Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes. (1/1134)

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.  (+info)

Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. (2/1134)

The irreversible G1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21(Sdi1,Cip1,Waf1), which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16(Ink4a), suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E- and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by +info)

Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice. (3/1134)

The response of the uterine epithelium to female sex steroid hormones provides an excellent model to study cell proliferation in vivo since both stimulation and inhibition of cell proliferation can be studied. Thus, when administered to ovariectomized adult mice 17beta-estradiol (E2) stimulates a synchronized wave of DNA synthesis and cell division in the epithelial cells, while pretreatment with progesterone (P4) completely inhibits this E2-induced cell proliferation. Using a simple method to isolate the uterine epithelium with high purity, we have shown that E2 treatment induces a relocalization of cyclin D1 and, to a lesser extent, cdk4 from the cytoplasm into the nucleus and results in the orderly activation of cyclin E- and cyclin A-cdk2 kinases and hyperphosphorylation of pRb and p107. P4 pretreatment did not alter overall levels of cyclin D1, cdk4, or cdk6 nor their associated kinase activities but instead inhibited the E2-induced nuclear localization of cyclin D1 to below the control level and, to a lesser extent, nuclear cdk4 levels, with a consequent inhibition of pRb and p107 phosphorylation. In addition, it abrogated E2-induced cyclin E-cdk2 activation by dephosphorylation of cdk2, followed by inhibition of cyclin A expression and consequently of cyclin A-cdk2 kinase activity and further inhibition of phosphorylation of pRb and p107. P4 is used therapeutically to oppose the effect of E2 during hormone replacement therapy and in the treatment of uterine adenocarcinoma. This study showing a novel mechanism of cell cycle inhibition by P4 may provide the basis for the development of new antiestrogens.  (+info)

Functions of cyclin A1 in the cell cycle and its interactions with transcription factor E2F-1 and the Rb family of proteins. (4/1134)

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. Here, we show that the expression of human cyclin A1 and cyclin A1-associated kinase activities was regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein were present at very low levels in cells at the G0 phase. They increased during the progression of the cell cycle and reached the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaked at the G2/M phase. We report that cyclin A1 could bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 was greatly enhanced when cyclin A1 was complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 were observed in vivo in several cell lines. When cyclin A1 was coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex had kinase activities for histone H1, E2F-1, and the Rb family of proteins. Our results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells.  (+info)

Heparin inhibits proliferation of myometrial and leiomyomal smooth muscle cells through the induction of alpha-smooth muscle actin, calponin h1 and p27. (5/1134)

Mast cells are widely distributed in human tissues, including the human uterus. However, the function of mast cells in uterine smooth muscle has not been clearly established. Mast cells possess secretory granules containing such substances as heparin, serotonin, histamine and many cytokines. To help establish the role of mast cells in the human myometrium, the action of heparin was investigated using smooth muscle cells (SMC) from normal myometrium and from leiomyoma. The proliferation of cultured myometrial and leiomyomal SMC was inhibited by heparin treatment. Flow cytometric analysis showed that the population in the G1 phase of the cell cycle increased under heparin treatment. Western blotting analysis showed that markers of SMC differentiation such as alpha-smooth muscle actin (alpha-SMA), calponin h1 and cyclin-dependent kinase inhibitor p27 were induced by heparin, whereas cell-cycle-related gene products from the G1 phase of the cell cycle, such as cyclin E and cdk2, were not changed. Taken together, these results indicate that heparin inhibits the proliferation of myometrial and leiomyomal SMC through the induction of alpha-SMA, calponin h1 and p27. We suggest that heparin from mast cells may induce differentiation in uterine SMC and may influence tissue remodelling and reconstruction during physiological and pathophysiological events.  (+info)

Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells. (6/1134)

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.  (+info)

A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells. (7/1134)

BACKGROUND: The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably normal when grown in vitro. One explanation may be the operation of compensatory pathways that control CDK activity and cell proliferation when normal pathways are inactivated. We have used mice lacking the CKIs p21(Cip1) and p27(Kip1) to investigate this issue, specifically with respect to CDK regulation by mitogens. RESULTS: We show that p27 is the major inhibitor of Cdk2 activity in mitogen-starved wild-type murine embryonic fibroblasts (MEFs). Nevertheless, inactivation of the cyclin E-Cdk2 complex in response to mitogen starvation occurs normally in MEFs that have a homozygous deletion of the p27 gene. Moreover, CDK regulation by mitogens is also not affected by the absence of both p27 and p21. A titratable Cdk2 inhibitor compensates for the absence of both CKIs, and we identify this inhibitor as p130, a protein related to the retinoblastoma gene product Rb. Thus, cyclin E-Cdk2 kinase activity cannot be inhibited by mitogen starvation of MEFs that lack both p27 and p130. In addition, cell types that naturally express low amounts of p130, such as T lymphocytes, are completely dependent on p27 for regulation of the cyclin E-Cdk2 complex by mitogens. CONCLUSIONS: Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.  (+info)

Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27(Kip1). (8/1134)

Proliferation and apoptosis are increased in many types of inflammatory diseases. A role for the cyclin kinase inhibitor p27(Kip1) (p27) in limiting proliferation has been shown. In this study, we show that p27(-/-) mesangial cells and fibroblasts have strikingly elevated rates of apoptosis, not proliferation, when deprived of growth factors. Apoptosis was rescued by restoration of p27 expression. Cyclin A-cyclin-dependent kinase 2 (CDK2) activity, but not cyclin E-CDK2 activity, was increased in serum-starved p27(-/-) cells, and decreasing CDK2 activity, either pharmacologically (Roscovitine) or by a dominant-negative mutant, inhibited apoptosis. Our results show that a new biological function for the CDK inhibitor p27 is protection of cells from apoptosis by constraining CDK2 activity. These results suggest that CDK inhibitors are necessary for coordinating the cell cycle and cell-death programs so that cell viability is maintained during exit from the cell cycle.  (+info)

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