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

The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. Treatment with 5 microM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48 hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5 microM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.
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PMID:SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells. 1223 12

Apoptotic cell death is an important mode of cell loss contributing to heart dysfunction. To analyze the importance of the E2F-dependent regulation of gene transcription in cardiomyocyte apoptosis, the function of cell cycle factors impinging on the retinoblastoma protein (pRb)/E2F pathway was investigated. In isolated neonatal ventricular myocytes, apoptotic cell death induced by hypoxia (deferoxamine, 100 micro mol/L) specifically activated cyclin-dependent kinases (cdks) 2 and 3. Apoptotic cell death was inhibited by ectopic expression of cdk inhibitors p21(CIP) and p27(KIP1) but not p16(INK4). In addition, apoptosis was also abrogated by forced expression of kinase dead mutant proteins of cdk2/3 but not of cdk4/6. Introduction of cdk inhibitors or dominant-negative cdk2/3 blocked pRb hyperphosphorylation and abrogated E2F-dependent gene transcription, including that of the E2F-responsive genes of proapoptotic caspase 3 and caspase 7. Moreover, introduction of constitutively active pRb and transcriptionally inert mutant E2F1/DP1 efficiently protected cardiomyocytes from apoptosis. In conclusion, these data demonstrate that cdk-specific inactivation of pRb and the subsequent activation of E2F-dependent gene transcription are required for cardiomyocyte apoptosis.
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PMID:Inhibition of hypoxia-induced apoptosis by modulation of retinoblastoma protein-dependent signaling in cardiomyocytes. 1241 92

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.
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PMID:Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed, oncogene-transformed, and human cancer cells. 1241 22

Apart from their coordinated inactivation by DNA tumor viral oncoproteins, the pRB and p53 tumor suppressor pathways were not known to be connected ten years ago. Within the last decade, our appreciation of how these pathways are interconnected has grown substantially. The checks and balances that exist between pRB and p53 involve the regulation of the G1/S transition and its checkpoints, and much of this is under the control of the E2F transcription factor family. Following DNA damage, the p53-dependent induction of p21CIP1 regulates cyclin E/Cdk2 and cyclin A/Cdk2 complexes both of which phosphorylate pRB, leading to E2F-mediated activation. Similarly, E2F1-dependent induction of p19ARF antagonizes the ability of mdm2 to degrade p53, leading to p53 stabilization and potentially p53-mediated apoptosis or cell cycle arrest. From the existing mouse models discussed above, we also know that proliferation, cell death and differentiation of distinct tissues are also intimately linked through entrance and exit from the cell cycle, and thus through pRB and p53 pathways. Virtually all human tumors deregulate either the pRB or p53 pathway, and often times both pathways simultaneously, which is critical for crippling cellular defense against neoplasia. The next decade of cancer research will likely see these two tumor suppressor pathways only merge even more.
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PMID:Role of the RB tumor suppressor in cancer. 1261 99

Neuronal apoptosis plays a critical role in the normal development of the mammalian brain and is thought to contribute to the pathogenesis of several neurologic disorders. However, the intracellular mechanisms underlying apoptosis of neurons remain incompletely understood. In the present study, we characterized a cell-cycle-based mechanism by which neuronal activity deprivation induces apoptosis of postmitotic neurons. Activity deprivation, but not growth factor withdrawal, was found to induce Cdc2 expression and consequent Cdc2-mediated apoptosis in granule neurons of the developing rat cerebellum. We found that activity deprivation induces cdc2 transcription in neurons via an E2F-binding element (EBE) within the cdc2 promoter. The transcription factor E2F1 that is expressed in granule neurons was found in DNA binding assays to bind to the EBE of the cdc2 gene. In chromatin immunoprecipitation analysis, endogenous E2F1 forms a complex with the promoter of the endogenous cdc2 gene in granule neurons, indicating that endogenous E2F1 is poised to activate transcription of the endogenous cdc2 gene in neurons. Consistent with this conclusion, a dominant interfering form of E2F, when expressed in granule neurons, blocked activity deprivation-induced cdc2 transcription. In other experiments, we found that the expression of E2F1 in granule neurons induces Cdc2 expression and promotes neuronal apoptosis via the activation of Cdc2. Remarkably, in contrast to inducing the E2F-mediated expression and activation of Cdc2 in granule neurons, activity deprivation fails to stimulate the expression of E2F-target genes that trigger DNA synthesis and replication. Together, our findings define a novel apoptotic mechanism whereby E2F selectively couples an activity deprivation-induced signal to cdc2 transcription in the absence of stimulating DNA synthesis and thus culminating in Cdc2-mediated apoptosis of postmitotic neurons.
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PMID:The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. 1262 69

NPAT is an in vivo substrate of cyclin E-Cdk2 kinase and is thought to play a critical role in coordinated transcriptional activation of histone genes during the G(1)/S-phase transition and in S-phase entry in mammalian cells. Here we show that NPAT transcription is up-regulated at the G(1)/S-phase boundary in growth-stimulated cells and that the NPAT promoter responds to activation by E2F proteins. We demonstrate that endogenous E2F proteins interact with the promoter of the NPAT gene in vivo and that induced expression of E2F1 stimulates NPAT mRNA expression, supporting the idea that the expression of NPAT is regulated by E2F. Consistently, we find that the E2F sites in the NPAT promoter are required for its activation during the G(1)/S-phase transition. Moreover, we show that the expression of NPAT accelerates S-phase entry in cells released from quiescence. The inhibition of NPAT expression by small interfering RNA duplexes impedes cell cycle progression and histone gene expression in tissue culture cells. Thus, NPAT is an important E2F target that is required for cell cycle progression in mammalian cells. As NPAT is involved in the regulation of S-phase-specific histone gene transcription, our findings indicate that NPAT links E2F to the activation of S-phase-specific histone gene transcription.
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PMID:NPAT expression is regulated by E2F and is essential for cell cycle progression. 1266 81

Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with myostatin treatment, consistent with D-type-cyclin-Cdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by myostatin.
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PMID:Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway. 1472 80

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation.
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PMID:Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7. 1502 26

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16(Ink4a)-cycD/cdk4-pRB-E2F and p19(Arf)-mdm2-p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21(Cip1). Thus, E2F3 is a key repressor of the p19(Arf)-p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21(Cip1) in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3(-/-) cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation.
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PMID:Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics. 1517 42

The activities of cell cycle regulatory proteins have been reported to be associated with the development of pathological lesions in glomerulonephritis. To assess the cellular mechanisms underlying the mesangial cell proliferation and glomerulosclerosis in progressive human IgA nephropathy (IgAN), we examined the expression of E2F1, Rb, c-Myc, proliferating cell nuclear antigen (PCNA), cyclins (D1, E and A), cyclin-dependent kinase 2 (CDK2) and CDK inhibitors (p21(waf1), p27(kip1), 57(kip2) and p16(ink4a)) by immunohistochemistry in renal biopsy specimens. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was also performed to detect the presence of apoptosis. In total, 51 cases of IgAN were categorized into four subgroups according to histological severity. A dramatic upregulation of E2F1 expression in mesangial cells was identified in proliferating glomeruli, which correlated well with the proliferation index. High endogenous expression of p27(kip1) and p57(kip2) by podocytes in normal glomeruli and glomeruli with minor lesions was observed to decrease in proliferating and sclerosing glomeruli; this pattern displayed a strong inverse correlation with the mean glomerulosclerosis score and the index of glomerular lesion. Increased apoptotic activity was identified in progressive glomerular lesions of advanced IgAN, which correlated with the proliferative activity in these lesions as assessed by total expression levels of PCNA and CDK2 in glomeruli, E2F1 expression levels in the mesangium, cyclin D1 expression levels in endothelium and the c-Myc glomerular staining score. Our results suggest that the onset and magnitude of mesangial cell proliferation and glomerulosclerosis is associated with the upregulation of E2F1 by mesangial cells and the downregulation of p27(kip1) and p57(kip2) by glomerular epithelial cells. The cell type-specific and coordinated regulation of proliferative and proapoptotic activities of cell cycle regulatory proteins may play an important role in mediating progressive glomerular injury in human IgAN.
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PMID:Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy. 1520 47


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