathway and regulating the expression of sex hormones as well as their receptors. However, the mechanism of its anti-BPH activity has remained to be fully elucidated. The present study aimed to investigate the mechanism underlying the anti-proliferative effect of QC in vivo and in vitro. Castrated male Sprage-Dawley (SD) rats where subcutaneously injected with testosterone propionate and the WPMY-1 cell line was stimulated with basic fibroblast growth factor in order to generate BPH in vivo and in vitro separately, both of which were then subjected to QC treatment. Finasteride was used as a positive control drug for the in vivo study. In the present study, it was found that treatment with QC or finasteride significantly reduced the prostatic index (PI=prostate wet weight/body weight x 100) in a rat model of BPH (P<0.05). In addition, reverse transcription quantitative polymerase chain reaction (RT-PCR) and western blot analyses showed that QC or finasteride treatment significantly inhibited model construction-induced upregulation of expression of proliferating cell nuclear antigen, cyclin D1 and cyclin-dependent kinase 4 in prostatic tissues of rats with BPH (P<0.05). The in vitro study further proved that QC exhibited anti-proliferative properties via G1/S cell cycle arrest in the WPMY-1 cell line, as evidenced by colony formation, flow cytometric cell cycle, immunoblot and RT-PCR analyses. In conclusion, the present study demonstrated that inhibition of cell proliferation via G1/S cell cycle arrest may be one of the underlying mechanisms of the effect of QC on BPH.

Topics: Animals; Capsules; Cell Line; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Drugs, Chinese Herbal; Fibroblast Growth Factor 2; G1 Phase Cell Cycle Checkpoints; Male; Proliferating Cell Nuclear Antigen; Prostate; Prostatic Hyperplasia; Rats; Rats, Sprague-Dawley; S Phase Cell Cycle Checkpoints; Signal Transduction

Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC.. Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated up-regulation of NOR1 protein as well as the cell-cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1, and hyperphosphorylation of the retinoblastoma protein were prevented by DNA-PK inhibition. Co-immunoprecipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in post-transcriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury.. DNA-PK directly phosphorylates NOR-1 and, this way, modulates SMC proliferation. These data add to our understanding of vascular remodelling processes and opens new avenues for treatment of vascular proliferative diseases.

Topics: Animals; Atherosclerosis; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; DNA-Activated Protein Kinase; DNA-Binding Proteins; Enzyme Inhibitors; Femoral Artery; Humans; Male; Membrane Transport Proteins; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Nuclear Proteins; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Stability; Proteolysis; Retinoblastoma Protein; RNA Interference; Signal Transduction; Time Factors; Transfection; Ubiquitination; Vascular Remodeling; Vascular System Injuries

Epithelial cells affected by somatic mutations undergo transition from a tumour-suppressive to a carcinogenic Smad pathway during sporadic colorectal carcinogenesis, and the specific linker threonine phosphorylation of Smad2/3 in colon epithelial cells indicates stem-like cells. This study extends previous observations to a model of colitis-associated colorectal cancer.. After Crl:CD-1 mice received an administration of azoxymethane [AOM], the mice were given dextran sodium sulfate [DSS] for 7 days. AOM/DSS-treated mice [AOM/DSS mice] were killed at 10 or 20 weeks. After macroscopic observations, a histopathological analysis was conducted. Immunohistochemical staining was performed using the avidin-biotin immunoperoxidase method [pSmad3C-Ser, pSmad3L-Ser, c-Myc] and immunofluorescent methods [Ki67, β-catenin, CDK4, cyclin D1, Sox9, pSmad2/3L-Thr].. The colons from AOM/DSS mice were shorter than those from control mice. The number of colon tumours at Week 20 was higher than at Week 10. The inflammation scores for AOM/DSS mice were greater than those for control mice. Immunostaining-positive cells (staining by Ki67, β-catenin [nuclear and cytoplasmic], cyclin D1, and Sox9) were diffusely distributed in colon tumours. The percentage of pSmad3L-Ser-positive cells in colon tumours was higher than in sites of pre-neoplastic colitis, and that in sites of pre-neoplastic colitis was higher than in control mice. pSmad2/3L-Thr-positive cells were sparsely detected around crypt bases in non-neoplastic colon epithelia and at the tops of tumours, and immunohistochemical co-localisation of pSmad2/3L-Thr with Ki67 was not observed. Immunohistochemical co-localisation of pSmad2/3L-Thr with β-catenin and CDK4 was observed.. pSmad3L-Ser signalling is an early event in colitis-associated colorectal cancer, and pSmad2/3L-Thr immunostaining-positive cells might be cancer stem cells.

Topics: Animals; Azoxymethane; beta Catenin; Biomarkers, Tumor; Carcinogenesis; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Ki-67 Antigen; Male; Mice; Neoplastic Stem Cells; Phosphorylation; Proto-Oncogene Proteins c-myc; Serine; Signal Transduction; Smad2 Protein; Smad3 Protein; SOX9 Transcription Factor

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3' UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Carcinoma, Hepatocellular; cdc25 Phosphatases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; E2F2 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms, Experimental; Mice; MicroRNAs; RNA Interference; RNA, Messenger; Xenograft Model Antitumor Assays

Liver cancer is a major health-care concern and its oncogenic mechanisms are still largely unclear. Persistent hepatocyte cell death is a common feature among various chronic liver diseases, the blocking of which presents as logical treatment. Therefore, we aimed at investigating tumor development in mice with hepatocyte-specific Bid depletion--a BH3-only Bcl-2 family member that amplifies apoptotic death signals. Hepatocyte-specific conditional Bid-knockout mice (Bid(Δhep)) were injected with 25 mg/kg diethylnitrosamine (DEN) at 14 days of age, and liver tumorigenesis was investigated 9 months later. Additionally, different models of acute liver injury were used including: acute high-dose DEN challenge, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and carbon tetrachloride (CCL4) injection. Bid(Δhep) mice developed significantly fewer tumors, showed smaller maximal and average tumor size and reduced tumor incidence. In the acute DEN model, 48 h post injection we observed a significant reduction in liver injury in Bid(Δhep) animals, assessed via serum transaminases and liver histopathology. Furthermore, TNF-α, IL-1ß, cJUN and IL-6 mRNA expression was reduced. These findings were accompanied by reduced compensatory hepatocyte proliferation in Bid(Δhep) mice when compared with controls by immunohistochemistry for Ki67 and proliferating cell nuclear antigen 48 h after DEN injection. In the acute CCL4 model, Bid(Δhep) mice displayed reductions in liver injury and inflammation when compared with controls. No differences in liver injury and serum bilirubin levels were detected in Bid(Δhep) and Bid(flo/flo) mice fed with DDC, which induces bile duct injury and a ductular reaction. Our study demonstrates that in DEN-induced hepatocellular carcinoma, the inhibition of hepatocyte death pathways through Bid deletion protects animals from tumorigenesis. These results suggest that reducing hepatocyte cell death, liver inflammation and compensatory proliferation has a stronger beneficial effect than the potential side effect of enhancing tumor cell survival.

Topics: Animals; BH3 Interacting Domain Death Agonist Protein; Carbon Tetrachloride; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cyclin D1; Diethylnitrosamine; Disease Models, Animal; Hepatocytes; Hydrogen Peroxide; Inflammation; Interleukin-6; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Non-alcoholic Fatty Liver Disease; Pyridines; Tumor Necrosis Factor-alpha

Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/β-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3β in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/β-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.

Topics: Administration, Oral; Animals; Antineoplastic Agents; beta Catenin; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HCT116 Cells; Hexanones; Humans; Mice, Transgenic; Oxidative Stress; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction; Transcription Factor 7-Like 2 Protein; Wnt Signaling Pathway

It has recently been shown that JARID2 contributes to the malignant character of solid tumors, such as epithelial-mesenchymal transition in lung and colon cancer cell lines, but its role in leukemia progression is unexplored. In this study, we explored the effect and underlying molecular mechanism of JARID2 on leukemia cell proliferation. Real-time PCR and Western assay were carried out to detect JARID2 and CCND1 expression. Cell number and cell cycle change were detected using hemocytometer and flow cytometry, and a ChIP assay was utilized to investigate JARID2 and H3K27me3 enrichment on the CCND1 promoter. JARID2 is down-regulated in B-chronic lymphocytic leukemia (B-CLL) and acute monocytic leukemia (AMOL), and knockdown of JARID2 promotes leukemia cell proliferation via acceleration of the G1/S transition. Conversely, ectopic expression of JARID2 inhibits these malignant phenotypes. Mechanistic studies show that JARID2 negatively regulates CCND1 expression by increasing H3K27 trimethylation on the CCND1 promoter. Our findings indicate that JARID2 is a negative regulator of leukemia cell proliferation, and functions as potential tumor suppressor in leukemia.

Topics: Adult; Aged; Aged, 80 and over; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromosome Aberrations; Cyclin D1; Disease Models, Animal; Down-Regulation; Female; Gene Expression Regulation, Leukemic; Heterografts; Histones; Humans; Leukemia; Male; Mice; Middle Aged; Polycomb Repressive Complex 2; Tumor Burden; Young Adult

The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct β-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and β-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes β-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active β-catenin were markedly increased in the absence of VDR. In vitro, VDR activation in breast cancer cells reduced β-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of β-catenin with the cyclin D1 promoter. Expression of a stabilized form or β-catenin ablated the protective effects of VDR activation.Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of β-catenin activation.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cholecalciferol; Cyclin D1; Disease Models, Animal; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Intercellular Signaling Peptides and Proteins; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Knockout; Neoplasm Invasiveness; Promoter Regions, Genetic; Receptor Protein-Tyrosine Kinases; Receptors, Calcitriol; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Wnt Proteins

MicroRNA-99a (miR-99a) has been reported to function as a tumor suppressor through regulating cell cycle and apoptosis. But its clinical significance in ischemic stroke and its function in cerebral ischemia-reperfusion (I/R) injury remained unknown. Herein transient middle cerebral artery occlusion was built on C57BL/6 mice, followed by intracerebroventricular injection of miR-99a agomir or antagomir before reperfusion for 24h. Our clinical analysis indicates that plasma miR-99a level was significantly decreased in ischemic stroke patients as compared to healthy subjects, and a significant correlation was observed between miR-99a and clinical parameters. And miR-99a overexpression mitigated I/R injury in mice, as evidenced by reduced brain infarct volume and neural apoptosis, whereas miR-99a downregulation aggravates brain injury. In vitro, miR-99a protected neuro-2a cells against hydrogen peroxide-induced oxidative stress injury, by improving cell viability, suppressing LDH release and cell apoptosis. In addition, miR-99a overexpression inhibited H2O2 induced G1/S phase transition in neuro-2a cells, accompanied by a significant decrease in cyclin D1 level and a tendency of down-regulation of CDK6. It was further proved in mice that miR-99a inhibited cyclin D1 and CDK6 expressions following cerebral I/R injury. These findings indicate that miR-99a reduces neuronal damage following cerebral I/R through regulating cell cycle progression and preventing apoptosis, suggesting that miR-99a could be used as a new therapeutic agent targeting neuronal cell cycle re-entry following stroke.

Topics: Animals; Brain Ischemia; Caspase 3; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 6; Disease Models, Animal; Gene Expression Regulation; Hydrogen Peroxide; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Neuroblastoma; Neuroprotective Agents; Oxidants; Reperfusion Injury; Statistics, Nonparametric; Transfection

Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.

Topics: Animals; Cerebral Cortex; Cyclin D1; Disease Models, Animal; Down Syndrome; Dyrk Kinases; G1 Phase; Gene Dosage; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neural Stem Cells; Neurogenesis; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Telencephalon; Trisomy

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Topics: Alleles; Animals; Cell Line, Tumor; Chromosome Breakpoints; Cyclin D1; Disease Models, Animal; DNA-Binding Proteins; Female; Gene Expression Profiling; Genome, Human; Genomics; Humans; Lung Neoplasms; Male; Mice; Mutation; Neurosecretory Systems; Nuclear Proteins; Receptors, Notch; Retinoblastoma Protein; Signal Transduction; Small Cell Lung Carcinoma; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Early brain injury (EBI) plays a crucial role in the pathological progress of subarachnoid hemorrhage (SAH). This study was designed to determine whether rosiglitazone protects the brain against EBI in rats, and discuss the role of the anti-apoptotic mechanism mediated by Bcl-2 family proteins in this neuroprotection. 86 male Sprague-Dawley rats were divided into the sham group, the SAH+ vehicle group and the SAH+ rosiglitazone group. SAH was induced via an endovascular perforation technique and rosiglitazone (3mg/kg) or vehicle was administered. Mortality, neurological scores, brain water content, Evans blue dye assay, TUNEL stain assay, Gelatin zymography, and western blot analysis were performed. Rosiglitazone significantly improved mortality, neurological scores, brain water content, blood brain barrier (BBB) and apoptosis compared with the vehicle group within 24h after SAH. The TUNEL staining assay demonstrated that apoptosis was ameliorated. Cleaved Caspase-3 and MMP-9 expression was reduced, whereas Bcl-2 and p-Bad was markedly preserved by rosiglitazone. A significant elevation of p-Akt was detected after rosiglitazone treatment. Our study demonstrated that rosiglitazone plays a neuroprotective role in EBI after SAH via attenuation of BBB disruption, brain edema and apoptosis.

Topics: Analysis of Variance; Animals; Blood-Brain Barrier; Brain Edema; Brain Injuries; Caspase 3; Cyclin D1; Disease Models, Animal; In Situ Nick-End Labeling; Male; Matrix Metalloproteinase 9; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Rosiglitazone; Subarachnoid Hemorrhage; Thiazolidinediones

Small-cell neuroendocrine differentiation in prostatic carcinoma is an increasingly common resistance mechanism to potent androgen deprivation therapy (ADT), but can be difficult to identify morphologically. We investigated whether cyclin D1 and p16 expression can inform on Rb functional status and distinguish small-cell carcinoma from adenocarcinoma.. We used gene expression data and immunohistochemistry to examine cyclin D1 and p16 levels in patient-derived xenografts (PDX), and prostatic small-cell carcinoma and adenocarcinoma specimens.. Using PDX, we show proof-of-concept that a high ratio of p16 to cyclin D1 gene expression reflects underlying Rb functional loss and distinguishes morphologically identified small-cell carcinoma from prostatic adenocarcinoma in patient specimens (n = 13 and 9, respectively). At the protein level, cyclin D1, but not p16, was useful to distinguish small-cell carcinoma from adenocarcinoma. Overall, 88% (36/41) of small-cell carcinomas showed cyclin D1 loss by immunostaining compared with 2% (2/94) of Gleason score 7-10 primary adenocarcinomas at radical prostatectomy, 9% (4/44) of Gleason score 9-10 primary adenocarcinomas at needle biopsy, and 7% (8/115) of individual metastases from 39 patients at autopsy. Though rare adenocarcinomas showed cyclin D1 loss, many of these were associated with clinical features of small-cell carcinoma, and in a cohort of men treated with adjuvant ADT who developed metastasis, lower cyclin D1 gene expression was associated with more rapid onset of metastasis and death.. Cyclin D1 loss identifies prostate tumors with small-cell differentiation and may identify a small subset of adenocarcinomas with poor prognosis. Clin Cancer Res; 21(24); 5619-29. ©2015 AACR.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinoma, Small Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Gene Expression; Gene Expression Profiling; Heterografts; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Mice; Neoplasm Grading; Neoplasm Metastasis; Prognosis; Prostatic Neoplasms; Retinoblastoma Protein

It was reported that the receptor tyrosine kinase (RTK) family often highly expressed in several mucinous carcinomas. In the present study, we established a murine model of colorectal mucinous adenocardinoma (CRMAC) by treating C57 mice [both wild type (WT) and loss-of-function c-kit mutant type (Wads-/-)] with AOM+DSS for 37 weeks and found that c-kit, a member of RTK family, clearly enhanced the tumor cell proliferation by decreasing p53 and increasing cyclin D1 through AKT pathway. Significantly, c-kit strongly promoted tumor cell invasiveness by increasing ETV4, which induced MMP7 expression and epithelial-mesenchymal transition (EMT) via ERK pathway. In vitro up- or down-regulating c-kit activation in human colorectal cancer HCT-116 cells further consolidated these results. In conclusion, our data suggested that the c-kit signaling obviously promoted proliferation and invasion of CRMAC. Therefore, targeting the c-kit signaling and its downstream molecules might provide the potential strategies for treatment of patients suffering from CRMAC in the future.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Animals; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Genotype; HCT116 Cells; Humans; Lentivirus; Matrix Metalloproteinase 7; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-kit; Signal Transduction; Tumor Suppressor Protein p53

Condensed-bicyclic triazolo-thiadiazoles were synthesized via an efficient "green" catalyst strategy and identified as effective inhibitors of PTP1B in vitro. The lead compound, 6-(2-benzylphenyl)-3-phenyl-[1,2,4]triazolo[3][1,3,4]thiadiazole (BPTT) was most effective against human hepatoma cells, inhibits cell invasion, and decreases neovasculature in HUVEC and also tumor volume in EAT mouse models. This report describes an experimentally unidentified class of condensed-bicyclic triazolo-thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds.

Topics: Animals; Antineoplastic Agents; Benzofurans; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Movement; Chromones; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; G1 Phase Cell Cycle Checkpoints; Hep G2 Cells; Human Umbilical Vein Endothelial Cells; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Mice; Models, Molecular; Neoplasm Invasiveness; Neovascularization, Pathologic; Poly(ADP-ribose) Polymerases; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; STAT3 Transcription Factor; Structure-Activity Relationship; Survivin; Thiadiazoles; Triazoles

Endometrial cancer is one of the most common gynecologic malignancies. Phosphatase and tensin homologue (PTEN)-mutation is frequently identified in endometrial cancer patients. Although high dietary intake of ω-3 polyunsaturated fatty acids (PUFAs) has been associated with reduced risk of endometrial cancer, the underlying mechanisms is still unknown. To this end, we evaluated the impact of ω-3 PUFAs using several endometrial cancer cellular and animal models. While ~27% and 40% of heterozygotic PTEN mutant mice developed endometrial cancer and atypical complex hyperplasia, respectively, none of the PTEN(+/-) mice developed cancer when we overexpressed an mfat-1 transgene, which allowed endogenous production of ω-3 PUFAs. Fish oil-enriched diet or expression of mfat-1 transgene significantly inhibited the growth of xenograft tumor derived from RL95-2 cells bearing a PTEN null mutation. At cellular level, ω-3 PUFAs treatment decreased the viability of RL95-2 cells, AKT phosphorylation, and cyclin D1 expression. These molecular events are primarily mediated through reduction of cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. Exogenous PGE2 treatment completely blunted the impact of ω-3 PUFAs on endometrial cancer. Thus, we revealed the direct inhibitory effects of ω-3 PUFAs on endometrial cancer development and the underlying mechanisms involving reduction of COX-2 and PGE2.

Topics: Animals; Cadherins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Eicosanoids; Endometrial Neoplasms; Fatty Acids, Omega-3; Female; Gene Expression; Heterografts; Humans; Metabolomics; Mice; Mice, Knockout; Mice, Transgenic; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.

Topics: Animals; Bacterial Adhesion; Biomarkers; Cattle; Cell Division; Cell Line; Colon; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Enterohemorrhagic Escherichia coli; Gene Expression; Humans; Intestinal Mucosa; Karyotype; Male; Mutation; Phenotype; Salmonella enterica; Telomerase; Toll-Like Receptors

To preliminarily investigate the temporal patterns of the endogenous mRNA expression for members of the Wnt signaling and a series of genes regulating bone formation during the development of traumatic temporomandibular joint (TMJ) bony ankylosis in a sheep model.. Six sheep were used for the induction of bony ankylosis of TMJ. We performed a condylar fracture, excision of the lateral 2/3 disc and serious injury to the glenoid fossa to induce bony ankylosis on the right TMJ. An isolated condylar fracture was performed on the left side. Two sheep were sacrificed at 1 month, 3 months, and 6 months after surgery, respectively. The specimens from the ankylosed joint and the condylar fracture were harvested for RNA extraction respectively. In this report (Part I), only the bony ankylosed samples were used for analysis of gene expressions. The specimens 1 month postoperatively were taken as the control, and the changes of expression of target genes over time were examined by real-time PCR.. mRNA expression of Wnt1, Wnt2b, Wnt3a, β-catenin, Sfrp1, Lrp6, Lef1, CyclinD1, and Runx2 was up-regulated at 3 and 6 months compared with 1 month. The expression of Wnt5a, Sox9, and Osterix was up-regulated with a peak at 3 months, and then fell back to the basal levels at 6 months. The expression of Ocn began to up-regulate until 6 month postoperatively.. Our findings suggested that Wnt signaling was involved in the formation of traumatic TMJ bony ankylosis and thus may be a potential therapeutic target for the treatment of the disease in the future.

Topics: Animals; Ankylosis; beta Catenin; Core Binding Factor Alpha 1 Subunit; Cyclin D1; Disease Models, Animal; Gene Expression Profiling; Glycoproteins; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Low Density Lipoprotein Receptor-Related Protein-6; Lymphoid Enhancer-Binding Factor 1; Mandibular Condyle; Mandibular Fractures; Osteocalcin; Osteogenesis; Pilot Projects; Proto-Oncogene Proteins; Sheep; SOX9 Transcription Factor; Temporal Bone; Temporomandibular Joint; Temporomandibular Joint Disc; Temporomandibular Joint Disorders; Transcription Factors; Wnt Proteins; Wnt Signaling Pathway; Wnt1 Protein; Wnt3A Protein

Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been fully investigated. We have previously described the FCPIK3ca* murine colon cancer model, which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas. These cancers form via a non-canonical mechanism of tumor initiation that is mediated through activation of PI3K and not through aberrations in WNT signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the majority of human colon cancers and often occurs simultaneously with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. In this study, we have generated mice in which the expression of a constitutively active PI3K and the loss of APC occur simultaneously in the distal small intestine and colon. Here, we demonstrate that expression of a dominant active PI3K synergizes with loss of APC activity resulting in a dramatic change in tumor multiplicity, size, morphology and invasiveness. Activation of the PI3K pathway is not able to directly activate WNT signaling through the nuclear localization of CTNNB1 (β-catenin) in the absence of aberrant WNT signaling. Alterations at the transcriptional level, including increased CCND1, may be the etiology of synergy between these activated pathways.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Cell Nucleus; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Epistasis, Genetic; Female; Gene Expression; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microsatellite Instability; Phosphatidylinositol 3-Kinases; Tumor Burden; Wnt Signaling Pathway

The purpose of the study was to preliminarily explore the differential expressions of a series of genes regulating bone formation in temporomandibular joint (TMJ) fibrous ankylosis, bony ankylosis and condylar fracture healing.. The cDNA from either the bony ankylosed callus or fracture callus of the 6 sheep, as described in the part I, were both used in the study. The differences of gene expressions between bony ankylosis and condylar fracture at 1, 3, and 6 months postoperatively were measured by real-time PCR, with 2 samples at each time point. In addition, another 2 sheep were added to have fibrous ankylosis induced on the right TMJ, and 1 sheep was sacrificed at 3 and 6 months after surgery, respectively. The differences of gene expressions between fibrous and bony ankylosis at 3 and 6 months postoperatively were measured by real-time PCR.. Bony ankylosis showed higher mRNA expression trends in Wnt2b, Wnt5a, β-Catenin, Lef1, CyclinD1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn, Bmp2, and Bmp7 compared to fibrous ankylosis, although no statistical analysis was performed due to the very small sample size. Whereas bony ankylosis showed a significant lower expression of Wnt5a, β-Catenin, Lef1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn and Bmp4 compared to condylar fracture at several time points (P < 0.05).. Our data provided a preliminary molecular evidence for the hypothesis that the development of traumatic TMJ bony ankylosis was the course of delayed bone healing or hypertrophic nonunion, and deserved to be further studied.

Topics: Alkaline Phosphatase; Animals; Ankylosis; beta Catenin; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bony Callus; Collagen Type X; Core Binding Factor Alpha 1 Subunit; Cyclin D1; Disease Models, Animal; Fibrosis; Fracture Healing; Gene Expression Profiling; Gene Expression Regulation; Lymphoid Enhancer-Binding Factor 1; Mandibular Condyle; Mandibular Fractures; Osteocalcin; Pilot Projects; Proto-Oncogene Proteins; Sheep; SOX9 Transcription Factor; Temporomandibular Joint; Temporomandibular Joint Disorders; Transcription Factors; Wnt Proteins

Liver X receptors (LXRs), including LXRα and LXRβ isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expression of cyclin D1 and cyclin B1, and thus suppresses the proliferation of multiple cancer cells, but the relevant mechanism is not well known. Forkhead box M1 (FOXM1) is a proliferation-specific member of forkhead box family, which is highly expressed in proliferating normal cells and numerous cancer cells. FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the enhancement of cell cycle progression and cell proliferation. However, it is unclear whether LXRs are involved in the regulation of FOXM1. In this study, we demonstrated that specific LXRs agonists downregulated expression of FOXM1, cyclin D1 and cyclin B1 in hepatocellular carcinoma (HCC) cells, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly alleviated LXRs activation-mediated cell cycle arrest and cell growth suppression. Reporter assays showed that the activation of LXRs significantly reduced the transcriptional activity of FOXM1 promoter. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that LXRα but not LXRβ could bind to an inverted repeat IR2 (-52CCGTCAcgTGACCT-39) in the promoter region of FOXM1 gene. Moreover, the xenograft tumor growth and the corresponding FOXM1 expression in nude mice were dramatically repressed by LXRs agonists. Taken together, we conclude that LXRα but not LXRβ functions as a transcriptional repressor for FOXM1 expression. The pathway 'LXRα-FOXM1-cyclin D1/cyclin B1' is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for HCC treatment.

Topics: Animals; Base Sequence; Benzoates; Benzylamines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Disease Models, Animal; Down-Regulation; Forkhead Box Protein M1; Forkhead Transcription Factors; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatocytes; Heterografts; Humans; Liver Neoplasms; Liver X Receptors; Mice; Molecular Sequence Data; Orphan Nuclear Receptors; Promoter Regions, Genetic; Protein Binding; Transcription, Genetic

Hyperammonaemia is a serious metabolic disorder commonly observed in patients with hepatic failure. However, it is unknown whether hyperammonaemia has a direct adverse effect on the hepatocytes and thereby serves as both a cause and effect of hepatic failure.. The purposes were to determine whether hepatic injury can be caused by hyperammonaemia, and if so, screen the key genes involved in hyperammonaemia.. Hyperammonaemic rats were established via intragastric administration of the ammonium chloride solution. The liver tissues were assessed via biochemistry, histology, immunohistochemistry and microarray analysis. Selected genes were confirmed by quantitative RT-PCR.. Administration of the ammonium chloride caused the hyperammonaemia, accompanied with the changes of plasma markers indicating hepatic injury. A pathological assessment demonstrated increased apoptosis and higher level of cyclin D1 and cyclin A in hyperammonaemic rat liver. Microarray was performed on the liver samples and 198 differentially expressed genes were identified in hyperammonaemic rats and validated by quantitative RT-PCR. These genes were associated with many vital functional classes and belonged to different signal transduction pathways.. This study demonstrates that hyperammonaemia can directly induce hepatic injury via the hepatocyte apoptosis. Gene expression profile may provide the possible explanations and mechanisms for the hepatic injury induced by hyperammonaemia.

Topics: Ammonium Chloride; Animals; Apoptosis; Cyclin A; Cyclin D1; Disease Models, Animal; Gene Expression Profiling; Hyperammonemia; Liver; Male; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Rats, Sprague-Dawley

We investigated the role of matrix metalloproteinase-8 (MMP8) in neointima formation and in vascular smooth muscle cell (VSMC) migration and proliferation.. After carotid artery wire injuring, MMP8(-/-)/apoE(-/-) mice had fewer proliferating cells in neointimal lesions and smaller lesion sizes. Ex vivo assays comparing VSMCs isolated from MMP8 knockout and wild-type mice showed that MMP8 knockout decreased proliferation and migration. Proteomics analysis revealed that a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) had lower concentrations in MMP8 knockout VSMC culture media than in MMP8 wild-type VSMC culture media. Western blot, flow cytometric, and immunocytochemical analyses showed that MMP8 knockout VSMCs contained more pro-ADAM10 but less mature ADAM10, more N-cadherin, and β-catenin in the plasma membrane but less β-catenin in the nucleus and less cyclin D1. Treatment of MMP8 wild-type VSMCs with an ADAM10 inhibitor, GI254023X, or siRNA knockdown of ADAM10 in MMP8 wild-type VSMCs inhibited proliferation and migration, increased N-cadherin and β-catenin in the plasma membrane, reduced β-catenin in the nucleus, and decreased cyclin D1 expression. Incubation of MMP8 knockout VSMCs with a recombinant ADAM10 rescued the proliferative and migratory ability of MMP8 knockout VSMCs and increased cyclin D1 expression. Furthermore, immunohistochemical analyses showed colocalization of ADAM10 with VSMCs and N-cadherin, and nuclear accumulation of β-catenin in the neointima in apoE(-/-)/MMP8(+/+) mice.. MMP8 enhances VSMC proliferation via an ADAM10, N-cadherin, and β-catenin-mediated pathway and plays an important role in neointima formation.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; Animals; Apolipoproteins E; beta Catenin; Cadherins; Carotid Artery Injuries; Cell Movement; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Cyclin D1; Disease Models, Animal; Matrix Metalloproteinase 8; Matrix Metalloproteinase Inhibitors; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Proteomics; RNA Interference; Time Factors; Transfection; Wnt Signaling Pathway; Wnt1 Protein

Clinical responses to the immmunomodulatory drug lenalidomide have been observed in patients with relapsed/refractory mantle cell lymphoma (MCL), although its mechanism of action remains partially unknown. We investigated whether the expression and subcellular localization of cyclin D1, a major cell-cycle regulator overexpressed in MCL, and the cyclin-dependent kinase inhibitor p27(KIP1), could identify MCL cases sensitive to lenalidomide, and whether the compound could modulate cyclin D1/p27(KIP1) complexes in MCL cells.. MCL primary samples and cell lines were analyzed for subcellular levels of cyclin D1/p27(KIP1) complexes by Western blot, immunohistochemistry, immunoprecipitation, and flow cytometry. Activity of lenalidomide in vitro and its effect on cyclin D1/p27(KIP1) complexes were evaluated by real-time PCR, immunoprecipitation, immunofluorescence, and Western blot. In vivo validation was carried out in a mouse xenograft model of human MCL.. We found cyclin D1 and p27(KIP1) to be coordinately expressed in all the MCL samples tested. Immunoprecipitation analyses and siRNA assays suggested a direct role of cyclin D1 in the regulation of p27(KIP1) levels. The nuclear accumulation of both proteins correlated with MCL cell tumorigenicity in vivo, and sensitivity to lenalidomide activity in vitro and in vivo. Lenalidomide mechanism of action relied on cyclin D1 downregulation and disruption of cyclin D1/p27(KIP1) complexes, followed by cytosolic accumulation of p27(KIP1), cell proliferation arrest, apoptosis, and angiogenesis inhibition.. These results highlight a mechanism of action of lenalidomide in MCL cases with increased tumorigenicity in vivo, which is mediated by the dissociation of cyclin D1/p27(KIP1) complexes, and subsequent proliferation blockade and apoptosis induction.

Topics: Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Female; Gene Expression; Heterografts; Humans; Lenalidomide; Lymphoma, Mantle-Cell; Male; Mice; Middle Aged; Protein Binding; Protein Stability; Thalidomide; Tumor Burden

MicroRNAs are small RNA molecules that post-transcriptionally regulate gene expression. MicroRNA-122 is the most abundant and specific liver microRNA. Hepatotoxicity involves a significant alteration of liver gene expression. The aim of this work was to evaluate the microRNA-122 regulatory network in models of hepatotoxicity induced by thioacetamide or carbon tetrachloride. We report that the toxins decreased the expression of microRNA-122, which corresponded with an increase in two target genes: Cyclin G1 and the cationic amino acid transporter CAT-1. We found a decreased expression of its precursor, pri-microRNA-122, and of the transcription factors that specifically bind its promoter: CCAAT/enhancer-binding protein alpha, and members of the hepatocyte nuclear factor family. Therefore, microRNA-122 expression levels are under transcriptional control during hepatotoxicity. We propose that the changes observed are associated with the liver response to cope with the injury caused by the hepatotoxins, likely through a cell proliferation process to repair the damaged tissue.

Topics: Animals; Carbon Tetrachloride; Cationic Amino Acid Transporter 1; CCAAT-Enhancer-Binding Protein-alpha; Chemical and Drug Induced Liver Injury; Cyclin D1; Cyclin G1; Cytochrome P-450 CYP2E1; Disease Models, Animal; Glutamate-Ammonia Ligase; Hepatocyte Nuclear Factor 4; Liver; Male; MicroRNAs; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; RNA, Messenger; Thioacetamide; Transcription, Genetic

The key role of the Wnt/β-catenin signaling in colorectal cancer (CRC) insurgence and progression is now recognized and several therapeutic strategies targeting this pathway are currently in developing. Wnt/β-catenin signaling not only dominates the early stages of sporadic colorectal cancer (SCC), but could also represent the connection between inflammatory bowel diseases (IBD) and increased risk of developing SCC. The knowledge on the sequential molecular events of Wnt-signaling cascade in IBD and during colorectal carcinogenesis, might provide new diagnostic/prognostic markers and could be helpful for optimizing the treatment protocols, thus improving the efficacy of Wnt-targeting therapies. We performed a comparative evaluation of the expression of some crucial molecules participating to Wnt signaling in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC. Specifically, we analyzed upstream events of Wnt signaling including β-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 expression. We demonstrated that these crucial components of the Wnt/β-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analyses of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and SCC, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Furthermore, we showed that the animal model of chemically-induced CRC mimics the molecular events of Wnt signaling during IBD and SCC development in humans and may therefore be suitable for testing chemopreventive or therapeutic drugs targeting this pathway.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Cadherins; Carcinogenesis; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Disease Progression; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Paraffin Embedding; Proto-Oncogene Proteins c-myc; Rats; Signal Transduction; Wnt Signaling Pathway

Hypothyroidism affects neuron population dynamics in the hippocampus of the adult rat, with neuronal damage as the main feature of its effect. This effect is prevented by the blockade of NMDA receptors, which suggests that glutamatergic activity mediates cell death in this condition. Glutamate can also stimulate cell proliferation and survival of newborn neurons, indicating that it can affect different stages of the cell cycle. In this work we measured the expression of specific proteins that control cell proliferation (cycline-D1), cell arrest (p21), damage (p53) or apoptosis (Bax and Bcl2) in the hippocampus of hypothyroid rats treated with the NMDA receptor (NMDAR) blocker MK-801 during the induction of hypothyroidism. The results show that hypothyroidism increases the expression of markers of DNA damage, cell arrest, and apoptosis, but does not affect the marker of cell proliferation. NMDAR blockade prevents the increase on markers of DNA damage and apoptosis, but does not influence cell arrest or cell proliferation. This suggests that hypothyroidism promotes cell death mainly by an excitotoxic effect of glutamate.

Topics: Analysis of Variance; Animals; Antithyroid Agents; Apoptosis; bcl-2-Associated X Protein; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; Hypothyroidism; Methimazole; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Suppressor Protein p53

Chemokine (C-C motif) receptor 7 (CCR7) is involved in lymph-node homing of naive and regulatory T cells and lymphatic metastasis of cancer cells. Sialic acids comprise a group of monosaccharide units that are added to the terminal position of the oligosaccharide chain of glycoproteins by sialyation. Recent studies suggest that aberrant sialylation of receptor proteins contributes to proliferation, motility, and drug resistance of cancer cells. In this study, we addressed whether CCR7 is a sialylated receptor protein and tried to elucidate the effect of sialylation in the regulation of signal transduction and biological function of CCR7. Our results demonstrated that α-2, 3-sialyltransferase which catalyze sialylation reaction in vivo was overexpressed in breast tumor tissues and cell lines. Lectin blot analysis clearly demonstrated that CCR7 receptor was sialyated in breast cancer cells. Chemokine (C-C motif) ligand 19 (CCL19), the cognate ligand for CCR7, induced the activation of extracellular signal-regulated kinase (ERK) and AKT signaling and increased the expression of cell cycle regulatory proteins and proliferation of breast cancer cells. When cells were pre-treated with a sialyltransferase inhibitor AL10 or sialidase, CCL19-induced cell growth was significantly suppressed. CCL19 also increased invasion and prevented anoikis by up-regulating pro-survival proteins Bcl-2 and Bcl-xL. Inhibition of sialylation by AL10 totally abolished these effects. Finally, we showed that AL10 inhibited tumorigenicity of breast cancer in experimental animals. Taken together, we demonstrate for the first time that CCR7 receptor is a sialylated protein and sialylation is important for the paracrine stimulation by its endogenous ligand CCL19. In addition, inhibition of aberrant sialylation of CCR7 suppresses proliferation and invasion and triggers anoikis in breast cancer cells. Targeting of sialylation enzymes may be a novel strategy for breast cancer treatment.

Topics: Animals; Anoikis; beta-Galactoside alpha-2,3-Sialyltransferase; Breast Neoplasms; Cell Line; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Chemokine CCL19; Cyclin D1; Disease Models, Animal; Epithelial Cells; Female; Gene Expression; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heterografts; Humans; Receptors, CCR7; RNA Processing, Post-Transcriptional; Sialyltransferases; Signal Transduction; Tumor Burden

The aim of this study was to investigate the renoprotective activity of chlorogenic acid (CA) in a murine model of cisplatin (CP)-induced kidney injury. Male BALB/cN mice were gavaged daily with CA at 3, 10 and 30mg/kg for two successive days, 48h after intraperitoneal injection of CP (13mg/kg). On the fifth day, serum creatinine and blood urea nitrogen (BUN) levels were significantly increased in CP-intoxicated mice, which was recovered by CA. Renal oxidative stress, evidenced by increased 4-hydroxynonenal (4-HNE) expression, was significantly reduced with CA. Simultaneously, the overexpression of heme oxygenase 1 (HO-1) and cytochrome P450 E1 (CYP2E1) was attenuated. The inhibition of inflammatory response by CA was achieved through the reduction of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) expression. Additionally, CA significantly suppressed p53, Bax active caspase-3, cyclin D1 and microtubule-associated protein 1 light chain 3 isoform B (LC3B) expression, suggesting the inhibition of both apoptosis and autophagy. The expression of multidrug resistance-associated proteins (Mrp1 and Mrp2) increased and organic cation transporter 2 (Oct2) decreased by CP, protecting the kidneys from nephrotoxicity by reducing the burden of tubular cells. CA dose-dependently restored Mrp1, Mrp2 and Oct2 expression. The recovery of kidney tissue form CP injury was accompanied by increased proliferating nuclear cell antigen (PCNA) expression. The results of this study suggest that CA attenuates CP-induced kidney injury through suppression of oxidative stress, inflammation, apoptosis and autophagy, with the improvement in kidney regeneration.

Topics: Acute Kidney Injury; Aldehydes; Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Biomarkers; Blood Urea Nitrogen; Caspase 3; Chlorogenic Acid; Cisplatin; Cyclin D1; Cyclooxygenase 2; Cytochrome P-450 CYP2E1; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Heme Oxygenase-1; Inflammation Mediators; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Organic Cation Transport Proteins; Organic Cation Transporter 2; Oxidative Stress; Proliferating Cell Nuclear Antigen; Protective Agents; Regeneration; Time Factors; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Prostate cancer incidence and mortality rates have increased over the past years. The purpose of the present study was to examine the molecular mechanism underlying the chemopreventive effects of quercetin on prostate cancer in an in vivo model. Sprague-Dawley male rats were divided into four groups, Group I: vehicle control (propylene glycol), Group II: chemically induced cancer model (MNU + T); Group III: chemically induced cancer model + quercetin (200 mg per kg b.w.); Group IV: quercetin (200 mg per kg b.w.). Serum levels of quercetin were assessed by high performance liquid chromatography (HPLC). EGFR, PI3K/Akt protein levels were significantly increased in chemically induced cancer rats, which were brought back to normalcy in both DLP & VP (dorsolateral prostate & ventral prostate) by quercetin supplementation. Also, the protein expression levels of proliferating cell nuclear antigen (PCNA), N-cadherin, vimentin, and cyclin D1 exhibited a significant increase in both DLP & VP of chemically induced cancer rats. However, simultaneous quercetin supplementation significantly decreased PCNA, N-cadherin, vimentin, and cyclin D1 protein levels compared to chemically induced cancer rats. The E-cadherin expression was decreased in chemically induced cancer animals. Simultaneous quercetin supplementation prevented it. Real time PCR was used to study the mRNA expression of snail, slug and twist. Quercetin significantly decreased snail, slug, and twist mRNA levels in chemically induced cancer rats. To conclude from the present study, quercetin was effective in preventing prostate cancer progression by inhibiting the EGFR signaling pathway and by regulating cell adhesion molecules in Sprague Dawley rats.

Topics: Animals; Anticarcinogenic Agents; Cadherins; Cell Adhesion; Chemoprevention; Chromatography, High Pressure Liquid; Cyclin D1; Disease Models, Animal; ErbB Receptors; Male; Phosphatidylinositol 3-Kinases; Polyphenols; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quercetin; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Vimentin

Myxofibrosarcomas frequently display arm-level gains on 5p. We characterized the pathogenetic and therapeutic relevance of the α-methylacyl coenzyme A racemase (AMACR) at 5p13.3.. AMACR mRNA expression in myxofibrosarcomas was analyzed using the public transcriptome and laser-microdissected sarcoma cells. We performed florescence in situ hybridization (FISH) and immunohistochemistry in independent samples for clinical correlates. In AMACR-overexpressing myxofibrosarcoma cells and xenografts, we elucidated the biologic function of AMACR using RNA interference and explored the therapeutic effect and mechanism of an AMACR inhibitor, ebselen oxide.. AMACR protein overexpression and gene amplification were significantly associated with each other (P < 0.001), with higher tumor grades (both P ≤ 0.002), and univariately with worse metastasis-free survival (MFS; both P < 0.0001) and disease-specific survival (DSS; P = 0.0002 for overexpression; P = 0.0062 for amplification). AMACR protein overexpression also independently portended adverse outcome (DSS, P = 0.007; MFS, P = 0.001). However, 39% of AMACR-overexpression cases did not show gene amplification, implying alternative regulatory mechanisms. In myxofibrosarcoma cell lines, stable AMACR knockdown suppressed cell proliferation, anchorage-independent growth, and expression of cyclin D1 and cyclin T2. These growth-promoting attributes of AMACR were corroborated in the AMACR-silenced xenograft model and AMACR-underexpressed myxofibrosarcomas, showing decreased labeling for cyclin D1, cyclin T2, and Ki-67. Compared with fibroblasts, AMACR-expressing myxofibrosarcoma cells were more susceptible to ebselen oxide, which not only decreased viable cells, promoted proteasome-mediated degradation of AMACR protein, and induced cellular apoptosis in vitro, but also dose-dependently suppressed xenografted tumor growth in vivo.. Overexpressed AMACR in myxofibrosarcomas can be amplification-driven, associated with tumor aggressiveness, and may be relevant as a druggable target.

Topics: Adult; Aged; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cluster Analysis; Comparative Genomic Hybridization; Cyclin D1; Cyclin T; Datasets as Topic; Disease Models, Animal; Female; Fibrosarcoma; Gene Amplification; Gene Dosage; Gene Expression; Gene Expression Profiling; Heterografts; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; Proteasome Endopeptidase Complex; Racemases and Epimerases; RNA, Messenger; Tumor Burden

To investigate esophageal Helicobacter pylori (H. pylori) colonization on esophageal injury caused by reflux and the related mechanisms.. An esophagitis model, with acid and bile reflux, was surgically produced in male rats. The rats were randomly divided into either: (1) an esophagogastroduodenal anastomosis (EGDA) group; (2) an EGDA with H. pylori infection group; (3) a pseudo-operation with H. pylori infection group; or (4) a pseudo-operation group. All rats were kept for 36 wk. Based on the location of H. pylori colonization, the EGDA rats with H. pylori infection were subdivided into those with concomitant esophageal H. pylori colonization or those with only gastric H. pylori colonization. The esophageal injuries were evaluated grossly and microscopically. The expressions of CDX2 and MUC2 were determined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Ki-67 antigen expression was determined by immunohistochemistry. The mRNA levels of cyclin D1, c-Myc, Bax and Bcl-2 were determined by RT-PCR. Cell apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method.. Esophagitis, Barrett's esophagus (BE), and esophageal adenocarcinoma (EAC) developed in rats that underwent EGDA. When comparing rats with EGDA and concomitant esophageal H. pylori colonization to EGDA-only rats, the severity of injury (87.9 ± 5.2 vs 77.2 ± 8.6, macroscopically, 92.5 ± 8.0 vs 83.8 ± 5.5, microscopically, both P < 0.05) and the incidences of BE (80.0% vs 33.3%, P = 0.055) and EAC (60.0% vs 11.1%, P < 0.05) were increased. These increases were associated with upregulation of CDX2 and MUC2 mRNA (10.1 ± 5.4 vs 3.0 ± 2.9, 8.4 ± 4.6 vs 2.0 ± 3.2, respectively, Ps < 0.01) and protein (8.1 ± 2.3 vs 3.3 ± 3.1, 7.3 ± 4.0 vs 1.8 ± 2.7, respectively, all P < 0.05). The expression of Ki-67 (8.9 ± 0.7 vs 6.0 ± 1.7, P < 0.01) and the presence of apoptotic cells (8.3 ± 1.1 vs 5.3 ± 1.7, P < 0.01) were also increased significantly in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only. The mRNA levels of cyclin D1 (5.8 ± 1.9 vs 3.4 ± 1.3, P < 0.01), c-Myc (6.4 ± 1.7 vs 3.7 ± 1.2, P < 0.01), and Bax (8.6 ± 1.6 vs 5.1 ± 1.3, P < 0.01) were significantly increased, whereas the mRNA level of Bcl-2 (0.6 ± 0.3 vs 0.8 ± 0.3, P < 0.01) was significantly reduced in rats with EGDA and concomitant esophageal H. pylori colonization compared with rats with EGDA only.. Esophageal H. pylori colonization increases esophagitis severity, and facilitates the development of BE and EAC with the augmentation of cell proliferation and apoptosis in esophageal mucosa.

Topics: Adenocarcinoma; Animals; Apoptosis; Barrett Esophagus; bcl-2-Associated X Protein; CDX2 Transcription Factor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Esophageal Neoplasms; Esophagitis, Peptic; Esophagus; Gastroesophageal Reflux; Gene Expression Regulation; Helicobacter Infections; Helicobacter pylori; Homeodomain Proteins; Ki-67 Antigen; Male; Mucin-2; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Rats, Sprague-Dawley; RNA, Messenger; Severity of Illness Index; Transcription Factors

The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells.. Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry.. Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant.. Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Cell Transplantation; Cyclin D1; Disease Models, Animal; Epithelial Cells; Female; Genetic Engineering; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Oncogenes; Polycomb Repressive Complex 1; Precancerous Conditions; Proto-Oncogene Proteins c-myc; Receptor, ErbB-3; Receptor, ErbB-4; RNA, Small Interfering; Telomerase; Transgenes; Tumor Suppressor Protein p53

Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action.. MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis.. Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.

Topics: Animals; Apoptosis; Calcium; Calcium-Binding Proteins; Carotid Artery Injuries; Carotid Artery, External; Cell Dedifferentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; Humans; Male; Membrane Glycoproteins; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Phenotype; Rats; Rats, Wistar; Signal Transduction; Stromal Interaction Molecule 1; Time Factors; Transfection; Up-Regulation

Colorectal cancer (CRC) is the second leading cause of death from cancer in the United States. Colorectal cancers have a prolonged latency following initiation that may span decades providing ample time for implementing a chemoprevention strategy that could block or reverse the progression to CRC. Cdk4 pathway alterations have been linked to a number of cancers including CRC. In these experiments we focused on the Cdk4 pathway and its role in intestinal tumorigenesis as a possible target in chemoprevention strategies.. We evaluated the effect of Cdk4 blockade on the prevention of intestinal tumor formation by crossing Cdk4(-/-) mice to Apc(-/+) mice. In addition, we tested the effect of the dietary compound silibinin on the Cdk4 pathway in Apc(-/+) mice and HT-29 colon cancer cells in culture.. Cdk4(-/-) mice backcrossed to Apc(-/+) mice reduced intestinal adenoma formation compared to Apc(-/+) controls. Silibinin effectively targeted the Cdk4 pathway causing hypophosphorylation of the retinoblastoma protein, inhibited cell growth, and induced apoptosis. As a result silibinin blocked the development of intestinal adenomas by 52% in this genetic model (Apc(-/+) mice) of early events in colorectal cancer formation. No toxic abnormalities were detected in mice which received silibinin.. Modification of the Cdk4 pathway using a natural plant-derived compound such as silibinin may be a useful chemopreventive strategy for colorectal carcinomas.

Topics: Adenoma; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemoprevention; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Female; Genes, APC; Humans; Ki-67 Antigen; Mice; Mice, Knockout; Retinoblastoma Protein; Signal Transduction; Silybin; Silymarin

In the present study, we determined whether Phosphoinositide 3-kinase (PI3K) and Notch signal pathways are involved in the expression of cyclinD1, cyclinA and p27kip1 which were key molecules in controlling cell cycling from CD4(+) T lymphocyte in animal model of asthma.. Ovalbumin (OVA) sensitized murine model of asthma was used to investigate the expression of cyclin D1, cyclin A, and p27kip1 by splenic CD4(+) T lymphocytes. We further observed the effect of specific inhibitor of PI3K(LY294002) and specific inhibitor of Notch(DAPT) on the proliferation of such CD4(+) T lymphocytes.. We found that the expression of cyclinD1 and cyclinA was upregulated at both protein and mRNA levels in asthma group while p27kip1 was down-regulated. Both LY294002 and DAPT inhibit the proliferation of CD4(+) T lymphocytes in a time- and dose-dependent manner. Furthermore, LY294002 and DAPT have additive effect in down-regulation of cyclinD1 and upregulation of p27kip1. An upregulation of cyclinA, although not statistically significant, was also observed.. These data suggested that PI3K signal pathway and Notch signal pathway may coordinately regulate the cell proliferation and differentiation processes through up-regulating cyclinD1 and down-regulating p27kip1 of CD4(+) T lymphocytes.

Topics: Animals; Asthma; CD4-Positive T-Lymphocytes; Cell Proliferation; Chromones; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Male; Mice; Mice, Inbred BALB C; Morpholines; Ovalbumin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptors, Notch; RNA, Messenger; Signal Transduction; Spleen; Time Factors; Up-Regulation

Evidence is accumulating that hematologic malignancies develop following acquisition of multiple genetic changes. Despite providing many insights into the way by which given genetic changes contribute to the development of disease, the generation of animal models is often laborious. We show a simplified method that allows the retroviral transduction of genes of interest into mouse B or T cells, thus leading to the rapid generation of models of lymphoid neoplasm in mice. Specifically, germinal center B cells induced in vitro from naive mouse B cells and infected with retroviruses for Myc and Bcl2 rapidly developed a neoplasm of immunoglobulin-expressing mature B cells in transplanted mice. Likewise, T cells induced in vitro from immature hematopoietic cells and infected with retroviruses for Myc, Bcl2, and Ccnd1 rapidly developed CD4(+)CD8(-) and CD4(+)CD8(+) T cell neoplasm in transplanted mice. These findings support the use of our simplified method as a versatile tool for lymphoma research.

Topics: Animals; B-Lymphocytes; Cyclin D1; Disease Models, Animal; Genes, bcl-2; Genes, myc; Genetic Vectors; In Vitro Techniques; Lymphoma; Mice; Retroviridae; T-Lymphocytes; Transduction, Genetic

Signal transducer and activator of transcription 3 (STAT3) is persistently activated in cancer cells and contributes to malignant progression in various types of cancer. The Janus-activated kinase (JAK) family phosphorylates STAT3 in response to stimulation by cytokines or growth factors. The JAK1-STAT3 signaling pathway plays an important role in cell proliferation and apoptosis. Nitidine chloride (NC) is a benzophenanthridine alkaloid that has been reported as an antitumor agent due to its its inhibitory effects on topoisomerase I. Using a mouse xenograft model of hepatocellular carcinoma (HCC), this study aimed to evaluate the effects of NC on tumor growth in vivo and to elucidate the underlying mechanisms. The analysis of the effects of NC on apoptosis in HCC tumor xenografts in mice was carried out by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expression of Bcl-2, Bax, cyclin-dependent kinase (CDK)4, cyclin D1, p21 and proliferating cell nuclear antigen (PCNA) was analyzed by immunohistochemistry; and the protein expression of JAK1 and STAT3 was examined by western blot analysis. Our results revealed that treatment with NC decreased the tumor volume and tumor weight, suggesting that NC inhibits HCC cell growth in vivo. In addition, NC blocked the activation of JAK1-STAT3 in the tumor tissues, which in turn resulted in the induction of cancer cell apoptosis and the inhibition of proliferation. Consequently, treatment with NC downregulated the expression of cyclin D1, CDK4 and Bcl-2 and increased the level of p21 and Bax. Our data provide a molecular basis for the antitumor activity of NC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Benzophenanthridines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 1; Liver Neoplasms; Mice; p21-Activated Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Tumor Burden; Xenograft Model Antitumor Assays

Histone deacetylase inhibitor (HDACi; vorinostat) responses were studied in murine and human lung cancer cell lines and genetically engineered mouse lung cancer models. Findings were compared with a window of opportunity trial in aerodigestive tract cancers. In human (HOP62, H522, and H23) and murine transgenic (ED-1, ED-2, LKR-13, and 393P, driven, respectively, by cyclin E, degradation-resistant cyclin E, KRAS, or KRAS/p53) lung cancer cell lines, vorinostat reduced growth, cyclin D1, and cyclin E levels, but induced p27, histone acetylation, and apoptosis. Other biomarkers also changed. Findings from transgenic murine lung cancer models were integrated with those from a window of opportunity trial that measured vorinostat pharmacodynamic responses in pre- versus posttreatment tumor biopsies. Vorinostat repressed cyclin D1 and cyclin E expression in murine transgenic lung cancers and significantly reduced lung cancers in syngeneic mice. Vorinostat also reduced cyclin D1 and cyclin E expression, but increased p27 levels in post- versus pretreatment human lung cancer biopsies. Notably, necrotic and inflammatory responses appeared in posttreatment biopsies. These depended on intratumoral HDACi levels. Therefore, HDACi treatments of murine genetically engineered lung cancer models exert similar responses (growth inhibition and changes in gene expression) as observed in lung cancer cell lines. Moreover, enhanced pharmacodynamic responses occurred in the window of opportunity trial, providing additional markers of response that can be evaluated in subsequent HDACi trials. Thus, combining murine and human HDACi trials is a strategy to translate preclinical HDACi treatment outcomes into the clinic. This study uncovered clinically tractable mechanisms to engage in future HDACi trials.

Topics: Aged; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Neoplasm Staging; Oncogene Proteins; Vorinostat

Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying isorhapontigenin anticancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that isorhapontigenin showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G(0)-G(1) arrest as well as downregulation of cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that isorhapontigenin downregulated cyclin D1 gene transcription via inhibition of specific protein 1 (SP1) transactivation. Moreover, ectopic expression of GFP-cyclin D1 rendered UMUC3 cells resistant to induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth by isorhapontigenin treatment. Together, our studies show that isorhapontigenin is an active compound that mediates Gnetum Cleistostachyum's induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth through downregulating SP1/cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anticancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate isorhapontigenin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Promoter Regions, Genetic; Sp1 Transcription Factor; Stilbenes; Transcription, Genetic; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Recent epidemiological studies provide strong evidence suggesting obesity is a risk factor in several cancers, including thyroid cancer. However, the molecular mechanisms by which obesity increases the risk of thyroid cancer are poorly understood. In this study, we evaluated the effect of diet-induced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer (Thrb(PV/PV)Pten(+/-) mice). These mice harbor a mutated thyroid hormone receptor-β (denoted as PV) and haplodeficiency of the Pten gene. A high-fat diet (HFD) efficiently induced the obese phenotype in Thrb(PV/PV)Pten(+/-) mice after 15 weeks. Thyroid tumor growth was markedly greater and survival was significantly lower in Thrb(PV/PV)Pten(+/-) mice fed an HFD than in controls fed a low-fat diet (LFD). The HFD increased thyroid tumor cell proliferation by increasing the protein levels of cyclin D1 and phosphorylated retinoblastoma protein to propel cell cycle progression. Histopathological analysis showed that the frequency of anaplasia of thyroid cancer was significantly greater (2.6-fold) in the HFD group than the LFD group. The HFD treatment led to an increase in parametrial/epididymal fat pad and elevated serum leptin levels in Thrb(PV/PV)Pten(+/-) mice. Further molecular analyses indicated that the HFD induced more aggressive pathological changes that were mediated by increased activation of the Janus kinase 2-signaling transducer and activator of transcription 3 (STAT3) signaling pathway and induction of STAT3 target gene expression. Our findings demonstrate that diet-induced obesity exacerbates thyroid cancer progression in Thrb(PV/PV)Pten(+/-) mice and suggest that the STAT3 signaling pathway could be tested as a potential target for the treatment of thyroid cancer.

Topics: Animals; bcl-X Protein; Cell Cycle; Cell Proliferation; Cyclin D1; Diet, High-Fat; Disease Models, Animal; Female; Heterozygote; Janus Kinase 2; Leptin; Male; Mice; Mutation; Obesity; Phosphorylation; Proto-Oncogene Proteins c-myc; PTEN Phosphohydrolase; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Thyroid Gland; Thyroid Hormone Receptors beta; Thyroid Neoplasms

Little is known about the molecular mechanisms driving proliferation of glial cells after an insult to the central nervous system (CNS). To test the hypothesis that the G1 regulator cyclin D1 is critical for injury-induced cell division of glial cells, we applied an injury model that causes brain damage within a well-defined region. For this, we injected the neurotoxin ibotenic acid into the prefrontal cortex of adult mice, which leads to a local nerve cell loss but does not affect the survival of glial cells. Here, we show that cyclin D1 immunoreativity increases drastically after neurotoxin injection. We find that the cyclin D1-immunopositive (cyclin D1+) cell population within the lesioned area consists to a large extent of Olig2+ oligodendrocyte progenitor cells. Analysis of cyclin D1-deficient mice demonstrates that the proliferation rate of Olig2+ cells diminishes upon loss of cyclin D1. Further, we show that cyclin-dependent kinase (cdk) 4, but not cdk6 or cdk2, is essential for driving cell division of Olig2-expressing cells in our injury model. These data suggest that distinct cell cycle proteins regulate proliferation of Olig2+ progenitor cells following a CNS insult.

Topics: Adult Stem Cells; Analysis of Variance; Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Injuries; Bromodeoxyuridine; Cell Proliferation; Cerebral Cortex; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Disease Models, Animal; Gene Expression Regulation; Ibotenic Acid; In Situ Nick-End Labeling; Mice; Mice, Knockout; Nerve Tissue Proteins; Neurotoxins; Oligodendrocyte Transcription Factor 2; Time Factors

Activation of the innate and acquired immune systems plays an important role in chronic inflammatory diseases and conditions such as obesity, insulin resistance, type 2 diabetes mellitus and atherosclerosis, with additional roles in regulation of cell proliferation and survival. Here, we provide evidence that TLR3 can respond to nutrient signals and induce loss of β-cell mass through induction of G1 cycle arrest. Activation of TLR3 by polyinosinic-polycytidylic acid [poly (I:C)] was shown to trigger the decline of cyclin D1/2 protein levels in pancreatic β-cell lines, which could be reversed by the proteasome inhibitor MG132. P38 was also found to interfere with this degradation which may be associated with G1 cycle arrest. Moreover, inhibitory effects of TLR3 on β-cell growth were supported by gene silencing of TRIF, which could inhibit p38 activity in response to poly (I:C) stimuli. These results support a role for TLR3 in β-cell mass loss in metabolic surplus and raise the possibility that TRIF/p38 signaling may be involved in G1 phase cycle arrest through ubiquitin/proteasome-dependent degradation of cyclin D.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Cell Line; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin D2; Disease Models, Animal; G1 Phase Cell Cycle Checkpoints; Glucose; Insulin-Secreting Cells; Lipids; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Models, Biological; p38 Mitogen-Activated Protein Kinases; Palmitic Acid; Poly I-C; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteolysis; Signal Transduction; Toll-Like Receptor 3; Up-Regulation

To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher therapeutic efficacy of IR-overexpressing β-cells.

Topics: Animals; beta Catenin; Blood Glucose; Cell Line; Cyclin D1; Diabetes Mellitus, Experimental; Disease Models, Animal; Gene Expression; Gene Expression Regulation; Glucokinase; Hyperglycemia; Insulin; Insulin-Secreting Cells; Male; Rats; Receptor, Insulin; Transcription, Genetic; Wnt Signaling Pathway

Our previous study has demonstrated that a plasmid-based short hairpin RNA (shRNA) against cyclin D1 could attenuate the pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling in smoking rats. In this report, we examined the efficiency of this shRNA plasmid in monocrotaline-induced pulmonary vascular remodeling. A single injection of monocrotaline induced pulmonary vascular remodeling and cyclin D1 over-expression in pulmonary vascular smooth muscle. The shRNA successfully suppressed the up-regulation of cyclin D1 in pulmonary vessels of monocrotaline-treated rats. Moreover, this shRNA decreased the percentage of muscularized vessels and the wall thickness of pulmonary vessels. So, we concluded that plasmid-based shRNA against cyclin D1 ameliorated pulmonary vascular remodeling in monocrotaline-treated rats. Cyclin D1 might be a potential target for the therapy of pulmonary vascular remodeling and pulmonary hypertension.

Topics: Animals; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; Genetic Therapy; Genetic Vectors; Hypertension, Pulmonary; Male; Monocrotaline; Muscle, Smooth, Vascular; Pulmonary Artery; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Transfection

Integrin-linked kinase (ILK) is a serine-threonine kinase that transduces extracellular matrix-related cues into intracellular signals, with fundamental roles in cell motility, development and cancer. Recently ILK been shown to have an important role in bacterial epithelial cell attachment, through ILK-bacterial OspE binding. Here we report on the role of epithelial derived ILK in response to Citrobacter rodentium infection.. C. rodentium was administered to both control and intestinal epithelial cell ILK knockout mice. Histological inflammatory scores were assessed, and cytokines measured by ELISA as well as RT-PCR, in mouse colons. Bacterial colonization was determined by plating homogenates onto MacConkey agar, and immunofluorescence microscopy performed using anti-LPS and anti-Tir antibodies.. ILK-ko mice exhibited reduced weight loss at 15 days post-infection (p < 0.01) and demonstrated reduced histological inflammatory scores (p < 0.01), reduced CCL2 and pro-inflammatory cytokines. This was not due to reduced colonization, but was associated with an altered pattern of C. rodentium bacterial migration. Attenuated fibronectin expression was found in the ILK-ko mice. C. rodentium exposure was shown to increase ILK expression in cell lines, and in murine epithelium in vivo. In ILK-ko mice reduced activation of ser473Akt and reduced crypt proliferation, together with reduced cyclin D1 expression were observed.. ILK influences the host response to C. rodentium -induced infection, independently of reduced colonization in the ILK knockout mice. The reduced inflammation and dramatically attenuated hyperplastic cryptal response to infection in this group, are at least in part the result of, the reduction in CCL2 and cyclin D1 expression respectively.

Topics: Animals; Chemokine CCL2; Citrobacter rodentium; Colitis; Cyclin D1; Cytokines; Disease Models, Animal; Enterobacteriaceae Infections; Fibronectins; HCT116 Cells; Humans; Intestinal Mucosa; Mice; Mice, Knockout; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Translation initiation plays a critical role in the regulation of cell growth and tumorigenesis. We report here that inhibiting translation initiation through induction of eIF2α phosphorylation by small-molecular-weight compounds restricts the availability of the eIF2.GTP.Met-tRNAi ternary complex and abrogates the proliferation of cancer cells in vitro and tumor growth in vivo. Restricting the availability of the ternary complex preferentially down-regulates the expression of growth-promoting proteins and up-regulates the expression of ER stress response genes in cancer cells as well as in tumors excised from either animal models of human cancer or cancer patients. These findings provide the first direct evidence for translational control of gene-specific expression by small molecules in vivo and indicate that translation initiation factors are bona fide targets for development of mechanism-specific anti-cancer agents.

Topics: Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chromans; Clotrimazole; Cyclin D1; Disease Models, Animal; Eicosapentaenoic Acid; Eukaryotic Initiation Factor-2; Female; Humans; Mice; Mice, Inbred DBA; Peptide Chain Initiation, Translational; Phosphorylation; Protein Biosynthesis; Random Allocation; Thiazolidinediones; Troglitazone; Xenograft Model Antitumor Assays

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.

Topics: Animals; Anthocyanins; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Proteolysis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Somatic mutations or loss of expression of tumor suppressor VHL happen in the vast majority of clear cell Renal Cell Carcinoma, and it's causal for kidney cancer development. Without VHL, constitutively active transcription factor HIF is strongly oncogenic and is essential for tumor growth. However, the contribution of individual HIF-responsive genes to tumor growth is not well understood. In this study we examined the contribution of important HIF-responsive genes such as VEGF, CCND1, ANGPTL4, EGLN3, ENO2, GLUT1 and IGFBP3 to tumor growth in a xenograft model using immune-compromised nude mice. We found that the suppression of VEGF or CCND1 impaired tumor growth, suggesting that they are tumor-promoting genes. We further discovered that the lack of ANGPTL4, EGLN3 or ENO2 expression did not change tumor growth. Surprisingly, depletion of GLUT1 or IGFBP3 significantly increased tumor growth, suggesting that they have tumor-inhibitory functions. Depletion of IGFBP3 did not lead to obvious activation of IGFIR. Unexpectedly, the depletion of IGFIR protein led to significant increase of IGFBP3 at both the protein and mRNA levels. Concomitantly, the tumor growth was greatly impaired, suggesting that IGFBP3 might suppress tumor growth in an IGFIR-independent manner. In summary, although the overall transcriptional activity of HIF is strongly tumor-promoting, the expression of each individual HIF-responsive gene could either enhance, reduce or do nothing to the kidney cancer tumor growth.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Glucose Transporter Type 1; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor-Proline Dioxygenases; Insulin-Like Growth Factor Binding Protein 3; Mice; Phosphopyruvate Hydratase; Receptor, IGF Type 1; Transcriptional Activation; Tumor Burden; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

The effects of C-phycocyanin (C-PC) on atherosclerosis and the regulatory effects of CD59 gene on anti-atherosclerotic roles of C-PC were investigated. Apolipoprotein E knockout (ApoE(-/-)) mice were randomly divided into four groups: control group, C-PC treatment group, CD59 transfection group and C-PC+CD59 synergy group. The mice were fed with high-fat-diet and treated with drug intervention at the same time. Results showed the atherosclerotic mouse model was successfully established. CD59 was over-expressed in blood and tissue cells. Single CD59 or C-PC could reduce blood lipid levels and promote the expression of anti-apoptotic Bcl-2 but inhibit pro-apoptotic Fas proteins in endothelial cells. The expression levels of cell cycle protein D1 (Cyclin D1) and mRNA levels of cyclin dependent protein kinase 4 (CDK4) in smooth muscle cells were restrained by CD59 and C-PC. CD59 or C-PC alone could inhibit the formation of atherosclerotic plaque by suppressing MMP-2 protein expression. In addition, C-PC could promote CD59 expression. So both CD59 and C-PC could inhibit the progress of atherosclerosis, and the anti-atherosclerotic effects of C-PC might be fulfilled by promoting CD59 expression, preventing smooth muscle cell proliferation and the apoptosis of endothelial cells, reducing blood fat levels, and at last inhibiting the development of atherosclerosis.

Topics: Animals; Apolipoproteins E; Atherosclerosis; CD59 Antigens; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Dietary Fats; Disease Models, Animal; Endothelial Cells; fas Receptor; Gene Expression Regulation; Mice; Mice, Knockout; Myocytes, Smooth Muscle; Phycocyanin; Plaque, Atherosclerotic; Proto-Oncogene Proteins c-bcl-2

The present study aimed to explore the role(s) of the soya isoflavone genistein (GEN) in preventing the development of colon pre-neoplasia, using Wingless/int (WNT)/β-catenin as a molecular marker of colon abnormality. Specifically, the effects on the WNT/β-catenin signalling pathway from GEN were examined by using an azoxymethane (AOM)-induced rat colon cancer model. Male Sprague-Dawley rats were fed a control (CTL), a soya protein isolate (SPI) or a GEN diet from gestation to 13 weeks of age. The first sampling was conducted at 7 weeks of age for pre-AOM analysis. The remaining rats were injected with AOM at 7 weeks of age. The descending colon was collected 6 weeks later for the evaluation of aberrant crypt foci (ACF), gene expression and nuclear protein accumulation. AOM injection induced aberrant nuclear accumulation of β-catenin in the CTL group but not in the SPI or GEN group. Moreover, the WNT target genes Cyclin D1 and c-Myc were repressed by SPI and GEN. Meanwhile, SPI and GEN suppressed the expression of WNT signalling genes including Wnt5a, Sfrp1, Sfrp2 and Sfrp5 to the similar level to that of the pre-AOM period. Rats fed SPI and GEN had a decreased number of total aberrant crypts. GEN feeding also resulted in a reduced number of ACF with N = 3 per foci. The reduction of WNT/β-catenin signalling was correlated with the decrease in total aberrant crypts. By testing WNT/β-catenin signalling as a biomarker of colon carcinogenic potential, we showed the novel role of GEN as a suppressor of carcinogen-induced WNT/β-catenin signalling in preventing the development of early colon neoplasia.

Topics: Aberrant Crypt Foci; Animals; Azoxymethane; Biomarkers; Carcinogens; Colon, Descending; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Down-Regulation; Female; Genistein; Lactation; Male; Maternal Nutritional Physiological Phenomena; Precancerous Conditions; Pregnancy; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Soybean Proteins; Wnt Signaling Pathway

Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma, and new therapeutic strategies are urgently needed.. The effects of ON 013105, a novel benzylstyryl sulfone kinase inhibitor, alone or with doxorubicin or rituximab, were examined in Granta 519 and Z138C cells. For in vivo studies, CB17/SCID mice were implanted subcutaneously with Z138C cells and treated with various combinations of ON 013105, doxorubicin, and rituximab. Tumor burden and body weight were monitored for 28 days.. ON 013105 induced mitochondria-mediated apoptosis in MCL cells. Death was preceded by translocation of tBid to the mitochondria and cytochrome c release. In addition, ON 013105-treated cells exhibited reduced levels of cyclin D1, c-Myc, Mcl-1, and Bcl-xL. Using nuclear magnetic resonance (NMR) spectroscopy, we showed specific binding of ON 013105 to eIF4E, a critical factor for the initiation of protein translation. We proffer that this drug-protein interaction preferentially prevents the translation of the aforementioned proteins and may be the mechanism by which ON 013105 induces apoptosis in MCL cells. Efficacy studies in a mouse xenograft model showed that ON 013105 inhibited MCL tumor growth and that combining ON 013105 with rituximab reduced tumor burden further with negligible unwanted effects.. Our findings suggest that ON 013105, alone or in combination with rituximab, may be a potent therapeutic agent to treat MCLs.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Doxorubicin; Eukaryotic Initiation Factor-4E; Female; Gene Expression; Lymphoma, Mantle-Cell; Mice; Mitochondria; Protein Binding; Proto-Oncogene Proteins c-myc; Rituximab; Styrenes; Sulfones; Tumor Burden

DJ-1/park7 is a ubiquitously expressed multifunctional protein that plays essential roles in a variety of cells. However, its function in the vascular system has not been determined. We investigated the protective roles of DJ-1/park7 in vascular disorders, especially in neointimal hyperplasia.. DJ-1/park7 was strongly expressed in the neointimal layer, in which its oxidized form was predominant. Treatment of vascular smooth muscle cells (VSMCs) from the mouse aorta with H(2)O(2) increased the oxidation of DJ-1/park7 visualized on two-dimensional electrophoresis gels. The growth of VSMCs in FBS-containing media and the release of H(2)O(2) were significantly increased in DJ-1/park7(-/-) knockout mice compared with DJ-1/park7(+/+) wild-type mice. The expression of cyclin D1 and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were greater in VSMCs from the DJ-1/park7(-/-) aorta than from the DJ-1/park7(+/+) aorta. Both of these measures were inhibited by treatment with an ERK1/2 inhibitor or antioxidants and in DJ-1/park7-overexpressing cells. VSMC proliferation, cyclin D1 expression, and ERK1/2 phosphorylation in response to platelet-derived growth factor-BB were upregulated in DJ-1/park7(-/-) compared with DJ-1/park7(+/+) mice. VSMCs of DJ-1/park7(-/-) mice exhibited higher levels of sprout outgrowth of aortic strips and neointimal plaque formation elicited by carotid artery ligation compared with those of DJ-1/park7(+/+) mice.. These results indicate that DJ-1/park7 is involved in the growth of VSMCs, thereby inhibiting neointimal hyperplasia, and suggest that it might play protective roles in vascular remodelling.

Topics: Animals; Aorta; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; In Vitro Techniques; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Neointima; Neovascularization, Pathologic; Oncogene Proteins; Peroxiredoxins; Phosphorylation; Protein Deglycase DJ-1; Proto-Oncogene Proteins c-sis; Reactive Oxygen Species

We evaluated the antitumoral properties of the multikinase inhibitor sorafenib in mantle cell lymphoma (MCL), an aggressive B lymphoma for which current therapies have shown limited efficacy.. Sensitivity to sorafenib was analyzed in MCL cell lines and primary samples in the context of BCR and microenvironment simulation. Sorafenib signaling was characterized by quantitative PCR, Western blotting, immunofluorescence, and protein immunoprecipitation. Migration analysis included flow cytometric counting, actin polymerization assays, and siRNA-mediated knockdown of focal adhesion kinase (FAK). In vivo antitumor effect of sorafenib and bortezomib was analyzed in an MCL xenograft mouse model.. Sorafenib rapidly dephosphorylates the BCR-associated kinases, Syk and Lyn, as well as FAK, an Src target involved in focal adhesion. In this line, sorafenib displays strong synergy with the Syk inhibitor, R406. Sorafenib also blocks Mcl-1 and cyclin D1 translation, which promotes an imbalance between pro- and antiapoptotic proteins and facilitates Bax release from cyclin D1, leading to the induction of mitochondrial apoptosis and caspase-dependent and -independent mechanisms. Moreover, sorafenib inhibits MCL cell migration and CXCL12-induced actin polymerization. FAK knockdown partially prevents this inhibitory effect, indicating that FAK is a relevant target of sorafenib. Furthermore, sorafenib enhances the antitumoral activity of bortezomib in an MCL xenograft mouse model as well as overcomes stroma-mediated bortezomib resistance in MCL cells.. We show for the first time that sorafenib interferes with BCR signaling, protein translation and modulates the microenvironment prosurvival signals in MCL, suggesting that sorafenib, alone or in combination with bortezomib, may represent a promising approach to treat patients with MCL.

Topics: Actins; Animals; Antineoplastic Agents; Apoptosis; Caspases; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Cyclin D1; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Humans; Lymphoma, Mantle-Cell; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Multimerization; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell; Signal Transduction; Sorafenib; Stromal Cells; Transplantation, Heterologous

Short-term β-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term β-adrenergic drive on CPC survival and proliferation are unknown.. We sought to determine the relationship between β-adrenergic activity and regulation of CPC function.. Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein-coupled receptor kinase 2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium.. β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors.

Topics: Adrenergic beta-2 Receptor Agonists; Adrenergic beta-2 Receptor Antagonists; Animals; Cell Death; Cell Proliferation; Cell Survival; Cells, Cultured; Coculture Techniques; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; G-Protein-Coupled Receptor Kinase 2; Humans; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Myocytes, Cardiac; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-kit; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; RNA Interference; Signal Transduction; Stem Cell Transplantation; Stem Cells; Time Factors; Transfection

To determine if the NADPH oxidase isoform Nox4 contributes to increased H(2)O(2) generation in persistent pulmonary hypertension of the newborn (PPHN) pulmonary arteries (PA), and to identify downstream signaling targets of Nox4 that contribute to vascular remodeling and vasoconstriction.. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs, PA, and PA smooth muscle cells (PASMC) were isolated from control and PPHN lambs. Increased expression of p22(phox) and Nox4 in PPHN lungs, PA, and PASMC was associated with increased reactive oxygen species in PPHN PA, increased protein thiol oxidation in PPHN PASMC, and a decreased activity of extracellular superoxide dismutase (ecSOD) in the lungs and PASMC. Nox4 small interfering RNA (siRNA) decreased Nox4 expression and thiol oxidation and increased the ecSOD activity in PPHN PASMC. An increased activity of nuclear factor-kappa B (NFκB) and expression of its target gene cyclin D1 were detected in PPHN lungs, PA, and PASMC. Nox4 siRNA and catalase attenuated these increases in PASMC, and catalase decreased cyclin D1 expression in PPHN lungs.. This study demonstrates for the first time that Nox4 expression is elevated in a lamb model of neonatal pulmonary hypertension. It identifies increased NFκB and cyclin D1 expression and a decreased ecSOD activity as targets of increased Nox4 signaling.. PPHN increases p22(phox) and Nox4 expression and activity resulting in elevated H(2)O(2) levels in PPHN PA. Increased H(2)O(2) induces vasoconstriction via mechanisms involving ecSOD inactivation, and stimulates vascular remodeling via NFκB activation and increased cyclin D1 expression. Approaches that inhibit the pulmonary arterial Nox4 activity may attenuate vasoconstriction and vascular remodeling in PPHN.

Topics: Animals; Animals, Newborn; Cyclin D1; Disease Models, Animal; Fluoresceins; Gene Expression; Hydrogen Peroxide; Hypertension, Pulmonary; NADPH Oxidases; NF-kappa B; Proliferating Cell Nuclear Antigen; Pulmonary Artery; RNA Interference; Sheep; Superoxide Dismutase

Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression.. In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed.. Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression.. In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Topics: Animals; Aorta; Apoptosis; Blotting, Western; Cell Line; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Glucose; Glucose Transporter Type 1; Hyperplasia; Hypertrophy; Immunohistochemistry; Indican; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nephrectomy; Phloretin; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Renal Insufficiency; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases; RNA, Messenger; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation; Uremia

Alzheimer's disease (AD) is characterized by accumulation and deposition of Aβ peptides in human brains. The present study aimed to determine the protective effect of HBO and EGB761 on Aβ25-35 peptides induced cognitive impairment and neuronal toxicity in rats. Characteristics of AD were induced in rats by the administration of Aβ25-35 in hippocampus. Rats were treated with HBO (2ATA 60min/day), EGB761 (20mg/kg/day), and the combination of HBO+EGB761 (20mg/kg/day+2ATA). The Morris water maze was used to detect the protective effects of HBO and EGB761 against cognitive impairment. The activities of SOD and GSH, the apoptosis-related genes and proteins and the apoptosis rate of hippocampus were detected. Compared to the model group, EGB761 and HBO treatments synergistically improved the escape latency. Furthermore, the activities of SOD and GSH in rat hippocampal tissue were found to have increased with a concomitant reduction in MDA levels, Bax expression, cytochrome c release, and the activity of caspase-9/3. Accordingly, a significant reduction was observed in the apoptosis rate following the treatment with EGB761 and HBO in this model of AD. Our findings suggest that HBO and EGB761 reduce cell toxicity and oxidative stress by blocking mitochondria-mediated apoptosis signaling in AD, and the combined treatment of HBO and Ginkgo further enhances these effects.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Caspases; Cyclin D1; Disease Models, Animal; Gene Expression Regulation; Ginkgo biloba; Hippocampus; Hyperbaric Oxygenation; In Situ Nick-End Labeling; Male; Maze Learning; Motor Activity; Neurons; Neuroprotective Agents; Oxidative Stress; Peptide Fragments; Plant Extracts; Rats; Rats, Sprague-Dawley; Reaction Time; Reactive Oxygen Species; RNA, Messenger; Time Factors

There is an association between chronic inflammation and cancer, including colon cancer. Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelial cells causing several parasitological and pathological changes. It is incriminated in the development of colorectal cancer in immunosuppressed individuals. Cyclin D1 expression is essential for cell cycle progression and its overexpression has been reported in colorectal cancer. This work aimed to study the gastrointestinal changes, including parasitological and pathological changes, induced by C. parvum infection in both immunocompetent and in chemically immunosuppressed mice, together with immunohistochemical assessment of cyclin D1 expression in infected tissues. In addition, the effectiveness of nitazoxanide (NTZ) in the treatment of cryptosporidiosis was evaluated.. This study included six groups of mice: group I, infected; group II, infected and immunosuppressed; group III, infected and treated with NTZ; group IV, infected, immunosuppressed, and treated with NTZ; and groups V and VI representing non-infected controls. Mice were subjected to stool examination for oocyst counts and were later sacrificed for intestinal dissection and routine histopathological examination of pathological changes; the endogenous developmental stages of the parasite were counted and immunohistochemical staining was carried out for the determination of cyclin D1.. Group II showed the highest numbers of oocysts shed and endogenous developmental stages compared to the other groups. Intestinal dysplastic changes were seen only in groups I and II, where these changes were in favor of group II compared to group I. High-grade dysplasia was seen in four out of 20 mice in group II and was significantly associated with the number of endogenous developmental stages of C. parvum. NTZ was effective in the treatment of Cryptosporidium infection, with a greater effect in group III than in group IV.. C. parvum is one of the infectious agents that may induce intestinal dysplasia, including the high-grade category, which occurs particularly in the presence of immune suppression states and elevated endogenous parasite loads. Cyclin D1 is a good and useful marker for the detection of intestinal dysplasia. The effectiveness of NTZ is dependent on the immune status of the infected host.

Topics: Animals; Antiparasitic Agents; Cryptosporidiosis; Cryptosporidium parvum; Cyclin D1; Disease Models, Animal; Female; Immunocompromised Host; Intestinal Mucosa; Intestines; Liver; Mice; Nitro Compounds; Oocysts; Thiazoles

The acute inhibitory effects of parathyroid hormone (PTH) on proximal tubule Na(+)-K(+)-ATPase (Na-K) and sodium-dependent phosphate (NaPi) transport have been extensively studied, while little is known about the chronic effects of PTH. Patients with primary hyperparathyroidism, a condition characterized by chronic elevations in PTH, exhibit persistent hypophosphatemia but not significant evidence of salt wasting. We postulate that chronic PTH stimulation results in differential desensitization of PTH responses. To address this hypothesis, we compared the effects of chronic PTH stimulation on Na-P(i) cotransporter (Npt2a) expression and Na-K activity and expression in Sprague Dawley rats, transgenic mice featuring parathyroid-specific cyclin D1 overexpression (PTH-D1), and proximal tubule cell culture models. We demonstrated a progressive decrease in brush-border membrane (BBM) expression of Npt2a from rats treated with PTH for 6 h or 4 days, while Na-K expression and activity in the basolateral membranes (BLM) exhibited an initial decrease followed by recovery to control levels by 4 days. Npt2a protein expression in PTH-D1 mice was decreased relative to control animals, whereas levels of Na-K, NHERF-1, and PTH receptor remained unchanged. In PTH-D1 mice, NpT2a mRNA expression was reduced by 50% relative to control mice. In opossum kidney proximal tubule cells, PTH decreased Npt2a mRNA levels. Both actinomycin D and cycloheximide treatment prevented the PTH-mediated decrease in Npt2a mRNA, suggesting that the PTH response requires transcription and translation. These findings suggest that responses to chronic PTH exposure are selectively regulated at a posttranscriptional level. The persistence of the phosphaturic response to PTH occurs through posttranscriptional mechanisms.

Topics: Animals; Cells, Cultured; Cyclin D1; Disease Models, Animal; Hypophosphatemia; Kidney Cortex; Kidney Tubules, Proximal; Mice; Mice, Transgenic; Opossums; Parathyroid Hormone; Phosphoproteins; Rats; Rats, Sprague-Dawley; Receptor, Parathyroid Hormone, Type 1; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger; Sodium-Hydrogen Exchangers; Sodium-Phosphate Cotransporter Proteins, Type IIa

p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor β, tumor necrosis factor-α, interleukin-1β, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice.. Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis.

Topics: Animals; Apoptosis; Cell Proliferation; Chronic Disease; Cyclin B1; Cyclin D1; Cytokinesis; Disease Models, Animal; Disease Progression; Hepatocytes; Liver; Liver Cirrhosis, Biliary; Male; MAP Kinase Kinase 2; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase 14; Oxidative Stress; Signal Transduction; Survival Rate

βKlotho is a regulator in multiple metabolic processes, while its role in cancer remains unclear. We found the expression of βKlotho was down-regulated in human hepatocellular carcinoma tissues compared with that in paired adjacent non-tumourous liver tissues. Hepatoma cells also showed decreased expression of βKlotho compared with normal hepatocyte cells. Reintroduction of βKlotho into hepatoma cells inhibited their proliferation. The anti-proliferative effect of βKlotho might be linked with G1 to S phase arrest, which was mediated by Akt/GSK-3β/cyclin D1 signaling, since forced expression βKlotho reduced the phosphorylation level of Akt and GSK-3β and induced down-regulation of cyclin D1. Furthermore, βKlotho overexpression could inhibit tumorgenesis, while constitutively activated Akt could override the suppressive effects of βKlotho in vivo. These data suggest βKlotho suppresses tumor growth in hepatocellular carcinoma.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Glucuronidase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Klotho Proteins; Liver Neoplasms; Male; Mice; Middle Aged; Proto-Oncogene Proteins c-akt; Signal Transduction; Transplantation, Heterologous

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell-cycle activation (CCA), which lead to neuronal cell death, microglial activation, and neurologic dysfunction. Here, we show progressive neurodegeneration associated with microglial activation after TBI induced by controlled cortical impact (CCI), and also show that delayed treatment with the selective cyclin-dependent kinase inhibitor roscovitine attenuates posttraumatic neurodegeneration and neuroinflammation. CCI resulted in increased cyclin A and D1 expressions and fodrin cleavage in the injured cortex at 6 hours after injury and significant neurodegeneration by 24 hours after injury. Progressive neuronal loss occurred in the injured hippocampus through 21 days after injury and correlated with a decline in cognitive function. Microglial activation associated with a reactive microglial phenotype peaked at 7 days after injury with sustained increases at 21 days. Central administration of roscovitine at 3 hours after CCI reduced subsequent cyclin A and D1 expressions and fodrin cleavage, improved functional recovery, decreased lesion volume, and attenuated hippocampal and cortical neuronal cell loss and cortical microglial activation. Furthermore, delayed systemic administration of roscovitine improved motor recovery and attenuated microglial activation after CCI. These findings suggest that CCA contributes to progressive neurodegeneration and related neurologic dysfunction after TBI, likely in part related to its induction of microglial activation.

Topics: Animals; Apoptosis; Behavior, Animal; Blotting, Western; Brain Injuries; Carrier Proteins; Cell Cycle; Cyclin A; Cyclin D1; Cyclin-Dependent Kinases; Disease Models, Animal; Immunohistochemistry; Maze Learning; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Motor Activity; Neurons; Neuroprotective Agents; Purines; Roscovitine; Time Factors

Spinal cord injury (SCI) is a traumatic event that causes a secondary and extended inflammation characterized by infiltration of immune cells, including T lymphocytes, release of pro-inflammatory mediators in the lesion site, and tissue degeneration. Current therapeutic approaches for SCI are limited to glucocorticoids (GC) due to their potent anti-inflammatory activity. GC efficacy resides, in part, in the capability to inhibit NF-κB, T lymphocyte activation, and the consequent cytokine production. In this study, we performed experiments aimed to test the susceptibility of glucocorticoid-induced leucine zipper (GILZ) transgenic (GILZ(TG)) mice, in which GILZ is selectively over-expressed in T lymphocytes, to SCI induction. Consistent with a decreased inflammatory response, GILZ(TG) were less susceptible to SCI as compared to wild-type littermates. Notably, inhibition of NF-κB activation and nuclear translocation, diminished T lymphocytes activation and tissue infiltration, as well as decreased release of cytokines were evident in GILZ(TG) as compared to wild-type mice. Moreover, GILZ(TG) showed a reduced tumor necrosis factor-α, IL-1β, Inductible nitric oxide synthase (iNOS) and nytrotyrosine production, apoptosis, and neuronal tissue damage. Together these results indicate that GILZ mimics the anti-inflammatory effect of GC and represents a potential pharmacological target for modulation of T lymphocyte-mediated immune response in inflammatory disorders, such as SCI.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cyclin D1; Cytokines; Dinoprostone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Inflammation; Mice; Mice, Transgenic; Nitric Oxide Synthase Type II; Peroxidase; Signal Transduction; Spinal Cord Injuries; T-Lymphocytes; Transcription Factors

Multiple genetic mutations with subsequent molecular events are required for progression of normal epithelial cells to cancer, with p53 mutations being a very common event in squamous carcinogenesis. Upregulation of nuclear factor kappa B (NF-κB) is an associated feature of malignancy, however studies have not examined purposeful overexpression of the NF-κB p65 subunit in in vitro models of oral carcinogenesis. Our objective is to demonstrate that NF-κB p65 transfection into p53 deficient Rhek keratinocytes produces carcinogenic progression. We constitutively over-expressed NF-κB p65 in Rhek keratinocytes, previously immortalized by SV 40 thus inactivating p53, and studied NF-κB dependent events. NF-κB p65 overexpression provided functional upregulation of NF-κB and produced cyclin D1-mediated proliferation and interleukin 8 transcription and secretion. Consequently, we demonstrated tumorigenesis in athymic mice with NF-κB p65 overexpressing cells. We conclude NF-κB p65 overexpression in p53 inactivated immortalized keratinocytes produces tumorigenesis, and that this single alteration in NF-κB expression on a p53 inactivated background is sufficient for squamous carcinogenesis features, thus providing evidence that p65 may act as a gain of function oncogene in this setting.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Female; Humans; Interleukin-8; Keratinocytes; Mice; Mice, Nude; NF-kappa B; Vascular Endothelial Growth Factor A

Epidemiologic studies suggest that intake of high-fat diet (HFD) promotes colon carcinogenesis. Epithelial-mesenchymal transition (EMT) and inflammation play important roles during tumor progression of colorectal cancer (CRC). Oncogenic pathways such as phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK)/ERK signaling cascades induce EMT and inflammation in cancer. No experimental evidence has been demonstrated regarding HFD-mediated tumor progression including EMT in CRC so far. Our results demonstrated that HFD consumption could induce tumor growth and progression, including EMT and inflammation, in a mouse xenograft tumor model. The molecular mechanisms were through activation of MAPK/ERK and PI3K/Akt/mTOR signaling pathways. HFD induced up-regulation of cyclooxygenase-2, cyclin D1 and proliferating cell nuclear antigen proteins concomitant with increases in expression of nuclear factor-κB p65 (RelA) and β-catenin proteins. Surprisingly, HFD consumption could suppress p21(CIP1/WAF1) expression through increases in nuclear histone deacetylase complex (HDAC). Moreover, HFD could mediate the disassembly of E-cadherin adherent complex and the up-regulation of Vimentin and N-cadherin proteins in tumor tissues. Taken together, our novel findings support evidence for HFD-mediated modulation of HDAC activity and activation of oncogenic cascades, which involve EMT and inflammation in CRC, playing important roles in tumor growth and progression in a mouse xenograft model.

Topics: Animals; beta Catenin; Cadherins; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Diet, High-Fat; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation; Vimentin

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a monoclonal proliferation of lymphocytes with the co-expression of CD5 and CD43, but not of CD23. Typical MCL is associated with overexpression of cyclin D1, and blastoid MCL variants are associated with Myc (alias c-myc) translocations. In this study, we developed a murine model of MCL-like lymphoma by crossing Cdk4(R24C) mice with Myc-3'RR transgenic mice. The Cdk4(R24C) mouse is a knockin strain that expresses a Cdk4 protein that is resistant to inhibition by p16(INK4a) as well as other INK4 family members. Ablation of INK4 control on Cdk4 does not affect lymphomagenesis, B-cell maturation, and functions in Cdk4(R24C) mice. Additionally, B cells were normal in numbers, cell cycle activity, mitogen responsiveness, and Ig synthesis in response to activation. By contrast, breeding Cdk4(R24C) mice with Myc-3'RR transgenic mice prone to develop aggressive Burkitt lymphoma-like lymphoma (CD19(+)IgM(+)IgD(+) cells) leads to the development of clonal blastoid MCL-like lymphoma (CD19(+)IgM(+)CD5(+)CD43(+)CD23(-) cells) in Myc/Cdk4(R24C) mice. Western blot analysis revealed high amounts of Cdk4/cyclin D1 complexes as the main hallmark of these lymphomas. These results indicate that although silent in nonmalignant B cells, a defect in the INK4-Cdk4 checkpoint can participate in lymphomagenesis in conjunction with additional alterations of cell cycle control, a situation that might be reminiscent of the development of human blastoid MCL.

Topics: Animals; B-Lymphocytes; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Gene Expression Profiling; Genes, myc; Immunoglobulins; Immunophenotyping; Lymphocyte Activation; Lymphoma, Mantle-Cell; Lymphopoiesis; Mice; Mice, Transgenic; Neoplasm Proteins; Protein Isoforms; Somatic Hypermutation, Immunoglobulin

Because of poor prognosis and development of resistance against chemotherapeutic drugs, the existing treatment modalities for gastric cancer are ineffective. Hence, novel agents that are safe and effective are urgently needed. Whether γ-tocotrienol can sensitize gastric cancer to capecitabine in vitro and in a xenograft mouse model was investigated.. The effect of γ-tocotrienol on proliferation of gastric cancer cell lines was examined by mitochondrial dye uptake assay, apoptosis by esterase staining, NF-κB activation by DNA-binding assay, and gene expression by Western blotting. The effect of γ-tocotrienol on the growth and chemosensitization was also examined in subcutaneously implanted tumors in nude mice.. γ-Tocotrienol inhibited the proliferation of various gastric cancer cell lines, potentiated the apoptotic effects of capecitabine, inhibited the constitutive activation of NF-κB, and suppressed the NF-κB-regulated expression of COX-2, cyclin D1, Bcl-2, CXCR4, VEGF, and matrix metalloproteinase-9 (MMP-9). In a xenograft model of human gastric cancer in nude mice, we found that administration of γ-tocotrienol alone (1 mg/kg body weight, intraperitoneally 3 times/wk) significantly suppressed the growth of the tumor and this effect was further enhanced by capecitabine. Both the markers of proliferation index Ki-67 and for microvessel density CD31 were downregulated in tumor tissue by the combination of capecitabine and γ-tocotrienol. As compared with vehicle control, γ-tocotrienol also suppressed the NF-κB activation and the expression of cyclin D1, COX-2, intercellular adhesion molecule-1 (ICAM-1), MMP-9, survivin, Bcl-xL, and XIAP.. Overall our results show that γ-tocotrienol can potentiate the effects of capecitabine through suppression of NF-κB-regulated markers of proliferation, invasion, angiogenesis, and metastasis.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Capecitabine; Cell Line, Tumor; Cell Proliferation; Chromans; Cyclin D1; Cyclooxygenase 2; Deoxycytidine; Disease Models, Animal; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Intercellular Adhesion Molecule-1; Ki-67 Antigen; Matrix Metalloproteinase 9; Mice; Mice, Nude; Mitochondria; Neovascularization, Pathologic; NF-kappa B; Platelet Endothelial Cell Adhesion Molecule-1; Proto-Oncogene Proteins c-bcl-2; Receptors, CXCR4; Repressor Proteins; Stomach Neoplasms; Survivin; Vascular Endothelial Growth Factor A; Vitamin E; Xenograft Model Antitumor Assays

Dedifferentiation, migration, and proliferation of resident vascular smooth muscle cells (SMCs) are key components of neointima formation after vascular injury. Activation of signal transducer and activator of transcription-3 (STAT3) is suggested to be critically involved in this process, but the complex regulation of STAT3-dependent genes and the functional significance of inhibiting this pathway during the development of vascular proliferative diseases remain elusive. In this study, we demonstrate that STAT3 was activated in neointimal lesions following wire-induced injury in mice. Phosphorylation of STAT3 induced trans-activation of cyclin D1 and survivin in SMCs in vitro and in neointimal cells in vivo, thus promoting proliferation and migration of SMCs as well as reducing apoptotic cell death. WP1066, a highly potent inhibitor of STAT3 signaling, abrogated phosphorylation of STAT3 and dose-dependently inhibited the functional effects of activated STAT3 in stimulated SMCs. The local application of WP1066 via a thermosensitive pluronic F-127 gel around the dilated arteries significantly inhibited proliferation of neointimal cells and decreased the neointimal lesion size at 3 weeks after injury. Even though WP1066 application attenuated the injury-induced up-regulation of the chemokine RANTES at 6 h after injury, there was no significant effect on the accumulation of circulating cells at 1 week after injury. In conclusion, these data identify STAT3 as a key molecule for the proliferative response of SMC and neointima formation. Moreover, inhibition of STAT3 by the potent and specific compound WP1066 might represent a novel and attractive approach for the local treatment of vascular proliferative diseases.

Topics: Animals; Apoptosis; Binding Sites; Cardiovascular Agents; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokine CCL5; Cyclin D1; Disease Models, Animal; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Phosphorylation; Promoter Regions, Genetic; Pyridines; Repressor Proteins; STAT3 Transcription Factor; Survivin; Time Factors; Tyrphostins

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however, the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, β-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/β-catenin signaling pathway. We determined the expression of Wnt3a, β-catenin, and Cyclin D1 in the adult spinal cord of SOD1(G93A) ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, β-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, β-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, β-catenin or Cyclin D1 in mature GFAP(+) astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that neurodegeneration activates the Wnt/β-catenin signaling pathway, which is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. This mechanism may be significant in clinical gene therapy.

Topics: Active Transport, Cell Nucleus; Amyotrophic Lateral Sclerosis; Animals; beta Catenin; Cell Membrane; Cell Nucleus; Cell Proliferation; Cyclin D1; Disease Models, Animal; Humans; Mice; Mice, Transgenic; Neuroglia; Spinal Cord; Superoxide Dismutase; Up-Regulation; Wnt Signaling Pathway; Wnt3A Protein

Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury.. Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used.. SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05).. Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.

Topics: Alanine Transaminase; Animals; Apoptosis; Axin Protein; beta Catenin; Blotting, Western; Cell Proliferation; Cyclin D1; Disease Models, Animal; Gene Expression Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunohistochemistry; Indoles; Inhibitor of Apoptosis Proteins; Liver; Liver Regeneration; Male; Maleimides; Mice; Mice, Inbred C57BL; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Repressor Proteins; Signal Transduction; Survivin; Time Factors

The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP-induced ICC and included phosphorylation of p38 mitogen-activated proteinbase (MAPK) and p44/42 mitogen-activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF-β1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF-β1 by in vivo morpholinos injections.. These results reveal that TGF-β1 plays an important role in HBx- and HCP-induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection.

Topics: Animals; Animals, Genetically Modified; Anti-Bacterial Agents; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Connective Tissue Growth Factor; Cyclin D1; Disease Models, Animal; Doxycycline; Gene Expression; Hepacivirus; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Smad2 Protein; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta1; Up-Regulation; Vascular Endothelial Growth Factor A; Viral Core Proteins; Viral Regulatory and Accessory Proteins; Zebrafish; Zebrafish Proteins

Interleukin (IL)-33 is a recently identified member of the IL-1 family that binds to the receptor, ST2L. In the current study, we sought to determine whether IL-33 is an important regulator in the hepatic response to ischemia/reperfusion (I/R). Male C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia, followed by up to 8 hours of reperfusion. Some mice received recombinant IL-33 (IL-33) intraperitoneally (IP) before surgery or anti-ST2 antibody IP at the time of reperfusion. Primary hepatocytes and Kupffer cells were isolated and treated with IL-33 to assess the effects of IL-33 on inflammatory cytokine production. Primary hepatocytes were treated with IL-33 to assess the effects of IL-33 on mediators of cell survival in hepatocytes. IL-33 protein expression increased within 4 hours after reperfusion and remained elevated for up to 8 hours. ST2L protein expression was detected in healthy liver and was up-regulated within 1 hour and peaked at 4 hours after I/R. ST2L was primarily expressed by hepatocytes, with little to no expression by Kupffer cells. IL-33 significantly reduced hepatocellular injury and liver neutrophil accumulation at 1 and 8 hours after reperfusion. In addition, IL-33 treatment increased liver activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB), p38 mitogen-activated protein kinase (MAPK), cyclin D1, and B-cell lymphoma 2 (Bcl-2), but reduced serum levels of CXC chemokines. In vitro experiments demonstrated that IL-33 significantly reduced hepatocyte cell death as a result of increased NF-κB activation and Bcl-2 expression in hepatocytes.. The data suggest that IL-33 is an important endogenous regulator of hepatic I/R injury. It appears that IL-33 has direct protective effects on hepatocytes, associated with the activation of NF-κB, p38 MAPK, cyclin D1, and Bcl-2 that limits liver injury and reduces the stimulus for inflammation.

Topics: Analysis of Variance; Animals; Blotting, Western; Cells, Cultured; Cyclin D1; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Hepatocytes; Interleukin-33; Interleukins; Kupffer Cells; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Random Allocation; Receptors, Interleukin-1; Reperfusion; Reperfusion Injury

The four and a half LIM-only protein 2 (FHL2) is upregulated in diverse pathological conditions. Here, we analyzed the effects of FHL2 overexpression in the liver of FHL2 transgenic mice (Apo-FHL2).. We first examined cell proliferation and apoptosis in Apo-FHL2 livers and performed partial hepatectomy to investigate high FHL2 expression in liver regeneration. Expression of FHL2 was then analyzed by real time PCR in human hepatocellular carcinoma and adjacent non-tumorous livers. Finally, the role of FHL2 in hepatocarcinogenesis was assessed using Apo-FHL2;Apc(lox/lox) mice.. Six-fold increase in cell proliferation in transgenic livers was associated with concomitant apoptosis, resulting in normal liver mass. In Apo-FHL2 livers, both cyclin D1 and p53 were markedly increased. Evidence supporting a p53-dependent cell death mechanism was provided by the findings that FHL2 bound to and activated the p53 promoter, and that a dominant negative p53 mutant compromised FHL2-induced apoptosis in hepatic cells. Following partial hepatectomy in Apo-FHL2 mice, hepatocytes displayed advanced G1 phase entry and DNA synthesis leading to accelerated liver weight restoration. Interestingly, FHL2 upregulation in human liver specimens showed significant association with increasing inflammation score and cirrhosis. Finally, while Apo-FHL2 mice developed no tumors, the FHL2 transgene enhanced hepatocarcinogenesis induced by liver-specific deletion of the adenomatous polyposis coli gene and aberrant Wnt/β-catenin signaling in Apc(lox/lox) animals.. Our results implicate FHL2 in the regulation of signaling pathways that couple proliferation and cell death machineries, and underscore the important role of FHL2 in liver homeostasis and carcinogenesis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Female; Hepatectomy; Homeostasis; Humans; LIM-Homeodomain Proteins; Liver; Liver Neoplasms; Liver Regeneration; Male; Mice; Mice, Transgenic; Muscle Proteins; Transcription Factors; Tumor Suppressor Protein p53

Proliferation and migration of vascular smooth muscle cells (VSMCs) can cause atherosclerosis and neointimal formation. MicroRNAs have been shown to regulate cell proliferation and phenotype transformation. We discovered abundant expression of microRNA-195 in VSMCs and conducted a series of studies to identify its function in the cardiovascular system.. MicroRNA-195 expression was initially found to be altered when VSMCs were treated with oxidized low-density lipoprotein (oxLDL) in a non-replicated microRNA array experiment. Using cellular studies, we found that microRNA-195 reduced VSMC proliferation, migration, and synthesis of IL-1β, IL-6, and IL-8. Using bioinformatics prediction and experimental studies, we showed that microRNA-195 could repress the expression of Cdc42, CCND1, and FGF1 genes. Using a rat model, we found that the microRNA-195 gene, introduced by adenovirus, substantially reduced neointimal formation in a balloon-injured carotid artery. In situ hybridization confirmed the presence of microRNA-195 in the treated arteries but not in control arteries. Immunohistochemistry experiments showed abundant Cdc42 in the neointima of treated arteries.. We showed that microRNA-195 plays a role in the cardiovascular system by inhibiting VSMC proliferation, migration, and proinflammatory biomarkers. MicroRNA-195 may have the potential to reduce neointimal formation in patients receiving stenting or angioplasty.

Topics: Adenoviridae; Animals; Carotid Arteries; Carotid Artery Injuries; cdc42 GTP-Binding Protein; Cell Movement; Cell Proliferation; Cells, Cultured; Computational Biology; Cyclin D1; Disease Models, Animal; Fibroblast Growth Factor 1; Gene Expression Profiling; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Humans; Immunohistochemistry; In Situ Hybridization; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Lipoproteins, LDL; Male; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Oligonucleotide Array Sequence Analysis; Phenotype; Rats; Rats, Sprague-Dawley; Time Factors; Transfection

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27(Kip1). A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Topics: Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Elongation Factor 2 Kinase; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, src; Humans; Mice; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA Interference

Phosphorylation of Tyr-88/Tyr-89 in the 3(10) helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 3(10) helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.

Topics: Amino Acid Sequence; Animals; Cell Proliferation; Central Nervous System Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; HEK293 Cells; Humans; Mice; Molecular Sequence Data; Mutation; Phosphorylation; Tyrosine

Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G(2)/M and the accumulation of cardiomyocytes in the G(0)/G(1) phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27(Kip1), possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27(Kip1). These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies.

Topics: 14-3-3 Proteins; Animals; Base Sequence; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA Primers; Female; Fetal Heart; Gene Expression Regulation, Developmental; Heart Defects, Congenital; Heart Ventricles; Humans; Male; Mice; Mice, 129 Strain; Mice, Knockout; Myocytes, Cardiac; Oncogene Proteins

Increasing evidence indicates that various cancer cell types are capable of producing IgG. The exact function of cancer-derived IgG has, however, not been elucidated. Here we demonstrated the expression of IgG genes with V(D)J recombination in 80 cases of colorectal cancers, 4 colon cancer cell lines and a tumor bearing immune deficient mouse model. IgG expression was associated with tumor differentiation, pTNM stage, lymph node involvement and inflammatory infiltration and positively correlated with the expressions of Cyclin D1, NF-κB and PCNA. Furthermore, we investigated the effect of cancer-derived IgG on the malignant behaviors of colorectal cancer cells and showed that blockage of IgG resulted in increased apoptosis and negatively affected the potential for anchor-independent colony formation and cancer cell invasion. These findings suggest that IgG synthesized by colorectal cancer cells is involved in the development and growth of colorectal cancer and blockage of IgG may be a potential therapy in treating this cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin G; Immunohistochemistry; In Situ Hybridization; Jurkat Cells; Mice; Mice, SCID; NF-kappa B; Proliferating Cell Nuclear Antigen

Cyclin D1, a member of the G1 cyclin family, plays a critical role in the progression of the cell cycle. In the present study, we investigated chronological alterations in cyclin D1 immunoreactivity and its protein levels in the gerbil hippocampus after ischemia/reperfusion.. Chronological alterations in cyclin D1 immunoreactivity and its levels were examined in the gerbil hippocampus after ischemia/reperfusion using immunohistochemistry and western blot analysis.. Changes in cyclin D1 immunoreactivity in the ischemic hippocampus were distinct in pyramidal neurons of the CA1 region and granule cells of the dentate gyrus. Cyclin D1 immunoreactivity in pyramidal neurons of the CA1 region was increased up to 1 day after ischemia/reperfusion, although a transient decrease of cyclin D1 immunoreactivity was detected at 12 hour after ischemia/reperfusion. Thereafter, cyclin D1 immunoreactivity in the CA1 pyramidal neurons was very weak 2 days and disappeared nearly 4 and 7 days after ischemia/reperfusion. However, 4 days after ischemia/reperfusion, the cyclin D1 immunoreactivity in non-pyramidal neurons of the CA1 region was very strong. In the CA2/3 region, cyclin D1 immunoreactivity was higher than that in the CA1 region and not changed after ischemia/reperfusion. In the dentate gyrus, chronological change in cyclin D1 immunoreactivity was observed. Cells in the granule cell layer showed distinct change in cyclin D1 immunoreactivity after ischemia/reperfusion: the cyclin D1 immunoreactivity was lowest at 12 hours and strong 1 and 4 days after ischemia/reperfusion. In addition, change in cyclin D1 protein level was found in the ischemic hippocampus.. Our results indicate that cyclin D1 may play an important role in cellular events related with neuronal damage following ischemia/reperfusion.

Topics: Animals; CA1 Region, Hippocampal; Cell Death; Cyclin D1; Dentate Gyrus; Disease Models, Animal; Gerbillinae; Ischemic Attack, Transient; Male; Neurons; Prosencephalon; Pyramidal Cells; Reperfusion

In this study we hypothesised that proliferation, and the increased expression of G(1)-phase cyclins (D1, E) and phosphorylated retinoblastoma protein (p-Rb) is restricted to the early period of synchronized cyst growth in autosomal-recessive polycystic kidney disease (ARPKD).. Lewis polycystic kidney disease (lpk) rats (model of ARPKD; postnatal weeks 1, 3, 6, 12 and 24; n = 6 each) as well as human juvenile cystic renal disease tissue (n = 2) were examined.. Between weeks 1 and 3, the percentage cyst area increased 6-fold in lpk rats, followed by a more progressive rise (1.5-fold increase) until week 24. The number of Ki-67-, cyclin D1- and p-Rb-positive cells increased in lpk rats and peaked at week 3, declining thereafter. By serial sections, cysts co-expressed Ki-67, cyclin D1 and p-Rb. The expression of cyclin E was variable, and peaked at week 24. In human tissue, small cysts had a higher expression of p-Rb.. Proliferation and the increased nuclear expression of cyclin D1 and p-Rb coincide with the early phase of cyst growth in rats and humans, suggesting that there might be a therapeutic window in which cyclin-dependent kinase inhibitors are most effective in preventing kidney enlargement in ARPKD.

Topics: Animals; Cell Nucleus; Cell Proliferation; Cyclin D1; Cysts; Disease Models, Animal; Humans; Immunohistochemistry; Ki-67 Antigen; Polycystic Kidney, Autosomal Recessive; Rats; Rats, Inbred Lew; Retinoblastoma Protein; Time Factors

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular functions and are highly active in many types of human cancers. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream MAPK involved in apoptosis, inflammation, and carcinogenesis. This study investigated the role of ASK1 in the development of gastric cancer. In human gastric cancer specimens, we observed increased ASK1 expression, compared to nontumor epithelium. Using a chemically induced murine gastric tumorigenesis model, we observed increased tumor ASK1 expression, and ASK1 knockout mice had both fewer and smaller tumors than wild-type (WT) mice. ASK1 siRNA inhibited cell proliferation through the accumulation of cells in G1 phase of the cell cycle, and reduced cyclin D1 expression in gastric cancer cells, whereas these effects were uncommon in other cancer cells. ASK1 overexpression induced the transcription of cyclin D1, through AP-1 activation, and ASK1 levels were regulated by cyclin D1, via the Rb-E2F pathway. Exogenous ASK1 induced cyclin D1 expression, followed by elevated expression of endogenous ASK1. These results indicate an autoregulatory mechanism of ASK1 in the development of gastric cancer. Targeting this positive feedback loop, ASK1 may present a potential therapeutic target for the treatment of advanced gastric cancer.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinase 5; Mice; Mice, Inbred C57BL; Mice, Transgenic; Signal Transduction; Stomach Neoplasms

Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive.. In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury.. These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Topics: Acetylation; Animals; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Chromatin Assembly and Disassembly; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; E2F Transcription Factors; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Hydroxylamines; Hyperplasia; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Quinolines; Rats; Retinoblastoma Protein; RNA Interference; Time Factors; Transcription, Genetic; Tunica Media; Vascular System Injuries

We previously reported that the abnormal BTG2 expression was related to genesis/development of hepatocellular carcinoma (HCC). The aim of this study was to evaluate the BTG2 expression in HCC compared with p53, cyclin D1, and cyclin E. For this purpose, modified diethylnitrosamine (DEN)-induced primary HCC rat model was established. Target proteins and mRNAs were measured by western blot and RT-PCR/northern blot, respectively. In rat liver, expression of BTG2 and other proteins was determined by western blot, and BTG2 mRNA in HCC/normal tissues was detected by high-flux tissue microarray (TMA) and in situ hybridization (ISH). BTG2 mRNA/protein expression was increased in fetal liver, 7701, and LO2 cell lines but decreased in HepG2 cells. BTG2/p53 were expressed early after DEN treatment, peaked at 5 weeks and decreased gradually thereafter. Cyclin-D1/Cyclin-E expression increased significantly with the tumor progression. BTG2 mRNA was expressed in 71.19% HCC by ISH and correlated with differentiation. Expression of p53/cyclin D1/cyclin E was positive in 82.35/94.12/76.47% BTG2 mRNA-negative tissues, respectively. BTG2 protein expression was lost in 32.2% (19/59) HCC tissues, and the mRNA/protein expression correlated significantly with the increasing tumor grade (P < 0.05). In conclusion, BTG2 expression is commonly impaired in HCC which may be a factor involved in deregulation of cyclin-D1/cyclin-E expression during hepatocarcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin E; Diethylnitrosamine; Disease Models, Animal; Female; Humans; Immediate-Early Proteins; Liver Neoplasms; Male; Middle Aged; Rats; Rats, Wistar; RNA, Messenger; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Syndecan-4 (Syn4) is a heparan sulfate proteoglycan and works as a coreceptor for various growth factors. We examined whether Syn4 could be involved in the development of neointimal formation in vivo.. Wild-type (WT) and Syn4-deficient (Syn4-/-) mice were subjected to wire-induced femoral artery injury. Syn4 mRNA was upregulated after vascular injury in WT mice. Neointimal formation was attenuated in Syn4-/- mice, concomitantly with the reduction of Ki67-positive vascular smooth muscle cells (VSMCs). Basic-fibroblast growth factor- or platelet-derived growth factor-BB-induced proliferation, extracellular signal-regulated kinase activation, and expression of cyclin D1 and Bcl-2 were impaired in VSMCs from Syn4-/- mice. To examine the role of Syn4 in bone marrow (BM)-derived vascular progenitor cells (VPCs) and vascular walls, we generated chimeric mice by replacing the BM cells of WT and Syn4-/- mice with those of WT or Syn4-/- mice. Syn4 expressed by both vascular walls and VPCs contributed to the neointimal formation after vascular injury. Although the numbers of VPCs were compatible between WT and Syn4-/- mice, mobilization of VPCs from BM after vascular injury was defective in Syn4-/- mice.. Syn4 deficiency limits neointimal formation after vascular injury by regulating VSMC proliferation and VPC mobilization. Therefore, Syn4 may be a novel therapeutic target for preventing arterial restenosis after angioplasty.

Topics: Animals; Apoptosis; Becaplermin; Bone Marrow Transplantation; Cell Movement; Cell Proliferation; Cyclin D1; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Femoral Artery; Fibroblast Growth Factor 2; Hyperplasia; Ki-67 Antigen; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Platelet-Derived Growth Factor; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-sis; Signal Transduction; Stem Cells; Syndecan-4; Time Factors; Tunica Intima; Vascular System Injuries

Cholestasis contributes to hepatocellular injury and promotes liver carcinogenesis. We created a mouse model of chronic cholestasis to study its effects on progression of cholangiocarcinoma and the oncogenes involved.. To induce chronic cholestasis, Balb/c mice were given 2 weekly intraperitoneal injections of diethylnitrosamine (DEN); 2 weeks later, some mice also received left and median bile duct ligation (LMBDL) and, then 1 week later, were fed DEN, in corn oil, weekly by oral gavage (DLD). Liver samples were analyzed by immunohistochemical and biochemical assays; expression of Mnt and c-Myc was reduced by injection of small inhibitor RNAs.. Chronic cholestasis was induced by DLD and accelerated progression of cholangiocarcinoma, compared with mice given only DEN. Cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma developed in the DLD group at weeks 8, 12, 16, and 28, respectively. LMBDL repressed expression of microRNA (miR)-34a and let-7a, up-regulating Lin-28B, hypoxia-inducible factor (HIF)-1α, HIF-2α, and miR-210. Up-regulation of Lin-28B might inhibit let-7a, which is associated with development of cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma. Knockdown of c-Myc reduced progression of cholangiocarcinoma, whereas knockdown of Mnt accelerated its progression. Down-regulation of miR-34a expression might up-regulate c-Myc. The up-regulation of miR-210 via HIF-2α was involved in down-regulation of Mnt. Activation of the miR-34a-c-Myc and HIF-2α-miR-210-Mnt pathways caused c-Myc to bind the E-box element of cyclin D1, instead of Mnt, resulting in cyclin D1 up-regulation.. DLD induction of chronic cholestasis accelerated progression of cholangiocarcinoma, which is mediated by down-regulation of miR-34a, up-regulation miR-210, and replacement of Mnt by c-Myc in binding to cyclin D1.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic Helix-Loop-Helix Transcription Factors; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Cholestasis; Cyclin D1; Diethylnitrosamine; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Hyperplasia; Hypoxia-Inducible Factor 1, alpha Subunit; Ligation; Liver; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Proto-Oncogene Proteins c-myc; Repressor Proteins; RNA Interference; RNA-Binding Proteins; Signal Transduction; Time Factors; Transcription Factors

During the disease process of mesangial proliferative glomerulonephritis, the expression of various factors that influence mesangial proliferation is altered. MAX interactor 1 (Mxi1) antagonizes the transcription factor Myc and is believed to be a tumor suppressor. However, no studies have investigated its effect on mesangial cell proliferation.. To investigate the effect of Mxi1 on renal mesangial cell proliferation, we established a classic rat anti-Thy1 mesangial proliferative glomerulonephritis model. Mesangial proliferation was estimated by immunohistochemical analysis of Ki67. Mxi1 expression at each time point was assessed by real-time RT-PCR and Western blot analyses. Furthermore, we altered the expression level of Mxi1 by a plasmid and siRNA to detect its effect on rat mesangial cell proliferation in vitro.. Mxi1 expression decreased significantly during the proliferative period of anti-Thy1 nephritis model and then gradually increased as proliferation declined, indicating that Mxi1 may be linked to mesangial cell proliferation. Upregulation of Mxi1 expression via plasmid transfection in vitro reduced the expression of the positive-acting cell cycle regulatory proteins cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; significantly reduced mesangial cell proliferation; reduced the percentage of S phase cells; and increased the percentage of G2/M phase cells. Inhibition of Mxi1 expression by siRNA in vitro produced the opposite effects: increased expression of cyclin B1, cyclin D1, cyclin E, CDC2 and CDK2; markedly increased cell proliferation; higher percentage of S phase cells; and dramatically lower percentage of G2/M phase cells. Transcription factor c-myc protein expression showed no obvious difference after Mxi1 plasmid and siRNA transfection. The expressions of cell cycle regulatory proteins mentioned above were negative correlated with Mxi1 expression in anti-Thy1 nephritis model.. These results suggest that Mxi1 expression levels were inversely correlated with proliferation in anti-Thy1 nephritis rats and it may influence cell cycle progression and thus the rate of mesangial cell proliferation by regulating the expression of c-myc target cell cycle regulatory proteins.

Topics: Animals; Antibodies; Basic Helix-Loop-Helix Transcription Factors; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Division; Cell Line; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Disease Models, Animal; G2 Phase; Glomerulonephritis; Male; Mesangial Cells; Proto-Oncogene Proteins c-myc; Rats; Rats, Wistar; RNA Interference; RNA, Small Interfering; S Phase; Thy-1 Antigens; Transfection; Tumor Suppressor Proteins

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer(32/36) in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr(393) and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β(Ser9). Dietary DMF (100mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Casein Kinase II; Caspase 3; Cyclin D1; Cyclooxygenase 2; Diethylnitrosamine; Disease Models, Animal; Flavones; Flavonoids; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; NF-kappa B; Nitric Oxide Synthase Type II; Precancerous Conditions; Rats; Rats, Wistar; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation; Wnt Proteins

The cyclin-dependent kinase 5 activator p35 can be cleaved into p25. Formation of p25 has been suggested to contribute to neurodegeneration in Alzheimer's disease (AD). However, overexpression of low levels of p25 in mice enhances memory formation. Therefore, it has been suggested that p25 formation might be an event early in AD to compensate for impairments in synaptic plasticity. Ongoing p25 formation has been hypothesized to contribute to neurodegeneration at the later stages of AD.. Here, we tested the early compensation hypothesis by analyzing the levels of p25 and its precursor p35 in AD postmortem samples from different brain regions at different stages of tau pathology, using quantitative Western blots. Furthermore, we studied p35 and p25 during spatial memory formation. By employing quantitative mass spectrometry, we identified proteins downstream of p25, which were then studied in AD samples.. We found that p25 is generated during spatial memory formation. Furthermore, we demonstrate that overexpression of p25 in the physiological range increases the expression of two proteins implicated in spine formation, septin 7 and optic atrophy 1. We show that the expression of p35 and p25 is reduced as an early event in AD. Moreover, expression of the p25-regulated protein optic atrophy 1 was reduced in a time course similar to p25 expression.. Our findings suggest that p25 generation is a mechanism underlying hippocampal memory formation that is impaired in the early stages of AD. Our findings argue against the previously raised early compensation hypothesis and they propose that p25-mediated neurotoxicity does not occur in AD.

Topics: Alzheimer Disease; Animals; Brain; Cyclin D1; Disease Models, Animal; Down-Regulation; GTP Phosphohydrolases; Humans; Mass Spectrometry; Maze Learning; Memory; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron, Transmission; Mutation; Nerve Tissue Proteins; Phosphopyruvate Hydratase; Septins; Synapses; Synaptosomes; tau Proteins

The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1-driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1-expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G(1)-S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2's antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1-expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1-expressing tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Disease Models, Animal; Gene Amplification; Genes, bcl-2; Humans; Lymphoma, Mantle-Cell; Mice; Nitrophenols; Piperazines; Sulfonamides; Xenograft Model Antitumor Assays

Idiopathic pulmonary fibrosis (IPF) may be triggered by epithelial injury that results in aberrant production of growth factors, cytokines, and proteinases, leading to proliferation of myofibroblasts, excess deposition of collagen, and destruction of the lung architecture. The precise mechanisms and key signaling mediators responsible for this aberrant repair process remain unclear. We assessed the importance of matrix metalloproteinase-3 (MMP-3) in the pathogenesis of IPF through i) determination of MMP-3 expression in patients with IPF, ii) in vivo experiments examining the relevance of MMP-3 in experimental models of fibrosis, and iii) in vitro experiments to elucidate possible mechanisms of action. Gene expression analysis, quantitative RT-PCR, and Western blot analysis of explanted human lungs revealed enhanced expression of MMP-3 in IPF, compared with control. Transient adenoviral vector-mediated expression of recombinant MMP-3 in rat lung resulted in accumulation of myofibroblasts and pulmonary fibrosis. Conversely, MMP-3-null mice were protected against bleomycin-induced pulmonary fibrosis. In vitro treatment of cultured lung epithelial cells with purified MMP-3 resulted in activation of the β-catenin signaling pathway, via cleavage of E-cadherin, and induction of epithelial-mesenchymal transition. These processes were inhibited in bleomycin-treated MMP-3-null mice, as assessed by cytosolic translocation of β-catenin and cyclin D1 expression. These observations support a novel role for MMP-3 in the pathogenesis of IPF, through activation of β-catenin signaling and induction of epithelial-mesenchymal transition.

Topics: Adenoviridae; Animals; beta Catenin; Bleomycin; Cadherins; Cyclin D1; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Enzymologic; Genetic Vectors; Humans; Lung; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Protein Transport; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer.. Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA.. Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model.. Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

Topics: Animals; Antineoplastic Agents; Aza Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Curcumin; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Immunohistochemistry; Mice; Mice, Inbred C57BL; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Notch receptors play an essential role in cellular processes during embryonic and postnatal development, including maintenance of stem cell self-renewal, proliferation, and determination of cell fate and apoptosis. Deregulation of Notch signaling has been implicated in some genetic diseases and tumorigenesis. The function of Notch signaling in a variety of tumors can be either oncogenic or tumor-suppressive, depending on the cellular context. In this study, Notch1 overexpression was observed in the majority of 45 astrocytic gliomas with different grades and in U251MG glioma cells. Transfection of siRNA targeting Notch1 into U251 cells in vitro downregulated Notch1 expression, associated with inhibition of cell growth, arrest of cell cycle, reduction of cell invasiveness, and induction of cell apoptosis. Meanwhile, tumor growth was delayed in established subcutaneous gliomas in nude mice treated with Notch1 siRNA in vivo. These results suggest that Notch1 plays an important oncogenic role in the development and progression of astrocytic gliomas. Furthermore, knockdown of Notch1 expression by siRNA simultaneously downregulated the expression of EGFR and the important components of its downstream pathways, including PI3K, p-AKT, K-Ras, cyclin D1 and MMP9, indicating the crosstalk and interaction of Notch and EGFR signaling pathways.

Topics: Animals; Annexin A5; Apoptosis; Astrocytoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; ErbB Receptors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Matrix Metalloproteinase 9; Mice; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins p21(ras); Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Transfection

The alteration of the beta-cell population in the Goto-Kakizaki rat (GK/Par line), a model of spontaneous type 2 diabetes, has been ascribed to significantly decreased beta-cell replication and neogenesis, while beta-cell apoptosis is surprisingly not enhanced and remains in the normal range. To gain insight into the mechanisms by which those beta-cells are protected from death, we studied ex vivo the apoptotic activity and the expression of a large set of pro/antiapoptotic and pro/antioxidant genes in GK/Par islet cells. This was done in vitro in freshly isolated islets as well as in response to culture conditions and calibrated reactive oxygen species (ROS) exposure (i.e., H2O2). We also investigated the intracellular mechanisms of the diabetic beta-cell response to ROS, the role if any of the intracellular cAMP metabolism, and finally the kinetic of ROS response, taking advantage of the GK/Par rat normoglycemia until weaning. Our results show that the peculiar GK/Par beta-cell phenotype was correlated with an increased expression of a large panel of antioxidant genes as well as pro/antiapoptotic genes. We demonstrate that such combination confers resistance to cytotoxic H2O2 exposure in vitro, raising the possibility that at least some of the activated stress/defense genes have protective effects against H2O2-triggered beta-cell death. We also present some evidence that the GK/Par beta-cell resistance to H2O2 is at least partly cAMP dependent. Finally, we show that such a phenotype is not innate but is spontaneously acquired after diabetes onset as the result of an adaptive response to the diabetic environment.

Topics: Adaptation, Physiological; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Division; Cells, Cultured; Cyclic AMP; Cyclin D1; Diabetes Mellitus, Type 2; Disease Models, Animal; Heme Oxygenase-1; Hydrogen Peroxide; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin-Secreting Cells; Male; Oxidants; Proto-Oncogene Proteins c-myc; Rats; Rats, Mutant Strains; Rats, Wistar; Reactive Oxygen Species; RNA, Messenger

Cyclin D1 overexpression is associated with mantle cell lymphoma and multiple myeloma. In myeloma, it often results from chromosomal translocations linking the CCND1 gene to the 3' part of the IgH locus constant region. This region includes a single and potent transcriptional regulatory region (RR) 3' of the Calpha gene mostly active in mature B-cells. To check whether this RR alone was sufficient to deregulate CCND1, we generated mice carrying a 3'IgH RR-driven human CCND1 transgene and specifically up-regulating cyclin D1 expression in B-cells. In transgenic B-cells, cyclin D1 enforced cell cycle entry in response to various stimuli (LPS, anti-IgM, anti-CD40) but also increased cell death, so that exaggerated proliferation did not result in peripheral lymphocytosis. Despite exaggerated B-cell entry into G(1) phase, malignant lymphoproliferation did not occur either. Crossing of CCND1-3'IgH RR mice with c-myc-3'IgH RR mice did not reveal accelerated tumorigenesis as compared with c-myc-3'IgH RR mice alone. The data presented here demonstrate that the 3'IgH RR-mediated deregulation of CCND1 in mature B-cells cannot by itself trigger the development of lymphomas and strengthen the concept that cyclin D1 per se is not an armful proto-oncogene. Rather its overexpression in several malignancies might be only a stigma of lymphomagenesis or represent a single hit within a multiple hit process.

Topics: Animals; Apoptosis; B-Lymphocytes; Blotting, Western; Bone Marrow; Cell Differentiation; Cell Proliferation; Cyclin D1; Disease Models, Animal; Flow Cytometry; Genetic Vectors; Immunoenzyme Techniques; Immunoglobulin Heavy Chains; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proto-Oncogene Mas; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen

Cholangiocyte proliferation is necessary for biliary recovery from cold ischemia and reperfusion injury (CIRI), but there are few studies on its intracellular mechanism. In this process, the role of rapamycin, a new immunosuppressant used in liver transplantation, is still unknown. In order to determine whether rapamycin can depress cholangiocyte regeneration by inhibiting signal transducer and activator of transcription 3 (STAT3) activation, rapamycin (0.05 mg/kg) was administered to rats for 3 days before orthotopic liver transplantation. The results indicated that cholangiocytes responded to extended cold preservation (12 hours) with severe bile duct injures, marked activation of the interleukin-6 (IL-6)/STAT3 signal pathway, and increased expression of cyclin D1 until 7 days after transplantation, and this was followed by compensatory cholangiocyte regeneration. However, rapamycin treatment inhibited STAT3 activation and resulted in decreased cholangiocyte proliferation and delayed biliary recovery after liver transplantation. On the other hand, rapamycin showed no effect on the expression of IL-6. We conclude that the IL-6/STAT3 signal pathway is involved in initiating cholangiocytes to regenerate and repair CIRI. Rapamycin represses cholangiocyte regeneration by inhibiting STAT3 activation, which might have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation. Insights gained from this study will be helpful in designing therapy using rapamycin in clinical patients after liver transplantation.

Topics: Animals; Bile Ducts, Intrahepatic; Cell Division; Cryopreservation; Cyclin D1; Disease Models, Animal; Graft Rejection; Immunosuppressive Agents; Interleukin-6; Liver Regeneration; Liver Transplantation; Male; Phosphorylation; Rats; Rats, Wistar; Reperfusion Injury; Sirolimus; STAT3 Transcription Factor

Chemoprevention is a practical and translational approach to reduce the risk of various cancers including colorectal cancer (CRC), which is a major cause of cancer-related deaths in the United States. Accordingly, here we assessed chemopreventive efficacy and associated mechanisms of long-term silibinin feeding on spontaneous intestinal tumorigenesis in the APC(min/+) mice model. Six-week-old APC(min/+) mice were p.o. fed with vehicle control (0.5% carboxymethyl cellulose and 0.025% Tween 20 in distilled water) or 750 mg silibinin/kg body weight in vehicle for 5 d/wk for 13 weeks and then sacrificed. Silibinin feeding strongly prevented intestinal tumorigenesis in terms of polyp formation in proximal, middle, and distal portions of small intestine by 27% (P < 0.001), 34% (P < 0.001), and 49% (P < 0.001), respectively. In colon, we observed 55% (P < 0.01) reduction in number of polyps by silibinin treatment. In size distribution analysis, silibinin showed significant decrease in large-size polyps (>3 mm) by 66% (P < 0.01) and 88% (P < 0.001) in middle and distal portions of small intestine, respectively. More importantly, silibinin caused a complete suppression in >3 mm sized polyps and 92% reduction in >2 to 3 mm sized polyps in colon. Molecular analyses of polyps suggested that silibinin exerts its chemopreventive efficacy by inhibiting cell proliferation, inflammation, and angiogenesis; inducing apoptosis; decreasing beta-catenin levels and transcriptional activity; and modulating the expression profile of cytokines. These results show for the first time the efficacy and associated mechanisms of long-term p.o. silibinin feeding against spontaneous intestinal tumorigenesis in the APC(min/+) mice model, suggesting its chemopreventive potential against intestinal cancers including CRC.

Topics: Adenomatous Polyposis Coli; Animals; Antioxidants; Apoptosis; beta Catenin; Cell Growth Processes; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Intestinal Polyps; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Silybin; Silymarin; Transcriptional Activation

MicroRNAs, by modulating gene expression, have been implicated as regulators of various cellular and physiological processes, including differentiation, proliferation, and cancer. Here, we study the role of microRNAs in Schwann cell (SC) differentiation by conditional removal of the microRNA processing enzyme Dicer1. We reveal that both male and female mice lacking Dicer1 in SC (Dicer1 conditional knock-outs) display a severe neurological phenotype resembling congenital hypomyelination. Ultrastructural analyses show that many SC lacking Dicer1 are stalled in differentiation at the promyelinating state and fail to myelinate axons. Gene expression analyses reveal a failure to extinguish genes characteristic of the undifferentiated state such as Sox2, Jun, and Ccnd1. Sox2 and Jun are well characterized negative regulators of SC differentiation. Consistent with Sox2/Jun maintenance, Egr2, a master regulator of the myelinating program, is drastically downregulated and likely accounts for the myelination defect. We posit a model wherein microRNAs are critical for downregulation of antecedent programs of gene expression. In SC differentiation, this is particularly relevant in the key developmental transition from a promyelinating to myelinating SC.

Topics: Animals; Animals, Newborn; Bromodeoxyuridine; Cell Differentiation; Cyclin D1; DEAD-box RNA Helicases; Demyelinating Diseases; Disease Models, Animal; Early Growth Response Protein 2; Endoribonucleases; Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Indoles; Male; Mice; Mice, Knockout; MicroRNAs; Microscopy, Electron, Transmission; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-jun; Ribonuclease III; Schwann Cells; Sciatic Nerve; SOXB1 Transcription Factors

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-kappaB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biological Availability; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Deoxycytidine; Disease Models, Animal; Down-Regulation; Drug Synergism; Gemcitabine; Humans; Matrix Metalloproteinase 9; Mice; Nanoparticles; Neoplasm Metastasis; NF-kappa B; Pancreatic Neoplasms; Polymers; Subcutaneous Tissue; Xenograft Model Antitumor Assays

1. Pravastatin is best known for its antilipidemic action. Recent studies have shown that statins have immunomodulatory and anti-inflammatory effects. The present study aimed to determine whether or not pravastatin can attenuate acute lung injury and fibrosis in a mouse model. 2. Bleomycin was given to C57BL6 mice through intratracheal instillation. Pravastatin was given through intraperitoneal injection. To study the effect of pravastatin on the early inflammatory phase and the late fibrotic phase, mice were killed on days 3, 7, 14 and 21. 3. Pravastatin attenuated the histopathological change of bleomycin-induced lung injury and fibrosis. The accumulation of neutrophils and increased production of tumor necrosis factor-α in bronchoalveolar lavage fluid were inhibited in the early inflammatory phase. Pravastatin effectively inhibited the increase of lung hydroxyproline content induced by bleomycin. Furthermore, pravastatin reduced the increased expression of transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), RhoA and cyclin D1. The increased levels of TGF-β1 and CTGF mRNA expression were also significantly inhibited by pravastatin. 4. Pravastatin effectively attenuated bleomycin-induced lung injury and pulmonary fibrosis in mice. Our results provide evidence for the therapeutic potential of pravastatin in the treatment of acute lung injury and pulmonary fibrosis.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Bleomycin; Blotting, Western; Bronchoalveolar Lavage Fluid; Connective Tissue Growth Factor; Cyclin D1; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Hydroxyproline; Mice; Mice, Inbred C57BL; Pravastatin; Pulmonary Fibrosis; Respiratory Distress Syndrome; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Transforming Growth Factor beta1

Gallbladder carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. We previously reported that norcantharidin (NCTD) is useful against growth, proliferation, and invasion of human gallbladder carcinoma GBC-SD cells in vitro. In this study, we further studied the inhibitory effect of NCTD on the growth of xenografted tumors of human gallbladder carcinoma in nude mice in vivo and the underlying mechanisms.. The tumor xenograft model of human gallbladder carcinoma in nude mice in vivo was established with subcutaneous GBC-SD cells. The experimental mice were randomly divided into control, 5-FU, NCTD, and NCTD+5-FU groups which were given different treatments. Tumor growth in terms of size, growth curve, and inhibitory rate was evaluated. Cell cycle, apoptosis, and morphological changes of the xenografted tumors were assessed by flow cytometry and light/electron microscopy. The expression of the cell cycle-related proteins cyclin-D1 and p27 as well as the apoptosis-related proteins Bcl-2, Bax, and survivin were determined by the streptavidin-biotin complex (SABC) method and RT-PCR.. NCTD inhibited the growth of the xenografted tumors in a dose- and time-dependent manner. Tumor volume decreased (5.61+/-0.39 vs. 9.78+/-0.61 cm3, P=0.000) with an increased tumor inhibitory rate (42.63% vs. 0%, P=0.012) in the NTCD group compared with the control group. The apoptosis rate increased (15.08+/-1.49% vs. 5.49+/-0.59%, P=0.0001) along with a decreased percentage of cells in S phase (43.47+/-2.83% vs. 69.85+/-1.96%, P=0.0001) in the NTCD group compared with the control group. The morphological changes of apoptosis such as nuclear shrinkage, chromatin aggregation, chromosome condensation, and typical apoptosis bodies in the xenografted tumor cells induced by NCTD were observed by light and electron microscopy. The expression of cyclin-D1, Bcl-2 and survivin proteins/mRNAs decreased significantly, with increased expression of p27 and Bax proteins/mRNAs in the NCTD group compared with the control group.. NCTD inhibits the growth of xenografted tumors of human gallbladder carcinoma in nude mice by inducing apoptosis and blocking the cell cycle in vivo.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gallbladder Neoplasms; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Time Factors; Xenograft Model Antitumor Assays

Proliferation of vascular smooth muscle cells (VSMCs) is a crucial event in the pathogenesis of intimal hyperplasia, the main cause of restenosis following vascular reconstruction. Here, the impact of sonic hedgehog (Shh)/Gli family zinc finger 2 (Gli2) signaling on VSMC proliferation was assessed.. Increased Shh signaling was detected in VSMCs in the neointima of vein grafts obtained from mice undergoing restenosis. Comparable results were found in primary cultured human VSMCs (hVSMCs) obtained from patients undergoing coronary bypass surgery, which were used to further assess the impacts of Shh signaling on VSMC proliferation. Inhibition of Shh signaling in hVSMCs through treatment with cyclopamine or knockdown of Gli2 results in G(1) arrest and reduced cyclin D1, cyclin E, and phosphorylated retinoblastoma (pRB) levels. In contrast, activation of Shh/Gli2 signaling in hVSMCs results in increased levels of G(1) cyclins and promotes G(1)-S transition. Stimulation of hVSMC proliferation by Shh is abolished by cyclin D1 knockdown.. Combined, these results demonstrate that Shh/Gli2 signaling stimulates VSMC proliferation via regulation of the G(1) cyclin-retinoblastoma axis and suggest that antagonists that target the Shh pathway may be therapeutically beneficial in the prevention of intimal hyperplasia.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin E; Disease Models, Animal; G1 Phase; Graft Occlusion, Vascular; Hedgehog Proteins; Humans; Hyperplasia; Jugular Veins; Kruppel-Like Transcription Factors; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Phosphorylation; Recombinant Fusion Proteins; Retinoblastoma Protein; RNA Interference; S Phase; Saphenous Vein; Signal Transduction; Veratrum Alkaloids; Zinc Finger Protein Gli2

α(2)-Adrenoceptors belong to the family of adrenergic receptors, which regulate the neuronal release of norepinephrine as part of a negative feedback loop. Among the α(2)-adrenoceptors, the α(2B)-subtype may also influence developmental signaling pathways involved in angiogenesis of the placenta. Thus, the aim of the present study was to determine whether α(2B)-adrenoceptors are also involved in other developmental processes beyond placental angiogenesis. Ablation of α(2B)-adrenoceptors led to lethality of mutant mice during the first hours after birth. Despite normal breathing and drinking behavior, mutant mice developed cyanosis, which could be traced back to a defect in lung morphology with significantly reduced alveolar volume and thickened interalveolar septi. In α(2B)-deficient lungs and in isolated alveolar type II cells, expression of sonic hedgehog (SHH) was significantly increased, resulting in mesenchymal proliferation. In vitro α(2B)-adrenoreceptor stimulation suppressed expression of sonic hedgehog and the cell cycle genes cyclin D1 and Ki67. In vivo inhibition of enhanced SHH signaling by the smoothened antagonist cyclopamine partially rescued perinatal lethality, lung morphology, and altered gene expression in mutant mice. Thus, α(2B)-adrenoceptors in lung epithelia play an important role in suppressing sonic hedgehog-mediated proliferation of mesenchymal cells and thus prevent respiratory failure.

Topics: Animals; Cyclin D1; Disease Models, Animal; Epithelium; Hedgehog Proteins; Heterozygote; Ki-67 Antigen; Lung; Lung Diseases; Mesoderm; Mice; Mice, Inbred C57BL; Neurotransmitter Agents; Receptors, Adrenergic, alpha-2; Veratrum Alkaloids

Increased proliferation of airway smooth muscle cells (ASMCs) are observed in asthmatic patients and smoking can accelerate proliferation of ASMCs in asthma. To elucidate the molecular mechanisms leading to these changes, we studied in vitro the effect of cigarette smoke extract (CSE) on the proliferation of ASMCs and the expression of cyclin D1, an important regulatory protein implicated in cell cycle.. ASMCs cultured from 8 asthmatic Brown Norway rats were studied. Cells between passage 3 and 6 were used in the study and were divided into control group, pcDNA3.1 group, pcDNA3.1-antisense cyclin D1 (ascyclin D1) group, CSE group, CSE + pcDNA3.1 group and CSE + pcDNA3.1-ascyclin D1 group based on the conditions for intervention. The proliferation of ASMCs was examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expression of cyclin D1 was detected by reverse transcriptase-PCR (RT-PCR) and Western blotting.. (1) The percentage of S + G2M phase, absorbance value at 490 nm wavelength (A(490)) and the expression rate of PCNA protein in CSE group were (31.22 +/- 1.17)%, 0.782 +/- 0.221, (90.2 +/- 7.0)% respectively, which were significantly increased compared with those of control group ((18.36 +/- 1.02)%, 0.521 +/- 0.109, and (54.1 +/- 3.5)%, respectively) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the percentage of S + G2M phase, A(490) and the expression rate of PCNA protein in ASMCs were much lower than in untreated cells (P < 0.01). (2) The ratios of A(490) of cyclin D1 mRNA in CSE group was 0.288 +/- 0.034, which was significantly increased compared with that of control group (0.158 +/- 0.006) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 mRNA in ASMCs was much lower than in untreated cells (P < 0.01). (3) The ratios of A(490) of cyclin D1 protein expression in CSE group was 0.375 +/- 0.008, which was significantly increased compared with that of control group (0.268 +/- 0.004) (P < 0.01). After the transfection with antisense cyclin D1 plasmid for 30 hours, the ratios of A(490) of cyclin D1 protein expression in ASMCs was much lower than in untreated cells (P < 0.01).. CSE may increase the proliferation of ASMCs in asthmatic rats via regulating cyclin D1 expression.

Topics: Animals; Asthma; Blotting, Western; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Models, Animal; Female; Flow Cytometry; Immunohistochemistry; Microscopy, Phase-Contrast; Myocytes, Smooth Muscle; Nicotiana; Plant Extracts; Rats; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Smoking

Patched1 heterozygous mice (Ptch1(+/-)) are useful for basal cell carcinoma (BCC) studies, being remarkably susceptible to BCC induction by ultraviolet or ionizing radiation. Analogously, skin carcinogenesis-susceptible (Car-S) mice are elective for studies of papilloma and squamous cell carcinoma (SCC) induction. We previously reported a striking effect of gender on BCC induction in Ptch1(+/-) mice, with total resistance of females; likewise, Car-S females show increased skin tumor resistance relative to males. Here, we investigated the protective role of endogenous estrogen in skin keratinocyte tumorigenesis. Control (CN) and ovariectomized Ptch1(+/-) or Car-S females were irradiated for BCC induction or topically treated with chemical carcinogens for SCC induction. Susceptibility to BCC or SCC was dramatically increased in ovariectomized Ptch1(+/-) and Car-S females and restored to levels observed in males. Remarkably, progression of initially benign papillomas to malignant SCC occurred only in ovariectomized Car-S females. We explored the mechanisms underlying tumor progression and report overexpression of estrogen receptor (ER)-alpha, downregulation of ERbeta and upregulation of cyclin D1 in papillomas from ovariectomized Car-S relative to papillomas from CN females. Thus, an imbalanced ERalpha/ERbeta expression may be associated with estrogen-mediated modulation of non-melanoma skin carcinogenesis, with a key role played by cyclin D1. Our findings underscore a highly protective role of endogenous estrogen against skin tumorigenesis by diverse agents in two independent mouse models of skin cancer.

Topics: Animals; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Female; Male; Mice; Neoplasms, Radiation-Induced; Ovariectomy; Papilloma; Patched Receptors; Patched-1 Receptor; Receptors, Cell Surface; Skin Neoplasms; Ultraviolet Rays

Inactivation and silencing of PTEN have been observed in multiple cancers, including follicular thyroid carcinoma. PTEN (phosphatase and tensin homologue deleted from chromosome 10) functions as a tumour suppressor by opposing the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. Despite correlative data, how deregulated PTEN signalling leads to thyroid carcinogenesis is not known. Mice harbouring a dominant-negative mutant thyroid hormone receptor beta (TRbeta(PV/PV) mice) spontaneously develop follicular thyroid carcinoma and distant metastases similar to human cancer. To elucidate the role of PTEN in thyroid carcinogenesis, we generated TRbeta(PV/PV) mice haploinsufficient for Pten (TRbeta(PV/PV)Pten(+/-) mouse). PTEN deficiency accelerated the progression of thyroid tumour and increased the occurrence of metastasis spread to the lung in TRbeta(PV/PV)Pten(+/-) mice, thereby significantly reducing their survival as compared with TRbeta(PV/PV)Pten(+/+) mice. AKT activation was further increased by two-fold in TRbeta(PV/PV)Pten(+/-) mice thyroids, leading to increased activity of the downstream mammalian target of rapamycin (mTOR)-p70S6K signalling and decreased activity of the forkhead family member FOXO3a. Consistently, cyclin D1 expression was increased. Apoptosis was decreased as indicated by increased expression of nuclear factor-kappaB (NF-kappaB) and decreased caspase-3 activity in the thyroids of TRbeta(PV/PV)Pten(+/-) mice. Our results indicate that PTEN deficiency resulted in increased cell proliferation and survival in the thyroids of TRbeta(PV/PV)Pten(+/-) mice. Altogether, our study provides direct evidence to indicate that in vivo, PTEN is a critical regulator in the follicular thyroid cancer progression and invasiveness.

Topics: Animals; Apoptosis; Carrier Proteins; Caspase 3; Cell Proliferation; Cell Survival; Chromosomes, Mammalian; Cyclin D1; Disease Models, Animal; Enzyme Activation; Forkhead Box Protein O3; Forkhead Transcription Factors; Lung Neoplasms; Mice; Mice, Mutant Strains; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Thyroid Hormone Receptors beta; Thyroid Neoplasms; TOR Serine-Threonine Kinases

Consumption of green tea polyphenols (GTPs) in drinking water prevents photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. Using IL-12p40 knockout (KO) mice and their wild-type counterparts and an established photocarcinogenesis protocol, we found that although administration of GTPs (0.2%, w/v) in drinking water significantly reduced UVB-induced tumor development in wild-type mice, this treatment had a nonsignificant effect in IL-12-KO mice. GTPs resulted in reduction in the levels of markers of inflammation (cyclooxygenase-2, prostaglandin E(2), proliferating cell nuclear antigen, and cyclin D1) and proinflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta) in chronically UVB-exposed skin and skin tumors of wild-type mice but less effective in IL-12p40-KO mice. UVB-induced DNA damage (cyclobutane pyrimidine dimers) was resolved rapidly in GTPs-treated wild-type mice than untreated wild-type mice and this resolution followed the same time course as the GTPs-induced reduction in the levels of inflammatory responses. This effect of GTPs was less pronounced in IL-12-KO mice. The above results were confirmed by treatment of IL-12-KO mice with murine recombinant IL-12 and treatment of wild-type mice with neutralizing anti-IL-12 antibody. To our knowledge, it is previously unreported that prevention of photocarcinogenesis by GTPs is mediated through IL-12-dependent DNA repair and a subsequent reduction in skin inflammation.

Topics: Administration, Oral; Animals; Beverages; Cyclin D1; Cyclooxygenase 2; Cytokines; Dinoprostone; Disease Models, Animal; DNA Repair; Female; Flavonoids; Inflammation; Interleukin-12; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Neoplasms, Radiation-Induced; Phenols; Plant Extracts; Polyphenols; Proliferating Cell Nuclear Antigen; Skin Neoplasms; Ultraviolet Rays

Toxic bile acids induce hepatocyte apoptosis, for which p53 and cyclin D1 have been implicated as underlying mediators. Both p53 and cyclin D1 are targets of c-Myc, which is also up-regulated in cholestasis. Myc and Mnt use Max as a cofactor for DNA binding. Myc-Max typically activates transcription via E-box binding. Mnt-Max also binds the E-box sequence but serves as a repressor and inhibits the enhancer activity of Myc-Max. The current work tested the hypothesis that the switch from Mnt-Max to Myc-Max is responsible for p53 and cyclin D1 up-regulation and apoptosis during cholestasis. Following common bile duct ligation or left hepatic bile duct ligation, the expression of p53, c-Myc, and cyclin D1 increased markedly, whereas Mnt expression decreased. Nuclear binding activity of Myc to the E-box element of p53 and cyclin D1 increased, whereas that of Mnt decreased in a time-dependent fashion. Lithocholic acid (LCA) treatment of primary human hepatocytes and HuH-7 cells induced a similar switch from Mnt to Myc and increased p53 and cyclin D1 promoter activity and endogenous p53 and cyclin D1 expression and apoptosis. Blocking c-Myc induction in HuH-7 cells prevented the LCA-mediated increase in p53 and cyclin D1 expression and reduced apoptosis. Lowering Mnt expression further enhanced LCA's inductive effect on p53 and cyclin D1. Bile duct-ligated mice treated with a lentivirus harboring c-myc small interfering RNA were protected from hepatic induction of p53 and cyclin D1, a switch from Mnt to Myc nuclear binding to E-box, and hepatocyte apoptosis.. The switch from Mnt to Myc during bile duct ligation and in hepatocytes treated with LCA is responsible for the induction in p53 and cyclin D1 expression and contributes to apoptosis.

Topics: Animals; Apoptosis; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cell Line, Tumor; Cells, Cultured; Cholestasis; Cyclin D1; Disease Models, Animal; Hepatocytes; Humans; Ligation; Lithocholic Acid; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-myc; Repressor Proteins; Signal Transduction; Tumor Suppressor Protein p53

Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

Topics: Animals; beta Catenin; Cell Transformation, Neoplastic; Cyclin D1; Cyclin D2; Cyclins; Disease Models, Animal; Female; Humans; Liver Neoplasms, Experimental; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins c-met; Transfection; Up-Regulation

In this study, the potential role of Stat3 in UVB-induced skin carcinogenesis was examined using skin-specific gain and loss of function transgenic mice, that is, K5.Stat3C and K5Cre.Stat3(fl/fl) mice, respectively. The epidermis of Stat3-deficient mice was highly sensitive to UVB-induced apoptosis, whereas the epidermis of K5.Stat3C mice was more resistant to UVB-induced apoptosis. In particular, the status of Stat3 influenced the survival of ultraviolet-photoproduct cells, including those located in the hair follicles. K5.Stat3C mice exhibited significantly increased epidermal proliferation and hyperplasia in response to UVB irradiation, whereas Stat3-deficient mice showed reduced epidermal proliferation and hyperplasia. Expression of target genes regulated by Stat3, such as cyclin D1 and Bcl-x(L), was increased in epidermis of both control and UVB-irradiated K5.Stat3C mice, and downregulated in epidermis of both control and UVB-irradiated K5Cre.Stat3(fl/fl) mice. Following UVB irradiation, the formation of skin tumors in K5.Stat3C mice was accelerated and both the incidence and multiplicity of skin tumors were significantly greater than wild-type controls. In contrast, Stat3-deficient mice were resistant to UVB skin carcinogenesis. These results show that Stat3 plays an important role in the development of UVB-induced skin tumors through its effects on both survival and proliferation of keratinocytes during carcinogenesis.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; Cyclins; Disease Models, Animal; Epidermis; Immunoenzyme Techniques; Integrases; Keratinocytes; Mice; Mice, Knockout; Mice, Transgenic; Neoplasms, Radiation-Induced; Skin Neoplasms; STAT3 Transcription Factor; Ultraviolet Rays

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells. In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2 antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were also tolerable in mice and superior to either agent alone. As DTX is the standard first-line chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest in this disease stage. Our results provide a rationale for clinical trials on combination treatments with azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Azacitidine; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Proliferation; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Docetaxel; Drug Synergism; Drug Therapy, Combination; Flow Cytometry; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Taxoids; Xenograft Model Antitumor Assays

Stem cell transplantation is a possible therapeutic option to repair ischemic damage to the heart. However, it is faced with a number of challenges including the survival of the transplanted cells in the ischemic region. The present study was designed to use stem cells preconditioned with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine; TMZ), a widely used anti-ischemic drug for treating angina in cardiac patients, to increase the rate of their survival after transplantation. Bone marrow-derived rat mesenchymal stem cells (MSCs) were subjected to a simulated host tissue environment by culturing them under hypoxia (2% O(2)) and using hydrogen peroxide (H(2)O(2)) to induce oxidative stress. MSCs were preconditioned with 10 microM TMZ for 6 h followed by treatment with 100 microM H(2)O(2) for 1 h and characterized for their cellular viability and metabolic activity. The preconditioned cells showed a significant protection against H(2)O(2)-induced loss of cellular viability, membrane damage, and oxygen metabolism accompanied by a significant increase in HIF-1alpha, survivin, phosphorylated Akt (pAkt), and Bcl-2 protein levels and Bcl-2 gene expression. The therapeutic efficacy of the TMZ-preconditioned MSCs was evaluated in an in vivo rat model of myocardial infarction induced by permanent ligation of left anterior descending coronary artery. A significant increase in the recovery of myocardial function and up-regulation of pAkt and Bcl-2 levels were observed in hearts transplanted with TMZ-preconditioned cells. This study clearly demonstrated the potential benefits of pharmacological preconditioning of MSCs with TMZ for stem cell therapy for repairing myocardial ischemic damage.

Topics: Animals; Blotting, Western; Cell Hypoxia; Cell Survival; Cyclin D1; Disease Models, Animal; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Oxidative Stress; Oxygen Consumption; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; Trimetazidine; Vasodilator Agents

The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus, and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect of cyclin D1 overexpression on susceptibility of mice to carcinogen-induced tumorigenesis, using 4-nitroquinoline-1-oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and nontransgenic littermates were administered 4NQO (20 or 50 parts per million (ppm) in the drinking water) for 8 wk and monitored for an additional 16 wk. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with nontransgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro-esophageal epithelia. Examination of the dysplastic and neoplastic lesions revealed abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen-induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero-digestive cancer.

Topics: 4-Nitroquinoline-1-oxide; Animals; Blotting, Northern; Carcinogens; Cell Differentiation; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Epithelium; Female; Gene Expression; Genetic Predisposition to Disease; Immunohistochemistry; Keratin-5; Ki-67 Antigen; Male; Mice; Mice, Transgenic; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Transgenes

The onset of adult hypothyroidism causes neuronal damage in the CA3 hippocampal region, which is attenuated by T(4) administration. We analyzed the expression of molecular proliferation markers (Cyclin D1 and PCNA), cellular damage-arrest (p53 and p21), and apoptosis (Bax/Bcl-2 index) in the hippocampus of hypothyroid (methimazole; 60 mg/kg) or thyroid replaced (T(4), 20 microg/kg; MMI+T(4) or T(3), 20 microg/kg; MMI+T(3)) adult male rats. Histological analysis showed that hypothyroid animals exhibit significant neuronal damage in all regions of the hippocampus accompanied by the triggering of the apoptotic pathway (increases in p53, p21 and the Bax/Bcl-2 index) and no changes in proliferation (Cyclin D1 and PCNA). MMI+T(4) replaced animals were completely protected with no changes in molecular markers. In contrast, MMI+T(3) replaced animals showed partial protection in which, although pro-apoptotic effects remained (increase in the Bax/Bcl-2), proliferative mechanisms were triggered (increase in p53, Cyclin D1 and PCNA expression). Our results indicate that thyroid hormones participate in the maintenance of the hippocampal neuronal population even in adulthood, suggesting that THs have different physiological roles as neuronal survival factors: T(4) prevents the activation of apoptotic pathways, whereas T(3) activates cell differentiation and proliferation mechanisms.

Topics: Animals; Antithyroid Agents; bcl-2-Associated X Protein; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Hippocampus; Hypothyroidism; Male; Methimazole; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Pyramidal Cells; Rats; Rats, Wistar; Thyroid Gland; Thyroid Hormones; Tumor Suppressor Protein p53

Asian ginseng (Panax ginseng C. A. Meyer) has been used in Chinese medicine for two thousand years. The root of ginseng contains several saponins (ginsenosides) which are biologically active compounds. Individual ginsenosides suppress tumor cell growth, induce cell differentiation, regulate apoptosis and inhibit metastasis formation. The aim of this study was to evaluate its chemo-preventive effects in an animal test model, through its regulatory effects on apoptosis and the cell cycle.The expression of genes (Bcl-2, Bcl-x and Cyclin D1) which affect apoptosis were examined, in different organs of animals which had consumed a ginseng-containing diet in the presence of a known carcinogen (DMBA). The pattern of gene expression was determined by Q-RT-PCR. The increase of antiapoptotic gene expression after carcinogenic exposure was suppressed by consumption of ginseng which promoted apoptosis.The population is exposed to numerous physical and chemical insults in the modern environment and these include compounds which are known carcinogens. Research has shown that it is possible to interfere with the multi-step process of carcinogenesis through the use of compounds with chemo-preventive effects, such as the inhibition of the activation of antiapoptotic genes.These results support the efficacy of ginseng-containing diets and dietary supplements in the prevention of cancerous diseases.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Carcinogens; Cyclin D1; Disease Models, Animal; Female; Gene Expression; Mice; Mice, Inbred AKR; Neoplasms; Panax; Phytotherapy; Plant Preparations; Plant Roots; Powders; Proto-Oncogene Proteins c-bcl-2

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; ras Proteins; Receptor, ErbB-2; RNA Interference; Simian virus 40; Telomerase; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Topics: Administration, Oral; Animals; Beverages; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; DNA Repair; Flavonoids; Inflammation; Interleukin-12; Mice; Mice, Knockout; Neoplasms, Radiation-Induced; Phenols; Plant Extracts; Polyphenols; Proliferating Cell Nuclear Antigen; Prostaglandins; Skin Neoplasms; Ultraviolet Rays

We investigated the possible mechanisms of inhibition of colorectal carcinogenesis by green tea (GT) in azoxymethane-treated (AOM) Apc(Min/+) mice. Mice received water or a 0.6% (w/v) solution of GT as the only source of beverage. GT treatment commenced at the 8th week of age and lasted for 8 wk. The treatment caused a statistically significant reduction in the number of newly formed tumors (28%, P < 0.05). Immunohistochemical analysis showed that GT decreased the levels of beta-catenin and its downstream target cyclin D1. To probe a mechanism, we further investigated the expression of retinoic X receptor alpha (RXR alpha) in AOM/Apc(Min/+) tumors. Our results show that RXR alpha is selectively downregulated in AOM/Apc(Min/+) mouse intestinal tumors. In contrast, other retinoic receptors including retinoic acid receptor alpha (RAR alpha), RAR beta, RXR beta, and RXR gamma were all expressed in Apc(Min/+) adenomas. Furthermore, our results show that RXR alpha downregulation is an early event in colorectal carcinogenesis and is independent of beta-catenin expression. GT significantly increased the protein levels of RXR alpha. In addition, RT-PCR analysis showed that GT induced a similar increase in the levels of RXR alpha mRNA. Genomic bisulfite treatment of colonic DNA followed by pyrosequencing of 24 CpG sites in the promoter region of RXR alpha gene showed a significant decrease in CpG methylation with GT treatment. The results suggest that a low concentration of GT is sufficient to desilence RXR alpha and inhibit intestinal tumorigenesis in the Apc(Min/+) mouse.

Topics: Adenoma; Animals; Azoxymethane; beta Catenin; Camellia sinensis; Carcinogens; Cyclin D1; Disease Models, Animal; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Female; Genes, APC; Immunoenzyme Techniques; Intestinal Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Promoter Regions, Genetic; Retinoid X Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tea

Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11-14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.

Topics: Animals; Capillaries; Cardiotonic Agents; CDC2 Protein Kinase; Cell Proliferation; Cyclin D1; Disease Models, Animal; Erythropoietin; Injections, Intralesional; Ki-67 Antigen; Male; Myocardial Contraction; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats; Rats, Inbred Lew; Recovery of Function; Time Factors; Ventricular Function, Left

This study aimed to evaluate cell cycle regulation in acute kidney injury after intraperitoneal sepsis in rats.. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in rats. At 0, 6, 12, 24, 48, and 72 h after CLP, serum creatinine was evaluated. DNA content of isolated kidney cells was analyzed using flow cytometer. Furthermore, the expression of p21, p53, cyclin D1, cyclin E, CDK2, CDK4 and P-pRb was also measured by western blot.. After sepsis-induced by CLP, kidney injury of rat was associated with G1 cell cycle arrest, however, recovery of renal function related to cell cycle progression 48h after CLP. Results also showed that the upregulation of p53 and p21 was correlated with G1 cell arrest in 48h after CLP. Nevertheless, upregulation of cyclin D1/CDK4 and cyclin E/CDK2 induced pRb phosphorylation, which resulted in the G1/S transition 48 h after CLP.. The data suggest that G1 cell cycle arrest may play a role in the initiation of kidney injury, whereas, through regulating cell cycle, p53, p21, CDKs, cyclins and P-pRb may be involved in the injury or recovery of renal function after intraperitoneal sepsis.

Topics: Acute Kidney Injury; Animals; Cell Cycle; Creatinine; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Models, Animal; DNA; Flow Cytometry; G1 Phase; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; S Phase; Sepsis; Tumor Suppressor Protein p53; Up-Regulation

We have recently reported that antioxidant liposomes can be used as antidotes for mustard gas induced lung injury in guinea pigs. The maximum protection was achieved with a liposome composed of tocopherols (alpha, gamma, delta) and N-acetylcysteine (NAC) when administered after 5 min of exposure of 2-chloroethyl ethyl sulfide (CEES), a half sulfur mustard gas. We also reported an association of mustard gas-induced lung injury with an activation of MAPK/AP-1 signaling pathway and cell proliferation. The objective of the present study was to investigate whether CEES-induced MAPKs/AP-1 signaling pathway is influenced by antioxidant liposome therapy. A single dose (200 microl) of the antioxidant liposome was administered intratracheally after 5 min of exposure of CEES (0.5 mg/kg). The animals were sacrificed after 1h and 30 days of CEES exposure. Although the liposome treatment did not have any significant effect on the activation of the MAPKs family (ERK1/2, p38 and JNK1/2), it significantly counteracted the CEES-induced activation of AP-1 transcription factors and corresponding increase in the protein levels of Fos, ATF and Jun family members. The liposome treatment significantly blocked the CEES-induced increase in the protein levels of cyclin D1, a cell cycle protein and PCNA, a cell differentiation marker. Furthermore, it protected lung against CEES-induced inflammation and infiltration of neutrophils, eosinophils and erythrocytes in the alveolar space. This suggests that the protective effect of antioxidant liposome against CEES-induced lung damage is mediated via control of AP-1 signaling.

Topics: Acetylcysteine; Activating Transcription Factors; Animals; Antidotes; Antioxidants; Blood Proteins; Cell Proliferation; Cyclin D1; Disease Models, Animal; Erythrocytes; Guinea Pigs; Liposomes; Lung; Lung Injury; Male; Mitogen-Activated Protein Kinases; Mustard Gas; Neutrophil Infiltration; Phosphorylation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Pulmonary Eosinophilia; Time Factors; Tocopherols; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Gliomas are the most common adult primary brain tumors, and the most malignant form, glioblastoma multiforme, is invariably fatal. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is altered in most glioblastoma multiforme. PTEN, an important negative regulator of the PI3K-Akt pathway, is also commonly mutated in glioma, leading to constitutive activation of Akt. One ultimate consequence is phosphorylation and inactivation of FOXO forkhead transcription factors that regulate genes involved in apoptosis, cell cycle arrest, nutrient availability, DNA repair, stress, and angiogenesis. We tested the ability of a mutant FOXO1 factor that is not subject to Akt phosphorylation to overcome dysregulated PI3K-Akt signaling in two PTEN-null glioma cell lines, U87 and U251. Adenovirus-mediated gene transfer of the mutant FOXO1 successfully restored cell cycle arrest and induced cell death in vitro and prolonged survival in vivo in xenograft models of human glioma (33% survival at 1 year of animals bearing U251 tumors). However, U87 were much more resistant than U251 to mutant FOXO1-induced death, showing evidence of increased nuclear export and Akt-independent phosphorylation of FOXO1 at S249. A cyclin-dependent kinase 2 inhibitor decreased phosphorylation of S249 and rendered U87 cells significantly more susceptible to mutant FOXO1-induced death. Our results indicate that targeting FOXO1, which is at the convergence point of several growth factor receptor tyrosine kinase pathways, can effectively induce glioma cell death and inhibit tumor growth. They also highlight the importance of Akt-independent phosphorylation events in the nuclear export of FOXO1.

Topics: Adenoviridae; Animals; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 2; Cyclins; Disease Models, Animal; DNA Repair; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Neovascularization, Pathologic; Phosphorylation

Cyproheptadine, an inhibitor of the H1 histamine receptors, has recently shown activity in models of leukaemia and myeloma, presumably through inhibition of cyclin-D expression. Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin-D1. We investigated the effect of cyproheptadine alone and in combination with the proteasome inhibitor bortezomib in models of MCL. The combination of these drugs was mathematically synergistic, producing significant reductions in the mitochondrial membrane potential leading to apoptosis. In a severe combined immunodeficient beige mouse model, cyproheptadine plus bortezomib demonstrated a statistically significant advantage compared to either agent alone.

Topics: Actins; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cyclin D1; Cyproheptadine; Disease Models, Animal; Histamine H1 Antagonists; Histone Deacetylase Inhibitors; Lymphoma, Mantle-Cell; Mice; Protease Inhibitors; Pyrazines

Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell carcinomas. The carcinogenic 4-nitroquinoline-1-oxide (4NQO) model has proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has a suitable volume for scientific treatment. For this end we applied 4NQO 3 times a week during 16 weeks and measured the volume of tumor tissue each week until the end of the experiment at 40 weeks. Concurrent histopathology at different time points up to the end of the experiment revealed that all mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. The 4NQO model is a suitable alternative for preclinical gene therapy experiments with primary oral tumors. Future survey of therapeutic options in the carcinogenic 4NQO model should be conducted around 40 weeks after the start of the treatment.

Topics: 4-Nitroquinoline-1-oxide; Animals; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cyclin D1; Disease Models, Animal; Immunohistochemistry; Male; Mice; Mice, Inbred CBA; Mouth Neoplasms; Tongue; Tumor Burden

To investigate the inhibitory effects of Kangjia Pill (KJP) on the cell proliferation in rat goiter model induced by methimazole (MMI).. Fifty-six Wistar rats were randomly divided into four groups: the normal group, MMI model group (MMI), low dose of KJP group (LKJP), and high dose of KJP (HKJP). Except the normal group (20 rats), the other groups (12 rats in each) were given 0.04% (w/v) MMI through the drinking water until the end of the experiment. One week later, the rats in the LKJP and HKJP groups were given KJP by gastrogavage at the dose of 250 mg/(kg x d) and 1,000 mg/(kg x d), respectively for 12 weeks. The relative thyroid weight (mg/100 g body weight) of each rat was accessed. The expression of proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry, and the correlation analysis between the PCNA positive thyrocytes and the relative thyroid weight was performed. The expressions of PCNA and cyclin D1 were examined with Western blotting.. After KJP treatment for 12 weeks, compared with the MMI group, the relative thyroid weight of the HKJP group decreased significantly, and the positive thyrocyte populations of PCNA in the two KJP groups reduced markedly (all P<0.05). The correlation analysis showed that PCNA was closely correlated with thyrocyte proliferation (r=0.685, P<0.05). KJP significantly decreased the protein expression of PCNA and cyclin D1 in the thyroid specimens (P<0.05), the high dose showed better effects.. KJP played a therapeutic role via inhibiting cell proliferation in the rat goitrous glands.

Topics: Animals; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Goiter; Male; Methimazole; Organ Size; Proliferating Cell Nuclear Antigen; Random Allocation; Rats; Rats, Wistar; Tablets; Thyroid Gland

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G(1) in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G(1) accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G(1)/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3' untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease.

Topics: Animals; Antineoplastic Agents; Apoptosis; Base Sequence; Cyclin D1; Disease Models, Animal; DNA Primers; Drug Screening Assays, Antitumor; Genistein; Imidazoles; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Inbred C57BL; MicroRNAs; Piperazines; RNA, Messenger

We investigated whether postinfarct treatment with oxytocin (OT) improves left ventricular (LV) function and remodeling via cardiac repair of myocardial ischemia-reperfusion injury.. Experiments were performed with 30 minutes of coronary occlusion and 2 or 14 days of reperfusion rabbit model of myocardial infarction. LV function and remodeling were significantly improved in the OT group. The infarct size was significantly reduced in the OT group. The number of CD31-positive microvessels was increased significantly in the OT group. There were no Ki67-positive myocytes in either group. The expression of the OT receptor, phosphorylated (p)-Akt protein kinase, p-extracellular signal-regulated protein kinase, p-enodthelial NO synthase, p-signal transducer and activator of transcription 3, vascular endothelial growth factor, B-cell lymphoma 2, and matrix metalloproteinase-1 (MMP-1) were markedly increased in the OT group days 2 and 14 post myocardial infarction.. Postinfarct treatment with OT reduces myocardial infarct size and improves LV function and remodeling by activating OT receptors and prosurvival signals and by exerting antifibrotic and angiogenic effects through activation of MMP-1, endothelial NO synthase, and vascular endothelial growth factor. These findings provide new insight into therapeutic strategies for ischemic heart disease.

Topics: Animals; Blood Pressure; Cyclin D1; Disease Models, Animal; Echocardiography; Extracellular Signal-Regulated MAP Kinases; Heart; Heart Rate; Male; Matrix Metalloproteinase 1; Microvessels; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Oxytocin; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Proto-Oncogene Proteins c-akt; Rabbits; Receptors, Oxytocin; Signal Transduction; STAT3 Transcription Factor; Stroke Volume; Vascular Endothelial Growth Factor A; Ventricular Dysfunction, Left; Ventricular Function, Left; Ventricular Remodeling

Low levels of serum adiponectin have been reported to be associated with obesity, diabetes, and non-alcoholic steatohepatitis (NASH), as well as several malignancies. Adiponectin knockout (KO) mice have been reported to cause insulin resistance and neointimal formation of the artery. We used adiponectin KO mice fed a high fat (HF) diet, and investigated the effect of adiponectin on the progression of steatohepatitis and carcinogenesis in vivo.. Adiponectin KO mice and wild type (WT) mice were fed a HF diet or normal chow for the periods of 24 and 48 weeks. The HF diet contained 60% of calories from fat.. The adiponectin KO mice on the HF diet showed obesity, marked elevation of serum transaminase levels, and hyperlipidemia. At 24 weeks, hepatic expression of tumor necrosis factor-alpha and procollagen alpha (I) was higher in KO mice as compared with WT mice. At 48 weeks, liver triglyceride contents in KO mice on normal chow were significantly higher than those in WT mice. Hepatocyte ballooning, spotty necrosis, and pericellular fibrosis around central veins were observed in KO mice on the HF diet. The pericellular fibrosis was more severe in KO mice on the HF diet than that in WT mice (1.62% vs 1.16%, P = 0.033). Liver adenoma and hyperplastic nodules developed in a KO mouse on the HF diet at 48 weeks (12.5%, n = 1/8), whereas no tumor was detected in WT mice (n = 10).. Adiponectin may play a protective role in the progression of NASH in the early stages by suppressing tumor necrosis factor-alpha expression and liver fibrosis.

Topics: Adenoma; Adiponectin; Alanine Transaminase; Animals; Aspartate Aminotransferases; Collagen Type I; Collagen Type I, alpha 1 Chain; Cyclin D1; Dietary Fats; Disease Models, Animal; Disease Progression; Fatty Liver; Hyperlipidemias; Hyperplasia; Liver; Liver Cirrhosis, Experimental; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Obesity; RNA, Messenger; Time Factors; Triglycerides; Tumor Necrosis Factor-alpha

The hepatic stellate cell (HSC) is the primary cell type in the liver responsible for excess collagen deposition during fibrosis. Following a fibrogenic stimulus the cell changes from a quiescent vitamin A-storing cell to an activated cell type associated with increased extracellular matrix synthesis and increased cell proliferation. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been shown to regulate several aspects of HSC activation in vitro, including collagen synthesis and cell proliferation. Using a targeted approach to inhibit PI3K signaling specifically in HSCs, we investigated the role of PI3K in HSCs using a rodent model of hepatic fibrosis. An adenovirus expressing a dominant negative form of PI3K under control of the smooth muscle alpha-actin (alphaSMA) promoter was generated (Ad-SMAdnPI3K). Transducing HSCs with Ad-SMAdnPI3K resulted in decreased proliferation, migration, collagen expression, and several additional profibrogenic genes, while also promoting cell death. Inhibition of PI3K signaling was also associated with reduced activation of Akt, p70 S6 kinase, and extracellular regulated kinase signaling as well as reduced cyclin D1 expression. Administering Ad-SMAdnPI3K to mice following bile duct ligation resulted in reduced HSC activation and decreased extracellular matrix deposition, including collagen expression. A reduction in profibrogenic mediators, including transforming growth factor beta, tissue inhibitor of metalloproteinase 1, and connective tissue growth factor was also noted. However, liver damage, assessed by alanine aminotransferase levels, was not reduced.. Inhibition of PI3K signaling in HSCs during active fibrogenesis inhibits extracellular matrix deposition, including synthesis of type I collagen, and reduces expression of profibrogenic factors. These data suggest that targeting PI3K signaling in HSCs may represent an effective therapeutic target for hepatic fibrosis.

Topics: Actins; Adenoviridae; Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Cyclin D1; Disease Models, Animal; Disease Progression; Extracellular Matrix; Hepatic Stellate Cells; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction

Transplantation of bone marrow stromal cells (BMSCs) improves animal neurological functional recovery after stroke. But the mechanism remains unclear. As cell cycle machinery plays an important role in stroke, we investigated the dynamic changes of cell cycle elements in a rat model of middle cerebral artery occlusion. We found the cell cycle markers, cdk4 along with its activator cyclin D1, and proliferating cell nuclear antigen (PCNA), increased after brain ischemia-reperfusion. Phosphorylation of the retinoblastoma protein (pRb, on ser-795), the cyclin D/cdk4 complex mutual target, was upregulated accordingly. However, intravenously administrated BMSCs facilitated cyclin D1, cdk4, and PCNA decrease in the ischemic cortex. Meanwhile, phospho-pRb (ser-795) was completely inhibited. On the contrary, endogenous cdk inhibitor p27 reduced before but enhanced after BMSCs treatment. These findings suggested BMSCs might modulate cell cycle progression in injured brain via downregulation of the cyclin D1/cdk4/pRb pathway as well as upregulation of p27 level. These results provide another way by which BMSCs may contribute to the recovery from stroke.

Topics: Animals; Bone Marrow Transplantation; Brain Ischemia; Cell Cycle; Cerebral Cortex; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Infarction, Middle Cerebral Artery; Male; Phosphorylation; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Retinoblastoma Protein; Signal Transduction; Stroke; Stromal Cells

The 22q11 deletion (or DiGeorge) syndrome (22q11DS), the result of a 1.5- to 3-megabase hemizygous deletion on human chromosome 22, results in dramatically increased susceptibility for "diseases of cortical connectivity" thought to arise during development, including schizophrenia and autism. We show that diminished dosage of the genes deleted in the 1.5-megabase 22q11 minimal critical deleted region in a mouse model of 22q11DS specifically compromises neurogenesis and subsequent differentiation in the cerebral cortex. Proliferation of basal, but not apical, progenitors is disrupted, and subsequently, the frequency of layer 2/3, but not layer 5/6, projection neurons is altered. This change is paralleled by aberrant distribution of parvalbumin-labeled interneurons in upper and lower cortical layers. Deletion of Tbx1 or Prodh (22q11 genes independently associated with 22q11DS phenotypes) does not similarly disrupt basal progenitors. However, expression analysis implicates additional 22q11 genes that are selectively expressed in cortical precursors. Thus, diminished 22q11 gene dosage disrupts cortical neurogenesis and interneuron migration. Such developmental disruption may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.

Topics: Animals; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Cerebral Cortex; Chromosome Deletion; Chromosomes, Human, Pair 21; Chromosomes, Mammalian; Cyclin D1; DiGeorge Syndrome; Disease Models, Animal; Gene Expression Regulation, Developmental; Histones; Humans; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoproteins; Reverse Transcriptase Polymerase Chain Reaction; Synteny; T-Box Domain Proteins

G1-phase cyclin D1 (cD1) expression has been documented in post-mitotic neurons undergoing apoptosis, leading others to propose that attempted cell cycle re-entry may induce cell death. Here, cD1 immunoreactivity was found in a subpopulation of healthy excitatory neurons throughout the brain. Most striking was the selective cD1 expression in hippocampal pyramidal neurons, an especially vulnerable cell group. Seizure threshold, cD1 induction and CA1 neuron death were examined following application of kainate (KA) or pentylenetetrazole (PTZ) in cD1 heterozygous (+/-) and wildtype mice to determine whether baseline cD1 correlates with pathology. cD1+/- mice displayed resistance to KA, but not PTZ-induced seizures and had reduced or equivalent cytotoxicity respectively, compared with wildtype. KA administration, but not PTZ, induced cD1 expression. These findings suggest that basal cD1 expression may render hippocampal circuits more susceptible to particular epileptogenic agents and excitotoxic cell death, though cD1 is not a direct precipitant in apoptosis.

Topics: Adult; Animals; Apoptosis; Convulsants; Cyclin D1; Disease Models, Animal; Epilepsy; Female; Hippocampus; Humans; Immunohistochemistry; Kainic Acid; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neurotoxins; Pentylenetetrazole; Pyramidal Cells; Rats; Rats, Sprague-Dawley

Cyclins and other cell-cycle regulators have been used in several studies to regenerate cardiomyocytes in ischaemic heart failure. However, proliferation of cardiomyocytes induced by nuclear-targeted cyclin D1 (D1NLS) stops after one or two rounds of cell cycles due in part to accumulation of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK). Thus, expression of S-phase kinase-associated protein 2 (Skp2), a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on cardiomyocyte proliferation in vitro. Here, we examined whether Skp2 can also improve cardiomyocyte regeneration and post-ischaemic cardiac performance in vivo.. Wistar rats underwent ischaemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs. 4.00%), and mitotic phosphorylated histone H3 (0.24% vs. 0.58%). Compared with rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio.. Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischaemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischaemic regeneration of cardiomyocytes in vivo.

Topics: Adenoviridae; Animals; Animals, Newborn; Apoptosis; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Heart Failure; Mitosis; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley; Regeneration; S-Phase Kinase-Associated Proteins; Time Factors; Ventricular Function, Left

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

Topics: Animals; Asthma; Cell Cycle; Cell Proliferation; Codon; Cyclin D1; Disease Models, Animal; DNA, Antisense; Gene Expression; Genetic Vectors; Male; Myocytes, Smooth Muscle; Rats; Rats, Sprague-Dawley; Recombination, Genetic; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Transduction, Genetic; Transfection

Tracheal agenesis/atresia (TA) is a rare but fatal congenital disease in which the breathing tube fails to grow. The etiology of this serious condition remains largely unknown. We found that Bmp signaling is prominently present in the anterior foregut where the tracheal primordium originates and targeted ablation of Bmp4 (Bmp4(cko)) resulted in a loss-of-trachea phenotype that closely resembles the Floyd type II pathology, the most common form of TA in humans. In Bmp4(cko) embryos, tracheal specification was not affected; however, its outgrowth was severely impaired due to reduced epithelial and mesenchymal proliferation. In agreement, we also observed significant reduction in the expression of Cyclin D1, a key cell cycle regulator associated with cellular proliferation. However, the proliferative effect of Bmp signaling appears to be independent of Wnt signaling. Interestingly, we found significantly reduced expression of activated extracellular signal-regulated kinase (Erk) in the Bmp4(cko) ventral foregut, suggesting that Bmp signaling promotes Erk phosphorylation which has been associated with cellular proliferation. This study provides the first evidence linking Bmp signaling to tracheal formation by regulating the proliferative response of the anterior ventral foregut. Our finding sheds light on human tracheal malformations by providing a novel mouse model implicating Bmp signaling, non-canonical Erk activation and cellular proliferation.

Topics: Animals; beta Catenin; Body Patterning; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Proliferation; Cyclin D1; Digestive System; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Forkhead Transcription Factors; Gene Expression Regulation, Developmental; Gene Targeting; Genes, Reporter; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Transgenic; Nerve Tissue Proteins; Signal Transduction; Trachea; Tracheal Diseases; Wnt Proteins

Both epidemiological and experimental findings have indicated that components of Western diets influence colonic tumorigenesis. Among dietary constituents, calcium and cholecalciferol have emerged as promising chemopreventive agents. We have demonstrated that a Western-style diet (WD) with low levels of calcium and cholecalciferol and high levels of (n-6) PUFA, increased the incidence of neoplasia in mouse intestine compared with a standard AIN-76A diet; models included wild-type mice and mice with targeted mutations. In the present study, adenomatous polyposis coli (Apc)(1638N/+) mice carrying a heterozygous Apc mutation were fed either an AIN-76A diet, a WD, or a WD supplemented with calcium and cholecalciferol (WD/Ca/VitD3). Diets were fed for 24 wk and effects on cellular and molecular events were assessed by performing immunohistochemistry in colonic epithelium along the crypt-to-surface continuum. Feeding WD to Apc(1638N/+) mice not only enhanced cyclin D1 expression in colonic epithelium compared with AIN-76A treatment as previously reported but also significantly increased the expression of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) concomitantly with a decrease in the proapoptotic Bcl2-associated X protein and the number of apoptotic epithelial cells. WD treatment enhanced mutant Apc-driven small intestinal carcinogenesis and also resulted in the formation of a small number of colonic adenomas (0.16 +/- 0.09; P < 0.05). By contrast, the WD/Ca/VitD3 diet reversed WD-induced growth, promoting changes in colonic epithelium. Importantly, Apc(1638N/+) mice fed the WD/Ca/VitD3 diet did not develop colonic tumors, further indicating that dietary calcium and cholecalciferol have a key role in the chemoprevention of colorectal neoplasia in this mouse model of human colon cancer.

Topics: Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Apoptosis; bcl-2-Associated X Protein; Body Weight; Calcium, Dietary; Carcinogenicity Tests; Cholecalciferol; Colon; Cyclin D1; Diet; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Male; Mice; Mutation; Proto-Oncogene Proteins c-bcl-2; Random Allocation

This study was undertaken to investigate the potential role of cell cycle re-entry in an experimental model of Huntington's disease and in human brain samples. We found that after treatment of rats with the mitochondrial neurotoxin 3-nitropropionic acid, the expression of cell cycle markers of G1 phase measured by immunohistochemistry was induced in the striatal brain region. Furthermore, we detected an increase in the nuclear and also cytoplasmatic E2F-1 expression, suggesting that this protein could activate the apoptotic cascade in rat brain. Western blot analysis of post-mortem brain samples from patients also showed an increase in the expression of E2F-1 and cyclin D1 in comparison with control samples. These results indicate that cell cycle re-entry is activated in Huntington's disease and may contribute to the neurodegenerative process.

Topics: Animals; Apoptosis; Cell Cycle Proteins; Convulsants; Corpus Striatum; Cyclin D1; Disease Models, Animal; Dyskinesia, Drug-Induced; E2F1 Transcription Factor; Female; G1 Phase; Gene Expression Regulation; Genes, cdc; Humans; Huntington Disease; Male; Nerve Degeneration; Neurons; Neurotoxins; Nitro Compounds; Propionates; Rats; Rats, Sprague-Dawley; Up-Regulation

Age-dependent epithelial cell hyperplasia in the dorsal and lateral lobes of Brown Norway rats is analogous to benign prostatic hyperplasia in aging men. A major question is whether differential lobe-specific and age-dependent proliferation of cells, rather than cell survival, contributes to the hyperplasia. Although serum testosterone (T) levels decline in aged rats, active cell proliferation was detected as Ki67-positive cells in the dorsal and lateral lobes. We determined whether androgens differentially affect cell proliferation and cell-cycle regulatory proteins in the prostate lobes of young and aged rats. Castrated rats were treated with different doses of T to restore serum levels to those of intact young or aged rats. Rates of cell proliferation, measured by 5-bromodeoxyuridine labeling, peaked after 3-d T treatment in all lobes. 5-bromodeoxyuridine-labeling indices were higher in the dorsal and lateral lobes of aged than of young rats with equivalent serum T levels. No age-dependent difference was seen in the ventral lobe. Cell proliferation was marked by increased levels of cyclins D1 and E and cyclin-dependent kinases 4 and 6, decreased p27 and increased phosphorylation of Rb. Levels of cyclins D1 and E were higher in the dorsal and lateral lobes of intact and T-treated aged than young rats. Confocal immunofluorescent microscopy documented changes in cyclin-dependent kinase 4 and cyclin D1 subcellular localization. Cyclin D1 nuclear localization correlated with the time frame for cell proliferation. In conclusion, rates of cell proliferation and levels of cell-cycle regulatory proteins that control the G1/S transition exhibit lobe-specific and age-dependent differences in response to androgens.

Topics: Age Factors; Animals; Castration; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Epithelial Cells; G1 Phase; Gene Expression Regulation; Male; Organ Size; Organ Specificity; Prostate; Prostatic Hyperplasia; Rats; Rats, Inbred BN; S Phase; Testosterone; Tissue Distribution

The mechanism that drives the growth of some colonic adenomas towards malignancy, while permitting others to remain for decades in quiescence, remains unknown. Diets can alter the growth rate of intestinal tumours but it is still unknown whether diets are able to alter the molecular biology of these adenomas in a way that predicts further outcome. To address this issue we fed Min/+ mice with two diets known to lead to different adenoma outcomes: a high-fat control diet (n 15) or a high-fat inulin-enriched (10 % w/w) diet (n 13). To study the effect of diet on cell signalling during adenoma growth, the adenomas of each Min/+ mouse were divided into three size-categories, and the levels of beta-catenin, E-cadherin, cyclin D1 and matrix metalloproteinase-9, which are known to be involved in colon tumorigenesis, were determined. The growth-promoting inulin diet resulted in more large adenomas than the control feeding (P = 0.003) and doubled the total area of the adenomas (P = 0.008). The inulin diet increased the expression of nuclear beta-catenin (P = 0.004) and its target cyclin D1 (P = 0.017) as the adenomas increased in size from small to large, indicating the presence of an accelerated cancerous process. Neither phenomenon was seen in the control group during adenoma growth. Our results suggest that in addition to the number, size, and growth rate of adenomatous polyps, the signalling pattern of the adenomas should also be considered when evaluating preventive dietary strategies.

Topics: Adenomatous Polyposis Coli; Animals; beta Catenin; Cadherins; Cyclin D1; Diet; Dietary Fats; Disease Models, Animal; Disease Progression; Inulin; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Signal Transduction

We have shown previously that beta-catenin and cyclin D1 are up-regulated in cortical neurons from homozygous mice carrying the familial Alzheimer's disease (FAD) presenilin-1 M146V mutation in a knock-in model (PS1 KI(M146V) mice), leading to cell cycle-associated apoptosis. Here, we have aimed to determine (i) whether this phenotype is present in heterozygous PS1 KI(M146V) mice, which reflects more accurately the PS1 FAD condition in humans and (ii) whether Abeta(1-42), which is invariably present in the PS1 FAD brain and is thought to affect neuronal cell cycle kinetics, may contribute to the abnormal cell cycle/cell death phenotype seen in PS1 KI(M146V) mice. We demonstrate that cell cycle-linked apoptosis occurs in heterozygous PS1 KI(M146V) post-mitotic neurons. In addition, there is a significant Abeta-associated increase in cell cycle and cell death that is not further modified by the PS1 KI(M146V) mutation. Our results are consistent with a cell cycle-associated neurodegeneration model in the PS1 FAD brain in which the loss of PS1-dependent beta-catenin regulatory function is sufficient to commit susceptible neurons to an abortive cell cycle, and may act synergistically with the Abeta cytotoxic challenge present in the PS1 FAD brain to expand the neuronal population susceptible to cell cycle-driven apoptosis.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Analysis of Variance; Animals; Apoptosis; beta Catenin; Bromodeoxyuridine; Cell Cycle; Cells, Cultured; Cerebral Cortex; Cyclin D1; Disease Models, Animal; Embryo, Mammalian; Hippocampus; Humans; Mice; Mice, Transgenic; Mutation; Neurons; Peptide Fragments; Presenilin-1; Up-Regulation

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.

Topics: Adenoma; Animals; Azoxymethane; beta Catenin; Carcinogens; Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; ErbB Receptors; Gefitinib; Genes, ras; Male; Mice; Mice, Inbred A; Mutation; Quinazolines; Signal Transduction; Up-Regulation

Astrogliosis occurs in a variety of neuropathological disorders and injuries, and excessive astrogliosis can be devastating to the recovery of neuronal function. In this study, we asked whether reactive astrogliosis can be suppressed in the lesion area by cell cycle inhibition and thus have therapeutic benefits. Reactive astrogliosis induced in either cultured astrocytes by hypoxia or scratch injury, or in a middle cerebral artery occlusion (MCAO) ischemia model were combined to address this issue. In the cultured astrocytes, hypoxia induced a cell cycle activation that was associated with upregulation of the proliferating cell nuclear marker (PCNA). Significantly, the cell cycle inhibitor, olomoucine, inhibited hypoxia-induced cell cycle activation by arresting the cells at G1/S and G2/M in a dose-dependent manner and also reversed hypoxia-induced upregulation of PCNA. Also in the cultured astrocytes, scratch injury induced reactive astrogliosis, such as hypertrophy and an increase in BrdU(+) astrocytes, both of which were ameliorated by olomoucine. In the MCAO ischemia mouse model, dense reactive glial fibrillary acidic protein and PCNA immunoreactivity were evident at the boundary zone of focal cerebral ischemia at days 7 and 30 after MCAO. We found that intraperitoneal olomoucine administration significantly inhibited these astrogliosis-associated changes. To demonstrate further that cell cycle regulation impacts on astrogliosis, cyclin D1 gene knockout mice (cyclin D1(-/-)) were subjected to ischemia, and we found that the percentage of Ki67-positive astrocytes in these mice was markedly reduced in the boundary zone. The number of apoptotic neurons and the lesion volume in cyclin D1(-/-) mice also decreased as compared to cyclin D1(+/+) and cyclin D1(+/-) mice at days 3, 7, and 30 after local cerebral ischemia. Together, these in vitro and in vivo results strongly suggest that astrogliosis can be significantly affected by cell cycle inhibition, which therefore emerges as a promising intervention to attenuate reactive glia-related damage to neuronal function in brain pathology.

Topics: Analysis of Variance; Animals; Astrocytes; Brain Injuries; Brain Ischemia; Cell Cycle; Cell Death; Cells, Cultured; Cyclin D1; Disease Models, Animal; Enzyme Inhibitors; Gliosis; Ki-67 Antigen; Kinetin; Male; Mice; Mice, Knockout; Middle Cerebral Artery; Rats; Rats, Wistar; Statistics, Nonparametric; Wounds and Injuries

Interleukin (IL)-6 induced vascular smooth muscle cell (VSMC) motility in a dose-dependent manner. In addition, IL-6 stimulated tyrosine phosphorylation of gp130, resulting in the recruitment and activation of STAT-3. IL-6-induced VSMC motility was found to be dependent on activation of gp130/STAT-3 signaling. IL-6 also induced cyclin D1 expression in a time- and gp130/STAT-3-dependent manner in VSMCs. Suppression of cyclin D1 levels via the use of its small interfering RNA molecules inhibited IL-6-induced VSMC motility. Furthermore, balloon injury induced IL-6 expression both at mRNA and protein levels in rat carotid artery. Balloon injury also caused increased STAT-3 phosphorylation and cyclin D1 expression, leading to smooth muscle cell migration from the media to the intimal region. Blockade of gp130/STAT-3 signaling via adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury-induced STAT-3 phosphorylation and cyclin D1 induction, resulting in reduced smooth muscle cell migration from media to intima and decreased neointima formation. Together, these observations for the first time suggest that IL-6/gp130/STAT-3 signaling plays an important role in vascular wall remodeling particularly in the settings of postangioplasty and thereby in neointima formation.

Topics: Animals; Carotid Artery Injuries; Cell Movement; Cells, Cultured; Cyclin D1; Cytokine Receptor gp130; Disease Models, Animal; Gene Transfer Techniques; Interleukin-6; Muscle, Smooth, Vascular; Rats; Signal Transduction; STAT3 Transcription Factor; Transcription Factors; Tunica Intima

To investigate the mechanisms of androgen-independent growth in prostate cancer (PCa), we established two PCa cell lines, LN-REC4 and LNCaP-SF, from the androgen-dependent PCa cell line, LNCaP.. LN-Pre and LN-REC4 cells were generated from LNCaP tumors grown on intact and castrated severe combined immunodeficient (SCID) mouse, respectively. After we cultured LNCaP cells under a steroid-free conditions for 6 months in vitro, LNCaP-SF cells were established. To show the character of LN-REC4 and LNCaP-SF cells, androgen sensitivity was investigated through examination of growth rate, and prostate-specific antigen (PSA), androgen receptor (AR), p21, p27, and cyclin D1 expression were examined by reverse transcription-polymerase chain reaction (RT-PCR). Angiogenesis assay in vitro was carried out using conditioned medium. To examine the expression level of vascular endothelial growth factor (VEGF), RT-PCR and enzyme-linked immunosorbent assay were also done.. LN-REC4 cells proliferated better than LNCaP cells in castrated mice and did well irrespective of castration, although responsiveness for androgen of LN-REC4 cells attenuated less than that of LNCaP cells in vitro. LNCaP-SF cells in castrated mice proliferated more rapidly than in normal mice. The PSA expression in LNCaP-SF cells was still induced by androgen. Expression of AR, p21, p27 and cyclin D1 were not changed in LN-REC4 and LNCaP-SF cells. Angiogenesis assay showed that both cells stimulated angiogenesis. LN-REC4 induced VEGF more than LNCaP and LN-Pre cells. However, expression of VEGF per cell in LNCaP-SF was lower than LNCaP cells, suggesting that other factors might be involved in angiogenesis. These cell lines might be a useful tool for researching androgen-independent growth and treatments of recurred PCa.

Topics: Androgens; Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Vascular Endothelial Growth Factor A

Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)-mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protect TRAMP mice from developing prostate cancer.. Eight-week-old TRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules.. We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylation-defective CREB inhibited cyclin D1 transcriptional activity.. The current study shows that Nexrutine-mediated targeting of Akt/CREB-induced activation of cyclin D1 prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.

Topics: Adenocarcinoma; Animals; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Dietary Supplements; Disease Models, Animal; Male; Mice; Mice, Transgenic; Phosphorylation; Plant Extracts; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Member 25

We investigated the effects of 9trans,11trans (9t,11t)-conjugated linoleic acid (CLA) isomer on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in rats. Male F344 rats were given 2 weekly subcutaneous injections of AOM (20 mg/kg bw) to induce colonic ACF. They also were fed a diet containing either 0.01%, 0.1%, or 1% 9t,11t-CLA for 4 wk starting 1 wk before the first dosing of AOM. The group that received a diet supplemented with 9t,11t-CLA had a significantly lower number of ACF/colon in comparison to the AOM alone group in a dose-dependent manner up to 0.1%. Furthermore, treatment with 9t,11t-CLA induced apoptosis and suppressed cell proliferation activity in the non-lesional crypts. The downregulation of cyclooxygenase-2 and cyclin D1 and the activation of peroxisome proliferators activated receptor gamma were observed in the colonic mucosa of rats fed a diet supplemented with 9t,11t-CLA. Our findings thus provide some novel insight into the chemopreventive effect of 9t,11t-CLA against preinitiation as well as postinitiation stages of colorectal carcinogenesis.

Topics: Animals; Apoptosis; Azoxymethane; Biomarkers, Tumor; Cell Division; Colon; Colonic Neoplasms; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Dose-Response Relationship, Drug; Immunohistochemistry; Intestinal Mucosa; Linoleic Acids, Conjugated; Lipids; Male; PPAR gamma; Precancerous Conditions; Random Allocation; Rats; Rats, Inbred F344

Mouse models of hepatocellular carcinoma (HCC) simulate specific subgroups of human HCC. We investigated hepatocarcinogenesis in Mdr2-knockout (Mdr2-KO) mice, a model of inflammation-associated HCC, using gene expression profiling and immunohistochemical analyses. Gene expression profiling showed that although Mdr2-KO mice differ from other published murine HCC models, they share several important deregulated pathways and many coordinately differentially expressed genes with human HCC data sets. Analysis of genome positions of differentially expressed genes in liver tumors revealed a prolonged region of down-regulated genes on murine chromosome 8 in three of the six analyzed tumor samples. This region is syntenic to human chromosomal regions that are frequently deleted in human HCC and harbor multiple tumor suppressor genes. Real-time reverse transcription-PCR analysis of 16 tumor samples confirmed down-regulation of several tumor suppressors in most tumors. We show that in the aged Mdr2-KO mice, cyclin D1 nuclear level is increased in dysplastic hepatocytes that do not form nodules; however, it is decreased in most dysplastic nodules and in liver tumors. We found that this decrease is mostly at the protein, rather than the mRNA, level. These findings raise the question on the role of cyclin D1 at early stages of hepatocarcinogenesis in the Mdr2-KO HCC model. Furthermore, we show that most liver tumors in Mdr2-KO mice were characterized by the absence of beta-catenin activation. In conclusion, the Mdr2-KO mouse may serve as a model for beta-catenin-negative subgroup of human HCCs characterized by low nuclear cyclin D1 levels in tumor cells and by down-regulation of multiple tumor suppressor genes.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; beta Catenin; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Gene Expression Profiling; Genes, Tumor Suppressor; Hepatocytes; Liver Neoplasms; Mice; Mice, Knockout

The roles of the extracellular signal-regulated kinase (ERK) pathway in the expression of cyclin D1 and p27(KIP1), the phosphorylation of p27(KIP1), and proliferation activity were examined after retinal detachment.. Normal eyes and eyes at 15 min, 2 and 4 days after retinal detachment in C57Bl6 mice were examined by immunohistochemistry using anti-phosphorylated (p) ERK1/2, anti-cyclin D1, anti-p27(KIP1), anti-p27(KIP1) phosphorylated at serine 10 (S10-phospho-p27), and anti-proliferating cell nuclear antigen (PCNA) antibodies with or without treatment with a specific ERK inhibitor, PD98059. Mouse Müller cells were isolated and examined for alteration of p27(KIP1) and cyclin D1 after exposure of basic fibroblast growth factor (bFGF) with and without treatment of PD98059 using Western blotting.. In the normal retina, nuclear immunoreactivity for p27(KIP1), but not S10-phospho-p27 or pERK1/2, was observed in the middle sublayer of the inner nuclear layer (INL), where Müller glial cells are situated. At 15 min after the retinal detachment, p27(KIP1), S10-phospho-p27 and pERK1/2-positive nuclei were noted in the INL, whereas immunoreactivity for pERK1/2 or S10-phospho-p27 was not observed after treatment with PD98095. Cyclin D1 was induced in the INL 2 days after the retinal detachment, and the induction was inhibited by PD98059. At 4 days after the detachment, p27(KIP1) immunoreactivity was not observed, and cyclin D1 and PCNA were expressed. The disappearance of p27(KIP1) was suppressed, whereas expression of cyclin D1 and PCNA was not observed in mice treated with PD98059. Exposure of bFGF relatively decreased the expression level of p27(KIP1) and increased the level of cyclin D1 in mouse Müller cells, compared with control level. Induction of cyclin D1 and decrease in p27(KIP1) were inhibited with treatment of PD98059.. Phosphorylation of ERK and expression of p27(KIP1) and cyclin D1 are involved in the proliferation of Müller cells after retinal detachment.

Topics: Animals; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Cell Culture Techniques; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Flavonoids; Fluorescent Antibody Technique, Indirect; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroglia; Phosphorylation; Proliferating Cell Nuclear Antigen; Retinal Detachment; Serine

Chronic ethanol consumption inhibits liver regeneration. We examined the effects of chronic ethanol consumption on two mitogen-activated protein kinases in relation to induction of cell cycle proteins after partial hepatectomy (PH).. Male Wistar rats were ethanol-fed (EF) or pair-fed (PF) for 16 weeks before PH. Hepatic activation of extracellular signal regulated kinase (ERK)1/2, p38 kinase and expression of cyclinD1, cyclin-dependent kinase-4 (cdk4) and proliferating cell nuclear antigen (PCNA) were studied.. In PF rats, PH-induced p38 activation was evident at 2h and was maximal at 12h. There was a close temporal relationship between p38 activation, cyclin D1 and PCNA expression. Alcohol exposure reduced p38 activation, cyclin D1 and PCNA, each by approximately 50%. ERK1/2 activation occurred during the first 2h post-PH in both EF and PF rats, and there was no later increase in PF rats. In vivo inhibition of p38 suppressed PCNA expression whereas the effect of ERK1/2 inhibition was inconsistent.. p38 kinase activation is linked temporally with cyclin D1 expression after PH and appears to exert cell cycle control in the adult liver. p38 signaling also appears to be a target for the inhibitory effect of chronic alcohol on liver regeneration.

Topics: Alcoholism; Animals; Blotting, Western; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Enzyme Activation; Ethanol; Gene Expression; Hepatectomy; Liver; Liver Regeneration; Male; p38 Mitogen-Activated Protein Kinases; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Patients exhibiting hepatic iron overload frequently develop hepatocellular carcinoma. An impaired expression of hepatic genes could be involved in this phenomenon. Our aim was to identify, during iron overload, hepatic genes involved in cell cycle which are misregulated.. Mouse iron overload was obtained by carbonyl-iron supplementation or iron-dextran injection. As expected, liver iron overload was associated to both hepatomegaly and hepatocyte polyploidisation. Hepatic gene expression was investigated using macroarray hybridizations. Cyclin D1 mRNA was the only gene whose expression increased in both models. Its overexpression was confirmed by real-time quantitative PCR. Immunobloting analysis demonstrated a strong increase of Cyclin D1 protein expression in iron-overloaded hepatocytes. This overexpression was correlated with early abnormalities in their cell cycle progression judged, in vitro, on DNA synthesis and mitotic index increase.. Our data demonstrates that Cyclin D1, a protein involved in G1-phase of cell cycle, is overexpressed in the iron-overloaded liver. This iron-induced expression of Cyclin D1 may contribute to development of cell cycle abnormalities, suggesting a role of Cyclin D1 in iron-related hepatocarcinogenesis.

Topics: Animals; Blotting, Western; Cell Cycle; Cells, Cultured; Cyclin D1; Disease Models, Animal; Disease Progression; Gene Expression; Hepatocytes; Iron Overload; Liver; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Mutations in Ki-ras occur in approximately 30-50% of patients with adenocarcinoma (AC) of the lung. We previously reported the development of a bitransgenic mouse model that expressed the human Ki-ras(G12C) allele in a lung-specific, tetracycline-inducible manner and gave rise to benign lung tumors. In the current study, these benign tumors, which represent relatively early lesions in neoplastic progression, were analyzed for molecular alterations secondary to mutant Ki-ras expression to determine the gene(s) that contribute to adenoma (AD) development. Tumors were removed following doxycycline (DOX) treatment for 9 and 12 mo and examined for alterations in cell-cycle regulatory genes. Quantification of mRNA expression for cyclin D1, retinoblastoma, p16(Ink4a), p19(Arf), and survivin was carried out by real-time PCR. All of the tumors examined exhibited a mean reduction of approximately fivefold for the retinoblastoma gene (P < 0.02). Increased expression of both p19(Arf) and survivin were detected in a majority of the tumors examined (P < 0.01 and 0.001, respectively), but no change in cyclin D1 RNA expression was observed. A subset of the lung tumors (8/28) displayed reduced levels of p16(Ink4a) expression (P = 0.02). Immunohistochemical analysis confirmed the upregulation of p19(Arf) and survivin in all 10 of the lung tumors examined. However, increased staining for cyclin D1 was observed in the tumor tissue. In addition, increased levels of activated p53 were found in lung tumor tissues stained with an anti-phospho-p53 antibody, while an absence of staining was observed with an anti-phospho-pRb antibody in both normal control and tumor tissue. Analysis of the methylation status of p16(Ink4a) by methylation-specific PCR (MSP) demonstrated that seven of eight tumors exhibiting decreased expression of p16(Ink4a) had at least partial methylation of the promoter region. Single stranded conformational polymorphism (SSCP) analysis demonstrated that neither exons 1 or 2 of p16(Ink4a) nor exons 5-8 of p53 exhibited mutations. These data thus identify alterations in specific genes and pathways that combine with the mutation in Ki-ras to promote the formation of benign lung tumors and suggest potential targets for the development of novel chemotherapeutic and chemopreventive agents during the early stages of lung tumor progression.

Topics: Animals; Base Sequence; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; DNA Primers; DNA, Neoplasm; Genes, ras; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Polymorphism, Single Nucleotide; ras Proteins; Retinoblastoma Protein

Topics: Age Factors; Animals; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Flow Cytometry; Genetic Predisposition to Disease; Injections, Intraperitoneal; Lymphoma, Mantle-Cell; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Pilot Projects; Sensitivity and Specificity; Terpenes

Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice.. A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided.. Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression.. Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinogens; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Fibroblast Growth Factor 2; Immunohistochemistry; Inflammation; Lung Neoplasms; Mice; Microcirculation; Neovascularization, Pathologic; Nitric Oxide Synthase Type II; Platelet Endothelial Cell Adhesion Molecule-1; Proliferating Cell Nuclear Antigen; Silybin; Silymarin; Urethane; Vascular Endothelial Growth Factor A

Small-for-size liver grafts have decreased survival compared to full-size grafts. This study investigated mechanisms of suppression of liver regeneration in small-for-size grafts.. Rat liver explants were reduced in size to 50% and implanted into recipients of different body weights, resulting in graft weight/standard liver weights of approximately 50% (half-size) and approximately 25% (quarter-size).. Hepatic cellular 5-bromo-2'-deoxyuridine (BrdU) incorporation increased from 0.2% after sham operation to 2%, 18%, and 1.2% in full-size, half-size, and quarter-size grafts, respectively. Graft weight did not increase in full- and quarter-size grafts but increased 40% in half-size grafts. By contrast, apoptosis remained low (< or =0.7%) and stem cells did not increase in all conditions. Phospho-c-Jun increased 27-fold in half-size grafts but only sevenfold in quarter-size grafts. Activating protein-1 activation increased 14-fold in half-size grafts but only fivefold in quarter-size grafts. Cyclin D1 (CyD1), which was barely detectable in full- and quarter-size grafts, increased 8.3-fold in half-size grafts. Adenosine 5'-triphosphate (ATP) per gram tissue decreased 70% in quarter-size grafts. Treatment of quarter-size grafts with radical scavenging C. sinenesis polyphenols (20 microg/ml) increased BrdU labeling and weight gain to 35% and 56%, respectively, reversed inhibition of CyD1 expression, c-Jun phosphorylation, and AP-1 activation in quarter-size grafts compared to half-size grafts, and restored ATP levels to 75%.. Liver regeneration is stimulated in half-size grafts but suppressed in quarter-size grafts. Defective liver regeneration in small grafts is associated with an inhibition of the c-Jun N-terminal kinase/c-Jun and CyD1 pathways and compromised energy production.

Topics: Animals; Cyclin D1; Disease Models, Animal; Energy Metabolism; Flavonoids; Hepatocyte Growth Factor; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Liver Regeneration; Liver Transplantation; Male; Phenols; Polyphenols; Portal System; Rats; Rats, Inbred Lew; Stem Cells; Tumor Necrosis Factor-alpha

Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth.. We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation.. The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug.. Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Endothelial Cells; Female; Humans; Mice; Mice, Nude; Neovascularization, Pathologic; Oligodeoxyribonucleotides, Antisense; RNA, Small Interfering; Structure-Activity Relationship; Transplantation, Heterologous; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be approximately 2 x 10(3) cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse.

Topics: Animals; Antigens, Polyomavirus Transforming; Avian Sarcoma Viruses; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Models, Animal; Epithelial Cells; Genetic Vectors; Mammary Glands, Animal; Mice; Mice, Transgenic; Oncogenic Viruses; Proto-Oncogene Proteins c-myc; Survival Rate

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF-kappaB) is a common characteristic of MM cells. In our study we successfully target NF-kappaB with a novel NF-kappaB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF-kappaB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF-kappaB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF-kappaB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl-XL and c-FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ-mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/gammac(null) (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF-kappaB, DHMEQ is a unique compound that blocks the translocation of NF-kappaB p65 into the nucleus and selectively targets NF-kappaB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Benzamides; Calcium-Binding Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cyclin D; Cyclin D1; Cyclins; Cyclohexanones; Disease Models, Animal; Down-Regulation; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Nerve Tissue Proteins; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Synaptotagmin I; Synaptotagmins; Translocation, Genetic; Vascular Endothelial Growth Factor A

The mechanisms through which beta-catenin signaling is inhibited during colorectal cancer chemoprevention by nonsteroidal anti-inflammatory agents is incompletely understood. We report that nabumetone decreased uninvolved intestinal mucosal beta-catenin levels in the MIN mouse with a concomitant increase in glycogen synthase kinase (GSK)-3beta levels, an enzyme that targets beta-catenin for destruction. However, in the azoxymethane-treated rat, where beta-catenin is frequently rendered GSK-3beta-insensitive, nabumetone failed to alter beta-catenin levels but did decrease beta-catenin nuclear localization and transcriptional activity as gauged by cyclin D1. In conclusion, we demonstrate that the differential mechanisms for beta-catenin suppression may be determined, at least partly, by GSK-3beta.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Azoxymethane; beta Catenin; Blotting, Western; Butanones; Cadherins; Carcinogens; Colonic Neoplasms; Cyclin D1; Cytoskeletal Proteins; Disease Models, Animal; Glycogen Synthase Kinase 3; Immunohistochemistry; Male; Mice; Nabumetone; Rats; Signal Transduction; Trans-Activators

This study aims to identify a novel molecule that may contribute to hepatocarcinogenesis in a rat orthotopic hepatocellular carcinoma model. The hepatocellular carcinoma model was generated by injection of tumor cells into the left lobe of the liver. Proteomic approaches, including ProteinChip and two-dimensional electrophoresis, were used to identify proteins from serially collected rat serum samples. By both ProteinChip and two-dimensional electrophoresis techniques, the level of a 27-kDa protein was found to be augmented in serum samples during tumor development, decreased after left lobectomy, and reincreased at the time of tumor recurrence. The protein was identified to be brain-derived neurotrophic factor (BDNF). By using specific primers and monoclonal antibody, the expression pattern of BDNF was confirmed in tumor tissue but not in the adjacent nontumorous liver tissue. In addition, the truncated isoform of BDNF receptor-tyrosine protein kinase receptor B was only found in tumor tissue. An in vitro study showed that exogenous BDNF could induce tumor cell proliferation predominantly in relatively small numbers of inoculated cells. Administration of BDNF to tumor cell lines induced significantly increased expression of heat shock protein 90 (Hsp90) and cyclin D1, and blocking the activity of Hsp90 could reverse the up-regulation of cyclin D1 induced by BDNF. The present study revealed that BDNF and its receptor were uniquely expressed in tumor tissue and cell lines of hepatocellular carcimona but not in nontumorous liver tissue and normal cell line. BDNF could stimulate tumor cell proliferation in a Hsp90-dependent manner.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Division; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; HSP90 Heat-Shock Proteins; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Protein Array Analysis; Rats; Rats, Inbred BUF; Receptor, trkB; Reverse Transcriptase Polymerase Chain Reaction

Squamous cancers of the oral cavity and esophagus are common worldwide. A number of environmental factors as well as genetic alterations have been identified. However, the specific combination of genetic events and their interplay with environmental carcinogens are largely un-known. Furthermore, no good animal model existed to study the molecular changes important in the induction and progression of the disease. Here we summarize the efforts made to establish a mouse model of oral-esophageal carcinogenesis. Cyclin D1 overexpressing(L2D1+) mice were generated using an EBV promoter to specifically target the oral cavity and the esophageal squamous epithelium. Besides analyzing different environmental factors, such as nitrosamines and zinc deficiency, cyclin D1 transgenic mice were crossbred with p53-deficient mice. While L2D1+ mice exhibited a phenotype of dysplasia, different combinations of mice result-ed in invasive oral-esophageal cancer. This mouse model provides a well-defined and reproducible model of oral-esophageal cancer that should be useful for testing chemopreventive, diagnostic, and therapeutic strategies.

Topics: Animals; Carcinogenicity Tests; Cyclin D1; Disease Models, Animal; Esophageal Neoplasms; Genetic Engineering; Humans; Mice; Mice, Transgenic; Mouth Neoplasms; Transfection

Expression of the p27kip1 cell cycle inhibitor is downregulated in a wide range of carcinomas, yet it is rarely inactivated completely. Our recent studies of a mouse model of prostate carcinogenesis have revealed that cancer progression is enhanced by a two-fold reduction in p27kip1 gene dosage, but is unexpectedly inhibited by further decrease in p27kip1 activity. This paradoxical finding may explain the unusual features of p27kip1 downregulation in human cancer, and also suggests a potential route for therapeutic intervention.

Topics: Animals; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; Down-Regulation; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Transgenic; Mutation; Phenotype; Prostatic Neoplasms

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Topics: Amides; Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Blotting, Northern; Blotting, Western; Brain Injuries; Bromodeoxyuridine; Butadienes; Cell Count; Cell Movement; Cell Proliferation; Cells, Cultured; Coculture Techniques; Colforsin; Cyclin D1; Disease Models, Animal; Drug Interactions; Enzyme Inhibitors; Functional Laterality; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; MAP Kinase Kinase Kinases; Mice; Mice, Knockout; Microglia; Nitriles; Oligopeptides; Pyridines; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombin; Time Factors

The mechanisms responsible for impaired regenerative ability after hepatic resection observed in chronic liver disease are not fully understood. We have examined the relationships between an altered expression of cell cycle-related proteins in regenerating liver after partial hepatectomy and the impaired regenerative process observed in fibrotic and cirrhotic rats.. We performed 70% partial hepatectomy in both control and porcine serum-induced fibrotic rats, and 45% partial hepatectomy in thioacetamide-induced cirrhotic rats because of the high mortality associated with 70% partial hepatectomy. Liver regeneration was monitored by proliferating cell nuclear antigen labeling index and the expression of G1 regulatory cell cycle-related proteins was determined by immunoblot analysis.. Compared with controls, hepatocyte DNA synthesis, and induction of cyclin D1 and p21(CIP1) proteins were delayed but not suppressed in porcine serum-induced fibrotic rats and markedly inhibited in thioacetamide-induced cirrhotic rats. p27(KIP1) protein levels were unaffected by partial hepatectomy and did not differ among all three groups.. Two distinct rat models of liver fibrosis and cirrhosis showed markedly different proliferative responses after partial hepatectomy. The delay or failure of cyclin D1 induction, but not the increase of p21(CIP1) or p27(KIP1) might be responsible for their impaired liver regeneration.

Topics: Animals; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; DNA; Hepatectomy; Liver; Liver Cirrhosis; Liver Regeneration; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Inbred F344; Serum; Thioacetamide

Previously we reported that an ordinarily nonlethal dose of thioacetamide (300 mg/kg) causes liver failure and 90% mortality in type 1 diabetic rats, primarily because of inhibited tissue repair. On the other hand, the diabetic rats receiving 30 mg thioacetamide/kg exhibited equal initial liver injury and delayed tissue repair compared to nondiabetic rats receiving 300 mg thioacetamide/kg, resulting in a delay in recovery from that liver injury and survival. These data indicate that impaired tissue repair in diabetes is a dose-dependent function of diabetes. The objective of the present study was to test the hypothesis that disrupted nuclear factor-kappaB (NF-kappaB)-regulated cyclin D1 signaling may explain dose-dependent impaired tissue repair in the thioacetamide-treated diabetic rats. Administration of 300 mg thioacetamide/kg to nondiabetic rats led to sustained NF-kappaB-regulated cyclin D1 signaling, explaining prompt compensatory tissue repair and survival. For the first time, we report that NF-kappaB-DNA binding is dependent on the dose of thioacetamide in the liver tissue of the diabetic rats. Administration of 300 mg thioacetamide/kg to diabetic rats inhibited NF-kappaB-regulated cyclin D1 signaling, explaining inhibited tissue repair, liver failure and death, whereas remarkably higher NF-kappaB-DNA binding but transient down regulation of cyclin D1 expression explains delayed tissue repair in the diabetic rats receiving 30 mg thioacetamide/kg. These data suggest that dose-dependent NF-kappaB-regulated cyclin D1 signaling explains inhibited versus delayed tissue repair observed in the diabetic rats receiving 300 and 30 mg thioacetamide/kg, respectively.

Topics: Alanine Transaminase; Analysis of Variance; Animals; Cyclin D1; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; Dose-Response Relationship, Drug; I-kappa B Kinase; I-kappa B Proteins; Liver; Liver Regeneration; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Streptozocin; Thioacetamide; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages.. To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days.. During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation.. TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.

Topics: Animals; Cell Proliferation; Cleft Lip; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation, Developmental; Mesoderm; Mice; Mice, Mutant Strains; Neovascularization, Physiologic; Organ Culture Techniques; Platelet Endothelial Cell Adhesion Molecule-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta3; Vascular Endothelial Growth Factor Receptor-2

Canine mammary gland tumor (MGT) is the commonest tumor in female dogs and a good animal model of human breast cancer. A group of newly identified genes encoding secreted frizzled-related proteins (SFRP) have been implicated in apoptosis regulation and tumorigenesis. Canine mammary tissues from 50 spontaneous MGTs and 10 normal mammary glands (MGs) were obtained from surgically excised specimens and analyzed for expression of SFRP2, beta-catenin, and cyclin D1. By RT-PCR and in situ hybridization, SFRP2 gene was found abundantly expressed in neoplastic mammary tissues but not in normal mammary tissues, suggesting that SFRP2 may contribute as a tumor marker in canine MGTs. By immunohistochemical staining, the immunoreactivity of the SFRP2 protein was detected in more diverse areas than SFRP2 mRNA expression, including nuclei or/and cytoplasm and extracellular matrix of the tumor. In tumor masses, beta-catenin lost its tight association with the membrane and diffused into the nucleus. The expression of beta-catenin (79.4% positive) and cyclin D1 (71.4% positive) was also increased in MGTs. In the course of tumor progression, SFRP2 mRNA ( p < 0.05) and beta-catenin protein ( p < 0.01) steadily increased but not in cyclin D1. The level of SFRP2 was linearly correlated with its downstream target beta-catenin ( p < 0.05), but not correlated with cyclin D1 ( p < 0.5). As revealed in this study, the exclusive overexpression of SFRP2 in canine MGTs suggests that SFRP2 is a potential candidate gene for further investigation of mammary tumorigenesis and complex etiology of the canine model of mammary neoplasms.

Topics: Animals; beta Catenin; Biomarkers, Tumor; Cyclin D1; Cytoskeletal Proteins; Disease Models, Animal; Dogs; Female; Gene Expression Regulation, Neoplastic; Immunohistochemistry; In Situ Hybridization; Mammary Glands, Animal; Mammary Neoplasms, Animal; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators

Certain cells of the human retina are extremely sensitive to loss of function of the retinoblastoma tumor suppressor gene RB. Retinoblastomas develop early in life and at high frequency in individuals heterozygous for a germ-line RB mutation, and sporadic retinoblastomas invariably have somatic mutation in the RB gene. In contrast, retinoblastomas do not develop in Rb+/- mice. Although retinoblastoma is thought to have developmental origins, the function of Rb in retinal development has not been fully characterized. Here we studied the role of Rb in normal retinal development and in retinoblastoma using conditional Rb mutations in the mouse. In late embryogenesis, Rb-deficient retinas exhibited ectopic S-phase and high levels of p53-independent apoptosis, particularly in the differentiating retinal ganglion cell layer. During postnatal retinal development, loss of Rb led to more widespread retinal apoptosis, and adults showed loss of photoreceptors and bipolar cells. Conditional Rb mutation in the retina did not result in retinoblastoma formation even in a p53-mutant background. However, on a p107- or p130-deficient background, Rb mutation in the retina caused retinal dysplasia or retinoblastoma.

Topics: Animals; Apoptosis; Blotting, Western; Crosses, Genetic; Cyclin D1; Disease Models, Animal; Genes, Retinoblastoma; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mutation; Nuclear Proteins; Proteins; Retina; Retinal Ganglion Cells; Retinoblastoma; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and implicated in oncogenesis. However, the role of Pin1 in oncogenesis in vivo is not known. We have shown that Pin1 ablation is highly effective in preventing oncogenic Neu or Ras from inducing cyclin D1 and breast cancer in mice, although it neither affects transgene expression nor mammary gland development. Moreover, we have developed an ex vivo assay to uncover that a significant fraction of primary mammary epithelial cells from Neu or Ras mice display various malignant properties long before they develop tumors in vivo. Importantly, these early transformed properties are effectively suppressed by Pin1 deletion, which can be fully rescued by overexpression of cyclin D1. Thus, Pin1 is essential for tumorigenesis and is an attractive anticancer target. Our ex vivo assay can be used to study early events of breast cancer development in genetically predisposed mice.

Topics: Animals; Breast Neoplasms; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Male; Mammary Glands, Animal; Mice; Mice, Nude; Mice, Transgenic; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Phenotype; Proto-Oncogene Proteins c-myc; ras Proteins; Receptor, ErbB-2; Sexual Abstinence

Lactoferrin, an iron-binding glycoprotein, exhibits suppressive effects on development of azoxymethane (AOM)-induced tumors in the rat colon, but the mechanisms are largely unknown. In this study, we investigated the effect of lactoferrin on the gene expression of 10 apoptosis-related molecules in colon mucosa of AOM-treated rats during early and late stages of colon carcinogenesis by reverse transcription PCR. Here we document that a death-inducing receptor, Fas, and a pro-apoptotic Bcl-2 family member, Bid, are increased in the colon mucosa in proportion to decreases in AOM-induced aberrant crypt foci by lactoferrin. Similarly, increased expression of the pro-apoptotic Bcl-2 family member, Bax, was also observed in AOM-induced tumors in rats fed by lactoferrin. These results indicate that Fas and pro-apoptotic Bcl-2 members participate in the lactoferrin action and may contribute to suppressive effects on tumor development in the rat colon.

Topics: Administration, Oral; Animals; Apoptosis; Azoxymethane; Carcinogens; Cell Transformation, Neoplastic; Chemoprevention; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Fas Ligand Protein; Gene Expression Regulation; Lactoferrin; Male; Membrane Glycoproteins; Rats; Rats, Inbred F344; Up-Regulation

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of neuronal cell death in ischemia and other diseases, but the mechanism by which COX-2 exacerbates cell death is unknown. COX-2 activity is known to induce expression of cyclin D1 in neoplastic cells, and cyclin D1 expression can induce cell death in postmitotic neurons. In the present study, the role of COX-2 and cyclin D1 in neuronal cell death induced by anoxia and ischemia was examined. Treatment with the COX-2 specific inhibitor (NS 398 25 microM) and cyclin D1 inhibitor (flavopiridol 1 microM) increased neuronal survival and inhibited DNA fragmentation after anoxia. NS-398 suppressed anoxia-induced expression of cyclin D1. Flavopiridol inhibited the anoxia-induced increased expression of cyclin D1, but had no effect on COX-2 expression. Treatment with the selective COX-2 inhibitor, SC58125, had no affect on COX-2 expression but partially suppressed cyclin D1 expression in the cortex following middle cerebral artery occlusion in vivo. These results show that COX-2 activity is required for cyclin D1 expression after ischemia in vivo and anoxia in vitro. These data provide support for the hypothesis that cyclin D1 expression is an important mechanism by which COX-2 activity exacerbates ischemic neuronal death.

Topics: Animals; Cell Survival; Cells, Cultured; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Hypoxia-Ischemia, Brain; Infarction, Middle Cerebral Artery; Neurons; Neuroprotective Agents; Nitrobenzenes; Piperidines; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Rats, Sprague-Dawley; Sulfonamides

In human prostate cancer, the frequent down-regulation of p27(kip1) protein expression is correlated with poor clinical outcome, yet p27(kip1) rarely undergoes mutational inactivation. Here, we investigate the consequences of reducing or eliminating p27(kip1) function for prostate carcinogenesis in the context of a mouse modeling lacking the Nkx3.1 homeobox gene and the Pten tumor suppressor. Unexpectedly, we find that triple mutant mice heterozygous for a p27(kip1) null allele (Nkx3.1(+/- or -/-); Pten(+/-); p27(+/-)) display enhanced prostate carcinogenesis, whereas mice that are homozygous null for p27(kip1) (Nkx3.1(+/- or -/-); Pten(+/-); p27(-/-)) show inhibition of cancer progression. Expression profiling reveals that Cyclin D1 is highly up-regulated in compound p27(kip1) heterozygotes, but is down-regulated in the compound p27(kip1) homozygous mutants. Using RNA interference in prostate cancer cell lines with distinct p27(kip1) gene doses, we show that prostate tumorigenicity depends on levels of p27(kip1) and that the consequences of p27(kip1) gene dosage can be attributed, in part, to altered levels of Cyclin D1. Our findings suggest that p27(kip1) possesses dosage-sensitive positive as well as negative modulatory roles in prostate cancer progression.

Topics: Animals; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; Gene Dosage; Gene Expression Regulation, Neoplastic; Genotype; Homeodomain Proteins; Male; Mice; Mice, Knockout; Mice, Mutant Strains; Prostatic Neoplasms; Protein Tyrosine Phosphatases; PTEN Phosphohydrolase; Transcription Factors; Tumor Suppressor Proteins

Insufficient cell number is a primary cause of failed retinal development in the Chx10 mutant mouse. To determine if Chx10 regulates cell number by antagonizing p27(Kip1) activity, we generated Chx10, p27(Kip1) double null mice. The severe hypocellular defect in Chx10 single null mice is alleviated in the double null, and while Chx10-null retinas lack lamination, double null retinas have near normal lamination. Bipolar cells are absent in the double null retina, a defect that is attributable to a requirement for Chx10 that is independent of p27(Kip1). We find that p27(Kip1) is abnormally present in progenitors of Chx10-null retinas, and that its ectopic localization is responsible for a significant amount of the proliferation defect in this microphthalmia model system. mRNA and protein expression patterns in these mice and in cyclin D1-null mice suggest that Chx10 influences p27(Kip1) at a post-transcriptional level, through a mechanism that is largely dependent on cyclin D1. This is the first report of rescue of retinal proliferation in a microphthalmia model by deletion of a cell cycle regulatory gene.

Topics: Animals; Cell Count; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; G1 Phase; Gene Expression Regulation, Developmental; Genes, bcl-1; Homeodomain Proteins; Mice; Mice, Knockout; Microphthalmos; Phenotype; Retina; RNA Processing, Post-Transcriptional; RNA, Messenger; Stem Cells; Transcription Factors; Tumor Suppressor Proteins

Apoptosis may contribute to the myocardial dysfunction associated with heart failure (HF). Activation of the p38 MAPK cascade can induce apoptosis in non-cardiac cells through increased expression of Fas-L, or through decreased expression of cyclin D(1).. We tested the hypothesis that hypoxia (HX), angiotensin-II (A-II) and norepinephrine (NEPI) can mediate apoptosis by activating p38 MAPK, and thus initiating stimulus specific changes in Fas-L and cyclin D(1) expression in failing cardiomyocytes.. Cardiomyocytes isolated from ten dogs with HF induced by coronary microembolizations were subjected to HX or A-II or NEPI with and without a p38 MAPK inhibitor (SB 203580). TUNEL staining for DNA fragmentation and Western blots for p38 MAPK, Fas-L and cyclin D(1) detection were performed. HX-induced apoptosis was associated with increased Fas-L expression, A-II-induced apoptosis was associated with increased Fas-L and decreased cyclin D(1) expression, and NEPI-induced apoptosis was associated with decreased cyclin D(1) expression. Inhibition of p38 MAPK activity attenuated stress-induced apoptosis in all experiments and reversed changes in Fas-L and cyclin D(1) expression.. HX, A-II and NEPI mediate apoptosis in failing cardiomyocytes via different effects on Fas-L and cyclin D(1) expression. Inhibition of p38 MAPK reversed these effects, suggesting that apoptosis induced by HX, A-II and NEPI involves activation of p38 MAPK upstream from Fas-L and cyclin D(1).

Topics: Angiotensin II; Animals; Apoptosis; Cyclin D1; Disease Models, Animal; Dogs; Fas Ligand Protein; Follow-Up Studies; Heart Failure; Hypoxia; Incidence; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Models, Cardiovascular; Myocytes, Cardiac; Norepinephrine; p38 Mitogen-Activated Protein Kinases

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Topics: Animals; Apoptosis; Cartilage; Cell Adhesion; Cell Differentiation; Cell Division; Chondrocytes; Chondrogenesis; Collagen Type II; Cyclin D1; Disease Models, Animal; Down-Regulation; Dwarfism; Exostoses, Multiple Hereditary; Female; Gene Expression Regulation, Developmental; Growth Plate; Integrases; Integrins; Male; Mice; Mice, Knockout; Osteogenesis; Protein Serine-Threonine Kinases; Transgenes; Viral Proteins

UVB radiation is a complete carcinogen able to initiate, promote, and progress keratinocyte cells toward carcinogenesis. Exposure to UVB leads to the propagation of a number of signal transduction pathways resulting in increased DNA binding of transcription factors, including activator protein-1 (AP-1), and subsequent gene expression. To test the hypothesis that AP-1 activation plays a role in the promotion of UVB-induced skin tumors, a dominant negative c-jun (TAM67) mutant transgene was expressed in the epidermis of SKH-1 hairless mice and bred with mice expressing an AP-1 luciferase reporter gene. Single UVB exposure experiments showed a significant decrease in AP-1 activity, as measured by luciferase levels, in mice expressing TAM67 72 h postexposure. Transgenic and nontransgenic littermates were placed into a chronic UVB exposure experiment, three exposures per week for 25 weeks. Expression of TAM67 reduced the number of tumors per mouse by 58% and tumor sizes were 79% smaller than the tumors present in the nontransgenic study group. These tumors were histologically identified as squamous cell carcinomas. TAM67 had no effect on UVB-induced hyperplasia because comparable epidermal thickening was observed in both study groups over a 5-day period post-UVB exposure. Immunohistochemical analysis showed a reduction in the number of cyclin D(1)-expressing cells in squamous cell carcinoma samples removed from the TAM67 study group. These data show that TAM67 can inhibit UVB-induced squamous cell carcinoma formation, suggesting that AP-1 is a good candidate target for the development of new chemoprevention strategies to prevent sunlight-induced skin cancers.

Topics: Animals; Carcinoma, Squamous Cell; Cyclin D1; Disease Models, Animal; Genes, jun; Hyperplasia; Mice; Mice, Inbred Strains; Mice, Transgenic; Neoplasms, Radiation-Induced; Peptide Fragments; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Time Factors; Transcription Factor AP-1; Transgenes; Ultraviolet Rays

Deregulation of beta-catenin activity is an important step in the development of colorectal cancers. One consequence of this is transcriptional activation of cyclin D1, an oncogene known to be overexpressed in colorectal cancers. We tested the hypothesis that cyclin D1 gene activation is important for intestinal tumorigenesis. Multiple intestinal neoplasia mice (a model for human familial adenomatous polyposis) were crossed with cyclin D1 knockout (Ccnd1(-/-)) mice. Despite the absence of cyclin D1, intestinal tumors still developed. However, Ccnd1(-/-) multiple intestinal neoplasia mice developed significantly fewer tumors than Ccnd1(+/-) or Ccnd1(+/+) mice (P = 0.003). We conclude that cyclin D1 is not essential for intestinal tumorigenesis, but it may act as a modifier gene.

Topics: Animals; beta Catenin; Cyclin D1; Cytoskeletal Proteins; Disease Models, Animal; Female; Gene Expression Regulation; Intestinal Neoplasms; Male; Mice; Mice, Knockout; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Transcriptional Activation

The neu (c-erbB-2, HER2) proto-oncogene encodes a receptor tyrosine kinase that is a member of an important growth factor receptor family which includes the epidermal growth factor receptor (EGFR, ErbB1), ErbB3 and ErbB4. The neu is found over-expressed in 20-30% of human breast tumors. The c-erbB-2 is sufficient for the induction of mammary tumorigenesis in transgenic mice and the pathology of these mammary tumors strongly resembles human breast cancer. Murine transgenic models engineered to recapitulate human breast cancer provide an excellent and straightforward approach to dissect the molecular mechanisms governing the onset and progression of this disease. The molecular mechanisms by which ErbB-2 transforms cells involves direct effects on components of the cell-cycle regulatory apparatus. Recent studies have demonstrated a key role for components of the cell-cycle, in particular cyclin D1 and p27Kip1 (p27) in the onset and progression of ErbB-2-induced murine mammary tumorigenesis. Such studies have provided further impetus to therapeutics targeting these cell-cycle proteins.

Topics: Animals; Carcinogenicity Tests; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Female; Genes, Tumor Suppressor; Mammary Neoplasms, Animal; Mice; Mice, Knockout; Proto-Oncogene Mas; Receptor, ErbB-2; Tumor Suppressor Proteins

Apc (adenomatous polyposis coli) encodes a tumour suppressor gene that is mutated in the majority of colorectal cancers. Recent evidence has also implicated Apc mutations in the aetiology of breast tumours. Apc is a component of the canonical Wnt signal transduction pathway, of which one target is Tcf-1. In the mouse, mutations of both Apc and Tcf-1 have been implicated in mammary tumorigenesis. We have conditionally inactivated Apc in both the presence and absence of Tcf-1 to examine the function of these genes in both normal and neoplastic development. Mice harbouring mammary-specific mutations in Apc show markedly delayed development of the mammary ductal network. During lactation, the mice develop multiple metaplastic growths which, surprisingly, do not spontaneously progress to neoplasia up to a year following their induction. However, additional deficiency of Tcf-1 completely blocks normal mammary development and results in acanthoma.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Breast; Carcinoma, Acinar Cell; Carcinoma, Squamous Cell; Cyclin D1; Cytoskeletal Proteins; Disease Models, Animal; DNA-Binding Proteins; Female; Gene Silencing; Genes, myc; Genotype; Germ-Line Mutation; Hepatocyte Nuclear Factor 1-alpha; Immunoenzyme Techniques; Integrases; Lac Operon; Lymphoid Enhancer-Binding Factor 1; Mammary Neoplasms, Experimental; Metaplasia; Mice; Mice, Inbred Strains; Phenotype; Skin Neoplasms; T Cell Transcription Factor 1; Trans-Activators; Transcription Factors; Viral Proteins

Squamous cancers of the oral cavity and esophagus are common worldwide, but no good genetically based animal model exists. A number of environmental factors as well as genetic alterations have been identified in these cancers, yet the specific combination of genetic events required for cancer progression remains unknown. The Epstein-Barr virus ED-L2 promoter (L2) can be used to target genes in a specific fashion to the oral-esophageal squamous epithelium. To that end, we generated L2-cyclin D1 (L2D1(+)) mice and crossbred these with p53-deficient mice. Whereas L2D1(+) mice exhibit a histologic phenotype of oral-esophageal dysplasia, the combination of cyclin D1 expression and p53 deficiency results in invasive oral-esophageal cancer. The development of the precancerous lesions was significantly reversed by the application of sulindac in the drinking water of the L2D1(+)/p53(+/-) mice. Furthermore, cell lines derived from oral epithelia of L2D1(+)/p53(+/-) and L2D1(+)/p53(-/-) mice, but not control mice, formed tumors in athymic nude mice. These data demonstrate that L2D1(+)/p53(+/-) mice provide a well-defined, novel, and faithful model of oral-esophageal cancer, which allows for the testing of novel chemopreventive, diagnostic, and therapeutic approaches.

Topics: Animals; Antineoplastic Agents; Cyclin D1; Disease Models, Animal; ErbB Receptors; Esophageal Neoplasms; Genotype; Herpesvirus 4, Human; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Mouth Neoplasms; Neoplasms, Squamous Cell; Promoter Regions, Genetic; Sulindac; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Intoxication of rats with thioacetamide (TAA) is a model system to investigate mechanisms involved in liver cell death and tissue reconstitution. Our study was undertaken to determine by immunohistochemistry the expression pattern of the cytoprotective chaperone proteins HSC70 and HSP25 and proliferation markers cyclin D1 and PCNA in livers of Wistar rats intraperitoneally injected with TAA at a single dose of 50 mg/kg. For each protein studied we observed distinct dynamic changes in appearance and localization in liver lobules. During 24-36 h after TAA injection the HSC70 cytoplasmic immunoreaction gradually disappeared from hepatocytes localized around central veins and a shift of immunostaining to cell nuclei took place. Then, 36-48 h after TAA injection the HSC70 cytoplasmic immunoreaction reappeared with the highest intensity in hepatocytes surrounding the areas of inflammatory cells. HSP25, undetectable in control hepatocytes began to appear at approximately 36 h after TAA injection and HSP25-immunopositive cells formed a characteristic ring around areas of inflammation. Of the proteins studied, the most rapid reaction to TAA was observed for cyclin D1. As early as 15 min after TAA administration cyclin D1-positive hepatocytes appeared in intermediate and periportal areas of liver lobules and a subsequent shift of staining to centrilobular hepatocytes took place at 36 and 48 h. There was no correlation of cyclin D1 localization either with PCNA-positive cells or mitotic cells. Our observations suggest that in TAA-treated livers HSP25 and HSC70 proteins can play an anti-inflammatory role, and the early and distinct cyclin D1 expression is not related to proliferation of hepatocytes.

Topics: Animals; Blotting, Northern; Blotting, Western; Chemical and Drug Induced Liver Injury; Cyclin D1; Disease Models, Animal; Heat-Shock Proteins; HSC70 Heat-Shock Proteins; HSP27 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Immunoenzyme Techniques; Injections, Intraperitoneal; Liver; Male; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar; Thioacetamide; Time Factors

To determine the role of cell-cycle proteins in regulating pathological renal hypertrophy, diabetes was induced in mice expressing a human retinoblastoma (RB) transgene and in wild-type littermates. Whole-kidney and glomerular hypertrophy caused by hyperglycemia was associated with specific G1 phase cell-cycle events: early and sustained increase in expression of cyclin D1 and activation of cyclin D1-cdk4 complexes, but no change in expression of cyclin E or cdk2 activity. Overexpression of RB alone likewise caused hypertrophy and increased only cyclin D1-cdk4 activity; these effects were not further augmented by high glucose. Identical observations were made when isolated mesangial cells conditionally overexpressing RB from a tetracycline-repressible system hypertrophied in response to high glucose. A mitogenic signal in the same cell-culture system, in contrast, transiently and sequentially activated both cyclin D1-cdk4 and cyclin E-cdk2. In vivo and in cultured mesangial cells, high glucose resulted in persistent partial phosphorylation of RB, an event catalyzed specifically by cyclin D1-cdk4. These data indicate that mesangial hypertrophy caused by hyperglycemia in diabetes results in sustained cyclin D1-cdk4-dependent phosphorylation of RB and maintenance of mesangial cells in the early-to-middle G1 phase of the cell cycle.

Topics: Animals; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Glomerular Mesangium; Humans; Hypertrophy; Mice; Mice, Transgenic; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein

The present study was conducted to assess the inhibitory effects of EGCG (epigallocatechin-3-gallate) on NMBA-induced rat esophageal tumorigenesis and to seek the potential mechanisms. In experiment I, 81 F344 rats were randomly divided into seven experimental groups according to the different regiments of NMBA 1 mg/kg subcutaneously (s.c.) and EGCG 4 mg/kg or 10 mg/kg orally or intraperitoneally (i.p.). The experiment was terminated at 24 weeks. In experiment II, 48 rats were allocated into two groups, each group contained 24 rats, in which the rats were injected with NMBA 1 mg/kg only or a combination of NMBA 1 mg/kg and EGCG 4 mg/kg i.p. Six rats from each group were sacrificed at the 12th, 16th, 20th and 24th week, respectively. The expression of cyclin D1 and cyclooxygenases (COX-2 and COX-1) was detected using semi-quantitative RT-PCR, and the production of prostaglandin E2 (PGE2) was measured by ELISA. In the groups which were treated with EGCG at a dose of 4 mg/kg i.p., or 10 mg/kg both orally and i.p., the mean number of tumors per rat was significantly reduced to 48, 56 and 61%, respectively (p<0.05). The incidence rate of esophageal carcinomas in the rats that were treated with EGCG 4 mg/kg i.p., was significantly lower than that in the rats which only received NMBA 1 mg/kg (p<0.05). The expression of cyclin D1 and COX-2, and the levels of PGE2 were also decreased by EGCG treatment. These results indicated that EGCG significantly inhibits the NMBA-induced rat esophageal carcinogenesis and it inhibitory effects may partly target cyclin D1 and COX-2 expression, and PGE2 production.

Topics: Animals; Anticarcinogenic Agents; Catechin; Cyclin D1; Cyclooxygenase 1; Cyclooxygenase 2; Dimethylnitrosamine; Dinoprostone; Disease Models, Animal; DNA Primers; Enzyme-Linked Immunosorbent Assay; Esophageal Neoplasms; Esophagus; Isoenzymes; Male; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred F344; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Urokinase-Type Plasminogen Activator

To explore the influence of different nutritional support routes on the intestinal mucosal epithelial cell cycle in burned rats.. Sixty-six Wistar rats inflicted with 30% TBSA III degree burns on the back were employed as the model and were randomly divided into enteral feeding group (EF) and intravenously parenteral nutrition group (PN). Equal volume of nutritional support fluid containing predetermined equal amount of calories and nitrogen was applied via feeding or intravenously infusion through external jugular vein. The indices were observed on 6, 12, 24, 48 and 72 postburn hours (PBHs) with the reference to those in 6 normal rats. The intestinal epithelial cell cycle in jejunal and ileal mucous membrane was analyzed by flow cytometry. Western blotting method was employed in the examination of the expression of cyclin D1, E and that of cyclin dependent kinase (CDK)2 and CDK4.. (1) lntestinal mucosal epithelial G0/G1 ratio in jejunum in EF group was significantly lower than that in PN group at 72 PBHs (P < 0.05). While the ratio in ileum in EF was obviously higher than that in PN groups at 6, 12, 48 and 72 PBHs (P < 0.05). (2) The cell percentage of S phase in EF group was evidently higher than that in PN group (P < 0.05 - 0.01) at 48 and 72 PBHs. (3) Intestinal mucosal cyclin D1 expression increased significantly in EF group at 24 PBHs and in PN group at 48 PBHs (P < 0.05) and which in EF group was obviously higher than that in PN group at 72 PBHs (P < 0.05). (4) The expression of the intestinal mucosal cyclin E in EF group at 72 PBHs was evidently higher than the control value and that in PN group (P < 0.05). (5) The expression of CDK2 exhibited no obvious difference among PN,EF and control group (P < 0.05). The CDK4 expression in EF group increased obviously at 72 PBHs (P < 0.05).. Early postburn enteral feeding was beneficial to the progression of intestinal mucosal epithelial cell cycle and to the repairing and renovation of injured intestinal mucosal membrane. Cyclin and CDK might be important in the modulation of the intestinal mucosal epithelial cell cycle.

Topics: Animals; Burns; CDC2-CDC28 Kinases; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Disease Models, Animal; Enteral Nutrition; Female; G1 Phase; Intestinal Mucosa; Male; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Rats; Rats, Wistar; Resting Phase, Cell Cycle; S Phase

The effects of tea polyphenols and tea pigments on rat liver precancerous lesions and some cell cycle regulators were studied. A modified Solt-Farber model in rats was established by multiple low-dosage of N-nitrosodiethylamine (NDEA) i.p. injections, followed by i.p. CCl(4) injection and partial hepatectomy. Sixty male Wistar rats were randomly divided into four groups: positive control group, two tea-treated groups, and negative control group. Rats in tea-treated groups were given tea polyphenols (0.1%) and tea pigments (0.1%) in drinking fluid during the whole experiment. The number and area of glutathione S-transferase P (GST-P)-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western blotting assay was carried out to detect the expression of cyclin D1, CDK4, and P21(WAF1/CIP1) on whole liver extract. At the end of the experiment (56 days), the number and area of GST-P-positive foci in liver increased significantly in carcinogen-administered positive control group, whereas no GST-P-positive foci were found in the negative control group in which animals did not receive carcinogen exposure. The number and area of GST-P-positive foci in tea-treated, carcinogen-exposed groups were significantly reduced as compared with the positive control group. It was also found that the expression of P21(WAF1/CIP1) was significantly induced and the expression of cyclin D1 and CDK4 was significantly inhibited in tea-treated groups. These results suggest that tea polyphenols and tea pigments are effective in preventing the precancerous liver lesions in rats, and modulation of cell cycle by regulating cell cycle regulators may be a possible mechanism.

Topics: Animals; Biomarkers, Tumor; Biopsy, Needle; Blotting, Western; Cell Cycle; Culture Techniques; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Disease Models, Animal; Flavonoids; Glutathione Transferase; Immunohistochemistry; Liver Neoplasms, Experimental; Male; Phenols; Plant Extracts; Polymers; Polyphenols; Precancerous Conditions; Proto-Oncogene Proteins; Random Allocation; Rats; Rats, Wistar; Reference Values; Sensitivity and Specificity; Tea

We have previously demonstrated that ursodeoxycholic acid(UDCA) and a fluorinated analogue of vitamin D(3), F(6)-D(3),inhibited colonic carcinogenesis in the azoxymethane (AOM) model. Generalized colonic mucosal hyperproliferation and aberrant crypt foci (ACF) are intermediate biomarkers of colon cancer. Using these biomarkers, in this study we examined the anticarcinogenic mechanisms of these chemopreventive agents. Rats were maintained on AIN-76A chow or supplemented with 0.4% UDCA or F(6)-D(3) (2.5 nmol/kg chow) and treated weekly with AOM 20 mg i.p./kg wt or saline x 2 weeks. F(6)-D(3) was continued for an additional 2 weeks and UDCA for the duration of the study. At 40 weeks, animals received bromodeoxyuridine (BrdUrd) i.p. 2 h before sacrifice. A portion of each tumor was fixed in formalin and the remainder flash frozen. Colons were divided longitudinally and half-fixed in formalin and half in ethanol. The size and location of methylene blue-stained ACF were recorded. Cell proliferation (BrdUrd labeling) and apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay) were measured in colonic crypts and tumors. Protein expression levels of several regulators of cell proliferation were analyzed by immunostaining and Western blotting. Colonic crypt cyclin D1 and E-cadherin mRNA levels were measured by real-time PCR. In saline injected controls, neither UDCA nor F(6)-D(3) alone had any effect on cytokinetic parameters or on the expression of mitogenic regulators. AOM significantly increased the proliferation (percentage of BrdUrd-positive cells) of both ACF (23.1 +/- 1.7%) and non-ACF crypts (17.6 +/- 1.6%), compared with normal colonic crypts (4.5 +/- 0.8%; P < 0.05). This hyperproliferation was accompanied by a 5-fold increase in cyclin D1 and >50% decrease in E-cadherin protein (P < 0.05) in ACF, both of which are predicted to be growth-enhancing alterations. UDCA and F(6)-D(3) significantly (P < 0.05) inhibited AOM-induced crypt cell hyperproliferation, ACF development, and tumor burden. These chemopreventive agents also significantly blocked AOM-induced alterations in cyclin D1 and E-cadherin protein in ACF and tumors. In ACF, changes in mRNA levels of cyclin D1, but not E-cadherin, paralleled alterations in protein expression. Cyclooxygenase-2 and inducible nitric oxide synthase were increased in AOM tumors but not in ACF, and these changes were blocked by UDCA and F(6)-D(3). UDCA and F(6)-D(3) significantly inhibited ACF de

Topics: Animals; Azoxymethane; Base Sequence; Biomarkers, Tumor; Biopsy, Needle; Blotting, Western; Cadherins; Cell Division; Cholecalciferol; Colonic Neoplasms; Cyclin D1; Disease Models, Animal; Immunohistochemistry; Injections, Intraperitoneal; Intestinal Mucosa; Male; Molecular Sequence Data; Neoplasms, Experimental; Polymerase Chain Reaction; Random Allocation; Rats; Rats, Inbred F344; Reference Values; RNA, Messenger; Sensitivity and Specificity; Ursodeoxycholic Acid

Trauma triggers diffuse apoptotic neurodegeneration in the developing rat brain. To explore the pathogenesis of this phenomenon we investigated the involvement of three possible mechanisms: death receptor activation, activation of the intrinsic apoptotic pathway by cytochrome c release into the cytoplasm, and changes in trophic support provided by endogenous neurotrophins. We detected a decrease in the expression of bcl-2 and bcl-x(L), two antiapoptotic proteins that decrease mitochondrial membrane permeability, an increase in cytochrome c immunoreactivity in the cytosolic fraction, and an activation of caspase-9 in brain regions which show apoptotic neurodegeneration following percussion brain trauma in 7-day-old rats. Increase in the expression of the death receptor Fas was revealed by RT-PCR analysis, Western blotting, and immunohistochemistry, as was activation of caspase-8 in cortex and thalamus. Apoptotic neurodegeneration was accompanied by an increase in the expression of BDNF and NT-3 in vulnerable brain regions. The pancaspase inhibitor z-VAD.FMK ameliorated apoptotic neurodegeneration with a therapeutic time window of up to 8 h after trauma. These findings suggest involvement of intrinsic and extrinsic apoptotic pathways in neurodegeneration following trauma to the developing rat brain. Upregulation of neurotrophin expression may represent an endogenous mechanism that limits this apoptotic process.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; bcl-X Protein; Brain; Brain Injuries; Brain-Derived Neurotrophic Factor; Caspase 9; Caspases; Cyclin D1; Cytochrome c Group; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; fas Receptor; Immunohistochemistry; Nerve Degeneration; Neurons; Neurotrophin 3; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction

Overexpression of PMP22 is responsible for the most common form of inherited neuropathy, Charcot-Marie-Tooth disease (CMT) type 1A. The PMP22-transgenic rat (CMT rat) is an animal model of CMT1A, and its peripheral nerves show the characteristic features of ongoing demyelination and remyelination that is also seen in CMT1A patients. Since Schwann cell proliferation is a prominent feature of peripheral nerves in inherited peripheral neuropathies, we examined proliferation and the expression of cyclin D1 in CMT rats. D-type cyclins are required for the initial steps in cell division and nuclear import is crucial for the function of cyclin D1 in promoting cell proliferation. Like normal myelinating Schwann cells in wild-type rats, remyelinating Schwann cells in CMT rats show perinuclear cyclin D1 expression. Schwann cells with nuclear cyclin D1 expression, as well as proliferating Schwann cells, were both associated with demyelinated axonal segments. Supernumerary onion bulb Schwann cells, however, do not express cyclin D1 and were not proliferating. Thus, cyclin D1 expression and its subcellular localization correlate directly with distinct physiological states of Schwann cells in this animal model of CMT1A.

Topics: Animals; Animals, Genetically Modified; Axons; Cell Compartmentation; Cell Division; Cell Nucleus; Charcot-Marie-Tooth Disease; Cyclin D1; Disease Models, Animal; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Histones; Myelin Proteins; Nerve Fibers, Myelinated; Peripheral Nerves; Rats; Schwann Cells

Beta-catenin and T cell factor (Tcf) are distal components of the highly conserved Wnt pathway that govern cell fate and proliferation in lower organisms. Thus, we hypothesized that the regulation of beta-catenin and Tcf played a critical role in vascular remodeling. The first objective was to define beta-catenin expression in vascular smooth muscle cells (VSMCs) after balloon injury. Indeed, beta-catenin mRNA and protein were significantly elevated 7 days after balloon injury in the rat carotid artery. We hypothesized that beta-catenin accumulation in response to vascular injury inhibited VSMC apoptosis. In line with our hypothesis, transfection of a degradation-resistant beta-catenin transgene into rat VSMCs significantly inhibited apoptosis. Accumulation of beta-catenin also resulted in a 10-fold increase in the activation of Tcf. To test if Tcf was necessary to confer beta-catenin-induced survival, loss of function studies were carried out with a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, Tcf4(N31). Indeed, loss of Tcf-4 activity abolished beta-catenin-induced survival. We further postulated that beta-catenin and Tcf promoted cell cycle progression by activating cyclin D1, a target gene of Tcf-4. Beta-catenin activated cyclin D1, and this activation was partially blocked with loss of Tcf-4. In parallel, blockade of Tcf-4 resulted in inhibition of [3H]thymidine incorporation and partial blockade of the G1-S phase transition. In conclusion, beta-catenin and Tcf-4 play a dual role in vascular remodeling by inhibiting VSMC apoptosis and promoting proliferation.

Topics: Amino Acid Substitution; Animals; Apoptosis; beta Catenin; Calcium-Calmodulin-Dependent Protein Kinases; Carotid Arteries; Carotid Stenosis; Catheterization; Cell Cycle; Cell Survival; Cells, Cultured; Cyclin D1; Cytoskeletal Proteins; Disease Models, Animal; Genes, Dominant; Genes, Reporter; Glycogen Synthase Kinase 3; Male; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transgenes; Up-Regulation

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16(INK4A), and p27(KIP1) did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16(INK4A) overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.

Topics: Animals; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; G1 Phase; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Liver; Liver Neoplasms, Experimental; Male; Phosphorylation; Proto-Oncogene Proteins c-myc; Rats; Rats, Inbred BN; Rats, Inbred F344; Rats, Wistar; Retinoblastoma Protein; S Phase; Transcription Factor DP1; Transcription Factors

This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.

Topics: Animals; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Disease Models, Animal; Disease Progression; Female; G1 Phase; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Pancreatic Neoplasms; Phenotype; Proto-Oncogene Proteins; ras Proteins; S Phase; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

After mild ischemic insults, many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in various conditions including ischemia. We demonstrate that loss of endogenous cyclin-dependent kinase (Cdk) inhibitor p16(INK4a) is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27(Kip1), another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen-glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. Most neurons undergo cell death before entering S-phase, albeit a small number ( approximately 1%) do progress to the S-phase before their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Topics: Animals; Brain Ischemia; Bromodeoxyuridine; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Death; Cell Hypoxia; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Disease Models, Animal; Enzyme Inhibitors; Glucose; In Situ Nick-End Labeling; Kinetin; Mice; Mice, Inbred Strains; Microtubule-Associated Proteins; Neurons; Oxygen; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Purines; Rats; Rats, Wistar; Tumor Suppressor Proteins

The vitamin D receptor knockout (VDR-KO) mouse presents with a skeletal phenotype typical for complete lack of genomic 1,25-dihydroxycholecalciferol effects. Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression. In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon, we characterized the amount and site-specific distribution of the VDR along the large intestine in wild-type (WT), heterozygote (HT) and KO mice. We also evaluated expression of proliferating cell nuclear antigen (PCNA), of cyclin D1 and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress. In colon ascendens, proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice. A decrease of VDR expression did not affect expression significantly. In colon descendens, however, fewer proliferative cells were solely located in the lower third of the crypt, and an inverse relationship between VDR reduction, PCNA positivity and cyclin D1 expression was found in HT and KO mice. In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred. Therefore, the sigmoid colon of VDR-KO mice, fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes, is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Calcium; Colon; Colorectal Neoplasms; Cyclin D1; Deoxyguanosine; Disease Models, Animal; DNA Damage; Female; Homeostasis; Immunohistochemistry; Male; Mice; Mice, Knockout; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Receptors, Calcitriol

Huntington's disease is a progressive, inherited neurodegenerative disorder characterized by the loss of subsets of neurons primarily in the striatum. In this study, we assessed the neuroprotective effect of lithium against striatal lesion formation in a rat model of Huntington's disease in which quinolinic acid was unilaterally infused into the striatum. For this purpose, we used a dopamine receptor autoradiography and glutamic acid decarboxylase mRNA in situ hybridization analysis, methods previously shown to be adequate for quantitative analysis of the excitotoxin-induced striatal lesion size. Here we demonstrated that subcutaneous injections of LiCl for 16 days prior to quinolinic acid infusion considerably reduced the size of quinolinic acid-induced striatal lesion. Furthermore, these lithium pre-treatments also decreased the number of striatal neurons labeled with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Immunohistochemistry and western blotting demonstrated that lithium-elicited neuroprotection was associated with an increase in Bcl-2 protein levels. Our results raise the possibility that lithium may be considered as a neuroprotective agent in treatment of neurodegenerative diseases such as Huntington's disease.

Topics: Animals; Antimanic Agents; Benzazepines; Cell Death; Cyclin D1; Disease Models, Animal; Dopamine Antagonists; Glutamate Decarboxylase; Huntington Disease; Immunohistochemistry; In Situ Nick-End Labeling; Isoenzymes; Lithium; Lithium Chloride; Male; Neostriatum; Neurons; Neuroprotective Agents; Neurotoxins; Quinolinic Acid; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D1; RNA, Messenger

Cisplatin is used in the treatment of ovarian and testicular cancer. Twenty percent of patients cannot be optimally treated because of sensory neurotoxicity. Human and animal studies demonstrate that the dorsal root ganglion neuron is the primary target of drug injury. We have previously demonstrated that cisplatin causes neuronal apoptosis in vitro. We now report a reproducible animal model of cell death induced by cisplatin. Drug was administered for 1 or 2 cycles of 5 days separated by 5 days. Total dose administered was 0, 5, 7.5, 10, or 15 mg/kg. Ganglia from 34 animals were processed and examined using in situ hybridization for cyclin D1 messenger RNA and digoxigenin coupled TUNEL staining. Overall, 2.9 +/- 3.9% of neurons were TUNEL positive in treated rats compared with 0.2 +/- 0.3% in controls (P <.005). There was a strong positive correlation (r2 = 0.88; P = 0.018) between percentage of TUNEL stained DRG and cumulative dose of cisplatin. Two independent approaches to quantitation of in situ cyclin D1 hybridization were used; blinded grading by an observer and measurement of color density using digital image analysis. Both demonstrated dramatic upregulation of expression of cyclin D1 mRNA in treated compared with control rats. This demonstrates that apoptosis of neurons is preceded by aberrant reentry into G1 phase of the cell cycle in an animal model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cisplatin; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Ganglia, Spinal; Immunohistochemistry; In Situ Hybridization; In Situ Nick-End Labeling; Nerve Degeneration; Neurons, Afferent; Neurotoxins; Pain Measurement; Peripheral Nervous System Diseases; Platinum; Rats; Rats, Sprague-Dawley; Renal Insufficiency; RNA, Messenger; Up-Regulation

Portal hypertensive (PHT) gastric mucosa has increased susceptibility to injury and impaired mucosal healing. Our previous study demonstrated increased ERK activation and MAP kinase phosphatase-1 (MKP-1) overexpression in PHT gastric mucosa. However, it remains unknown which tyrosine kinase receptors are involved in ERK activation and whether ERK activation results in increased cell proliferation. We examined whether EGF receptor (EGF-R) is involved in ERK activation and whether ERK activation triggers epithelial proliferation in PHT gastric mucosa. In gastric mucosa of PHT and sham-operated (SO) rats we studied: (1) EGF-R mRNA and protein expression as well as phosphorylation and membrane protein tyrosine kinase (PTK) activity; (2) ERK2 phosphorylation and activity; (3) MKP-1 mRNA and protein; (4) c-fos, c-myc and cyclin D1 mRNAs, and gastric epithelial proliferation. In PHT gastric mucosa: (1) EGF-R mRNA, protein and phosphorylation and membrane PTK activity were all significantly increased by 38%, 49%, 43% and 49%, respectively; (2) ERK2 phosphorylation and activity were significantly increased by 40% and 50 %, respectively; (3) MKP-1 mRNA and protein expression were significantly increased by 27% and 34%, respectively. In contrast, (4) c-fos, c-myc, and cyclin D1 mRNAs expression were all significantly decreased in PHT gastric mucosa by 36%, 33%, and 49%, respectively, and cell proliferation was significantly lower that in SO rats (11% in PHT vs. 18% in SO). These results suggest that in PHT gastric mucosa, ERK activation is mediated through EGF-R upregulation, but the gastric epithelial proliferation is impaired, possibly by MKP-1 overexpression, leading to reduction of c-fos, c-myc and cyclin D1.

Topics: Animals; Cell Cycle Proteins; Cell Division; Cyclin D1; Disease Models, Animal; DNA Primers; DNA-Binding Proteins; Dual Specificity Phosphatase 1; Epithelial Cells; ErbB Receptors; Gastric Mucosa; Hypertension, Portal; Immediate-Early Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation