Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture

Epithelial cells dissociated from mammary glands of midpregnant mice and cultured with lactogenic hormones on plastic or collagen gel substrates have been shown to vary in their extent of differentiation, as identified by the presence of secretory organelles and accumulation and secretion of casein. Morphological and biochemical differentiation was obtained on floating collagen gels. At least four unique factors provided by the floating collagen gel substrates are not found on plastic substrates: access of nutrients to basolateral cell surfaces, close proximity of cells to the medium surface and gas phase, interaction of epithelial cells with stromal elements, and substrate flexibility permitting cell shape change. In this study, we have attempted to assess the relative contributions of these factors in the ultrastructural differentiation of mammary cells in culture. None of these factors alone is responsible for the differentiation achieved when all are present. The novel aspect of this research is the identification of the cells' apparent requirements for basolateral access to nutrients and for freedom to assume a preferred shape in order to achieve differentiation.     

Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix

Collagen gel matrix has been used successfully to promote sustained growth of human normal mammary epithelial cells in primary culture using serum-containing medium supplemented with hormones and growth factors (Nandi et al., 1932). Sustained growth can now be accomplished in a serum-free medium consisting of a 1:1 mixture of Ham's F12 and DMEM supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, cortisol, and BSA. Human normal mammary epithelial cells derived from reduction mammoplasties can be routinely propagated in this serum-free medium. The extent of growth and the resulting three-dimensional outgrowths in this serum-free medium, using the collagen gel matrix system, are comparable to those seen in serum-containing medium. This is the first demonstration of sustained growth of human normal mammary epithelial cells in serum-free primary culture.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Growth of normal human mammary cells in culture

Summary 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. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

A cell cycle study of human mammary epithelial cells.

The growth regulation of human mammary epithelial cells (HMEC) cultured in a growth factor/hormone-enriched (e.g. EGF, insulin) medium with bovine pituitary extract as the only undefined supplement was studied. The doubling times of the cultures, in which the cells appear in colonies, was 55-72 h, and a considerable intercolonial heterogenecity in proliferative activity could be demonstrated. However, every colony, irrespective of the size of the growth fraction, comprised a sub-population of rapidly growing cells which had a mean generation time of approximately 22 h. When insulin was removed from the culture medium, HMEC proliferation was inhibited. This growth inhibition was shown to be a result of a cell cycle-specific block.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Theophylline effects on normal uveal melanocytes in culture: an ultrastructural study

Theophylline enhances maturation and differentiation of uveal melanocytes. By electron microscopy, we showed that theophylline changes small, dendritic melanocytes into large, platelike cells; it also enhances DOPA reaction as evidenced by increased deposition of DOPA reaction products in dilated cisternae and vesicles around the Golgi region. The effect is partially reversible in choroidal melanocytes but irreversible in iridial cells. It appears that theophylline, in addition to inducing tyrosine activity, accelerates the maturation and/or aging that normally occurs in cultured melanocytes when incubation is prolonged.     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.     

Differential expression of cholinephosphotransferase in normal and cancerous human mammary epithelial cells

Membrane phospholipids as well as fatty acid profile of cell membrane phospholipids are altered in tumorigenicity and malignancy. Synthesis of total cellular phosphatidylcholine (PC) can be used as a marker for membrane proliferation in neoplastic mammary gland tissues. Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of PC, has an important role in regulating the acyl group of PC in mammalian cells. In this study, the effect of neoplasia on CPT was examined. The gene shows an elevated expression in cancerous (11-9-14) breast epithelial cell line when compared to that of normal non-tumorigenic (MCF-12A) breast epithelial cell line. Four nucleotide substitutions are observed in the cancer cell line. Of these, three are null mutations, but the third one shows an interesting serine to tyrosine substitution (at amino acid position 89 of our partial sequence which corresponds to position 323 of the CPT sequence reported as NM_020244 in GenBank) in 11-9-14 cells. The tyrosine is present in the right context of KSELYQDT, which directs tyrosine phosphorylation at the tyrosine site. Biochemical approach also reveals a 1.5-fold stimulation in CPT activity in 11-9-14 cells compared to that of the MCF-12A cells.     

Absence of fibronectin and presence of plasminogen activator in both normal and malignant human mammary epithelial cells in culture

Primary monolayer cultures of normal and malignant human mammary epithelial cells were tested for fibronectin by indirect immunofluorescence using antisera specific for fibronectin. This protein was not detectable on either the normal or malignant epithelial cells. Similar results were obtained for normal and malignant mouse mammary epithelial cell cultures. Control normal and transformed fibroblasts exhibited the expected result: the normal cells were positive and the transformed cells were negative. With the use of supernatant fluids from the same cultures or an agar-overlay assay on viable cells, high levels of plasminogen-dependent fibrinolytic activity were detectable in both the normal and malignant mammary cells. Thus, two characteristics that distinguish normal from transformed fibroblasts do not serve as markers of malignancy in mammary epithelial/carcinoma systems.     

Differentiation of mammary epithelial stem cells to alveolar-like cells in culture: cellular pathways and kinetics of the conversion process

The cuboidal epithelial stem cell line Rat Mammary (Rama) 25 can differentiate in culture to droplet, alveolar-like cells that form domes, secrete small amounts of casein, and bind peanut lectin after treatment with neuraminidase. Differentiation to droplet cells is accelerated by dimethyl sulfoxide (DMSO). Morphologically intermediate states (gray and dark) which occur in the order: cuboidal----gray----dark----dark droplet----doming cells have been identified along this pathway by time-lapse cinematography. The dark and dark droplet states are associated with increased peanut lectin binding capacity whereas casein is secreted mainly by cells in domes. Cells in cultures containing low concentrations of DMSO (less than 56 mM) acquire droplets predominantly in the dark state, whereas with higher concentrations of DMSO droplet formation is seen mainly in the gray state. Kinetic analysis both from time-lapse films and conventional microscopy, shows that increasing the concentration of DMSO prolongs the time spent in the gray state, decreases the time of initial appearance of droplet cells, and increases their subsequent rate of formation, without detectable effects on the rates of the remaining morphological transitions. DMSO also reduces the average rate of DNA synthesis and increases the average cell cycle time, particularly in the second (and subsequent) cell cycles after its addition. However, neither droplet nor doming cells are terminally differentiated. Thus a linear sequence of morphological states exists between the Rama 25 stem cells and the alveolar-like or more probably alveolar bud cells in vitro, and DMSO accelerates the overall conversion predominantly by truncating one of the steps in this pathway.     

To grow mouse mammary epithelial cells in culture

Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures.