Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0029463 (osteosarcoma)
 16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarial osteoblasts. Three developmental stages were examined: (a) proliferation; (b) monolayer confluency; and (c) mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and D1 persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor beta for 48 h during the proliferation period renders the cells incompetent for differentiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of ROS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cytoplasmic cyclin B when compared to the proliferation period. This observation is consistent with abrogation of both growth control and differentiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation.
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PMID:Expression of cell cycle regulatory factors in differentiating osteoblasts: postproliferative up-regulation of cyclins B and E. 758 45

Protein phosphatase 1 and protein phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle protein phosphatase 1, as well as recombinant protein phosphatase 1 alpha and protein phosphatase 1 gamma 1, but not protein phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine. Neither cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active form of protein phosphatase 1 alpha in which Thr-320 had been mutated to alanine, indicating that the phosphorylation occurred at this threonine residue. Furthermore, protein phosphatase 1, but not protein phosphatase 2A, activity was found to change during the cell cycle of human MG-63 osteosarcoma cells. The observed oscillations in protein phosphatase 1 activity during the cell cycle may be due, at least in part, to phosphorylation of protein phosphatase 1 by cyclin-dependent kinases. Together, the results suggest a mechanism for direct regulation of protein phosphatase 1 activity.
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PMID:Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases. 802 97

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.
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PMID:Biochemical characterization of the human cyclin-dependent protein kinase activating kinase. Identification of p35 as a novel regulatory subunit. 855 Jun 4

Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E-associated substrates of 78 and 105 kD. Activity of the differentiation-related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx-i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx-i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin-dependent kinase inhibitor p21CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation-related cyclin E-associated kinase activity.
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PMID:Post-proliferative cyclin E-associated kinase activity in differentiated osteoblasts: inhibition by proliferating osteoblasts and osteosarcoma cells. 921 16

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.
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PMID:Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex. 929 75

A mathematical model integrating the roles of cyclin D, cdk4, cyclin E, cdk2, E2F and RB in control of the G1 phase of the cell cycle is described. Experimental results described with murine embryo fibroblasts (MEFs), either Rb+/+ or Rb-/-, and with the RB-deficient osteosarcoma cell line, Saos-2, served as the basis for the formulation of this mathematical model. A model employing the known interactions of these six proteins does not reproduce the experimental observations described in the MEFs. The appropriate modelling of G1 requires the inclusion of a sensing mechanism which adjusts the activity of cyclin E/cdk2 in response to both RB concentration and growth factors. Incorporation of this sensing mechanism into the model allows it to reproduce most of the experimental results observed in Saos-2 cells, Rb-/- MEFS, and Rb+/+ MEFs. The model also makes specific predictions which have not been tested experimentally.
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PMID:A mathematical model of the regulation of the G1 phase of Rb+/+ and Rb-/- mouse embryonic fibroblasts and an osteosarcoma cell line. 937 29

Osteosarcomas often suffer mutations of the RB (retinoblastoma) gene, with resultant inactivation of the pRb protein. pRb is one component in a cell-cycle control pathway that includes the p16 (encoded by the CDKN2A gene) and cyclin-dependent kinase 4 (cdk4, encoded by the CDK4 gene) proteins. We therefore sought to determine whether the CDKN2A and CDK4 genes were altered in those osteosarcomas that lacked RB inactivation. Twenty-one osteosarcomas (2 low-grade and 19 high-grade) were evaluated for homozygous deletion of the CDKN2A gene, CDK4 amplification, and allelic loss of the RB gene, as well as for expression of p16 and pRb proteins. Five high-grade osteosarcomas showed loss of p16 expression; four of these had homozygous CDKN2A deletions, and the fifth had a probable deletion obscured by numerous nonneoplastic, p16-immunopositive multinucleated giant cells. Thus, p16 immunohistochemistry may provide a sensitive means for assessing CDKN2A status. Twelve tumors (including the two low-grade osteosarcomas) were immunopositive for pRb, and nine tumors were immunonegative for pRb. Of the five cases with CDKN2A/p16 alterations, none had allelic loss of the RB gene and all expressed pRb, suggesting that each of these tumors had an intact RB gene. None of the tumors showed CDK4 amplification. No alterations were detected in the two low-grade osteosarcomas. This study suggests that CDKN2A is a tumor suppressor inactivated in osteosarcomas that lack RB mutations and that the p16-pRb cell-cycle control pathway is deregulated in a large number of high-grade osteosarcomas.
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PMID:CDKN2A gene deletions and loss of p16 expression occur in osteosarcomas that lack RB alterations. 966 76

We previously demonstrated that P16Ink4a (p16) expression in p16-deficient U343 astrocytoma cells causes a G1 cell cycle arrest, profound changes in cytoskeletal proteins and alterations in expression and activity of the pRB and E2F family proteins. We examine here the effects of expressing wild type or mutant versions of the downstream targets of p16 in U343 astrocytomas. We first attempted to block proliferation of U343 cells using the dominant mutant of pRB, deltap34. Expression of this mutant in the human osteosarcoma, SAOS-2, potently blocked proliferation but did not affect the cell cycle of U343 cells. We next showed that expression of E2F-1, E2F-2, E2F-3 and E2F-4 are each able to overcome this p16-dependent cell cycle arrest but exhibit distinct biological activities. Adenoviral-mediated expression of E2F-1, E2F-2, E2F-3, or E2F-4 overcame the p16-dependent cell cycle block and induced alterations in cell morphology. E2F-5, only in conjunction with DP1, promoted cell cycle progression. For both E2F-1 and E2F-2, but not E2F-3 or E2F-5/DP1, cell cycle re-entry was associated with almost quantitative cell death. Only small numbers of dying cells were observed in E2F-4-expressing cultures. Expression of the different E2F's altered the expression of distinct sets of cell cycle regulatory proteins. E2F-1 induced endogenous E2F-4 expression and also caused an increase in pRB, p107 and cyclin E levels. Expression of E2F-4 caused a weak increase in E2F-1 levels but also strongly induced pRB, p107, p130 and cyclin E. However, E2F-1 and E2F-4 clearly regulate expression of distinct genes, demonstrated when E2F-4 caused a threefold increase in the levels of cdk2 whereas E2F-1 failed to increase in this cyclin dependent kinase. Similarly, expression of E2F-1 or E2F-2 were shown to have distinct effects on the expression of cdk2, cyclin E and pRB despite both of these closely related E2F-family members potently inducing cell death. Thus, E2F-1, E2F-2, E2F-3 and E2F-4 are able to overcome the p16-dependent proliferative block in U343 astrocytoma cells. While overcoming this cell cycle block, each of the E2F's uniquely affect the expression of a number of cell cycle regulatory proteins and have distinct abilities to promote cell death.
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PMID:The E2F-family proteins induce distinct cell cycle regulatory factors in p16-arrested, U343 astrocytoma cells. 978 3

The tumor suppressor p16(INK4a) inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27(KIP1), and p21(WAF1/CIP1). Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14(ARF)/p53 tumor suppressor pathways.
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PMID:Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a). 1020 15

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.
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PMID:Expression of G1 phase regulators in MG-63 osteosarcoma cell line. 1033 67


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