43210 Transforming growth factor- β (TGF- β ) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF- β are mediated by the TGF- β receptor complex, a multimer composed of TGF- β receptor type I ( T β R-I ) and TGF- β receptor type II ( T β R-II ) subunits. Evidence suggests that loss of expression of T β R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF- β -mediated growth inhibition. A panel of human breast cell PCR designed according to GenBank accession numbers: L11695 ( T β R-I ), L07594 ( T R-II and M26434 ( HPRT annealing temperature ( T and range of amplification empirically bromide

Transforming growth factor-beta receptor (TbetaR)-dependent signals are critical for cell growth and differentiation and are often disrupted during tumorigenesis. The entire coding region of TbetaR-I and flanking intron sequences from 30 head and neck carcinomas were examined for alterations using "Cold" SSCP and direct sequencing. No somatic point mutations were found in the TbetaR-I gene. In contrast, 14 polymorphic sequence changes were detected in TbetaR-I in 13 (43%) of the samples, including eight (27%) nucleotide alterations identified as polymorphisms in an exon-1 (GCG)(9) microsatellite repeat, a previously reported tumor susceptibility allele. A nine base pair deletion was found in 23% of the samples including five heterozygous and two homozygous deletions as well as single homozygous 12bp deletion. Additionally, six heterozygous polymorphisms in intronic sequences were determined, including one heterozygous C/A genotype at the +82 nucleotide position of the intron-5 intervening sequence (IVS), and five heterozygous G/A genotypes within intron-7 at the +24 nucleotide position. Exon-1 polymorphisms in the (GCG)(9) microsatellite region of the TbetaR-I gene and their association with head/neck cancers, suggest that development of these cancers may be a direct consequence of loss of responsiveness to TGF-beta mediated growth inhibition.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines.

In the present study, we evaluated a series of sporadic ovarian carcinomas for mutations within the entire coding region of TbetaR-II. Using reverse transcription-PCR and "Cold" single-strand conformational polymorphism analysis, 6 of 24 samples (25%) were found to contain code-altering mutations in TbetaR-II: (a) four mutations resulting in amino acid substitutions in the highly conserved serine/threonine kinase domain; (b) one mutation resulting in a conservative amino acid change in the transmembrane domain; and (c) a 1-bp insertion in the polyadenylic acid microsatellite region resulting in a reading frameshift. In addition, six cases (25%) exhibited a common bp substitution (C-->T at nucleotide 1322) in both tumor and patient-matched normal tissues. This is the first report of such TbetaR-II mutations in primary human ovarian carcinomas. Immunohistochemical analysis demonstrated a loss of expression of TbetaR-II in 5 of 22 available tumors (23%; 4 of which also had mutations in the coding region) and decreased expression of TbetaR-II in 10 of 22 available tumors (44%; 1 of which had a mutation in the coding region). Thus, the loss or decreased expression of TbetaR-II seems to be a common event in sporadic ovarian carcinomas, and mutational inactivation, due to either frameshift mutations in the polyadenylic acid microsatellite region or point mutations in conserved functional domains, is one mechanism by which this occurs.