HeLa cells are human cervical cancer cells with HR HPV-18 genes integrated in the genome. The functions of tumor suppressor proteins p53 and pRB are abrogated and cell cycle regulation becomes nonfunctional. The aim of the present study was to investigate whether the CDK inhibitor R-Roscovitine would allow the G1/S blocked HeLa cells to enter into mitosis prematurely and induce apoptosis. HeLa cells blocked in G1/S border were treated with different concentrations of Roscovitine for 4 and 18 h respectively. Induction of apoptosis was studied by FACS and DNA fragmentation. Presence of γH2AX in the treated cells was studied by confocal microscopy. Expression levels of CASP3, CDKN1A i.e. p21 (Cip1/Waf1) and Bcl2 were studied by semi-quantitative RT-PCR to analyze the role played by these proteins in Roscovitine induced apoptosis in G1/S blocked HeLa cells. Results indicate that the Roscovitine allowed the thymidine blocked HeLa cells to enter into mitosis prematurely. Presence of γH2AX loci in treated cells indicates DNA damage in prematurely mitotic cells. Analysis of DNA fragmentation and chromatin condensation confirmed apoptosis as the possible mechanism of Roscovitine induced cell death. Our results also reveal that Roscovitine induced apoptosis is associated with the overexpression of CASP3, p21 (cip1/waf1) and Bcl2.
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This work was supported by grants from Indian Council of Medical Research (ICMR, IRIS Cell No. 2005-02090). The authors are thankful to the Department of Botany, CAS; Department of Biotechnology and Department of Biochemistry, University of Calcutta for the instrument facilities provided.
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Misra, K., Paul, S. & Kundu, R. Induction of Apoptosis in G1/S Blocked HeLa Cells by R-Roscovitine: A Preliminary Study. Proc Zool Soc 67, 114–125 (2014). https://doi.org/10.1007/s12595-013-0075-2
- p21 (Cip1/Waf1)