In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G1 arrest imposed by either the p16INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.
- MeSH
- cyklin D1 MeSH
- cykliny genetika metabolismus MeSH
- DNA vazebné proteiny * MeSH
- fibroblasty cytologie MeSH
- fosforylace MeSH
- G1 fáze fyziologie MeSH
- interfáze fyziologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- luciferasy genetika izolace a purifikace MeSH
- mikroinjekce MeSH
- mutace MeSH
- myši MeSH
- onkogenní proteiny genetika metabolismus MeSH
- proteiny buněčného cyklu * MeSH
- reportérové geny MeSH
- retinoblastomový protein metabolismus MeSH
- S fáze fyziologie MeSH
- transfekce MeSH
- transkripční faktor DP1 MeSH
- transkripční faktory E2F MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * MeSH
- vazebný protein 1 retinoblastomu MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- srovnávací studie MeSH
The p16Ink4/CDKN2, D-type cyclins, their partners Cdk4/Cdk6, and pRb constitute a G1 regulatory pathway commonly targeted in tumorigenesis. Genetic, immunochemical, and functional cell cycle analyses showed abnormalities of this pathway in each of 22 human melanoma cell lines examined. Normal melanocytes and all melanoma lines expressed Cdk4, Cdk6, and cyclins D1 and D3. The tumor suppressors p16Ink4/CDKN2 and pRb were lost in 17 and 4 cases, respectively, due to various genetic mechanisms, including transcriptional block of p16 and nonsense mutations of RB1. Ectopic expression of p16 prevented S-phase entry of Rb+/p16- but not Rb-deficient melanoma lines. The SK29-MEL-1 cell line harboring an R24C mutation in Cdk4 expressed wild-type pRb and overabundant p16, the latter preventing endogenous Cdk6 but not Cdk4 from associating with cyclin D1. Microinjection of cyclin D1-neutralizing antibody arrested the SK29-MEL-1 cells in G1, whereas pl6 did not, indicating that the cyclin D1/Cdk4-R24C complex is required for G1 progression, and the resistance of the complex to p16 in vivo. These data strongly support the candidacy of Cdk4 as a novel proto-oncogene, provide further evidence for the p16-cyclin D/Cdk-pRb pathway as a functional unit, and suggest that deregulation of this checkpoint may represent a common step in the multistep progression of sporadic malignant melanomas.
- MeSH
- cyklin D MeSH
- cyklin-dependentní kinasa 4 MeSH
- cyklin-dependentní kinasy genetika fyziologie MeSH
- cykliny genetika fyziologie MeSH
- delece genu MeSH
- G1 fáze fyziologie MeSH
- geny retinoblastomu MeSH
- inhibitor p16 cyklin-dependentní kinasy MeSH
- lidé MeSH
- melanom etiologie genetika patofyziologie MeSH
- molekulární sekvence - údaje MeSH
- nádorová transformace buněk genetika MeSH
- nádorové buňky kultivované MeSH
- polymerázová řetězová reakce MeSH
- progrese nemoci MeSH
- protoonkogenní proteiny * MeSH
- protoonkogeny MeSH
- retinoblastomový protein fyziologie MeSH
- sekvence nukleotidů MeSH
- signální transdukce fyziologie MeSH
- transportní proteiny genetika fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.
- MeSH
- biologické modely MeSH
- buněčné klony MeSH
- cyklin E genetika metabolismus MeSH
- cyklin-dependentní kinasa 2 MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- DNA vazebné proteiny * MeSH
- fosfatasy cdc25 metabolismus MeSH
- G1 fáze fyziologie MeSH
- interfáze * fyziologie MeSH
- kinasy CDC2-CDC28 * MeSH
- nádorové buňky kultivované MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny buněčného cyklu * metabolismus MeSH
- protoonkogenní proteiny c-myc genetika metabolismus MeSH
- regulace genové exprese MeSH
- replikace DNA MeSH
- retinoblastomový protein * metabolismus MeSH
- S fáze fyziologie MeSH
- transgeny MeSH
- transkripční faktor DP1 MeSH
- transkripční faktor E2F1 MeSH
- transkripční faktory E2F MeSH
- transkripční faktory * metabolismus MeSH
- transportní proteiny * MeSH
- vazebný protein 1 retinoblastomu MeSH
p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.
- MeSH
- cyklin-dependentní kinasa 2 MeSH
- cyklin-dependentní kinasy biosyntéza metabolismus MeSH
- G1 fáze genetika fyziologie MeSH
- inhibitor p16 cyklin-dependentní kinasy biosyntéza genetika fyziologie MeSH
- inhibitory růstu genetika fyziologie MeSH
- kinasy CDC2-CDC28 * MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- nádorová transformace buněk genetika metabolismus MeSH
- nádorové buňky kultivované MeSH
- osteosarkom MeSH
- protein-serin-threoninkinasy biosyntéza metabolismus MeSH
- retinoblastomový protein biosyntéza genetika fyziologie MeSH
- S fáze genetika MeSH
- technika přenosu genů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The commitment of mammalian cells in late G1 to replicate the genome and divide in response to mitogenic growth factors operating via tyrosine kinase receptors depends on phosphorylation of the retinoblastoma protein (pRb), a process controlled by cyclin D-associated cyclin-dependent kinases (cdks) and their inhibitors. This study addressed the issue of whether also other mitogenic signalling cascades require activation of cyclin D-associated kinases or whether any mitogenic pathway can bypass the cyclin D-pRb checkpoint. We show that mitogenic signal transduction pathways from three classes of receptors, the membrane tyrosine kinase receptors activated by serum mitogens or epidermal growth factor, estrogen receptors triggered by estradiol, and the cyclic AMP-dependent signalling from G-protein-coupled thyrotropin receptors, all converge and strictly require the cyclin D-cdk activity to induce S phase in human MCF-7 cells and/or primary dog thyrocytes. Combined microinjection and biochemical approaches showed that whereas these three mitogenic cascades are sensitive to the p16 inhibitor of cdk4/6 and/or cyclin D1-neutralizing antibody and able to induce pRb kinase activity, their upstream biochemical routes are distinct as demonstrated by their differential sensitivity to lovastatin and requirements for mitogen-activated protein kinases whose sustained activation is seen only in the growth factor-dependent pathway. Taken together, these results support the candidacy of the cyclin D-cdk-pRb interplay for the convergence step of multiple signalling cascades and a mechanism contributing to the restriction point switch.
- MeSH
- buněčný cyklus * MeSH
- cyklin D MeSH
- cyklin-dependentní kinasy * metabolismus MeSH
- cykliny * metabolismus MeSH
- G1 fáze * MeSH
- lidé MeSH
- mitogeny * metabolismus MeSH
- nádorové buňky kultivované MeSH
- psi MeSH
- retinoblastomový protein * metabolismus MeSH
- signální transdukce * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH