Cell Organization and Responsiveness to Hormones in Vitro: Genesis of Domes in Mammary Cell Cultures

Transcription

1 AMER. ZOOL., 15: (1975). Cell Organization and Responsiveness to Hormones in Vitro: Genesis of Domes in Mammary Cell Cultures CHARLES M. MCGRATH Division of Biology, Michigan Cancer Foundation, Detroit, Michigan SYNOPSIS. Domes are multicellular structures generated from confluent monolayers of mammary epithelium under the influence of insulin and a corticosteroid hormone. The hemicyst structure and occurrence patterns of domes suggest an in vitro analogy to organized acini of mammary parenchyma. Two activities of dome cells, vegetative replication of the mammary tumor virus and synthesis of casein, suggest a functional analogy between domes and acini. The corticosteroid hormone is considered the primary hormonal stimulant for dome formation. Evidence is presented that RNA and protein synthesis is required for the corticosteroid effect, as are intact active transport functions of epithelial cells. Domes were first observed in mass cultures of neoplastic mouse mammary epithelial cells (McGrath and Blair, 1970). Due largely to the focal piled-up growth of cells above the plane of the monolayer in domes, their occurrence in cultures of neoplastic cells, and localization of the mammary tumor oncornavirus (MMTV) in the foci, cells in these structures were considered candidates for viral-induced transformants. More extensive studies have revealed that they are not foci of cellular transformants, but organized cellular structures which are generated under the influence of hormones. In this communication I have attempted to summarize present knowledge concerning dome genesis and the activities of cells in domes, which suggest that these structures represent a model system for studies of hormonal induced changes in organization of cells in vitro which lead to secretory acinar differentiation. STRUCTURE OF DOMES Domes are hollow three-dimensional cellular structures which are generated Much of the work described in this report was conducted in the laboratories of Drs. P. B. Blair, S. Nandi, and K. B. DeOme. The author is grateful for the support received therefrom. I also acknowledge support from an institutional grant to the Michigan Cancer Foundation from the United Foundation of Detroit, Michigan. under the influence of insulin and a glucocorticoid hormone in monolayer cultures of mammary epithelial cells (McGrath, 1970, 1971). Figure 1 is a schematic illustration of dome topography, based on a number of light and electron microscopic observations of murine mammary epithelial cell cultures by Pitelka and her colleagues (see McGrath, 1970; Pitelka et al., 1974). They are composed of a shell of epithelium in a single (occasionally partially double) layer of epithelial cells surrounding a fluid-filled lumen which is ionically insulated from the bulk culture fluid. Microvillae project from the mediumbathed surface of cells in domes as well as in cells confined to the monolayer. Cells in domes are 1.5 to 2.0 times larger than cells in the monolayer. The base of the dome is the culture vessel substrate itself with large, flat, functionally undefined cells attached thereto (McGrath, 1970). Domes range in size from 75/u. to 1 mm in basal diameter. Cells in domes (as well as in the monolayer) are linked at their upper (medium-bathed) edges by tight junctions (Pitelka et al., 1973). It is likely that these junctions function as a permeability barrier between the two fluid compartments, which accounts for the ionic insulation of the inner fluid compartment from the bulk culture media (McGrath, 1970). Low resistance junctions are also functional between epithelial cells in domes as shown by ionic coupling data (McGrath, 1970). These low 231

2 232 CHARLES M. MCGRATH G.RM DC BC. M.V. T.J. MC FIG. 1. Dome structure schematized. M.V., microvillae; L.R.J., low resistancejunctions; G.P.M., glycoprotein matrix; D.C., dome cell; B.C., basal cell; T.J., tight resistance pathways between cells may correspond to the gap junctions observed by Pitelka et al. (1973) in monolayer cultures of mouse mammary epithelium. An extracellular glycoprotein matrix is exuded from dome cells. This surface covering extends 10 to 20 cell diameters from the base of the dome into the monolayer. The significance of this material as it relates to glucocorticoid effects on mammary cells will be presented elsewhere (McGrath et al., unpublished). OCCURRENCE OF DOMES Only those mammary cells which organize into acinar structures in vivo have exhibited the capability to organize into domes in vitro. These include epithelial cells from virgin and midpregnant mammary glands (McGrath et al., 1972), preneoplastic alveolar nodules (unpublished) and acinar mammary tumors (McGrath et al., 1972). Both MMTV-producing and non-producing cells from the same mouse strain exhibit equal propensity for dome formation (McGrath, 1971; Soule et al., 1973&). Dome cells in tumor cell cultures are truly neoplastic, suggesting that neither the capability of cells to organize into discrete acini nor the capability of the cells to form domes are lost as a consequence of neoplastic transformation. The implications of these findings for models of mammary cell carcinogenesis have been discussed (McGrath et al., 1972). Cells which do not organize into acini in vivo, including fibroblasts, fat cells, epidermal cells (unpublished), and non-acinar junctions; M.C., monolayer cell. See text for further details. neoplastic mammary epithelial cells (McGrath et al., 1972) exhibit no propensity to organize into domes in vitro. Acinar mammary epithelial cells from mouse (Yagi, 1973), rat (unpublished), cow (Ebner et al., 1961), and man (Soule et al., 1973a) all have exhibited dome-forming capabilities. Structures which bear topographic resemblance to domes and also exhibit some secretory activity have been observed in monolayer cultures of normal thyroid and salivary epithelial cells (unpublished), as well as neoplastic kidney (Leighton et al., 1969) and cervical (Auersperg, 1969) epithelial cells, suggesting the possibility that all secretory acinar cell derivatives have some dome-forming capability. ACINAR ACTIVITIES OF DOME CELLS Differentiated activities of acinar (or alveolar) mammary cells in monolayer culture have been studied almost exclusively in mouse systems. Two such activities have been attributed to dome cells: (i) vegetative replication of the mouse mammary tumor virus; (ii) synthesis of casein. Vegetative replication (replication through maturation and release of virus particles from cells) of MMTV takes place almost exclusively in cells organized as alveoli or lobules in the intact mouse mammary gland or mammary tumors (Nakayama, 1968; McGrath etal., 1972). In that sense, then, vegetative replication of MMTV is a marker for epithelial cells at that stage of differentiation. When epithelial cells from either midpregnant normal mammary glands or acinar tumors, which

3 DOMES IN MAMMARY EPITHELIAL CELL CULTURES 233 are infected with the MMTV, are plated in primary monolayer culture, the cells which organize into domes after treatment with a corticosteroid (cortisol or dexamethasone) are the primary foci of virus release, while cells confined to the monolayer are much less active (McGrath, 1971; McGrath et al., 1972). This suggests that in terms of MMTV replicational functions, cells organized as domes may be analogous to alveolar cells in vivo. Only 40 to 60% of domes contain cells active in release of MMTV (McGrath et al., 1972). The significance of this heterogeneity in activity is unknown. It may be due to the fact that many dome cells are in equilibrium with the monolayer cells interconverting with cyclic regularity (Visser and Prop, 1974), since the percentage of MMTV-releasing cells in domes is significantly elevated when cells are incubated at lower temperatures than 37 C, incubation conditions which stabilize the equilibrium toward dome maintenance (McGrath et al., unpublished). There is also some evidence that cells in domes respond to prolactin to synthesize casein. The conditions under which these results were obtained have been reported (McGrath, 1970). We have recently established a radioimmunoassay for both casein (kappa casein) and a-lactalbumin (Rose and McGrath, unpublished). Application of this quantitative technique to assays for differentiative functions of dome and nondome cells is in progress. Confirmation of the earlier data would be a strong suggestion that domes are functional analogues of mammary alveoli. HORMONAL FACTORS IN DOME GENESIS Domes are generated from confluent, non-dividing monolayers of mammary epithelial cells. Both insulin and a corticosteroid hormone are necessary for dome formation. In limiting concentrations of serum (High concentrations [> 10%] of certain lots of calf or fetal calf sera will replace both insulin and the steroid for dome genesis. It is not known if this effect is due to the presence of threshold concentrations of the hormones in the sera or to unknown factors in some sera which act like insulin and cortisol), insulin stimulates mitosis of the epithelial cells during preconfluent stages of culture growth. Though the insulin-stimulated division of epithelial cells appears necessary for subsequent dome formation in stationary cultures, domes are not formed by dividing cells (Table 1) (McGrath, 1971). Whether the insulin-stimulated cell division per se is fundamental to dome formation or is only necessary to establish the crowded cell conditions at saturation required for dome formation in response to the corticosteroid hormone (McGrath, 1970) is not presently known. The crowded conditions are not Inhibitors None (control) Actinomycin D (10~ 6 M) Puromycin (10" 5 M) Cytosine arabinaside (10 /xg/ml) Ouabain (10-4 M) TABLE 1. Effect offour inhibitors on dome formation. Percentage RNA, protein and DNA synthesis" 100% 2% 7% 5%" Not Tested Dome formation (# domes/30 cm 2 ) Neoplastic mouse mammary epithelium was plated and grown in primary culture as described (McGrath, 1971). After 4 days growth in medium containing 10 A*g/ml insulin, inhibitors were added for 4 hr. Cortisol (5 /xg/ml) was then added, with the inhibitors still present. Domes were counted 12 hr after addition of cortisol, an interval coincident with maximum dome formation. a Inhibitory effects of RNA and protein synthesis were measured at the time of cortisol addition by measuring incorporation of 3 H-Uridine or 3 H-Leucine into TCA precipitable cell material during an 1-hr incubation period. Control values for RNA were 46,000 cpm/mg protein; for protein, 31,000 cpm/mg protein. b Effectiveness of cytosine arabinoside as an inhibitor of DNA synthesis was measured by 3 H-Thymidine incorporation into DNA of log phase cells. The control value for DNA was 2,200 cpm/mg cell protein. c The inhibitory effect of cytosine arabinoside on dome formation was measured in an identical way as for actinomycin D and puromycin (in stationary cultures)

4 234 CHARLES M. MCGRATH established, however, if insulin is excluded from the culture media (unpublished). The corticosteroid hormone is the prime mover in dome genesis. Cortisol has been used most extensively (McGrath, 1971), but dexamethasone is as efficient in dome formation in murine cultures at a fourfold lower concentration than the most effective dose of cortisol (Soule and McGrath, unpublished). As stated above, only crowded monolayers will respond to the steroid hormone. Crowded conditions were also reported as necessary for "domes" to form in cervical carcinoma cell cultures (Auersperg, 1969). The extent of cell crowding is influenced by the density of cell plating. A fivefold difference in plated cell density results in a two- to threefold difference in saturation density (Hosick, 1974). This difference is sufficient for a qualitative difference in responsiveness to cortisol for dome formation (McGrath, 1970). Reorganization of monolayer cells into dome cells begins as early as 1 hr after addition of cortisol to non-dividing cultures of mouse mammary epithelial cells, reaching maximum numbers by 12 hr after addition. Addition of inhibitors of RN A and protein synthesis 4 hr prior to the addition of cortisol have shown that cortisol-stimulated new RNA and protein synthesis is required for dome formation (Table 1). Addition of cytosine arabinoside in concentrations (10 /u.g/ml) sufficient to block 95% of DNA synthesis had no effect on cortisol-stimulated formation of domes (Table 1), confirming earlier observations that cell division is not involved in their formation. Ouabain (10~ 4 M) also inhibits cortisolstimulated dome formation when added 4 hr prior to the hormone (Table 1), suggesting that active transport functions of epithelial cells (Sharp and Leaf, 1966), modified by cortisol, are important in the genesis of domes. The involvement of solute and solvent movement in dome genesis can also be inferred from the fact that intact domes are in equilibrium with the monolayer, rising and falling with cyclic periodicity (Visser and Prop, 1974). THE MECHANICS OF DOME FORMATION: A SPECU- LATION At present, one can only speculate on the mechanics of dome formation by cortisol or other corticosteroids. Fluid transport is undoubtedly a major feature in generating and maintaining domes. It has been shown that stimulation of Na + and water transport across toad bladder epithelial membranes by the corticosteroid aldosterone, a function sensitive to Ouabain, requires RNA and DNA synthesis which is stimulated by the steroid (Sharp and Leaf, 1966). The requirement for RNA and protein synthesis for dome formation may be explicable in those terms. Furthermore, occluding junctions between cells in the dome are likely important in maintaining solute and solvent differentials in the lumen of a dome. The focal occurrence of domes may be explicable by differentials in anchorage of cells to the culture vessel. In this regard, the basal cell(s) of domes and the glycoprotein matrix surrounding domes may play a role, if epithelial cells adhere more strongly to plastic or glass than to the basal cell and if the matrix confers more substrate anchorage to those cells in the matrix. The cells in the glycoprotein matrix do adhere to the substrate more strongly than other epithelial cells in a culture, and recent results suggest that this feature is as important as fluid transport and permeability barriers in dome formation, since in low saturation density epithelial cultures, wherein no domes are formed (McGrath, 1970), both tight junctions and Na + transport functions are intact, but no glycoprotein matrix is synthesized and all cells exhibit equal adherence to the substrate (unpublished). This may be an explanation for the fact that low saturation density monolayers do not form domes. Clearly, more data are needed for an understanding of the mechanics of dome formation. The advantage of the culture system is in the ease with which the various elements in hormone-induced dome formation can be separated and studied independently. DISCUSSION Present information suggests that domes represent in vitro analogues of mammary

5 DOMES IN MAMMARY EPITHELIAL CELL CULTURES 235 secretory acini. This suggestion is based on: (i) the occurrence of domes only in cultures of mammary epithelium derived from acini-forming parenchyma; (ii) the hemicyst-like structure of domes, expecially the shell of epithelium, linked by tight junctions, surrounding a lumen; (iii) vegetative replication and secretion of MMTV in dome cells; and (iv) preliminary data on responsiveness to prolactin to synthesize casein. Quantitative data on differentiative functions of dome cells are needed to confirm the functional analogy between domes and acini. Toward this end, we have constructed a radioimmunoassay for casein and a-lactalbumin for mouse and human systems. Application of this technology to cultures of mouse and human mammary epithelial cultures have shown that not only normal, but neoplastic cells synthesize a-lactalbumin in the appropriate hormonal mileu and secrete this protein into culture media (Rose and McGrath, unpublished). Studies are in progress to evaluate the contribution of dome and non-dome cells in the synthesis and secretion of this protein. The changes in cell organization brought about by corticosteroid hormones which require RNA and protein synthesis and involve modification in active transport properties of mammary cells as well as glycoprotein metabolism make this culture system a desirable one to study molecular aspects of corticosteroid action on those functions of mammary cells. Studies on modification of transcriptional controls, for example, as performed by Tomkins and his colleagues (Levinson et al., 1971) on hepatoma cells, would provide information on the mechanics of controls active in those events which culminate in organizational changes. Glucocorticoid receptors have recently been detected in normal (Shyamala, 1973) and neoplastic (Shyamala, 1974) mammary cell membranes. Studies should be conducted to determine if cortisol or dexamethasone act through the receptor to modify the functional and topographic organization of mammary epithelium. The effectiveness of mineralocorticoid and glucocorticoid hormones as well as other steroid hormones could also be tested in this system. Finally, the retention of normal acinar organizational capabilities of neoplastic mammary epithelium must be considered in constructing models of mammary cell carcinogenesis. These cells stop dividing in culture at low (comparable to normal) saturation densities (McGrath et al., 1972; Hosick, 1974); they synthesize products of differentiated mammary epithelium such as casein (McGrath, 1970), and.a-lactalbumin (Rose and McGrath, unpublished); and they form essentially normal intercellular junctional complexes (Pitelka et al., 1973). Observations on primary mouse mammary tumors by Pierce and his colleagues (Wylie et al., 1973) have suggested that cells with histogenetic capabilities represent partially or fully differentiated, non-dividing progeny of malignant stem cells in a tumor. These authors suggest further that the non-dividing differentiated cell pool is senescent and does not reinitiate growth during the course of tumor growth. We have recently isolated clones of neoplastic mammary epithelial cells both exhibiting and not exhibiting histogenetic capabilities in cell culture. We have initiated studies to evaluate the malignant and differentiative potential and stability of these cells in an effort to clarify the contributions of dedifferentiation and multipotential stem cell growth to tumor growth and progression. REFERENCES Auersperg, N Histogenetic behavior of tumors. I. Morphologic variation in vitro and in vivo of two related human carcinoma cell lines. J. Nat. Cancer Inst. 43: Ebner, K. D., C. R. Hoover, E. C. Hageman, and B. L. Larson Cultivation and properties of bovine mammary cell cultures. Exp. Cell Res. 23: Hosick, H. L A note on growth patterns of epithelial tumor cells in primary culture. Cancer Res. 34: Leighton, J., Z. Brada, L. W. Estes, and G. Justh Secretory activity and oncogenicity of a cell line (MDCK) derived from canine kidney. Science 163: Levinson, B., G. Tomkins and R. Stellwagen Regulation of tyrosine aminotransferase synthesis. Studies in HTC cells with inhibitors of RNA synthesis. J. Biol. Chem. 246: McGrath, C. M Analysis of mammary tumor virus replication in tumor cell culture. Ph.D. Diss. Univ. California, Berkeley.

6 236 CHARLES M. MCGRATH McGrath, C. M Replication of mammary tumor virus in tumor cell cultures: dependence on hormone-induced cellular organization. J. Nat. Cancer Inst. 47: McGrath, C. M., and P. B. Blair Immunofluorescent localization of mammary tumor virus antigens in mammary tumor cells in culture. Cancer Res. 30: McGrath, C. M., S. Nandi, and L. Young Relationship between organization of mammary tumors and the ability of tumor cells to replicate mammary tumor virus and to recognize growth-inhibitory contact signals in vitro. J. Virol. 9: Nakayama, P Occurrence and distribution of B particles in Balb/cfc3H mammary gland. Master's Thesis, Univ. California, Berkeley. Pitelka, D. R., S. T. Hammamoto, and P. Pickett Ultrastructure and distribution of contacts between mouse mammary epithelial cells in vivo and in vitro. Page 24 in Proc. Vlllth Meeting on Mammary Cancer in Experimental Animals and Man. Sharp, G., and A. Leaf Studies on the mode of action of aldosterone. Recent Progr. Hormone Res. 22: Shyamala, G Specific cytoplasmic glucocorticoid hormone receptors in lactating mammary glands. J. Biol. Chem. 12: Shyamala, G Glucocorticoid receptors in mouse mammary tumors. J. Biol. Chem. 249: Soule, H. D., J. Vasquex, A. Long, S. Albert, and M. Brennan. 1973a. A human cell line from a pleural effusion derived from a breast carcinoma. J. Nat. Cancer Inst. 51: Soule, H., T. Moloney, J. Vazquez, and A. Long A cell line from a primary tumor arising in a D2 hyperplastic nodule. Proc. Amer. Ass. Cancer Res. 14:90. Visser, A. S.,and F. J. A. Prop "Domes", periodically expanding and collapsing secretory structures in cell cultures of mouse mammary tumors. J. Nat. Cancer Inst. 52: Wylie, C. V., P. Nakane, and G. B. Pierce Degree of differentiation in nonproliferating cells of mammary carcinoma. Differentiation 1: Yagi, M. J Cultivation and characterization of Balb/cfc3H mammary tumor cell lines. J. Nat. Cancer Inst. 51: