Induction of Apoptosis in G1/S Blocked HeLa Cells by R-Roscovitine: A Preliminary Study

Abstract

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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References

  1. Asada, M., T. Yamada, H. Ichijo, D. Delia, K. Miyazono, K. Fukumuro, and S. Mizutani. 1999. Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. European Molecular Biology Organization Journal 18: 1223–1234.

    Article  CAS  Google Scholar 

  2. Attardi, L.D. 2005. The role of p53-mediated apoptosis as a crucial anti-tumor response to genomic instability: lessons from mouse models. Mutation Research 569: 145–157.

    PubMed  Article  CAS  Google Scholar 

  3. Avramis, V.I., P. Nandy, R. Kwock, M.M. Solorzano, S.K. Mukherjee, P. Danenberg, and L.J. Cohen. 1998. Increased p21/WAF-1 and p53 protein levels following sequential three drug combination regimen of fludarabine, cytarabine and docetaxel induces apoptosis in human leukemia cells. Anticancer Research 18: 2327–2338.

    PubMed  CAS  Google Scholar 

  4. Bissonnette, N., and D.J. Hunting. 1998. p21-Induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage. Oncogene 16: 3461–3469.

    PubMed  Article  CAS  Google Scholar 

  5. Chanoux, R.A., B. Yin, K.A. Urtishak, A. Asare, C.H. Bassing, and E.J. Brown. 2009. ATR and H2AX cooperate in maintaining genome stability under replication stress. Journal of Biological Chemistry 284: 5994–6003.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  6. Chen, J., T. Willingham, M. Shuford, D. Bruce, E. Rushing, Y. Smith, and P.D. Nisen. 1996. Effects of ectopic overexpression of p21(WAF1/CIP1) on aneuploidy and the malignant phenotype of human brain tumor cells. Oncogene 13: 1395–1403.

    PubMed  CAS  Google Scholar 

  7. Chen, Y.Q., S.C. Cipriano, J.M. Arenkiel, and F.R. Miller. 1995. Tumor suppression by p21WAF1. Cancer Research 55: 4536–4539.

    PubMed  CAS  Google Scholar 

  8. Cheng, E.H., D.G. Kirsch, R.J. Clem, R. Ravi, M.B. Kastan, A. Bedi, K. Ueno, and J.M. Hardwick. 1997. Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278: 1966–1968.

    PubMed  Article  CAS  Google Scholar 

  9. Chinnaiyan, A.M., K. Orth, K. O’Rourke, H. Duan, G.G. Poirier, and V.M. Dixit. 1996. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. The Journal of Biological Chemistry 271: 4573–4576.

    PubMed  Article  CAS  Google Scholar 

  10. Crescenzi, E., G. Palumbo, and H.J. Brady. 2005. Roscovitine modulates DNA repair and senescence: implications for combination chemotherapy. Clinical Cancer Research 11: 8158–8171.

    PubMed  Article  CAS  Google Scholar 

  11. David-Pfeuty, T. 1999. Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells. Oncogene 18: 7409–7422.

    PubMed  Article  CAS  Google Scholar 

  12. DeFilippis, R.A., E.C. Goodwin, L. Wu, and D. DiMaio. 2003. Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. Journal of Virology 77: 1551–1563.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  13. Duttaroy, A., J.F. Qian, J.S. Smith, and E. Wang. 1997. Up-regulated P21CIP1 expression is part of the regulation quantitatively controlling serum deprivation-induced apoptosis. Journal of Cellular Biochemistry 64: 434–446.

    PubMed  Article  CAS  Google Scholar 

  14. Eastham, J.A., S.J. Hall, I. Sehgal, J. Wang, T.L. Timme, G. Yang, L. Connell-Crowley, S.J. Elledge, W.W. Zhang, J.W. Harper, et al. 1995. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Research 55: 5151–5155.

    PubMed  CAS  Google Scholar 

  15. Ekholm, S.V., and S.I. Reed. 2000. Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle. Current Opinion in Cell Biology 12: 676–684.

    PubMed  Article  CAS  Google Scholar 

  16. Eliyahu, D., D. Michalovitz, S. Eliyahu, O. Pinhasi-Kimhi, and M. Oren. 1989. Wild-type p53 can inhibit oncogene-mediated focus formation. Proceedings of the National Academy of Sciences USA 86: 8763–8767.

    Article  CAS  Google Scholar 

  17. Federico, M., C.E. Symonds, L. Bagella, F. Rizzolio, D. Fanale, A. Russo, and A. Giordano. 2010. R-Roscovitine (Seliciclib) prevents DNA damage-induced cyclin A1 upregulation and hinders non-homologous end-joining (NHEJ) DNA repair. Molecular Cancer 9(208): 1–14.

    Google Scholar 

  18. Finlay, C.A., P.W. Hinds, and A.J. Levine. 1989. The p53 proto-oncogene can act as a suppressor of transformation. Cell 57: 1083–1093.

    PubMed  Article  CAS  Google Scholar 

  19. Givol, I., D. Givol, S. Rulong, J. Resau, I. Tsarfaty, and S.H. Hughes. 1995. Overexpression of human p21waf1/cip1 arrests the growth of chicken embryo fibroblasts transformed by individual oncogenes. Oncogene 11: 2609–2618.

    PubMed  CAS  Google Scholar 

  20. Gorospe, M., C. Cirielli, X. Wang, P. Seth, M.C. Capogrossi, and N.J. Holbrook. 1997. p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene 14: 929–935.

    PubMed  Article  CAS  Google Scholar 

  21. Gorospe, M., X. Wang, K.Z. Guyton, and N.J. Holbrook. 1996. Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells. Molecular and Cellular Biology 16: 6654–6660.

    PubMed  CAS  PubMed Central  Google Scholar 

  22. Hockenbery, D.M., Z.N. Oltvai, X.M. Yin, C.L. Milliman, and S.J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241–251.

    PubMed  Article  CAS  Google Scholar 

  23. Hsu, S.L., M.C. Chen, Y.H. Chou, G.Y. Hwang, and S.C. Yin. 1999. Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells. Experimental Cell Research 248: 87–96.

    PubMed  Article  CAS  Google Scholar 

  24. Ianari, A., T. Natale, E. Calo, E. Ferretti, E. Alesse, I. Screpanti, K. Haigis, A. Gulino, and J.A. Lees. 2009. Proapoptotic function of the retinoblastoma tumor suppressor protein. Cancer Cell 2009(15): 184–194.

    Article  Google Scholar 

  25. Kondo, Y., S. Kondo, J. Liu, T. Haqqi, G.H. Barnett, and B.P. Barna. 1997. Involvement of p53 and WAF1/CIP1 in gamma-irradiation-induced apoptosis of retinoblastoma cells. Experimental Cell Research 236: 51–56.

    PubMed  Article  CAS  Google Scholar 

  26. Li, L., M. Ljungman, and J.E. Dixon. 2000. The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53. The Journal of Biological Chemistry 275: 2410–2414.

    PubMed  Article  CAS  Google Scholar 

  27. Li, R., G.J. Hannon, D. Beach, and B. Stillman. 1996. Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage. Current Biology 6: 189–199.

    PubMed  Article  CAS  Google Scholar 

  28. Li, R., S. Waga, G.J. Hannon, D. Beach, and B. Stillman. 1994. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371: 534–537.

    PubMed  Article  CAS  Google Scholar 

  29. Ljungman, M., and M.T. Paulsen. 2001. The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382. Molecular Pharmacology 60: 785–789.

    PubMed  CAS  Google Scholar 

  30. Martinez, L.A., J. Yang, E.S. Vazquez, C. Rodriguez-Vargas Mdel, M. Olive, J.T. Hsieh, C.J. Logothetis, and N.M. Navone. 2002. p21 Modulates threshold of apoptosis induced by DNA-damage and growth factor withdrawal in prostate cancer cells. Carcinogenesis 23: 1289–1296.

    PubMed  Article  CAS  Google Scholar 

  31. Matsushita, H., R. Morishita, I. Kida, M. Aoki, S. Hayashi, N. Tomita, K. Yamamoto, A. Moriguchi, A. Noda, Y. Kaneda, J. Higaki, and T. Ogihara. 1998. Inhibition of growth of human vascular smooth muscle cells by overexpression of p21 gene through induction of apoptosis. Hypertension 31: 493–498.

    PubMed  Article  CAS  Google Scholar 

  32. Meijer, L., A. Borgne, O. Mulner, J.P. Chong, J.J. Blow, N. Inagaki, M. Inagaki, J.G. Delcros, and J.P. Moulinoux. 1997. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. European Journal of Biochemistry 243: 527–536.

    PubMed  Article  CAS  Google Scholar 

  33. Meijer, L., and E. Raymond. 2003. Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials. Accounts of Chemical Research 36: 417–425.

    PubMed  Article  CAS  Google Scholar 

  34. Morgan, D.O. 1995. Principles of CDK regulation. Nature 374: 131–134.

    PubMed  Article  CAS  Google Scholar 

  35. Poluha, W., D.K. Poluha, B. Chang, N.E. Crosbie, C.M. Schonhoff, D.L. Kilpatrick, and A.H. Ross. 1996. The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells. Molecular and Cellular Biology 16: 1335–1341.

    PubMed  CAS  PubMed Central  Google Scholar 

  36. Reed, J.C. 1994. Bcl-2 and the regulation of programmed cell death. The Journal of Cellular Biology 124: 1–6.

    Article  CAS  Google Scholar 

  37. Scheffner, M., K. Munger, J.C. Byrne, and P.M. Howley. 1991. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proceedings of National Academy of Sciences USA 88: 5523–5527.

    Article  CAS  Google Scholar 

  38. Sherr, C.J. 1993. Mammalian G1 cyclins. Cell 73: 1059–1065.

    PubMed  Article  CAS  Google Scholar 

  39. Tsao, Y.P., S.J. Huang, J.L. Chang, J.T. Hsieh, R.C. Pong, and S.L. Chen. 1999. Adenovirus-mediated p21(WAF1/SDII/CIP1) gene transfer induces apoptosis of human cervical cancer cell lines. Journal of Virology 73: 4983–4990.

    PubMed  CAS  PubMed Central  Google Scholar 

  40. Waldman, T., Y. Zhang, L. Dillehay, J. Yu, K. Kinzler, B. Vogelstein, and J. Williams. 1997. Cell-cycle arrest versus cell death in cancer therapy. Nature Medicine 3: 1034–1036.

    PubMed  Article  CAS  Google Scholar 

  41. Wang, Y., S.J. Decker, and J. Sebolt-Leopold. 2004. Knockdown of Chk1, Wee1 and Myt1 by RNA interference abrogates G2 checkpoint and induces apoptosis. Cancer Biology & Therapy 3: 305–313.

    Article  CAS  Google Scholar 

  42. Werness, B.A., A.J. Levine, and P.M. Howley. 1990. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248: 76–79.

    PubMed  Article  CAS  Google Scholar 

  43. Wesierska-Gadek, J., A. Borza, O. Komina, and M. Maurer. 2009a. Impact of roscovitine, a selective CDK inhibitor, on cancer cells: bi-functionality increases its therapeutic potential. Acta Biochimica Polonica 56: 495–501.

    PubMed  CAS  Google Scholar 

  44. Wesierska-Gadek, J., A. Borza, E. Walzi, V. Krystof, M. Maurer, O. Komina, and S. Wandl. 2009b. Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment. Journal of Cellular Biochemistry 106: 937–955.

    PubMed  Article  CAS  Google Scholar 

  45. Yang, H.L., J.X. Pan, L. Sun, and S.C. Yeung. 2003. p21 Waf-1 (Cip-1) enhances apoptosis induced by manumycin and paclitaxel in anaplastic thyroid cancer cells. The Journal of Clinical Endocrinology and Metabolism 88: 763–772.

    PubMed  Article  CAS  Google Scholar 

  46. Yin, X.M., Z.N. Oltvai, D.J. Veis-Novack, G.P. Linette, and S.J. Korsmeyer. 1994. Bcl-2 gene family and the regulation of programmed cell death. Cold Spring Harbor Symposia on Quantitative Biology 59: 387–393.

    PubMed  Article  CAS  Google Scholar 

  47. Yu, D., T. Jing, B. Liu, J. Yao, M. Tan, T.J. McDonnell, and M.C. Hung. 1998. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Molecular Cell 2: 581–591.

    PubMed  Article  CAS  Google Scholar 

  48. Zhang, F., T. Zhang, T. Jiang, R. Zhang, Z.H. Teng, C. Li, Z.P. Gu, and Q. Mei. 2009. Wortmannin potentiates roscovitine-induced growth inhibition in human solid tumor cells by repressing PI3K/Akt pathway. Cancer Letters 286: 232–239.

    PubMed  Article  CAS  Google Scholar 

  49. Zharskaya, O.O., A.S. Barsukova, and O.V. Zatsepina. 2008. Effect of roscovitine, a selective cyclin B-dependent kinase 1 inhibitor, on assembly of the nucleolus in mitosis. Biochemistry (Moscow) 73: 411–419.

    Article  CAS  Google Scholar 

  50. Zheng, P.W., L.C. Chiang, and C.C. Lin. 2005. Apigenin induced apoptosis through p53-dependent pathway in human cervical carcinoma cells. Life Sciences 76: 1367–1379.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

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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Correspondence to Rita Kundu.

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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

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Keywords

  • Roscovitine
  • Apoptosis
  • Caspase
  • p21 (Cip1/Waf1)
  • Bcl2
  • γH2AX