Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa ), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Su rgical specimens from 12 patients with sporadic colon cancer and normal muc osae from the same patients were investigated. In addition, we analyzed 3 h uman colon cancer cell lines that exhibited defects in mismatch repair (DLD -1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29 , SW480 and SW620), For detection of mutations, we used reverse transcripti on of mRNA, amplification of cDNAs by PCR, analysis of single-strand confor mation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon ca ncer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in I colon carcinoma and in cell line HCT116, Two sequence alterations of the 70 kDa subunit of replication factor A were id entified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carci nomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange, In carcinomas CC5MA and CC 8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The devi ations in codons 351 and 352 occurred in both cancer tissues and normal muc osae, suggesting a genetic polymorphism. No mutation was found in the 140 k Da subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the P53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens, We did not find tumor-associated DNA sequence alterations that resulted in ami no acid changes in the DNA replication genes analyzed. We infer that the sc arcity of mutations found is due to stringent selection, eliminating functi onally impaired replication proteins. Int. J, Cancer 86:318-324, 2000. (C) 2000 Wiley-Liss, Inc.