p16INK4A mediates cyclin dependent kinase 4 and 6 inhibition in senescent prostatic epithelial cells.
(33/363)
The senescence checkpoint constrains the proliferative potential of normal cells in culture to a finite number of cell doublings. In this study, we investigated the mechanism of cyclin dependent kinase (cdk) inhibition in senescent human prostatic epithelial cells (HPECs). Progression of HPECs from early passage to senescence was accompanied by a gradual loss of cells in S phase and an accumulation of cells containing 2N DNA. Furthermore, G1-S phase-associated kinase activities progressively diminished with increasing cell passage. In senescent HPECs, cdk4 and cyclin E1- and A-associated kinases were catalytically inactive. In contrast to observations in senescent fibroblasts, levels of the kinase inhibitor protein (KIP) inhibitor p21CIP1 diminished over the proliferative life span of HPECs. p27KIP1 levels fell as cells approached senescence, and the association of both p21CIP1 and p27KIP1 with cdk4/6 complexes was decreased. However, the level of cyclin E1-associated KIP molecules was unaltered as cells progressed into senescence. Progression to senescence was accompanied by a progressive increase in both the level of p16(INK4A) and in its association with cdk4 and cdk6. As HPECs approached senescence, cdk4- and cdk6-bound p16(INK4A) showed a shift to a slower mobility due to a change in its phosphorylation profile. As p16(INK4A) increased in cdk4 and cdk6 complexes, there was a loss of cyclin D1 binding. The altered phosphorylation of p16(INK4A) in senescent prostatic epithelial cells may facilitate its association with cdk4 and cdk6 and play a role in the inactivation of these kinases. (+info)
Stem cell factor inhibits erythroid differentiation by modulating the activity of G1-cyclin-dependent kinase complexes: a role for p27 in erythroid differentiation coupled G1 arrest.
(34/363)
Terminal erythroid differentiation is accompanied by decreased expression of c-Kit and decreased proliferation of erythroid progenitor cells. Using a newly established erythroleukemia cell line HB60-5, which proliferates in response to erythropoietin (Epo) and stem cell factor (SCF) and differentiates when stimulated with Epo alone, we characterized several events associated with the cell cycle during erythroid differentiation. Forty-eight h after SCF withdrawal and Epo stimulation, there was strong inhibition of cyclin-dependent kinase (cdk) 4 and cdk6 activities, associated with an increase in the binding of p27 and p15 to cdk6. A significant increase in the binding of p27 to cyclin E- and cyclin A-associated cdk2 correlated with the inhibition of these kinases. In addition, the expression of c-Myc and its downstream transcriptional target Cdc25A were found to be down-regulated during Epo-induced terminal differentiation of HB60-5 cells. The loss of Cdc25A was associated with an increase in the phosphotyrosylation of cyclin E-associated cdk2, which may contribute to cell cycle arrest during differentiation. Although overexpression of p27 in HB60-5 cells caused G1 arrest, it did not promote terminal erythroid differentiation. Thus, the cell cycle arrest that involves p27 is part of a broader molecular program during HB60-5 erythroid differentiation. Moreover, we suggest that SCF stimulation of erythroblasts, in addition to inhibiting erythroid differentiation, activates parallel or sequential signals responsible for maintaining cyclin/cdk activity. (+info)
Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6.
(35/363)
Because ribosome biogenesis plays an essential role in cell proliferation, control mechanisms may have evolved to recognize lesions in this critical anabolic process. To test this possibility, we conditionally deleted the gene encoding 40S ribosomal protein S6 in the liver of adult mice. Unexpectedly, livers from fasted animals deficient in S6 grew in response to nutrients even though biogenesis of 40S ribosomes was abolished. However, liver cells failed to proliferate or induce cyclin E expression after partial hepatectomy, despite formation of active cyclin D-CDK4 complexes. These results imply that abrogation of 40S ribosome biogenesis may induce a checkpoint control that prevents cell cycle progression. (+info)
Cdk2-dependent phosphorylation and functional inactivation of the pRB-related p130 protein in pRB(-), p16INK4A(+) tumor cells.
(36/363)
The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G(1) phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16(INK4A) in most pRB(-) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G(1)-S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(-), p16(+) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G(1) and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(-), p16(+) cells as compared with pRB(+), p16(-) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E- and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins. (+info)
Changes in E2F binding after phenylbutyrate-induced differentiation of Caco-2 colon cancer cells.
(37/363)
Differentiation agents use existing cellular systems to induce neoplastic cells to regain a normal phenotype and/or to cause growth arrest and therefore may offer novel chemotherapeutic approaches to treating solid tumors. In this study, we demonstrate in Caco-2 colon cancer cells that the differentiation agent phenylbutyrate (PB) causes a decrease in viable cells, an increase in cell differentiation, and a G1-S-phase block. The mechanism of this last effect is related to a PB-induced increase in p27Kip1, leading to a decrease in the activity of cyclin-dependent kinase 2 (CDK2), a positive regulator of the G1-S-phase cell cycle transition. Consistent with the decreased CDK2 kinase activity, we also observed a decrease in the phosphorylation state of the retinoblastoma protein after PB treatment. This was associated with increased binding and consequent inactivation of E2F, a transactivator of genes that regulate the G1 to S phase cell cycle transition. These data suggest that the differentiation agent PB inhibits tumor growth by limiting the availability of active E2F, with a subsequent G1-S-phase block. Additional studies should show whether PB is a clinically effective therapeutic agent against colorectal cancer. (+info)
The Rb-CDK4/6 signaling pathway is critical in neural precursor cell cycle regulation.
(38/363)
The tumor suppressor, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. We hypothesized that activation of the Rb pathway would induce cell cycle arrest in primary neural precursor cells, independent of the proposed function of cyclin-dependent kinases 4/6 (CDK4/6) to sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27 from CDK2. We expressed dominant negative adenovirus mutants of CDKs 2, 4, and 6 (dnCDK2, dnCDK4, and dnCDK6) in neural progenitor cells derived from E12.5 wild type and Rb-deficient mouse embryos. In contrast to previous studies, our results demonstrate that in addition to dnCDK2, the dnCDK4/6 mutants can induce growth arrest. Moreover, the dnCDK4/6-mediated inhibition is Rb-dependent. The dnCDK2 partially inhibited cell growth in Rb-deficient cells, suggesting that CDK2 may have additional targets. A previously proposed function of CDK4/6 is CKI sequestration, thereby preventing the resulting inhibition of CDK2, believed to be the key regulator of cell cycle. However, our immunoprecipitations revealed that the dominant negative CDK mutants could arrest cell growth despite their interaction with p21 and p27. Taken together, our results demonstrate that both CDK2 and CDK4/6 are crucial for cell cycle regulation. Furthermore, our data underscore the importance of the Rb regulatory pathway in neuronal development and cell cycle regulation, independent of CKI sequestration. (+info)
Inhibition of Hsp90 function by ansamycins causes retinoblastoma gene product-dependent G1 arrest.
(39/363)
The ansamycin antibiotics, herbimycin A (HA) and geldanamycin (GM), bind to a conserved pocket in heat shock protein 90 (Hsp90) and alter the function of this chaperone protein. Occupancy of this pocket results in the degradation of a subset of signaling molecules. These include proteins known to associate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family. In a variety of tumor cell lines, treatment with HA potently inhibited cellular proliferation by inducing G1 arrest. This arrest was accompanied by hypophosphorylation of the retinoblastoma gene product (RB) and rapid down-regulation of cyclin D- and E-associated kinase activities. Inhibition of kinase activity was found to result from loss in expression of cyclins D1, D3, and E, as well as the associated cyclin-dependent kinases, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, HA treatment also caused a late induction of p27(Kip1) protein. The loss of cyclin D preceded the other effects of HA, suggesting that it might be the primary cause of G1 arrest. To determine whether the effects of HA are mediated by selective inhibition of the cyclin D-RB pathway, HA was added to tumor cell lines lacking functional RB. HA treatment of Rb-negative tumor cell lines failed to elicit a G1 arrest. In addition, after release from synchronization with nocodazole, Rb-negative but not Rb-positive cell lines were able to progress through G1 into S phase in the presence of HA. Together, these findings suggest that induction of G1 arrest by HA results from down-regulation of cyclin D expression and its associated kinase activity. Furthermore, these findings imply that Hsp90 selectively regulates signaling pathways upstream of RB. (+info)
Adenovirus-mediated overexpression of p15INK4B inhibits human glioma cell growth, induces replicative senescence, and inhibits telomerase activity similarly to p16INK4A.
(40/363)
The genes encoding the cyclin-dependent kinase inhibitors p16INK4A (CDKN2A) and p15INK4B (CDKN2B) are frequently homozygously deleted in a variety of tumor cell lines and primary tumors, including glioblastomas in which 40-50% of primary tumors display homozygous deletions of these two loci. Although the role of p16 as a tumor suppressor has been well documented, it has remained less well studied whether p15 plays a similar growth-suppressing role. Here, we have used replication-defective recombinant adenoviruses to compare the effects of expressing wild-type p16 and p15 in glioma cell lines. After infection, high levels of p16 and p15 were observed in two human glioma cell lines (U251 MG and U373 MG). Both inhibitors were found in complex with CDK4 and CDK6. Expression of p16 and p15 had indistinguishable effects on U251 MG, which has homozygous deletion of CDKN2A and CDKN2B, but a wild-type retinoblastoma (RB) gene. Cells were growth-arrested, showed no increased apoptosis, and displayed a markedly altered cellular morphology and repression of telomerase activity. Transduced cells became enlarged and flattened and expressed senescence-associated beta-galactosidase, thus fulfilling criteria for replicative senescence. In contrast, the growth and morphology of U373 MG, which expresses p16 and p15 endogenously, but undetectable levels of RB protein, were not affected by exogenous overexpression of either inhibitor. Thus, we conclude that overexpression of p15 has a similar ability to inhibit cell proliferation, to cause replicative senescence, and to inhibit telomerase activity as p16 in glioma cells with an intact RB protein pathway. (+info)