Chemotactic response of rat mammary adenocarcinoma cell clones to tumor-derived cytokines

A cytokine with an apparent molecular weight of 53,000 daltons was isolated from serum-free medium conditioned by MTLn3 cells or from homogenates of MTLn3 cells, a highly metastatic variant of the rat 13762NF mammary adenocarcinoma. The chemotactic responses of MTLn3 and the low metastatic variant MTLn2 cells to this cytokine were tested in vitro using modified Boyden chambers. Both the chemotactic and chemokinetic movements of MTLn3 cells were stimulated by the MTLn3-derived cytokine. In addition, the MTLn3-derived cytokine stimulated a relatively small, but significant chemotactic migration of MTLn2 tumor cells, while these cells did not respond to medium conditioned by MTLn2 cells. MTLn3 cells themselves did not respond chemotactically to type I collagen or medium conditioned by MTLn2 cells. These results suggest that the chemotactic response may be a function of metastatic potential of the invading tumor cells. The production of tumor cytokines that enhance tumor cell motility may thus represent a phenotypic difference between 13762NF tumor cell subpopulations of high and low metastatic potential.     

Selection of highly metastatic rat MTLn2 mammary adenocarcinoma cell variants using in vitro growth response to transferrin

We previously found that the proliferative response to transferrin and the expression of transferrin receptors (TfR) on the cell surface of various rat 13762NF mammary adenocarcinoma cell sublines correlated with their spontaneous metastatic capability. To further assess the involvement of transferrin and TfR in metastasis, transferrin-responsive cells were selected from the poorly-metastatic, low-transfferin responsive 13762NF MTLn2 subline. When maintained in low serum (0.3%) conditions, MTLn2 cells failed to survive. However, if like medium was supplemented with 0.5 μmg/ml rat transferrin, some colonies emerged, presumably due to their ability to proliferate in response to the added transferrin. The surviving cells were expanded and exposed to ten or 20 similar cycles of transferrin growth selection to obtain the sublines MTLn2-Tf10 and MTLn2-Tf20, respectively. The MTLn2-Tf20 cells proliferated in response to transferrin at a rate similar to that of the high metastatic 13762NF sublines. Using immunofluorescent staining, Scatchard analysis, and affinity isolation of TfR, we discovered that the MTLn2-Tf20 cells had 5 to 6 times more TfR than did the parental MTLn2 line. When injected into the mammary fat pads of rats, the MTLn2-Tf20 line metastasized to the axillary lymph node in seven out of ten animals and to the lungs in six out of ten (median number = 13). No metastases were seen in the MTLn2 parental line. The MTLn2-Tf10 cells showed intermediate properties compared with the MTLn2 and MTLn2-Tf20 cells. The results indicate that variant cells with a high response to transferrin may be more metastatic than the bulk cells in a poorly metastatic population. The selection of cells with high levels of TfR and a higher proliferative response to transferrin results in sublines with greater potentials for spontaneous metastasis. J. Cell. Physiol. 174:48–57, 1998. © 1998 Wiley-Liss, Inc.     

Lung-derived growth factor that stimulates the growth of lung-metastasizing tumor cells: identification as transferrin.

We have previously shown that culture medium conditioned by lung fragments contains mitogenic activity for lung-metastasizing tumor cells but not for their non-metastatic counterparts. The growth-promoting component from media conditioned by rat and porcine lungs has been purified and partially characterized as a Mr approximately 66,000 (unreduced) or Mr approximately 72,000 (reduced) glycoprotein [Cancer Res 49:3928, 1989; J Cell Biochem 43:127, 1990]. Here we report that this factor is the iron transport protein transferrin. Migration distances in sodium dodecyl sulfate and native gel polyacrylamide electrophoresis systems were similar, as were the specific activities and spectrum of mitogenic activities of the lung-derived growth factor and transferrin. Electrophoretically separated holo-rat transferrin and rat lung-derived growth factor displayed similar positive stains for iron. A polyclonal antibody generated against the lung-derived growth factor cross-reacted with human and rat transferrin in Western blots, and anti-human transferrin cross-reacted with rat lung-derived growth factor. All of the mitogenic activity contained in crude lung conditioned media could be removed by antibody-mediated transferrin depletion. The putative cell receptor molecular weights for the lung-derived growth factor and transferrin were similar as were the molecular weights of polypeptides produced by partial trypsin cleavage of the two. Finally, the amino acid sequence of certain regions of rat lung-derived growth factor demonstrated a high degree of homology to human transferrin. The physical and biochemical properties, antigenicity, and mitogenic activity of a previously unidentified lung-derived growth factor for lung-metastasizing tumor cells indicate that it is transferrin.     

Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

Ligand-induced activation of epidermal growth factor receptor in intact rat mammary adenocarcinoma cells without detectable receptor phosphorylation.

Expression and function of epidermal growth factor receptor (EGFR) was investigated in a metastatic cell clone (MTLn3) derived from the 13762NF rat mammary adenocarcinoma. No receptor phosphorylation could be identified in intact cells or in membrane preparations, while EGF-dependent phosphorylation of substrates occurred in intact cells. Indications for active suppression of receptor phosphorylation came from the fact that EGFRs bound in immunocomplexes or associated with the cytoskeleton of detergent treated cells were able to undergo basal and EGF-induced phosphorylation in vitro. Cross-linking experiments with 125I-EGF, as well as [35S]methionine labeling followed by immunoprecipitation with receptor specific antibodies readily detected in MTLn3 cells the expected 170-kDa EGFR protein. In addition, two proteins with molecular masses of 420-480 and 95 kDa specifically bound 125I-EGF on intact MTLn3 and sparse cultures of A431 cells. Phosphorylation of the 420-480 kDa molecule could be identified in immunocomplexes of EGFRs isolated from MTLn3 and sparse A431 cells, but the 95-kDa receptor molecule was never phosphorylated. While the presence of alternative forms of EGFR in the highly metastatic cell clone MTLn3 was unexpected, our observations of inefficient receptor autophosphorylation are in agreement with other recent reports and suggest that in MTLn3 cells EGFR-mediated signal transduction can be an event independent from receptor autophosphorylation.     

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

The expression of a high-Mr sialogalactoprotein (gp580) on rat 13762NF mammary adenocarcinoma cells was identified and correlated with spontaneous metastatic potential to colonize lung [Steck & Nicolson (1983) Exp. Cell Res. 147, 255-267]. Using a highly metastatic tumour-cell clone, MTLn3, we isolated and characterized gp580 from cells growing in vitro and in vivo in the mammary fat-pads of Fischer 344 rats. The glycoprotein was extracted with 4 M-guanidinium chloride/4% Zwittergent 3-12 solution in the presence of proteinase inhibitors. The extracts were then subjected to dissociative CsCl-density-gradient centrifugation, gel filtration on Sepharose CL-2B columns and ion-exchange chromatography on DEAE-Sephacel. The isolated glycoprotein possessed low electrophoretic mobility in SDS/polyacrylamide gels, and after desialylation bound 125I-labelled peanut agglutinin. Electrophoresis of gp580 in polyacrylamide-gradient gels resulted in a diffuse but homogeneous migrating band of Mr approx. 55,000. After removal of carbohydrate, gp580 was demonstrated to have a protein core of Mr approx. 150,000. The gp580 had a high density (1.430 g/ml) on isopycnic centrifugation in 4 M-guanidinium chloride and was resistant to most proteinases and other degradative enzymes, suggesting a mucin-like structure. Amino acid and carbohydrate analyses revealed that gp580 has high contents of serine, threonine, glutamic acid, aspartic acid, glucosamine and galactosamine; several acidic and neutral oligosaccharides were obtained from alkaline-borohydride digests. Cellular localization studies suggested that gp580 is associated mainly with the cell-surface and extracellular-matrix fractions of MTLn3 cells.     

Pyrimido-pyrimidine modulation of EGF growth-promoting activity and p21ras expression in rat mammary adenocarcinoma cells

RA 233, a pyrimido-pyrimidine analogue developed originally as an antiplatelet agent, has reduced the incidence of tumor metastases in clinical trials. However, in animal tumor models antimetastatic therapy using RA 233 has been inconsistent. We therefore tested RA 233 for additional effects, such as its direct action on tumor cells. Using the rat 13726NF mammary adenocarcinoma tumor system, low, nontoxic concentrations of RA 233 had pleiotropic and differential effects on two 13762NF tumor cell clones. The growth of MTC cells (low spontaneous metastatic potential) was not affected by low concentrations of RA 233 (50 microM) or epidermal growth factor (EGF) (up to 10 ng/ml) for 3 days in 0.5-10% fetal bovine serum. In contrast, MTLn3 (high spontaneous metastatic potential) cell cultures maintained for 3 days in low (0.5-1%) serum in the presence of 1.25-10 ng/ml EGF doubled in cell numbers compared with control cultures, and addition of 50 microM RA 233 abrogated the growth-stimulatory effect of EGF. The inhibitory effect of RA 233 on MTLn3 cells was dose dependent and not due to cell toxicity as determined by cell viability, cell growth, and colony formation properties after drug removal. In addition, incubation of MTLn3 cells with 50 microM RA 233 resulted in an increase of p21ras protein expression, whereas there was no effect on the level of p21ras in identically treated MTC cells or when either clone was treated with 10 ng/ml EGF. The results suggest that among the heterogeneous effects of RA 233 on tumor cells, modulation of growth factor responses and regulatory molecules may be important.     

A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells.

Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.     

Differential adhesion of metastatic rat mammary carcinoma cells to organ-derived microvessel endothelial cells and subendothelial matrix

The in vitro adhesion rates of rat 13762NF mammary adenocarcinoma cell clones of different spontaneous metastatic potentials to cloned microvessel endothelial cell monolayers and their subendothelial extracellular matrix were investigated. In this system, high rates of adhesion of the cloned tumor cell lines to syngeneic target (lung) organ-derived subendothelial matrix correlated with spontaneous metastatic potential, whereas adhesion to the lung microvessel endothelial cell apical surfaces occurred at lower rates and was not highly significantly different among the tumor cell lines. Adhesion rates to bovine aortic large vessel, and human brain and human meningeal microvessel endothelial cell monolayers were, in general, lower than those found with syngeneic lung microvessel endothelial cells, and did not correlate with spontaneous metastatic potential. Growth of endothelial cells in fetal bovine serum or platelet-poor horse serum did not affect the results, suggesting that in this system metastasis-associated organ-adhesive specificity is determined at the level of the subendothelial matrix.     

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.     

Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.     

An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.     

Distinct T-cell proliferative responses to 13762A rat mammary adenocarcinoma and derived clones

We examined the in vitro responses of immune lymphocytes to the tumor antigens of the syngeneic rat mammary adenocarcinoma 13762A. This tumor readily metastasizes to lymph node and lungs and is poorly immunogenic. Rats were immunized with a highly immunogenic clone (18A) which was isolated as a spontaneous variant from the parental 13762A tumor. Clone 18A grew progressively in irradiated rats but regressed completely in normal rats. Animals immune to 18A tumor were also immune to parental 13762A. Lymphocytes obtained from the spleen and peritoneum of immune rats were tested for specific proliferation to parental 13762A tumor and clone 18A to determine whether similar cross-reactivity to these tumors occurred in vitro. We found an anatomical difference in localization of immune lymphocytes which reacted to the two tumor cell lines. Immune peritoneal exudate cells (PEC) responded strongly to clone 18A but poorly to 13762A, while immune spleen cells from the same animals responded predominantly to 13762A tumor. After 7 days culture, PEC proliferating in response to clone 18A contained 84-95% W3/25+ T-helper cells, and only 5-8% OX8+ cytotoxic/suppressor cells, while analogous cultures of spleen cells responding to parental 13762A tumor consisted of 60-80% W3/25+ cells and 20-23% OX8+ cells. Immune spleen cell cultures stimulated with 13762A tumor generated cytotoxic lymphocytes which specifically lysed both parental 13762A and clone 18A cells. We conclude that despite cross-reactivity in vivo and in vitro, antigens present on 13762A and 18A tumor cells stimulated different subsets of immune T cells.     

Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases.     

Modulation of extracellular proteolytic activity and anchorage-independent growth of cultured cells by sarcoma cell-derived factors: relationships to transforming growth factor-beta

We have previously described a factor(s) produced by 8387 fibrosarcoma cells, which can affect plasminogen activator (PA) activity of cultured cells. Since then, transforming growth factor-beta (TGF beta) has been established as a major growth factor/growth inhibitor that regulates both the expression and activity of PAs and their endothelial-type inhibitor (PAI-1). The present study was undertaken to characterize the 8387 fibrosarcoma cell-derived factor(s) and to investigate its relationships to TGF beta by analysis of modulation of PA activity and cell growth. The fibrosarcoma cell-derived proteins were partially purified from serum-free conditioned culture medium using Bio-Gel P-10 chromatography. Two separate fractions with apparent molecular weights of 16,000 and 12,000 contained activities that both decreased the secretion of PA activity by human lung fibroblasts and inhibited the soft agar growth of A549 lung adenocarcinoma cells. Both factors affected similarly the production of urokinase-type PA and PAI-1 in various cell lines and enhanced anchorage-independent growth of murine AKR-2B fibroblasts. The effects of these factors thus resembled those of TGF beta. The immunological relationships between the Mr 16,000 and Mr 12,000 factors and TGF beta were therefore studied using neutralizing anti-TGF beta antibodies. The TGF beta antibodies efficiently inhibited the effects of the Mr 16,000 factor but not those of the Mr 12,000 factor in cell culture assays. The results suggest that 8387 fibrosarcoma cells produce two major growth inhibitors, one of which is closely related to TGF beta.     

Fibroblast-Derived Extracellular Matrices: An Alternative Cell Culture System That Increases Metastatic Cellular Properties

Poor survival rates from lung cancer can largely be attributed to metastatic cells that invade and spread throughout the body. The tumor microenvironment (TME) is composed of multiple cell types, as well as non-cellular components. The TME plays a critical role in the development of metastatic cancers by providing migratory cues and changing the properties of the tumor cells. The Extracellular Matrix (ECM), a main component of the TME, has been shown to change composition during tumor progression, contributing to cancer cell invasion and survival away from the primary cancer site. Although the ECM is well-known to influence the fate of tumor progression, little is known about the molecular mechanisms that are affected by the cancer cell-ECM interactions. It is imperative that these mechanisms are elucidated in order to properly understand and prevent lung cancer dissemination. However, common in vitro studies do not incorporate these interactions into everyday cell culture assays. We have adopted a model that examines decellularized human fibroblast-derived ECM as a 3-dimensional substrate for growth of lung adenocarcinoma cell lines. Here, we have characterized the effect of fibroblast-derived matrices on the properties of various lung-derived epithelial cell lines, including cancerous and non-transformed cells. This work highlights the significance of the cell-ECM interaction and its requirement for incorporation into in vitro experiments. Implementation of a fibroblast-derived ECM as an in vitro technique will provide researchers with an important factor to manipulate to better recreate and study the TME.     

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.