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The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Furth-Wistar rats. Transfection of this stably diploid cell line with genomic DNA fragments from a human metastasizing breast cancer cell line yields cells which, when injected subcutaneously in syngeneic rats, give rise to secondary tumours in a number of the animals. From one such secondary lung tumour, a cell line was established designated Ca2-5-LT1. This cell line, when introduced into the syngeneic rat host, also showed the ability to metastasise. To determine key changes in gene expression that occur during the progression from Rama 37, the benign tumour-inducing cell line, to the metastatic derivative Ca2-5-LT1, a general method of subtractive hybridization has been employed. This procedure in conjunction with Northern blotting and nucleic acid sequencing has been used to identify mRNAs expressed differentially between the metastatic and nonmetastatic cell lines described above. So far, of the subtracted cDNAs that have been identified which represent differentially expressed mRNAs, a large proportion of these cDNAs corresponded to the mRNA for rat osteopontin (OPN). The mRNA for OPN was expressed at a ninefold higher level in the metastatic Ca2-5-LT1 cell line when compared to the nonmetastatic parental Rama 37 cell line. Rama 37 cells transfected with DNA from a human benign cell line failed to show elevated levels of OPN mRNA. Following transfection of Rama 37 cells with an expression-construct producing elevated levels of OPN, the newly-transfected cells, when introduced into the rat host, developed metastases in 55% of the animals that produced primary tumours. These experiments show that increasing the expression of OPN in a previously benign cell tine is sufficient to produce a metastatic phenotype in this particular rat mammary model.

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08-0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0-50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required.

The S-100 proteins are a structurally related family displaying diverse intracellular and extracellular interactions. One such protein p9Ka (also known as calvasculin), or its mRNA (also known as CAPL, 42A, 18A2, mts, pEL 98), becomes elevated upon changes in the growth and differentiation of cells. Overexpression of p9Ka in benign rat mammary cells induces the metastatic phenotype. In order to help understand the role of p9Ka in these processes, the molecular properties of recombinant rat p9Ka have been studied. Recombinant p9Ka forms multimers in vitro, which are not due to intermolecular disulfide bridges, it binds 2 mol of calcium ions/mol of protein, and the binding of calcium ions is strongly antagonized by monovalent and divalent cations tested. Immunofluorescence studies indicate that p9Ka is located on cytoskeletal elements in a pattern which is identical to actin filaments stained with phalloidin. In vitro, it is shown that recombinant p9Ka binds to sites on at least two intracellular polypeptides. These sites display the same binding capacity for p9Ka in extracts of cultured rat mammary cells which show widely differing levels of expression of natural p9Ka. The results suggest that the production of p9Ka, and not of its target molecules, may be associated with the changes seen in cultured cells.

Transfection of the stably diploid rat mammary epithelial cell line, Rama 37, which yields nonmetastasizing, adenomatous tumors in syngeneic rats with HindIII-fragmented DNA from malignant or nonmalignant human breast epithelial cell lines and the drug-resistance plasmid pSV2neo, yields transformants with a frequency of 10(-4) to 10(-5). The resultant cell lines form tumors with varying frequencies when injected s.c. into the mammary fat pads of syngeneic rats. Cells transfected with DNA from the malignant human breast carcinoma cell line, Ca2-83, or DNA from the human pleural effusion-derived cell lines, MCF-7 or ZR-75-1, yield transformants which metastasize to lungs and/or lymph nodes at high frequency, whereas transfection of HindIII-fragmented DNA from nonmetastatic human mammary epithelial cell lines, transfection of the drug-resistance plasmid pSV2neo alone, or nonspecific DNA such as salmon sperm DNA fails to yield transformants expressing the metastatic phenotype. Transfectants which metastasized were reestablished in culture and reinjected into syngeneic rats to confirm their metastatic properties. These transfectants yield rapidly growing tumors with reduced latent periods, which give rise to significant numbers of metastases. The karyotype of selected transfectants after passage in vivo remains stably diploid. Hybridization of a 32P-labeled oligonucleotide probe specific for the human Alu family of sequences to DNA from these transfectants reveals the presence of human-specific DNA sequences integrated into the genome. It is suggested that transfection of specific genomic DNA sequences from the malignant human cell lines can induce the metastatic phenotype in the nonmetastatic Rama 37 cell line in a genetically dominant manner, whereas genomic DNA from the nonmetastatic cells cannot confer metastatic properties to the Rama 37 cell line.

The messenger RNAs for the angiogenic acidic and basic fibroblast growth factors are expressed at a significantly higher level in samples of human benign neoplastic and hyperplastic tissue than in samples from breast cancers. However, approximately one in four malignant breast cancer samples contain basic fibroblast growth factor mRNA at the same level as in the benign lesions when basic fibroblast growth factor mRNA levels are corrected with respect to levels of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. A similar proportion of human malignant breast cancer cell lines express a high level of basic fibroblast growth factor mRNA. The results suggest that some malignant breast cancers and their constitutive carcinoma cells express abundant levels of basic fibroblast growth factor mRNA. The resultant production of basic fibroblast growth factor by breast cancer cells within some tumours may contribute to their development.

The secretion of transforming growth factor alpha (TGF alpha) and the expression of cell-surface receptors for epidermal growth factor (EGF) were measured in a series of human mammary cell lines. The amount of TGF alpha secreted by the cells did not correlate with the phenotype of the cells (epithelial or myoepithelial), the mechanism of immortalization of the cells (SV40 or spontaneous) or the source of the cells (normal mammary gland, benign hyperplastic lesion, malignant tumour). The level of expression of cell-surface receptors for EGF was markedly increased as a consequence of SV40-immortalization of mammary cells, but otherwise did not correlate with the phenotype of the cells or the source of the cells. Much of the increase was accounted for by the appearance of a large number of low-affinity receptors for EGF in the SV40-immortalized cells. It is suggested that one of the mechanisms whereby SV40-immortalization suppresses the senescence of primary cultures of human mammary epithelial cells involves increasing the level of expression of receptors for EGF. In contrast the level of secretion of TGF alpha by cells in culture is probably a consequence of the mechanisms of adaptation of each cell line to culture conditions, and does not reflect the level of secretion of TGF alpha by cells in vivo.

A rat mammary (Rama) epithelial cell line, Rama 704, derived from normal rat mammary gland does not possess any detectable cell-surface receptors for basic fibroblast growth factor (bFGF), produces a barely detectable level of bFGF mRNA and does not contain detectable levels of bFGF-like activity. Similar results have been obtained with the Rama 37 epithelial cells derived from benign tumours. However, 4 independently isolated epithelial cell lines derived from malignant rat mammary tumours and their metastases possess receptors for bFGF and contain between 2 ng and 9 ng heparin-binding, growth-stimulatory activity per 10(6) cells. The weakly metastatic Rama 600 cells possess high- and low-affinity receptors for bFGF, (KD 20 pM and 8 nM, respectively), while the moderately metastatic Rama 800 cells possess only high-affinity receptors (KD 40 pM). The moderately metastatic C18PLN and 267LU cells, derived from metastases arising from benign Rama 37 cells which had been transfected with DNA from the malignant Rama 800 cells, also possess only high-affinity receptors (KD 36 pM and 80 pM, respectively). Our results show that within the Rama system there is a correlation between the appearance of heparin-binding growth factors and of high-affinity but not low-affinity receptors for bFGF with the malignant phenotype.

The developing breasts of peripubescent girls consist of ducts and budded structures, which can subdivide to alveolar buds/lobules with advancing maturity and finally to secretory alveoli during pregnancy and lactation. Immunochemical reagents have been used to visualize the three major cell types in histological sections of mature/pregnant breasts, the epithelial cells which line ducts/ductules, the smooth muscle-like myoepithelial cells and the casein-secretory alveolar cells. Ductal budded structures contain basal cells intermediate in immunocytochemical staining characteristics between epithelial and myoepithelial cells. Immortalization of primary epithelial cultures of normal breasts by simian virus 40 yields epithelial cell lines that can differentiate to myoepithelial-like and to secretory alveolar-like cells; similar cell types are identifiable in primary cultures. Immunocytochemical staining shows that both hyperplastic and neoplastic benign lesions contain myoepithelial-like cells, and, under suitable hormonal conditions, alveolar-like cells, but invasive carcinomas contain neither differentiated cell type. Primary cell cultures of benign hyperplastic and neoplastic lesions contain epithelial, myoepithelial-like and presumptive alveolar-like cells whilst malignant cell fractions of invasive carcinomas contain only epithelial cells. Spontaneously-immortalized epithelial cell lines from hyperplastic benign breast disease can generate myoepithelial-like and alveolar-like cells, whilst standard epithelial cell lines from pleural effusions and novel epithelial cell lines from primaries of invasive carcinomas fail to differentiate to either cell type. It is suggested that epithelial/intermediate stem cells exist in a basal position predominantly in terminal structures of growing breasts, and that they are the major cell type involved in benign hyperplastic, benign neoplastic and malignant breast diseases. The acquisition of the malignant phenotype is associated with the carcinoma cells having a greatly impaired ability to differentiate to myoepithelial and to alveolar cells.

The expression of the protease cathepsin-D has been evaluated using an immunohistochemical technique with a polyclonal antibody in paraffin-embedded tissue from 359 patients treated between the years 1975-1981 for Stage I and II breast cancer. One hundred and twenty seven patients (35%) have strongly positive, granular staining, 138 (38%) are intermediately stained in the cytoplasm, and in 94 (26%) no staining is observed. There is a strong positive association between expression of cathepsin-D and the presence of tumour in axillary lymph nodes (P < 0.006). Expression of the protease is associated with significantly poorer survival of patients in univariate analysis (P = 0.025); however, this is not independent of other tumour variables.

The rat mammary epithelial cell line, Rama 37, yields benign, non-metastasizing adenomatous tumours in syngeneic Wistar-Furth rats. Transfection of this line with a drug resistance plasmid containing both the gene for resistance to Geneticin (neo) and the gene for p9Ka (pSV2neo-p9Ka), a rat calcium-binding protein, or with a similar plasmid containing neo and the oncogene EJ-ras-1 (pSV2neo-ras) yields drug-resistant transformants that express high levels of the p9Ka or EJ-ras-1 mRNAs and proteins. These transfected cells all produce tumours when injected at subcutaneous sites with a shorter median latent period than the tumours produced by the parental untransfected Rama 37 cells in syngeneic hosts. Cells transfected with pSV2neo-p9Ka yield a higher incidence of tumours than untransfected Rama 37 cells, many of which metastasize to lungs and/or lymph nodes in syngeneic rats. However, cells transfected with pSV2-neo-ras or pSV2neo plasmid alone yield tumours that fail to metastasize. Immunofluorescent studies suggest an association of p9Ka with the cytoskeleton, as depicted by F-actin staining with the reagent phalloidin. It is suggested that the transfection of copies of the gene for the rat calcium-binding protein p9Ka can enhance the tumorigenic potential and induce the metastatic phenotype in this rat mammary model, whereas transfection of control plasmid DNA or the oncogene EJ-ras-1 fails to induce the metastatic phenotype, although EJ-ras-1 transfectants, like those containing p9Ka, possess increased growth properties in vivo.

The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli.

Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work. The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands.

The rat mammary myoepithelial-like cell line Rama 401 possesses 46,000 high-affinity receptors (Kd 52 pM) and 2.8 x 10(6) low-affinity receptors (Kd 24 nM) for basic fibroblast growth factor (bFGF) per cell. Heparin or heparinase pretreatment of the cells inhibits the specific binding of [125I]-bFGF by over 70%, and abolishes binding to the low-affinity sites. Dissociation experiments suggest that there are three kinetically distinct low-affinity receptors, with dissociation rate constants of 3.8 s-1, 0.067 s-1 and 0.0018 s-1. Consistent with the presence of low-affinity receptors possessing a slow dissociation rate constant, exogenously added bFGF bound to the low-affinity receptor can stimulate DNA synthesis in Rama 401 cells without being released into the bulk of the culture medium. These results suggest that the low-affinity receptors on Rama 401 cells are heparan sulfate glycosaminoglycans (HSGAGs) and that their ability to modulate the action of bFGF may result from their diverse range of dissociation rate constants. A cell line, Rama 401ts, derived from Rama 401 by transformation with a temperature sensitive src gene, deposits less extracellular matrix at the permissive temperature of 34 degrees C than at the non-permissive temperature of 41 degrees C. Whilst the binding of [125I]-bFGF to Rama 401ts cells at 41 degrees C is identical to that observed with the parental Rama 401 cells, at 34 degrees C there are fewer low-affinity receptors. These results suggest the (HSGAGs) low-affinity receptors on Rama 401 cells are associated at least in part with the extracellular matrix.

In developing human breast, terminal end buds (TEBs), lateral buds (LBs), and lobules of three to five alveolar buds (ABs) predominate in prepubertal females, whereas lobules of ABs and lobules of up to 60 ductules predominate in pubertal females. The appearance of clefts in TEBs and LBs suggests that they are precursors of ABs. In histological sections the ductal buds are composed of a heterogeneous collection of cells that include cortical and peripheral cells. The cortical cells can line small lumina in TEBs/LBs, whereas the peripheral cells which cap their distal tips are more irregular and loosely packed. Monoclonal antibodies (MAb) to epithelial milk-fat globule membranes and antiserum to epithelial membrane antigen immunocytochemically stain the cortical cells, particularly where such cells line lumina, and weakly stain the peripheral cap cells. Similar histochemical staining patterns are observed in desialylated sections with peanut lectin. Antiserum and MAb to smooth muscle actin moderately stain the peripheral cap cells, and this staining increases the closer the peripheral cells become to the myoepithelial cells of the subtending duct. Similar but weaker staining patterns are observed with antibodies to vimentin. Keratin MAb PKK2 and LP34, which stain myoepithelial cells in preference to epithelial cells in main ducts, as well as MAb to epithelium-specific keratin 18, all stain many of the cortical/luminal cells in buds and lobules of developing breast; the peripheral cap cells are relatively unstained. It is suggested that the undifferentiated peripheral cap cells show transitional forms both to the cortical epithelial cells that eventually line the lumina and to the myoepithelial cells of the subtending duct.

Human breast cancer cells are used extensively for the study of steroid hormone action. It is known that in both receptor positive and receptor negative cell lines there is considerable metabolism of the natural estrogens, estradiol (E2) and estrone (E1) with interconversion of the two steroids and formation of sulphate and glucuronide conjugates. The aim of the present work was to see if the commonly used oral contraceptive steroids (OCS) ethynylestradiol (EE2) and norgestimate (Ngmate) were metabolized in human breast cancer cell lines (MCF-7 and ZR-75-1) and a normal breast cell line (Huma 7). MCF-7, ZR-75-1 and Huma 7 cells were maintained in Dulbeccos Modified Eagles Medium (DMEM) containing foetal calf serum (FCS) insulin and hydrocortisone. In addition, ZR-75-1 cells required epidermal growth factor (EGF) and E2 while MCF-7 cells required only EGF. On reaching confluence cells were transferred to DMEM containing charcoal-stripped FCS, insulin and hydrocortisone. 48 h later this medium was renewed, radiolabelled steroid ([3H]E1; [3H]E2; [3H]EE2, [3H]Ngmate; [3H]E1-SO4; 1 nM; 0.2 microCi) was added and incubation was for 24 or 48 h. Following incubation, the medium was removed and radioactive steroid extracted with ether. Metabolites were analysed by on-line radiometric HPLC. All the cell lines were able to interconvert E1 and E2; the equilibrium favouring the formation of E2 in MCF-7 and ZR-75-1 and E1 in Huma 7 cells. E1 and E2 also underwent phase II metabolism to form their respective estrogen sulphates, this activity being most marked in the Huma 7 cell line. In addition to sulphotransferase activity, the study with E1 sulphate demonstrated sulphatase activity in both normal and cancer cells. There appeared to be no difference in extent of hydrolysis, with both E1 and E2 formed. With EE2 as substrate there was no evidence of phase I metabolism in any of the cell lines but there was conversion to the presumed 3-sulphate conjugate. The percentage formation of this metabolite was very much greater in Human 7 cells (64.1 +/- 9.6% after 24 h) than in MCF-7 and ZR-75-1 cells (7.4 +/- 5.3% and 10.6 +/- 4.1%, respectively after 24 h). In all the cell lines deacetylation of the progestogen Ngmate to norgestrel oxime was complete within 24 h. In addition there was evidence of loss of the oxime moiety to give norgestrel.(ABSTRACT TRUNCATED AT 400 WORDS)

Implantation of excised bud-free ductal fragments (DUCTS), terminal end buds (TEBs), or alveolar buds (ABs) from virgin mammary glands of Wistar-Furth rats into interscapular fat pads of syngeneic female rats produces, after 16 weeks, complete ductal outgrowths including TEBs and ABs. Treatment of the recipient rats with perphenazine for 1 day or mating them after 12 weeks and then isolating the resultant outgrowths after 16 weeks produces significantly larger outgrowths than those from untreated hosts. The outgrowths consist of distended ducts and lobules or distended ducts and alveoli, respectively. Histochemical and immunocytochemical staining of the outgrowths with reagents that depict epithelial, myoepithelial, and lactating alveolar cells (peanut lectin alone, monoclonal and polyclonal antibodies to rat caseins) indicate similar cell compositions and arrangements for all outgrowths irrespective of their source; these are also similar to the mammary glands of the perphenazine-stimulated or lactating hosts. There is one major difference: the degree of staining of peanut lectin alone and the anti-caseins is greater for outgrowths produced by the ABs and TEBs than for those produced by the DUCTs. DUCT implants left for 1 year after cessation of lactation of the hosts are still stained appreciably by peanut lectin alone and by the anti-caseins, particularly the luminal secretions. Therefore, the complete morphogenetic and cell differentiating ability for generating mammary glands is present in bud-free ducts, but this ability can be enhanced in TEBs/ABs or abnormally expressed at ectopic sites.

The expression of the c-erbB-2 oncogene has been evaluated using an immunohistochemical technique with the 21N polyclonal antibody in paraffin embedded tissue from 465 patients treated between the years 1975-1981 for Stage I and II breast cancer. One hundred and four (22%) patients exhibited positive staining. This was not associated with any other variables. Expression of the oncogene was associated with significantly poorer survival which was independent of other tumour variables.

Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial-related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast.

Individual single-cell-cloned cell lines of the different rat mammary (Rama) cell types have been tested for their ability to bind the lectins Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) using fluorescent, histochemical, and radioactive assays. Myoepithelial-like cell lines isolated from neonatal rat mammary glands and from nonmetastasizing tumors strongly bind GS-1 and PWM, whereas the corresponding epithelial and fibroblastic cell lines do not. When the epithelial cell lines are grown on floating gels of polymerised rat tail collagen, the basally situated or peripheral cells are stained strongly with peroxidase-conjugated lectins, whereas the apically or luminally situated cells are unstained. The capacity of cell lines intermediate in morphology between epithelial and myoepithelial-like cells to bind to GS-1 is as follows: Rama 25 epithelial less than Rama 25-12 less than Rama 25-11 less than Rama 25-14 less than Rama 29 myoepithelial-like cells, the same order as for other markers of myoepithelial cells. Conjugated PWM, however, binds only to the myoepithelial-like cell lines. Treatment of Rama 25 epithelial cells with agents that disrupt microtubules accelerates their conversion to elongated, myoepithelial-like cells in culture. The binding of cells to GS-1 is observed prior to, and that to PWM after, the major morphological change. It is suggested that the stepwise appearances of carbohydrate receptors for GS-1 and PWM mark discrete stages in the differentiation of epithelial to myoepithelial-like cells in culture, in the same way that they mark similar differentiation stages in ductal development in mammary glands of prepubertal rats.

The different mammary cell lines described herein appear to be representative of the cell types found in both normal glands and benign tumors of rats and humans. The epithelial cell lines can differentiate to both alveolar-like and myoepithelial-like cells in culture. The epithelial cell lines and particularly those cell lines representing intermediate stages in the myoepithelial differentiation pathway are candidates for the epithelial stem cells found in rat and possibly in human terminal ductal structures. The systemic mammatrophic hormones that are thought to control the growth of the mammary gland in vivo have little or no stimulatory effect alone on the growth of normal and neoplastic rat mammary cells in culture. The pituitary growth factors (fibroblast growth factor [FGF] and pituitary-derived mammary growth factor [PMGF],) and the growth factors released from the different cell lines, (stromal prostaglandin E2 [PGE2] and myoepithelial transforming growth factor alpha [TGF-alpha]) are much more potent mitogenic agents for the mammary cell lines. The ability of FGF and epidermal growth factor (EGF) -related molecules to simulate growth of the different mammary cell types in culture correlates with the presence of their high-affinity receptors. Thus these growth factors are promising candidates for some of the primary effectors of mammary growth in vivo. Malignant mammary epithelial cells have a greatly reduced rate of growth compared to their normal and benign counterparts. They also fail to differentiate or to respond to PMGF but can still respond to PGE2 and TGF-alpha. In addition, highly malignant variants appear capable of adapting to a new growth environment in vivo. This suggests that simple molecular explanations based solely on the autostimulation of cell growth may not be sufficient to explain some of the properties of the slowly growing, highly malignant cells.

Transfection of rat mammary (Rama) 37 epithelial cells which yield non-metastasizing adenomas in syngeneic Wistar-Furth rats with a drug resistance plasmid containing both the neo gene and EJ-ras-1 (pSV2neo.ras) or with pSV2neo and a plasmid encoding the large T Antigen (pLT214) of polyoma virus yields drug-resistant transformants with a frequency of 10(-5). Representative transformants have been propagated in neo-selecting medium to yield various cell lines. The 7 lines transfected with pSV2neo.ras (EJ1 set) and the 10 lines co-transfected with pSV2neo and pLT214 (LT1 set) all produce tumours at subcutaneous (s.c.) sites with a shorter median latent period than tumours produced by the parental Rama 37 cells. In addition, the LT1 set of transformants yields a higher incidence of tumours than the Rama 37 cells. No metastases are produced when any of the oncogene transformants are inoculated s.c. into rats. However, when an EJ1 representative is inoculated intravenously (i.v.), tumour deposits are found in the lungs of the host animals. In contrast, other Rama 37 variants that metastasize from s.c. sites fail to produce any metastases when inoculated i.v. The oncogene transfectants contain integrated DNA that hybridizes to neo and to the requisite oncogenic DNAs; the pattern of hybridizing bands to the transfected genes and their expression as mRNA is complex, and is presented in detail.

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.

The histochemical binding of peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) to methacarn-preserved and paraffin-embedded female rat mammary glands at different developmental stages has been undertaken with a view to investigating the ontogeny of the myoepithelial cell. Conjugated GS-1 fails to stain the outer layer of ductal cells in neonatal rats up to 3 days old, but thereafter the staining increases so that all such cells are intensely stained in rats 5 days old and in mature, pregnant, and lactating rats. Conjugated GS-1 also stains most of the inner epithelial cells that line the ducts in neonatal rats up to about 5 days after birth; thereafter no such cells are stained in mature, pregnant, and lactating rats. Conjugated PWM stains both the inner and outer cell layers of ducts in neonatal rats up to 5 days old; thereafter the fraction of strongly stained cells declines rapidly in both cell layers so that in 6-day-old rats only weak staining is visible. Staining with PWM continues to decline for the inner epithelial cells of the ducts until it ceases when the rats mature; in contrast, that for the outer cell layer of the ducts increases so that all such cells are stained intensely when the rats mature. This pattern of ductal staining with PWM is maintained for pregnant and lactating rats. In terminal end buds of mammary ducts of prepubertal rats, GS-1 binds mainly to the peripheral or cap cells, the staining intensity increasing from cap cells at the distal tip to myoepithelial cells of the subtending duct. PWM binds to many more of the cortical epithelial cells and fewer of the cap cells. At the ultrastructural level, cap cells and adjacent immature myoepithelial cells both bind GS-1 and PWM to their surfaces, but basal clear cells do not. In alveolar buds and in alveoli, both conjugated lectins GS-1 and PWM bind to myoepithelial cells but not to epithelial cells of the rat mammary gland. We suggest that the appearance of carbohydrate receptors for GS-1 and PWM marks specific stages of myoepithelial cell differentiation in developing rat mammary glands.

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.

The rat mammary 37 epithelial cell line yields non-metastasizing adenomas in syngeneic rats. On cellular DNA transfection, a series of cell lines have been isolated that grow in drug-selective medium. Representative transfected cell lines all yield tumors in rats that consist predominantly of spindle cells, but two also contain epithelial-like cells and glandlike elements (C18P, C19P). Immunocytochemical staining for milk fat globule membrane antigens, human callus keratin, and laminin confirms the identity of the epithelial cells and suggests a (myo)epithelial origin for the spindle cells. Some of the transfected cell lines also generate well-differentiated metaplastic elements in their tumors. One cell line (CT4-41) produces rhabdomyoblastic and possibly smooth-muscle-related elements; two (C18P, C19P) produce squamous metaplasia and sebaceous elements; and two (CL1-31, C11P) produce cartilaginous elements. The identities of the heterologous elements are confirmed by immunocytochemical staining for myoglobin, actin (CT4-41), keratin (C18P, C19P), type II collagen, and type II keratan sulfate (CL1-31). Those cell lines that have acquired the ability to metastasize from subcutaneous sites (CT4-41, C18P) reproduce the same metaplastic elements in their metastases. Thus, a cloned mammary epithelial cell line can be made to generate many of the well-differentiated, heterologous elements observed in human breast carcinomas, and this change is often associated with the rat cells acquiring metastatic properties.

Although the tumorigenic phenotype can be transferred by transfecting DNA from a variety of human tumours into 3T3 mouse fibroblasts, the ability to transfer the metastatic phenotype in a similar manner in syngeneic animals is largely unknown. We now report that transfection of a rat mammary epithelial cell line Rama 37, which produces nonmetastasizing adenomatous tumours in syngeneic rats, with DNA from a metastasizing rat mammary cell line, Rama 800, yields some transfectants with reproducible metastatic capabilities. Nonspecific DNA or the oncogenes EJ-ras-1 or Polyoma Large T Antigen fail in this respect, although their transfectants often possess increased tumorigenic potential.

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.

p9Ka is a polypeptide of apparent molecular mass 9 kDa, present in cultured rat mammary myoepithelial-like cells, but virtually absent in their parental epithelial cells. mRNA for p9Ka is present in normal rodent tissues. The amino acid sequence of a protein of molecular mass 12 KDa, derived from the nucleotide sequence of the p9Ka gene, is related to that of S-100 protein, a calcium-ion-binding protein. p9Ka, isolated from cultured rat mammary myoepithelial-like cells is now shown to bind calcium ions in vitro suggesting that the derived amino acid sequence is correct, and that an apparent discrepancy between the molecular masses of the predicted and isolated p9Ka does not affect this activity.

Cloned simian virus 40 (SV40)-transformed human breast epithelial cell lines can differentiate to myoepithelial-like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor-nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial-like cells and cell lines. The myoepithelial-like cell lines produce 4-6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5-5-fold in the myoepithelial-like cell lines when analysed by dot-blot or by Northern hybridisations. Thus, differentiation along the myoepithelial-like cell pathway is associated in these SV40-transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.

The binding of [125I]-epidermal growth factor (EGF) and [125I]-basic fibroblast growth factor (bFGF) to a number of single-cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high-affinity [125I]-EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial-like cell lines. In contrast, high-affinity (KD 35-276 pM) [125I]-bFGF binding sites were present on fibroblastic and myoepithelial-like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial-like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity-cross-linked to [125I]-bFGF were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.

Transfection of rat mammary (Rama) 37 epithelial cells, which yield nonmetastasizing adenomas in syngeneic Wistar-Furth rats, with HindIII-fragmented cellular DNA and the drug-resistance plasmids pSV2gpt or pSV2neo yields drug-resistant transformants with a frequency of 10(-4)-10(-5). Transformant cell lines transfected with the following, pSV2gpt alone, pSV2gpt and Rama 37 DNA, pSV2gpt and DNA from a metastasizing cell line Rama 800 (CT set), pSV2neo and salmon sperm DNA, pSV2neo and Rama 800 DNA (C set), all yield tumors when injected subcutaneously into syngeneic rats. A few transformants obtained by cotransfection with DNA from Rama 800 cells produce metastases in lungs and/or lymph nodes. The incidence of such metastases for two transfectants, termed CT4-41 and C18P, is significant at 20 and 24%, respectively, but only half (48%) that achieved with Rama 800 cells. Reintroduction into rats of cells cultured from a metastatic tumor of CT4-41 and of C18P, and from their lung or lymph node metastases, produces either a similar incidence (20-24%) or a significantly higher (48-52%) incidence of metastasis than that of the original transfectants. Cells cultured from nonmetastatic tumors fail to produce any metastatic lesions. When [32P]-labeled gpt or neo DNAs are hybridized to EcoRI-digested cellular DNA of the CT4-41 or C18P series of cell lines, tumors or metastases, gpt binds to one major fragment of 3,800 basepairs, and neo to two major fragments of 5,700 and 4,200 basepairs. The same cell lines produce hybridizing mRNAs of 1,500 and 1,900 bases for the CT4-41 series and 2,000-2,400 bases for the C18P series. It is suggested that transfection of DNA from the metastatic cells causes the nonmetastatic cells to become metastatic, in a genetically dominant manner, but additional steps are required for this process to become established and expressed at a level equivalent to that of the original, metastasizing donor cells.

The metastasizing rat mammary cell strain from the Ludwig Institute for Cancer Research (London Branch) which was originally developed from a benign rat mammary tumor induced by N-methyl-N-nitrosourea (CAS: 684-93-5), yielded single-cell-cloned lines of isometric epithelial cells [rat mammary (Rama) 600-Rama 621] and one line of elongated cells (Rama 622); the former had a higher estrogen receptor content than the latter. All the representative epithelial cell lines tested (Rama 600, 603, and 617) failed to convert to elongated, myoepithelial-like cells or droplet cell/doming, alveolar-like cells in vitro. All representative cell lines tested induced tumors in syngeneic F344/N rats and CBA nu/nu mice, but only the epithelial lines metastasized to lungs and local lymph nodes in rats and to lungs in nude mice. The involved lungs and lymph nodes contained mainly intravascular thrombi and deposits in the subcapsular sinus, respectively. Tumors and metastases from the representative epithelial cell lines contained acinar and glandular structures together with an elongated cellular component. The Rama 622 tumors contained mainly spindle cells. Antisera to rat milk fat globule membranes and human keratins stained some of the epithelial and elongated cells in the Rama 600 tumors; less staining was observed in the Rama 622 tumors. None of the tumor cells stained with antiserum to myosin. Anti-laminin serum delineated a fragmented basement membrane in glandular elements and stained weakly the cytoplasm of the more elongated tumor cells. Ultrastructural analysis confirmed the identity of epithelial cells in the Rama 600 tumors, but no well-differentiated myoepithelial cells were seen in either type of tumor. Since nonmetastasizing epithelial cells isolated directly from carcinogen-induced benign rat mammary tumors can differentiate to myoepithelial-like cells in vitro or when growing as tumors in animals, it is suggested that the development of the malignant phenotype is associated with a loss of this differentiating ability.

Fibroblast Growth Factors obtained from bovine brain and pituitary glands were compared. They were shown to behave identically on three high performance liquid chromatographic systems, which separate proteins by the independent criteria of size, charge and hydrophobicity. They also migrated similarly on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate with an apparent molecular weight of 12,000. It was concluded that the two factors were very similar, or identical. They were active at promoting DNA synthesis in rat mammary fibroblasts (Rama 27) at a concentration of 0.1 ng/ml.

Various epidermal growth factor preparations obtained from the mouse submaxillary gland (mEGF), have been separated into a number of components by reversed-phase high-performance liquid chromatography (HPLC). It is shown here, however, that when the mEGF is isolated rapidly, using only reversed-phase HPLC for trace enrichment and high-resolution fractionation, it is a single molecular species as determined with several ion-pairing solvent systems, provided that proteolysis is inhibited in the original extracts. This indicates that the minor components of mEGF that have been reported are artefacts formed during the isolation procedure, and are of no biological significance. The products of deliberate mild degradation of mEGF are shown to produce similar chromatographic profiles to those observed in samples of mEGF prepared in the absence of proteolytic inhibitors. Rat EGF has been isolated in a similar manner, and is shown to share many of the properties of the major tryptic digestion product of mEGF.

Single-cell-cloned cell lines intermediate in morphology between the cuboidal epithelial and fully elongated myoepithelial-like cells have been isolated from the single-cell-cloned epithelial stem cell lines Rama 25 and Rama 37 originally obtained from dimethylbenz(a)anthracene-induced mammary tumors from Sprague-Dawley and Wistar-Furth rats, respectively. These are designated Rama 25-l1, Rama 25-l2, Rama 25-l4 (Sprague-Dawley) and Rama 50-55, Rama 59, and Rama 60 (Wistar-Furth), respectively. When growing as tumors in nude mice or syngeneic Wistar-Furth rats, respectively, many of the newly cloned cell lines give rise to spindle and giant, multinucleated cells which stain immunocytochemically with antisera to myoglobin and myosin and contain longitudinal fibrils, some of which contain phosphotungstic acid-hematoxylin-staining cross-striations. Ultrastructural analysis demonstrates the presence of A-, l-, and H-bands and Z-discs and the hexagonal arrangement of thick and thin filaments characteristic of skeletal muscle. Similar results are obtained with selected cloned cell lines growing on floating collagen gels in vitro. Thus, a developmentally committed mammary epithelial cell can give rise, under suitable conditions, to a well-differentiated mesenchymal lineage, that of skeletal muscle. It is suggested that such cells may be responsible for the generation of the well-differentiated mesenchymal elements seen in the mixed (epithelial and myoepithelial) tumors of glandular origin.

A rat mammary myoepithelial-like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative "dot" hybridization techniques, the level of poly (A)-containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5-fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 71% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.

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.

Epithelial cell lines isolated from benign rat mammary tumors converted to elongated cells that showed some aspects of myoepithelial differentiation. This cellular conversion was blocked in cells isolated from malignant tumors. For investigation of the pathway of the conversion, a rat mammary epithelial cell line (Rama 25) that converted to elongated cells through a series of morphologically distinct intermediates was isolated. These intermediates formed a series in the order: Rama 25, Rama 25-I2, Rama 25-I1, Rama 25-I4, and elongated cells. These cell lines were examined for aspects of myoepithelial or mesenchymal differentiation with the use of a polyclonal antibody to type IV collagen and a keratin monoclonal antibody, LP34 (myoepithelial markers), or a polyclonal antibody to type I procollagen (mesenchymal marker) for cells grown on plastic or as tumors in nude mice. The more epithelial-like cell lines Rama 25 and Rama 25-I2 produced relatively small amounts of type IV collagen and did not stain with LP34 or anti-type I procollagen. The flatter, polygonal cell line Rama 25-I1 stained more strongly with the antibody to type IV collagen but did not stain with anti-type I procollagen. Rama 25-I1 cells, and to a lesser extent Rama 25-I4 cells in tumors, contained a network of cytoplasmic filaments that stained strongly with LP34. The elongated cells, Rama 25-I4 and Rama 25-floaters (Rama 25-FL), did not stain with LP34 in vitro but produced an extracellular matrix that stained with antibodies to both type I procollagen and type IV collagen. The results obtained with these marker proteins suggested that, in this series of morphologic intermediates, the myoepithelial phenotype was best expressed in Rama 25-I1 cells and to a lesser extent in 25-I4 cells. However, this phenotype was relatively unstable, converting to elongated cells, some of which have decreased myoepithelial and increased mesenchymal characteristics. Such a pathway may explain the mixed population of cells seen in some types of benign mammary tumor.

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.

A review is presented of the use of immunohistochemical probes in the study of the cellular pathology of the breast. Using a combination of monoclonal and polyclonal antisera it has been possible to investigate the relationship between myoepithelial cells and basement membrane components in benign breast disease and the disturbance of this relationship in early invasive and infiltrating ductal carcinomas. The use of immunohistochemical techniques at the ultrastructural level provides the methodology for future detailed analyses. The heterogeneous expression of antigenic determinants characteristic of epithelial cells in the normal breast is considered in conjunction with its relevance to the heterogeneity seen in the invasive tumours.

With the use of immunofluorescent and immunocytochemical techniques on histologic sections of mammary glands from inbred WF rats, the thymocyte differentiation antigen (Thy-1) was identified partially on and immediately adjacent to the myoepithelial cells of ducts and alveoli. This antigen was absent from epithelial cells lining such structures. Some fibroblastic cells external to these structures also bore Thy-1. During glandular development the intensity of fluorescence due to the binding of Thy-1 antibodies increased as the myoepithelial cells matured. Similarly, mammary adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and N-methyl-N-nitrosourea contained elongated, presumptive myoepithelial cells that demonstrated varying degrees of Thy-1 immunofluorescence. This phenomenon was qualitatively mimicked by cultured cell lines from normal and DMBA-induced tumors. Elongated myoepithelial-like and fibroblast-like cells possessed Thy-1, whereas the cuboidal epithelial cell lines failed to express this antigen on the cell surface. Measurement of the relative number of Thy-1 molecules per cell by antiserum absorption techniques suggested that a neoplastic stem cell line, rat mammary (Rama) 25, contained about 3 X 10(5) Thy-1 molecules per cell in a form not directly accessible at the cell surface to anti-Thy-1 antibodies; the number of cryptic Thy-1 molecules was reduced when this cell line differentiated to alveolar-like cells in culture. However, when this cell line differentiated to myoepithelial-like cells, approximately 5 X 10(6) molecules per cell were exposed to anti-Thy-1 antibodies with a concomitant reduction of the cryptic pool. Morphologic maturation of elongated, myoepithelial-like cell line variants was also accompanied by increased surface Thy-1 fluorescence. Thus some of the myoepithelial cells in normal glands and tumors may arise by differentiation of epithelial stem cells and a spectrum of maturation states of the myoepithelial cell may exist as monitored by cellular morphology and surface Thy-1 expression.

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.

The cloned cuboidal epithelial cell line Rat Mammary (Rama) 25 converts at low frequency in culture to elongated cells that possess some of the properties of myoepithelial cells; one such clonal cell line is termed Rama 29. Three morphologically intermediate clonal cell lines have been isolated from Rama 25 which form a morphological series in the order: Rama 25 cuboidal cells, Rama 25-Intermediate 2(I2), Rama 25-I1, Rama 25-I4, and Rama 29 elongated cells. This same order is largely maintained for increasing percentages of elongated cells, decreasing percentages of cuboidal cells, decreasing tubular structures on collagen gels, and increasing times of appearance of tumors in nude mice. The fully elongated cells fail to revert to cuboidal cells and to form tumors. Binding of antisera to epithelial-specific milk fat globule membranes and human keratin declines whereas binding of antisera to myoepithelial-associated laminin, vimentin, and Thy-1 increases in the cell lines in the same order. Similarly 7 polypeptides characteristic of elongated cells increase and 4 polypeptides characteristic of cuboidal cells decrease in the cell lines in the same way. Anti-actin serum binds equally to all cell lines grown on plastic, except for Rama 25-I4, where its binding is increased. Rama 25-I1 and Rama 25-I4 cells also give rise to anti-actin, anti-myoglobin, and phosphotungstic acid hematoxylin-staining giant, striated cells on collagen gels and in tumors that also have ultrastructural characteristics of skeletal muscle. Fresh elongated converts of Rama 25 bind appreciably more anti-actin serum than many of the clonal elongated cell lines such as Rama 29. Ultrastructural analysis confirms the gradual loss of epithelial characteristics and the acquisition of immature myoepithelial characteristics in the same sequence of cell lines. It is suggested that such a linear sequence of intermediate morphological states occurs between the Rama 25 cuboidal cells and the elongated myoepithelial-like cells in vitro, and that a similar morphological sequence may exist in terminal ductal structures in vivo.

The myoepithelial-type cell line, Rama 712, derived from a normal rat mammary gland, deposits an extracellular matrix containing type-IV collagen and other basement membrane proteins round its cellular periphery. After transformation with a temperature-sensitive mutant of Rous sarcoma virus (tsRSV) the cells fail to deposit an extracellular matrix at the permissive temperature (35 degrees C), but retain the capacity to do so at the non-permissive temperature (41 degrees C). The synthesis of type-IV collagen is not affected by the temperature shift. Rama 712 cells fail to form tumours in syngeneic rats. However, Rama 712-tsRSV cells form tumours that are locally invasive but fail to metastasize. In histological sections, the tumour cells stain with an antibody to type-IV collagen, but do not deposit any extracellular type-IV collagen. Cells isolated from the tumours (Rama 712T) remain temperature-sensitive for the extracellular deposition of type-IV collagen when grown in vitro. Rama 712, Rama 712-tsRSV and Rama 712T fail to produce any detectable type-I or type-IV collagenase at either 35 degrees C or 41 degrees C. These results show that in this system extracellular deposits of basement membrane proteins are lost from invasive tumours produced by myoepithelial-type cells by mechanisms other than those due to the production of collagenolytic enzymes.

A number of single-cell-cloned cell lines have been used to examine the growth-promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary-derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial-like cells. Epidermal growth factor (EGF) has a widespread but variable growth-stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 micrograms/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial-like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.