cyclin-d1 and Necrosis

The rexinoid bexarotene represses cyclin D1 by causing its proteasomal degradation. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib represses cyclin D1 via different mechanisms. We conducted a preclinical study and 2 clinical/translational trials (a window-of-opportunity and phase II) of bexarotene plus erlotinib. The combination repressed growth and cyclin D1 expression in cyclin-E- and KRAS/p53-driven transgenic lung cancer cells. The window-of-opportunity trial in early-stage non-small-cell lung cancer (NSCLC) patients (10 evaluable), including cases with KRAS mutations, repressed cyclin D1 (in tumor biopsies and buccal swabs) and induced necrosis and inflammatory responses. The phase II trial in heavily pretreated, advanced NSCLC patients (40 evaluable; a median of two prior relapses per patient (range, 0-5); 21% with prior EGFR-inhibitor therapy) produced three major clinical responses in patients with prolonged progression-free survival (583-, 665-, and 1,460-plus days). Median overall survival was 22 weeks. Hypertriglyceridemia was associated with an increased median overall survival (P = 0.001). Early PET (positron emission tomographic) response did not reliably predict clinical response. The combination was generally well tolerated, with toxicities similar to those of the single agents. In conclusion, bexarotene plus erlotinib was active in KRAS-driven lung cancer cells, was biologically active in early-stage mutant KRAS NSCLC, and was clinically active in advanced, chemotherapy-refractory mutant KRAS tumors in this study and previous trials. Additional lung cancer therapy or prevention trials with this oral regimen are warranted.

Topics: Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Female; Humans; Immunoblotting; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Mouth Mucosa; Mutation; Necrosis; Neoplasm Recurrence, Local; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; Salvage Therapy; Survival Rate; Tetrahydronaphthalenes; Treatment Outcome; Tumor Cells, Cultured

Chemotherapy failure is often caused by drug resistance, for which no effective treatment strategy has been established. Many studies have been undertaken with the aim of overcoming drug resistance using natural products. Arctigenin (ATG), a natural product, has been investigated for its anti-cancer effects in HER2-overexpressing, ER-positive, and triple-negative breast cancer cells. We investigated the efficacy of ATG against self-established doxorubicin (DOX)-resistant breast cancer cells (MCF-DR and MDA-DR cells) derived from MCF-7 and MDA-MB-231 cells, respectively. ATG was found to increase DOX intracellular levels by downregulating multidrug Resistance 1 (MDR1) mRNA expression in DOX-resistant cells. In addition, combined treatment with DOX and ATG (DOX/ATG) reduced the viability of and colony formation by DOX-resistant cells. DOX/ATG also significantly induced G2/M cell cycle arrest by suppressing the Cyclin D1/CDK4/RB pathways and suppressed the expressions of MDR1 and Cyclin D1 by inhibiting the Mitogen-activated protein kinase (MAPK)/Activating protein-1 (AP-1) signaling pathways. Furthermore, DOX/ATG induced DNA damage and attenuated the expressions of RAD51 and Ku80. However, PARP1 (Poly [ADP-ribose] polymerase1) cleavage and AIF (Apoptosis-inducing factor) induced apoptosis did not occur despite DNA damage-induced cell death. Rather, flow cytometry showed that DOX/ATG caused necrosis. In summary, DOX/ATG increased intracellular DOX levels by inhibiting MDR1 and inducing G2/M arrest by inhibiting the Cyclin D1/CDK4/RB pathways and causing necrosis by damaging DNA. Our results suggest that ATG might be used as an adjuvant to enhance the efficacy of DOX in DOX-resistant breast cancer.

Topics: Apoptosis; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Doxorubicin; Drug Resistance, Neoplasm; Female; G2 Phase Cell Cycle Checkpoints; Humans; Necrosis

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare soft tissue tumor with a predilection for the distal extremities and a tendency for local recurrence. Morphologically, MIFS consists of spindle and bizarre epithelioid cells resembling virocytes embedded in a fibrous to myxoid stroma with an abundant inflammatory infiltrate. Importantly, the molecular landscape of MIFS is wide and includes: VGLL3 amplification, BRAF fusion/amplification and OGA/TGFBR3 rearrangements. In this study, we describe a variant of MIFS showing a frequent nodular configuration associated with necrosis and recurrent YAP1::MAML2 fusions. The cohort consisted of 7 patients (4 females and 3 males) ranging in age from 21 to 71 years (median: 47 years). Two tumors (28%) occurred in acral locations while the remaining cases were more widely distributed (thigh, n = 2; arm, n = 1; neck; n = 1; chest-wall, n = 1). Tumor size ranged from 10 to 38 mm (median: 20 mm). Histologically, lesions frequently presented as nodules with central areas of necrosis, and were predominantly composed of sheets of epithelioid cells with large vesicular nuclei and prominent nucleoli (Reed-Sternberg-like cells or virocytes). The stroma was mostly fibrous and showed a polymorphous inflammatory infiltrate. Myxoid stromal changes were focally seen in one case, and pseudolipoblasts were absent. The immunophenotype was nonspecific, with only pan-keratin (AE1-AE3) and cyclin D1 expression in a subset of cases. RNA-Sequencing detected YAP1::MAML2 fusions in 3/7 cases; aCGH showed no significant gene copy number variations in 4 tested cases, and FISH analysis showed no VGLL3 amplification in 1 tested case. Follow-up was available for 6 cases, ranging from 7 to 63 months (median: 42 months). Local recurrence and metastasis were not seen and one tumor showed spontaneous regression following initial biopsy. In conclusion, we describe a novel variant of MIFS with distinctive clinicopathological and molecular features for which we propose the term "nodular necrotizing" MIFS.

Topics: Cyclin D1; DNA Copy Number Variations; Female; Fibrosarcoma; Humans; Keratins; Male; Necrosis; Proto-Oncogene Proteins B-raf; RNA; Skin Neoplasms; Soft Tissue Neoplasms; Trans-Activators; Transcription Factors; YAP-Signaling Proteins

Ependymal tumors are pathologically defined intrinsic neoplasms originating in the intracranial compartments or the spinal cord that affect both children and adults. The recently integrated classification of ependymomas based on both histological and molecular characteristics is capable of subgrouping patients with various prognoses. However, the application of histological and molecular markers in Chinese patients with ependymomas has rarely been reported. We aimed to demonstrate the significance of histological characteristics, the v-relavian reticuloendotheliosis viral oncogene homolog A (RELA) fusions and other molecular features in ependymal tumors.. We reviewed the histological characteristics of ependymal tumors using conventional pathological slides and investigate the RELA fusions and Cylclin D1 (CCND1) amplification by Fluorescence in situ hybridization (FISH) and trimethylation of histone 3 lysine 27 (H3K27me3) expression by immunohistochemistry (IHC) methods. SPSS software was used to analyze the data.. We demonstrated that hypercellularity, atypia, microvascular proliferation, necrosis, mitosis, and an elevated Ki-67 index, were tightly associated with an advanced tumor grade. Tumor location, necrosis, mitosis and the Ki-67 index were related to the survival of the ependymomas, but Ki67 was the only independent prognostic factor. Additionally, RELA fusions, mostly presented in pediatric grade III intracranial ependymomas, indicated decreased survival times of patients, and closely related to the patients' age, tumor grade, cellularity, cellular atypia, necrosis and Ki67 index in the intracranial ependymal tumors, whereas reduction of H3K27me3 predicted the worse prognosis in ependymal tumors.. Histological and molecular features facilitate tumor grading and prognostic predictions for ependymal tumors in Chinese patients.

Topics: Adolescent; Adult; Biomarkers, Tumor; Brain Neoplasms; Child; China; Cyclin D1; Ependymoma; Female; Follow-Up Studies; Histones; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Ki-67 Antigen; Male; Necrosis; Neoplasm Grading; Prognosis; Spinal Cord Neoplasms; Transcription Factor RelA; Young Adult

Spirulina is a well-described and popular dietary supplement derived from Arthrospira algae. In the present study, the anticancer potential of a water extract of a commercial Spirulina product (SE) against the human non-small-cell lung carcinoma A549 cell line was evaluated. After qualitative analysis, we investigated the effect of SE on cell viability, proliferation, and morphology. Furthermore, the influence of SE on regulation of the cell cycle, induction of apoptosis in lung cancer cells, and expression of cell cycle/apoptosis-related proteins was evaluated. Additionally, we examined the cytotoxic effect of SE on normal human skin fibroblasts (HSF). Our studies revealed that SE significantly reduced cancer cell viability and proliferation, which was accompanied by cell cycle inhibition in the G

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Fibroblasts; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Necrosis; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Signal Transduction; Solvents; Spirulina; Water

The present study aimed to investigate the molecular mechanisms underlying the anticancer properties of ginger extract (GE) in mice bearing solid Ehrlich carcinoma (SEC) and to evaluate the use of GE in combination with doxorubicin (DOX) as a complementary therapy against SEC.. SEC was induced in 60 female mice. Mice were divided into four equal groups: SEC, GE, DOX and GE + DOX. GE (100 mg/kg orally day after day) and DOX (4 mg/kg i.p. for 4 cycles every 5 days) were given to mice starting on day 12 of inoculation. On the 28th day, blood samples were collected, mice were scarified, tumor volume was measured, and tumor tissues were excised.. The anti-cancer effect of GE was mediated by activation of adenosine monophosphate protein kinase (AMPK) and down-regulation of cyclin D1 gene expression. GE also showed pro-apoptotic properties as evidenced by elevation of the P53 and suppression of nuclear factor-kappa B (NF-κB) content in tumor tissue. Co-administration of GE alongside DOX markedly increased survival rate, decreased tumor volume, and increased the level of phosphorylated AMPK (PAMPK) and improved related pathways compared to DOX group. In addition, the histopathological results demonstrated enhanced apoptosis and absence of multinucleated cells in tumor tissue of GE + DOX group.. AMPK pathway and cyclin D1 gene expression could be a molecular therapeutic target for the anticancer effect of GE in mice bearing SEC. Combining GE and DOX revealed a greater efficacy as anticancer therapeutic regimen.

Topics: AMP-Activated Protein Kinases; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; Biomarkers, Tumor; Carcinoma, Ehrlich Tumor; Combined Modality Therapy; Cyclin D1; Dietary Supplements; Doxorubicin; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Necrosis; Neoplasm Proteins; Plant Extracts; Rhizome; Survival Analysis; Tumor Burden; Zingiber officinale

A subset of patients with ductal carcinoma in situ (DCIS) experience recurrence or progression to invasive cancer. Current clinical practice is not reliably guided by DCIS recurrence prediction, although recurrence risk for invasive breast cancer can now be assessed. We analyzed a panel of biomarkers (estrogen receptor, Her2, Ki67, p53, cyclin D1, COX-2, caveolin-1, survivin, and PPAR-γ) and DCIS histologic and clinical features to determine associations with DCIS recurrence.. Seventy DCIS cases diagnosed between 1995 and 2010 were divided into 2 groups: 52 had DCIS without known recurrence after excision and 18 had DCIS with subsequent recurrence after excision as DCIS or invasive carcinoma in the ipsilateral or contralateral breast. Tissue microarrays were prepared, immunohistochemistry performed, and expression of the biomarkers scored semiquantitatively. Variables analyzed included age, tumor size, margin status, DCIS grade, necrosis, histologic type, and immunohistochemistry scores. Differences between groups were evaluated using t tests for continuous variables and Fisher exact tests for categorical variables.. Intraductal necrosis was associated with increased recurrence risk: 46% of nonrecurrent cases showed necrosis compared with 83% of those who recurred (P=0.007). Her2 (human epidermal growth factor receptor 2) and Ki67 expression distributions were significantly different between nonrecurrent and recurrent cases. Her2 was overexpressed in 14% of nonrecurrent cases compared with 50% in the recurrent cases (P=0.03). A total of 87% of nonrecurrent cases had low Ki67 staining (0% to 10%) compared with 50% among the recurrent cases (P=0.002).. Our results suggest that Her2 and Ki67 immunohistochemistry and the presence of intraductal necrosis aid in DCIS risk stratification.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Caveolin 1; Cyclin D1; Cyclooxygenase 2; Estrogen Receptor alpha; Female; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Middle Aged; Necrosis; PPAR gamma; Prognosis; Receptor, ErbB-2; Recurrence; Retrospective Studies; Risk Factors; Survivin; Treatment Outcome; Tumor Suppressor Protein p53

This study was designed to evaluate the effect of zearalenone (ZEA) on cell cycle checkpoints cyclin D1 and E2f1 expression at mRNA level and to analyze the estrogen like impact of ZEA on male reproductive system. Thirty mature male rats were randomly assigned into five groups as; control (received 0.5 mL saline normal, i.p.), ZEA-received groups (1, 2 and 4 mg/kg, b.w., i.p.) and 17 β-estradiol-received group (0.1 mg/kg, i.p.). All animals received chemicals for 28 days. Cyclin D1 expression was evaluated both by using PCR and immunohistochemistry staining. The mRNA level of E2f1 was also assessed by PCR. Cellular apoptosis was evaluated by using TUNEL and DNA laddering tests. ZEA, at low and medium doses increased the cellular apoptosis and DNA fragmentation. However, at high dose-received animals, severe necrosis was revealed in germinal epithelium. The medium dose of ZEA exhibited the same phenotype as 17 β-estradiol-showed. Accordingly, the medium dose of ZEA-and 17 β-estradiol significantly up-regulated the expression of cyclin D1 and E2f1 in the testis. In contrast, high dose of ZEA down regulated the expression of both genes at mRNA level. The histomorphometric analyses showed that ZEA in medium and high doses remarkably lowered the tubular differentiation and spermiogenesis indices. In conclusion, our data suggest that ZEA enhances the cellular apoptosis likely via oppositional roles of cyclin D1 and E2F1 checkpoint machineries for cell cycles in testicular tissue. Moreover, at medium dose level ZEA exerts the same pathological phenotype as 17 β-estradiol induces.

Topics: Animals; Apoptosis; Cyclin D1; DNA Fragmentation; Down-Regulation; E2F1 Transcription Factor; Estradiol; Immunohistochemistry; In Situ Nick-End Labeling; Male; Necrosis; Organ Size; Phenotype; Polymerase Chain Reaction; Random Allocation; Rats; Rats, Wistar; RNA, Messenger; Spermatogenesis; Testis; Zearalenone

Although the aberrant activation of cell cycle proteins has a critical role in neuronal death, effectors or mediators of cyclin D1/cyclin-dependent kinase 4 (CDK4)-mediated death signal are still unknown. Here, we describe a previously unsuspected role of LIM kinase 2 (LIMK2) in programmed necrotic neuronal death. Downregulation of p27(Kip1) expression by Rho kinase (ROCK) activation induced cyclin D1/CDK4 expression levels in neurons vulnerable to status epilepticus (SE). Cyclin D1/CDK4 complex subsequently increased LIMK2 expression independent of caspase-3 and receptor interacting protein kinase 1 activity. In turn, upregulated LIMK2 impaired dynamic-related protein-1 (DRP1)-mediated mitochondrial fission without alterations in cofilin phosphorylation/expression and finally resulted in necrotic neuronal death. Inhibition of LIMK2 expression and rescue of DRP1 function attenuated this programmed necrotic neuronal death induced by SE. Therefore, we suggest that the ROCK-p27(Kip1)-cyclin D1/CDK4-LIMK2-DRP1-mediated programmed necrosis may be new therapeutic targets for neuronal death.

Topics: Animals; CA1 Region, Hippocampal; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dynamins; Gene Expression; Lim Kinases; Male; Mitochondrial Dynamics; Necrosis; Protein Serine-Threonine Kinases; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; rho-Associated Kinases; Status Epilepticus

Baicalin, the main active ingredient in the Scutellaria baicalensis (SB), is prescribed for the treatment of various inflammatory diseases and tumors in clinics in China. In the present study, we evaluated the antitumor activity of baicalin for gallbladder carcinoma and the underlying mechanisms both in vitro and in vivo. Our results indicate that baicalin induced potent growth inhibition, cell cycle arrest, apoptosis and colony-formation inhibition in a dose-dependent manner in vitro. We observed inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, as well as increased caspase-3 and caspase-9 expression after baicalin treatment in vitro and in vivo, which indicates that the mitochondrial pathway was involved in baicalin-induced apoptosis. In addition, daily intraperitoneally injection of baicalin (15, 30 and 60 mg/kg) for 21 days significantly inhibited the growth of NOZ cells xenografts in nude mice, which improved the survival of baicalin-treated mice. In summary, baicalin exhibited a significant anti-tumor effect by suppressing cell proliferation, promoting apoptosis, and inducing cell cycle arrest in vitro, and by suppressing tumor growth and improving survival in vivo, which suggested that baicalin represents a novel therapeutic option for gallbladder carcinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B1; Cyclin D1; Flavonoids; Gallbladder Neoplasms; Heterografts; Humans; Membrane Potential, Mitochondrial; Mice, Nude; Mitochondria; Necrosis; Signal Transduction

Melanoma is a type of cancer that reaches more people in the world, characterized by genetic mutations that trigger the growth of disorganized cells. The diagnosis of skin tumors by invasive techniques has become a risk to the patients, so the search for new non-invasive techniques has been the subject of research in recent years. The objective of this work is to propose a non-invasive method prognosis based on the identification of specific biomarkers of the cancer, known as metabolomics analysis. For this study, we used B16F10 melanoma tumor cells and metabolic profiles were obtained at three time-periods by (1)HNMR and comparison with the cell cycle, apoptosis pathways and proliferation index. The metabolic profiles show the relationship between the metabolites found with energy metabolism, pathways of apoptosis and proliferation, which showed increases in proportion during growth and progression. Were found 29 metabolites, of which the differentially expressed are: lactate, aspartate, glycerol, lipids, alanine, myo-inositol, phosphocholine, choline, acetate, creatine and taurine. Choline and creatine are closely related with tumor progression, and are inversely expressed in later stages of tumor growth, which demonstrates the ability to be markers of tumor progression or monitoring the pharmacological efficacy when combined with other therapies. We conclude that the metabolome appeared as effective non-invasive technique predicts, besides providing possible biomarkers of melanoma.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Choline; Creatine; Cyclin D1; Disease Progression; Energy Metabolism; Female; Melanoma, Experimental; Metabolome; Metabolomics; Mice; Mice, Inbred C57BL; Necrosis; Prognosis; Skin Neoplasms

Cyclin D1 is important for pancreatic cancer growth. Our aim was to determine the effects of cyclin D1 inhibition on the growth of established pancreatic tumors.. PANC-1 cells harboring cyclin D1 antisense cDNA in a tetracycline-inducible vector system were prepared. The effects of cyclin D1 inhibition after tumor development were characterized in a mouse model.. In vitro removal of tetracycline induced cyclin D1 antisense cDNA expression and inhibited cyclin D1 expression and cyclin D1-associated kinase activity as well as anchorage-dependent and -independent growth. After establishment of xenograft tumors in the presence of tetracycline (2 mg/mL) in the drinking water, animals were assigned to either control (tetracycline remained in the drinking water) or to the group without tetracycline for which tetracycline was removed from the drinking water. Tumor growth was significantly inhibited after removal of tetracycline. Microscopic analysis revealed that the area of central necrosis was significantly increased in the group without tetracycline paralleled by a reduction of the vital peripheral area of proliferating cells.. Our results confirmed that cyclin D1 plays an important role in the growth of pancreatic cancer cells and may be an attractive molecular target for the treatment of human pancreatic cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA, Antisense; Female; Gene Expression Regulation; Genes, Reporter; Genetic Therapy; Humans; Mice; Mice, Nude; Necrosis; Pancreatic Neoplasms; Tetracycline; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N,N-dimethylamino-benzo[b]furans, and among these 3-iodoacetylamino-6-methoxybenzofuran-2-yl(3,5-trimethoxyphenyl)methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Benzamides; Benzofurans; Benzophenones; Bone Marrow Cells; Cell Line, Tumor; Colony-Forming Units Assay; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Fusion Proteins, bcr-abl; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Genes, bcl-2; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Necrosis; Neoplasm Proteins; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Resting Phase, Cell Cycle; STAT5 Transcription Factor

Nasopharyngeal carcinoma (NPC) is a highly malignant and frequently metastasized tumor, and the prognosis is very poor when distant metastases occur. Recently, immunotherapy is becoming a promising therapeutic approach. Interferon-α (IFN-α) represents the cytokines exhibiting the longest record of use in clinical oncology. In this study, we examined the antitumor effects of IFN-α1b on NPC. The results showed that recombinant human IFN-α1b (hIFN-α1b) suppressed cell growth, induced a G1-phase cell cycle arrest in vitro, increased the expression of p16 and pRb, and decreased the expression of CCND1 and CDK6. In vivo analyses showed that either recombinant adeno-associated virus (rAAV)-IFN-α1b or hIFN-α1b treatment inhibited tumor growth and metastasis, reduced intratumoral microvessel density, increased cell apoptosis and necrosis, and induced prolonged survival. Notably, rAAV-IFN-α1b or hIFN-α1b treatment led to significantly higher serum levels of IL-12 and GM-CSF in mice compared to respective controls. Our findings suggest that IFN-α1b acts as a multifunctional antitumor agent in NPC, which may have important therapeutic implications.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; G1 Phase; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-alpha; Interleukin-12; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Necrosis; Neoplasm Metastasis; Recombinant Proteins; Retinoblastoma Protein

Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 μg/ml in A549 cells over 22 h and at 10 μg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results.

Topics: Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Thin Layer; Chrysobalanaceae; Cyclin D1; DNA Fragmentation; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Euphorbia; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Necrosis; Neoplasms, Squamous Cell; Plant Extracts; Staurosporine; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Medulloblastoma (MB) is the most common malignant brain tumor in children. CyclinD1 is strongly implicated in the control of G1 progression and the phosphorylation of the retinoblastoma protein; p16 could specifically interact with cyclinD1 to inhibit the activity of cyclin-dependent kinase 4. The purpose of this study was to investigate whether quantitative assessment of cyclinD1 and p16 may predict the clinical prognosis in MB. We analyzed the expression of cyclinD1 and p16 antigens in a series of 42 MB with various grades and pathological types by immunohistochemical analysis on paraffin-embedded sections. Then, the correlation of cyclinD1 and p16 expression patterns with clinical-pathological features of patients and their prognostic relevance were determined. Compared with the normal cerebellum, expression of cyclinD1 in MB was significantly higher [69.05% (29/42) vs. 9.5% (4/42), P = 0.002], expression of p16 was significantly lower [42.9% (18/42) vs. 85.7% (36/42), P = 0.01]. CyclinD1 expression in MB was up-regulated in metastatic stage (P = 0.01), pathological type (P = 0.02), necrosis (P = 0.009), differentiation level (P = 0.01) as well as with differentiation direction (P = 0.03); p16 expression in MB was down-regulated with metastatic stage (P = 0.01), pathology type (P = 0.02), necrosis (P = 0.009) as well as with differentiation level (P = 0.01); negative significant correlation between p16 immunostaining and cyclinD1 was observed in MB (rs = -0.36, P = 0.02). We also found a statistically significant relationship between the percent of cyclinD1-positive cells and p16-positive cells in the tumors and clinical outcome, with higher levels of cyclinD1 and lower levels of p16 correlating with a worse prognosis. CyclinD1 and p16 may be independent biomarkers for clinical prognosis in MB. A combined detection of cyclinD1+/p16- expression may benefit us in prediction of a poor survival of MB.

Topics: Biomarkers, Tumor; Cell Differentiation; Cerebellar Neoplasms; Cerebellum; Child; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Down-Regulation; Female; Fourth Ventricle; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Genes, p16; Humans; Infant; Male; Medulloblastoma; Necrosis; Neoplasm Metastasis; Neoplasm Proteins; Prognosis; Up-Regulation

The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC(50) =13 microM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G(1)/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8h, baseline levels by 24h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21(cip-1), which suppressed the levels of cyclin D1 that controls the G(1)/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21(cip-1) was induced. When COX-2 expression was knocked down using siRNA against cox-2, the expression of p21(cip-1) was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS-->MAPKs-->p21(cip-1)-->cyclin D1-->cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Inhibitory Concentration 50; Necrosis; Nitric Oxide; Oxidation-Reduction; Pancreatic Neoplasms; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

Resolution of alveolar epithelial/capillary membrane damage after acute lung injury requires coordinated and effective tissue repair to reestablish a functional alveolar epithelial/capillary membrane barrier. We hypothesized that signaling pathways important in lung alveolar bud ontogeny are activated in the recovery and remodeling phases after profound oxidant stress lung injury in a murine model. To test this, we characterized the expression of noncanonical beta-catenin pathway proteins E-cadherin, integrin-linked kinase-1, and beta-catenin in mice undergoing normoxic recovery after exposure to butylated hydroxytoluene (BHT, ionol) and concomitant sublethal (75% O2) hyperoxia. Mice developed early acute lung injury with subsequent inflammation, collagen deposition, interstitial cellular proliferation, and lung architectural distortion. Reduced E-cadherin expression after 6 d of BHT and hyperoxia was accompanied by enhanced expression and nuclear localization of beta-catenin and increased integrin-linked kinase-1 expression during subsequent normoxic recovery. This resulted in increased expression of the cotranscriptional regulators TCF-1 and -3 and cyclin D1. Proliferation of murine lung epithelial-12 cells in vitro after 8 h of treatment with BHT quinone-methide and hyperoxia and 48 h of normoxic recovery was enhanced 2.7-fold compared with vehicle-treated control mice at the same time point. BHT/hyperoxia-exposed mice treated with the pan-caspase inhibitor z-ASP had increased acute lung injury and reduced survival despite the presence of TUNEL-positive cells, suggesting enhanced lung cell necrosis. Beta-catenin expression was reduced in z-ASP-co-treated lungs after BHT/hyperoxia. The noncanonical cadherin-beta-catenin axis is associated with fibroproliferative repair after BHT/hyperoxia exposure and may regulate epithelial proliferation and lung matrix remodeling and repair in response to lung injury.

Topics: Animals; Apoptosis; Asparagine; beta Catenin; Butylated Hydroxytoluene; Cadherins; Caspase Inhibitors; Caspases; Cell Line, Transformed; Cell Proliferation; Collagen; Cyclin D1; Epithelial Cells; Lung; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Oxidative Stress; Oxygen; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Respiratory Distress Syndrome

The diagnosis of hidradenocarcinoma is difficult due to a combination of factors including inconsistent nomenclature/ classification, rarity of the neoplasm, and variable morphology of cells composing the neoplasm. Immunohistochemistry has not been previously performed on a series of hidradenocarcinomas. We evaluated six cases of hidradenocarcinoma histologically and immunohistochemically using antibodies to gross cystic disease fluid protein-15 (GCDFP-15), carcino-embryonic antigen (CEA), epithelial membrane antigen (EMA), S-100 protein, keratin AE1/3, cytokeratin 5/6, p53, bcl-1, bcl-2, and Ki67. Histology suggested concurrent eccrine and apocrine differentiation of the cases. Ki67 and p53 staining was strongly positive in five of six tumors. The neoplasms stained with antibodies to CEA, S-100 protein, GCDFP-15, EMA, bcl-1, and bcl-2 in no consistent pattern. All tumors studied stained positively for keratin AE1/3 and cytokeratin 5/6. In making the diagnosis of hidradenocarcinoma, it may be unnecessary to separate hidradenocarcinoma into eccrine and apocrine categories, and although Ki67 and p53 may be helpful, histological parameters remain paramount.

Topics: Adenoma, Sweat Gland; Adult; Aged; Carcinoembryonic Antigen; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Keratins; Ki-67 Antigen; Male; Middle Aged; Mitosis; Mucin-1; Necrosis; Proto-Oncogene Proteins c-bcl-2; S100 Proteins; Sweat Gland Neoplasms; Tumor Suppressor Protein p53

Alvocidib (Flavopiridol, HMR1275) is a potent inhibitor of multiple cyclin-dependent kinases and has been identified recently as an antitumor agent in several cancers. Previous studies have shown that alvocidib could potentially treat esophageal cancer in vitro. This study evaluates alvocidib for its ability to suppress tumor growth in severe combined immunodeficiency (SCID) mice bearing TE8 human esophageal squamous cell carcinoma (SCC) xenografts. Alvocidib treatment of 10mg/kg body weight reduced tumor volume significantly. Immunohistochemistry analysis of alvocidib-treated tumor sections showed significant reductions in cyclin D1, VEGF, and Rb levels. Alvocidib treatment did not cause a marked increase in apoptotic tumor cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis, yet hematoxylin and eosin staining revealed tumor necrosis. In vivo investigation of alvocidib treatment confirmed antitumor activity in TE8 esophageal xenografts. These findings suggest that alvocidib could be a useful anti-cancer agent for esophageal cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclin D1; Esophageal Neoplasms; Female; Flavonoids; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, SCID; Necrosis; Neoplasm Transplantation; Piperidines; Retinoblastoma Protein; Vascular Endothelial Growth Factor A

The mechanisms by which polyglutamine expansion causes common features of neuronal death remain unclear. Here we describe an approach for delivering polyglutamine expansions directly into cultured sympathetic neurons. Glutamine (Q) residues (n = 10, 22, 30) were conjugated with a peptide possessing translocation properties across plasma membranes (PDP) and a nuclear localization signal (NLS). These peptides were rapidly incorporated into sympathetic neurons and showed neurotoxicity in a length- and dose-dependent manner. A robust induction of c-jun and cyclin D1 occurred following treatment with PDP-Q22-NLS. Enhanced c-Jun phosphorylation showed c-Jun N-terminal kinase (JNK) activation. Coincidentally, TrkA tyrosine phosphorylation was decreased in association with loss of phospho-Akt, the downstream target of PI-3 kinase. Despite such proapoptotic signals, neither release of cytochrome c from mitochondria nor caspase-3/7 activation was detected. TdT-mediated dUTP nick-end labeling-positive nuclear condensation, but no fragmentation, occurred. At 24 hr of treatment, cytoplasmic Ca2+ levels began to become elevated, and the cellular level of ATP was decreased. Cytoplasmic Ca2+ responses to KCl depolarization displayed a delayed recovery, providing evidence for lack of Ca2+ homeostasis. The neurons became committed to death at about 36 hr when mitochondrial Ca2+ uptake declined concurrently with loss of mitochondrial membrane potential. Collectively, these results show that, despite induction of early apoptotic signals, nonapoptotic neuronal cell death occurred via perturbed Ca2+ homeostasis and suggest that mitochondrial permeability transition may play important roles in this model of neuronal death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Caspase 3; Caspase 7; Caspases; Cell Count; Cells, Cultured; Colforsin; Cyclin D1; Cycloheximide; Dihydrotachysterol; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Homeostasis; Immunohistochemistry; In Situ Nick-End Labeling; Ionophores; Lactic Acid; Mitochondria; Models, Biological; Necrosis; Nerve Growth Factor; Neurons; Neuroprotective Agents; Peptides; Permeability; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-jun; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staurosporine; Superior Cervical Ganglion; Time Factors

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are active in cancer therapy. Mechanisms engaged during these clinical responses need to be determined. We reported previously that epidermal growth factor stimulation markedly increased cyclin D1 protein expression in human bronchial epithelial (HBE) cells, and this was opposed by chemoprevention with all-trans-retinoic acid. The current study sought to determine whether the EGFR TKI erlotinib repressed cyclin D1 protein expression in immortalized HBE cells, lung cancer cell lines, and clinical aerodigestive tract cancers.. The BEAS-2B immortalized HBE cell line was exposed to varying concentrations of erlotinib, and effects on proliferation, cell cycle distribution, G1 cyclin expression, and cyclin D1 reporter activity were measured. Non-small-cell lung cancer cell lines were also evaluated for changes in proliferation and cyclin protein expression after erlotinib treatments. A proof of principle clinical trial was conducted. During this study, patients underwent a 9-day course of erlotinib treatment. Pretreatment and posttreatment tumor biopsies were obtained, and changes in candidate biomarkers were determined by immunostaining. Plasma pharmacokinetics and tumor tissue erlotinib concentrations were measured.. Erlotinib, at clinically achievable dosages, repressed BEAS-2B cell growth, triggered G1 arrest, and preferentially reduced cyclin D1 protein expression and transcriptional activation. Erlotinib also preferentially repressed proliferation and cyclin D1 protein expression in responsive, but not resistant, non-small-cell lung cancer cell lines. This occurred in the presence of wild-type EGFR sequence at exons 18, 19, and 21. Five patients were enrolled onto an erlotinib proof of principle clinical trial, and four cases were evaluable. Pharmacokinetic studies established therapeutic erlotinib plasma levels in all patients, but tissue levels exceeding 2 micromol/L were detected in only two cases. Notably, these cases had pathological evidence of response (necrosis) in posttreatment biopsies as compared with pretreatment biopsies. In these cases, marked repression of cyclin D1 and the proliferation marker Ki-67 was detected by immunohistochemical assays. Cases without pathological response to erlotinib did not exhibit changes in cyclin D1 or Ki-67 immunohistochemical expression and had much lower erlotinib tissue levels than did responding cases.. Taken together, these in vitro and in vivo findings provide direct evidence for repression of cyclin D1 protein as a surrogate marker of response in aerodigestive tract cancers to erlotinib treatment. These findings also provide a rationale for combining an EGFR TKI with an agent that would cooperatively repress cyclin D1 expression in clinical trials for aerodigestive tract cancer therapy or chemoprevention.

Topics: Biomarkers, Tumor; Bronchi; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Cyclin D1; DNA; Dose-Response Relationship, Drug; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Exons; G1 Phase; Gastrointestinal Neoplasms; Gastrointestinal Tract; Humans; Immunoblotting; Immunohistochemistry; Ki-67 Antigen; Kinetics; Luciferases; Necrosis; Neoplasms; Quinazolines; Sequence Analysis, DNA; Time Factors; Transcriptional Activation

Patients diagnosed with ductal carcinoma in situ (DCIS) at a young age appear to have a different natural history and biology, including a higher local relapse rate, than patients diagnosed later in life. The current study compared various pathologic and molecular features of DCIS arising in a cohort of young women with those of DCIS arising in a cohort of older women to identify potential biologic differences between these two populations of patients.. The study population consisted of 20 patients age < 42 years and 34 patients age > 60 years who were treated at Yale University School of Medicine with breast-conserving therapy (BCT) and whose archival paraffin blocks were available and had sufficient tumor for staining. The original slides from each case were reviewed and the most representative specimen block from each case was processed for immunohistochemical staining. Pathologic characteristics evaluated for each case included histology, grade, and presence of necrosis. Paraffin-embedded sections were immunohistochemically evaluated for expression of HER-2/neu, estrogen receptor (ER), progesterone receptor (PR), bcl-2, cyclin D1, Ki-67, and p53.. Although there was no difference in pathologic features of the tumors between the two groups, HER-2/neu was found to be overexpressed in a greater percentage of the younger population (P = 0.06). There was no apparent difference in expression of the other markers. Of note, HER-2/neu expression was correlated with high nuclear grade (P = 0.004), necrosis (P = 0.06), and ER and PR negativity (P = 0.01 and P = 0.03, respectively) in the combined population.. The current study data suggested that HER-2/neu overexpression in younger patients may characterize a biologic difference in their tumor and may partially contribute to their higher risk of recurrence. Further studies are needed to assess whether this difference holds independent of grade and to evaluate the prognostic significance of HER-2/neu overexpression in DCIS.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cohort Studies; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Necrosis; Neoplasm Recurrence, Local; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Risk Factors; Tumor Suppressor Protein p53

Profound impairment of liver regeneration in rodents with dysfunctional leptin signaling has been attributed to non-alcohol-induced fatty liver disorders (NAFLD). Our aim was to establish whether defective liver regeneration in ob/ob mice is a direct consequence of leptin-dependent, intracellular signaling mechanisms controlling cell-cycle regulation in hepatocytes.. After exposure to a single hepatotoxic dose of (CCl(4)), the regenerative response to hepatic injury was studied in leptin-deficient ob/ob and control mice. The effects of leptin supplementation (100 microg x kg(-1) x day(-1)) were examined. We assessed entry into and progression through the cell cycle and activation of key signaling intermediates and transcriptional regulators.. CCl(4)-induced liver injury was equally severe in ob/ob and control mice. In leptin-deficient mice, it was associated with exaggerated activation of NF-kappa B and STAT3 during the priming phase, abrogation of tumor necrosis factor (TNF) and interleukin (IL)-6 release at the time of G1/S transition, and failure of hepatocyte induction of cyclin D1 and cell cycle entry. Leptin replacement corrected these defects in ob/ob mice by restoring TNF and IL-6 release and inducing cyclin D1. Hepatocytes entered S phase and progressed, as in wild-type mice, to vigorous mitosis and normal hepatic regenerative response. In ob/ob mice, low doses of TNF before CCl(4) also were associated with restitution of TNF release and proliferative capabilities.. Impaired liver regeneration in ob/ob mice is caused by leptin deficiency. We propose that altered cytokine production in ob/ob mice is part of the mechanisms responsible for impaired proliferation in response to hepatic injury.

Topics: Animals; Carbon Tetrachloride; Cell Division; Chemical and Drug Induced Liver Injury; Cyclin D1; Fatty Liver; Interleukin-6; Leptin; Liver; Liver Regeneration; Mice; Mice, Inbred C57BL; Mice, Obese; Necrosis; Proliferating Cell Nuclear Antigen; Recombinant Proteins; Signal Transduction; Tumor Necrosis Factor-alpha

To determine the role of Bone morphogenetic protein (BMP) signaling in murine limb development in vivo, the keratin 14 promoter was used to drive expression of the BMP antagonist Noggin in transgenic mice. Phosphorylation and nuclear translocation of Smad1/5 were dramatically reduced in limbs of the transgenic animals, confirming the inhibition of BMP signaling. These mice developed extensive limb soft tissue syndactyly and postaxial polydactyly. Apoptosis in the developing limb necrotic zones was reduced with incomplete regression of the interdigital tissue. The postaxial extra digit is also consistent with a role for BMPs in regulating apoptosis. Furthermore, there was persistent expression of Fgf8, suggesting a delay in the regression of the AER. However, Msx1 and Msx2 expression was unchanged in these transgenic mice, implying that induction of these genes is not essential for mediating BMP-induced interdigital apoptosis in mice. These abnormalities were rescued by coexpressing BMP4 under the same promoter in double transgenic mice, suggesting that the limb abnormalities are a direct effect of inhibiting BMP signaling.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Carrier Proteins; Cyclin D1; DNA-Binding Proteins; Extremities; Female; Fibroblast Growth Factor 8; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Hedgehog Proteins; Homeodomain Proteins; Keratin-14; Keratins; Male; Mice; Mice, Transgenic; MSX1 Transcription Factor; Necrosis; Phosphorylation; Proteins; Signal Transduction; Syndactyly; Trans-Activators; Transcription Factors; Zebrafish Proteins

Abnormalities of the G1 cell-cycle checkpoint are commonly reported in cancers at various anatomic sites. pRB, p16(INK4a) and cyclin D1 are critical G1-checkpoint proteins responsible for maintaining the balance of cellular proliferation. We examined a series of 38 pediatric osteosarcomas for abnormal expression of pRB, p16(INK4a) and cyclin D1 by immunohistochemical analysis of archival biopsy specimens. Overall, 17/38 (45%) osteosarcomas showed evidence of G1-checkpoint abrogation, including 11/38 (29%) with loss of pRB expression and 6/38 (16%) with loss of p16(INK4a) expression. Cyclin D1 over-expression was not detected. There was an inverse correlation between loss of pRB and p16(INK4a) expression (p = 0.07). pRB and p16(INK4a) abnormalities were independent of site of disease, presence of metastasis at diagnosis and percentage of tumor necrosis in the resection specimen. Clinical follow-up was available on all patients (median 31.6 months, range 5.9-116 months). Absence of p16(INK4a) expression significantly correlated with decreased survival in univariate analysis (p = 0.03), while loss of pRB expression did not affect survival. Immunohistochemical analysis of p16(INK4a) expression in pediatric osteosarcomas may be a useful adjunctive marker of prognosis.

Topics: Adolescent; Adult; Biopsy; Bone Neoplasms; Child; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Down-Regulation; Female; G1 Phase; Humans; Immunohistochemistry; Male; Necrosis; Neoplasm Metastasis; Osteosarcoma; Prognosis; Retinoblastoma Protein; Time Factors

Sepsis is the leading cause of death in surgical intensive care units. Although both mild sepsis secondary to cecal ligation and single puncture (CLP) and fulminant, double puncture CLP (2CLP) may provoke hepatocyte death, we hypothesize that regeneration compensates for cell death after CLP but not 2CLP. In male Sprague-Dawley rats, hepatic necrosis, as determined by serum alpha-glutathione S-transferase (alpha-GST) levels, was significantly but equally elevated over time after both CLP and 2CLP. Apoptosis, evaluated using both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and morphological examination, was minimal after both CLP and 2CLP. Regeneration, assayed by staining tissue for incorporation of exogenously administered bromodeoxyuridine, was present after CLP but not after 2CLP. To further substantiate impaired regeneration, steady-state levels of mRNAs encoding JunB, LRF-1, and cyclin D1 were determined. After 2CLP, the absence of JunB, LRF-1, and cyclin D1 mRNAs confirmed failed activation of the mitogen-activated protein kinase-linked proliferative pathway and progression through the cell cycle. Therefore, failed hepatocyte regeneration may be a manifestation of hepatic dysfunction in fulminant sepsis.

Topics: Activating Transcription Factor 3; Animals; Apoptosis; Biomarkers; Cecum; Cyclin D1; DNA-Binding Proteins; Glutathione Transferase; In Situ Nick-End Labeling; Liver; Liver Regeneration; Male; Necrosis; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sepsis; Time Factors; Transcription, Genetic

In the present study, we report the cyclin-dependent kinase (Cdk)-inhibitory activity of a series of p21waf1/cip1 (p21) peptide fragments spanning the whole protein against the cyclin D1/Cdk4 and cyclin E/Cdk2 enzymes. The most potent p21 peptide tested in our initial peptide series, designated W10, spanned amino acids 139 to 164, a region of p21 that has been found independently to bind to proliferating cell nuclear antigen and also to inhibit Cdk activity. We go on to report the importance of putative beta-strand and 3(10)-helix motifs in the W10 peptide for cyclin-dependent kinase-inhibitory activity. We also describe the cellular activity of W10 and derivatives that were chemically linked to an antennapedia peptide, the latter segment acting as a cell membrane carrier. We found that the W10AP peptide exhibited growth inhibition that resulted from necrosis in human lymphoma CA46 cells. Furthermore, regions in the W10 peptide responsible for Cdk-inhibition were also important for the degree of this cellular activity. These studies provide insights that may eventually, through further design, yield agents for the therapy of cancer.

Topics: Amino Acid Sequence; Antennapedia Homeodomain Protein; Burkitt Lymphoma; Cell Membrane; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Flow Cytometry; Homeodomain Proteins; Humans; Microscopy, Electron; Molecular Sequence Data; Necrosis; Neoplasm Proteins; Nuclear Proteins; Peptide Fragments; Proliferating Cell Nuclear Antigen; Structure-Activity Relationship; Transcription Factors; Tumor Cells, Cultured

In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and low differentiation degree in large cell carcinoma (LCLC) and is regulated by bcl-2 family proteins, we analysed the extent of apoptosis and tumor necrosis and their relation to the expression of bcl-2, bax, bak and mcl-1 in 35 LCLCs, of which 20 were classified as large cell neuroendocrine lung carcinomas (LCNEC) and 15 as large cell non-neuroendocrine lung carcinomas (LCNNEC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using in situ 3 -end labelling of the apoptotic DNA. The extent and intensity of expression of the bcl-2, bax, bak and mcl-1 proteins were studied by immunohistochemistry. Also the relative volume density of necrosis was evaluated and correlated with the other parameters. Finally, all the parameters were evaluated as prognostic markers and correlated with data on the survival of the patients. Relatively high apoptotic indices were seen in both tumor types (average for both 2.53%, range 0.09 27.01%). Significantly higher bcl-2 and bak indices were detected more often in LCNECs than in LCNNECs. Immunohistochemically detected bax, bcl-2 and bak expression was independent of apoptotic index in both tumor types, while there was a statistically significant positive association between mcl-1 expression and apoptotic index in LCNNEC but not in LCNEC. There was a statistically significant association between high apoptotic index and shortened survival in LCLC. However, no association was found between tumor stage and apoptosis. The patients with LCNEC and low bcl-2 protein expression had a significantly shorter survival time than those with high bcl-2 indices. There was also a clear association between shortened survival and necrotic LCNNEC. LCLCs show relatively high apoptotic activity, which is associated with shortened survival. The expression of bcl-2, bak and mcl- 1 is associated with neuroendocrine differentiation in LCLC. Finally, our results support some previous reports suggesting that bcl-2 expression in combination with some other markers involved in apoptosis and/or proliferation may be of prognostic value in cases of lung carcinoma with neuroendocrine differentiation.

Topics: Adult; Aged; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Neuroendocrine; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cyclin D1; Female; Follow-Up Studies; Humans; Immunohistochemistry; Lung Neoplasms; Male; Membrane Proteins; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Necrosis; Neoplasm Proteins; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Survival Rate

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle and it is characterized by increased expression of cyclins, such as cyclin D1, prior to death. Cyclin D1 increases to permit transition from growth phase (G0/G1) to synthesis phase (S) during normal development but there is controversy as to which of the cyclins are activated prior to apoptotic cell death. We looked at the expression of cyclin D1 in cortical neuronal cultures treated with either staurosporine to produce apoptotic death, or with glutamate, to produce a non-apoptotic death. Cyclin D1 immunoreactivity was observed in the cytoplasm and nucleus of virtually all neurons under control conditions. Following the addition of either staurosporine or glutamate, cyclin D1 immunoreactivity did not change within 4 h. The cyclin D1 immunoreactivity was lost by 6 h with the appearance of either staurosporine-induced fragmented nuclei or glutamate-induced pyknotic nuclei. These immunocytochemical observations were confirmed with immunoblot analysis. Therefore, cyclin D1 is not a reliable indicator of apoptosis in cortical neuronal cultures and should not be used as an indicator of apoptotic cell death.

Topics: Animals; Apoptosis; Cell Cycle; Cells, Cultured; Cerebral Cortex; Cyclin D1; Fetus; G1 Phase; Glutamic Acid; Kinetics; Models, Neurological; Necrosis; Nerve Degeneration; Neurons; Rats; Resting Phase, Cell Cycle; S Phase; Staurosporine

Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response.

Topics: Animals; Cyclin D1; Cyclins; DNA; DNA-Binding Proteins; G1 Phase; Gene Expression Regulation; Gene Targeting; Genes, Immediate-Early; Hepatectomy; Interleukin-6; Liver; Liver Failure; Liver Regeneration; Mice; Mice, Inbred C57BL; Mitosis; Mutation; Necrosis; Oncogene Proteins; Proto-Oncogene Proteins c-myc; STAT3 Transcription Factor; Trans-Activators; Transcription Factor AP-1