Identification of amino acid residues in bone morphogenetic protein-1 important for procollagen C-proteinase activity.

Bone morphogenetic protein (BMP)-1, which belongs to the tolloid subgroup of astacin-like zinc metalloproteinases, cleaves the C-propeptides of procollagen at the physiologic site and is, therefore, a procollagen C-proteinase (PCP). Cleavage occurs between a specific alanine or glycine residue (depending on the procollagen chain) and an invariant aspartic acid residue in each of the three chains of procollagen. To learn more about how BMP-1 exhibits PCP activity we mapped the primary structure of BMP-1 onto the x-ray crystal structure of astacin and identified residues in the metalloproteinase domain of BMP-1 for subsequent site-directed mutagenesis studies. Recombinant wild-type and mutant BMP-1 were expressed in COS-7 cells and assayed for PCP activity using type I procollagen as the substrate. We showed that substitution of alanine for Glu(94), which occurs in the HEXXH zinc-binding motif of BMP-1, abolishes PCP activity. Furthermore, mutation of residues Lys(87) and Lys(176), which are located in the S1' pocket of the enzyme and are therefore adjacent to the P1' residue in the substrate, reduced the proteolytic activity of BMP-1 by approximately 50%. A surprising observation was that mutation of Cys(66) reduced the activity to 20%, suggesting that this residue is crucial for activity. Further experiments showed that Cys(66) and Cys(63), which are located in the tolloid-specific sequence Cys(63)-Gly(64)-Cys(65)-Cys(66) in the active site, most likely form a disulfide bridge.

Transforming growth factor beta type I receptor kinase mutant associated with metastatic breast cancer.

Malignant breast carcinoma cell lines are frequently refractory to transforming growth factor beta (TGF-beta)-mediated cell cycle arrest. To identify molecular mechanisms of TGF-beta resistance, we have conducted a comprehensive structural analysis of the TGF-beta receptor types I (TbetaR-I) and II (TbetaR-II) genes in primary human breast carcinomas and associated axillary lymph node metastases. No evidence for loss of expression (n=14) or structural alterations of the TbetaR-II gene (n=30) were identified. However, 2 of 31 primary carcinomas and 5 of 12 lymph node metastases carried a C to A transversion mutation resulting in a serine to tyrosine substitution at codon 387 (S387Y) of the TbetaR-I receptor gene. This TbetaR-I mutant has a diminished ability to mediate TGF-beta-dependent effects on gene expression as compared with wild-type TbetaR-I. S387Y is the first reported mutation in the TbetaR-I gene in human cancer that was primarily associated with lymph node metastases in the present series.

Functional characterization of transforming growth factor beta type II receptor mutants in human cancer.

We recently identified missense mutations at amino acid residues 526 and 537 located within the highly conserved subdomain XI of the transforming growth factor beta type II receptor (TbetaR-II) serine-threonine kinase in two human squamous carcinoma cell lines. These cell lines are resistant to transforming growth factor beta-mediated inhibition of growth. Moreover, treatment with transforming growth factor beta fails to increase the levels of type 1 plasminogen activator inhibitor and fibronectin synthesis. To test the effects of the mutations on receptor function, mutant TbetaR-II cDNAs were expressed in TbetaR-II-deficient T47D cells. Cyclin A promoter activity was reduced by 50% in cells expressing wild-type TbetaR-II but increased 2-fold in cells transfected with either of the two mutant receptors. Conversely, plasminogen activator inhibitor type 1 promoter activity was increased 6-fold in cells transfected with wild-type receptor but not with either of the two mutant receptors. Moreover, the activity of both mutant serine-threonine kinases was strongly reduced compared to that of the wild-type receptor. Thus, the amino acid residues at positions 526 and 537 seem to be essential for kinase function and signaling activity of the TbetaR-II.

Frequent inactivation of the transforming growth factor beta type II receptor in small-cell lung carcinoma cells.

Small-cell lung cancer (SCLC) has a significantly worse prognosis than other forms of bronchogenic carcinoma. Because transforming growth factor beta (TGF-beta) appears to play an important role in the pathogenesis of SCLC, we examined the status of the TGF-beta receptor system in a series of 11 human small-cell carcinoma cell lines. None of these cell lines expressed more than one-tenth the level of TGF-beta type II receptor (T beta R-II) gene mRNA produced by TGF-beta-sensitive normal epithelial cells. In addition, one of the cell lines expressed a second truncated T beta R-II transcript, which is predicted to encode a protein that lacks the terminal two-thirds of the serine-threonine kinase domain. No other structural alterations in the promoter or coding sequences of the T beta R-II gene were found in any of the cell lines, nor could the loss of T beta R-II mRNA expression be ascribed to de novo hypermethylation of promoter/enhancer sequences. These findings indicate that inactivation of the TGF-beta signaling pathway caused by the loss of T beta R-II gene expression is a common and, therefore, probably pathogenetically important feature of small-cell lung carcinoma.

Alterations of transforming growth factor-beta 1 receptor type II occur in ulcerative colitis-associated carcinomas, sporadic colorectal neoplasms, and esophageal carcinomas, but not in gastric neoplasms.

BACKGROUND & AIMS: Gastric cancers, sporadic colorectal cancers, and ulcerative colitis (UC)-associated colorectal carcinomas and dysplasias manifest microsatellite instability (MI); however, esophageal carcinomas rarely exhibit MI. Recently, a transforming growth factor-beta 1 type II receptor (TGF-beta 1RII) mutation in a coding microsatellite was described in primary colorectal carcinomas demonstrating MI. No previous studies of TGF-beta 1RII have addressed mechanisms of inactivation other than MI in human tumors; furthermore, MI-negative tumors have not been examined for TGF-beta 1RII mutation. We evaluated 138 primary human neoplasms for mutation in the poly-A microsatellite tract of TGF-beta 1RII. Additionally, a group of esophageal tumors was evaluated for the expression of TGF-beta 1RII messenger RNA (mRNA). METHODS: First, we determined whether MI was present at other chromosomal loci in these lesions. The poly-deoxyadenine (poly-A) microsatellite tract within the TGF-beta 1RII coding region was then PCR-amplified. In a group of MI-negative esophageal tumors, RT-PCR was performed to determine the expression of TGF-beta 1RII mRNA. RESULTS: Among 17 MI+ UC specimens, 3 (18%) demonstrated TGF-beta 1RII poly-A tract mutation (2 cancers and 1 dysplasia), while 2 (4%) of 44 MI-negative UC specimens (1 dysplasia and 1 tumor), and 13 (81%) of 16 MI+ sporadic colorectal cancers, contained TGF-beta 1RII poly-A mutation. No gastric or esophageal tumors contained TGF-beta 1RII mutation. Among 21 MI-negative esophageal carcinomas. 6 cases (28.5%) had TGF-beta 1RII transcripts that were low or undetectable by RT-PCR. CONCLUSIONS: Mutation within the poly-A microsatellite tract of TGF-beta 1RII occurs early in a subset of UC-neoplasms and commonly in sporadic colorectal cancers, but may be rare in MI+ gastric tumors. Diminished expression of TGF-beta 1RII mRNA in esophageal tumors suggests that mechanisms of inactivation in this gene other than MI play a role in esophageal carcinogenesis.

Loss of transforming growth factor-beta type II receptor gene expression in primary human esophageal cancer.

Cell lines derived from carcinomas of the upper aero-digestive tract are typically refractory to transforming growth factor beta-mediated cell cycle arrest. Recently, we reported that the type II transforming growth factor beta receptor (T beta R-II) gene can be inactivated on the basis of missense mutations in such cell lines. These findings prompted us to investigate the molecular status of the T beta R-II gene in primary tumor specimens. Among 21 of 24 evaluable primary esophageal carcinomas, there were 6 cases (28.5%; 95% confidence interval, 11% to 52%) in which T beta R-II transcripts were low or undetectable by a reverse transcription PCR assay. In one of these cases, we were able to ascribe the loss of T beta R-II gene expression to high-density methylation of promoter sequences. We failed to detect any mutations within the open reading frame of the remaining tumors that expressed T beta R-II mRNA. In this relatively small series of cases, loss of T beta R-II expression was independent of pathologic tumor stage, histologic subtype, or outcome of patients with esophageal cancer. Thus, loss of expression of the T beta R-II gene appears to be the predominant mechanism through which this gene is inactivated in esophageal cancer.

Optimisation of CCL64-based bioassay for TGF-beta.

Transforming growth factor beta (TGF-beta) is released by a variety of cells and known to be involved in many different processes including the immune response, wound healing and carcinogenesis. As most experimental investigations have been based on quantitative analysis of TGF-beta production using a bioassay, it seemed important to test the validity and limitations of this method. This paper analyses several parameters that may impair TGF-beta quantification by bioassay. Recommendations are made concerning the influence of technical parameters and the presence of other cytokines (EGF and bFGF) commonly released by cultured cells to which the Mv1Lu mink lung epithelial cell line (CCL64) is sensitive.

Missense mutations of the transforming growth factor beta type II receptor in human head and neck squamous carcinoma cells.

In this study, we report the occurrence of missense mutations of the transforming growth factor beta (TGF beta) type II receptor gene in two human squamous head and neck carcinoma cell lines. Both mutations are G:C-->C:G transversions, which result in the replacement of a glutamic acid by a glutamine, and of an arginine by a proline residue, respectively. Moreover, both are located at highly conserved sites within the serine-threonine kinase domain. One of the mutants appears to be defective in its autophosphorylation as well as in the transphosphorylation of the TGF beta type 1 receptor protein, whereas the second mutant appears to be constitutively activated. These are the first reported naturally occurring nucleotide substitution mutations in the T beta R-11 gene in human head and neck cancer cells, which may explain their resistance to TGF beta 1-mediated cell cycle arrest.