Skip to main content
SpringerLink
Log in

Synergistic Cytotoxicity, Apoptosis and Protein-linked DNA Breakage by Etoposide and Camptothecin in Human U87 Glioma Cells: Dependence on Tyrosine Phosphorylation

  • Published:
Journal of Neuro-Oncology Aims and scope Submit manuscript

Abstract

In this study, simultaneous administration of certain inhibitors of topoisomerase I and topoisomerase II produced synergistic cytotoxicity in a series of human glioma cell lines. Camptothecin (CPT) and etoposide (VP-16) produced combination indices (CI) <1.0 in all glioma cell lines tested, including those that were relatively resistant to the two topoisomerase inhibitors individually. In contrast, CPT and VP-16 produced additive cytotoxicity in HT-29 and SW-620 colon carcinoma cell lines. To explore the molecular basis for synergy in glioma cells, we focused on one glioma cell line (U87) in which even sub-cytotoxic doses of CPT potentiated the action of VP-16. Except for genistein (a topo II agent with tyrosine kinase inhibitory function), all topo II inhibitors tested (doxorubicin, ellipticine, and m-AMSA) were synergistic with CPT. While CPT and VP-16 produced cytotoxicity and protein-linked DNA breaks (PLDB) that were supra-additive in U87 glioma cells, CPT and genistein produced additive results. Pretreatment of U87 cells with the tyrosine kinase inhibitor tyrphostin-A23 or the tyrosine phosphatase activator O-phospho-L-tyrosine (OPLT) reduced combination PLDB from synergistic to additive levels, but had no effect on the formation of PLDB induced by either CPT or VP-16 alone. CPT and VP-16 also produced a synergistic accumulation of sub-G0 (apoptotic) cells which was blocked by tyrphostin-A23. No significant increase in topoisomerase protein levels could be detected in response to combination treatment. Thus, synergistic effects between topoisomerase I and topoisomerase II inhibitors in U87 glioma cells may depend upon phosphorylation of cellular proteins other than the topoisomerases themselves.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Chen AY, Liu LF: DNA topoisomerases. Essential enzymes and lethal targets. Annu Rev Pharmacol Toxicol 34: 191–218, 1994

    PubMed  Google Scholar 

  2. Wang JC, Caron PR, Kim RA: The role of DNA topoisomerases in recombination and genome stability: a double edged sword. Cell 62: 403–406, 1990 233

    PubMed  Google Scholar 

  3. Akihiko T, Kohn K, Kohlhagen G, Leteurtre F, Pommier Y: Differential stabilization of eukaryotic DNA topoisomerase I cleavable complexes by camptothecin derivatives. Biochemistry 34: 7200–7206, 1995

    PubMed  Google Scholar 

  4. Ryan AJ, Shoshana S, Strutt HL, Johnson RT: Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistant double strand breaks in replicating DNA. Nucleic Acids Research 19(12): 3295–3300, 1991

    PubMed  Google Scholar 

  5. Pantazis P, Kozielski AJ, Vardeman DM, Petry ER, Giovanella BC: Efficacy of camptothecin congeners in the treatment of human breast carcinoma xenografts. Oncol Res 5(8): 273–281, 1993

    PubMed  Google Scholar 

  6. Giovanella BC, Stehlin JS, Wall ME, Mansukh C, Wani AW, Nicholas LF, Lui LF, Silber R, Potmesil M: DNA topoisomerase-I-targeted chemotherapy of human colon cancer in xenografts. Science 246: 1046–1048, 1989

    PubMed  Google Scholar 

  7. Burris HA, Rothenberg ML, Kuhn JG, Von Hoff DD: Clinical trials with the topoisomerase I inhibitors. Sem Oncology 19(6): 663–669, 1992

    Google Scholar 

  8. Blaney SM, Phillips PC, Packer RJ, Heideman RL, Berg SL, Adamson PC, Allen JC, Sallan SE, Jakacki RI, Lange BJ, Reaman GH, Horowitz ME, Poplack DG, Balis FM: Phase II evaluation of topotecan for pediatric central nervous system tumors. Cancer 78(3): 527–531, 1996

    PubMed  Google Scholar 

  9. Macdonald D, Cairncross G, Stewart D, Forsyth P, Sawka C, Wainman N, Eisenhauer E: Phase II study of topotecan in patients with recurrent malignant glioma. National Cancer Institute of Canada Clinical Trials Group. Ann Oncol 7(2): 205–207, 1996

    PubMed  Google Scholar 

  10. Chamberlain MC, Grafe MR: Recurrent chiasmatichypothalamic glioma treated with oral etoposide. JClin Oncol 13(8): 2072–2076, 1995

    Google Scholar 

  11. Fulton D, Urtasun R, Forsyth P: Phase II study of prolonged oral therapywith etoposide (VP16) for patients with recurrent malignant glioma. J Neuro-Oncol 27(2): 149–155, 1996

    Google Scholar 

  12. Stevnsner T, Bohr VA: Studies on the role of topoisomerases in general, gene-and strand-specific DNA repair. Carcinogenesis 14(9): 1841–1850, 1993

    PubMed  Google Scholar 

  13. Chattergee S, Cheng MF, Berger NA:Hypersensitivity to clinically useful alkylating agents and radiation in poly(ADPribose) polymerase-deficient cell lines. Cancer Commun 2: 401–407, 1990

    PubMed  Google Scholar 

  14. Lelievre S, Benchokroun Y, Larsen AK: Altered topoisomerase I and II activities in suramin-resistant lung fibrosarcoma cells. Mol Pharmacol 47: 898–906, 1995

    PubMed  Google Scholar 

  15. Suglimoto Y, Tsukahara S, Oh-Hara T, Liu LF, Tsuoruo T: Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines. Cancer Res 50: 7962–7965, 1990

    PubMed  Google Scholar 

  16. Thrash C, Voekel K, Dinardo S: Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem 259: 1375–1377, 1984

    PubMed  Google Scholar 

  17. Taudou G, Portemer C, Jaxel C, Duguet M: Inhibition ofDNA synthesis and DNA fragmentation in stimulated splenocytes by the concerted action of topoisomerase I and II poisons. Biochem Pharmacol 45(2): 331–337, 1993

    PubMed  Google Scholar 

  18. Bertrand R, O'Connor PM, Kerrigan D, Pommier Y: Sequential administration of camptothecin and etoposide circumvents the antagonistic cytotoxicity of simultaneous drug administration in slowly growing human colon carcinoma HT-29 cells. Eur J Cancer 28A(4–5): 743–748, 1992

    Google Scholar 

  19. Sorensen M, Sehested M, Jensen PB: Characterisation of a human small-cell lung cancer cell line resistant to the DNA topoisomerase I-directed drug topotecan. Br J Cancer 72: 399–404, 1995

    PubMed  Google Scholar 

  20. Whitacre CM, Zborowska E, Gordon NH, Mackay W, Berger NA: Topotecan increases topoisomerase II_levels and sensitivity to treatment with etoposide in scheduledependent process. Cancer Res 57: 1425–1428, 1997

    PubMed  Google Scholar 

  21. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Viscita D, Warren JT, Bokesch H, Kenney S, Boyd MR: New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82(13): 1107–1112, 1990

    PubMed  Google Scholar 

  22. Chou TC, Talalay P: Applications of the median-effect principle for the assessment of low-dose risk of carcinogens and for the quantitation of synergism and antagonism of chemotherapeutic agents, in New Avenues in Developmental Cancer Chemotherapy. Academic Press, Orlando, FL, 1987, pp 36–64

    Google Scholar 

  23. Zwelling LA, Hinds M, Chan D, Mayes J, Sie KL, Parker E, Silberman L, Radcliffe A, Beran M, Blick M: Characterization of an amsacrine-resistant line of human leukemia cells. J Biol Chem 264(28): 16411–16420, 1989

    PubMed  Google Scholar 

  24. Aflalo E, Iftach S, Segal S, Gazit A, Priel E: Inhibition of topoisomerase I activity by tyrphostin derivatives, protein tyrosine kinase blockers: mechanism of action. Cancer Res 54: 5168–5142, 1994

    Google Scholar 

  25. Mishra S, Hamburger AW: O-Phospho-L-tyrosine inhibits cellular growth by activating protein tyrosine phosphatases. Cancer Res 53: 557–563, 1993

    PubMed  Google Scholar 

  26. Karato A, Sasaki Y, Shinkai T, Eguchi K, Tamura T, Ohe Y, Oshita F, Nishio M, Kunikane H, Ohmatsu H, Nakashima H, Shiraishi J, Saijo N: Phase I study of CPT-11 and etoposide in patients with refractory solid tumors. J Clin Oncol 11: 2030–2035, 1993

    PubMed  Google Scholar 

  27. Masuda N, Fukuoka M, Kudoh S, Matsui K, Kusunoki Y, Takada M, Nakagawa K, Hirashima T, Tsukada H, Yana T, Yoshikawa A, Kubo A, Matsuura E, Nitta T, Takifuji N, Terakawa K, Negoro S: Phase I and pharmacologic study of irinotecan and etoposide with recombinant human granulocyte colony-stimulating factor support for advanced lung cancer. J Clin Oncol 12: 1833–1841, 1994

    PubMed  Google Scholar 

  28. Kaufmann SH:Antagonism between camptothecin and topoisomerase II-directed chemotherapeutic agents in a human leukemia cell line. Cancer Res 51(4): 1129–1136, 1991

    PubMed  Google Scholar 

  29. Cortes F, Pinero J: Synergistic effect of inhibitors of topoisomerase I and II on chromosome damage and cell killing in cultured Chinese hamster ovary cells. Cancer Chemo Pharmacol 34: 411–415, 1994

    Google Scholar 

  30. Bonner JA, Kozelsky TF: The significance of the sequence of administration of topotecan and etoposide. Cancer Cemother Pharmacol 39: 109–112, 1996

    Google Scholar 

  31. Gazit A, Yaish P, Gilon C, Levitzki A: Tyrphostins I: Synthesis and biological activity of protein tyrosine kinase inhibitors. J Med Chem 32: 2344–2352, 1989

    PubMed  Google Scholar 

  32. Pantazis P, Vardeman D, Mendoza J, Early J, Kozielski A, DeJesus A, Giovanella B: Sensitivity of camptothecinresistant human leukemia cells and tumors to anticancer drugs 234 with diverse mechanisms of action. Leukemia Res 19(1): 43–55, 1995

    Google Scholar 

  33. Madden KR, Champoux JJ: Overexpression of human topoisomerase I in baby hamster kidney cells: hypersensitivity of clonal isolates to camptothecin. Cancer Res 52: 525–530, 1992

    PubMed  Google Scholar 

  34. Kaufmann SH:Antagonism between camptothecin and topoisomerase II-directed chemotherapeutic agents in a human leukemia cell line. Cancer Res 51(4): 1129–1136, 1991

    PubMed  Google Scholar 

  35. Sorenson BS, Jensen PB, Sehested M, Jensen PS, Kjeldsen E, Nielsen OF, Alsner J: Antagonistic effect of aclarubicin on camptothecin induced cytotoxicity: role of topoisomerase I. Biochem Pharmacol 47(11): 2105–2110, 1994

    PubMed  Google Scholar 

  36. Kano Y, Suzuki K, Akutsu M, Suda K, Inoue Y, Yoshida M, Sakamoto S, Miura Y:Effects of CPT-11 in combination with other anti-cancer agents in culture. Int J Cancer 50(4): 604–610, 1992

    PubMed  Google Scholar 

  37. Lopez R, Peters GJ, Smitskamp-Wilms E, Virizuela JA, van Ark-Otte J, Pinedo HM, Giaccone G: In vitro sequencedependent synergistic effect of suramin and camptothecin. Eur J Cancer 30A(11): 1670–1674, 1994

    PubMed  Google Scholar 

  38. Samuels DS, Shimuzu Y, Nakabayashi T, Shimizu N: Phosphorylation of DNA topoisomerase I is increased during the response of mammalian cells to mitogenic stimuli. Biochimic Biophys Acta 1223: 77–83, 1994

    Google Scholar 

  39. Alghisi GC, Roberts E, Cardenas ME, Gasser SM: The regulation of DNA topoisomerase II by casein kinase II. Cell Mol Biol Res 40(5–6): 563–571, 1994

    PubMed  Google Scholar 

  40. Chresta CM, Francis GE: Retinoic acid and phorbol ester induced hyperphosphorylation of topoisomerase II-_is an early event in HL-60 human leukaemia cell differentiation: effect on topoisomerase activity and etoposide sensitivity. Leukemia 9: 1373–1381, 1995

    PubMed  Google Scholar 

  41. Kordiyak GJ, Jakes S, Ingebritsen TS, Benbow RM: Casein kinase II stimulates Xenopus laevis DNA topoisomerase by physical association. Biochemistry 33(45): 1344–1391, 1994

    Google Scholar 

  42. Ritke MK, Murray NR, Allan WP, Fields AP, Yalowich JC: Hypophosphorylation of topoisomerase II in etoposide (VP-16)-resistant human leukemia K562 cells associated with reduced levels of beta II protein kinase C. Mol Pharmacol 48(5): 798–805, 1995

    PubMed  Google Scholar 

  43. Takano H, Kohno K, Ono M, Uchida Y, Kuwano M: Increased phosphorylation of DNA topoisomerase II in etoposideresistant mutants of human cancer KB cells. Cancer Res 51: 2156–2161, 1991

    Google Scholar 

  44. DeVore RF, Corbett AH, Osheroff N: Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: Effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 40–(9–acridinylamino) methane-sulfon-m-aniside. Cancer Res 52: 2156–2161, 1992

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Neurosurgery, Roswell Park Cancer Institute, Buffalo, New York, USA

    Michael J. Ciesielski & Robert A. Fenstermaker

  2. Buffalo, New York, USA

    Robert A. Fenstermaker

Authors
  1. Michael J. Ciesielski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert A. Fenstermaker
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ciesielski, M.J., Fenstermaker, R.A. Synergistic Cytotoxicity, Apoptosis and Protein-linked DNA Breakage by Etoposide and Camptothecin in Human U87 Glioma Cells: Dependence on Tyrosine Phosphorylation. J Neurooncol 41, 223–234 (1999). https://doi.org/10.1023/A:1006129119460

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1006129119460

Search

Navigation