Vasopressin: action on WRK-1 rat mammary tumor cells.

WRK-1, a cell line in long-term culture derived from a 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumor, responds to physiologic concentrations of vasopressin with increased precursor incorporation into phospholipids and with increased protein accumulation. Because vasopressin has been reported to be a potent mitogen for Hela cells and 3T3 cells, a study was conducted to determine whether it could act as a mitogen for WRK-1 cells. Under no conditions was a clear-cut mitogen response to vasopressin demonstrated.

Characterization of rat mammary tumor cell populations.

Nodules of tumor cells have been isolated from 7, 12-dimethylbenz (alpha)anthracene-induced rat mammary tumors. These nodules consist of at least two cell populations which can be subfractionated by selective attachment in calcium-free medium. One cell population which attaches in the absence of calcium (basal cells) stains intensively with antibodies against type IV collagen and also with antibodies against keratin. The nonattaching population, the epithelial cells, stains much more weakly with either antibody. Biochemical analyses indicate that 27% of the protein labeled in basal cell culture is type IV collagen, while 8% is keratin. With epithelial cells, only 0.35% of the protein made is collagen, and only 2.4% is keratin. Basal cells contain about 2 times as much calmodulin as epithelial cells but only about one-tenth as many estrogen receptors. In culture, the basal cells stimulate the attachment and/or division of the epithelial cell population. The epithelial cells have little effect on the division or attachment of the basal cells. Interaction between the two cell populations may be important for tumor growth in vivo.

Unsaturated fatty acid requirements for growth and survival of a rat mammary tumor cell line.

A cell line, the growth and survival of which is markedly affected by linoleic acid, has been established from a carcinogen-induced rat mammary tumor. The cells have been continuously passaged in 5% rat serum plus 10% fetal calf serum-supplemented medium. The rat serum component was found to be indispensalbe, for when it was omitted the growth rate rapidly declined and the cells died by 5 to 7 days. Removal of the rat serum from the growth medium also resulted in a dramatic loss of Oil Red O-positive droplets in the cells, suggesting that the lipid component of rat serum might be a major growth-promoting principle in rat serum. This is likely since the total lipid fraction, but not the delipidized protein fraction, could largely supplant requirement of the cells for rat serum. Pure linoleic acid was found to be effective in maintaining the cell growth in delipidized serum or in whole fetal calf serum-supplemented medium. Fatty acid analysis revealed a 19-fold higher amount of linoleic acid in rat serum than in fetal calf serum.

Vasopressin stimulation of acetate incorporation into lipids in a dimethylbenz(a)anthracene-induced rat mammary tumor cell line.

In a preliminary report we described the effects of rat prolactin on the incorporation of [14C]acetate into lipids by a cell line from a dimethylbenz(a)anthracene-induced rat mammary tumor. The characteristics of the response to prolactin were very similar to those described for the normal rat mammary gland; namely, insulin was required for full expression of the response, maximal activity was not seen until 36 hr after the addition of the hormones, and growth hormone was able to elicit the same response. However, we were unable to detect binding of 125I-labeled prolactin to these cells, and furthermore, other more purified prolactin preparations were inactive. Upon further investigation we discovered that the activity resided in a low-molecular-weight fraction of the rat prolactin B-1 preparation and was probably either vasopressin or oxytocin or both. These data suggest the possibility that vasopressin may play a role in rodent mammary tumorigenesis.

Partial purification of transforming growth factors from human milk.

Crude, delipidated milk and the acid:ethanol extracts of primary human breast tumors contain several activities that biologically resemble transforming growth factors (TGFs) in that they promote the anchorage-independent growth of normal rat kidney and Mm5mt/c1 mouse mammary tumor cells in soft agar. Three major TGF species with isoelectric points (pl) of about 4.0, 6.0-6.5, and 7.0 have been detected in both tumors and milk. The pl 4.0 species from milk has been purified about 10,000-fold by isoelectric focusing and high-performance liquid chromatography. This species, designated milk-derived growth factor II (MDGFII), coelutes from gel filtration columns with an authentic human epidermal growth factor standard when using a low ionic strength eluting buffer. However, on the same column, MDGFII is completely resolved from human epidermal growth factor with high ionic strength eluting buffers. Nevertheless, MDGFII purified by the latter technique still competes with 125I-epidermal growth factor for receptor binding to A431 cell membranes. Additionally the TGF activity of MDGFII present in the pl 4.0 fraction of milk is markedly inhibited by anti-epidermal growth factor receptor antibody preparations. Consequently MDGFII appears to be an alpha-TGF. MDGFII is a pepsin-sensitive, disulfide reducing agent-sensitive, heat-stable protein that may be physiologically important for the mammary gland or the neonate.

Presence of transforming growth factors in human breast cancer cells.

Conditioned medium (CM) from a human mammary carcinoma cell line, MCF-7, and ten individual clones derived from these cells was examined for the presence of transforming growth factors (TGFs). Concentrated CM from all of the MCF-7 cell lines was found to stimulate the anchorage-independent growth of normal rat kidney cells in soft agar and to inhibit the binding of epidermal growth factor (EGF) to mouse NIH/3T3 fibroblasts and to A431 human epidermoid carcinoma cell membranes. The soft agar stimulating activity was heat stable but sensitive to treatment with dithiothreitol. EGF receptors were measured on the MCF-7 cell lines to determine whether the amount of TGFs associated with the CM from the various cell lines was correlated with the level of EGF receptors being expressed on these cells. Moreover, the intrinsic cloning efficiency of these lines in soft agar was measured to ascertain if any correlation might exist between the level of TGFs associated with these cells and the ability of these cell lines to form colonies in soft agar. Although all the MCF-7 cell lines had approximately the same number of EGF receptors per cell, ranging from 3 to 6 X 10(3) sites/cell, CM from these lines varied in potency with respect to inducing the growth of normal rat kidney cells as colonies in soft agar and in inhibiting the binding of EGF to NIH/3T3 cells. Likewise, the level of TGFs associated with the CM from the various clones showed no correlation with the ability of these individual lines to grow as colonies in soft agar. TGF activity was also detected in acid-ethanol extracts prepared from MCF-7 cells propagated in nude mice as tumors and in the extracts from two transplantable human mammary adenocarcinomas, Clouser I and II. In addition, approximately 50% of the normal rat kidney colonies formed in response to the Clouser II tumor extracts exhibited a branching morphology in contrast to spherical colonies produced by Clouser I or MCF-7 extracts. These results demonstrate that human mammary carcinoma cells from both established cell lines and cells maintained in nude mice as tumors contain TGF-like activities. Furthermore, the variation in TGFs associated with the CM from the MCF-7 clones suggests that the parent MCF-7 cell line contains a heterogeneous population of cells.

Anchorage-independent growth-conferring factor production by rat mammary tumor cells.

Conditioned medium from cultures of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells contain factors that resemble sarcoma growth factor and other transforming growth factors in biological activity but differ in their physical properties. The mammary tumor factors (MTF) are acid stable and heat and protease sensitive. They inhibit the binding of epidermal growth factor, but not insulin, to mouse embryonal carcinoma cells. MTF confers upon normal rat kidney and BALB/c-3T3 cells the ability to grow in soft agar. This effect is enhanced synergistically by high concentrations of fetal calf serum but not by epidermal growth factor. Anchorage-independent growth promotion, however, is not seen with normal mammary epithelial cells, although MTF is mitogenic for these cells as well as normal rat kidney cells, BALB/c-3T3 cells, and chick embryo fibroblasts in monolayer culture, MTF is not mitogenic for primary cultures of the tumor cells from which the factors are derived. Two major molecular weight species of MTF, eluting at Mr 6,000 and 65,000 to 70,000 on Bio-Gel P-100 columns, are present in acid-ethanol extracts of 7,12-dimethylbenz(a)anthracene- and nitrosomethylurea-induced rat mammary tumors. Transplantable tumors derived from primary 7,12-dimethylbenz(a)anthracene- or nitrosomethylurea-induced tumors have little or no MTF activity. These results demonstrate that different chemically induced rat mammary tumors contain transforming growth factor-like activities. Furthermore, it is possible that MTF is unnecessary for the maintenance of tumorigenicity, since some tumors contain no detectable MTF.

Sensitivity of N-nitrosomethylurea-induced rat mammary tumors to cis-hydroxyproline, an inhibitor of collagen production.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Differential growth sensitivity to 4-cis-hydroxy-L-proline of transformed rodent cell lines.

The effect of 4-cis-hydroxy-L-proline (CHP), a proline analogue, on the anchorage-dependent and -independent growth of several transformed rodent cell lines was studied. Mouse NIH-3T3 fibroblasts transformed by a variety of different oncogenes (Ki-ras, mos, src, fms, fes, met, and trk) by a DNA tumor virus (SV40) or by a chemical carcinogen (N-methylnitrosourea) were all found to be more sensitive (50% inhibitory dose, 20 to 55 micrograms/ml) to the dose-dependent inhibitory effects of CHP on growth in monolayer culture than were NIH-3T3 cells (50% inhibitory dose, 120 micrograms/ml). CHP was generally found to be even more effective in inhibiting the growth of these transformed cells as colonies in soft agar than in monolayer cultures. In addition, rat embryo fibroblasts (CREF) and normal rat kidney fibroblasts (NRK) after transformation with a Ki-ras oncogene exhibit a similar increase in their sensitivity to CHP-induced growth inhibition. Treatment of NRK cells with transforming growth factor alpha (TGF-alpha) and beta (TGF-beta), which reversibly induces phenotypic transformation of these cells, increases their sensitivity to CHP to a level comparable with that observed in Ki-ras-transformed NRK cells (K-NRK). The growth inhibitory effects of CHP are reversible, since removal of CHP results in a normal resumption of cell growth. CHP uptake occurs primarily through the Na+- and energy-dependent neutral amino acid transport A system, which is 6- to 7-fold more elevated in K-NRK cells compared with NRK cells. Treatment of NRK cells with TGF-alpha and/or -beta increases the uptake of [3H]methylaminoisobutyric acid on the A system to a level that is similar to that found in K-NRK cells. The functions of the Na+/K+ and Na+/H+ exchange systems are apparently necessary for the enhanced A system activity, since ouabain and amiloride can inhibit the uptake of [3H]methylaminoisobutyric acid in K-NRK cells and in NRK cells treated with TGF-alpha and/or -beta. The activity of the A system is specifically increased in K-NRK and in TGF-alpha- and/or -beta-treated NRK cells, since the other two major neutral amino acid uptake systems, the ASC and the L systems, and the Ly+ system for basic amino acid uptake show no apparent changes in their activity in NRK cells after treatment with TGF-alpha and/or -beta or in these cells after transformation with the Ki-ras oncogene. These results suggest that the differential growth sensitivity to CHP of transformed rodent cells and of normal fibroblasts treated with TGF-alpha and/or -beta is due in part to an elevated uptake of this amino acid analogue on the neutral amino acid transport A system.

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production.

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene.

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.

Blocking basement membrane collagen deposition inhibits the growth of 7,12-dimethylbenzanthracene-induced rat mammary tumors.

7,12-Dimethylbenzanthracene-induced rat mammary tumors contain basement membrane collagen in vivo and in vitro in primary culture, as determined by immunofluorescence. The proline analogue dis-hydroxyproline, which selectively blocks collagen accumulation, inhibits tumor cell growth in vitro. In vivo cis-hydroxyproline blocks tumor growth and produces epithelial cell degeneration and tumor necrosis at a non-toxic dose. This compound has no effect on the growth of the mammary carcinoma cell line 64/24, which makes no basement membrane collagen. As we have previously described for the normal proliferating rat mammary gland, basement membrane collagen deposition may be necessary for the growth of some rat mammary tumors.