Changes in in vitro growth behaviour of the mammary epithelial cell line NMuMG caused by the v-fos oncogene

A defective retrovirus was constructed to investigate the effect of the expression of the v-fos oncogene from FBJ-MSV on the in vitro growth properties of the mammary epithelial cell line NMuMG. Clearly visible areas of overgrowth in monolayer cultures of NMuMG were seen in cells infected with the v-fos-containing retrovirus but not in cells infected with control virus which did not contain an oncogene. Two cell lines, representing two morphological types of infected cell, were isolated from a morphologically altered region and further characterized. Fos.3.1.NMuMG grew as very spindly cells, achieving a higher density than control cells in 5% foetal calf serum (FCS) but growing very poorly in 1% FCS or in soft agar. Fos.3.3.NMuMG grew to a high density in 5% FCS and to a limited extent in low serum. This cell line also grew in soft agar. Fos.3.3.NMuMG seemed to be more transformed than fos.3.1.NMuMG using the criteria of growth in soft agar and low serum. All the cells used in this study were shown to retain epithelial characteristics by staining for cytokeratins and to contain at least one viral genome by Southern blotting. fos mRNA expression was raised over control levels in the two transformed cell lines.     

In vitro differentiation and progression of mouse mammary tumor cells

We have isolated clonal cell lines from a transplanted adenocarcinoma induced by the RIII strain of mouse mammary tumor virus in a BALB/c mouse. Three major morphological cell types of these lines are developmentally linked; polygonal cells give rise to cuboidal and then to elongated cells. All cell lines expressed markers that are characteristic of mammary basal cells. In addition, the polygonal lines contained cells that have cell markers and ultrastructural features of epithelial cells; in these lines an occasional cell was found with myoepithelial features. The cuboidal and elongated lines lacked many epithelial differentiation characteristics and showed no myoepithelial differentiation. The cell lines contained variable numbers of acquired mouse mammary tumor virus and ecotropic murine leukemia virus proviruses. The various subclones derived from the original cell lines contained, in addition to the acquired proviruses of the parental line, one or more unique proviruses of either mouse mammary tumor virus or ecotropic murine leukemia virus origin. These unique insertions were used as genotypic markers to demonstrate the clonal relationship of the cell lines. Both polygonal and elongated cells are tumorigenic and give rise to adenocarcinomas and sarcoma-like tumors, respectively. In contrast, the cuboidal cells are poorly tumorigenic. Since cuboidal cells are derived from the polygonal cells, this suggests that tumor progression in this system proceeds via intermediates that are either poorly or nontumorigenic.     

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.     

Identification of genes preferentially expressed in mammary epithelial cells of Copenhagen rat using subtractive hybridization and microarrays

Rats, like humans, vary considerably in susceptibility for mammary cancer development among different strains. The Copenhagen (Cop) rat is extremely resistant to mammary cancer development induced by a variety of carcinogens. Multiple genetic loci have been linked to the resistant phenotype, but the genes have yet to be cloned and the mechanisms underlying the resistance still remain unknown. Transplantation experiments, however, have demonstrated that these genes act only in the epithelial cells of mammary parenchyma; they do not act systemically. In the present study, we analyzed genes differentially expressed in mammary epithelial cells obtained from pubescent female Cop and susceptible Buffalo (Buf) rats, using PCR-based suppressive subtractive hybridization and cDNA microarray approaches. Our results showed a high degree of similarity in the expression profiles of about 4000 genes between Cop and Buf rats, with a few exceptions. We found that the interleukin-2 receptor alpha (IL-2Ralpha) chain gene and claudin-6 gene were preferentially expressed in mammary epithelial cells purified from Cop rats. We further demonstrated that IL-2Ralpha message was undetectable in two rat mammary cancer cell lines and in two human breast cancer cell lines. The level of claudin-6 mRNA was undetectable in two rat mammary cancer cell lines and was lower in two human breast cancer cell lines and one breast cancer sample than that in normal breast tissues. These results suggest that IL-2Ralpha and claudin-6 may function as tumor suppressors, particularly for breast cancer. However, this possibility needs further investigation.     

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal–epithelial interactions

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for α-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.     

Development of an in vitro model of tumor progression using v-raf and v-raf/v-myc transformed rat liver epithelial cells: correlation of tumorigenicity with the downregulation of specific proteins

A series of clonal cell lines were derived from rat liver epithelial cells after being infected with a defective retrovirus containing either v-raf (3611-MSV) or v-raf/v-myc(J2) together with a helper virus. These clones exhibited a different morphology from the regular cuboid shape of the control cells, infected only with the helper virus. All of the infected cell lines contained at least one full length copy of appropriate proviral DNA and expressed comparable levels of v-raf mRNA, although only the cells transformed with the v-raf/v-myc combination were capable of anchorage-independent growth in soft agar. All of the clones except the controls formed tumors in nude mice but with markedly different latency periods and growth rates. Thus, these cell lines represent an in vitro model for tumor progression. Two-dimensional polyacrylamide electrophoresis was used to investigate changes in cellular protein expression related to malignant conversion. The expression of three proteins of pI/Mr x 10(-3) 5.9-7.2/205 (RP1), 6.5-7.5/160 (RP2) and 4.0/85 (RP3) consistently matched the transformed phenotype. In particular the expression of RP1 and RP2 correlated with the relative tumorigenicity of the cell lines. Rat liver epithelial cell lines transformed by other protocols that did not involve v-raf also showed downregulation of these three polypeptides. Crude fractionation studies determined RP1 to be soluble and RP2 and RP3 to be membrane associated. RP2 was shown to be a glycoprotein containing mannose and galactose residues. These three proteins are consistent markers for the tumorigenic potential of rat liver epithelial cells.     

Markers to distinguish normal and neoplastic mammary epithelial cells in vitro: comparison of saturation density, morphology and concanavalin A reactivity.

Normal and premalignant mouse mammary epithelial cells can be prepared in high yields by collagenase dissociation of minced glands followed by a brief, differential centrifugation to remove contaminating fibroblasts and fat cells. The major difficulties in preparing pure cultures in quantity are 1) incomplete dissociation of gland material, and 2) cell death during enzymatic digestion. These problems are eliminated by careful selection of collagenases for dissociation. Normal and premalignant mammary epithelial cells are morphologically indistinguishable from malignant mouse mammary epithelial cells in primary monolayer cultures. In addition, the growth rates and saturation densities achieved by normal mammary epithelial cells are indistinguishable from those of malignant mammary epithelial cells in primary culture. In both cases, a monolayer of cells is preserved with no evidence of focal overgrowth. Malignant adenocarcinoma mammary cells can however be distinguished from normal mammary epithelial cells by virtue of differences in their surface interactions with concanavalin A. A hemadsorption assay using Con-A-coated erythrocytes was the most sensitive indicator for these differences. In hemadsorption assays malignant mammary epithelial cells were half-maximally reactive with 2.5 mug/ml concanavalin A, while normal cells were completely unreactive even at concanavalin A concentrations five-times higher. Premalignant mammary epithelial cells were as reactive as malignant mammary epithelial cells in the hemadsorption assays. Hemadsorption of malignant cells was observed in primary and secondary cultures of epithelium as well as in cell lines. Malignant cells forming mammary adenocarcinomas were as highly reactive as malignant cells forming scirrhous carcinomas. Malignant cells not releasing mammary tumor virus (MuMTV) were as reactive as cells releasing that virus. Adsorption of concanavalin-A-coated erythrocytes to normal mammary epithelial cells could be induced by brief treatment of cell monolayers with hyaluronidase. Exposure of active sites was not affected with either trypsin or collagenase. Our results show that while the growth of malignant cells does not serve to distinguish them from normal cells in monolayer culture, surface changes do exist which can be identified by differences in concanavalin A reactivity. Since the earliest transformants identifiable in vivo (premalignant) have undergone conversion of the surface marker, concanavalin-A-mediated hemadsorption provides a sensitive measure for mammary epithelial cell transformants in vitro.     

Constitutive activation of pp125fak in newly isolated human breast cancer cell lines

Our laboratory has developed twelve human breast cancer cell lines from primary and metastatic sites. In this report we demonstrate that eight of eight breast cancer cell lines examined exhibit constitutively tyrosine phosphorylated and enzymatically active endogenous pp125fak when grown in monolayer. The activation status of pp125fak in breast cancer cells in monolayer is significantly elevated over that exhibited by normal mammary epithelial cells cultured under the same conditions. Constitutive activation of pp125fak is the only characteristic so far studied that all of these breast cancer cell lines have in common. In contrast to HBC cells, tyrosine phosphorylation of pp125fak in HME cells was low or absent in monolayer culture but was induced to high levels by culturing the cells in Matrigel. Thus tyrosine phosphorylation and activation of pp125fak is a regulated process in normal mammary epithelial cells, but is constitutive in breast cancer cells. Finally, analysis of the ability of normal human mammary epithelial cells and breast cancer cell lines to grow under anchorageindependent conditions indicated that normal human mammary epithelial cells rapidly and uniformly lost viability when not substrateattached, whereas all of the breast cancer cell lines survived for a 3week culture period. Furthermore, a subset of the breast cancer cell lines grew to form large colonies under anchorageindependent conditions. Interestingly, pp125fak activation decreased dramatically in HBC cells cultured for two weeks in suspension, suggesting that activation of this kinase is not necessary for longterm growth under anchorageindependent conditions. These results suggest that constitutive activation of pp125fak results in preferential survival of human breast cancer cells under anchorageindependent conditions but that activation of pp125fak is not the sole mediator of anchorageindependent colony formation.     

Suppression of cellular aggregation by high levels of episialin.

Episialin is a mucin-like molecule located at the apical surface of most glandular epithelial cells. It is present at increased levels in carcinomas, where the molecule is often distributed over the entire cell surface. We have simulated this overproduction of episialin by transfecting a normal mammary epithelial cell line and a melanoma cell line with full-length complementary DNA encoding episialin. Transfectants of both cell lines containing episialin at levels similar to that of carcinoma cell lines do not aggregate as efficiently as their control cells, which do not express exogenous episialin. In mixing experiments, episialin transfectants are excluded from aggregates formed by these control cells, indicating that high levels of episialin on one of the interacting cells is sufficient to inhibit aggregation. The effect of episialin overexpression on aggregation is probably not only due to the negative charge of its numerous sialic acid residues, since neuraminidase treatment only partially restored the aggregation capacity of the transfectants. We propose that episialin, as a result of its large, extended, and rigid structure, can mask most cell surface molecules in its immediate surroundings and that a high density of episialin can severely disturb the interaction of cell surface proteins with macromolecules on adjacent cell membranes.     

Mitochondrial amplification selectively increases doxorubicin sensitivity in breast cancer cells with acquired antiestrogen resistance

The metabolic phenotype of cancer, characterized by uncoupled mitochondrial respiration and increased mitochondrial oxidative stress, is an attractive pharmacological target for sensitizing cancer cells to therapies that rely on oxidative stress for their tumor specific cytotoxicity. The identification of specific cancer sub-types for which metabolic priming of tumors prior to chemotherapy is beneficial is critical, particularly in heterogeneous diseases such as breast cancer. The effects of the thiazolidinedione drug troglitazone were examined in normal mammary epithelial cells and cancer cell lines representing three clinically relevant breast cancer phenotypes. Endpoints measured were PGC1α mRNA expression, proliferation, cell cycle phase distribution, mitochondrial capacity and superoxide generation, and sensitivity to the chemotherapy drug doxorubicin. Troglitazone increases expression of PGC1α, a key mediator of mitochondrial biogenesis, in normal mammary epithelial cells and in breast cancer cell lines. The induction of PGC1α mRNA is at least partially dependent on PPARγ activation. In estrogen receptor negative cells and cells with acquired antiestrogen resistance, troglitazone treatment increased mitochondrial superoxide production and mitochondrial capacity. At pharmacologically achievable doses, troglitazone pretreatment significantly enhanced the sensitivity of cancer cells to the chemotherapy agent doxorubicin. This effect was most dramatic in estrogen receptor positive cells with acquired antiestrogen resistance, in which troglitazone and doxorubicin combined had superadditive effects compared to treatment with either agent alone. In contrast, troglitazone treatment did not appreciably sensitize non-malignant mammary epithelial cells to doxorubicin induced cytotoxicity, despite increasing PGC1α mRNA. These data suggest that troglitazone or a similarly acting compound could be used to selectively prime tumor cells to the cytotoxic effects of anticancer agents such as doxorubicin and ionizing radiation. This novel treatment strategy may be most effective in women with antiestrogen insensitive tumors, a patient population with historically poor response to traditional therapies.     

Reducing mammary cancer risk through premature stem cell senescence

The reproductive capacity of the mammary epithelial stem cell is reduced coincident with the number of symmetric divisions it must perform. In a study of FVB/N mice with the transgene, WAP-TGFbeta1, we discovered that mammary epithelial stem cells were prematurely aged due to ectopic expression of TGF-beta1. To test whether premature aging of mammary epithelial stem cells would have an impact on susceptibility or resistance to mammary cancer, female littermates from FVB/N x WAP-TGF-beta1 mating were injected with mouse mammary tumor virus (MMTV) at 8-10 weeks of age. A total of 44 females were inoculated, maintained as breeders and observed for tumor development for up to 18 months. Only one mammary tumor appeared in 17 TGF-beta1 females while 15 were collected from 29 wild type sisters. Premalignant mammary epithelial cells in infected glands were identified by transplantation of single cell (1 x 10(5)) suspensions into nulliparous hosts and testing for hyperplastic outgrowth. Although the number of positive takes was significantly reduced with TGF-beta1 cells, both MMTV-infected TGF-beta1 and wild type cells produced hyperplastic outgrowths suggesting that premalignant transformation was achieved in each group. The results suggest a positive correlation between the procreative life-span of mammary epithelial stem cells and mammary cancer risk.     

Growth requirements of human mammary epithelial cells in culture.

Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.     

Isolation of two syngeneic cell lines from a rat mammary carcinoma: growth factor production by neoplastic epithelial cells

Two distinctly different clonal cell lines were isolated from a mammary tumor induced by ingestion of 7,12-dimethylbenz[a]anthracene (CAS: 57-97-6) in a SD rat. Cells of one of the clones, RMT-1 clone E4, showed typical epithelial characters; it was concluded that they were derived from neoplastic mammary epithelial cells. The other clone, M2, exhibited characters consistent with its derivation from the normal mammary myoepithelial component. The 2 cell lines had different proliferative responses to growth factors (GF) such as cholera toxin, dexamethasone, and epidermal GF. The epithelioid E4 cells were found to produce potent growth-promoting activity in culture medium that stimulated proliferation of myoepithelial M2 cells as well as that of stromal fibroblasts. The present work provides supporting evidence that the mechanism of "paracrine stimulation" is operative in the mammary tumorigenesis of the rat.     

Mitochondrial inclusions in selenium-treated mouse mammary epithelial cell lines

The effects of selenium on three mammary epithelial cell lines (YN-4, WAZ-2t, and CL-S1) grown in vitro were examined by immunocytochemical and transmission electron microscopy technique. The primary effect of selenium at the ultrastructural level was the appearance of electron-dense inclusions within the mitochondrial matrix. The mitochondrial inclusions were seen in all three cell lines, although most readily induced in YN-4 cells, the cell line which is most sensitive to selenium-mediated growth inhibition. Selenium at 5 x 10(-8) and 5 x 10(-6) M did not alter cytoplasmic microtubules or intermediate filament networks, as determined by immunocytochemical staining. Immunocytochemical staining for cytoplasmic filaments and microtubules, and transmission electron microscopy observations, supported the contention that cells from all three cell lines were epithelial in origin, since they contained abundant desmosomes and were uniformly positive for keratin intermediate filaments. Whereas line YN-4 was negative for vimentin intermediate filaments, a minority (5 to 24%) of the cells in lines CL-S1 and WAZ-2t stained positively. In addition, the tumorigenicity of these three cell lines was assessed by in vitro growth assays and in vivo transplantation assays. Cell lines YN-4 and WAZ-2t, but not line CL-S1, were tumorigenic in syngeneic mice. All tumors were mammary adenocarcinomas. Cytochalasin B-induced multinucleation assay and growth as multicellular spheroides correlated positively with in vivo tumorigenicity, whereas saturation density and growth in low Ca2+ medium were not correlated with tumorigenicity. It is speculated that one of the early effects of selenium-mediated growth inhibition may be a modulation of mitochondrial function.     

Growth regulation of mouse mammary tumor cells in collagen gel cultures by diffusible factors produced by normal mammary gland epithelium and stromal fibroblasts

We have reported previously that both normal mammary epithelium and stroma stimulate the growth of mouse mammary tumors in vivo. We have devised a method to investigate the role of diffusible factors in these growth interactions in vitro. Because an appropriate matrix, a particular cell shape, or multicellular organization may be required for the production of factors and/or for the response to such factors, the method assesses the expansion of boluses of cells in collagen gel matrix. Under these culture conditions, paracrine effects were detected which were not observed when cells were growing in monolayer on plastic. The growth of mammary tumor lines 66, 410.4, and D2A1 was stimulated by both mammary epithelial cells and fibroblasts prepared from midpregnant mouse mammary glands. In contrast, these mammary tumor lines were inhibited by coculturing with normal mammary cells in monolayer on plastic. Stimulation in collagen and inhibition in monolayer cultures were both dose dependent; increasing numbers of regulator mammary cells increased the effects on tumor growth. Additional tumor cells did not stimulate growth of target tumor cell boluses in collagen gel cultures. This attempt to model the in situ situation may be a more appropriate model for the detection and characterization of relevant diffusible growth regulatory factors than monolayers on plastic and/or colonies growing in agar.     

Loss of Fatty-Acid delta(6) desaturating ability in human mammary epithelial-cells that express an activated C-ha-ras oncogene

The synthesis of essential fatty acids (EFAs) that have been shown to inhibit breast cancer cell growth in vitro and tumor growth in animals requires desaturation at C-6 of linoleic or alpha-linolenic acid. This observation, combined with reports that many tumors and tumor cell lines are deficient in Delta(6) desaturation and/or contain low levels of 6-desaturated EFAs, has led to the suggestion that loss of Delta(6) desaturating ability is relevant to the process of malignant transformation. This study was undertaken in search of direct evidence that malignant transformation of mammary epithelial cells alters EFA metabolism. We used two cell lines derived from the spontaneously immortalized human mammary epithelial cell line MCF-10A and expressing either the c-Ha-ras protooncogene (MCF-10AneoN) or an activated c-Ha-ras oncogene (MCF-10AneoT), and a cell line immortalized by transfection of human mammary epithelial cells with SV40 T antigen. We compared these cell lines in terms of ability to convert EFAs (30 mu M) to other EFAs of the same family. MCF-10AneoT cells lose the ability to perform Delta(6) and Delta(4) desaturations, whereas MCF-10AneoN cells and the SV40 T antigen-transformed cell line do not. No significant changes in growth response to culture with 6-desaturated EFAs were noted for MCF-10AneoT cells compared with MCF-10AneoN and parental MCF-10A cells, suggesting that FA metabolism alone cannot account for the effects of EFAs on the growth of neoplastic and non-neoplastic mammary epithelial cells.