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Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

ABSTRACT: The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searching for a protein(s) which might interact with Nur77, an orphan steroid receptor required for activation-induced apoptosis of T-cell hybridomas, we have cloned a novel human and mouse CDK inhibitor, p19. The deduced p19 amino acid sequence consists of four ankyrin repeats with 48% identity to p16. The human p19 gene is located on chromosome 19p13, distinct from the positions of p18, p16, and p15. Its mRNA is expressed in all cell types examined. The p19 fusion protein can associate in vitro with CDK4 but not with CDK2, CDC2, or cyclin A, B, E, or D1 to D3. Addition of p19 protein can lead to inhibition of the in vitro kinase activity of cyclin D-CDK4 but not that of cyclin E-CDK2. In T-cell hybridoma DO11.10, p19 was found in association with CDK4 and CDK6 in vivo, although its association with Nur77 is not clear at this point. Thus, p19 is a novel CDK inhibitor which may play a role in the cell cycle regulation of T cells.

SUBMITTER: Chan FK

PROVIDER: S-EPMC230498 | biostudies-other | 1995 May

REPOSITORIES: biostudies-other

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Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

Chan F K FK Zhang J J Cheng L L Shapiro D N DN Winoto A A

Molecular and cellular biology 19950501 5

The cell cycle in mammalian cells is regulated by a series of cyclins and cyclin-dependent kinases (CDKs). The G1/S checkpoint is mainly dictated by the kinase activities of the cyclin D-CDK4 and/or cyclin D-CDK6 complex and the cyclin E-CDK2 complex. These G1 kinases can in turn be regulated by cell cycle inhibitors, which may cause the cells to arrest at the G1 phase. In T-cell hybridomas, addition of anti-T-cell receptor antibody results not only in G1 arrest but also in apoptosis. In searchi ...[more]

PMID: 7739548

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Project description:BackgroundOverexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear.MethodsLeukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis.ResultsOur studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2.ConclusionsWe demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Project description:Background CDK4 and CDK6 are frequently over expressed or hyper activated in human cancers. Due to the importance of CDK4/6 activity in cancer cells, CDK4/6 inhibitors have emerged as promising candidates for cancer treatment. LEE011, a novel inhibitor of CDK4/6, until now the molecular function of LEE011 in acute myeloid leukemia is still unknown. Methods AML cell growth was assessed by CCK-8 and annexin V/propidium iodide staining followed by flow cytometry detected apoptosis in cell culture. AML cell senescence was assessed by β-Galactosidase Staining, p16INK4a expression and HP1α Staining analysis. Gene expressions of LEE011-treated tumor cells were analyzed using an Arraystar Human LncRNA Array. The Multi Experiment View cluster software analyzed the expression data. Differentially expressed genes from the cluster analyses were imported into the GO and KEGG pathway analysis. Results Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and do not induce apoptosis. Hoechst 33342 staining analysis showed that DNA fragmentation and abnormal nuclear cells was observed after LEE011 treatment. Cell cycle analysis showed in these eight acute leukemia cells, LEE011 induce the cell cycle G1 arrest very significantly, except THP-1 cells. Cell senescence β-Galactosidase Staining analysis, p16INK4a expression and HP1α Staining analysis all showed that LEE011 treatment can induce the cell senescence of AML cells. LncRNA microarray was used to identify 2083 differently expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with control group. Molecular function analysis showed that LEE011 induced cellular senescence in leukemia cells partially through down regulation the transcriptional expression of MYBL2. Conclusions In this study, we showed for the first time that LEE011 treatment resulted in inhibition of cell proliferation and induction of G1 arrest and cellular senescence in AML cells. LncRNA microarray was used to identify differentially expressed genes and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induce cellular senescence partially through down regulation the expression of MYBL2. These results may provide new insights into the molecular mechanism of LEE011-induced cellular senescence; however, further research will be required to determine the underlying details. Taken together, our findings firstly suggest that LEE011 may act as new candidate’s drug for AML.

Dataset Information

Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

Publications

Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4.

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