The transforming growth-beta receptor type II (TGF-beta RII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF-beta RII gene, both located on the short arm of chromosome 3. Introduction of the wild-type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR-deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF-beta RII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF-beta-TGF-beta RII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild-type as well as mutated TGF-beta RII mRNA. In addition, phosphorylation of TGF-beta RI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF-beta. The amount of TGF-beta1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF-beta produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti-TGF-beta antibody. Taken together, these results strongly suggest that the TGF-beta RII gene defect in HCT116 is complemented by a wild-type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF-beta. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.
Copyright 2001 Wiley-Liss, Inc.