The estrogen-regulated 52K-cathepsin-D in breast cancer: From biology to clinical applications

We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer celllines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in λgt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness, In addition to other estrogen regulated growth factors, the precursor of lysosomal proteases has the potential to stimulate the proliferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.     

Estrogen-induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.     

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

Immunohistochemical distribution of the 52-kDa protein in mammary tumors: a marker associated with cell proliferation rather than with hormone responsiveness

We have previously described a secreted glycoprotein of mol. wt 52,000 (52-kDa protein) which is induced by estrogen in some human breast cancer cell lines. This protein has been identified as the proenzyme of a lysosomal cathepsin-D-like protease which is secreted in large proportions in breast cancer cells. To determine which information may be generated by this marker when detected in mammary tumors, in comparison with hormone receptors, we used monoclonal antibodies interacting specifically with the 52-kDa protein and its related cellular processed products (mols. wts 48 and 34 kDa). A high concentration of this protein has been shown in proliferative ductal mastopathies and cysts, suggesting its value in detecting high-risk mastopathies. We now present the immunoperoxidase distribution of this protein in breast carcinoma compared to the cytosolic hormone receptors assayed in parallel. In 232 breast cancers, no correlation was found between the cellular 52-kDa protein content and cytosolic estrogen or progesterone receptor concentrations. This absence of correlation was also shown by the constitutive production of this protein by estrogen-receptor-negative breast cancer cell lines and confirmed by double immunostaining of breast cancer cell aspirates showing a dissociation between the cytoplasmic staining of this 52-kDa lysosomal protease and the nuclear staining of the estrogen receptor. These clinical results, associated with the in vitro mitogenic and proteolytic activities of this protein, strongly suggest that the 52-kDa protein staining in tissue is associated with tumor proliferation and/or invasion, rather than with hormone responsiveness.     

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells

In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

p52 induction by cytochalasin D in rat kidney fibroblasts: homologies between p52 and plasminogen activator inhibitor type-1

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.     

Heterogeneity of the complex N-linked oligosaccharides at specific glycosylation sites of two secreted carrot glycoproteins

The N-linked glycans from the 52/54-kDa medium protein and cell wall beta-fructosidase, two glycoproteins secreted by carrot suspension culture cells, were characterized. Carrot cells were labelled with [3H]glucosamine or [3H]fucose. The 52/54-kDa medium protein was isolated from the culture medium and beta-fructosidase from cell walls. The purified proteins were digested with trypsin and glycopeptides were isolated and sequenced. Glycans obtained from individual glycopeptides were separated by gel filtration chromatography and characterized by concanavalin A chromatography, by treatments with exoglycosidases and by sugar composition analysis. The 52/54-kDa medium protein and cell wall beta-fructosidase have one high-mannose-type glycan similar to those from yeast and animal glycoproteins. In addition, the 52/54-kDa medium protein has three complex-type and cell wall beta-fructosidase two complex-type glycans per polypeptide. The complex-type glycans isolated from individual glycosylation sites are fairly large and very heterogeneous. The smallest of these glycans has the structure [Xyl](Man)3[Fuc](GlcNAc]2Asn (square brackets indicating branching) whereas the larger ones carry additional sugars like terminal N-acetylglucosamine and possibly rhamnose and arabinose in the case of the 52/54-kDa medium protein and only arabinose in the case of cell wall beta-fructosidase. These terminal sugars are linked to the alpha-mannose residues of the glycan cores. The 52/54-kDa medium protein is secreted with large and homogeneous complex glycans, their heterogeneity originates from slow processing after secretion. The complex glycans from cell wall beta-fructosidase are processed before the enzyme is integrated into the cell wall.     

Characterization of p51/52, a cell-growth regulated protein of WI-38 cells.

A number of proteins have been identified whose expression or activity is regulated by cell growth. We have produced a monoclonal antibody against a new cell-growth regulated protein found in normal human fibroblasts. We have shown that this antibody recognizes a 51/52-kDa doublet (p51/52) found mainly in normal cells. This doublet is sensitive to degradation by the calcium-activated protease, calpain, breaking down to a 37/38-kDa doublet. The relative amount of the two members of the 51/52-kDa doublet changes when serum-starved cells reenter the cell cycle. Quiescent cells express mainly the 51-kDa form; the 52-kDa form becomes more abundant upon refeeding serum-starved cells. Transformed cells express either very small amounts of this doublet, and then predominantly the 52-kDa form, or no detectable amount of either form. These characteristics distinguish this molecule from several other known growth-regulated proteins such as statin and the anti-oncogene p53.     

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell growth and the secretion of the estrogen-induced 34-, 52- and 160-kDa proteins in human breast cancer cells

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth of estrogen-responsive MCF-7 human breast cancer cells in the presence of 17 beta-estradiol was determined. After treatment with 17 beta-estradiol (1 nM), TCDD (10 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) the cells were monitored daily for cell growth and DNA content for 7 days. The results showed that TCDD inhibited cell proliferation and DNA content of untreated cells and inhibited the 17 beta-estradiol-stimulated cell proliferation and increase in cellular DNA content. In contrast, TCDD did not effect the growth of estrogen non-responsive MDA-MB-231 human breast cancer cells. TCDD (0.1-10 nM) also caused a concentration-dependent decrease in the 17 beta-estradiol-induced proliferation in MCF-7 cells. The effects of TCDD on the 17 beta-estradiol-induced secretion of the 52-kDa protein (i.e. procathepsin D), the 34-kDa (cathepsin D) and 160-kDa proteins were also determined in the MCF-7 and MDA-MB-231 human breast cancer cell lines. The levels of the proteins were determined by autoradiographic analysis of the incorporation of [35S]methionine into the secreted proteins which were separated by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of MCF-7 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in levels of the 52-kDa protein which were 497, 63.6, 98.1 and 66.3%, respectively, of the corresponding levels observed in control (untreated) cells. Using the same concentrations, the levels of the 34-kDa protein secreted into the media were 372, 42.3, 64.0 and 43.8% of control values, respectively, and the corresponding levels of the 160-kDa protein were 381, 52.9, 71.2 and 76.6% of the control values, respectively. In contrast, treatment of MDA-MB-231 cells with 17 beta-estradiol (1 nM), TCDD (10 and 100 nM) and 17 beta-estradiol (1 nM) plus TCDD (10 nM) resulted in a 31-39% reduction in the secretion of the 52-kDa protein however these effects were not statistically different from the control values. In addition, the treatments did not cause any significant effects on the secretion of the 34- and 160-kDa proteins by MDA-MB-231 cells. These results clearly confirm and extend the range of antiestrogenic effects caused by TCDD in estrogen-responsive MCF-7 cells and indicate that the MDA-MB-231 cells are not responsive to the antiestrogenic effects of TCDD.     

Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, 1-antitrypsin, and 2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins. protease; type 2 transmembrane serine protease; protease inhibitor; ST-14; hepatocyte growth factor activator inhibitor 1     

Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.     

Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo.

Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo, ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium.     

A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes

We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.     

Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Oviductin, the oviductal protease that mediates gamete interaction by affecting the vitelline coat in Bufo japonicus: its molecular cloning and analyses of expression and posttranslational activation

Previous studies indicated that the acquisition of egg fertilizability during transit through the pars recta portion of the oviduct in Bufo japonicus is accompanied by hydrolytic conversion of the vitelline coat 40- to 52-kDa components to 39-kDa components induced by a 66-kDa serine protease, "oviductin." In this study, we cloned a 3028-bp cDNA that contained an open reading frame encoding 974 amino acids with a calculated molecular mass of 107.6 kDa, including two protease domains and three repeats of CUB domains. Sequence analysis indicated that the catalytically active 66-kDa protein comprised an N-terminally located oviductin protease and two CUB domains. The oviductin gene was transcribed as a part of 6-kb mRNA that was expressed specifically in the cells lining the bottom of epithelial folds in the oviductal pars recta, and this expression was highly accelerated when the pars recta fragments were cultured in the presence of hCG. Western blot analyses using antibodies against a protease domain revealed that the catalytically inactive 102-kDa proteins in the pars recta granules yield 66-kDa catalytically active and 82- and 59-kDa inactive molecules. We propose that the oviductin translated as 107.6-kDa precursors are processed both N- and C-terminally to give rise to a 66-kDa active form comprising a serine protease and two CUB domains.     

Fibronectin Fibrils and Growth Factors Stimulate Anchorage-Independent Growth of a Murine Mammary Carcinoma

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol.13, 1189–1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-β (TGF-β) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-β) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.