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. Surgical specimens from 12 patients with sporadic
colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human
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 transcription of
mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and
DNA sequencing. Eleven
colon cancers and 6
colon cancer cell lines were analyzed for
DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon
carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of
replication factor A were identified in 15 specimens (9 colon
carcinomas and 6 cell lines). Colon
carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in
codon 351, which caused a Thr-->Ala exchange. In
carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in
codon 352 was observed. The deviations in
codons 351 and 352 occurred in both
cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa 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
amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication
proteins.