Abstract. Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF/31 cDNA contains high levels of latent TGF/31. The amino-terminal region of the TGF/~I precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical crosslinking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS 3) followed by immunoblot analyses indicates that latent recombinant TGF/31 contains both the cleaved amino-terminal glycopeptide and mature TGF/31 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF/3 binding protein(s) in latent recombinant TGF/31. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF/3 competing activity. In addition, immunoblot analysis of plasmintreated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF/31 is released. Thus, activation of latent TGFB1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF/~I. Acid activation of latent TGF/~, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

Proteolytic processing of the transforming growth factor beta precursor (pro-TGF beta) is an essential step in the formation of the biologically active TGF beta homodimeric protein (TGF beta). The 361-amino-acid precursor pro-TGF beta 1 has within its primary structure the R-H-R-R processing signal found in many constitutively secreted precursor proteins and potentially recognized by members of the mammalian convertase family of endoproteases. To determine whether cleavage of pro-TGF beta 1 can be achieved by the furin convertase in vitro, purified precursor was incubated in the presence of a truncated/secreted form of the enzyme. Immunoblots showed that the 55-kDa pro-TGF beta 1 was converted into the 44 and 12.5 kDa bands corresponding to the pro-region and the mature monomer, respectively. Treatment of pro-TGF beta 1 with furin resulted in a 5-fold increase in the production of biologically active TGF beta 1. Furthermore, when expressed in the furin-deficient LoVo cells, no processing of pro-TGF beta 1 was observed. In contrast, efficient processing was observed when pro-TGF beta was coexpressed with the furin convertase. Collectively, these results provide evidence that in our experimental systems the TGF beta 1 precursor is efficiently and correctly processed by human furin thus permitting release of the biologically active peptide.

Escape from negative growth regulation by transforming growth factor β (TGF-β) as a result of the loss of TGF-β type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-β 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-β 1 -neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-β autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Recently, the simian type 1 transforming growth factor beta (TGF-I1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene ampliffication (L. E. Gentry, N. R. Webb, G. J. Lim, A. M. Brunner, J. E. Ranchalis, D. R. Twardzik, M. N. Lioubin, H. Marquardt, and A. F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-I1, yielding pro-TGF-j11 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-II1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-,1 had the same specific biological activity as natural TGF-111. The concentration of TGF-j1 required for half-maximal inhibition of MvlLu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-I1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-01 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-j1l should prove useful for further structural and functional studies.Type 1 transforming growth factor beta (TGF-B1) belongs to a closely related family of polypeptides with potent cellular modulating activities (for reviews, see references 12 and 19). This growth factor molecule appears to be intimately associated with cell growth and differentiation and may play pivotal roles in the autocrine or paracrine regulation of these processes. Although numerous studies have addressed important cellular and physiological properties of TGF-pl, very little information is available concerning structural features and posttranslational processing events necessary for its expression and function.The molecular cloning of TGF-p1 cDNA from several species (4, 5, 18) and analysis of the predicted protein sequences has provided some insight into the structure and processing of this polypeptide growth factor. Sequence analysis has predicted that the mature 112-amino-acid chain of TGF-pl is derived from the C terminus of a 390-aminoacid pre-pro-TGF-,1 by proteolytic cleavage. Proteolytic cleavage is predicted to occur following a dibasic Arg-Arg sequence and immediately preceding the N-terminal Ala residue of the mature growth factor. Structural studies of mature TGF-13l have revealed that TGF-,1 is homodimeric and interlinked by disulfide bonds (1,6,16). The precursor for TGF-,1l contains a typical hyd...

Recombinant type 1 transforming growth factor beta (TGF-,) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-I and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-, nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants.The major proteins secreted by these cells consisted of latent as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-, precursor. Levels of recombinant TGF-0 protein secreted by these cells approached 30 ,ug/24 h per 107 cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-, which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-4. In addition to mature recombinant TGF-jA, site-specific antibodies demonstrated the existence of larger TGF-3 precursor polypeptides. The availability of biologically active recombinant type 1 TGF-I8 and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.Results of recent studies have suggested that transforming growth factor beta (TGF-P) consists of a family of closely related biologically active polypeptides with potent cellularmodulating activities. Three molecular forms of TGF-P (types 1, 2 and 1.2) have been identified and characterized.Type 1 TGF-P was the first growth factor isolated and, as a result, the best characterized (for reviews, see references 23 and 47). sources have been obtained. The predicted protein sequences derived from the cDNAs reveal a remarkable homology. The mature forms of the human and simian growth factor differ from the murine factor by one amino acid. Analysis of the various TGF-,B cDNA clones suggests that the mature 112 amino acid chain of type 1 TGF-P is derived from a much larger precursor polypeptide by proteolytic cleavage. The full-length precursor of TGF-,1 shows a high degree of structural conservation and constitutes 391 amino acids for human and 390 amino acids for both murine and simian precursors. A typical leader sequence is observed in the precursor, as well as three potential N-linked glycosylation sites. A dibasic sequence immediately precedes the amino-terminal alanine residue of the mature growth factor, indicating the involvement of a dibasic protease in processing of the growth factor.To understand in more detail the structure and processing of type 1 TGF-P, as well as to provide a source for large...