Different growth responses to agents which elevate cAMP in human melanoma cell lines of high and low experimental metastatic capacity.

Human melanoma variants of low and high experimental metastatic activity, which had been derived from the same parental line, showed markedly different growth responses to agents which elevated intracellular cAMP. The high metastatic line had a significant decrease in in vitro proliferation following treatment with cholera toxin (10(-9) M) and forskolin (100 microM), with both agents causing virtual cessation of cell growth after 3-5 days incubation. Pre-treatment with 10(-9) M cholera toxin reduced colony forming ability to 11-15 per cent of control values, saturation densities were decreased to 10-25 per cent of controls and these cytostatic responses were accompanied by changes in cellular morphology. Lung colonising capacity of this cell line after i.v. injection into athymic mice was reduced significantly by prior exposure to cholera toxin (a median of 2 lung nodules versus 26 lung nodules for untreated, control cells). In contrast, low metastatic cell lines showed no significant growth inhibition in the presence of these agents. Cholera toxin (10(-9) M) reduced colony forming ability of these cells to only 74 per cent of control values and there were no significant decreases in growth rate nor any morphological changes in response to either cholera toxin or forskolin. The variable response obtained in the cell lines appeared neither to be a consequence of variation in induced levels of intracellular cAMP nor in differences between the cell lines in response to the same agent; forskolin (100 microM) induced a maximal 25-fold elevation and cholera toxin (10(-9) M) a 2.5-fold elevation increase in cAMP. These data show that highly metastatic variants of a human melanoma cell line differ from their less metastatic counterparts in the way they respond to agents which elevate the second messenger molecule cAMP.

Adhesion characteristics of human melanoma cell lines of varying metastatic potential.

The adhesive behaviour of a series of human melanoma cell lines, of varying metastatic potential, to basement membrane and stromal components was investigated in vitro. Experimental metastatic propensity was assessed from the number of pulmonary nodules formed after i.v. injection of cells into BALB/c nude mice. All cell lines showed similar kinetics of attachment when tested on plastic, type-I collagen films, type-I collagen hydrated gels, fibronectin, laminin type-IV collagen substrates and bovine aortic endothelial monolayers. Fibronectin-coated plastic compared to plastic alone produced increased cell attachment and spreading to the same extent in all the cell lines. The melanoma lines attached preferentially to cryostat sections of lung compared to other organs reflecting the pattern of organ involvement of metastasis in vivo. However, no significant quantitative differences in attachment to lung sections were seen between melanoma variants of differing metastatic capacities. Cells labelled with [125I]iododeoxyuridine to determine their initial organ distribution following i.v. injection showed that tumour-cell arrest was not significantly changed enough to explain the differing metastatic capacities. Thus it appears that adhesive properties of these melanoma cells are not correlated with their capacity to form metastases in vivo.

Mammary gland morphogenesis in vitro: extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line.

The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel.

DNA methylation levels in human and murine melanoma cell lines of varying metastatic potential.

DNA methylation levels were measured in a series of murine and human melanoma cell lines consisting of matched variants of low and high experimental metastatic capacity. The percentage of cytosine residues modified to 5-methylcytosine ranged between 2.13-3.92% in these lines. Ten cell lines were established in culture from individual lung tumor nodules produced in nude mice by i.v. injection of DX-3 human melanoma cells. Upon reinjection into groups of nude mice the individual lines manifested marked diversity for lung nodule formation (median number of pulmonary tumor nodules ranging from less than 10/group-greater than 100/group). DNA methylation levels in these lines were also heterogeneous (range 1.59 +/- 0.13 (SD)-4.04 +/- 0.15%) but no correlation was detected between methylation status of the genomic DNA and metastatic capacity.

Regeneration of mammary glands in vivo from isolated mammary ducts.

Rat mammary ducts, free of buds, can alone regenerate complete mammary trees when transplanted into the interscapular fat pads of syngeneic host rats. All the main mammary cell types are identified within such outgrowths. Epithelial cells, which show the presence of milk fat globule membrane antigens and microvilli on their luminal surfaces, line the ducts. Basal cells surrounding the ducts show characteristic features of myoepithelial cells: immunoreactive actin and keratin within the cytoplasm, myofilaments, pinocytotic vesicles and hemidesmosomal attachments to the basement membrane. Cells within the end buds and lateral buds, however, show few if any cytoplasmic myofilaments and are relatively undifferentiated in appearance. Intermediate morphologies between these cells and myoepithelial cells are seen nearer the ducts. In this respect they exactly resemble the cap cells found in terminal end buds (TEBs) of normal mammary glands. Occasional epithelial cells within alveolar buds show the presence of immunoreactive casein, which is a product of secretory alveolar cells in the normal rat mammary gland. Dissected terminal end buds can regenerate similar ductal outgrowths. Thus, ductal tissue alone can generate all the major mammary cell types seen in the normal gland, including the cap cells.

Increased experimental metastatic capacity of a murine melanoma following induction of differentiation.

Because of the interest in possible links between defective differentiation and cellular malignancy, the effects were examined of induced cell differentiation upon the experimental metastatic potential of the sublines F1 and F10 of the B16 mouse melanoma. These cell lines normally have low and high rates, respectively, of colonization of the lungs of mice after i.v. injection. Cellular differentiation was assessed by pigmentation and tyrosinase activity. In both cell lines, low and high levels of differentiation could reproducibly be generated by culture, respectively, at a low extracellular pH and at a higher pH in the presence of a melanocyte-stimulating hormone. Surprisingly, in both lines the cells grown under conditions promoting differentiation showed a markedly higher rate of experimental metastasis, despite their slower proliferation in culture and in subcutaneous tumor implants, than the poorly differentiated cells. Radiolabeled well- and poorly pigmented cells were not initially deposited at significantly different rates in the lungs of mice after i.v. injection. However, subsequent retention in the lungs fell more quickly for the poorly differentiated cells. As indicated by tests in vitro, this difference appears not to be due to differential cytotoxicity by either host macrophages or natural killer cells, and it is under further study.

Enhanced experimental metastatic capacity of a human tumor line following treatment with 5-azacytidine.

We have studied the effect of the nucleoside analogue 5-azacytidine (5-azaC) a known activator of gene expression, on the metastatic behavior of the human melanoma cell line DX3. After exposure to 3 microM 5-azaC for 24 h cells were carried through five subcultures before being tested for their metastatic capacity. Following i.v. injection in BALB/c nude mice, cells treated with 5-azaC showed a 40-fold increase in the number of lung tumor nodules as compared to that obtained with control cells. Tumors resulting from 5-azaC-treated cells exhibited a 5-fold increase in mitotic figures, considerable cellular pleomorphism, and variation in the histological character of the individual nodules. These in vivo differences were not reflected in vitro where treated and untreated cells showed no significant differences in morphology, karyotypes, growth rates, saturation densities, or plating efficiencies. Cells populating the lung tumor nodules retained their increased metastatic capacity through successive cycles of growth in vitro followed by reinjection into nude mice and indeed the ability of the cell lines to form lung tumors was increased with the succeeding cycles in vivo. These cell lines, each established from individual tumor nodules, maintained a human karyotype and reacted positively with human-specific antiserum. Characterization of these lines showed that while there were no alterations in in vitro growth behavior the selected lines exhibited a decreased latency period for s.c. tumor formation, increased retention in the lungs following i.v. injection, and enhanced lung nodule formation (60- to 80-fold compared to control cells). Examination of genomic DNA showed that treatment with 5-azaC brought about a significant decrease (50%) in the percentage of methylated cytosine residues but this extensive hypomethylation was not maintained in the lung tumor nodule-derived cell lines.

Dedifferentiation of rat mammary myoepithelial-like cell lines after passage in vivo or cloning in vitro.

Myoepithelial-like cell lines from normal mammary glands of neonatal Ludwig Wistar rats, rat mammary (Rama) 401 and Rama 704E, were injected into fat pads of syngeneic animals or were single-cell cloned in vitro. Rama 401 produced tumors that were predominantly composed of elongated cells, while the subclones of both cell lines yielded multilayered structures of elongated cells when grown on floating 0.3% collagen gels in vitro. Immunocytochemical analysis of histologic sections for markers of myoepithelial cells revealed that anti-actin-myosin and human keratin sera failed to stain the Rama 401 tumor cells or subclones of both cell lines on collagen gels, but both were stained with antilaminin serum. Immunofluorescent analysis of cultures of Rama 401 tumors showed that the resulting elongated cells failed to stain with antikeratin serum, but abundant staining was observed with antilaminin and antivimentin sera, as in the tumors. Ultrastructural analysis of the Rama 401 tumor cells identified intermediate junctions and extracellular basement membrane-like material in the vicinity of plasma membrane-associated pinocytotic vesicles, but neither true desmosomes nor myofilamental bundles were observed. Thus growth of rat mammary myoepithelial-like cells as tumors in syngeneic animals or as subclones in vitro can lead to selective loss of myofilaments and prekeratin-containing intermediate filaments. Similar relatively undifferentiated elongated cells may be responsible for some of the cellular heterogeneity observed in certain carcinogen-induced rat mammary tumors.

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study.

Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumour cells surrounded by connective tissues. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions and reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.

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.

Isolation and differentiation of cloned epithelial cell lines from normal rat mammary glands.

Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels will form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cell lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pinocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.

Cellular composition and organization of ductal buds in developing rat mammary glands: evidence for morphological intermediates between epithelial and myoepithelial cells.

In the developing rat mammary gland, terminal end buds (TEBs), lateral buds and alveolar buds represent the major sites of morphogenetic activity and cellular differentiation. The morphology and cellular composition of these buds from 20-to 22-day-old rats and cycling rats have been studied by immunocytochemical and electron microscopic techniques. The mammary buds are composed of a heterogeneous collection of cells including epithelial and myoepithelial cells, irregular loosely adherent cells, and occasional large clear cells. The irregular, loosely packed cells or cap cells are mainly situated around the periphery of the TEBs and lateral buds. "Chains" of irregularly shaped cells also extend from the peripheral cap cell layer to the center of the TEB; and, where they converge on lumina, they display microvilli and junctional complexes. At the tips of the end buds, the cap cells are of undifferentiated appearance; however, similar cells situated toward the subtending mammary ducts show a gradation in ultrastructure to that of myoepithelial cells. This change is accompanied by an increase in the amounts of immunoreactive myosin and keratin seen within the cells and a 200-fold increase in the thickness of the basement membrane. In contrast, the peripheral cells of the alveolar buds are more closely packed, contain a greater number of myofilaments, and show increased staining with antisera to myosin. We suggest that the undifferentiated cap cells do not represent a discrete cell type, since they show transitional forms to myoepithelial cells within the subtending mammary ducts, and that the tendency toward the myoepithelial phenotype is predominant in the more differentiated structures, the alveolar buds.

Loss of myoepithelial cell characteristics in metastasizing rat mammary tumors relative to their nonmetastasizing counterparts.

A series of WF rat mammary tumors comprising one transplantable nonmetastasizing line (MT-W9), two predominantly lymphatic (SMT-2A and SMT-077) and one lymphatic and hematogenous (MT-450) metastasizing transplantable lines, and 7,12-dimethylbenz[a]anthracene (DMBA)-induced nonmetastasizing primary tumors was examined for the presence of epithelial and myoepithelial cell characteristics with the use of immunocytochemical techniques. Tumor cells staining for myosin were only occasionally observed in a basal orientation in glandular structures in sections of DMBA-induced and MT-W9 tumors; anti-laminin serum stained the peripheries of the glandular structures in the DMBA-induced and MT-W9 tumors but failed to stain the SMT-2A and SMT-077 tumor cells. In the nonmetastasizing tumors immunologically detectable keratin occurred mainly in the outer cellular layer of glandlike structures, whereas milk fat globule membrane immunoreactivity occurred primarily in the luminal cells. Both these types of immunoreactivity were observed in MT-450 tumor cells, but the pattern of keratin staining was random. No such immunoreactivity occurred in SMT-2A or SMT-077 tumors. No tumor cell-associated staining for fibronectin was seen in any of the tumors examined, although host stromal components stained intensely. The nonmetastasizing tumors contained cuboidal epithelial cells with lumen formation, surface microvilli, and intercellular junctional complexes, together with a relatively undifferentiated elongated cell component. Other elongated cells showed hemidesmosomes, pinocytotic vesicles, tonofilaments, and small bundles of cytoplasmic filaments, suggesting gradations toward a myoepithelial phenotype. The MT-450 tumors were ultrastructurally similar to the nonmetastasizing tumors, but no features of myoepithelial cells were seen, although some cuboidal epithelial cells exhibited prominent tonofilaments. The SMT-2A and SMT-077 tumors consisted of nests of cuboidal-like cells with highly pleomorphic nuclei and much intercellular collagen. The results indicate a progressive loss of cellular differentiation characteristics, particularly those of the myoepithelial cell with increasing malignancy in this system.

Synthesis of basement membrane proteins by rat mammary epithelial cells.

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.

Two forms of tumors in nude mice generated by a neoplastic rat mammary stem cell line.

The rat mammary (Rama) epithelial stem cell line Rama 25, isolated from a dimethylbenz[a]anthracene-induced tumor, differentiates in culture into elongated cells. The uncloned elongated cells or "floaters" and one cloned elongated cell line, Rama 29, have been identified previously as containing some cells with myoepithelial characteristics. Both Rama 29 and the floaters contained a spectrum of different morphological forms, the multipolar and bipolar representatives of which were isolated by cloning cell lines. After six passages in culture, the ratio of bipolar to multipolar forms, proliferation rates, and final saturation densities usually increased, and at still higher passages, morphologically transformed foci of criss-cross cells were observed in Rama 29 cultures. From these, two morphologically transformed cell lines were isolated, Rama 29:T and Rama 521. Rama 25 and a similar subclone Rama 259, Rama 29:T, and Rama 521 readily formed tumors when injected into nude mice, whereas both the cloned (Rama 29, Rama 502, and Rama 503) and uncloned elongated cell lines (floaters) obtained directly from Rama 25 failed to do so within 12 weeks. Rama 25 and Rama 259 formed tumors which contained glandular-like areas, sheets of cuboidal cells, and spindle-cell areas, whereas Rama 29:T and Rama 521 formed tumors which contained only spindle cells. Immunocytochemistry showed that glandular areas stained with antibodies to rat milk fat globule membranes; that spindle-cell areas stained with antibodies to laminin, type IV collagen, and myosin; and that Rama 25- and Rama 259-induced tumors contained spindle-cell areas which also stained with antibodies to keratins. Ultrastructural analysis confirmed the presence of glandular-like structures and showed that spindle-cell regions contained mesenchymal-like cells with varying myoepithelial characteristics. Thus, Rama 25 and Rama 259 cells yielded some glandular-like regions and some myoepithelial cells in their tumors, while Rama 29:T and Rama 521 cells yielded some myoepithelial-like cells in their tumors without any glandular elements.

Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland.

Using antisera to specific proteins, the localization of the rat mammary parenchymal cells (both epithelial and myoepithelial), the basement membrane, and connective tissue components has been studied during the four physiological stages of the adult rat mammary gland, viz. resting, pregnant, lactating, and involuting glands. Antisera to myosin and prekeratin were used to localize myoepithelial cells, antisera to rat milk fat globule membrane for epithelial cells, antisera to laminin and type IV collagen to delineate the basement membrane and antisera to type I collagen and fibronectin as markers for connective tissue. In the resting, virgin mammary gland, myoepithelial cells appear to form a continuous layer around the epithelial cells and are in turn surrounded by a continuous basement membrane. Antiserum to fibronectin does not delineate the basement membrane in the resting gland. The ductal system is surrounded by connective tissue. Only the basal or myoepithelial cells in the terminal end buds of neonatal animals demonstrate cytoplasmic staining for basement membrane proteins, indicating active synthesis of these proteins during this period. In the secretory alveoli of the lactating rat, the myoepithelial cells no longer appear to form a continuous layer beneath the epithelial cells and in many areas the epithelial cells appear to be in contact with the basement membrane. The basement membrane in the lactating gland is still continuous around the ducts and alveoli. In the lactating gland, fibronectin appears to be located in the basement membrane region in addition to being a component of the stroma. During involution, the alveoli collapse, and appear to be in a state of dissolution. The basement membrane is thicker and is occasionally incomplete, as also are the basket-like myoepithelial structures. Basement membrane components can also be demonstrated throughout the collapsed alveoli.

Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland.

A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes project from the cell surface and numerous membrane pinocytotic vesicles can be seen. The cytoplasm is filled with linear arrays of 5- to 7-nm filaments with occasional dense foci. Cell junctions with associated 8- to 11-nm tonofilaments are also observed. Immunofluorescence techniques reveal actin and myosin filaments and also intermediate filaments of both prekeratin and vimentin types. Rama 401 cells secrete an amorphous material that, when an immunoperoxidase technique is used, stains with antibodies to basement membrane-specific type IV collagen. Localized densities of the cell membrane, which resemble hemidesmosomes, are located adjacent to these extracellular deposits. Immunofluorescence staining and immunoprecipitation techniques reveal that the cells also synthesize two other basement membrane proteins, laminin and fibronectin. The type IV collagen consists of two chains with molecular weights of 195,000 and 185,000.