Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer. 相似文献
The novel cell-cycle regulator p12(CDK2AP1) (p12) gene encodes a cyclin-dependent kinase 2 (CDK2) partner that participates in cell-cycle regulation, apoptosis, and proliferation. CDK2 has been implicated in maintenance of gonadal homeostasis, as knockout mice display reproductive abnormalities. To investigate the role of p12 in homeostasis of gonadal tissues in vivo, we generated a transgenic mouse model driven by the human keratin 14 promoter, reported to target transgene expression to gonadal tissues and also stratified epithelia. Overexpression of the transgene was associated with a gonadal atrophy phenotype in mice of both sexes, yet fertility was not impaired. Histological evaluation of testes showed seminiferous tubule degeneration and decreased tubule diameter. Female transgenic mice had small ovaries, with a higher number of atretic follicles/mm(2) as compared to control nontransgenic mice. Also observed was increased germ cell apoptosis in both sexes (TUNEL). These results suggest that overexpression of p12 leads to testicular and ovarian abnormalities, a phenotype closely related to that of cdk2-/- mice. In combination, these observations suggest that the p12/CDK2 signaling pathways are carefully orchestrated to maintain proper gonadal tissue homeostasis. We suggest that the mechanisms of this regulation may be through p12-mediated altered expression of gonadal-specific genes and apoptotic pathways. 相似文献
The enzymatic activity of the CDK2/Cyclin A complex increases upon the specific phosphorylation of Thr160@CDK2. In the present study, we have performed a comparative molecular dynamics (MD) study of models of the complex CDK2/Cyclin A/Substrate, which differ for the presence or absence of the phosphate group bound to Thr160. The models are based on two X-ray structures available for CDK2/CyclinA and pCDK2/CyclinA/Substrate complexes. In this way, we analyze the influence of the phosphorylated Thr160 (pThr160) on both the flexibility of CDK2 activation loop (AL) and substrate binding in CDK2. Our calculations point to a decreased flexibility of the AL in the phosphorylated model, in fairly good agreement with experimental data, and to a key role of pThr160 for substrate recognition and stability. Multiple alignments of the CDKs sequences point to the very high conservation of the AL sequence among the CDKs, thus extending our results to all CDKs. 相似文献
Background information. Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin‐dependent kinase). Results. We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti‐PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. Conclusions. We conclude we have probably isolated a dinoflagellate CDK5‐like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families. 相似文献
In breast cancer, cyclins D1 and E and the cyclin-dependent kinase inhibitors p21 (Waf1/Cip1)and p27 (Kip1) are important in cell-cycle control and as potential oncogenes or tumor suppressor genes. They are regulated in breast cancer cells following mitogenic stimuli including activation of receptor tyrosine kinases and steroid hormone receptors, and their deregulation frequently impacts on breast cancer outcome, including response to therapy. The cyclin-dependent kinase inhibitor p16 (INK4A) also has a critical role in transformation of mammary epithelial cells. In addition to their roles in cell cycle control, some of these molecules, particularly cyclin D1, have actions that are not mediated through regulation of cyclin-dependent kinase activity but may be important for loss of proliferative control during mammary oncogenesis. 相似文献
Progression through the cell cycle relies on the activities of cyclin-dependent kinases (Cdk), which in turn are modulated by inhibitory proteins such as p21(waf1/cip1) that are induced when genomic damage occurs. In this study, we show that exposure of normal mammalian cells, such as NIH3T3 fibroblasts, to UVC (25 J/m2, at 254 nm) induces the expression of p21 without causing significant apoptosis, whereas similar treatment of Chinese hamster ovary (CHO-K1) cells with UVC causes apoptosis without inducing p21. The absence of p21 in UV-irradiated CHO-K1 cells is accompanied by the deregulation of Cdk2 activity. The elevation of Cdk2 activity correlates with the increase of UV-induced apoptosis, which can be suppressed by small-molecule Cdk2 inhibitors such as roscovitine and pyrrolidine dithiocarbamate. The results of this study suggest that the deregulation of Cdk2 activity may be critical to UV-induced apoptosis in CHO-K1 cells. 相似文献
Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N‐ and C‐terminal regions of CDC25B and requires CDC25B binding to its CDK—cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2‐containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components. 相似文献
We studied the effects of fibroblast growth factor (FGF-10) on H2O2-induced alveolar epithelial cell (AEC) G1 arrest and the role of G1 cyclins. FGF-10 prevented H2O2-induced AEC G1 arrest. FGF-10 induced 2-4-fold increase in cyclin E, cyclin A and CDKs (2,4) alone and in AEC treated with H2O2. H2O2 downregulated cyclin D1; FGF-10 blocked these effects. FGF-10 prevented H2O2-induced upregulation of CDK inhibitor, p21. SiRNAp21 blocked H2O2-induced downregulation of cyclins, CDKs and AEC G1 arrest. Accordingly, we provide first evidence that FGF-10 regulates G1 cyclins and CDKs, and prevents H2O2-induced AEC G1 arrest. 相似文献
CVT‐313 is a potent CDK2 inhibitor that was identified by screening a purine‐analogue library and is currently in preclinical studies. Since this molecule has the potential to be developed as a CDK2 inhibitor for cancer therapy, the potency of CVT‐313 to bind and stabilize CDK2 was evaluated, together with its ability to inhibit aberrant cell proliferation. CVT‐313 increased the melting temperature of CDK2 by 7°C in thermal stabilization studies, thus indicating its protein‐stabilizing effect. CVT‐313 inhibited the growth of human lung carcinoma cell line A549 in a dose‐dependent manner, with an IC50 of 1.2 µM, which is in line with the reported biochemical potency of 0.5 µM. To support the further chemical modification of CVT‐313 and to improve its biochemical and cellular potency, a crystal structure was elucidated in order to understand the molecular interaction of CVT‐313 and CDK2. The crystal structure of CDK2 bound to CVT‐313 was determined to a resolution of 1.74 Å and clearly demonstrated that CVT‐313 binds in the ATP‐binding pocket, interacting with Leu83, Asp86 and Asp145 directly, and the binding was further stabilized by a water‐mediated interaction with Asn132. Based on the crystal structure, further modifications of CVT‐313 are proposed to provide additional interactions with CDK2 in the active site, which may significantly increase the biochemical and cellular potency of CVT‐313. 相似文献
Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca2+ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca2+ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca2+‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca2+ influx, induced by membrane depolarization with high K+, was monitored with a fluorescent Ca2+ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca2+ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.
The present study was undertaken to investigate the regulation of P12(CDK2AP1) by miRNAs. A conserved target site for miR-21 within the CDK2AP1-3'-UTR at nt 349-370 was predicted by bioinformatics software and an inverse correlation of miR-21 and CDK2AP1 protein was observed. Highly specific amplification and quantification of miR-21 was achieved using real-time RT-PCR. Transfection of HaCaT cells with pre-miR-21 significantly suppressed a luciferase reporter including the CDK2AP1-3'-UTR, whereas transfection of Tca8113 with anti-miR-21 increased activity of this reporter. This was abolished when a construct mutated at the miR-21/nt 349-370 target site was used instead. Anti-miR-21-transfected Tca8113 cells showed an increase of CDK2AP1 protein and reduced proliferation and invasion. Resected primary tumors and tumor-free surgical margins of 18 patients with head and neck squamous cell carcinomas demonstrated an inverse correlation between miR-21 and P12(CDK2AP1). This study shows that P12(CDK2AP1) is downregulated by miR-21 and that miR-21 promotes proliferation and invasion in cultured cells. 相似文献
