Cyclin-dependent Kinase Inhibitor Proteins P21 Research Articles

Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways

BackgroundQuercetin (QCT) is a flavonol present in many vegetables, it is proved to show chemo preventive effect against lung, cervical, prostate, breast and colon cancer due to its anti-inflammatory, anti-tumor and anti-oxidant property. Looking into the reported chemo-preventive effect we speculated antitumor activity in retinoblastoma (RB) Y79 cells, we also studied the molecular mechanism for antitumor activity.MethodsThe effect of QCT on Y79 cell viability count was done by cell counting kit, cell cycle distribution, apoptosis studies and mitochondrial membrane potential was evaluated by flow cytometry. Protein expression was done by western blot analysis.ResultsThe outcomes of study showed that QCT reduced Y79 cell viability and caused arrest of G1 phase in cell cycle via decreasing the expression levels of cyclin-dependent kinase (CDK)2/6 and cyclin D3 and by increasing the levels of both CDK inhibitor proteins p21 and p27. Apoptosis of Y79 cells mediated by QCT occurred via activation of both caspases-3/-9. Flow cytometry studies showed that QCT caused collapse in mitochondrial membrane potential (ΔΨm) in Y79 cells. Western blot studies confirmed that QCT brought about phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). We also established that inhibitors of JNK and p38 MAPK suppressed QCT mediated activation of both caspases-3/-9 and subdued the apoptosis of cancerous Y79 cells.ConclusionAll the results of the study suggest that QCT induced the apoptosis of Y79 cells via activation of JNK and p38 MAPK pathways, providing a novel treatment approach for human RB.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

The NuRD complex-mediated p21 suppression facilitates chemoresistance in BRCA-proficient breast cancer

The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex play a role in silencing gene expression. CHD4, the core component of the NuRD complex, which cooperates with histone deacetylase in reducing tumor suppressor genes (TSGs). To dissect the mechanisms underlying cancer promotion, we clarify the role of CHD4 in cyclin-dependent kinase inhibitor protein p21. Here, our data indicates that CHD4 deficiency impairs the recruitments of HDAC1 to the p21 promoter. ~ 300bp proximal promoter region is responsible for CHD4-HDAC1 axis-mediated p21 transcriptional activity. For identifying the role of anti-cancer drug response, knockdown of p21 overcomes cisplatin and poly-(ADP-ribose) polymerase (PARP) inhibitor-mediated growth suppression in CHD4-depleted cells. Consistent with in vitro data, tissue of patients and bioinformatics approach also showed positive correlation between CHD4 and p21. Overall, our findings not only identify that CHD4 deficiency preferentially impairs cell survival via increasing the level of p21, but also establishes targeting CHD4 as a potential therapeutic implication in BRCA-proficient breast cancer treatment.

Curcumin exerts antitumor effects in retinoblastoma cells by regulating the JNK and p38 MAPK pathways.

Curcumin, a naturally occurring polyphenolic compound present in turmeric(Curcuma longa), exerts antitumor effects in various types of malignancy. However, the precise mechanisms responsible for the effects of curcumin on retinoblastoma(RB) cells have not been fully explored. In the present study, the molecular mechanisms by which curcumin exerts its anticancer effects in RB Y79cells were investigated. The results showed that curcumin reduced cell viability in Y79cells. Curcumin induced G1 phase arrest through downregulating the expression of cyclinD3 and cyclin-dependent kinase(CDK)2/6 and upregulating the expression of CDK inhibitor proteins p21 and p27. Curcumin-induced apoptosis of Y79cells occurred through the activation of caspases-9/-3. Moreover, flow cytometric analysis showed that curcumin induced mitochondrial membrane potential(∆Ψm) collapse in Y79cells. We also found that curcumin induced the phosphorylation of c-Jun N-terminal kinase(JNK) and p38 mitogen-activated protein kinase(MAPK). JNK and p38 MAPK inhibitors significantly suppressed curcumin‑induced activation of caspases-9/-3 and inhibited the apoptosis of Y79cells. Taken together, our results suggest that curcumin induced the apoptosis of Y79cells through the activation of JNK and p38 MAPK pathways. These findings provide a novel treatment strategy for human RB.

P58 * BRAIN TUMOUR CELLS CAN SURVIVE AND THRIVE IN THE HYPOXIC SOLID TUMOUR MICROENVIRONMENT

INTRODUCTION: Normal brain tissue is exposed to 5-8% oxygen whilst the average oxygen level within brain tumours is just 1% (hypoxia). Hypoxia is associated with the increased malignancy, invasiveness, and treatment resistance of brain tumours. Paradoxically, hypoxia has also been shown to induce cell cycle arrest in certain cells; however the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this project was to investigate the consequences of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an aggressive brain tumour with an extremely poor prognosis. METHOD: GBM cell lines were exposed to a range of oxygen tensions and their cell cycle distribution and survival was assessed using flow cytometry. Changes in the expression level of genes involved in regulation of the cell cycle were quantified by qRT-PCR. RESULTS: Results indicate that GBM proliferation and survival is unaffected by long-term exposure to pathophysiological levels of oxygen (1% O2). Cell cycle arrest and/or cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). Sensitivity to severe hypoxia was associated with increased expression of the cyclin-dependent kinase inhibitor protein p21. CONCLUSION: These findings demonstrate that the pro-tumorigenic effects of pathophysiological hypoxia are not balanced out by reduced cellular proliferation, strengthening the association between hypoxia and tumour malignancy. This helps to explain how a hypoxic tumour such as GBM can be incredibly fast growing and aggressive.

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.

Estrogen regulated expression of the p21Waf1/Cip1 gene in estrogen receptor positive human breast cancer cells

The cyclin-dependent kinase inhibitor protein p21(Waf1/Cip1) is a potent tumor suppressor. Here, we demonstrate that estradiol regulates the p21(Waf1/Cip1) gene. Estradiol induces p21(Waf1/Cip1) mRNA expression within 30-60 min independent of new protein synthesis in the estrogen receptor alpha (ER alpha) positive human breast cancer cell line MCF-7. Similar to other estradiol responsive promoters, the p21(Waf1/Cip1) upstream promoter region has several estrogen response element (ERE) half-sites nestled in AP-1 binding sites, which are positioned upstream to Sp1 binding sites. Using the chromatin immunoprecipitation (ChIP) assay, we show that estradiol stimulation resulted in the recruitment of transcription factors ER alpha, Sp1, and Sp3 to the p21(Waf1/Cip1) upstream promoter element. The Sp1 inhibitor mithramycin A abrogated Sp1, and to a lesser extent Sp3 binding, and markedly reduced the estradiol stimulated p21(Waf1/Cip1) gene expression. However, ER alpha binding was not affected in the mithramycin A and estradiol treated cells. On closer examination of the half-site ERE/AP-1 sites upstream to the Sp1 sites in a separate ChIP experiment, we found a pronounced association of ER alpha upon estradiol treatment compared to almost negligible binding of Sp1 or Sp3. Together these studies provide evidence that ER alpha is recruited to the half-site ERE/AP-1 sites in the p21(Waf1/Cip1) upstream promoter element. Although Sp1/Sp3 is not involved in the recruitment of ER alpha to the promoter, Sp1 is necessary for estrogen-induced p21(Waf1/Cip1) promoter activity.

Gold Nanoparticles Inhibit the Proliferation of Multiple Myeloma Cells

Gold nanoparticles significantly inhibit proliferation in multiple myeloma cell lines (see figure) in a dose-dependent manner. Gold-treated multiple myeloma cells show cell-cycle arrest in the G1-phase (the phase when chromosomes prepare for replication) by up-regulation of cell-cycle cyclin-dependent kinase inhibitor proteins p21 and p27. Gold nanoparticles do not affect normal peripheral blood mononuclear cells.

Separation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.

Cell proliferation and terminal differentiation are mutually exclusive in most cell lineages. The b-zip transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) induces proliferation arrest and differentiation in many cell types, suggesting that both activities are linked. Here we show that C/EBPalpha-mediated proliferation arrest and differentiation pathways can be separated by the E7 oncoprotein of the "high-risk" human papilloma virus 16. The E7 oncoprotein overrides C/EBPalpha-mediated cell cycle withdrawal without compromising the transactivation activity of C/EBPalpha or its ability to participate in differentiation. Uncoupling of both pathways depends on the casein kinase II site of the oncoprotein but not on its ability to neutralize pocket proteins or the cyclin-dependent kinase inhibitor protein p21. Our results suggest a bifurcation of C/EBPalpha-mediated proliferation arrest and differentiation pathways.

A role for the cyclin-dependent kinase inhibitor p21 in the G1 cell cycle arrest mediated by the type I interferons.

Type I interferons (IFN), such as IFN-alpha, are potent antiproliferative and antitumor agents. IFN-tau, originally identified as a pregnancy recognition hormone, is a type I IFN that is related to IFN-alpha. We examine here the mechanism of the antiproliferative effects of IFN-alpha and IFN-tau in terms of their effects on intracellular events that regulate the cell cycle. Both IFN inhibited proliferation of the human Burkitt lymphoma cell line, Daudi, causing accumulation of cells in the G1 phase of the cell cycle. IFN-alpha was more effective than IFN-tau in this regard. Both IFN were found to inhibit the kinase activity of the cyclin-dependent kinase cdk2 in a manner that correlated with their relative abilities to cause cells to accumulate in the G1 phase of the cell cycle. Further, IFN treatment did not affect the expression of cdk2 protein, suggesting that the IFN modulated cdk2 activity through a cdk inhibitor. Consistent with this conclusion, both IFN induced the expression of the cyclin-dependent kinase inhibitor protein p21. The levels of p21 induced also correlated with the relative abilities of the IFN to inhibit cdk2 activity and to arrest cell growth in the G1 phase of the cell cycle. Moreover, following IFN treatment, increased levels of p21 were found complexed with cdk2, consistent with its role in the inhibition of cdk2 activity. These data suggest that p21-mediated inhibition of cdk2 activity plays an important role in the antiproliferative activity of type I IFN. The findings highlight interesting similarities between these cytokines and the products of tumor suppressor genes, such as p53, and may indicate a mechanism for the antitumor effects of the type I IFN.

G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.