Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ceramide is known to induce pRb (retinoblastoma gene product) dephosphorylation through the activation of ceramide-activated protein phosphatase (CAPP) during G1 arrest, but other molecular mechanisms linked to regulation of pRb dephosphorylation during ceramide-induced G1 arrest are poorly understood. In this paper, we investigated whether p21, a cdk (cyclin-dependent kinase) inhibitor, is involved in the induction of pRb dephosphorylation during ceramide-induced G1 arrest. In SK-Hep-1 cells, the addition of ceramide resulted in pRb dephosphorylation and G1 arrest. The activity of cdk2 was inhibited in response to ceramide during this process. p21 protein and mRNA were remarkably induced, while the protein level of p53, known as a transcriptional activator of p21, was not elevated at the same condition. p21 induction was also observed in the Hep3B cells lacking a functional p53 after exposure to ceramide. Although p21 is induced in ceramide-treated Hep3B cells, Hep3B cells do not induce G1 arrest, because Hep3B cells are deficient in a functional pRb protein. To confirm that pRb is a critical target for the induction of G1 arrest by inhibiting cdk2 activity through p53-independent p21, pRb-expressing vector was transfected into Hep3B cells. After treatment with ceramide, pRb-expressing cells (pRb+/+), but not pRb-/- cells, were arrested in G1 phase. In pRb+/+ cells, ceramide-mediated G1 arrest was accompanied by the accumulation of hypophosphorylated pRb and p21 associated with cdk2. Together, these results suggest that p21, induced through p53-independent pathway, participates in the induction of pRb dephosphorylation by inhibiting cdk2 activity during ceramide-mediated G1 arrest in hepatocarcinoma cells.
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PMID:Induction of p53-independent p21 during ceramide-induced G1 arrest in human hepatocarcinoma cells. 1087 74

The incidence of cutaneous malignant melanoma is undergoing a dramatic increase in persons with light-color skin in all parts of the world. The prognosis for individuals with advanced disease is dismal due to the lack of effective treatment options. Thus, there is a need for new approaches to control tumor progression. Epidemiological, experimental, and mechanistic data implicate omega-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chain omega-3 PUFAs as inhibitors of development and progression of a range of human cancers, including melanoma. The aim of this study was to assess the mechanisms by which docosahexaenoic acid (DHA), an omega-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposed in vitro to DHA and then assessed for (a) inhibition of cell growth; (b) expression of cyclins and cyclin-dependent kinase inhibitors in individual cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to cyclin D1, cyclin E, p21WAF1/CIP1, or p27(KIP1); and (c) expression of total pRb(T) independent of phosphorylation state and hypophosphorylated pRb(P-) in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb(T) or pRb(P-), respectively. After treatment with increasing concentrations of DHA, cell growth in a majority of melanoma cell lines (7 of 12) was inhibited, whereas in 5 of 12 cell lines, cell growth was minimally affected. Two melanoma cell lines were examined in detail, one resistant (SK-Mel-29) and one sensitive (SK-Mel-110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 microg/ml DHA whether grown in the absence or presence of 1% fetal bovine serum (FBS). No appreciable change was observed in cell growth, cell cycle distribution, the status of pRb phosphorylation, cyclin D1 expression, or the levels of the cyclin-dependent kinase inhibitors p21 and p27. In contrast, SK-Mel-110 cell growth was inhibited by DHA with the cells accumulating either in G1 or S phase: 0% in SK-Mel-29 versus 13.3 or 41.2% in SK-Mel-110 in the absence or presence of FBS, respectively. In the absence of serum, considerable death occurred by apoptosis. In addition, DHA treatment resulted in increasing numbers of SK-Mel-110 cells (from 12 to >40%) expressing hypophosphorylated pRb, whereas the levels of cyclin D1 and p21 changed little. Expression of p27 in these cells increased >2.5 times when grown in the absence of FBS but not in the presence of 1% FBS. Thus, we show for the first time that DHA inhibits the growth of cultured metastatic melanoma cells. Furthermore, growth inhibition correlates with a quantitative increase in hypophosphorylated pRb in the representative sensitive melanoma cell line SK-Mel-110. Although multiple factors influence pRb phosphorylation, it appears that both cyclin D1 and p21 expression do not change in the presence of DHA, although p27 was strikingly increased in SK-Mel-110 cells in the absence of FBS. The fact that pRb became hypophosphorylated after exposure to DHA suggests a cross-talk mechanism between fatty acid metabolism and the pRb pathway. Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents.
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PMID:Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation. 1094 21

The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.
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PMID:Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry. 1096 47

The association between the phosphorylation status of the retinoblastoma protein, pRb and changes in cell cycle control caused by either protein kinase C (PKC) or protein kinase A (PKA) stimulation was evaluated in human myeloblastic leukaemia ML-1 cells. TPA-induced PKC activation resulted in dephosphorylation of pRb and subsequently induced ML-1 differentiation based on morphological changes and CD14 expression. In the present study, we showed that inhibition of protein phosphatases (PP-1 and PP-2a) prevented the TPA-induced differentiation in ML-1 cells. Preinhibition of PP-1 and PP-2a activities with 1-100 nM okadaic acid dose-dependently blunted the decrease in the phosphorylation status of pRb obtained with TPA and overrode cell cycle arrest. PKA stimulation with 8-chlorophenylthio-cAMP (100 microM) decreased cell proliferation by 65% and the distribution of cells in the G1 phase significantly increased from 38% to 83% concomitant with a 34% decline in the number of cells present in the S phase. In addition, PKA stimulation significantly decreased the pRb phosphorylation status but did not elicit CD14 expression, indicating that cAMP-induced dephosphorylation of pRb cannot by itself trigger differentiation in ML-1 cells.
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PMID:Okadaic acid suppresses TPA-induced differentiation by stimulating G1/S transition in human myeloblastic leukaemia ML-1 cells. 1104 Dec

In current models of cell cycle control, the transition between different cell cycle states is regulated at checkpoints. Transition through the cell-cycle is induced by a family of protein kinase holoenzymes, the cyclin-dependent kinases (CDKs) and their heterodimeric cyclin partner. Orderly progression through the cell-cycle involves co-ordinated activation of the CDKs, which in the presence of an associated CDK-activating kinase, phosphorylate target substrates including members of the 'pocket protein' family. This family includes the product of the retinoblastoma susceptibility gene (the pRb protein) and the related p107 and p130 proteins. Activity of these holoenzymes is regulated by post-translational modification. Phosphorylation of inhibitory sites on a conserved threonine residue within the activation segment is regulated by CDK7/cyclin H, referred to as CDK-activating kinase [1]. In addition, the cdc25 phosphatases activate the CDKs by dephosphorylating their inhibitory tyrosine and threonine phosphorylated residues [2,3]. Among the many roles for endogenous inhibitors (CDKIs), including members of the p21(CIP1/Waf1) family and the p16 family, one role is to regulate cyclin activity. Cellular neoplastic transformation is accompanied by loss of regulation of cell cycle checkpoints in conjunction with aberrant expression of CDKs and/or cyclins and the loss or mutation of the negative regulators (the CDKIs or the pocket protein pRb). One strategy to inhibit malignant cellular proliferation involves inhibiting CDK activity or enhancing function of the CDKI. Novel inhibitors of CDKs showing promise in the clinic include flavopiridol and UCN-01, which show early evidence of human tolerability in clinical trials. This review examines pertinent advances in the field of CDK inhibitors.
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PMID:Cyclin-dependent kinase inhibitors: novel anticancer agents. 1106 Jul 82

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.
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PMID:Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium. 1107 62

UCN-01 (7-hydroxystaurosporine) is a newly developed cell cycle inhibitor known to have several modes of action, including inhibition of cyclin-dependent kinase, induction of p21 and suppression of pRb phosphorylation. In order to test a combination therapy of UCN-01 and 5-fluorouracil (5-FU), growth inhibition of CRL 1420 (MIA PaCa-2; undifferentiated pancreatic carcinoma) by four different treatments was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The treatments used were UCN-01 alone, 5-FU alone, 5-FU followed by UCN-01 (5-FU/UCN-01) and UCN-01 followed by 5-FU (UCN-01/5-FU). We also assessed changes in thymidylate synthetase (TS) mRNA levels, TS activity, and 5-FU incorporation by RNA (F-RNA) for each treatment. Although treatment with UCN-01 alone, 5-FU alone, and 5-FU/UCN-01 inhibited CRL 1420 growth in a concentration-dependent manner, treatment with UCN-01/5-FU inhibited the growth of CRL 1420 synergistically at less than 1 microg/ml drug concentration. The down-regulation of TS mRNA by UCN-01 resulted in stable total TS and decreased free TS, and UCN-01/ 5-FU resulted in enhanced thymidylate synthetase inhibition rate (TSIR) compared to UCN-01 alone and 5-FU/UCN-01. This increased TSIR due to UCN-01 pretreatment was accompanied by elevated F-RNA concentrations in the UCN-01/5-FU treatment. The suppression of TS mRNA and TS activity by UCN-01 may lead to higher sensitivity of tumor cells to 5-FU and may explain the synergistic antitumor effect of UCN-01/5-FU. In conclusion, low concentrations of UCN-01 (from 0.01 to 1 microg/ml) may be clinically useful, affording low cytotoxicity of UCN-01, while enhancing the antitumor effect of 5-FU.
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PMID:UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA. 1109 86

Somatostatin, or its structural analog SMS 201-995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4-2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4-2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4-2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4-2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27(Kip1) down-regulation, as well as marginally induces p21(Cip) expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21(Cip) and p27(Kip1), leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.
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PMID:Somatostatin inhibits Akt phosphorylation and cell cycle entry, but not p42/p44 mitogen-activated protein (MAP) kinase activation in normal and tumoral pancreatic acinar cells. 1114 74

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.
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PMID:p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest. 1115 6

Normal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G(1)/S phase transition. Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours (Ta/T1 = 97; T2-T4 = 53) were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours (Ta/T1 = 28; T2-T4 = 26) was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2-T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2-T4 tumours (8%, P< 0.005) with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours (14%, P< 0.005). There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index > or = 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2-T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These results indicate that cyclin D1 expression is significantly higher in low-stage, well differentiated bladder tumours and strongly correlates with waf1 expression. In a multivariate analysis, cyclin D1 expression is an independent prognostic indicator of survival in those patients with muscle-invasive disease.
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PMID:Cyclin D1 expression in transitional cell carcinoma of the bladder: correlation with p53, waf1, pRb and Ki67. 1116 87


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