Common fragile sites in colon cancer cell lines: Role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1

Abstract

t r a c tCommon fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

“…An important step ensuring the fidelity of this process is the assessment of sequence identity between paired strands of heteroduplex DNA (31,53). A large body of evidence has suggested that the MMR machinery plays a central role in rejecting heteroduplex DNA containing a mismatch and that this role is particularly important during repair of highly repetitive DNA and of the common fragile sites (1,3,54). Here, we demonstrated that the first MMR responder in humans, hMSH2-hMSH6, is able to recognize a mismatch within the heteroduplex region of a D-loop recombination intermediate (Figs.…”

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan

“…HCT116+3 cells, transfected with pBabe vector only, were used as control (HCT116+3 Babe). The HCT116 SiP and Babe cell lines were previously described (23). Cells were maintained in the presence of 1 µg/ml puromycin (Sigma-Aldrich).…”

Integrated analysis of FHIT gene alterations in cancer

“…Whether microsatellite instability is the cause or consequence of FHIT loss, if they are both consequences of a common cause, or if one of the events is positively selected in the presence of the other one, is not known. Of note, it has been suggested that defective MMR, which generates MSI, can also cause deletions affecting genes in CFSs [ 81 , 82 ].…”