Jumpei Enami

Mammary Fibroblast-derived Hepatocyte Growth Factor Stimulates Growth and Morphogenesis of Mouse Mammary Tumor Cells in Primary Culture

We have recently isolated a mammary growth factor from the conditioned medium of mouse mammary stromal fibroblasts and identified it as a mouse homologue of human HGF (hepatocyte growth factor). To elucidate the role of HGF in mouse mammary tumorigenesis, we produced recombinant mouse HGF and examined its effects on primary cultures of mouse mammary tumor cells in this study. HGF at concentrations above 20 ng/ml maximally stimulated the growth of mammary tumor cells in primary monolayer culture. HGF also stimulated the three‐dimensional growth and branching morphogenesis of mammary tumor cells cultured inside collagen gels. A comparison of the growth‐stimulating activity of HGF with that of EGF (epidermal growth factor) and KGF (keratinocyte growth factor) revealed that HGF is the most potent growth factor among the three. Immunological studies using an antibody against mouse HGF demonstrated that 74% of the growth‐stimulating activity present in the mammary fibroblast‐conditioned medium was abolished by the antibody, indicating that HGF is the major growth factor produced by the fibroblasts. These observations thus suggest a role for HGF as a mammary stromal fibroblast‐derived factor which stimulates growth and morphogenesis of adjacent mammary tumor cells in vivo.

Hepatocyte growth factor supports androgen stimulation of growth of mouse ventral prostate epithelial cells in collagen gel matrix culture.

To assess the role of hepatocyte growth factor (HGF) and androgen in growth of prostate epithelial cells, we isolated mouse ventral prostate epithelial cells and cultured them in a three‐dimensional type I collagen gel matrix under serum‐free conditions. Although the prostate epithelial cells tended to die in the insulin‐supplemented basal medium, 5α‐dihydrotestosterone (DHT) prevented the cell death, and HGF slightly stimulated the growth. By contrast, coexistence of DHT and HGF greatly augmented the growth and branching morphogenesis of the epithelial cells. Some of the outgrowths formed under these conditions showed enlarged structures resembling the prostate ducts or alveoli. Examination of the stromal cell‐conditioned medium revealed that a growth‐stimulating activity is present in the conditioned medium. A major portion of this activity was abolished by anti‐HGF IgG. These observations suggest that HGF is produced by the stromal cells of the prostate gland and supports the androgen stimulation of growth of the epithelial cells.

Biochemical Changes during Growth and Regression of Pregnancy-Dependent Mammary Tumors of GR/A Mice

Abstract The weights of pregnancy-dependent mammary tumors (PDMT) of GR/A mice continued to increase until parturition and decreased soon after delivery; however, mitotic indices in epithelial cells and stromal cells of PDMT reached a maximum plateau on Day 18–19 of pregnancy and decreased thereafter. Growth of PDMT in progesterone-treated mice on Day 15 of pregnancy was higher than that in 17β-estradiol-treated mice and no treatment controls. DNA fragmentation was observed in PDMT on Day 20 of pregnancy and just after parturition. Two-dimensional gel electrophoresis of PDMT extracts revealed that five and six protein spots appeared newly on Day 20 of pregnancy and just after parturition, respectively. N-terminal amino acid sequences of two of the protein spots were identical to that of α-lactalbumin. PDMT on Day 15 and 20 of pregnancy and just after parturition secreted matrilysin, one of the matrix metalloproteinases, which was identified by Western blotting. However, matrilysin was not found in hormone-independent autonomous mammary tumors of the mouse. Estrogen receptor and c-fos mRNA expression levels in PDMT were high on Day 15 of pregnancy but low on Day 20 of pregnancy and just after parturition. These findings suggest that regression of PDMT is caused by apoptosis, and new proteins expressed on Day 20 may participate in the process of regression.

Hormone-Dependent Expression of γ-Casein mRNA in Mouse Mammary Epithelial Cells Cultured on Floating Collagen Gels

Abstract Mammary epithelial cells isolated from pregnant mice were plated on a collagen gel matrix and cultured in a serum-free medium supplemented with combinations of insulin, dexamethasone and prolactin (PRL). After the cells formed a monolayer, the collagen gel was detached from the culture dish and allowed to float in the medium. Quantification of &ggr;-casein mRNA by a competitive PCR method revealed that the cells on the floating gel accumulated considerably larger amounts of &ggr;-casein mRNA than the cells on the gel remained attached to the dish. Under these floating collagen gel culture conditions, addition of both dexamethasone and PRL to the insulin-supplemented basal medium maximally stimulated the accumulation of &ggr;-casein mRNA. These observations strongly suggest that the status of the extracellular matrix as well as hormones controls the differentiation of mouse mammary epithelial cells at the mRNA level.

Expression of Ly-6D on the surface of normal and neoplastic mammary epithelial cells of the mouse.

Rat were immunized with mouse mammary epithelial cells and monoclonal antibodies (MAbs) were obtained to identify antigens stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Examination of the reactivities of the MAbs by immunofluorescence staining of tissue sections showed that one of the antibodies, MAb 2A2, reacted with luminal epithelium of the mammary gland and spontaneous mammary carcinomas of SHN mice. Further examination of the tissue lysates by western blot analysis revealed that MAb 2A2 reacts with a 17‐kDa antigen expressed in normal mammary epithelial cells and mammary carcinoma cells. The antigen recognized by MAb 2A2 was absent in the lysates of liver, lung, salivary gland, kidney, small intestine, ovary and uterus. After immunoaffinity purification of the MAb 2A2‐ recognized antigen and determination of its N‐terminal amino acid sequence, we identified the antigen as Ly‐6D, also known as ThB, which belongs to a family of glycosyl‐phosphatidylinositol‐anchored cell surface proteins. Northern blot analysis further demonstrated that Ly‐6D mRNA is expressed in the mammary gland. Based on these observations we concluded that Ly‐6D is stably expressed on the surface of both normal and neoplastic mammary epithelial cells of the mouse. Ly‐6D will serve as a useful epithelial cell surface marker for the study of mammary gland development, as well as for breast cancer research.