Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Jun 11;93(12):5941–5946. doi: 10.1073/pnas.93.12.5941

G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.

J B Schnier 1, K Nishi 1, D W Goodrich 1, E M Bradbury 1
PMCID: PMC39167  PMID: 8650198

Abstract

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.

Full text

PDF
5941

Images in this article

5942Fig. 1
on p.5942
5943Fig. 2
on p.5943
5943Fig. 3
on p.5943
5944Fig. 4
on p.5944
5945Fig. 5
on p.5945

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe K., Yoshida M., Usui T., Horinouchi S., Beppu T. Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases. Exp Cell Res. 1991 Jan;192(1):122–127. doi: 10.1016/0014-4827(91)90166-r. [DOI] [PubMed] [Google Scholar]
  2. Akiyama T., Ohuchi T., Sumida S., Matsumoto K., Toyoshima K. Phosphorylation of the retinoblastoma protein by cdk2. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7900–7904. doi: 10.1073/pnas.89.17.7900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bookstein R., Shew J. Y., Chen P. L., Scully P., Lee W. H. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science. 1990 Feb 9;247(4943):712–715. doi: 10.1126/science.2300823. [DOI] [PubMed] [Google Scholar]
  4. Bruno S., Ardelt B., Skierski J. S., Traganos F., Darzynkiewicz Z. Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res. 1992 Jan 15;52(2):470–473. [PubMed] [Google Scholar]
  5. Buchkovich K., Duffy L. A., Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. 1989 Sep 22;58(6):1097–1105. doi: 10.1016/0092-8674(89)90508-4. [DOI] [PubMed] [Google Scholar]
  6. Chen P. L., Chen Y., Shan B., Bookstein R., Lee W. H. Stability of retinoblastoma gene expression determines the tumorigenicity of reconstituted retinoblastoma cells. Cell Growth Differ. 1992 Feb;3(2):119–125. [PubMed] [Google Scholar]
  7. Chen P. L., Scully P., Shew J. Y., Wang J. Y., Lee W. H. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation. Cell. 1989 Sep 22;58(6):1193–1198. doi: 10.1016/0092-8674(89)90517-5. [DOI] [PubMed] [Google Scholar]
  8. Crissman H. A., Gadbois D. M., Tobey R. A., Bradbury E. M. Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7580–7584. doi: 10.1073/pnas.88.17.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crissman H. A., Tobey R. A. Cell-cycle analysis in 20 minutes. Science. 1974 Jun 21;184(4143):1297–1298. doi: 10.1126/science.184.4143.1297. [DOI] [PubMed] [Google Scholar]
  10. DeCaprio J. A., Ludlow J. W., Lynch D., Furukawa Y., Griffin J., Piwnica-Worms H., Huang C. M., Livingston D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell. 1989 Sep 22;58(6):1085–1095. doi: 10.1016/0092-8674(89)90507-2. [DOI] [PubMed] [Google Scholar]
  11. Dulić V., Kaufmann W. K., Wilson S. J., Tlsty T. D., Lees E., Harper J. W., Elledge S. J., Reed S. I. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell. 1994 Mar 25;76(6):1013–1023. doi: 10.1016/0092-8674(94)90379-4. [DOI] [PubMed] [Google Scholar]
  12. Ewen M. E., Sluss H. K., Whitehouse L. L., Livingston D. M. TGF beta inhibition of Cdk4 synthesis is linked to cell cycle arrest. Cell. 1993 Sep 24;74(6):1009–1020. doi: 10.1016/0092-8674(93)90723-4. [DOI] [PubMed] [Google Scholar]
  13. Goodrich D. W., Chen Y., Scully P., Lee W. H. Expression of the retinoblastoma gene product in bladder carcinoma cells associates with a low frequency of tumor formation. Cancer Res. 1992 Apr 1;52(7):1968–1973. [PubMed] [Google Scholar]
  14. Goodrich D. W., Wang N. P., Qian Y. W., Lee E. Y., Lee W. H. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell. 1991 Oct 18;67(2):293–302. doi: 10.1016/0092-8674(91)90181-w. [DOI] [PubMed] [Google Scholar]
  15. Gross J. L., Herblin W. F., Do U. H., Pounds J. S., Buenaga L. J., Stephens L. E. Characterization of specific [3H]dimethylstaurosporine binding to protein kinase C. Biochem Pharmacol. 1990 Jul 15;40(2):343–350. doi: 10.1016/0006-2952(90)90697-j. [DOI] [PubMed] [Google Scholar]
  16. Gu Y., Rosenblatt J., Morgan D. O. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992 Nov;11(11):3995–4005. doi: 10.1002/j.1460-2075.1992.tb05493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  18. Herbert J. M., Seban E., Maffrand J. P. Characterization of specific binding sites for [3H]-staurosporine on various protein kinases. Biochem Biophys Res Commun. 1990 Aug 31;171(1):189–195. doi: 10.1016/0006-291x(90)91375-3. [DOI] [PubMed] [Google Scholar]
  19. Huang H. J., Yee J. K., Shew J. Y., Chen P. L., Bookstein R., Friedmann T., Lee E. Y., Lee W. H. Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells. Science. 1988 Dec 16;242(4885):1563–1566. doi: 10.1126/science.3201247. [DOI] [PubMed] [Google Scholar]
  20. Hunter T., Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994 Nov 18;79(4):573–582. doi: 10.1016/0092-8674(94)90543-6. [DOI] [PubMed] [Google Scholar]
  21. Koh J., Enders G. H., Dynlacht B. D., Harlow E. Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature. 1995 Jun 8;375(6531):506–510. doi: 10.1038/375506a0. [DOI] [PubMed] [Google Scholar]
  22. Kraemer P. M., Bradbury E. M. Abrogation of a kinase-mediated G1 cell cycle arrest point is a late event in the neoplastic progression of human fibroblasts transfected with the SV40 large T antigen gene. Exp Cell Res. 1993 Jul;207(1):206–210. doi: 10.1006/excr.1993.1183. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lee M. H., Reynisdóttir I., Massagué J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev. 1995 Mar 15;9(6):639–649. doi: 10.1101/gad.9.6.639. [DOI] [PubMed] [Google Scholar]
  25. Ludlow J. W., DeCaprio J. A., Huang C. M., Lee W. H., Paucha E., Livingston D. M. SV40 large T antigen binds preferentially to an underphosphorylated member of the retinoblastoma susceptibility gene product family. Cell. 1989 Jan 13;56(1):57–65. doi: 10.1016/0092-8674(89)90983-5. [DOI] [PubMed] [Google Scholar]
  26. Lukas J., Bartkova J., Rohde M., Strauss M., Bartek J. Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity. Mol Cell Biol. 1995 May;15(5):2600–2611. doi: 10.1128/mcb.15.5.2600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lukas J., Parry D., Aagaard L., Mann D. J., Bartkova J., Strauss M., Peters G., Bartek J. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature. 1995 Jun 8;375(6531):503–506. doi: 10.1038/375503a0. [DOI] [PubMed] [Google Scholar]
  28. Matsuoka S., Edwards M. C., Bai C., Parker S., Zhang P., Baldini A., Harper J. W., Elledge S. J. p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev. 1995 Mar 15;9(6):650–662. doi: 10.1101/gad.9.6.650. [DOI] [PubMed] [Google Scholar]
  29. Matsushime H., Quelle D. E., Shurtleff S. A., Shibuya M., Sherr C. J., Kato J. Y. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol. 1994 Mar;14(3):2066–2076. doi: 10.1128/mcb.14.3.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Medema R. H., Herrera R. E., Lam F., Weinberg R. A. Growth suppression by p16ink4 requires functional retinoblastoma protein. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6289–6293. doi: 10.1073/pnas.92.14.6289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meyerson M., Harlow E. Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Mol Cell Biol. 1994 Mar;14(3):2077–2086. doi: 10.1128/mcb.14.3.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miyazaki A., Kitamura Y., Nomura Y. Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain. Neurochem Int. 1993 May;22(5):455–464. doi: 10.1016/0197-0186(93)90040-c. [DOI] [PubMed] [Google Scholar]
  33. Morgan D. O. Principles of CDK regulation. Nature. 1995 Mar 9;374(6518):131–134. doi: 10.1038/374131a0. [DOI] [PubMed] [Google Scholar]
  34. Ohtsubo M., Theodoras A. M., Schumacher J., Roberts J. M., Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. doi: 10.1128/mcb.15.5.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Polyak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massagué J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994 Jul 15;78(1):59–66. doi: 10.1016/0092-8674(94)90572-x. [DOI] [PubMed] [Google Scholar]
  36. Schnier J. B., Gadbois D. M., Nishi K., Bradbury E. M. The kinase inhibitor staurosporine induces G1 arrest at two points: effect on retinoblastoma protein phosphorylation and cyclin-dependent kinase 2 in normal and transformed cells. Cancer Res. 1994 Nov 15;54(22):5959–5963. [PubMed] [Google Scholar]
  37. Sherr C. J. Mammalian G1 cyclins. Cell. 1993 Jun 18;73(6):1059–1065. doi: 10.1016/0092-8674(93)90636-5. [DOI] [PubMed] [Google Scholar]
  38. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  39. Sumegi J., Uzvolgyi E., Klein G. Expression of the RB gene under the control of MuLV-LTR suppresses tumorigenicity of WERI-Rb-27 retinoblastoma cells in immunodefective mice. Cell Growth Differ. 1990 May;1(5):247–250. [PubMed] [Google Scholar]
  40. Toyoshima H., Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994 Jul 15;78(1):67–74. doi: 10.1016/0092-8674(94)90573-8. [DOI] [PubMed] [Google Scholar]
  41. Weinberg R. A. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323–330. doi: 10.1016/0092-8674(95)90385-2. [DOI] [PubMed] [Google Scholar]
  42. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]
  43. Xiong Y., Zhang H., Beach D. Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation. Genes Dev. 1993 Aug;7(8):1572–1583. doi: 10.1101/gad.7.8.1572. [DOI] [PubMed] [Google Scholar]
  44. Zhang H., Hannon G. J., Beach D. p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 1994 Aug 1;8(15):1750–1758. doi: 10.1101/gad.8.15.1750. [DOI] [PubMed] [Google Scholar]
  45. Zhang H., Kobayashi R., Galaktionov K., Beach D. p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell. 1995 Sep 22;82(6):915–925. doi: 10.1016/0092-8674(95)90271-6. [DOI] [PubMed] [Google Scholar]
  46. Zhou Y., Li J., Xu K., Hu S. X., Benedict W. F., Xu H. J. Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4165–4169. doi: 10.1073/pnas.91.10.4165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES