Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cell cycle is governed by cyclin dependent kinases (cdks), which are activated by binding of cyclins, inhibited by cdk inhibitors and regulated by phosphorylation and dephosphorylation. Exposure to high dose dihydrotestosterone (DHT) inhibits population growth of the human prostate carcinoma cell line, LNCaP. To determine the mechanism of growth arrest by high dose DHT, we assayed the changes in cell cycle profile and the cell cycle regulators that mediate these effects. Treatment of asynchronously growing LNCaP cells with 100 nM DHT caused a G1 arrest. The proportion of cells in S phase fell from 22 to 2%, while the G1 fraction rose from 74 to 92% by 24 h. Loss of phosphorylation of the retinoblastoma protein was noted and cdk4 and cyclin E/ cdk2 activities fell. Inhibition of these G1 cyclin dependent kinases was not due to loss of either cyclin or cdk proteins nor to increases in the cdk inhibitors p16INK4A and p21CiP1. p21Cip1 protein levels remained constant, and cyclin E-associated p21CiP1 fell, suggesting that p21CiP1 is not relevant to this form of cyclin E/cdk2 inhibition. Of note, total p27KiP1 levels and cyclin E-associated p27Kip1 increased as cells arrested and the amount of the CAK activated cdk2 bound to cyclin E decreased. p27KiP1 immunodepletion experiments demonstrated that the DHT-mediated increase in p27Kip1 was sufficient to fully saturate and inhibit target cyclin E/ cdk2. The inhibition of cyclin E/cdk2 by p27Kip1 contributes to G1 arrest of LNCaP following high dose DHT. p27KiP1 may be a key effector of androgen dependent growth modulation in prostate cancer cells.
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PMID:Involvement of p27Kip1 in G1 arrest by high dose 5 alpha-dihydrotestosterone in LNCaP human prostate cancer cells. 1069 12

Ubiquitin-mediated proteolysis controls intracellular levels of various cell cycle regulatory proteins, and its inhibition has been shown to induce apoptosis in proliferating cells. In the present study, we examined induction of apoptosis in oral squamous cell carcinoma (OSCC) cells by treatment with specific proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal and lactacystin. In all three OSCC cell lines examined, apoptotic changes such as apoptotic body formation and DNA fragmentation were observed at various degrees after 24 h of the carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal or lactacystin treatment. HSC2 cells showed the most prominent apoptotic changes among the cell lines examined and demonstrated the highest level of accumulation of p27Kip1 protein after the treatment with proteasome inhibitor. Reduced expressions of cyclin D1 and phospho pRb were also observed after the treatment with proteasome inhibitor. Moreover, 12 h of treatment with the proteasome inhibitor inhibited cdk2/cyclin E kinase activity and increased the ratio of the cell cycle population at the G1 phase. The proteasome inhibitor led to inhibition of cell cycle progression. In addition, activation of CPP32 and reduced expression of Bcl-2 were observed. Because apoptosis induced by the proteasome inhibitor was inhibited by treatment with antisense p27Kip1 oligonucleotide, accumulation of the p27Kip1 protein might play an important role in the apoptosis induced by proteasome inhibitor. The present results suggest that inhibition of proteasome function may be used as a possible target of novel therapy for OSCC.
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PMID:p27Kip1 accumulation by inhibition of proteasome function induces apoptosis in oral squamous cell carcinoma cells. 1074 16

Phosphorylation/inactivation of RB is typically required for cell cycle progression. However, we have identified a tumor cell line, C33A, which progresses through the cell cycle in the presence of an active allele of RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, we compared RB signaling to downstream effectors in this resistant cell line to that of the RB-sensitive SAOS-2 cell line. Although introduction of PSM-RB repressed E2F-mediated transcription in both C33A and SAOS-2 cells, PSM-RB failed to repress Cyclin A promoter activity in C33A. Ectopic expression of PSM-RB in SAOS-2 cells resulted in a decrease in both Cyclin A and Cdk2 protein levels without affecting Cyclin E or Cdk4. In contrast, over-expression of PSM-RB in C33A cells did not alter endogenous Cyclin A, Cyclin E, or Cdk2 protein levels or impact Cdk2 kinase activity, indicating that signaling from RB to down-stream targets is abrogated in this cell line. The importance of Cdk2 activity was demonstrated by p27Kip1, which attenuated Cdk2 activity and inhibited cell cycle progression in C33A cells. Since RB signaling to Cdk2 is disrupted in these tumor cells, we co-expressed two proteins that cooperate with RB in transcriptional repression, AHR and BRG-1, in an attempt to correct this signaling dysfunction. Co-expression of AHR/BRG-1 with PSM-RB attenuated Cyclin A and Cdk2 expression as well as Cdk2-associated kinase activity, resulting in cell cycle inhibition of C33A cells. Importantly, ectopic expression of Cyclin A was able to reverse the arrest mediated by co-expression of AHR/BRG-1 with PSM-RB. These results indicate that down-regulation of Cdk2 activity is requisite for RB-mediated cell cycle arrest. Thus, this study reveals a new mechanism through which tumor cells evade anti-proliferative signals, and provides insight into how RB-signaling is mediated.
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PMID:Restoration of retinoblastoma mediated signaling to Cdk2 results in cell cycle arrest. 1077 75

Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context.
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PMID:Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1. 1080 68

The retinoblastoma family of proteins, pRb/p105, p107, and pRb2/ p130, cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Each of the family members realize their common goal of G1-S checkpoint regulation through overlapping and unique growth regulatory pathways. We took advantage of a tetracycline-regulated gene expression system to control the expression of RB2/p130 in JC virus-induced hamster brain tumor cells to study in vivo the molecular mechanisms used by pRb2/p130 to elicit its growth-suppressive function. We have previously used this system to demonstrate that induction of pRb/ p130 expression suppresses tumor growth in vivo by overcoming neoplastic transformation mediated by the large T-antigen oncoprotein of JCV (JCV TAg). Here we found that induction of pRb2/p130 in vivo specifically inhibits cyclin A- and cyclin E-associated kinase activity and by doing so induces p27Kip1 levels presumably by inhibiting p27Kip1-targeted proteolysis by cyclin E-Cdk2 phosphorylation of p27Kip1. RB2/p130 induction also decreased cyclin A and the transcription factor E2F-1 while increasing cyclin E at both the transcriptional and protein levels of expression. The growth inhibitory activity of pRb2/p130 also correlated with its E2F-binding capacity. Furthermore, p27Kip1 and pRb2/p130 were found to be targets of the JCV TAg oncoprotein and to interact in vivo with each other independently from the presence of TAg. Interestingly, pRb2/p130 expression negatively modulated the binding of p27Kip1 to JCV TAg. These data suggest that pRb2/p130 and p27Kip1 may cooperate in regulating cellular proliferation, and both may be involved in a negative feedback regulatory loop with cyclin E.
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PMID:Inducible pRb2/p130 expression and growth-suppressive mechanisms: evidence of a pRb2/p130, p27Kip1, and cyclin E negative feedback regulatory loop. 1082 49

An enhanced vasoconstriction and vascular smooth muscle cell proliferation are involved in pathogenesis of hypertension. Beta3-blockers are effective for treatment of hypertensive patients. Recently the new beta1-receptor blocker nebivolol showed a different hemodynamic profile from those of other classic beta-blockers. In this study we hypothesized that nebivolol may also have different effects on smooth muscle cell proliferation compared with other beta-blockers such as atenolol. Human aortic smooth muscle cells (SMCs) were cultured, and cell growth was determined by increase in cell number. Growth-signaling molecules such as mitogen-activated protein kinase (p42mapk) and S6-kinase (p70S6K) and cell-cycle regulatory proteins (i.e., Cdk2, p27Kip1, and pRb) were analyzed by immunoblotting. In cultured human aortic SMCs, cell number was markedly increased in response to 5% fetal calf serum (FCS) over 6 days (87 +/- 11 x 10(3)/well), which was inhibited by nebivolol (10(-8)-10(-5) M; 25 +/- 2 x 10(3)/well; n = 6; p < 0.05), but not by atenolol. 5% FCS activated p42mapk, S6K, and Cdk2, but downregulated p27Kip1 and hyperphosphorylated pRb. Nebivolol prevented Cdk2 activation without influencing p42mapk, S6K, pRB, and p27Kip1. Thus, the new beta1-blocker nebivolol exhibits antiproliferative effect on human SMC through inactivation of Cdk2. This effect of nebivolol may have advantages over other beta-blockers in treatment of patients with cardiovascular disease.
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PMID:Nebivolol inhibits human aortic smooth muscle cell growth: effects on cell cycle regulatory proteins. 1083 16

The Akt/PKB protein kinase is implicated in the control of cell cycle progression and the suppression of apoptosis in cancer cells. Here we describe the use of a conditionally active form of Akt/PKB (M+ Akt:ER*) to study the ability of this protein to influence biological processes that are central to the process of oncogenic transformation of mammalian cells. Activation of M+ Akt:ER* in Rat1 cells elicited alterations in cell morphology and promoted anchorage-independent growth in agarose with high efficiency. Consistent with these observations, activation of M+ Akt:ER* suppressed the apoptosis of Rat1 cells that occurs after the detachment of these cells from extracellular matrix. Furthermore, activation of M+ Akt:ER* was sufficient to promote the progression of quiescent Rat1 cells into the S and G2-M phases of the cell cycle. In accord with this is the observation that activation of M+ Akt:ER* led to decreased expression of the cyclin-dependent kinase inhibitor p27Kip1 with a concomitant increase in cyclin-dependent kinase-2 activity. Perhaps surprisingly, activation of M+ Akt:ER* or expression of a constitutively active form of Akt led to rapid activation of MAP/ERK Kinase (MEK) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases in Rat1 cells. However, pharmacological inhibition of MEK by PD098059 did not inhibit the morphological alterations of Rat1 cells that occur after M+ Akt:ER* activation. These data suggest that M+ Akt:ER* can activate a number of pathways in Rat1 cells, leading to significant alterations in a number of biological processes. The conditional transformation system described here will allow further elucidation of the ability of Akt to contribute to both the normal response of cells to mitogenic stimulation and the aberrant proliferation observed in cancer cells.
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PMID:Oncogenic transformation of cells by a conditionally active form of the protein kinase Akt/PKB. 1091 95

Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.
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PMID:Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells. 1091 38

Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well.
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PMID:A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1. 1108 22

Replicative senescence may be an important tumor suppressive mechanism for human cells. We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of 'self-selection', and as a consequence exhibit diminished p16INK4A levels. As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively. Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age. In contrast to observations in fibroblast, p21Cip1 was not increased at senescence in HMECs. There was no increase in p27Kip1 levels nor in KIP association with targets cdks. While p15INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15INK4B. The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A. Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence. Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2. In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction. Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase.
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PMID:Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescence in human mammary epithelial cells. 1110 32


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