Effect of Vitamin D on Retinoblastoma Protein in Prediabetic Individuals.

About 50.8 million people were diagnosed with diabetes in 2011; the count has increased by 10 million in the last five years. Type-1 diabetes could occur at any age, but predominantly in children and young adults. The risk of developing type II diabetes mellitus in the offspring of parents with DM II is 40% if one parent has DM II and approaches 70% if both parents have DM II. The process of developing diabetes from normal glucose tolerance is continuous, with insulin resistance being the first stage. As prediabetes progresses slowly to DM II, it may take approximately 15-20 years for an individual to become diabetic. This progression can be prevented or delayed by taking some precautions and making some lifestyle amendments, e.g., reducing weight by 5-7% of total body weight if obese, etc. Retinoblastoma protein is one of the pocket proteins that act as crucial gatekeepers during the G1/S transition in the cell cycle. A loss or defect in single- cell cycle activators (especially CDK4 and CDK6) leads to cell failure. In diabetic or stress conditions, p53 becomes a transcription factor, resulting in the transactivation of CKIs, which leads to cell cycle arrest, cell senescence, or cell apoptosis. Vitamin D affects insulin sensitivity by increasing insulin receptors or the sensitivity of insulin receptors to insulin. It also affects peroxisome proliferator-activated receptors (PPAR) and extracellular calcium. These influence both insulin resistance and secretion mechanisms, undertaking the pathogenesis of type II diabetes. The study confines a marked decrement in the levels of random and fasting blood glucose levels upon regular vitamin D intake, along with a significant elevation of retinoblastoma protein levels in the circulatory system. The most critical risk factor for the occurrence of the condition came out to be family history, showing that patients with first-degree relatives with diabetes are more susceptible. Factors such as physical inactivity or comorbid conditions further aggravate the risk of developing the disease. The increase in pRB levels caused by vitamin D therapy in prediabetic patients directly influences blood glucose levels. pRB is supposed to play a role in maintaining blood sugar levels. The results of this study could be used for further studies to evaluate the role of vitamin D and pRB in regeneration therapy for beta cells in prediabetics.

TTC39B destabilizes retinoblastoma protein promoting hepatic lipogenesis in a sex-specific fashion.

BACKGROUND & AIMS: Molecular mechanisms underlying the different susceptibility of men and women to non-alcoholic fatty liver disease (NAFLD) are poorly understood. The TTC39B locus encodes a scaffolding protein, associates with gynecological disorders and its deletion protects mice from diet-induced steatohepatitis. This study aimed to elucidate the molecular mechanisms linking TTC39B (T39) to the expression of lipogenic genes and to explore sex-specific effects. METHODS: Co-expression in HEK293A cells validated the novel T39/pRb interaction predicted by a protein-protein interaction algorithm. T39 was knocked down using an antisense oligonucleotide (ASO) in mice with dietary NAFLD and a genetic deficiency of pRb or its downstream effector E2F1, as well as in primary human hepatocytes. RESULTS: T39 interacts with pRb via its C-terminal TPR domain and promotes its proteasomal degradation. In female mice, T39 deficiency reduces the mRNA of lipogenic genes, especially Pnpla3, in a pRb- and E2F1-dependent manner. In contrast, in male mice, T39 deficiency results in a much smaller reduction in lipogenic gene expression that is independent of pRb/E2F1. T39 also interacts with VAPB via an N-terminal FFAT motif and stabilizes the interaction of VAPB with SCAP. Ovariectomy abolishes the effect of T39 knockdown on the hepatic pRb/E2F1/Pnpla3 axis. In both sexes T39 knockdown reduces SCAP independently of pRb. In primary human hepatocytes, T39 knockdown reduces expression of PNPLA3 and other lipogenic genes in women but not men. CONCLUSIONS: We have uncovered a conserved sexual dimorphism in the regulation of hepatic lipogenic genes, with effects of T39 mediated through pRb/E2F1 in females and VAPB/SCAP in both sexes. T39 inhibition could be a novel strategy to downregulate PNPLA3 and treat NAFLD in women. LAY SUMMARY: In females, the protein TTC39B degrades a tumor suppressor in the liver to promote the synthesis of new fat and the expression of a major genetic risk factor for non-alcoholic fatty liver disease. TTC39B is a potential therapeutic target for non-alcoholic fatty liver disease, especially in women.

Pharmacodynamic Modeling of CDK4/6 Inhibition-Related Biomarkers and the Characterization of the Relationship Between Biomarker Response and Progression-Free Survival in Patients With Advanced Breast Cancer.

Identification of a pharmacodynamic (PD) biomarker, which is predictive of the efficacy outcome, is of ultimate interest in drug development. The objectives of the current analyses are to develop the pharmacokinetic (PK)/PD model for biomarkers (thymidine kinase 1 [TK1] in serum and phosphor-retinoblastoma protein [pRb] and Ki67 in skin tissues) related to cyclin-dependent kinase (CDK) 4/6 inhibition by palbociclib and to explore the relationship of the biomarker response with the efficacy end point (progression-free survival). The data used for analysis consisted of extensive sampling of palbociclib PK and longitudinal rich sampling for the PD biomarkers TK1, pRb, and Ki67 in 26 patients. A 2-compartment model was used to describe the PK of palbociclib. A precursor-dependent indirect response PD model was developed to describe the pRb time course, whereas a similar PD model with an additional transit compartment to model the delayed effect on Ki67 and TK1 response was used to describe the Ki67 and TK1 time course. Palbociclib effect on biomarkers was modeled as a maximum inhibition model. A Cox proportional hazard model was used to assess the relationship of progression-free survival with the biomarker response. The PK/PD models adequately described the observed PK of palbociclib and the longitudinal change of pRb, Ki67, and TK1. Palbociclib exposure significantly correlated with the reduction of all 3 biomarkers, and the estimated concentration to achieve 50% inhibition of the synthesis rate values were 45.2, 42.4, 50.2 ng/mL, respectively, for pRb, Ki67, and TK1. The exploratory biomarker-response analyses showed that a longer PFS was associated with lower baseline TK1 and simulated minimum TK1. Such results may warrant further confirmation from future large-scale study. Clinical Trial Registration: NCT02499146.

Autophagy Inhibition Sensitizes Renal Tubular Epithelial Cell to G1 Arrest Induced by Transforming Growth Factor beta (TGF-ß).

BACKGROUND Cell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-ß-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-ß has not been explored well. MATERIAL AND METHODS The effects of autophagy inhibition on TGF-ß-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-ß with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf). RESULTS Autophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-ß-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-ß treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-ß treated HK-2 cells. Western blot analysis showed that TGF-ß-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors. CONCLUSIONS Inhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-ß that contributes to the induction of tubulointerstitial fibrosis.

Olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.

OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) is an important molecule in the early stress response of DNA damage, which is involved in DNA damage repair and cellular senescence. Olaparib, as PARP inhibitor, has an anti-tumor effect on high grade serous ovarian cancer, but its effects on cellular senescence have not been reported. This study intends to explore the role of olaparib in the regulation of senescence in ovarian cancer cells. METHODS: The effects of olaparib on the senescence of ovarian cancer cells were detected by using the senescence-associated ß-galactosidase (SA-ß-Gal) and senescence-associated heterochromatin aggregation (SAHF). Quantitative real-time polymerase chain reaction was used to analyze the senescence-associated secretory phenotype (SASP). Cell cycle and apoptosis were detected by flow cytometry. The effect of olaparib on tumor growth was analyzed in a nude mouse xenograft transplantation model. RESULTS: Long-term (6 days) treatment with olaparib (5 µ) significantly inhibited the growth of ovarian cancer cells, leading to arrest the cell cycle at G0/G1 phase, significant increase the number of positive SA-ß-Gal stained cells and positive SAHF cells. The expression of P16 and retinoblastoma protein (p-RB) were significantly enhanced in SKOV3 cells under olaparib treated, meanwhile, the expression of P53 and p-RB were upregulated in A2780 cells. In OVCAR-3 cells, the expression of P53 was downregulated and p-RB was upregulated. Mice with SKOV3 xenograft transplantation was given olaparib (10 mg/kg/day) via abdominal cavity administration, the tumor volume was reduced (p<0.01). CONCLUSION: Continuous low dosage administration of olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.

Carnosic acid induces proteasomal degradation of Cyclin B1, RB and SOX2 along with cell growth arrest and apoptosis in GBM cells.

BACKGROUND: Carnosic acid (CA) is a diterpenoid found in Rosmarinus officinalis L. and Salvia officinalis L. as well as in many other Lamiaceae. This compound is reported to have antioxidant and antimicrobial properties. In addition, a number of reports showed that CA has a cytotoxic activity toward several cancer cell lines. PURPOSE: The aim of this study was to establish whether CA has any specific antiproliferative effect toward human glioblastoma (GBM) cells and to analyze the molecular mechanisms involved. METHODS: We evaluated cell survival by MTT assay, apoptosis and DNA content by flow cytometry, protein expression and phosphorylation by immunoblot analyses. RESULTS: Our results showed that CA inhibited cell survival on both normal astrocytes and GBM cells. In GBM cells, in particular, CA caused an early G2 block, a reduction in the percentage of cells expressing Ki67, an enhanced expression of p21(WAF) and induced apoptosis. Furthermore, we showed that CA promoted proteasomal degradation of several substrate proteins, including Cyclin B1, retinoblastoma (RB), SOX2, and glial fibrillary acid protein (GFAP), whereas MYC levels were not modified. In addition, CA dramatically reduced the activity of CDKs. CONCLUSION: In conclusion, our findings strongly suggest that CA promotes a profound deregulation of cell cycle control and reduces the survival of GBM cells via proteasome-mediated degradation of Cyclin B1, RB and SOX2.

CDK4/6 Inhibition Controls Proliferation of Bladder Cancer and Transcription of RB1.

PURPOSE: The retinoblastoma signaling network is frequently altered in advanced bladder cancer. We investigated the potential of CDK4/6 as a therapeutic target and determined biomarkers for patient stratification. MATERIALS AND METHODS: Genetic alterations were analyzed using public databases, including TCGA (The Cancer Genome Atlas), COSMIC (Catalogue of Somatic Mutations in Cancer) and CCLE (Cancer Cell Line Encyclopedia). Effects of the CDK4/6-inhibitor PD-0332991 or LY2835219 were examined in 10 bladder cancer cell lines by immunoblot, cell viability, apoptosis and cell cycle progression. Efficacy of the PD-0332991 and cisplatin combination was analyzed using the combination index. Gene expression level was determined by quantitative polymerase chain reaction. Cytomegalovirus promoter regulated recombinant retinoblastoma was used for reconstitution. Three-dimensional xenografts were grown on chicken chorioallantoic membrane and analyzed by measuring tumor weight and immunohistochemical expression of total retinoblastoma and Ki-67. RESULTS: PD-0332991 treatment decreased the proliferation of retinoblastoma positive bladder cancer cell lines and was synergistic in combination with cisplatin. PD-0332991 or LY2835219 treatment decreased the phosphorylation, total protein and transcript level of retinoblastoma. Treatment resulted in a decrease in E2F target gene expression (CCNA2 and CCNE2) and cell cycle progression from G0/G1 to the S-phase but did not affect apoptosis. In retinoblastoma negative cells reconstituted with recombinant retinoblastoma PD-0332991 affected only phosphorylation and not the total retinoblastoma level. These cells remained resistant to treatment. In 3-dimensional retinoblastoma xenografts, treatment resulted in reduced tumor weight and decreased expression of total retinoblastoma and Ki-67. CONCLUSIONS: We provide preclinical evidence that CDK4/6 inhibition is a potential therapeutic strategy for retinoblastoma positive bladder cancer that probably acts by negatively regulating retinoblastoma transcription.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions.

BACKGROUND AND OBJECTIVE: Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. RESULTS: Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. CONCLUSION: In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.

BACKGROUND AND OBJECTIVE: Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS: Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS: Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION: The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

Isoprenoid geranylgeranylacetone inhibits human colon cancer cells through induction of apoptosis and cell cycle arrest.

Geranylgeranylacetone (GGA), an isoprenoid compound, is a widely used antiulcer drug developed in Japan. GGA is structurally similar to plaunotol and geranylgeraniol, another isoprenoid reported to exert strong anticancer effects. In an earlier study, GGA was shown to inhibit ovarian cancer invasion by attenuating not only Rho activation, but also Ras-MAPK activation. In this study, we aimed to test whether GGA could have a therapeutic effect on colon cancer cells. As a result, we found that GGA induced a dose-dependent decrease in the proliferative activity through induction of cell apoptosis and cell cycle arrest in the G1 phase. The induction of apoptosis was mediated by the activation of both caspase-8 and caspase-9 pathways. The induction of G1 arrest was mediated by the increase of p21 and p27, and also the decrease of phosphorylated retinoblastoma protein levels. This study showed the potential anticancer activity of GGA. As this drug is already available in Japan for clinical use as an antiulcer/antigastritis agent, clinical trials will be designed to confirm its potential usefulness for cancer patients.

The activated aryl hydrocarbon receptor synergizes mitogen-induced murine liver hyperplasia.

Mechanisms of hepatocyte proliferation triggered by tissue loss are distinguishable from those that promote proliferation in the intact liver in response to mitogens. Previous studies demonstrate that exogenous activation of the aryl hydrocarbon receptor (AhR), a soluble ligand-activated transcription factor in the basic helix-loop-helix family of proteins, suppresses compensatory liver regeneration elicited by surgical partial hepatectomy. The goal of the present study was to determine how AhR activation modulates hepatocyte cell cycle progression in the intact liver following treatment with the hepatomitogen, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Mice were pretreated with the exogenous AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24h prior to treatment with TCPOBOP (3 mg/kg).). In contrast to the suppressive effects of AhR activation observed during compensatory regeneration, TCDD pretreatment resulted in a 30-50% increase in hepatocyte proliferation in the intact liver of TCPOBOP-treated mice. Although pretreatment with TCDD suppressed CDK2 kinase activity and increased the association of CDK2 with negative regulatory proteins p21Cip1 and p27Kip1, a corresponding increase in CDK4/cyclin D1 association and CDK4 activity which culminated in enhanced phosphorylation of retinoblastoma protein, consistent with the increased proliferative response. These findings are in stark contrast to previous observations that the activated AhR can suppress hepatocyte proliferation in vivo and reveal a new complexity to AhR-mediated cell cycle control.

MLN8054, an inhibitor of Aurora A kinase, induces senescence in human tumor cells both in vitro and in vivo.

Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated beta-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft-bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated beta-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples.

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation.

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles.

Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1.

BACKGROUND AND PURPOSE: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation. EXPERIMENTAL APPROACH: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng.mL(-1)) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators. KEY RESULTS: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC(50)= 520 +/- 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 +/- 8%) and asthmatic subjects (57 +/- 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21(Waf/Cip-1), p27(Kip1), cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC(50)= 450 +/- 100 pM) PDGF-induced cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.

Low-penetrant RB allele in small-cell cancer shows geldanamycin instability and discordant expression with mutant ras.

Certain kindreds with low-penetrant (lp) retinoblastoma carry mutant alleles which retain partial tumor suppressor activity and we previously showed that these alleles exhibit defective, temperature-sensitive binding in yeast. To investigate the molecular basis for incomplete penetrance, we studied three recurrent lp alleles and observed approximately 50% of wildtype activity measured by (i) phosphorylation at key regulatory sites, S780, S795, S807/S811, (ii) transcriptional co-activation, and (iii) 'flat-cell' differentiation in mammalian cells in vivo. In addition, we studied a small-cell carcinoma that is homozygous for the R661W allele providing the first analysis of the effect of a naturally occurring lp allele in a human tumor. While we detected abundant expression of the R661W protein, we noted marked instability of both endogenous and recombinant R661W following treatment in vivo with the Hsp90 inhibitor, geldanamycin and stabilization of R661W following heat shock. In addition, we observed a discordant phenotype in the tumor cells with induction of p16 and loss of cyclin D1 consistent with a null RB status combined with homozygous expression of mutant ras which had not been reported previously for RB (-) small-cell cancer. These findings show that a recurrent missense lp allele retains greater functional activity in vivo than predicted from earlier in vitro assays, proposing a role for stabilizing chaperone-like activity in vivo. In addition, these data suggest that reversible protein instability and the requirement for a cooperating mutation may provide a stochastic explanation for the molecular basis of incomplete penetrance in kindreds carrying these alleles.

BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway: analyses using a breast cancer cell line and tumoral xenograft model.

It is well established that statins display potent anticancer activity in several types of proliferating tumor cells. However, how to promote the sensitivity of statins to mammary cancer is yet to be completely deciphered. The purpose of this study was to investigate whether breast cancer susceptibility gene 1 (BRCA1) overexpression sensitizes mammary cancer cells to statins. MCF-7 cells, which have only one wild-type BRCA1 allele, were transfected with pcDNA3-beta-HA-hsBRCA1 plasmids via liposomes to reconstitute BRCA1 overexpression human breast cancer cell line, and tumoral xenografts with BRCA1 overexpression were subsequently established in BALB/c nude mice. Then, the inhibitory activity of lovastatin on cancer cells and tumoral xenografts, and the underlying mechanism involving in cell-cycle regulatory proteins were analyzed. The proliferative ability of MCF-7 cells treated with lovastatin was reduced compared to normal, and further decreased in the presence of excess BRCA1, detected by methyl thiazolyl tetrazolium and flow cytometry techniques in vitro or by 5-bromodeoxyuridine incorporation in vivo. Additionally, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (pRb), was further down-regulated under exposure to lovastatin in condition of BRCA1 overexpression, but the expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor (CDKI), was further up-regulated, both in vitro and in vivo detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Moreover, we found the further reduced volume of tumoral xenografts treated with lovastatin in the presence of BRCA1 overexpression. Our results suggest that BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway, which will provide an innovative experimental framework to study control of breast cancer cell proliferation.

Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+ -induced neuronal death in cultured hippocampal neurons.

Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb(2+) causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb(2+). Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb(2+)-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb(2+) treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 microM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb(2+). And that Pb(2+)-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb(2+) and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.

Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro.

Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin's epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested.

Expression of survivin and p16(INK4a)/Cdk6/pRB proteins and induction of apoptosis in response to radiation and cisplatin in meningioma cells.

Although meningiomas represent the most common class of tumors of the central nervous system, the molecular events underlying their genesis and development are still not well defined, and therapeutic approaches based on the genetics of these tumors are currently lacking. In the present study we have used the immunoblotting technique to show that the p16(INK4A), Cdk6 and pRB proteins are differentially expressed in primary meningioma cells with 20-, 30- and 36-fold difference between the lowest and the highest levels of each protein, respectively. In addition, we present evidence that the level of the anti-apoptosis survivin protein is high in these benign tumors. Moreover, the annexin V-associated flow cytometry technique was used to show that 60% of meningioma cell cultures underwent apoptosis in response to both gamma-rays and cisplatin, and 50% of these cells exhibited significant sensitivity to hydroxyurea. These agents triggered apoptosis through the mitochondrial pathway, by increasing the Bax/Bcl-2 ratio. Interestingly, the induction of apoptosis following radiation and cisplatin was significant in all cells that expressed low levels of p16(INK4A), Cdk6 and pRB proteins. These data shed more light on the molecular biology of meningioma cells and suggest that survivin and proteins of the RB pathway could play a determinant role in the development and the treatment of meningiomas.

A plant oxylipin, 12-oxo-phytodienoic acid, inhibits proliferation of human breast cancer cells by targeting cyclin D1.

Cyclin D1 overexpression has been associated with poor prognosis and resistance to therapy in human breast cancer. Thus, the development of therapeutic agents that selectively target cyclin D1 activity is of clinical interest. This study demonstrates that 12-oxo-phytodienoic acid (OPDA), a phytohormone with critical functions in growth and development in plants, induces growth arrest in MDA-MB-231 and T47D breast cancer cells. In response to OPDA treatment, the human breast cancer cell lines exhibit a progressive decline in cyclin D1 expression, which is tightly associated with the accumulation of hypophosphorylated form of the retinoblastoma protein (Rb) and G1 arrest. The decrease in cyclin D1 protein expression accompanies a dramatic decline in nuclear but not membranous beta-catenin expression and activation of glycogen synthase kinase-3-beta (GSK3beta) caused by inhibition of its serine-9 phosphorylation. The proteasome inhibitor MG132 blocks OPDA-mediated decrease in cyclin D1. In addition, the overexpression of T286A, a cyclin D1 mutant which is refractory to phosphorylation by GSK3beta and proteosomal degradation, is resistant to OPDA-mediated Rb dephosphorylation as well as G(1) cell cycle arrest. Thus, our results demonstrate that degradation of cyclin D1 protein is a key event in OPDA induced growth inhibition in breast cancer cells. These data provide the basic foundation for future efforts to develop OPDA-based approaches in the prevention and treatment of breast cancer and other types of cancer.