Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1999 Sep;81(2):252–260. doi: 10.1038/sj.bjc.6690685

Involvement of bcl-2 and p21waf1 proteins in response of human breast cancer cell clones to Tomudex

L Orlandi 1, A Bearzatto 1, G Abolafio 1, C De Marco 1, M G Daidone 1, N Zaffaroni 1
PMCID: PMC2362863  PMID: 10496350

Abstract

Mechanisms of resistance to Tomudex include increased thymidylate synthase activity, as well as reduced intracellular drug uptake and polyglutamation. However, little is known about other mechanisms of resistance, such as a possible protection against Tomudex-induced apoptosis mediated by bcl-2. We transfected the MDA-MB-435 human breast cancer cell line, which is characterized by a mutated p53 gene, with cDNA of the bcl-2 gene and generated two clones (MDA-bcl4 and MDA-bcl7) characterized by bcl-2 expression twofold and fourfold that observed in the control cell clone (MDAneo). A concomitant overexpression of p21wafl was also detected in the MDA-bcl7 clone. The MDA-bcl4 clone was three times more resistant to a 24-h Tomudex exposure than the MDAneo clone, whereas the MDA-bcl7 clone was as sensitive to Tomudex as the control cell clone. A lower sensitivity of the MDA-bcl4 clone than MDAneo and MDA-bcl7 clones to 5-fluorouracil and gemcitabine was also observed. No significant difference was noted in the susceptibility of clones to fludarabine and methothrexate. Basal levels of thymidylate synthase activity were superimposable in the three clones. Tomudex induced a marked accumulation of cells in the S phase in all the clones. However, an apoptotic hypodiploid DNA peak and the characteristic nuclear morphology of apoptosis were observed only in the MDA-bcl7 clone after exposure to Tomudex. No difference in the treatment-induced modulation of proteins involved in cell cycle progression (cyclin A, cdk2, pRB, E2F-1) and apoptosis (bcl-2, bax) was observed in the three clones. The only exception was that the expression of p21wafl in the MDA-bcl4 clone was inducible at a Tomudex concentration much higher than that required to induce the protein in the other clones. Overall, the results indicate that bcl-2 and p21wafl proteins concur in determining the cellular profile of sensitivity/resistance to Tomudex. © 1999 Cancer Research Campaign

Keywords: Tomudex, resistance, bcl-2, p21wafl, breast cancer cells

Full Text

The Full Text of this article is available as a PDF (367.6 KB).

Selected References

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

  1. Blackledge G. New developments in cancer treatment with the novel thymidylate synthase inhibitor raltitrexed ('Tomudex'). Br J Cancer. 1998;77 (Suppl 2):29–37. doi: 10.1038/bjc.1998.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  3. Chen Y. Q., Cipriano S. C., Arenkiel J. M., Miller F. R. Tumor suppression by p21WAF1. Cancer Res. 1995 Oct 15;55(20):4536–4539. [PubMed] [Google Scholar]
  4. Cunningham D., Zalcberg J. R., Rath U., Olver I., Van Cutsem E., Svensson C., Seitz J. F., Harper P., Kerr D., Perez-Manga G. 'Tomudex' (ZD1694): results of a randomised trial in advanced colorectal cancer demonstrate efficacy and reduced mucositis and leucopenia. The 'Tomudex' Colorectal Cancer Study Group. Eur J Cancer. 1995 Nov;31A(12):1945–1954. doi: 10.1016/0959-8049(95)00502-1. [DOI] [PubMed] [Google Scholar]
  5. Drake J. C., Allegra C. J., Moran R. G., Johnston P. G. Resistance to tomudex (ZD1694): multifactorial in human breast and colon carcinoma cell lines. Biochem Pharmacol. 1996 May 17;51(10):1349–1355. doi: 10.1016/0006-2952(96)00057-3. [DOI] [PubMed] [Google Scholar]
  6. Fisher T. C., Milner A. E., Gregory C. D., Jackman A. L., Aherne G. W., Hartley J. A., Dive C., Hickman J. A. bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res. 1993 Jul 15;53(14):3321–3326. [PubMed] [Google Scholar]
  7. Freemantle S. J., Jackman A. L., Kelland L. R., Calvert A. H., Lunec J. Molecular characterisation of two cell lines selected for resistance to the folate-based thymidylate synthase inhibitor, ZD1694. Br J Cancer. 1995 May;71(5):925–930. doi: 10.1038/bjc.1995.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gartenhaus R. B., Wang P., Hoffmann P. Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):265–268. doi: 10.1073/pnas.93.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ikeda M. A., Jakoi L., Nevins J. R. A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3215–3220. doi: 10.1073/pnas.93.8.3215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jackman A. L., Calvert A. H. Folate-based thymidylate synthase inhibitors as anticancer drugs. Ann Oncol. 1995 Nov;6(9):871–881. doi: 10.1093/oxfordjournals.annonc.a059353. [DOI] [PubMed] [Google Scholar]
  11. Jackman A. L., Farrugia D. C., Gibson W., Kimbell R., Harrap K. R., Stephens T. C., Azab M., Boyle F. T. ZD1694 (Tomudex): a new thymidylate synthase inhibitor with activity in colorectal cancer. Eur J Cancer. 1995 Jul-Aug;31A(7-8):1277–1282. doi: 10.1016/0959-8049(95)00166-g. [DOI] [PubMed] [Google Scholar]
  12. Jackman A. L., Kelland L. R., Kimbell R., Brown M., Gibson W., Aherne G. W., Hardcastle A., Boyle F. T. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines. Br J Cancer. 1995 May;71(5):914–924. doi: 10.1038/bjc.1995.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jackman A. L., Marsham P. R., Moran R. G., Kimbell R., O'Connor B. M., Hughes L. R., Calvert A. H. Thymidylate synthase inhibitors: the in vitro activity of a series of heterocyclic benzoyl ring modified 2-desamino-2-methyl-N10-substituted-5,8-dideazafolates. Adv Enzyme Regul. 1991;31:13–27. doi: 10.1016/0065-2571(91)90006-8. [DOI] [PubMed] [Google Scholar]
  14. Jackman A. L., Taylor G. A., Gibson W., Kimbell R., Brown M., Calvert A. H., Judson I. R., Hughes L. R. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res. 1991 Oct 15;51(20):5579–5586. [PubMed] [Google Scholar]
  15. Judson I. R. 'Tomudex' (raltitrexed) development: preclinical, phase I and II studies. Anticancer Drugs. 1997 Aug;8 (Suppl 2):S5–S9. doi: 10.1097/00001813-199708002-00002. [DOI] [PubMed] [Google Scholar]
  16. Kelland L. R., Kimbell R., Hardcastle A., Aherne G. W., Jackman A. L. Relationships between resistance to cisplatin and antifolates in sensitive and resistant tumour cell lines. Eur J Cancer. 1995 Jun;31A(6):981–986. doi: 10.1016/0959-8049(95)00198-0. [DOI] [PubMed] [Google Scholar]
  17. Kinsella A. R., Smith D., Pickard M. Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage. Br J Cancer. 1997;75(7):935–945. doi: 10.1038/bjc.1997.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med. 1997 Jun;3(6):614–620. doi: 10.1038/nm0697-614. [DOI] [PubMed] [Google Scholar]
  19. Li W. W., Fan J., Hochhauser D., Bertino J. R. Overexpression of p21waf1 leads to increased inhibition of E2F-1 phosphorylation and sensitivity to anticancer drugs in retinoblastoma-negative human sarcoma cells. Cancer Res. 1997 Jun 1;57(11):2193–2199. [PubMed] [Google Scholar]
  20. Lu K., Yin M. B., McGuire J. J., Bonmassar E., Rustum Y. M. Mechanisms of resistance to N-[5-[N-(3,4-dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line. Biochem Pharmacol. 1995 Jul 31;50(3):391–398. doi: 10.1016/0006-2952(95)00135-m. [DOI] [PubMed] [Google Scholar]
  21. O'Connor P. M., Jackman J., Bae I., Myers T. G., Fan S., Mutoh M., Scudiero D. A., Monks A., Sausville E. A., Weinstein J. N. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res. 1997 Oct 1;57(19):4285–4300. [PubMed] [Google Scholar]
  22. Panadero A., Yin M. B., Voigt W., Rustum Y. M. Contrasting patterns of DNA fragmentation induced by thymidylate synthase inhibitors, ZD1694 and AG-331. Oncol Res. 1995;7(2):73–81. [PubMed] [Google Scholar]
  23. Pinard M. F., Jolivet J., Ratnam M., Kathmann I., Molthoff C., Westerhof R., Schornagel J. H., Jansen G. Functional aspects of membrane folate receptors in human breast cancer cells with transport-related resistance to methotrexate. Cancer Chemother Pharmacol. 1996;38(3):281–288. doi: 10.1007/s002800050483. [DOI] [PubMed] [Google Scholar]
  24. Prabhu N. S., Blagosklonny M. V., Zeng Y. X., Wu G. S., Waldman T., El-Deiry W. S. Suppression of cancer cell growth by adenovirus expressing p21(WAF1/CIP1) deficient in PCNA interaction. Clin Cancer Res. 1996 Jul;2(7):1221–1229. [PubMed] [Google Scholar]
  25. Russo T., Zambrano N., Esposito F., Ammendola R., Cimino F., Fiscella M., Jackman J., O'Connor P. M., Anderson C. W., Appella E. A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress. J Biol Chem. 1995 Dec 8;270(49):29386–29391. doi: 10.1074/jbc.270.49.29386. [DOI] [PubMed] [Google Scholar]
  26. Rustum Y. M., Harstrick A., Cao S., Vanhoefer U., Yin M. B., Wilke H., Seeber S. Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors. J Clin Oncol. 1997 Jan;15(1):389–400. doi: 10.1200/JCO.1997.15.1.389. [DOI] [PubMed] [Google Scholar]
  27. Sheikh M. S., Li X. S., Chen J. C., Shao Z. M., Ordonez J. V., Fontana J. A. Mechanisms of regulation of WAF1/Cip1 gene expression in human breast carcinoma: role of p53-dependent and independent signal transduction pathways. Oncogene. 1994 Dec;9(12):3407–3415. [PubMed] [Google Scholar]
  28. Sheikh M. S., Rochefort H., Garcia M. Overexpression of p21WAF1/CIP1 induces growth arrest, giant cell formation and apoptosis in human breast carcinoma cell lines. Oncogene. 1995 Nov 2;11(9):1899–1905. [PubMed] [Google Scholar]
  29. Sherr C. J. Cancer cell cycles. Science. 1996 Dec 6;274(5293):1672–1677. doi: 10.1126/science.274.5293.1672. [DOI] [PubMed] [Google Scholar]
  30. Silvestrini R., Zaffaroni N., Costa A., Orlandi L., Villa R., Hendriks H. R. Flunarizine as a modulator of doxorubicin resistance in human colon-adenocarcinoma cells. Int J Cancer. 1993 Oct 21;55(4):636–639. doi: 10.1002/ijc.2910550420. [DOI] [PubMed] [Google Scholar]
  31. Smith I., Jones A., Spielmann M., Namer M., Green M. D., Bonneterre J., Wander H. E., Hatschek T., Wilking N., Zalcberg J. A phase II study in advanced breast cancer: ZD1694 ('Tomudex') a novel direct and specific thymidylate synthase inhibitor. Br J Cancer. 1996 Aug;74(3):479–481. doi: 10.1038/bjc.1996.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Van Cutsem E. Future developments with 'Tomudex' (raltitrexed). Anticancer Drugs. 1997 Aug;8 (Suppl 2):S33–S38. doi: 10.1097/00001813-199708002-00007. [DOI] [PubMed] [Google Scholar]
  33. Van der Wilt C. L., Pinedo H. M., Smid K., Peters G. J. Elevation of thymidylate synthase following 5-fluorouracil treatment is prevented by the addition of leucovorin in murine colon tumors. Cancer Res. 1992 Sep 15;52(18):4922–4928. [PubMed] [Google Scholar]
  34. Westerhof G. R., Rijnboutt S., Schornagel J. H., Pinedo H. M., Peters G. J., Jansen G. Functional activity of the reduced folate carrier in KB, MA104, and IGROV-I cells expressing folate-binding protein. Cancer Res. 1995 Sep 1;55(17):3795–3802. [PubMed] [Google Scholar]
  35. 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]
  36. el-Deiry W. S., Harper J. W., O'Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994 Mar 1;54(5):1169–1174. [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES