Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: correlation with tumour type and progression.

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.

Effects of flutamide and hydroxy-flutamide on the growth of human benign prostatic hyperplasia cells in primary culture: a preliminary report.

Tissues from human benign prostatic hyperplasia [BPH] were collected from twelve patients undergoing routine transurethral resection of the prostate to relieve urine out-flow obstruction. Viable epithelial organoids were obtained after enzymatic digestion of the tissue. Primary cultures of epithelium were successfully maintained on collagen gel for up to 21 days. Immunocytochemical staining revealed that there was no expression of either desmin or vimentin in these cells; however, the anticytokeratin antibodies LP-34 (cytokeratins 4, 5, 6, 10, 13, 16, 17 and 18), LE-61 (cytokeratin 18) and CAM 5.2 (cytokeratins 7 and 8) all showed positive responses, indicating the epithelial nature of the cells. Cell growth was significantly increased in the presence of 3 x 10(-10) M testosterone propionate [TP] in the culture medium. The presence of the non-steroidal anti-androgens, Flutamide and Hydroxy-Flutamide [Flu-OH], in the concentration range 1.0-0.001 micrograms per ml of medium inhibited the growth in the presence of androgens in a dose-dependent manner. The anti-androgens failed to affect cell growth in the absence of TP. In view of these preliminary findings, it is postulated that the antiandrogens might be acting either by displacing the androgen from its receptor or alternately by inhibiting the activity of prostatic 5 alpha-reductase.

Divergent effects of interferons on the growth of human benign prostatic hyperplasia cells in primary culture.

Epithelial cells from human benign prostatic hyperplasia tissues were grown in primary cultures for up to 21 days and the effects of interferons on the growth of the cells were investigated. Interferon-alpha (Wellferon) showed growth inhibition both in the presence and in the absence of 3 x 10(-10)M testosterone propionate (TP) whereas interferon-gamma stimulated growth in a dose dependent manner under similar conditions. Interferon-beta had little effect on growth at the dose levels used in the study. The growth inhibition by interferon-alpha can be induced after stimulation of growth is achieved either with TP or with interferon-gamma. Implications of these findings for clinical use of these lymphokines is discussed.

The effects of interferons on the activity of alpha-glycerolphosphate dehydrogenase in benign prostatic hyperplasia cells in primary culture.

Human benign prostatic hyperplasia (BPH) tissues were obtained from patients undergoing transurethral resection of the prostate and viable cells from these were successfully maintained in primary cultures grown on collagen gel. The prostatic origin of the cells was confirmed by the measurement of prostate specific acid phosphatase and by scanning and transmission electron microscopy before and after immunostaining with human prostate specific antigen-antibody. The cell cultures were treated with various interferons (IFNs), both in the presence and absence of testosterone propionate (TP), for 72 hours and the activities of seven enzymes of carbohydrate metabolism were estimated in the cytosolic fraction of the cells. Treatment with TP induced a significant decrease in the activity of alpha-glycerolphosphate dehydrogenase (alpha-GPDH). Using this enzyme activity as a marker, the effects of various types of IFNs were investigated. IFN-alpha (wellferon) increased the activity of the enzyme both in the presence of one microgram./ml. of TP and in its absence whereas IFN-gamma inhibited the activity under similar conditions. The effect of treatment with IFN-beta in the presence of TP was biphasic in that there was an increase in the activity of the enzyme at the lowest concentration while at higher concentrations an inhibition of enzymic activity was observed. In the absence of TP IFN-beta inhibited the activity. The significance of these findings in terms of the clinical usefulness of IFNs is discussed and it is postulated that IFN-alpha (wellferon) might be effective in the treatment of metastatic carcinoma of the prostate in selected patients.

Cytochemical and biochemical evidence of cathepsin B in malignant, transformed and normal breast epithelial cells.

Human breast cancer cell lines, as well as transformed mammary epithelial cells (HBL-100) and growth-stimulated normal breast epithelial cells showed positive cytochemical reaction with the proteinase substrate 2-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methoxynapht halene, in the presence of 5-nitrosalicylaldehyde. The reaction product, small fluorescent granules, was distributed throughout the cytoplasm, in the perinuclear zone, in some cytoplasmic projections, and at the cell surface. Using a panel of various proteinase inhibitors, we found that the formation of the reaction product was an enzymic function of a cysteine proteinase. Using the substrate 7-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin, we evaluated some biochemical properties of the cysteine proteinase, including pH-activity profile, pH stability, apparent relative molecular mass and sensitivity toward various proteinase inhibitors. We found that the proteinase from the studied breast epithelial cells exhibited characteristics of a mature form of cathepsin B. Taken together, the cytochemical and biochemical data provide evidence that human breast epithelial cells of cancer origin, as well as in the transformed or growth-stimulated state express active cathepsin B and compartmentalize it into specific subcellular sites.

Loss of production of myoepithelial cells and basement membrane proteins but retention of response to certain growth factors and hormones by a new malignant human breast cancer cell strain.

Digestion of primary breast cancers and their metastases with collagenase yields cell clusters which can be selectively isolated from stromal cells and from the less malignant-looking epithelium of the primary tumors by their failure to attach as rapidly to collagen gel. Continued passage in culture of one preparation of cell clusters has yielded a continuously growing cell strain, termed Ca2-83. This strain continues to grow mainly as cell clusters with doubling times of 10 to 14 days, although some clusters eventually adhere to plastic substrata. Two morphological extremes of cell were observed, smaller polygonal or cuboidal cells and larger, often-multinucleated cells which contain fat droplets. Cell clusters grew in a gland-like pattern similar to those of the original carcinoma and formed small nodules in 50% of recipient nu/nu mice. Both morphological forms of Ca2-83 in culture or in tumor nodules stained immunocytochemically with epithelial cell-specific antisera to epithelial membrane antigens and to human keratins but not to laminin or actin. Cultures of Ca2-83 failed to synthesize laminin under conditions where its synthesis was observed in a rat myoepithelial cell line. Ultrastructural analysis of the cell clusters has identified microvilli coated with epithelial membrane antigens and junctional complexes typical of secretory epithelia in both morphological forms, but no characteristics of myoepithelial cells or basement membranes were observed. The DNA content of the cultures increased in response to serum, a bovine pituitary fraction, and insulin. Numbers of cell clusters were also increased in the presence of culture medium exposed to preadipocytes, myoepithelial- or mesothelial-like cells/stromal cells, or to prostaglandin E2.

A subclass of luminal epithelial cells in the human mammary gland, defined by antibodies to cytokeratins.

Two monoclonal antibodies, BA16 and BA17, have been developed using a detergent-insoluble extract of human mammary epithelial organoids as immunogen. Indirect immunofluorescent staining of cultured cells showed that the component reacting with the antibodies was filamentous and the intensity of staining was stronger in mitotic cells. Immunoblotting of cell extracts showed that both antibodies react with only one band of 40 X 10(3) molecular weight, which was present in keratin-enriched extracts of cells or organoids. Furthermore, the tissue distribution of the component reacting with the antibodies was that predicted for human keratin 19. The antibodies showed differences in the intensity of staining of cells or tissue sections fixed and prepared in different ways indicating that they reacted with different epitopes. The pattern of expression of the 40 X 10(3) Mr keratin by normal mammary epithelial cells was investigated by immunoperoxidase staining of tissue sections, cultured milk cells, and organoids of different sizes cultured in collagen gels. It was found that basal or myoepithelial cells did not express this keratin. Some heterogeneity of expression of this component was seen in luminal epithelial cells, found almost exclusively in the smaller structures. These cells did, however, express other keratins characteristic of luminal cells. The distribution in the mammary tree of the luminal cells that did not express the 40 X 10(3) Mr keratin appears to be similar to that expected for cells with the proliferative potential to produce new terminal ductal lobular units or an increase in branching of existing terminal ductal lobular units. It is shown that these cells have considerable proliferative potential by the fact that they form large colonies in milk cell cultures.

Effect of estradiol on nonmalignant human mammary cells in primary culture.

We have studied effects of estradiol on primary cultures of nonmalignant human mammary tissue collected surgically from fibroadenomas or during reduction mammoplasties. After enzymatic digestion, "organoids" made of epithelial cells organized in ductal or alveolar structure were grown in primary cultures (up to 12 days) on different substrata (glass, plastic, collagen-coated plastic, and floating collagen membranes). Transmission and scanning electron microscopy showed that these organoids were responsive to physiological concentrations of estradiol. Condensed chromatin of epithelial cells became dispersed following estrogen treatment. The plasma membrane of epithelial cells at the surface of the organoids was dramatically modified by estradiol, which increased the number and the length of the microvilli, as observed previously in the MCF7 breast cancer cell line (Vic et al., Cancer Res., 42: 667-673, 1982). This effect was not observed with the same concentrations of progesterone, dexamethasone, dihydrotestosterone, or 1 microM tamoxifen or in fibroblasts of the same tissue, demonstrating that epithelial mammary cells are specifically responsive to estradiol. By contrast, no effect of estradiol could be evidenced on the [35S]methionine-labeled proteins released into the medium by the organoids. The estrogen-regulated protein of Mr 52,000 was not found in the medium after purification by concanavalin A-sepharose or immunoprecipitation with specific antibodies to the Mr 52,000 protein from MCF7 cells. We conclude that nonmalignant mammary cells are responsive to estrogens in primary culture.

Growth of normal human mammary cells in culture.

Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times.

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands.

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.

Trypan blue: identification and teratogenic and oncogenic activities of its coloured constituents.

Three coloured substances frequently present as contaminants in commercial samples of trypan blue have been identified as those monoazo dyes in which 4-amino-3,3'-dimethyl-biphenyl, 4-amino-3,3'-dimethyl-4'-hydroxy-biphenyl or omicroc-tolidine are coupled to H-acid. These dyes have been synthesized and, together with purified samples of trypan blue, tested for teratogenic activity in mice and oncogenic activity in rats. Unpurified trypan blue was both teratogenic and oncogenic; purified trypan blue, was teratogenic but only weakly oncogenic; the monoazo dyes possessed neither activity. It is concluded that the main blue component of trypan blue is the teratogenic principle and that some as yet unidentified component of the purple fraction either is the main oncogenic principle or potentiates the action of the blue component.