cyclin-d1 and Retinoblastoma

Topics: Apoptosis; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Genes, p53; Humans; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins p21(ras); Retinoblastoma; Thrombospondins; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

This study aims to investigate cordycepin's effects on the proliferation, migration, and invasion of retinoblastoma (RB) cells and its regulatory mechanism. In this study, it was found that cordycepin inhibited RB cell proliferation, migration, and invasion in vitro, and pulmonary metastasis in vivo. c-Myc was a downstream target of cordycepin, and cordycepin significantly suppressed c-Myc expression, and c-Myc overexpression markedly counteracted the impacts of cordycepin on RB cell proliferation, migration, and invasion. c-Myc was positively correlated with the cell cycle pathway. Cordycepin restrained cyclin D1 expression, and c-Myc overexpression rescued this effect. In conclusion, cordycepin targets the c-Myc/cyclin D1 pathway, thereby suppressing the malignant biological behaviors of RB cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; Retinal Neoplasms; Retinoblastoma

Krüppel-like factor 2 (KLF2) belongs to the KLF family of zinc-finger transcription factors and mediates the occurrence and progression of various cancers. However, little is known about its expression pattern and biological role in retinoblastoma (RB). In the present study, we showed that KLF2 was markedly downregulated in human RB tissue compared with retina. KLF2 overexpression significantly inhibited RB cell proliferation and decreased proliferating cell nuclear antigen (PCNA) expression. Subsequently, we confirmed that KLF2 arrested cells at the G1-S phase transition, accompanied by the upregulation of p21 and downregulation of CyclinD1, as well as the activation of mitochondria-mediated apoptosis in RB cells. In addition, KLF2 overexpression contributed to suppressing RB cell migration and invasion by downregulating matrix metallopeptidase 9 (MMP9). On the contrary, KLF2 downregulation promoted RB cells proliferation, migration and invasion. Notably, the KLF2 expression pattern was opposite to that of C-X-C chemokine receptor 4 (CXCR4) in the two RB cell lines, KLF2 overexpression significantly decreased CXCR4 expression, silencing KLF2 had the opposite effect. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that KLF2 directly bound to the CXCR4 promoter and negatively regulated its expression in RB cells. Collectively, our results suggested that KLF2 function as a tumor suppressor in RB and may represent a potential therapeutic target for RB.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; In Situ Nick-End Labeling; Kruppel-Like Transcription Factors; p21-Activated Kinases; Plasmids; Proliferating Cell Nuclear Antigen; Real-Time Polymerase Chain Reaction; Retinal Neoplasms; Retinoblastoma; RNA, Small Interfering; Transfection; Tumor Suppressor Proteins

Topics: Antineoplastic Agents; Carboplatin; Caspase 3; Cell Line, Tumor; Child; Cyclin D1; Cyclin D3; Humans; Retinal Neoplasms; Retinoblastoma

Mesenchymal epithelial transition (C-MET) factor overexpression has been found in many types of cancer and has served as an important molecular target for therapeutic intervention. However, the role of C-MET in retinoblastoma remains largely unclear. The present study aimed to investigate the potential role and mechanism of C-MET in Y79 retinoblastoma cells. We found that C-MET was highly expressed in Y79 retinoblastoma cells, and, in addition, the levels of C-MET were positively correlated with cell proliferation and retinoblastoma growth. Inhibition of C-MET suppressed Y79 retinoblastoma cell proliferation and tumour growth. Mechanistically, we showed that HGF-induced C-MET-dependent signal transduction resulted in ERK 1/2 phosphorylation, which subsequently promoted the nuclear translocation of PKM2. Nuclear PKM2 further interacted with histone H3 and contributed to C-MET-dependent cyclin D1 and c-Myc expression and cell proliferation. These findings highlight the role of C-MET in Y79 retinoblastoma cells and reveal a C-MET-dependent signal transduction mechanism. C-MET may be a potential therapeutic target for retinoblastoma. SIGNIFICANCE OF THE STUDY: We demonstrated a new target of retinoblastoma, C-MET. C-MET-dependent signal transduction promotes Y79 retinoblastoma cell proliferation and tumour growth through ERK 1/2/PKM2/histone H3 signalling pathway. C-MET may be a potential target for retinoblastoma therapy.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Histones; Humans; Male; Membrane Proteins; Mice; Mice, Nude; Neoplasm Transplantation; Phosphorylation; Proto-Oncogene Proteins c-met; Proto-Oncogene Proteins c-myc; Pyruvate Kinase; Retinoblastoma; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones

Retinoblastoma is an ocular malignancy occurring in childhood. The current study evaluates the ability of silenced PRC1 on retinoblastoma cell proliferation, and angiogenesis via the Wnt/β-catenin signaling pathway. A total of 36 cases of retinoblastoma tissues (n = 36) and normal retinal tissues (n = 10) were selected in the current study. Retinoblastoma cells presenting with the high PRC1 messenger RNA (mRNA) expression were selected among the WERI-Rb-1, HXO-RB44, Y79, SO-Rb50, and SO-Rb70 cells lines, and were transfected with siRNA-PRC1 and LiCl (the activator of the Wnt/β-catenin pathway). The expressions of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin, and GSK-3β phosphorylation were evaluated. Cell proliferation, cell-cycle distribution, and cell invasion of retinoblastoma cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Transwell assay. The angiogenesis of retinoblastoma cells was detected by tube formation assay. HXO-RB44 and WERI-Rb-1 cells were selected owing to the highest PRC1 mRNA expression. Meanwhile, PRC2 gene silencing presented lower expression levels of PRC1, VEGF, Wnt1, β-catenin, CyclinD1, extent of β-catenin and GSK-3β phosphorylation, decreased proliferation and invasion abilities, extended G0/G1 phase, and shortened S and G2/M phases of HXO-RB44 and WERI-Rb-1 cells, suggesting the silenced PRC2 inactivated Wnt/β-catenin pathway, so as to further restrain the retinoblastoma cell proliferation, invasion, and angiogenesis. These results support the view that PRC1 gene silencing could suppress the proliferation, and angiogenesis of retinoblastoma cells by repressing the Wnt/β-catenin pathway.

Topics: beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphorylation; Retinal Neoplasms; Retinoblastoma; RNA Interference; RNA, Small Interfering; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway; Wnt1 Protein

Although the cure rate for retinoblastoma is high, surviving patients are at risk for developing secondary cancers and require life-long follow-up. It is imperative to discover and develop novel therapeutic agents with better efficiency and fewer adverse effects. Ginsenoside-Rg5 is an active derivate from ginseng and exerts anti-cancer activity in breast cancer cells. However, it is still unclear whether ginsenoside-Rg5 has similar anti-cancer functions in retinoblastoma.. Retinoblastoma cells were treated with ginsenoside-Rg5, followed by MTT assay analysis of the cell viability, cell number assay and colony formation assay analyses of cell proliferation, and flow cytometric analysis of apoptosis. Gene mRNA levels and protein levels were determined by quantitative real-time PCR and Western blot, respectively.. Ginsenoside-Rg5 inhibited retinoblastoma cell viability in a dose-dependent and time-dependent manner via preventing cell proliferation and inducing cell apoptosis. BCL2 expression was downregulated by ginsenoside-Rg5 treatment via inactivating the AKT signaling pathway. BCL2 overexpression completely eliminated the inhibitory effect of ginsenoside-Rg5 on cancer cell viability.. Ginsenoside-Rg5 inhibits cell proliferation and induces apoptosis in retinoblastoma cells by inactivating the AKT signaling pathway, thereby downregulating BCL2 expression.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Ginsenosides; Humans; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma

This study aimed to investigate the effects of SOST and the Wnt/β-catenin signaling pathway on the proliferation, migration, invasion, and apoptosis of human retinoblastoma cells. Fifty-five retinoblastoma and 21 normal retinal tissue samples were collected as the case group and control group, respectively. HXO-RB44 and SO-RB50 cells were selected and assigned into blank, negative control (NC), siRNA 1, siRNA 2, siRNA 3, IWR-1-endo 1, IWR-1-endo 2 and IWR-1-endo 3 groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of SOST, Wnt-1, and β-catenin in the collected tissue samples. MTT assay, flow cytometry, transwell assay and the starch test were employed to determine the cell proliferation, cell cycle, apoptosis, invasion and migration after transfection. The qRT-PCR and western blotting were also used to detect the mRNA and protein expressions of SOST, Wnt-1, β-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9. The tumor formation in nude mice was conducted to evaluate the effects of SOST on the growth of a transplanted tumor. Compared with normal retinal tissues, the retinoblastoma tissues exhibited a downregulation of SOST but an upregulation of Wnt-1 and β-catenin. The proliferation, invasion and migration of HXO-RB44 and SO-RB50 cells in the SOST-siRNA group were significantly higher than the cells in the blank and NC groups. The expressions of Wnt-1, β-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9 in the three SOST-siRNA groups were elevated, but the SOST decreased when compared with the blank and NC groups. SOST silencing promoted the growth of transplanted tumors in nude mice. These findings indicate that SOST silencing promotes the proliferation, invasion and migration, and decreases the apoptosis of human retinoblastoma cells by activating the Wnt/β-catenin signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; beta Catenin; Bone Morphogenetic Proteins; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Child; Child, Preschool; Cyclin D1; Female; Gene Silencing; Genetic Markers; Humans; Infant; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Retinoblastoma; Wnt Signaling Pathway; Wnt1 Protein

Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1-CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation-quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.

Topics: Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; E2F Transcription Factors; Humans; Mitogens; Mitosis; Retinoblastoma; Signal Transduction; Stress, Physiological; Tumor Suppressor Protein p53

Although the eukaryotic translation initiation factor 4E (eIF4E) has been shown to be critically involved in the transformation and progression of various tumors, little is known about the role of eIF4E in retinoblastoma. In this work, we report that ribavirin, a pharmacologic inhibitor of eIF4E function, effectively targets retinoblastoma and angiogenesis. Ribavirin treatment dose-dependently blocked the growth and stimulated apoptosis in various retinoblastoma cell lines, with IC

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Endothelial Cells; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Neovascularization, Pathologic; Proto-Oncogene Proteins c-myb; Retinoblastoma; Ribavirin; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

Euphorbia tirucalli is a long‑established treatment for a wide variety of cancers. However, the mechanism of its anticancer effect is yet to be elucidated. In the present study, we examined the anticancer effect of euphol, a tetracyclic triterpene alcohol isolated from the sap of Euphorbia tirucalli, in T47D human breast cancer cells. Following the treatment of cells with different doses of euphol for 24, 48 and 72 h, the cell proliferation, cell cycle, and mRNA and protein levels of cell cycle regulatory molecules were analyzed, respectively. Treatment of the cells with euphol resulted in decreased cell viability, which was accompanied by an accumulation of cells in the G1 phase. Further studies demonstrated that euphol treatment downregulated cyclin D1 expression and the hypophosphorylation of Rb. Furthermore, this effect was correlated with the downregulation of cyclin‑dependent kinase 2 (CDK2) expression and the upregulation of the CDK inhibitors p21 and p27. Reduced expression levels of cyclin A and B1 were also observed, corresponding to the decreased distribution of cells in the S and G2/M phases, respectively. These findings indicated that euphol is an active agent in Euphorbia tirucalli that exerts anticancer activity by arresting the cell cycle of cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Screening Assays, Antitumor; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lanosterol; Retinoblastoma; Transcription, Genetic

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal-regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant "low-AKT" myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in "high-AKT" myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Multiple Myeloma; Phosphorylation; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Retinoblastoma; Ribosomes; Sirolimus; TOR Serine-Threonine Kinases

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Topics: Animals; Brain Neoplasms; Cell Growth Processes; Child, Preschool; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Eye Proteins; Genes, bcl-1; Genes, p53; Humans; Hyperplasia; Infant; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroectodermal Tumors, Primitive; Phosphorylation; Pineal Gland; Retinoblastoma; Retinoblastoma Protein; Retinol-Binding Proteins

Matrix attachment region binding proteins have been shown to play an important role in gene regulation by altering chromatin in a stage- and tissue-specific manner. Our previous studies report that SMAR1, a matrix-associated protein, regresses B16-F1-induced tumors in mice. Here we show SMAR1 targets the cyclin D1 promoter, a gene product whose dysregulation is attributed to breast malignancies. Our studies reveal that SMAR1 represses cyclin D1 gene expression, which can be reversed by small interfering RNA specific to SMAR1. We demonstrate that SMAR1 interacts with histone deacetylation complex 1, SIN3, and pocket retinoblastomas to form a multiprotein repressor complex. This interaction is mediated by the SMAR1(160-350) domain. Our data suggest SMAR1 recruits a repressor complex to the cyclin D1 promoter that results in deacetylation of chromatin at that locus, which spreads to a distance of at least the 5 kb studied upstream of the cyclin D1 promoter. Interestingly, we find that the high induction of cyclin D1 in breast cancer cell lines can be correlated to the decreased levels of SMAR1 in these lines. Our results establish the molecular mechanism exhibited by SMAR1 to regulate cyclin D1 by modification of chromatin.

Topics: Binding Sites; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Size; Chromatin; Chromatin Immunoprecipitation; Cyclin D1; DNA Primers; DNA-Binding Proteins; G1 Phase; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Histone Deacetylase 1; Histone Deacetylases; Histones; Humans; Immunoblotting; Immunoprecipitation; Luciferases; Nuclear Proteins; Promoter Regions, Genetic; Protein Structure, Tertiary; Recombinant Fusion Proteins; Repressor Proteins; Retinoblastoma; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; S Phase; Saccharomyces cerevisiae Proteins; Sin3 Histone Deacetylase and Corepressor Complex; Time Factors; Transcription Factors

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

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis.

Topics: Animals; Blotting, Western; Cell Cycle; Cell Proliferation; Coculture Techniques; Culture Media; Cyclin D1; Flow Cytometry; Helminth Proteins; Mice; NIH 3T3 Cells; Opisthorchiasis; Opisthorchis; Retinoblastoma

Lymph node metastasis is a major prognostic factor for esophageal squamous cell carcinoma (ESCC). In recent years, endoscopic mucosal resection (EMR) has been developed with excellent results for the treatment of the superficial ESCC. To make the EMR treatment successful, it is important to establish a good indicator to identify ESCC patients at a high risk of lymph node metastasis. In this study, we examined clinicopathological and immunohistochemical factors to investigate the factors involved in lymph node metastasis of ESCC invading to the submucosal layer (sm-ESCC). Surgical specimens from 84 sm-ESCC patients were examined. Among 84 sm-ESCC patients, 33 (39.3%) had lymph node metastases. Clinicopathologically, tumor depth, lymphatic invasion and blood vessel invasion showed significant correlations with lymph node metastasis by univariate analysis. Tumor depth and lymphatic invasion showed significant correlations by multivariate analysis of these factors. Immunohistochemically, P53 accumulation was observed in 45 cases (53.6%), cyclin D1 overexpression in 25 (29.8%), and pRB in 65 (77.4%). P53 accumulation, cyclin D1 overexpression and MIB-1 Labeling Index were significantly associated with lymph node metastasis by univariate analysis, and P53 accumulation showed a significant correlation with lymph node metastasis by multivariate analysis. Among tumor depth, lymphatic invasion and P53 accumulation, tumor depth and lymphatic invasion were significantly correlated with lymph node metastasis (P = 0.0023 and P = 0.0092, respectively) by multivariate analysis. These data suggest that tumor depth and lymphatic invasion can be considered as good indicators for lymph node metastasis among patients with sm-ESCC. In addition, P53 accumulation could be helpful to identify the patients who need additional treatment after EMR.

Topics: Adult; Aged; Aged, 80 and over; Antigens, Nuclear; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cyclin D1; Esophageal Neoplasms; Female; Humans; Ki-67 Antigen; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Mucous Membrane; Neoplasm Invasiveness; Nuclear Proteins; Prognosis; Retinoblastoma; Survival Rate; Tumor Suppressor Protein p53

Second nonocular malignancies develop with increased incidence in patients with hereditary retinoblastoma. Osteosarcoma is by far the most common type with an incidence of up to 50%, followed by soft tissue sarcomas. Visceral leiomyosarcoma is extremely rare and only 2 cases have been reported in the past 2 decades, one in the liver and another one in the urinary bladder, both of which developed after cyclophosphamide therapy. Here we report a case of vesical leiomyosarcoma that was diagnosed in a 49-year-old woman 47 years after the diagnosis of a hereditary retinoblastoma. The patient's retinoblastoma was treated with unilateral enucleation without adjuvant radiation or chemotherapy. We believe that this is the first report of vesical leiomyosarcoma occurring in a patient with retinoblastoma without a prior history of radiation or chemotherapy. This report is significant not only because of the rarity of vesical leiomyosarcoma as a second nonocular tumor in retinoblastoma patients, but also because of the infrequency of vesical leiomyosarcoma in general. We also investigated the potential molecular pathogenesis of the leiomyosarcoma.

Topics: Actins; Adult; Cyclin D1; Cystectomy; Desmin; Eye Neoplasms; Female; Hematuria; Humans; Hysterectomy; Leiomyosarcoma; Neoplasms, Second Primary; Ovariectomy; Retinoblastoma; Retinoblastoma Protein; Survivors; Tomography, X-Ray Computed; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

The differentiation of neuronal cells in the developing mammalian retina is closely coupled to cell cycle arrest and proceeds in a highly organized manner. Cyclin D1, which regulates cell proliferation in many cells, also drives the proliferation of photoreceptor progenitors. In the mouse retina, cyclin D1 protein normally decreases as photoreceptors mature. To study the importance of the down-regulation of cyclin D1 during photoreceptor development, we generated a transgenic mouse in which cyclin D1 was persistently expressed in developing photoreceptor cells. We observed numerous abnormalities in both photoreceptors and other nonphotoreceptor cells in the retina of these transgenic mice. In particular, we observed delayed opsin expression in developing photoreceptors and alterations in their number and morphology in the mature retina. These alterations were accompanied by disorganization of the inner nuclear and plexiform layers. The expression of cyclin D1 caused excess photoreceptor cell proliferation and apoptosis. Loss of the p53 tumor suppressor gene decreased cyclin D1-induced apoptosis and led to microscopic hyperplasia in the retina. These findings are distinct from other mouse models in which the retinoblastoma gene pathway is disrupted and suggest that the IRBP-cyclin D1 mouse model may recapitulate an early step in the development of retinoblastoma.

Topics: Animals; Apoptosis; Cell Differentiation; Cell Division; Cyclin D1; Genes, p53; Hyperplasia; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retina; Retinoblastoma; Transgenes

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16(INK4a) cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.

Topics: Animals; BRCA1 Protein; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Keratinocytes; Mice; Mice, Transgenic; Ovarian Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins; Repressor Proteins; Retinoblastoma; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; RNA, Messenger; Skin; Transcription Factor DP1; Transcription Factors; Transcriptional Activation

Mammalian fibroblasts require mitogens in order to exit from G0 (quiescence) and progress through the G1 phase of the cell cycle, although once they pass the restriction point late in G1 they can enter S phase and complete the cell cycle without mitogens [1]. Mitogenic signals are integrated through the GTPase Ras, which regulates the levels of cyclin D1 [2-5], a component of the cell cycle machinery that operates during G1 phase by activating cyclin-dependent kinase 4 (Cdk4). The accumulation of active cyclin E-Cdk2 complexes also requires Ras [6]. These two G1 cyclin-Cdk complexes act on a family of E2F-associated transcriptional repressors typified by the retinoblastoma protein (Rb) to bring about a transcriptional program that promotes passage through S phase [7-9], but can also activate DNA replication independently of Rb-E2F [10-12]. Although G1 cyclin-Cdk complexes are required for S-phase entry and can shorten G1 phase when overexpressed [13-15], it is not known whether they are sufficient for this transition. Here, we report that serum-starved (G0) diploid human fibroblasts initiate DNA synthesis upon microinjection of active G1 cyclin-Cdk complexes, but not upon microinjection of an S-phase cyclin-Cdk complex. These data indicate that G1 Cdk activation is rate-limiting for S-phase entry, and that Cdk activation is likely to be the primary function of growth factor signalling pathways that lead to DNA synthesis.

Topics: Carrier Proteins; Cell Cycle Proteins; Cell Line; Cyclin D1; Cyclin E; Cyclin G; Cyclin G1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA Replication; DNA-Binding Proteins; E2F Transcription Factors; Fibroblasts; G1 Phase; Humans; Macromolecular Substances; Proto-Oncogene Proteins; Retinoblastoma; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors

The cell cycle-associated retinoblastoma protein (pRb) and p16 protein were demonstrated using immuno-histochemistry on paraffin sections from 192 cases of invasive breast carcinoma. Abnormal expression of pRb was defined as negative staining and was seen in 17% of tumours. Such abnormal expression was significantly more frequent in tumours with negative oestrogen receptor (ER) status. There was also a trend for tumours which were negative for pRb to be grade III ductal carcinomas. There was no association between p16 staining and any histopathological parameter, though, surprisingly, log-rank analysis showed that strong staining was associated with a poor outcome. There was a significant inverse relationship between pRb and p16 expression and a significant positive association between pRb and cyclin D1. In a Cox multivariate analysis, which included cyclin D1, neither pRb nor p16 was an independent predictor of patient outcome.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Immunohistochemistry; Multivariate Analysis; Prognosis; Receptors, Estrogen; Retinoblastoma; Survival Analysis

A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma.. 90 eyes were enucleated from 89 patients for retinoblastoma (n = 24) or for choroidal or ciliary body melanoma (n = 66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1.. Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed.. Retinoblastomas and uveal melanomas, two tumours of differing pRB status, differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Cyclin D1; Female; G1 Phase; Humans; Immunoenzyme Techniques; Infant; Male; Melanoma; Neoplasm Proteins; Retinal Neoplasms; Retinoblastoma; Retinoblastoma Protein; S Phase; Uveal Neoplasms

It has been demonstrated that protein expression of p16, the inhibitor of cyclin-dependent kinase 4 and 6, increases 4 fold at the G1/S transition when serum-arrested cells are restimulated to logarithmic growth. We examined the cell cycle regulation of this cyclin-dependent kinase inhibitor in cells separated according to their cell cycle phases by centrifugal elutriation. Neither p16 mRNA nor its protein expression are regulated during the cell cycle of normal phytohemagglutinin-stimulated lymphocytes, retinoblastoma protein-negative cells, papilloma virus-transformed cells, and acute promyelocytic leukemia cells. p16 mRNA is constitutively expressed in cells in which we detected the normal E2F-dependent S-phase specific expression of thymidine kinase mRNA. We further observed a G1-phase specific expression of cyclin D1 mRNA in the same cells separated by centrifugal elutriation.

Topics: Blotting, Western; Carrier Proteins; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; Enzyme Inhibitors; Eye Neoplasms; Gene Expression; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins; Protein Kinase Inhibitors; Retinoblastoma; Retinoblastoma Protein; RNA, Messenger; Thymidine Kinase; Tumor Cells, Cultured

Temperature-sensitive (ts) cell cycle mutant mouse cell, tsFT20, is deficient in DNA polymerase alpha activity to initiate DNA replication at replicon origins. Here, we analyzed phenotypes concerning growth control genes in the arrested tsFT20 cells. Analysis of cyclins showed that expression levels of cyclin D1, which is essential for G1/S transition, remarkably decreased in the mutant cells after temperature up-shift. Further we examined phosphorylation states of retinoblastoma protein (pRB) in the cells. Though the tsFT20 cells arrested in G1/S-S phase at nonpermissive temperature (Eki et al., (1990) J. Biol. Chem. 265 26-33), a large proportion of pRB was found as an underphosphorylated growth-suppressive form in the arrested cells. In revertant cell lines of tsFT20, pRB was not underphosphorylated even at nonpermissive temperature. The pRB underphosphorylation occurred later than the decrease of mRNA levels of cyclin D1, thus the underphosphorylation may be caused by the decrease in amount of cyclin D1 protein. These results indicated that the mutational inactivation of DNA polymerase alpha evokes phenotypes in which the inhibitory machinery of G1/S transition has been turned on.

Topics: Animals; Base Sequence; Cell Division; Cyclin D1; Cyclins; DNA Polymerase II; Mice; Molecular Sequence Data; Mutation; Oncogene Proteins; Retinoblastoma; Retinoblastoma Protein; RNA, Messenger; Temperature; Tumor Cells, Cultured