Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma

In previous studies, we established two camptothecin (CPT)-resistant sublin es, HT-29/CPT and St-4/CPT, from the human colon cancer cell line HT-29 and the human stomach cancer cell line St-4, respectively. Cellular contents o f DNA topoisomerase I (topo I) in the resistant cells were eight-fold less than those in the corresponding parental lines. In this study, we have show n expression of two species of the TOPI mRNA in HT-29/CPT. The longer mRNA (4.0 kh) is the wild-type TOPI mRNA, and the shorter mRNA (3.3 kh) proved t o have a deletion of 672 bp (nucleotides 58-729 or 59-730) that caused the in-frame deletion of amino acids 20-243 of human topo I. The deleted region is identical to exons 3-9 of the TOPI gene, The expression level of the 3. 3-kb mRNA was similar to that of the wild-type mRNA in HT-29/CPT St-4/CPT e xpressed only the wild-type TOPI mRNA in lesser amounts than did St-4, Mous e NIH3T3 cells transfected with the wild-type TOPI cDNA showed higher sensi tivity to CPT than the parental cells, whereas those transfected with the d eleted TOPI cDNA showed levels similar to those of the parental cells. Expr ession of the exogenous TOPI mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TO PI cDNA, These results suggest that the expression of the deleted TOPI mRNA led to the low expression of CPT-sensitive topo I in the resistant cells.