DNA damage signaling in response to 5-fluorouracil in three colorectal cancer cell lines with different mismatch repair and TP53 status

Abstract

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) G 1 /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 indicated 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.

MicroRNA-18a Attenuates DNA Damage Repair through Suppressing the Expression of Ataxia Telangiectasia Mutated in Colorectal Cancer

“…Huehls et al showed that depletion of ATM did not sensitize cells to 5-FU, which is the main regimen used in CRC [31]. Admansen et al also showed that at clinical relevant dosage of 5-FU, the ATM-pathway is not activated for DNA repair in CRC cells [32]. Therefore, though our in vitro data clearly demonstrated the suppression of ATM by miR-18a sensitized cancer cells to etoposide, the role of ATM role on chemo-resistance may vary in a chemotherapeutic-specific and tumor-specific manner in vivo .…”

The novel tankyrase inhibitor (AZ1366) enhances irinotecan activity in tumors that exhibit elevated tankyrase and irinotecan resistance

“…Irinotecan is used as standard therapy for the treatment of CRC [ 28 , 29 ]. Although treatment responses are observed in patients, drug resistance remains a major problem ultimately leading to disease recurrence and death [ 28 , 29 ]. It has been shown that Wnt pathway inhibition may facilitate sensitivity to irinotecan and overcome treatment resistance [ 30 ].…”

NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair

“…Cells with MMR deficiencies have been reported to be more resistant to these DNA-damaging drugs. 30 , 37 , 38 , 39 , 40 , 41 , 42 , 43 Thus, a restoration of sensitivity is associated with the return of MMR. In the context of MMR, MNU treatment creates O 6 -methylguanine lesions in the DNA that are mispaired with thymine (T), and the resulting O 6 -methylguanine:T mismatch is detected by MMR proteins.…”