Skip to main content
British Journal of Cancer logoLink to British Journal of Cancer
. 1999 Nov;81(6):959–965. doi: 10.1038/sj.bjc.6690793

Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts

L Enns 1, D Murray 1, R Mirzayans 1
PMCID: PMC2362948  PMID: 10576651

Abstract

Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3-kinase and PI 3-kinase-related proteins (e.g. ATM), but it does not inhibit the activity of purified calmodulin-dependent protein kinase II (CaMKII). In the present study, we compared the effects of wortmannin and the CaMKII inhibitor KN62 on the response of normal human dermal fibroblast cultures to γ radiation. We demonstrate that wortmannin confers a phenotype on normal fibroblasts remarkably similar to that characteristic of cells homozygous for the ATM mutation. Thus wortmannin-treated normal fibroblasts exhibit increased sensitivity to radiation-induced cell killing, lack of temporary block in transition from G1 to S phase following irradiation (i.e. impaired G1/S checkpoint), and radioresistant DNA synthesis (i.e. impaired S phase checkpoint). Wortmannin-treated cultures display a diminished capacity for radiation-induced up-regulation of p53 protein and expression of p21WAF1, a p53-regulated gene involved in cell cycle arrest at the G1/S border; the treated cultures also exhibit decreased capacity for enhancement of CaMKII activity post-irradiation, known to be necessary for triggering the S phase checkpoint. We further demonstrate that KN62 confers a radioresistant DNA synthesis phenotype on normal fibroblasts and moderately potentiates their sensitivity to killing by γ rays, without modulating G1/S checkpoint, p53 up-regulation and p21WAF1 expression following radiation exposure. We conclude that CaMKII is involved in the radiation responsive signalling pathway mediating S phase checkpoint but not in the p53-dependent pathway controlling G1/S checkpoint, and that a wortmannin-sensitive kinase functions upstream in both pathways. © 1999 Cancer Research Campaign

Keywords: wortmannin, KN62, ionizing radiation, radiosensitivity, cell cycle checkpoint, p53, p21WAF1

Full Text

The Full Text of this article is available as a PDF (128.1 KB).

Selected References

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

  1. Banin S., Moyal L., Shieh S., Taya Y., Anderson C. W., Chessa L., Smorodinsky N. I., Prives C., Reiss Y., Shiloh Y. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 1998 Sep 11;281(5383):1674–1677. doi: 10.1126/science.281.5383.1674. [DOI] [PubMed] [Google Scholar]
  2. Beamish H., Lavin M. F. Radiosensitivity in ataxia-telangiectasia: anomalies in radiation-induced cell cycle delay. Int J Radiat Biol. 1994 Feb;65(2):175–184. doi: 10.1080/09553009414550211. [DOI] [PubMed] [Google Scholar]
  3. Brugarolas J., Chandrasekaran C., Gordon J. I., Beach D., Jacks T., Hannon G. J. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature. 1995 Oct 12;377(6549):552–557. doi: 10.1038/377552a0. [DOI] [PubMed] [Google Scholar]
  4. Cliby W. A., Roberts C. J., Cimprich K. A., Stringer C. M., Lamb J. R., Schreiber S. L., Friend S. H. Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J. 1998 Jan 2;17(1):159–169. doi: 10.1093/emboj/17.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  6. Huang H., Li C. Y., Little J. B. Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation. Int J Radiat Biol. 1996 Aug;70(2):151–160. doi: 10.1080/095530096145148. [DOI] [PubMed] [Google Scholar]
  7. Jung M., Zhang Y., Lee S., Dritschilo A. Correction of radiation sensitivity in ataxia telangiectasia cells by a truncated I kappa B-alpha. Science. 1995 Jun 16;268(5217):1619–1621. doi: 10.1126/science.7777860. [DOI] [PubMed] [Google Scholar]
  8. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  9. Khanna K. K., Lavin M. F. Ionizing radiation and UV induction of p53 protein by different pathways in ataxia-telangiectasia cells. Oncogene. 1993 Dec;8(12):3307–3312. [PubMed] [Google Scholar]
  10. Komatsu K., Yoshida M., Okumura Y. Murine scid cells complement ataxia-telangiectasia cells and show a normal post-irradiation response of DNA synthesis. Int J Radiat Biol. 1993 Jun;63(6):725–730. doi: 10.1080/09553009314552121. [DOI] [PubMed] [Google Scholar]
  11. Mayford M., Wang J., Kandel E. R., O'Dell T. J. CaMKII regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP. Cell. 1995 Jun 16;81(6):891–904. doi: 10.1016/0092-8674(95)90009-8. [DOI] [PubMed] [Google Scholar]
  12. Meyn M. S. Ataxia-telangiectasia and cellular responses to DNA damage. Cancer Res. 1995 Dec 15;55(24):5991–6001. [PubMed] [Google Scholar]
  13. Mirzayans R., Aubin R. A., Bosnich W., Blattner W. A., Paterson M. C. Abnormal pattern of post-gamma-ray DNA replication in radioresistant fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome. Br J Cancer. 1995 Jun;71(6):1221–1230. doi: 10.1038/bjc.1995.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mirzayans R., Enns L., Paterson M. C. Inhibition of DNA synthesis and G1/S-phase transition in normal human fibroblasts elicited by a heat-labile trans-acting factor in gamma-irradiated HeLa cell extracts. Radiat Res. 1997 Jan;147(1):13–21. [PubMed] [Google Scholar]
  15. Mirzayans R., Famulski K. S., Enns L., Fraser M., Paterson M. C. Characterization of the signal transduction pathway mediating gamma ray-induced inhibition of DNA synthesis in human cells: indirect evidence for involvement of calmodulin but not protein kinase C nor p53. Oncogene. 1995 Oct 19;11(8):1597–1605. [PubMed] [Google Scholar]
  16. Mirzayans R., Smith B. P., Paterson M. C. Hypersensitivity to cell killing and faulty repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in human (ataxia-telangiectasia) fibroblasts treated with 4-nitroquinoline 1-oxide. Cancer Res. 1989 Oct 15;49(20):5523–5529. [PubMed] [Google Scholar]
  17. Morgan S. E., Lovly C., Pandita T. K., Shiloh Y., Kastan M. B. Fragments of ATM which have dominant-negative or complementing activity. Mol Cell Biol. 1997 Apr;17(4):2020–2029. doi: 10.1128/mcb.17.4.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nagasawa H., Li C. Y., Maki C. G., Imrich A. C., Little J. B. Relationship between radiation-induced G1 phase arrest and p53 function in human tumor cells. Cancer Res. 1995 May 1;55(9):1842–1846. [PubMed] [Google Scholar]
  19. Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem. 1992 Feb 5;267(4):2157–2163. [PubMed] [Google Scholar]
  20. Olivier M., Bautista S., Vallès H., Theillet C. Relaxed cell-cycle arrests and propagation of unrepaired chromosomal damage in cancer cell lines with wild-type p53. Mol Carcinog. 1998 Sep;23(1):1–12. [PubMed] [Google Scholar]
  21. Painter R. B. Inhibition of mammalian cell DNA synthesis by ionizing radiation. Int J Radiat Biol Relat Stud Phys Chem Med. 1986 May;49(5):771–781. doi: 10.1080/09553008514552981. [DOI] [PubMed] [Google Scholar]
  22. Powis G., Bonjouklian R., Berggren M. M., Gallegos A., Abraham R., Ashendel C., Zalkow L., Matter W. F., Dodge J., Grindey G. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994 May 1;54(9):2419–2423. [PubMed] [Google Scholar]
  23. Price B. D., Youmell M. B. The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. Cancer Res. 1996 Jan 15;56(2):246–250. [PubMed] [Google Scholar]
  24. Rosenzweig K. E., Youmell M. B., Palayoor S. T., Price B. D. Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. Clin Cancer Res. 1997 Jul;3(7):1149–1156. [PubMed] [Google Scholar]
  25. Savitsky K., Bar-Shira A., Gilad S., Rotman G., Ziv Y., Vanagaite L., Tagle D. A., Smith S., Uziel T., Sfez S. A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995 Jun 23;268(5218):1749–1753. doi: 10.1126/science.7792600. [DOI] [PubMed] [Google Scholar]
  26. Shao R. G., Cao C. X., Shimizu T., O'Connor P. M., Kohn K. W., Pommier Y. Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res. 1997 Sep 15;57(18):4029–4035. [PubMed] [Google Scholar]
  27. Taylor A. M. What has the cloning of the ATM gene told us about ataxia telangiectasia? Int J Radiat Biol. 1998 Apr;73(4):365–371. doi: 10.1080/095530098142185. [DOI] [PubMed] [Google Scholar]
  28. Tokumitsu H., Chijiwa T., Hagiwara M., Mizutani A., Terasawa M., Hidaka H. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem. 1990 Mar 15;265(8):4315–4320. [PubMed] [Google Scholar]
  29. Wang Y., Iliakis G. Prolonged inhibition by X-rays of DNA synthesis in cells obtained by transformation of primary rat embryo fibroblasts with oncogenes H-ras and v-myc. Cancer Res. 1992 Feb 1;52(3):508–514. [PubMed] [Google Scholar]
  30. Young B. R., Painter R. B. Radioresistant DNA synthesis and human genetic diseases. Hum Genet. 1989 May;82(2):113–117. doi: 10.1007/BF00284040. [DOI] [PubMed] [Google Scholar]
  31. 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 British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES