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

✍ Scribed by Odilia Popanda; Chun Zheng; J. Richard Magdeburg; Jens Büttner; Thomas Flohr; Egbert Hagmüller; Heinz Walter Thielmann

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: French
• Weight: 91 KB
• Volume: 86
• Category: Article
• ISSN: 0020-7136
• DOI: 10.1002/(sici)1097-0215(20000501)86:3<318::aid-ijc3>3.0.co;2-b

✦ Synopsis

We examined cDNAs of the catalytic subunit of DNA polymerase ␣ (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 ␣. 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 ACA3 GCA transition in codon 351, which caused a Thr3 Ala exchange. In carcinomas CC5MA and CC8MA, a TCC3 TCT (Ser3 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 tumorassociated 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.