Secreted ovarian stromal substance inhibits ovarian epithelial cell proliferation.

OBJECTIVE: Determine the effects of factors secreted by normal human ovarian stroma on the proliferation of benign and malignant ovarian epithelia, in vitro. METHODS: Primary cultures of normal human ovarian surface epithelium (HOSE), human ovarian stromal tissue (HOST), and epithelial ovarian carcinomas (CSOC) were established from surgical specimens and characterized immunohistochemically using anti-cytokeratin, vimentin, and Factor VIII antibodies. Stroma-conditioned media (SCM) were collected over 3 days from confluent HOST cultures. The SCM were dialyzed, lyophilized, resuspended, and added to HOSE, CSOC, SKOV-3, and Caov-3 ovarian cancer cell cultures and growth inhibitory effects were assayed by MTS and [3H]thymidine uptake. RESULTS: SCM inhibited the growth and DNA synthesis of normal HOSE cells and cancer cells by 79-99% in > 10-cell lines studied to date. The inhibitory effect was rapid in onset with 31-82% reduction in DNA synthesis at 1 hr and approximately 50% return of activity by 23 hr following a 1-hr SCM pulse treatment. The SCM inhibitory activity was not abolished by boiling or by absorption with heparin-agarose. Size exclusion filtration places the molecular weight of the inhibitory substance between 1 and 3 kDa. Neither trypsin nor proteinase K treatments altered the inhibitory activity of SCM, while a Bligh-Dyer organic extraction placed the activity in the aqueous phase. CONCLUSION: A heat-stable, non-heparin-binding, low-molecular-weight, water-soluble substance secreted by normal ovarian stroma significantly inhibits HOSE and ovarian cancer cell proliferation. Derangements in normal ovarian stroma-epithelial interactions may contribute to growth dysregulation of the surface epithelia and result in ovarian carcinogenesis.

Cloning and characterization of the human insulin-like growth factor-I receptor gene 5'-flanking region.

The insulin-like growth factor-I receptor (IGFIR) is a membrane-bound glycoprotein that mediates the action of insulin-like growth factors. The cDNAs for the human IGFIR have been cloned and expressed, but the structures of the gene and its promoter have not been elucidated. In this study, we isolated an IGFIR promoter clone from a human chromosome 15 library. This clone contained the promoter, first exon, and a portion of the first intron. Sequence analysis of the 5' region that contained the promoter revealed that it lacked both TATA and CAAT boxes. The promoter contained binding sites for the transcription factors Sp1, AP-2, and the epidermal growth factor receptor transcription factor (ETF). Primer extension analysis of IGFIR mRNA indicated the presence of a single transcription start site 1,012 bp upstream from the ATG. When the putative promoter was ligated into a promoterless CAT vector and transfected mto HEPG2 cells, CAT activity was expressed, indicating that promoter activity was contained in this fragment. Other constructs containing the promoter and portions of the 5' untranslated region were used in transfection studies, and indicated that the 5' untranslated regions may play a role in promoter activity. Comparison of the human IGFIR promoter with that of the rat IGFIR promoter revealed significant sequence homology. Comparison of the IGFIR promoter with that of the human insulin receptor (IR) revealed structural similarities, although the arrangement of promoter elements differed.

Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.

Regulating insulin-receptor-gene expression by differentiation and hormones.

Insulin regulates cell function by first binding to the insulin receptor (IR) localized on the cell surface. With the cloning of IR cDNA and the IR-gene promoter, the regulation of the IR gene during differentiation and by various hormones can be studied. Muscle is a major target tissue for insulin action. BC3H1 cells, a mouse muscle cell line in culture, are a model cell type for studying insulin action. Differentiation in these cells results in a 5- to 10-fold increase in IR binding and a 5- to 10-fold increase in IR content. Studies of IR mRNA by Northern and slot-blot analyses reveal a 10-fold increase in IR mRNA after differentiation. These studies indicate that there is a selective increase in IR-gene expression during muscle differentiation. A similar increase in IR-gene expression is observed for the IR during pancreatic acinar cell differentiation. Glucocorticoids increase IR content in several target tissues. Studies in cultured IM-9 lymphocytes indicate that glucocorticoids induce a 5-fold increase in IR mRNA levels. Studies of IR mRNA half-life indicate that glucocorticoids do not alter IR mRNA stability. When the transcription of the IR is measured by elongation assays, glucocorticoids directly stimulate IR transcription 5- to 10-fold. The effect is detectable within 30 min of glucocorticoid treatment and is maximal within 2 h. Therefore, these studies demonstrate that the IR gene is under the direct regulation of glucocorticoids. Insulin downregulates the IR in various target tissues. Prior studies indicate that this downregulation was partly because of accelerated IR degradation. Studying AR42J pancreatic acinar cells, we also found that insulin accelerates IR degradation. Moreover, in these cells, insulin decreases IR biosynthesis by approximately 50%. Studies of IR mRNA indicate there is a concomitant decrease in IR mRNA levels after insulin treatment. Thus, insulin decreases IR-gene expression. The genomic structure of the IR promoter has been elucidated. Primer extension and nuclease S1 analysis indicate that IR mRNA has multiple start sites. The promoter fragment was ligated to a promoterless "reporter" plasmid containing the bacterial gene chloramphenicol acetyltransferase (CAT). When this plasmid is transfected into cultured cells, CAT activity is detected, indicating promoter activity. Various portions of a genomic fragment were ligated to a promoter to study glucocorticoid regulation of the IR promoter. These studies indicate that IR-gene expression is regulated by differentiation and hormonal agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase.

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.

Sequence and analysis of promoter region of human insulin-receptor gene.

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.

Expression of human salivary protein genes.

Human proline-rich proteins (PRPs) are polymorphic, homologous in sequence, and linked in a cluster called the human salivary protein complex (SPC). Recently this complex was localized to human chromosome band 12p13.2 (Mamula et al., Cytogenet. Cell Genet. 39:279, 1985). We have isolated a PRP cDNA, EO27, from a human parotid gland library, identified it by DNA sequencing, and used it to study the molecular and cellular biology of PRP production. Cell-free translation and mRNA characterization with EO27 indicate that the numerous PRPs seen in saliva are produced from relatively few, large precursors, probably by posttranslational cleavage. This supports an hypothesis originally proposed by Friedman and Karn in 1977 (Am. J. Hum. Genet. 29:44 A; Biochem. Genet. 15:549) and later supported by biochemical studies (Karn et al., Biochem Genet. 17:1061, 1979) and molecular studies (Mamula et al., Fed. Proc. 43:1522, 1984; Maeda et al., J. Biol. Chem. 260:1123, 1985). EO27 was also used in this study to localize PRP mRNA production to the acinar cells of the parotid gland by in situ hybridization.

Localization of the human salivary protein complex (SPC) to chromosome band 12p13.2.

In situ hybridization of a 3H-labeled probe containing a fragment from PRP-1, a genomic clone with human salivary proline-rich protein gene sequences, revealed significant labeling on the short arm of human chromosome 12 in metaphase preparations from two individuals. Fifty-three percent of metaphases exhibited labeling on one or both chromosomes 12. Additional cells scored at the 850-1,000 band level revealed a significant proportion (52% [32/61] grains, p less than 0.005) of the labeled sites on chromosome 12 to be on band 12p13.2. This probe for a human salivary proline-rich protein gene fragment, probably PMS, is from a cluster of 13 linked genes designated as the human salivary protein complex (SPC). Studies of the DNA of human-mouse somatic-cell hybrids have assigned the SPC to chromosome 12, but have not provided a regional localization (Azen et al, 1985). This paper reports the localization of the SPC to a specific chromosomal band, 12p13.2.