Mutation analysis of the transforming growth factor-β type II receptor in human cell lines resistant to growth inhibition by transforming growth factor-β

Abstract

The transforming growth factor-b (TGF-b) binds the type II TGF-b growth factor receptor (RII) to inhibit the growth of most epithelial tissues. Most human colon and gastric cancers with microsatellite instability (MI) have frameshift mutations in polynucleotide repeats within the RII coding region; these mutations truncate the receptor protein and disable the serine/threonine kinase to produce TGF-b resistance. To further investigate the type, frequency and tissue distribution of RII mutations, we selected 24 human cancer cell lines from various tissues which were previously reported to be resistant to the inhibitory e ects of TGF-b. We developed protocols for non-isotopic SSCP analysis of PCR products from genomic DNA samples, and we tested them for microsatellite instability. PCR ± SSCP analysis followed by DNA sequencing identi®ed deletion mutations in the exon 3 poly-adenine tract in three colon tumor cell lines: LS174T and SW48 had a single base deletion and LS411 had a two base deletion. Among the 24 previously unreported cell lines, only these three demonstrated microsatellite instability. These and other recent data indicate that RII mutations are essentially con®ned to colon and gastric cancers with microsatellite instability. The narrow spectrum of tissues containing RII mutations illustrates the complexity of genetic checkpoints in human carcinogenesis.

Establishment and characterisation of six human biliary tract cancer cell lines

“…A significant number of human carcinomas and cancer cell lines lose sensitivity to negative growth regulation by transforming growth factor b (TGF-b) (Vincent et al, 1997). In biliary tract cancers, it was reported that 16% (five out of 32 primary biliary carcinomas) had point mutations in the DPC4 gene (Hahn et al, 1998).…”

Intrahepatic cholangiocarcinoma escapes from growth inhibitory effect of transforming growth factor-β1 by overexpression of cyclin D1

“…Most mutations of the TbR-II gene reported so far are located in exon 3, 5, and 7. 43 As microsatellite regions exist in exon 3 and 7 of the TbR-II gene, 44 somatic mutations in these regions may relate to microsatellite instability or replication error. 45,46 Mutations in exon 5 are sporadic and are not associated with replication error.…”

hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors

“…Likewise, there were no somatic mutations in a series of 51 human tumor cell lines which included 20 cell lines known to be resistant to TGF-b 1 . Although we recently reported mutations in the TGF-b type II receptor in three colon cell lines with microsatellite instability (Vincent et al, 1997), the mechanisms for TGF-b 1 resistance remain unknown for most human cell lines. Our results are consistent with the ®ndings of Riggins and coworkers (Riggins et al, 1997) who found no mutations in 167 early-passage human cell lines representing tumors of the colon, breast, brain, lung, pancreas, head and neck and others.…”

“…No homozygous deletions were detected in 40 primary gastric cancers from Chinese patients surgically resected at the School of Oncology, Beijing Medical University and 51 human cancer cell lines that included tumors of the head and neck, retina, breast, lung, esophagus, stomach, liver, colon, pancreas, cervix, skin, ovary, and blood (Table 1) (Gemma et al, 1996;Vincent et al, 1997) by PCR ampli®cation of each exon ( Figure 5). This set of cell lines included 20 that were reported to be completely or substantially resistant to growth inhibition by TGF-b 1 (Table 1) (Fynan and Reiss, 1993;Vincent et al, 1997). We did not ®nd aberrant bands in SSCP analysis of the same PCR products.…”

“…TGF-b 1 inhibits proliferation of epithelial cells and resistance to TGF-b 1 is a common feature of human cancers Lechner et al, 1983). For example, mutational inactivation of the TGF-b type II receptor interrupts TGF-b 1 regulation at the cell membrane, and these mutations occur commonly in colon (Vincent et al, 1997;Parsons et al, 1995;Takenoshita et al, 1997) and gastric cancers (Park et al, 1994) with microsatellite instability. In addition to type II receptor mutations, inactivation of downstream signal transduction proteins is a logical mechanism to mediate TGF-b 1 resistance, and there is some evidence to support this hypothesis.…”