The transforming growth factor-b (TGF-b receptor system has been implicated in the development of resistance to the growth-inhibitory e ects of TGF-b. It has been reported that resistance to TGF-b correlates with inactivation of the TGF-b type II receptor (RII). In the present report, we examine the genetic changes in the TGF-b RII gene of human gastric cancer cell lines, SNU-5 and SNU-668, which we had previously reported to express truncated TGF-b RII transcripts. By independent PCR and Southern hybridization analysis of genomic DNA, we found that the genomic sequence of TGF-b RII is truncated after exon 2 in SNU-5 and after exon 3 in SNU-668. This was con®rmed by sequencing the TGF-b RII cDNA cloned from a SNU-5 cDNA library. Predicted TGF-b RII protein of SNU-5 cells based on sequencing data contains only a part of extracellular domain of TGF-b RII. We demonstrate that cotransfection of 3TP-Lux and wild type TGF-b RII restores the TGF-b responsiveness in SNU-5 cells, suggesting that genetic changes in the TGF-b RII gene of SNU-5 cells are responsible for the loss of sensitivity to TGF-b. This is the ®rst report demonstrating that truncation of the TGF-b RII gene is an alternative mechanism to inactivate the TGF-b signal transduction pathways.

No abstract

Transforming growth factor ␤ (TGF-␤) exerts an inhibitory effect on the growth of most epithelial cell types, and the loss of responsiveness to this growth inhibition has been implicated in the development of a variety of human cancers. The genetic alteration of TGF-␤ receptors is known to play a critical role in this escape from growth regulation. We asked whether there is a correlation between TGF-␤ sensitivity and the genetic status of TGF-␤ type I and type II receptors (RI and RII, respectively) in human cervical carcinoma cell lines. Among 8 cell lines examined, 3 (ME-180, C-33A and HeLaS3) showed resistance to TGF-␤ and 3 (SiHa, CaSki and HeLa229) showed minimal response to the growth inhibitory effect of TGF-␤; the other cell lines (HeLa and HT-3) were sensitive. Northern blot analysis revealed that the RII mRNA was not expressed in 2 TGF-␤-resistant cell lines (ME-180 and C-33A) but was expressed in the other cell lines. Southern blot analysis of RI and RII revealed a homozygous deletion of the entire TGF-␤ RII gene in the cell line ME-180. We then asked whether the other TGF-␤-resistant or refractory cell lines had microsatellite instability and/or poly-adenine tract mutations of RII. We also checked for point mutations in the individual exons of the entire RII using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). Although C-33A exhibited poly-adenine microsatellite instability, its RII gene showed no signs of mutation. The molecular integrity of the TGF-␤ receptors in all cell lines, except ME-180 and C-33A, could be confirmed by examining the distinct transcriptional induction of plasminogen activator inhibitor-1 (PAI-1), p21 WAF1/CIP1 and, in some cases, the accompanying downregulation of c-myc in response to TGF-␤. Our observations, taken together, indicate that inactivation of the RII contributes to the resistance to TGF-␤ of some cervical carcinoma cell lines. Loss of or attenuated sensitivity to TGF-␤ growth inhibition in other cells may be attributed to the disruption of distal components in the TGF-␤ signal pathway, but not to the receptor system. Int.