Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

Department of Structural and Chemical Biology, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA.
Molecular cell (Impact Factor: 14.02). 06/2011; 42(5):624-36. DOI: 10.1016/j.molcel.2011.03.031
Source: PubMed


Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and nonphysiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a noncatalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and noninhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and nonredundant, rate-limiting roles in restriction point passage and S phase entry.

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    • "Even though knockout studies show that no individual iCdk is strictly essential, our data suggest that iCdks maintain cell proliferation and forestall aging features in postnatal somatic tissues, when expressed at normal levels. This observation dovetails well with recent findings using gene replacement in vitro with analog-sensitive forms that, when expressed at normal levels, Cdk2 is required for efficient cell cycle entry in human and mouse cells (Merrick et al., 2011). The phenotype of mice with p16 induction also matches well that recently reported in mice with induction of the iCdk inhibitor p27 (Pruitt et al., 2013). "
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    ABSTRACT: The cyclin-dependent kinase (Cdk) inhibitor p16(Ink4a) (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit-amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de-induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16-mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
    Full-text · Article · Dec 2014 · Aging cell
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    • "In untreated cells and in DMSO treated cells we detected similar expression levels of CDK2 independently of the cell cycle phases. It is possible that mammalian cells can tolerate loss of CDK2 (by additional support from other CDKs as CDK1, CDK4, or CDK6), but CDK2 activity is required for cell proliferation [46]. CDK1/cyclin A complexes have been specifically implicated in attenuating the expression of histone genes at the end of the S phase [47]. "
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    ABSTRACT: As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation.When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.
    Full-text · Article · Nov 2014 · International Journal of Cell Biology
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    • "In fact, Cdk2 knock-down seemed to have an effect on phosphorylation of band #2 only in asynchronized cells (no nocodazole treatment), with the signal coming from this form increasing after induction of G2/M arrest. This effect could be explained hypothesizing that additional CDKs might have substituted Cdk2 in its role of phosphorylating specific substrates [43], [44] or that additional residues (which could not be separated on phos- tag gels) were phosphorylated during G2/M. Further validation is required to confirm these results. "
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    ABSTRACT: Background 3′-Deoxy-3′-[18F]-fluorothymidine ([18F]FLT) is being investigated as a Positron Emission Tomography (PET) proliferation biomarker. The mechanism of cellular [18F]FLT retention has been assigned primarily to alteration of the strict transcriptionally regulated S-phase expression of thymidine kinase 1 (TK1). This, however, does not explain how anticancer agents acting primarily through G2/M arrest affect [18F]FLT uptake. We investigated alternative mechanisms of [18F]FLT cellular retention involving post-translational modification of TK1 during mitosis. Methods [18F]FLT cellular retention was assessed in cell lines having different TK1 expression. Drug-induced phosphorylation of TK1 protein was evaluated by MnCl2-phos-tag gel electrophoresis and correlated with [18F]FLT cellular retention. We further elaborated the amino acid residues involved in TK1 phosphorylation by transient transfection of FLAG-pCMV2 plasmids encoding wild type or mutant variants of TK1 into TK1 negative cells. Results Baseline [18F]FLT cellular retention and TK1 protein expression were associated. S-phase and G2/M phase arrest caused greater than two-fold reduction in [18F]FLT cellular retention in colon cancer HCT116 cells (p<0.001). G2/M cell cycle arrest increased TK1 phosphorylation as measured by induction of at least one phosphorylated form of the protein on MnCl2-phos-tag gels. Changes in [18F]FLT cellular retention reflected TK1 phosphorylation and not expression of total protein, in keeping with the impact of phosphorylation on enzyme catalytic activity. Both Ser13 and Ser231 were shown to be involved in the TK1 phosphorylation-modulated [18F]FLT cellular retention; although the data suggested involvement of other amino-acid residues. Conclusion We have defined a regulatory role of TK1 phosphorylation in mediating [18F]FLT cellular retention and hence reporting of antiproliferative activity, with implications especially for drugs that induce a G2/M cell cycle arrest.
    Full-text · Article · Jul 2014 · PLoS ONE
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