5-fluorouracil The Cell Cycle Regulatory Effects of High Dose 5-fluorouracil on Breast Cancer Cell Line Joung Soon Jang 1, Jung Ill Yang 1, Seho Chang 1, Won Sup Lee 1, Jong Seok Lee 1, Myung-Ju Ahn 2 and Byung-Kiu Park 3 1 Department of Internal Medicine and Gyeong Sang Institute of Health Science, Gyeong Sang National University College of Medicine and 2 Department of Internal Medicine, Hanyang University College of Medicine, 3 National Cancer Center ABSTRACT Background: Chemotherapy with 5-fluorouracil (5-FU) has been one of the mainstay in breast cancer treatment. The effects of high dose 5-FU on cell cycle regulation were studied in breast caner cells. Methods: A breast cancer cell line MCF-7 was used. Protein expressions of G1/S cyclins, p21 Waf1/Cip1, cdk2, E2F1 and retinoblastoma were tested by western blot analysis. Immunoprecipitation and immune complex kinase assay were done for the assessment of E2F1/RB interacton and the activity of cdk2 respectively. Results: p21 Waf1/Cip1 expression was barely detectable in control cells. With addition of 5-FU level of p21 Waf1/Cip1 were induced and cyclin D3 level was decreased as cell growth decreases. In accordance with increased expression of p21 Waf1/Cip1, cyclin E-associated cdk2 kinase activity was reduced. Retinoblastoma protein (RB) became dephosphorylated and E2F-1 binding activity with RB was increased. Conclusion: In this situation of high concentration of 5-FU breast cancer cells tend to be G1/S cell cycle arrested. Overexpression of p21 Waf1/Cip1 and dephosphorylation of RB may mediate the effectss of 5-FU by inhibiting E2F-1 activity, which contributes to G1/S cell cycle arrest. These results could be an indicating landmark for further study of high dose chemotherapy with 5-FU. (Immune Network 2002;2(1):60-64) Key Words: 5-FU, breast cancer, cell cycle regulation Immune Network 60
Effects of 5-FU on Breast Cancer Cells 61 μ μ μ μ μ β μ γ μ
62 Joung Soon Jang, et al. Figure 2. Induction of p21 Waf1/Cip1 with 5-FU. Cells were incubated with 1 mm 5-FU. The level of p21 protein was determined by Western blot analysis. h: hours of 5-FU treatment. Figure 1. A Protein levels of cyclin D3 determined by western blot analysis. B. Protein levels of cyclin E, A and cdk 2, 4 determined by Western blot analysis. Cells were treated with 1 mm 5-FU. h: hours of 5-FU treatment. Figure 4. Decreased cdk2 kinase activity with 5-FU. Cell lysates were immunoprecipitated with polyclonal anti-cdk2 antibody. Immune complexes were incubated with [γ-32p] ATP and histone H1. Phosphorylation of histone H1 was determined by autoradiography. Figure 3. A. Dephosphorylation of RB with 5-FU. Cells were treated with 5-FU and the level of RB was determined by western blot analysis. B. Increased binding between RB and E2F1 in the presence of 5-FU (36 h). Cell extracts were immunoprecipitated with polyclonal anti-e2f1 antibody and protein level of RB was determined by Western blot anlysis. h: hours of 5-FU treatment, IP: immunoprecipitation, RB: retinoblastoma protein.
Effects of 5-FU on Breast Cancer Cells 63 1. Harras A, Edwards BK, Blot WJ, Gloeckler Ries LA, eds: Cancer: Rates and Risks. 4th ed. Bethesda, NIH/NCI, 1996 2. Haskell CM: Systemic treatment of metastatic breast cancer. In: Haskell CM: Cancer Treatment. 354-368 Philadelphia Saunders, 1995 3. Allegra CJ, Grem JL: Antimetabolite. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer: principles and practice of oncology, 437-441 Philadelphia Lippincott, 1997 4. Grem JL: Current therapeutic strategies to enhance 5-fluorouracil activity. J Infusional Chemotherapy 2;138-142, 1992 5. Lokich J: Infusional 5-FU: historical evolution, rationale, and clinical experience. Oncology (Huntingt) 12;19-22, 1998 6. Leichman CG: Prolonged infusion of fluorinated pyrimidines in gastrointestinal malignancies: a review of recent clinical trials. Cancer Invest 12; 166-175, 1994 7. Jang JS, Lee SJ, Choi YH, Nguyen PM, Wu M, Takano E, Maki M, Henkart PA, Trepel JB: Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease. Oncogene 18;1789-1796, 1999 8. Parr AL, Drake JC, Gress RE, Schwarz G, Steinberg SM, Allegra CJ: 5-fluorouracil-mediated thymidylate synthase induction in malignant and nonmalignant human cells. Biochem Pharm 56; 231-235, 1998 9. Lilling G, Hacohen H, Nordenberg J, Livnat T, Rotter V, Sidi Y: Differential sensitivity of MCF- 7 and LCC2 cells, to multiple growth inhibitory agents: possible relation to high bcl-2/bax ratio. Cancer Lett 161;27-34, 2000 10. Sherr CJ: Cancer cell cycles. Science 274;1672-1677, 1996 11. Whyte P: The retinoblastoma protein and its relatives. Semin Cancer Biol 6;83-90, 1995 12. Li WW Fan J, Hochhauser D, Bertino JR: Overexpression of p21waf1 leads to increased inhibition of E2F-1 phosphorylation and sensitivity to anticancer drugs in retinoblastoma-negative human sarcoma cells. Cancer Res 1;2193-2199, 1997 13. Hopkin K: Regulating cellular processes: The power of protein degradation. J NIH Res 9;36-42, 1997 14. Carson B, Lahusen T, Singh S, Loaiza-Perez A, Pestell R, Sausville EA, Senderowicz AM: Downregulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 15;4634-4641, 1999 15. Russell A, Hendley J, Germain D: Inhibitory effect of p21 in MCF-7 cells is overcome by its coordinated stabilization with D-type cyclins. Oncogene 11;6454-6459, 1999 16. Brambilla E, Brambilla C: p53 and lung cancer. Pathol Biol 45;852-863, 1997 17. Sellers WR, Kaelin WG: Role of the retinoblastoma protein in the pathogenesis of human cancer. J Clin Oncol 15;3301-3312, 1997 18. Beijersbergen RL, Bernards R: Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins. Biochimi Biophysi Acta 1287;103-120, 1996 19. Johnson KR, Wang L, Miller MC, Willingham MC, Fan W: 5-fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells. Clin Cancer Res 3;1739-1745, 1997 20. Ardalan B, Luis R, Jaime M, Franceschi D: Biomodulation of Fluorouracil in colorectal cancer. Cancer Invest 16(4); 237-251, 1998
64 Joung Soon Jang, et al. 21. O Byrne KJ, Koukourakis MI, Saunders MP, Salisbury AJ, Isaacs R, Varcoe S, Taylor M, Ganesan TS, Harris AL, Talbot DC: Cyclophosphamide, methotrexate and infusional 5-fluorouracil (infusional CMF) in metastatic breast cancer. Br J Cancer 77;1950-1956, 1998