title: DNA damage signaling in response to 5-fluorouracil in three colorectal cancer cell lines with different mismatch repair and TP53 status.
abstract: We studied patterns of DNA damage signaling and cell cycle response to clinically-relevant (bolus) and high doses of 5-fluorouracil (5-FU) in three colorectal cancer cell lines with differing MMR and TP53 status in an attempt to better understand how 5-FU exerts its cytotoxicity. The ATM/CHEK2/ CHEK1 signaling pathway was not activated in response to bolus 5-FU in the MMR-deficient cell lines HCT116 (TP53-proficient or TP53-depleted) and HCT15 (TP53-deficient), consistent with negligible/reparable DNA damage and no cell death. The pattern of DNA damage checkpoint activation in bolus 5-FU-treated HT29 (TP53-deficient/MMR-proficient) cultures suggested SSB formation (CHEK1 activation) followed by DSB formation (CHEK2 activation and increased phospho-H2AX levels), but no cell death suggested that DNA repair capacity was not overwhelmed. High-dose 5-FU treatment led to activation of ATM/CHEK2/TP53 (not CHEK1) in TP53-proficient and TP53-depleted HCT116 (later CHEK2 activation relative to TP53-proficient) cultures; HCT15 cultures had ATM activation only. These data and increased phospho-H2AX levels indicated DSB formation; apoptosis was induced in both cell lines indicating irreparable DNA damage. TP53-depleted HCT116 cultures also had DSBs after high-dose 5-FU treatment but experienced a (transient) G1/S cell cycle arrest that protected them from apoptosis. TP53 phosphorylation at Ser20/33/37 was seen in TP53-proficient HCT116 cultures regardless of 5-FU concentration at ≥4 h following treatment, indicating TP53 stabilization/transcriptional activation. Overall, activation of ATM, CHEK1 and/or CHEK2 and phospho-H2AX levels reflected the nature of 5-FU-induced DNA damage and indi-cated when DNA damage was significant (5-FU-dose-dependent). DNA repair and cell cycle responses to 5-FU-induced DNA damage were distinctly affected by MMR and TP53 (role in BER/NER) functionalities, but MMR deficiency especially seemed to confer less overall sensitivity to 5-FU.
authors.affiliation: Department of Pathology, Clinic for Diagnostics and Intervention, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
authors.forename: Birgitte L
authors.initials: BL
authors.lastname: Adamsen
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Kravik
authors.forename: Paula M
authors.initials: PM
authors.lastname: De Angelis
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D048708:Cell Growth Processes / Q000187:drug effects; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004249:DNA Damage; D053843:DNA Mismatch Repair; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology*; D016158:Genes, p53; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008297:Male; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
chemical_list: D000964:Antimetabolites, Antineoplastic; D005472:Fluorouracil