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A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

ABSTRACT: Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retained the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquired phenotypic characteristics of differentiated macrophages and became arrested in the cell cycle with a 4N DNA content. AP also inhibited mitosis in Xenopus egg extracts, suggesting that it acted on an evolutionarily conserved cell cycle regulatory pathway. Affinity chromatography and biochemical reconstitution experiments with Xenopus egg extracts identified cyclin-dependent kinase (CDK) 1-cyclin B as a target of the compound. Although AP potently inhibited immunoprecipitates of both human CDK1 and CDK2 from human leukemic cell extracts, our results indicate that the compound preferentially targets the G2/M-phase transition in vivo.

SUBMITTER: Rosania GR

PROVIDER: S-EPMC21771 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

Rosania G R GR Merlie J J Gray N N Chang Y T YT Schultz P G PG Heald R R

Proceedings of the National Academy of Sciences of the United States of America 19990401 9

Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retained the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquired phenotypic characteristics of differentiated macrophages and became arrested in the ...[more]

PMID: 10220373

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Project description:AimsCyclin-dependent kinase inhibitors (CDKIs) play a critical role in negatively regulating the proliferation of cardiomyocytes, although their role in cardiac differentiation remains largely undetermined. We have shown that the most prominent CDKI in Xenopus, p27(Xic1)(Xic1), plays a role in neuronal and myotome differentiation beyond its ability to arrest the cell cycle. Thus, we investigated whether it plays a similar role in cardiomyocyte differentiation.Methods and resultsXenopus laevis embryos were sectioned, and whole-mount antibody staining and immunofluorescence studies were carried out to determine the total number and percentage of differentiated cardiomyocytes in mitosis. Capped RNA and/or translation-blocking Xic1 morpholino antisense oligonucleotides (Xic1Mo) were microinjected into embryos, and their role on cardiac differentiation was assessed by in situ hybridization and/or PCR. We show that cell-cycling post-gastrulation is not essential for cardiac differentiation in Xenopus embryos, and conversely that some cells can express markers of cardiac differentiation even when still in cycle. A targeted knock-down of Xic1 protein by Xic1Mo microinjection decreases the expression of markers of cardiac differentiation, which can be partially rescued by co-injection of full-length Xic1 RNA, demonstrating that Xic1 is essential for heart formation. Furthermore, using deleted and mutant forms of Xic1, we show that neither its abilities to inhibit the cell cycle nor the great majority of CDK kinase activity are essential for Xic1's function in cardiomyocyte differentiation, an activity that resides in the N-terminus of the molecule.ConclusionAltogether, our results demonstrate that the CDKI Xic1 is required in Xenopus cardiac differentiation, and that this function is localized at its N-terminus, but it is distinct from its ability to arrest the cell cycle and inhibit overall CDK kinase activity. Hence, these results suggest that CDKIs play an important direct role in driving cardiomyocyte differentiation in addition to cell-cycle regulation.

Dataset Information

A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

Publications

A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

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