Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed.
Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.
Peters
G J
GJ
Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. gj.peters@azvu.nl
van Triest
B
B
Backus
H H
HH
Kuiper
C M
CM
van der Wilt
C L
CL
Pinedo
H M
HM
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000008:administration & dosage; D005434:Flow Cytometry; D005472:Fluorouracil / Q000008:administration & dosage; D019254:Genes, bcl-2 / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D011799:Quinazolines / Q000008:administration & dosage; D013876:Thiophenes / Q000008:administration & dosage; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011799:Quinazolines; D013876:Thiophenes; D051028:bcl-2-Associated X Protein; D013940:Thymidylate Synthase; C068874:raltitrexed; D005472:Fluorouracil