Molecular events in cells transformed by Rous Sarcoma virus.

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src-protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.

Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin.

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF beta 1 cDNA contains high levels of latent TGF beta 1. The amino-terminal region of the TGF beta 1 precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical cross-linking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS3) followed by immunoblot analyses indicates that latent recombinant TGF beta 1 contains both the cleaved amino-terminal glycopeptide and mature TGF beta 1 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF beta binding protein(s) in latent recombinant TGF beta 1. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF beta competing activity. In addition, immunoblot analysis of plasmin-treated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF beta 1 is released. Thus, activation of latent TGF beta 1 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 beta 1. Acid activation of latent TGF beta, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide.

Site-specific increased phosphorylation of pp60v-src after treatment of RSV-transformed cells with a tumor promoter.

When vole cells that had been transformed by Rous sarcoma virus were treated with the tumor-promoting phorbol ester 12-O-tetradecanoyl-13-acetate (TPA), specific phosphorylation of pp60v-src was increased. Partial V8 protease mapping indicated that the increased phosphorylation occurred exclusively on serine residues located in the amino terminus of the molecule. Treatment of cells with dimethyl sulfoxide or 4 alpha-phorbol-12,13-didecanoate did not elicit this response. Two-dimensional tryptic phosphopeptide mapping of pp60v-src immunoprecipitated from untreated and TPA-treated cells indicated that a specific tryptic amino-terminal peptide was hyperphosphorylated.

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

pp60v-src, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp60v-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp60v-src-associated kinase activity are discussed.

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Novel serine phosphorylation of pp60c-src in intact cells after tumor promoter treatment.

Treatment of normal cells with the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and mezerein results in increased phosphorylation of pp60c-src. Two-dimensional tryptic phosphopeptide analysis of partial V8 protease fragments indicated that this phosphorylation takes place on a serine residue which lies within the amino-terminal 18 kilodaltons of pp60c-src and represents the major phosphorylation site following tumor promoter treatment. Untreated cells exhibited a low but detectable level of phosphorylation at this serine residue. The significance of these results with respect to the phosphoregulation of pp60c-src as well as tumor promotion is discussed.

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

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.

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

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.

The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha.

Degenerate oligonucleotide probes corresponding to a highly conserved region common to epidermal growth factor, transforming growth factor alpha, and vaccinia growth factor were used to identify a novel growth factor gene in the Shope fibroma virus genome. Sequence analysis indicates that the Shope fibroma growth factor is a distinct new member of this family of growth factors.

Stimulation of T cells through the CD3/T-cell receptor complex: role of cytoplasmic calcium, protein kinase C translocation, and phosphorylation of pp60c-src in the activation pathway.

Stimulation of T cells or the Jurkat T-cell line with soluble antibodies to the CD3/T-cell receptor complex causes mobilization of cytoplasmic Ca2+, which is blocked by pertussis toxin but not by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and translocation of protein kinase C activity from the cytoplasm to the membrane. Such stimulation also causes phosphorylation of pp60c-src at an amino-terminal serine residue. These activities are consistent with induction of phosphatidylinositol metabolism after antibody binding. Anti-CD3 stimulation with antibody in solution, however, does not cause Jurkat cells to release interleukin 2 and blocks rather than induces proliferation of T cells. Induction of interleukin 2 production by Jurkat cells and proliferation by normal T cells requires anti-CD3 stimulation with antibody on a solid support, such as Sepharose beads or a plastic dish. Thus, we examined phosphorylation of pp60c-src after stimulation of Jurkat cells with anti-CD3 in solution or on solid phase. Both of these caused serine phosphorylation of pp60c-src that was indistinguishable even after 4 h of stimulation. These results indicate that the mode of anti-CD3 stimulation (in solution or on solid phase) controls a cellular function that modifies the consequences of signal transduction through phosphatidylinositol turnover.

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

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.

Differential effects of transforming growth factor beta 1 on the growth of poorly and highly metastatic murine melanoma cells.

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth of paired murine melanoma cell clones that differ with respect to their experimental metastatic potential. Neither poorly (clone 16) nor highly (clone M2) metastatic cells were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium in the absence of serum. However, both clones were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium containing 10% calf serum. Colony formation in the presence of 10% calf serum was enhanced in a dose-dependent manner by TGF-beta 1 (half-maximal dose, 0.1 ng/ml) and was 5- to 10-fold greater than colony formation in the presence of 10% calf serum alone. Under anchorage-dependent (monolayer) conditions, neither clone grew in the absence of serum or in medium containing less than 1% calf serum. The monolayer growth of poorly metastatic cells (clone 16) was enhanced in a dose-dependent manner by TGF-beta 1 in medium supplemented with calf serum. Growth was 3.5-fold and 2.3-fold greater than untreated controls after 5 days in submitogenic (0.5%) and mitogenic (10%) concentrations of calf serum, respectively. In contrast, TGF-beta 1 had no effect on the monolayer growth of highly metastatic cells (clone M2) either in submitogenic (0.5%) or mitogenic (10%) concentrations of serum. TGF-beta 1 did not directly stimulate DNA synthesis by either poorly or highly metastatic cells when measured 24 h after TGF-beta 1 treatment. The ability of TGF-beta 1 to stimulate the anchorage-independent growth of metastatic melanoma cells suggests that this potent growth factor may play a role in the growth of these cells in vivo. In addition, the differential sensitivity of poorly and highly metastatic cells to TGF-beta 1 may be relevant to their metastatic potential in vivo. While the mechanism(s) by which TGF-beta 1 stimulates the growth of these cells remains unknown, these differentially metastatic clones of the K-1735 murine melanoma should provide a useful model in which to study the effects of transforming growth factor beta on the metastatic phenotype.

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion.

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.

Site-directed mutagenesis of glycosylation sites in the transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 (414) precursors and of cysteine residues within mature TGF beta 1: effects on secretion and bioactivity.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.

Beta IG-H3, a novel secretory protein inducible by transforming growth factor-beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro.

We have previously identified a gene, beta ig-h3, which is highly induced in A549 cells (human lung adenocarcinoma) after growth arrest by transforming growth factor-beta. The beta ig-h3 gene encodes a 683-amino-acid secretory protein termed beta IG-H3, and treatment of several cell lines with transforming growth factor-beta results in increased secretion of beta IG-H3 into cell culture supernatants. In this report, we further characterize beta IG-H3 with respect to its synthesis and function. Primary human foreskin fibroblasts grown in monolayer culture produced beta IG-H3 mRNA and secreted beta IG-H3 protein into the growth media. Treatment of these cells with transforming growth factor-beta led to an increase in beta IG-H3 mRNA and protein. Cells grown on three-dimensional scaffolds secreted beta IG-H3 into the extracellular matrix, as judged by immunostaining with anti-beta IG-H3 antibodies. beta IG-H3 was also detected in normal human skin, especially in the papillary dermis. Finally, we show that recombinant beta IG-H3 supported attachment and spreading of dermal fibroblasts, suggesting that beta IG-H3 may function as an extracellular attachment protein in skin.

Characterization of latent transforming growth factor-beta 2 from monkey kidney cells.

Serum-free medium conditioned by BSC-40 cells was analyzed for the presence of transforming growth factor-beta 2 (TGF beta 2)-related proteins. Western blot analysis was performed using site-specific antipeptide antibodies directed against the pro- and mature regions of the TGF beta 2 precursor. When conditioned medium was analyzed by polyacrylamide gel electrophoresis under reducing conditions, proteins with mol wt of 53 kDa (containing both mature and proregion sequences), 34-38 kDa (containing proregion sequences only), and 12 kDa (containing mature sequences) were detected. Under nonreducing conditions, complexes of 60- to 80-kDa, 160- to 200-kDa, as well as 24-kDa mature dimers were seen. Cleavage of mature TGF beta 2 from its precursor was inhibited by monensin and chloroquin, but not by ammonium chloride or methylamine. Two peaks of bioactivity were detected after fractionation on a TSK column corresponding to mol wt of 130 and 400 kDa. These peaks contained TGF beta 2 and pro-TGF beta 2 proteins. Partial purification of the 130-kDa complex followed by N-glyconase digestion indicated that the pro-TGF beta 2 proteins were glycosylated. These data demonstrate that BSC-40 cells secrete mature TGF beta 2 complexed with proregion-containing proteins and suggest that this association may contribute to the latency phenomena observed with respect to this growth regulator.

Ovarian transforming growth factor-alpha gene expression: immunohistochemical localization to the theca-interstitial cells.

Rat ovaries were examined for the presence of transforming growth factor-alpha (TGF-alpha). Immature diethylstilbestrol-primed rats were treated for 3 days prior to sacrifice with or without FSH. Their ovaries were either fixed in formaldehyde for immunohistochemistry or the RNA was extracted for northern blot analysis. Hybridization with a rat TGF-alpha cDNA probe revealed a 4.5 kilobase mRNA whose abundance was markedly stimulated in the rats treated with FSH. Immunohistochemical staining, using a sequence specific monoclonal antibody to TGF-alpha, MF9, detected immunoreactive TGF-alpha only in the interstitial and theca cells. FSH treatment resulted in no appreciable change in the immunostaining. These results suggest that TGF-alpha is synthesized in the ovary, perhaps in the theca-interstitial compartment.

Transforming growth factor-beta 1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action.

It is the aim of this study to establish ovarian transforming growth factor-beta 1 (TGF beta 1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF beta 1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF beta 1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 microgram/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements, TGF beta 1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF beta 1 to be a highly potent inhibitor (greater than 80%) of hCG hormonal action. This reversible TGF beta 1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF beta 1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF beta 1-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17 alpha-hydroxylation step, suggesting TGF beta 1-mediated blockade at the level of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF beta 1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF beta 1 action, as manifested by its diametrically opposed effects in different ovarian compartments.

Cloning the argF gene from Escherichia coli K-12 with simian virus 40.

We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus. In the first case, the E. coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells. The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells. In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences. One was a 3325 base pair segment from a HaeII + BamHI digest. The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper. These hybrid DNAs were replicated and packaged into virions. Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed.