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)

Drug resistance that occurs during cancer chemotherapy has been a major problem in controlling neoplastic progression. To study the cellular mechanisms of acquired drug resistance we developed 1,25-dihydroxyvitamin D3 (1,25D3)-resistant sublines of promyelocytic leukemia HL60 cells which have increased proliferation rates (Exp. Cell Res., 224, 312, 1996; Cancer Res., 50, 5513, 1996). We report here that the resistant sublines display varying degrees of shortening of the G1 phase as compared to the parental HL60-G cells. Protein levels of cyclins E, D1, D2 and D3 are elevated in these resistant cell lines, and cyclin D1 is especially high in 40AF cells, which has the shortest G1 length. The protein levels of cyclin-dependent kinase (Cdk)2, Cdk4 and Cdk6 are not altered in the resistant sublines. Both Cdk2 and Cdk6-associated kinase activites are increased in the resistant sublines, but not Cdk4 kinase activity. Protein levels of p27Kip1 are not consistently altered in the resistant sublines as compared to the parental HL60-G cells, but are reduced relative to HL60-G cells arrested by 96 h treatment with 1,25D3. Interestingly, the resistant cell lines constitutively express high levels of retinoblastoma protein (pRb), and pRb is highly phosphorylated, indicating that the G1 cyclin/Cdk complexes in the resistant cells are physiologically active. The results suggest that the increased activity of cyclin D/Cdk6, and perhaps cyclin E/Cdk2, lead to rapid hyperphosphorylation of pRb and consequently a shorter early G1 phase, and that in the resistant cells the increased ratio of cyclin E to p27Kip1 results in activation of Cdk2 and contributes to the abrogation of the 1,25D3-induced block to the S phase entry. Additionally, it is apparent that constitutively increased levels of pRb are compatible with increased rates of cell proliferation.
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PMID:Retinoblastoma protein-overexpressing HL60 cells resistant to 1,25-dihydroxyvitamin D3 display increased CDK2 and CDK6 activity and shortened G1 phase. 965 39

To explore the regulation and function of D-type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth-inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G1; association of D-type cyclins with cyclin-dependent kinase (cdk) 4 and cdk6; G1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D-type cyclins; and association of p16INK4a, p15INK4b, and p27Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G1 arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G1), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4-fold in treated versus untreated cells. At 5 h, cyclin D-associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4-associated p27Kip1 and, at 9 h, a fourfold increase in cdk6-associated p27Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cells but not their Rb-negative parental Du/145 cells. The data suggest that HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4- and cdk6-associated p27Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state.
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PMID:Interaction of retinoblastoma protein and D cyclins during cell-growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells. 965 57

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.
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PMID:Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. 965 99

Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A-associated cyclin-dependent kinase 2(cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21Cip1/Waf1 and p27Kip1 results in a substantial reduction in their association with nuclear cyclin-cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.
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PMID:Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade. 966 Sep 39

The E8 open reading frame of Bovine papillomavirus type 4 (BPV-4) encodes a small (42 amino acid) hydrophobic polypeptide localized to cellular membranes and capable of conferring an anchorage-independent (AI) growth phenotype on primary bovine cells co-transfected with BPV-4 E7 ORF and an activated ras gene. To further study the function of E8 independently of other viral gene products, we have expressed it in the murine fibroblast cell line, NIH3T3. Cells expressing E8 are capable of AI growth and escape growth arrest after serum withdrawal. E8 deregulates cyclin A expression, induces transactivation of the human cyclin A gene promoter and increases endogenous protein levels in cells maintained in short-term suspension culture and in low-serum (LS). Both these culture conditions promote downregulation of cyclin A in control cells. In LS growth conditions E8 permits sustained cyclin A-associated kinase activity but not cyclin E-cdk2 activity. Cyclin A-cdk2 activity and, in part, cyclin A gene expression are regulated by the cdk inhibitor p27Kip1. Expression of this cdk inhibitor is also de-regulated in E8 cells, with high levels being detected under all culture conditions tested. These data suggest that the ability of BPV-4 E8 to transform NIH3T3 cells is associated with upregulation of cyclin A-associated kinase activity and de-regulated expression of the cdk inhibitor p27Kip1 and does not rely on down-regulation of p27Kip1 expression. Analysis of E8 mutants indicate that the hydrophilic 'tail' of the molecule (residues 31-42) is required for cell transformation, as assessed by anchorage-independent growth, while a form of E8 with expression restricted to the Endoplasmic Reticulum/cis-Golgi membranes by addition of a 'KDEL' retention signal revealed that the sub-cellular localization is an important determinant of E8 biological activity.
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PMID:BPV-4 E8 transforms NIH3T3 cells, up-regulates cyclin A and cyclin A-associated kinase activity and de-regulates expression of the cdk inhibitor p27Kip1. 969 May 11

The cyclin dependent kinase holoenzymes (CDKs), composed of catalytic (cdk) and regulatory (cyclin) subunits, promote cellular proliferation and are inhibited by cyclin dependent kinase inhibitor proteins (CDKIs). The CDKIs include the Ink4 family (p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d) and the KIP family (p21Cip1 and p27Kip1). The sustained induction of p21 and p18 during myogenesis implicates these CDKI in maintaining cellular differentiation. Herein we examined the CDK (cyclin D1, cdk5) and CDKI expression profiles during the first 24 days of postnatal rat cerebella development. Cdk5 abundance increased and cyclin D1 decreased from day 9 through to adulthood. The CDKIs increased transiently during differentiation. p27 increased 20-fold between days 4 and 24, whereas p21 rose twofold between 6 to 11 days. p19, p18 and p16 increased approximately two- to threefold, falling to low levels in the adult. Immunostaining of cyclin D1 was localized in the external granular cells, whereas p27, was found primarily in the Purkinje cells. The period of maximal differentiation between days 9 to 13 was associated with a change in p21 and p16 staining from the external granular and Purkinje cells to a primarily Purkinje cell distribution. Protein-calorie malnutrition, which was previously shown to arrest rat cerebella development, reduced cyclin D1 kinase activity and p27 levels. However, p16 and p21 levels were unchanged. We conclude that the CDKIs are induced with distinct kinetics in specific cell types and respond differentially to growth factors during cerebella development, suggesting discrete roles for these proteins in normal cerebella development.
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PMID:Regulation of cyclin dependent kinase inhibitor proteins during neonatal cerebella development. 969 86

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.
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PMID:Molecular basis for the lack of T cell proliferation induced by an altered peptide ligand. 970 Oct 35

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.
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PMID:Role of p27Kip1 and cyclin-dependent kinase 2 in the proliferation of non-small cell lung cancer. 970 10

The chemotherapeutic agent and vitamin A metabolite retinoic acid (RA) has been used to treat many tumor types. The effects of RA are mediated by a family of ligand-dependent transcription factors, the RA receptors and the retinoid X receptors (RXR). Alterations in retinoid receptor expression have been implicated in tumorigenesis. Previous studies have shown lack of RXR-gamma expression in squamous cell carcinoma (SCC) lines. To begin to elucidate the role of RXR-gamma in the malignant transformation of SCCs, we expressed RXR-gamma in SCC lines by stable transfection. SCC lines expressing RXR-gamma produced large numbers of flat cells with abundant cytoplasm, which died and detached from the culture dish. These cells morphologically resembled the differentiated cells of normal stratified squamous epithelium in culture. These cells did not exhibit the characteristic DNA fragmentation pattern of apoptotic cells, nor did they label in a fluorescent apoptosis assay. RNase protection and Western blot analysis revealed induction of RA-responsive involucrin and keratin 10 expression, early markers of terminal differentiation. RXR-gamma expression produced significant reduction in levels of RA-responsive genes including the cyclin-dependent kinase inhibitors p21Cip1/WAF1 and p27Kip1, resulting in increased cdc2 and cdk2 kinase activity and RB phosphorylation. We concluded that RXR-gamma induced terminal differentiation in SCC lines, suggesting a potential tumor suppressor function for this transcription factor.
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PMID:Increased cdc2 and cdk2 kinase activity by retinoid X receptor gamma-mediated transcriptional down-regulation of the cyclin-dependent kinase inhibitor p21Cip1/WAF1 correlates with terminal differentiation of squamous cell carcinoma lines. 971 79

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.
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PMID:Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d. 975 Oct 51


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