scholarly journals Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells.

1987 ◽  
Vol 7 (10) ◽  
pp. 3418-3427 ◽  
Author(s):  
L E Gentry ◽  
N R Webb ◽  
G J Lim ◽  
A M Brunner ◽  
J E Ranchalis ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cho Cells ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Expression Plasmid ◽  
Site Specific ◽  
Recombinant Type ◽  
Growth Factor Beta

Recombinant type 1 transforming growth factor beta (TGF-beta) 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-beta 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-beta 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 TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) 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-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells

1987 ◽  
Vol 7 (10) ◽  
pp. 3418-3427
Author(s):  
L E Gentry ◽  
N R Webb ◽  
G J Lim ◽  
A M Brunner ◽  
J E Ranchalis ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cho Cells ◽  
Transforming Growth Factor ◽  
Tgf Beta ◽  
Expression Plasmid ◽  
Site Specific ◽  
Recombinant Type ◽  
Growth Factor Beta

Recombinant type 1 transforming growth factor beta (TGF-beta) 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-beta 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-beta 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 TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) 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-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

scholarly journals Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

1988 ◽  
Vol 8 (10) ◽  
pp. 4162-4168 ◽  
Author(s):  
L E Gentry ◽  
M N Lioubin ◽  
A F Purchio ◽  
H Marquardt
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cho Cells ◽  
Transforming Growth Factor ◽  
Proteolytic Processing ◽  
Tgf Beta ◽  
Recombinant Type ◽  
Growth Factor Beta

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (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-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 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-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu 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-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide

1988 ◽  
Vol 8 (10) ◽  
pp. 4162-4168
Author(s):  
L E Gentry ◽  
M N Lioubin ◽  
A F Purchio ◽  
H Marquardt
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Cho Cells ◽  
Transforming Growth Factor ◽  
Proteolytic Processing ◽  
Tgf Beta ◽  
Recombinant Type ◽  
Growth Factor Beta

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (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-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 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-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu 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-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.

scholarly journals Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated.

1988 ◽  
Vol 8 (5) ◽  
pp. 2229-2232 ◽  
Author(s):  
A M Brunner ◽  
L E Gentry ◽  
J A Cooper ◽  
A F Purchio
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Chinese Hamster Ovary ◽  
Transforming Growth Factor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tgf Beta ◽  
Ovary Cells ◽  
Growth Factor Beta

Analyses of cDNA clones coding for simian type 1 transforming growth factor beta (TGF-beta 1) suggest that there are three potential sites for N-linked glycosylation located in the amino terminus of the precursor region. Analysis of [3H]glucosamine-labeled serum-free supernatants from a line of Chinese hamster ovary cells which secrete high levels of recombinant TGF-beta 1 indicate that the TGF-beta 1 precursor, but not the mature form, is glycosylated. Digestion with neuraminidase resulted in a shift in migration of the two TGF-beta 1 precursor bands, which suggests that they contain sialic acid residues. Endoglycosidase H had no noticeable effect. Treatment with N-glycanase produced two faster-migrating sharp bands, the largest of which had a molecular weight of 39 kilodaltons. TGF-beta 1-specific transcripts produced by SP6 polymerase programmed the synthesis of a 42-kilodalton polypeptide which, we suggest, is the unmodified protein backbone of the precursor. Labeling with 32Pi showed that the TGF-beta 1 precursor was phosphorylated in the amino portion of the molecule.

scholarly journals Platelets stimulate endothelial cells to synthesize type 1 plasminogen activator inhibitor. Evaluation of the role of transforming growth factor beta

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1013-1019 ◽  
Author(s):  
SR Slivka ◽  
DJ Loskutoff
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Plasminogen Activator Inhibitor ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Activator Inhibitor ◽  
Pai 1

Abstract A model system consisting of thrombin-stimulated bovine platelet releasates (PRthr) and bovine aortic endothelial cells (BAEs) was developed to determine if the interaction between platelets and endothelial cells regulates fibrinolysis. Zymographic analysis indicated that PRthr treatment of BAEs decreases urokinase and increases type 1 plasminogen activator inhibitor (PAI-1) activity. Although PRthr did not affect the overall rate of BAE protein synthesis, it increased PAI-1 biosynthesis within 6 hours. This increase was complete by 12 hours, with maximum stimulation at 10 to 15 micrograms/mL PRthr (1 microgram approximately 10(7) platelets). Neutralizing antibodies to transforming growth factor beta (TGF beta) reduced this effect by 75%. Treatments that activate latent TGF beta (eg, acidification or plasmin) increased this effect approximately fivefold, suggesting that TGF beta in PRthr exists in both a latent (approximately 80%) and an active (approximately 20%) form. In contrast to PRthr, adenosine diphosphate-prepared platelet releasates did not increase PAI-1 synthesis before acidification, indicating that they contain only the latent form of TGF beta. These results suggest that platelets can modulate the fibrinolytic system of the endothelium through the release of TGF beta, and that the mechanism by which the platelets are activated can influence the relative amount of active TGF beta.

Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated

1988 ◽  
Vol 8 (5) ◽  
pp. 2229-2232
Author(s):  
A M Brunner ◽  
L E Gentry ◽  
J A Cooper ◽  
A F Purchio
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Chinese Hamster Ovary ◽  
Transforming Growth Factor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tgf Beta ◽  
Ovary Cells ◽  
Growth Factor Beta

Analyses of cDNA clones coding for simian type 1 transforming growth factor beta (TGF-beta 1) suggest that there are three potential sites for N-linked glycosylation located in the amino terminus of the precursor region. Analysis of [3H]glucosamine-labeled serum-free supernatants from a line of Chinese hamster ovary cells which secrete high levels of recombinant TGF-beta 1 indicate that the TGF-beta 1 precursor, but not the mature form, is glycosylated. Digestion with neuraminidase resulted in a shift in migration of the two TGF-beta 1 precursor bands, which suggests that they contain sialic acid residues. Endoglycosidase H had no noticeable effect. Treatment with N-glycanase produced two faster-migrating sharp bands, the largest of which had a molecular weight of 39 kilodaltons. TGF-beta 1-specific transcripts produced by SP6 polymerase programmed the synthesis of a 42-kilodalton polypeptide which, we suggest, is the unmodified protein backbone of the precursor. Labeling with 32Pi showed that the TGF-beta 1 precursor was phosphorylated in the amino portion of the molecule.

scholarly journals Platelets stimulate endothelial cells to synthesize type 1 plasminogen activator inhibitor. Evaluation of the role of transforming growth factor beta

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1013-1019 ◽  
Author(s):  
SR Slivka ◽  
DJ Loskutoff
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Plasminogen Activator Inhibitor ◽  
Tgf Beta ◽  
Growth Factor Beta ◽  
Activator Inhibitor ◽  
Pai 1

A model system consisting of thrombin-stimulated bovine platelet releasates (PRthr) and bovine aortic endothelial cells (BAEs) was developed to determine if the interaction between platelets and endothelial cells regulates fibrinolysis. Zymographic analysis indicated that PRthr treatment of BAEs decreases urokinase and increases type 1 plasminogen activator inhibitor (PAI-1) activity. Although PRthr did not affect the overall rate of BAE protein synthesis, it increased PAI-1 biosynthesis within 6 hours. This increase was complete by 12 hours, with maximum stimulation at 10 to 15 micrograms/mL PRthr (1 microgram approximately 10(7) platelets). Neutralizing antibodies to transforming growth factor beta (TGF beta) reduced this effect by 75%. Treatments that activate latent TGF beta (eg, acidification or plasmin) increased this effect approximately fivefold, suggesting that TGF beta in PRthr exists in both a latent (approximately 80%) and an active (approximately 20%) form. In contrast to PRthr, adenosine diphosphate-prepared platelet releasates did not increase PAI-1 synthesis before acidification, indicating that they contain only the latent form of TGF beta. These results suggest that platelets can modulate the fibrinolytic system of the endothelium through the release of TGF beta, and that the mechanism by which the platelets are activated can influence the relative amount of active TGF beta.

scholarly journals Effects of IGF-I and -II, IGF binding protein-3 (IGFBP-3), and transforming growth factor-beta (TGF-beta) on growth and apoptosis of human osteosarcoma Saos-2/B-10 cells: lack of IGF-independent IGFBP-3 effects

2001 ◽  
pp. 213-221 ◽  
Author(s):  
C Schmid ◽  
C Ghirlanda-Keller ◽  
J Zapf
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Thymidine Incorporation ◽  
Tgf Beta ◽  
Igf I ◽  
Growth Factor Beta ◽  
Igfbp 3

OBJECTIVE: Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits cell growth. Previous reports have suggested the existence of plasma membrane IGFBP-3 receptors that could mediate direct, IGF-independent effects. Thus far, however, the only well-defined putative IGFBP-3 receptor is the type V transforming growth factor-beta (TGF-beta) receptor, a membrane glycoprotein that mediates TGF-beta-induced growth inhibition in selected cells. The aim of the study was to test whether IGFBP-3 and TGF-beta exert short-term effects in an osteosarcoma cell line that produces no IGF but contains type 1 IGF receptors. DESIGN: DNA synthesis and apoptosis in Saos-2/B-10 cells were measured in response to IGF-I, IGF-II, IGFBP-3 and TGF-beta2, and to type 1 IGF receptor ligands with poor affinity for IGFBP-3 ([QAYL]-IGF-I and insulin). RESULTS: IGF-I and IGF-II stimulated thymidine incorporation into DNA and suppressed apoptosis in a dose-dependent manner with maximal effects at 1 and 3 nM respectively. TGF-beta2 slightly increased thymidine incorporation into DNA but had no effect on apoptosis. IGFBP-3 had no effect by itself. Whereas it blocked the above effects of 1 nmol/l IGF-I, it did not block those of 1 nmol/l [QAYL]-IGF-I or 100 nmol/l insulin. CONCLUSIONS: IGFBP-3 does not affect DNA synthesis or apoptosis in an IGF-independent manner in IGF-responsive osteosarcoma cells. It therefore appears to act essentially by sequestration of IGF.

scholarly journals Discovery of New Inhibitors of Transforming Growth Factor-Beta Type 1 Receptor by Utilizing Docking and Structure-Activity Relationship Analysis

2019 ◽  
Vol 20 (17) ◽  
pp. 4090 ◽  
Author(s):  
Jiang ◽  
Deng
Keyword(s):  
Molecular Docking ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Binding Modes ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Growth Factor Beta ◽  
New Compounds

The transforming growth factor-beta (TGF-β) plays an important role in pathological fibrosis and cancer transformation. Therefore, the inhibition of the TGF-β signaling pathway has therapeutic potential in the treatment of cancer. In this study, the binding modes between 47 molecules with a pyrrolotriazine-like backbone structure and transforming growth factor-beta type 1 receptor (TβR1) were simulated by molecular docking using Discovery Studio software, and their structure–activity relationships were analyzed. On the basis of the analysis of the binding modes of ligands in the active site and the structure–activity relationships, 29,254 new compounds were designed for virtual screening. According to the aforementioned analyses and Lipinski’s rule of five, five new compounds (CQMU1901–1905) with potential activity were screened through molecular docking. Among them, CQMU1905 is an attractive molecule composed of 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), and 5-azacytosine. Interestingly, 5-FU, 6-MP, and 5-azacytidine are often used as anti-metabolic agents in cancer treatment. Compared with existing compounds, CQMU1901–1905 can interact with target proteins more effectively and have good potential for modification, making them worthy of further study.

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