Metabolism of [3H]equilin in normal and malignant human endometrium and in endometrial adenocarcinoma transplanted into nude mice

One of the main components of conjugated equine estrogens is equilin sulfate and this estrogen in postmenopausal women is metabolized to 17 beta-dihydroequilin, 17 beta-dihydroequilenin and equilenin. To investigate the possibility that some of these estrogens may be formed directly in the target tissues, we studied the in vitro metabolism of [3H]equilin in various types of normal and malignant human endometrium, including adenocarcinoma grown in athymic nude mice. The results indicate that normal and neoplastic human endometrium can form the above three metabolites. The highest level of 17 beta-reduced products were isolated from the normal secretory endometrium. Equilenin was the most abundant metabolite isolated from both the normal and malignant endometrium. The formation of [3H]equilenin indicates the presence of a 6,8(9) steroid dehydrogenase-isomerase in the human endometrium. The formation of 17 beta-dihydroequilin in the endometrium may be of importance as this estrogen is 8 times more potent as a uterotrophic agent than equilin and estrone.     

Membrane properties of metastatic and non-metastatic cells cultured from C3H mice injected with LM fibroblasts

Little is known regarding the membrane properties of metastatic cells as compared to non-metastatic tumor cells. In order to remove variables such as site of growth and nutrition, C3H mice and LM fibroblasts were used as a model system to derive cell lines from local tumors and lung metastases. LM cells were injected subcutaneously into C3H mice and local skin tumors and secondary lung tumors were isolated, cultured in vitro and analyzed. The activities of lipid-sensitive membrane enzymes, membrane lipid composition, and membrane structure were correlated with metastatic ability. Plasma membranes and microsomes of the cultured metastatic cells had 3.8 +/- 0.5- and 5.4 +/- 0.6-fold elevated 5'-nucleotidase activity, respectively, as compared to plasma membranes and microsomes of cultured non-metastatic cells. The mitochondria of cultured metastatic cells had 3.5 +/- 0.5-fold decreased succinate-dependent cytochrome-c reductase activity as compared to mitochondria of the cultured non-metastatic cells. The lipids of plasma membranes from the metastatic cells had 30 +/- 2% and 46 +/- 7% lower phosphatidylinositol and sterol/phospholipid ratio, respectively, and 30 +/- 3% increased unsaturated/saturated fatty acid as compared to cultured non-metastatic cells. The lower sterol/phospholipid ratio correlated with a 30 +/- 1% lower level of cytosolic sterol carrier protein in the cultured metastatic cells as compared to cultured non-metastatic cells. Multifrequency phase and modulation fluorometry in conjunction with the fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, was used to determine the static and dynamic aspects of membrane fluidity. The plasma membranes and microsomes of cultured metastatic cells were more fluid than those of cultured non-metastatic cells as indicated by 24 +/- 3% and 7 +/- 1%, respectively, lower limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene in the membranes of the metastatic as compared to non-metastatic cells.     

The cell cycle in the small intestine transplanted beneath the kidney capsule in syngenic mice

Summary Transplantation of a small fragment of the ileum beneath the kidney capsule in syngenic mice results in the formation of a cyst lined with proliferating intestinal epithelium. The duration of the cell cycle in this epithelium was determined (using tritiated thymidine and the FLM method) as 14.5 h, as compared with 11.5 h in the intestinal epithelium in situ. We conclude that the intestinal content has little effect on the cell cycle of epithelial cells of the small intestine.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.     

Pulmonary calcification in C3H mice

C3H mice including aged retired breeding females, aged virginal females, young virginal females and young males were examined for the incidence and severity of pulmonary calcification. Pulmonary calcification appeared in aged females, but not in young mice of either sex, and the lesions in aged breeders were more severe and frequent than in aged virgins. These results indicate that spontaneous pulmonary calcification is observed in aged females of the C3H strain. The findings of increased incidence and severity of pulmonary lesions in aged breeders suggest that pregnancy, delivery and lactation are important enhancing factors. Calcified lesions were also observed in kidney, heart, brain, ovary, choroid plexus, cornea and artery in the animals examined.     

Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts

A set of complementary DNAs (cDNAs) that encode the complete 672-amino acid sequence for mouse protein kinase C (PKC) type alpha have been isolated from a mouse Swiss 3T3 cDNA library. Extensive rescreening of this cDNA library only resulted in the isolation of clones of the same PKC species, indicating that Swiss 3T3 fibroblasts express exclusively PKC-alpha. This enzyme is encoded by two different mRNAs that exhibit a substantial heterogeneity in their 3'-noncoding regions. This heterogeneity could have been derived from alternative splicing of the pre-mRNAs or be due to differential usage of the polyadenylation motif. The expression of PKC mRNA in fibroblasts is very low and it is not influenced by treatment with the tumor promoter 12-O-tetradecanoyl-13-phorbol-acetate.     

Azidocytidine is incorporated into RNA of 3T6 mouse fibroblasts

Earlier work has shown that azidocytidine inhibits the growth and DNA synthesis of 3T6 mouse fibroblasts by inactivation of the enzyme ribonucleotide reductase. RNA synthesis, as measured by incorporation of [3H]cytidine was not affected. Here I show that azidocytidine is incorporated into RNA, but not into DNA. Incorporation of the analogue into RNA may under special circumstances contribute to the biological effect of the nucleoside.     

Defective tumoricidal capacity of macrophages from C3H/HeJ mice

Peritoneal macrophages from C3H/HeN mice treated i.p. with T cell mitogens or viable BCG organisms were cytotoxic to syngeneic tumor cells in vitro. Macrophages from endotoxin-unresponsive C3H/HeJ mice treated with BCG or T cell mitogens, however, were not tumoricidal. Furthermore, unlike cells from C3H/HeN mice, macrophages from C3H/HeJ mice could not be activated for tumor cytotoxicity after in vitro treatment with bacterial endotoxins or with lymphokine-rich supernatants. The subnormal induction of cytotoxic macrophages after in vitro or in vivo treatments in C3H/HeJ mice appears to be a highly selective defect. Macrophage responses (yield, phagocytosis, or peroxidase staining) in inflammatory exudates induced by BCG, T cell mitogens, or heterologous serum in C3H/HeJ or C3H/HeN mice were identical. C3H/HeJ macrophages also responded normally in vitor to chemotactic lymphokines. Thus, C3H/HeJ macrophages possess a profound and selective defect in tumoricidal capacity. This defect was not dependent upon exogenous endotoxins. Defective macrophage cytotoxic responses may reflect non-LPS related functions regulated by the LPS gene.     

Microcell-mediated transfer of carcinogen-induced ouabain resistance from C3H/10T1/2 Cl 8 mouse fibroblasts to human cells

We previously developed a quantitative assay for measuring the induction of ouabain-resistant (Ouar) variants in transformable C3H/20T1/2 Cl 8 mouse fibroblasts following treatment of the cells with chemical carcinogens. To further define the nature of the Ouar phenotype, we conducted microcell-mediated chromosome transfer studies using Ouar cell lines induced by chemical carcinogens in C3H/10T1/2 Cl 8 cells as donors and 8-azaguanine-resistant (Azgr) derivatives of the human cell lines, D98/AH2 and HT 1080, as recipients. Microcells prepared from one spontaneous and two carcinogen-induced Ouar mouse cell lines were able to transfer resistance to 0.01 and 1 mM Oua to ouabain-sensitive D98 and HT 1080 cells. The frequency of microcell hybrid formation ranged from 10(-6) to 10(-5). Karyotypic analysis of the microcell hybrids indicated that the Ouar phenotype of C3H/10T1/2 Cl 8 derivatives mapped to mouse chromosome 3, the chromosome to which the wild-type murine Oua-1 allele had previously been assigned. These studies show that both spontaneous and chemically induced high level Ouar phenotypes of C3H/10T1/2 Cl 8 mouse fibroblasts can be transferred via microcell-mediated chromosome transfer, and provide strong genetic evidence that chemically induced Ouar phenotypes of C3H/10T1/2 Cl 8 cells arise from mutations at Oua-1. In addition, this study sufficiently standardizes microcell-mediated chromosome transfer in the C3H/10T1/2 Cl 8 cell line so that this technique can be used to investigate the nature of other phenotypic changes in these cells, such as the chemically transformed phenotype.     

Lateral hermaphroditism in a C3H mouse.

A spontaneous case of true lateral hermaphroditism was observed in one of approximately 1000 necropsies of 12-wk-old female C3Hf-Wg mice (a substrain of C3H/He). Both the right ovary and abdominal left testis were functional as evidenced by the presence of oocytes in graffian follicles and spermatocytes maturing on sertoli cells. Both gonads communicated, the ovary via an oviduct and normal right uterine horn and the testis via an epididymus and vas deferens, with a vagina which ended in a blind pouch and was filled with squamous debris.     

Multinucleate cells in thymus of C3H mice

Summary Multinucleate epithelial cells occur in the thymus of C3H mice. They are poorly differentiated and scarce, but are more numerous in the medulla than in the cortex. Their increase in number with age is particularly significant between the first and the third months especially for cells with a large number of nuclei, and may be related to thymic involution.Viral particles of type C, similar to those described in murine leukemias, are found in mono- and multinucleate medullary epithelial cells.Research supported by grant 10.013 of the Fonds de la Recherche Fondamentale Collective (Brussels)