Cyclin-dependent Kinase Inhibitor P27 Kip1 Research Articles

Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration

AbstractThe BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C−/−) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C−/− recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9–dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57Kip2, p21Cip1, p16Ink4a). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.

Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

IntroductionEarlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal transduction pathway involving ATM-AMPK-p53/p21cip1 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer.MethodsImmuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis.ResultsIR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls.ConclusionIR inhibits epithelial cancer tumour growth and results in sustained expression and activation of ATM-Chk2, and AMPK-p53/p21cip1/p27kip1 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt-mTOR pathways by novel therapeutics can sensitize lung tumours to radiation.

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

Calpain 2 Regulates Akt-FoxO-p27Kip1 Protein Signaling Pathway in Mammary Carcinoma

PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27 Kip1.

P27Kip1 (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91–120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.

Ganoderma lucidum polysaccharides prevent platelet‐derived growth factor‐stimulated smooth muscle cell proliferation in vitro and neointimal hyperplasia in the endothelial‐denuded artery in vivo

Ganoderma lucidum is used in traditional Chinese medicine to prevent or treat a variety of diseases, including cardiovascular disorders. We previously demonstrated that a glucan-containing extract of Reishi polysaccharides (EORP) has the potent anti-inflammatory action of reducing ICAM-1 expression in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and LPS-treated mice. In the present study, we examined whether EORP inhibited platelet-derived growth factor-BB (PDGF)-stimulated HASMC proliferation and the mechanism involved. EORP dose-dependently reduced cell numbers and DNA synthesis of PDGF-treated HASMCs in vitro. EORP also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). The anti-proliferative effect of EORP was partly mediated by downregulation of PDGF-induced JNK phosphorylation. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice were fed a diet containing 100 mg/kg/day of EORP. On day 14, both cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima and the neointima/media area ratio (0.67 ± 0.03 vs. 1.46 ± 0.30) were significantly reduced. Our data show that EORP interferes with the mitogenic activation of JNK, preventing entry of HASMCs into the cell cycle in vitro and reducing cell proliferation in the neointima and decreasing the neointimal area in vivo. Thus, EORP may represent a safe and effective novel approach to the prevention and treatment of vascular proliferative diseases.

Abstract 1480: Regulation of ovarian tumor growth and stromal cell infiltration by the HU177 biomechanical ECM switch

Abstract Accumulating evidence indicates that the malignant behavior of tumors depends not only on tumor cells themselves, but also on the stromal cells that comprise the malignant tumor mass. Experimental findings suggest that stromal cells such as cancer-associated fibroblast (CAF) may play important roles in promoting tumor growth and metastasis as well as regulating the efficacy of certain chemotherapeutic drugs. However, developing novel clinical strategies that selectively and simultaneously impacts tumor and stromal cells remains challenging. Alterations in the integrity and molecular composition of the extracellular matrix (ECM) are hallmarks of tumor progression. Our previous studies have shown that structural remodeling of the ECM can result in localized triggering of what we have termed “biomechanical ECM switches” or changes in the three-dimensional structure of pre-existing ECM molecules. A humanized antibody (TRC093/D93) specifically directed to the HU177 cryptic collagen epitope that is selectively exposed following triggering of a biomechanical ECM switch has been developed, and a human phase-I clinical trial was recently completed with encouraging results. Here we provide evidence that the HU177 biomechanical ECM switch is triggered within human ovarian tumors resulting in the exposure of the HU177 cryptic collagen epitope. The relative exposure of the HU177 cryptic site was significantly (P<0.05) enhanced in biopsies of malignant ovarian tumors as compared to benign ovarian lesions. Selective targeting of the HU177 cryptic collagen epitope by Mab D93 significantly (P<0.05) inhibited SKOV-3 tumor growth by approximately 70% as compared to controls. Tumors from these mice exhibited reduced angiogenesis, elevated levels of apoptosis and a significant reduction in infiltration of alpha smooth muscle cell actin (αSMC-actin) positive stromal fibroblasts. Importantly, while Mab D93 inhibited SKOV-3 tumor cell adhesion to denatured collagen type-I and enhanced the expression of the cyclin dependent kinase inhibitor P27KIP1, it also selectively inhibited (80%) fibroblast migration on denatured collagen type-I that was induced by either FGF-2 or SKOV-3 condition medium. Collectively these studies provide evidence that specific targeting of the HU177 cryptic collagen epitope may represent a highly selective strategy to inhibit ovarian tumor growth in part by limiting stromal cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1480. doi:1538-7445.AM2012-1480

Abstract 3043: A CDK-independent function of p27kip1 controls cell proliferation

Abstract The integrated regulation of different intracellular signaling pathways is fundamental to ensure appropriate timing of cell division. One critical mediator of this regulation is the CDK inhibitor p27kip1, via a mechanism that still presents many controversial aspects. p27kip1 (hereafter p27) is a tumor suppressor protein, implicated in the regulation of several biological activities, thus acting as a relay that integrates different signals and ensuring appropriate timing of cellular behaviors. Knock out of p27 (p27KO) in mice causes an increase in animal size due to increased proliferation and hypercellularity in all tissues. This phenotype seems not to be due to CDK-dependent activity of p27, since it is not rescued by concomitant genetic ablation of the major p27 target, CDK2. Mice double KO (DKO) for p27 and CDK2 still display multiorgan hyperplasia and gigantism. We recently demonstrated that p27 also controls cell morphology and motility, by interacting with stathmin in the cytoplasm. Stathmin is a cytosolic phospho-protein that plays an important role in the regulation of microtubule dynamics, particularly during mitosis. It also participates in many important biological functions during interphase, and human tumors frequently overexpress stathmin, especially in advanced invasive and metastatic stages. Thus, we decided to generate mice DKO for p27 and stathmin, to highlight the possible in vivo consequences of such interaction characterized in vitro. Unexpectedly, the characterization of mice WT, p27 KO and p27/stathmin DKO revealed an important rescue of the most significant phenotypes of p27 KO mice by the DKO ones. Not only body size and organomegalia, but also development of pituitary tumors and outgrowth of retina basal layer were reverted by loss of stathmin in p27 null background. These unpredictable findings clearly indicated that p27/stathmin interaction importantly affected cell proliferation, as well as migration, and we thus focused on the mechanism underlying these phenotypes. We here present evidences that p27kip1 is implicated in the control of the MAPK pathway, through a CDK-independent mechanism. Acting on the subcellular localization and activity of stathmin, p27kip1 controls Ras full activation, and as a consequence, the downstream MAPK cascade. In this way p27kip1 restrains the G1-S phase transition, eventually influencing cell proliferation in vitro, mice growth in vivo and tumorigenesis in humans. These data led us to hypothesize that p27 controls cell proliferation by two different mechanisms: one directly blocking CDK activity and the other interacting with stathmin and thus controlling MAPK pathway activation. Overall our work unveils a new mechanism that in mammalian cells contributes to the proper regulation of cell proliferation, whose alteration may contribute to tumor onset and progression, and that could be of great value in the design of innovative therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3043. doi:1538-7445.AM2012-3043

Impact of Eugenol and Isoeugenol on AhR Translocation, Target Gene Expression, and Proliferation in Human HaCaT Keratinocytes

The phenolic derivatives eugenol and isoeugenol, which are naturally found in essential oils of different spices, are commonly used as fragrances. Recently data demonstrated that growth suppression produced by these substances occurs in keratinocytes and that the effects may be mediated via aryl hydrocarbon receptor (AhR) interactions. In this study the effects of eugenol and isoeugenol were determined on intracellular localization of AhR, AhR target gene expression, AhR-dependent cell cycle regulation, and proliferation in HaCaT cells. Both compounds produced a rapid and marked translocation of AhR into the nucleus, induced the expression of the AhR target genes cytochrome P-450 1A1 (CYP1A1) and AhR repressor (AhRR), and inhibited proliferation of HaCaT cells. Among the G1 phase cell cycle-related proteins, levels of the retinoblastoma protein (RB), which is known to interact with AhR, and levels of the cyclin dependent kinase (CDK) 6 were reduced by eugenol and isoeugenol, whereas steady-state levels of CDK2 and CDK4 remained unaffected. Protein levels of CDK inhibitor (CKI) p27KIP1, known to be modulated in an AhR-dependent manner, were increased after treatment with both substances. In conclusion, data show that the antiproliferative properties of eugenol and isoeugenol in HaCaT cells are mediated through AhR, and thereby the molecular mechanisms of action in these cells were identified for the first time in this study.

Abstract 795: Epigenetics, cell cycle and drug-resistant ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal of the gynaecological malignancies and currently stage III/IV disease is associated with a 25% 5 year survival rate. A major factor associated with advanced EOC is the development of drug resistant disease, characterised by a lack of response to platinum and taxane based agents. In an effort to clarify mechanisms underlying chemorefractory EOC we have developed a number of drug resistant cancer cell lines. DNA microarray screening of drug resistant versus drug sensitive cell lines (combining a methylation reversal step using demethylating agents) indicated down-regulation of a number of genes with evidence of epigenetic silencing by promoter methylation in many cases. We have identified P57kip2 a cyclin dependent kinase inhibitor (CDKi) as a gene that is subjected to methylation in carboplatin-resistance, as shown in the PEO1CarbR line. Using a siRNA approach, silencing of the p57kip2 gene in drug sensitive parental PEO1 or SKOV-3 cells gave rise to a reduced sensitivity to platinum agents i.e. cisplatin or carboplatin as assessed using apoptosis with the annexinV assay and flow cytometry. Using chemosensitivity testing (MTT) of PEO1 parental and PEO1CarbR lines versus seliciclib (a CDK2 inhibitory drug) we saw an association with sensitivity to this agent with down-regulation of p57kip2. The IC50 values obtained for the human ovarian cancer cell line PEO1 and PEO1CarboR were 27.4 and 19.2μM, respectively (p = <0.05). Cell cycle analysis of seliciclib treated cells indicated a prolonged S-phase as a marker of increased sensitivity to the drug and an increased apoptotic response in p57kip2 silenced cells. Our data suggest that CDK inhibitors such as seliciclib may have utility in tumours that show downregulation of the endogenous CDKi p57kip2 and identification of such patients may indicate a sub-group that respond preferentially to seliciclib. The same screening platform also enabled us to identify wee1 and stratifin (14-3-3α) as genes significantly downregulated in taxane-resistant cell lines. The 2 gene products are important in maintaining genomic stability alongside other proteins such as Chk1 and ATR. Wee1 controls G2M by keeping CDK2/Cyclin B inactive whilst binding of 14-3-3 isoforms enhances its activity. Demethylation treatment using 5′aza-cytidine and Trichostatin A unmasked gene expression, indicating that these genes are hypermethylated in taxane-resistant cell line. Current work aims to confirm further the role of these 2 genes in drug resistance by a series of knock-in and knock-out experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 795. doi:1538-7445.AM2012-795

Abstract 3051: CDC28 protein kinase subunit 1B (CKS1B) is a potential therapeutic target in esophageal adenocarcinomas overexpressing the CDK inhibitor p21Cip1/Waf1

Abstract Background: The incidence of adenocarcinoma of the esophagus and gastroesophageal junction (EAC) has grown faster than any tumor type in the United States and westernized countries, outpacing the next closest cancer by almost 3 times. This rising incidence of EAC is associated with the increasing prevalence of obesity and gastroesophageal reflux disease (GERD). Due to the limited effect of current chemotherapy for this cancer, the search for new therapeutic targets is disparately needed. Chromosomal amplification is one mechanism by which cancer cells acquire and maintain neoplastic phenotype and genes that are amplified and overexpressed may represent potential therapeutic targets. Methods: DNA and RNA from a cohort of 116 patients were analyzed using SNP and mRNA expression microarrays respectively. Gene amplification was explored for correlation with expression and patient survival. Results: 25% of tumors harbor amplification at chromosome 1q which is significantly associated with poor survival. Although the amplicon is non-focal spanning almost the entire arm of chromosome 1q, only a subset of amplified genes (including The CDC28 protein kinase subunit 1B, CKS1B) are significantly overexpressed. CKS1B is an auxiliary protein required for ubiquitination and cytosomal degradation of the CDK inhibitors p27Kip1 and p21Cip1/Waf1 by the SCFSKP2 E3 ubiquitin ligase complex. Both p27Kip1 and p21Cip1/Waf1 inhibits CDK2/Cyclin E complex during the G1/S cell cycle transition. The endogenous protein expression of p27Kip1 is low among EAC cell lines. However the expression of p21Cip1/Waf1 is differential being very high in a subset of cells and low or undetectable in others. Knocking down CKS1B by siRNA in six EAC cell lines results in accumulation of p27 Kip1 with little or no effect on p21Cip1/Waf1 expression. CKS1B knockdown inhibits monolayer cell proliferation only in cells with high endogenous expression of p21Cip1/Waf1. Conclusion: We suggest that the combined action of accumulation of p27 Kip1 and the endogenously highly expressed p21Cip1/Waf1 triggers the response to CKS1B knockdown in EAC cell lines. We thus propose the potential use of CKS1B as a novel therapeutic target in esophageal adenocarcinomas with high p21Cip1/Waf1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3051. doi:1538-7445.AM2012-3051

Abstract 3062: Ada3, a component of STAGA HAT complex, controls cell cycle by regulating c-myc

Abstract The human homologue of the yeast Ada3 (alteration/deficiency in activation) protein is an essential component of various Histone acetyl transferase complexes (HATs). To gain insight into the physiological importance of Ada3, we established a conditional knockout mouse model and showed that loss of Ada3 led to embryonic lethality at an early stage of development. Using the derived Ada3lox/lox mouse embryonic fibroblast (MEF) cell lines we show that Ada3 deletion causes defects in histone acetylation. Also we show that Ada3 enhances catalytic activity of p300 HAT. We further showed that Ada3 plays an important role in cell proliferation; depletion of Ada3 from cells caused cell lethality. This proliferation defect was reversed by introduction of human Ada3 in Ada3 null MEFs. Ada3 null MEFs also showed delay in transition from G0-G1 phase of the cell cycle to the S phase. These cells also manifested decreased hyperphosphorylation of Retinoblastoma protein (Rb); an increased protein half life of CDK inhibitor p27Kip1 and decrease in Cdk2 kinase activity. The increase in p27 protein was a result of decreased Skp2 protein and mRNA levels which is a direct target of c-myc oncogene. We further show that mRNA levels of c-myc, a direct transcriptional target of Ada3 containing STAGA HAT complex, are reduced on deletion of Ada3. These data suggest an Ada3-myc-skp2-p27 pathway that controls mammalian cell cycle. Taken together, our data suggests an essential role of Ada3 in embryonic development and cell cycle progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3062. doi:1538-7445.AM2012-3062

Foxp-Mediated Suppression of N-Cadherin Regulates Neuroepithelial Character and Progenitor Maintenance in the CNS

Neuroepithelial attachments at adherens junctions are essential for the self-renewal of neural stem and progenitor cells and the polarized organization of the developing central nervous system. The balance between stem cell maintenance and differentiation depends on the precise assembly and disassembly of these adhesive contacts, but the gene regulatory mechanisms orchestrating this process are not known. Here, we demonstrate that two Forkhead transcription factors, Foxp2 and Foxp4, are progressively expressed upon neural differentiation in the spinal cord. Elevated expression of either Foxp represses the expression of a key component of adherens junctions, N-cadherin, and promotes the detachment of differentiating neurons from the neuroepithelium. Conversely, inactivation of Foxp2 and Foxp4 function in both chick and mouse results in a spectrum of neural tube defects associated with neuroepithelial disorganization and enhanced progenitor maintenance. Together, these data reveal a Foxp-based transcriptional mechanism that regulates the integrity and cytoarchitecture of neuroepithelial progenitors.

A natural mutation of the hepatitis B virus X gene affects cell cycle progression and apoptosis in Huh7 cells

Hepatocellular carcinoma (HCC), one of the most prevalent cancers, and HCC-associated diseases are caused by the hepatitis B virus (HBV). Viral sequences often contain mutations in the basal core promoter (BCP), which overlaps with the open reading frame encoding HBV X protein (HBx). HBx protein plays a very important role in the development of HCC. Influence of naturally-occurring mutation of HBx (BCP1) on cell cycle progression was investigated in the Huh7 HCC line. BCP1 mutation was cloned from clinical samples into recombinant green fluorescent protein-fusion expression plasmid constructs that were then transfected into Huh7 cells for either transient expression studies or isolation of stable transfectants, along with wild-type HBx and empty vector controls. The mutant protein was more effective than wild-type in reducing expression of cyclindependent kinase (CDK) inhibitor p27kip1, promoting cell cycle progression, inhibiting production of cleaved poly ADP ribose polymerase (PARP) (a marker for apoptosis), and down-regulating the multifunctional cytokine transforming growth factor-β (TGF-β). Since TGF-controls the cell cycle and apoptosis, the effect of BCP1 mutation on the enhanced activity of HBx to promote cell cycle progression and to inhibit apoptosis in Huh7 cells may result from down-regulation of TGF-β.

Loss of Protein-tyrosine Phosphatase α (PTPα) Increases Proliferation and Delays Maturation of Oligodendrocyte Progenitor Cells

Tightly controlled termination of proliferation determines when oligodendrocyte progenitor cells (OPCs) can initiate differentiation and mature into myelin-forming cells. Protein-tyrosine phosphatase α (PTPα) promotes OPC differentiation, but its role in proliferation is unknown. Here we report that loss of PTPα enhanced in vitro proliferation and survival and decreased cell cycle exit and growth factor dependence of OPCs but not neural stem/progenitor cells. PTPα(-/-) mice have more oligodendrocyte lineage cells in embryonic forebrain and delayed OPC maturation. On the molecular level, PTPα-deficient mouse OPCs and rat CG4 cells have decreased Fyn and increased Ras, Cdc42, Rac1, and Rho activities, and reduced expression of the Cdk inhibitor p27Kip1. Moreover, Fyn was required to suppress Ras and Rho and for p27Kip1 accumulation, and Rho inhibition in PTPα-deficient cells restored expression of p27Kip1. We propose that PTPα-Fyn signaling negatively regulates OPC proliferation by down-regulating Ras and Rho, leading to p27Kip1 accumulation and cell cycle exit. Thus, PTPα acts in OPCs to limit self-renewal and facilitate differentiation.

Effect of curcumin on proliferation, cell cycle, and caspases and MCF-7 cells

We examined the mechanisms by which curcumin inhibits the growth of breast cancer cells. First, we investigated its antiproliferative effects on MCF-7 cells exposed to 2.5 to 40 μM curcumin for 24, 48 or 72 h. Curcumin inhibited cell proliferation in a dose-dependent and time-dependent manner (p<0.05) and resulted in significant cell cycle arrest in G1 phase after 72 h of treatment at concentrations of over 10 and 20 μM in MCF-7 cells, respectively (p<0.05). In addition, curcumin caused the accumulation of cells in sub-G0 phase in a dose-dependent manner in MCF-7 cells (p<0.05). As biomarkers of apoptosis induction, caspase-3 activity and caspase-9 activity were increased by curcumin in MCF-7 cells. To investigate the effects of curcumin on the proteins regulating cell cycle arrest, cells were treated with 20 μM curcumin for 72 h. Similar changes in the expression of regulatory proteins were detected in MCF-7 cells. Curcumin treatment resulted in decreases in cyclin D, cyclin E, CDK2, CDK4 and CDK6. The protein expression of CDK2 related to the G1 phase increased markedly with curcumin treatment. Curcumin treatment increased the expression of the CDK inhibitors p19INK4, p21Cip1 and p27Kip1. Thus, curcumin exerts its anticancer effects in human breast cancer cells via cell cycle arrest at the G1 phase. Key words: Apoptosis, breast cancer, cell proliferation, cell cycle, caspase, curcumin.

Glioma-specific Cation Conductance Regulates Migration and Cell Cycle Progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.

Differential expression of forkhead box M1 and its downstream cyclin‐dependent kinase inhibitors p27kip1 and p21waf1/cip1 in the diagnosis of pulmonary neuroendocrine tumours

Pulmonary neuroendocrine (NE) tumours represent a spectrum of phenotypically distinct entities with different biological behaviours. Difficulties in classifying these tumours are frequently encountered in clinical practice. Forkhead box M1 (FoxM1) is essential for the development of various cancers and is a proliferation-specific transcription factor that regulates transcription of cell cycle genes, including cyclin-dependent kinase inhibitors p27(kip1) and p21(waf1/cip1) . This study was performed to determine the utility of FoxM1, p27(kip1) and p21(waf1/cip1) as immunomarkers for subtyping pulmonary NE tumours. FoxM1, p27(kip1) and p21(waf1/cip1) expression was evaluated by immunohistochemistry in 60 pulmonary NE tumours [19 typical carcinoids (TCs), six atypical carcinoids (ACs), 17 large cell neuroendocrine carcinomas (LCNECs) and 18 small cell lung cancers (SCLCs)]. The frequencies of FoxM1 and p21(waf1/cip1) expression were significantly different between TCs and ACs (each P = 0.009), and those of FoxM1 and p27(kip1) expression were significantly different between LCNECs and SCLCs (P = 0.012 and P = 0.002, respectively). The combined FoxM1((-)) /p21(waf1/cip1(-)) and FoxM1((+)) /p27(kip1(high)) phenotypes had the best diagnostic accuracy for distinguishing TCs from ACs, and SCLCs from LCNECs, respectively. FoxM1, p27(kip1) and p21(waf1/cip1) showed distinct immunoreactivity according to histological subtype, which may be of value as an ancillary test in the differential diagnosis of pulmonary NE tumours.

Signal transduction pathways in FSH regulation of rat Sertoli cell proliferation

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH is the major Sertoli cell mitogen; however, little is known about the signal transduction pathways that regulate the proliferation of Sertoli cells. The hypothesis of this investigation was that FSH regulates proliferation through a PI3K/Akt/mTORC1 pathway, and additionally, AMPK-dependent mechanisms counteract FSH proliferative effects. The present study was performed in 8-day-old rat Sertoli cell cultures. The results presented herein show that FSH, in addition to increasing p-Akt, p-mTOR, and p-p70S6K levels, increases p-PRAS40 levels, probably contributing to improving mTORC1 signaling. Furthermore, the decrease in FSH-stimulated p-Akt, p-mTOR, p-p70S6K, and p-PRAS40 levels in the presence of wortmannin emphasizes the participation of PI3K in FSH signaling. Additionally, the inhibition of FSH-stimulated Sertoli cell proliferation by the effect of wortmannin and rapamycin point to the relevance of the PI3K/Akt/mTORC1 signaling pathway in the mitotic activity of FSH. On the other hand, by activating AMPK, several interesting observations were made. Activation of AMPK produced an increase in Raptor phosphorylation, a decrease in p70S6K phosphorylation, and a decrease in FSH-stimulated Sertoli cell proliferation. The decrease in FSH-stimulated cell proliferation was accompanied by an increased expression of the cyclin-dependent kinase inhibitors (CDKIs) p19INK4d, p21Cip1, and p27Kip1. In summary, it is concluded that FSH regulates Sertoli cell proliferation with the participation of a PI3K/Akt/mTORC1 pathway and that AMPK activation may be involved in the detention of proliferation by, at least in part, a decrease in mTORC1 signaling and an increase in CDKI expression.

P57KIP2 regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.

The cyclin-dependent kinase inhibitor p57Kip2 is epigenetically regulated in carboplatin resistance and results in collateral sensitivity to the CDK inhibitor seliciclib in ovarian cancer

Background:Carboplatin remains a first-line agent in the management of epithelial ovarian cancer (EOC). Unfortunately, platinum-resistant disease ultimately occurs in most patients. Using a novel EOC cell line with acquired resistance to carboplatin: PEO1CarbR, genome-wide micro-array profiling identified the cyclin-dependent kinase inhibitor p57Kip2 as specifically downregulated in carboplatin resistance. Presently, we describe confirmation of these preliminary data with a variety of approaches.Methods:Cytotoxicity testing (MTT) and cell cycle blockade assessed drug responsiveness. Methylation specific PCR and pyrosequencing identified sites of promoter methylation in p57Kip2. siRNA to p57Kip2 was used to look at the changes in apoptosis of carboplatin treated EOC cells. EOC tissues (20 cases) were assessed for mRNA levels of p57Kip2.Results:Carboplatin resistance was reversed using 5-aza-cytidine in vitro. Promoter methylation sites and preferential sensitivity to seliciclib were seen in PEO1CarbR cells. Silencing p57Kip2 decreased the apoptotic response to the effects of platinum but produced sensitisation to seliciclib. EOC biopsies indicated an association of high levels of p57Kip2mRNA with complete responses to chemotherapy and improved outcome.Conclusion:We conclude that p57Kip2 is a candidate biomarker of platinum sensitivity/resistance in EOC and such cases may show preferential response to the cyclin-dependent kinase inhibitor seliciclib.