WT-1 is required for early kidney development.

In humans, germline mutations of the WT-1 tumor suppressor gene are associated with both Wilms' tumors and urogenital malformations. To develop a model system for the molecular analysis of urogenital development, we introduced a mutation into the murine WT-1 tumor suppressor gene by gene targeting in embryonic stem cells. The mutation resulted in embryonic lethality in homozygotes, and examination of mutant embryos revealed a failure of kidney and gonad development. Specifically, at day 11 of gestation, the cells of the metanephric blastema underwent apoptosis, the ureteric bud failed to grow out from the Wolffian duct, and the inductive events that lead to formation of the metanephric kidney did not occur. In addition, the mutation caused abnormal development of the mesothelium, heart, and lungs. Our results establish a crucial role for WT-1 in early urogenital development.

Skin graft rejection by beta 2-microglobulin-deficient mice.

Mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption lack beta 2-m protein and are deficient for functional major histocompatibility complex class I (MHC-I) molecules. The mutant mice have normal numbers of CD4+8- T helper cells, but lack MHC-I-directed CD4-8+ cytotoxic T lymphocytes (CTLs). In this study we used the beta 2-m mutant mice to study the importance of MHC-I-directed immunity in skin graft rejection. Our results indicate that MHC-I-directed CD8+ CTLs are not essential in the rejection of allografts with whole MHC or multiple minor H differences. However, the absence of MHC-I-guided immunity profoundly reduces the ability of mutant mice to reject H-Y disparate grafts. In addition, we show that natural killer cells which vigorously reject MHC-I-deficient bone marrow grafts, are not effective in the destruction of MHC-I-deficient skin grafts.

Blockade of transgenic gamma delta T cell development in beta 2-microglobulin deficient mice.

The gamma delta T cell receptor (TCR) of the hybridoma KN6 recognizes the self molecule encoded by a class I gene which maps within the TL region of the major histocompatibility complex (MHC) of H-2b mice. Mice transgenic (Tg) for this TCR were crossed with mice genetically deficient in beta 2-microglobulin (beta 2m). No mature Tg gamma delta T cells were detected in the thymus or the spleen of the beta 2m- gamma delta Tg mice. We conclude that interaction between the Tg gamma delta TCR and a beta 2m-associated molecule (probably an MHC class I molecule) is required for the generation of mature Tg gamma delta T cells.

Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells.

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4+8+ and CD4+8- T cells, but have no mature CD4-8+ T cells and are defective in CD4-8+ T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta+ CD4-8+ T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.

Acute and chronic effects of diethylstilbestrol on serum levels of alpha melanocyte stimulating hormone and prolactin in the hamster.

Continuous exposure to diethylstilbestrol (DES) induces renal tumors in male hamsters. The tumor formation is preceded by an increase in pituitary weight and elevation of the pituitary hormones-alpha melanocyte stimulating hormone (aMSH) and prolactin (Pr1). A decline in Pr1 (to normal levels) but not aMSH then accompanies the development of tumors and the enlargement of the intermediate lobe of the pituitary. Hypophysectomy, castration and thymectomy reduced serum levels of aMSH. DES administered for one week increased the serum levels of both hormones in normal and castrated animals, but not in hypophysectomized hamsters.

Control of 2,3,7,8-tetrachlorodibenzo-p-dioxin binding protein(s) in the hamster kidney.

Hamster renal cytosol binds [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high specificity. Sucrose density gradient centrifugation revealed two binding entities- one with a low sedimentation coefficient of 4-5 S which was displaced by neither TCDD nor other polycyclic aromatic hydrocarbons (PAHs) and another with a high sedimentation coefficient of 7-8 S which was displaced by TCDD, benzo[a]pyrene (BP), 2-methylcholanthrene (MC), and 7,12-dimethylbenzo[a]anthracene (DMBA) but not by estradiol (E), progesterone (P), cortisol (F), testosterone (T), 5 alpha-dihydrotestosterone (DHT), or methyltrienolone (R-1881), a synthetic androgen. Cytosol from intact male hamsters showed maximum binding of labelled TCDD to the 7-8 S binding site. Castration or hypophysectomy reduced this binding. Pretreatment with DMBA increased binding, whereas diethylstilbestrol (DES) decreased binding. Sex difference was observed in the binding capacity of renal cytosol. This is the first report of endocrine control over TCDD binding and its modulation by other PAHs and steroids.

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster.

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbecco's modified Eagle's medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.

Permissive role of the pituitary in the induction and growth of estrogen-dependent renal tumors.

Prolonged administration of estrogen to hamsters by implanted pellets induces not only renal adenocarcinomas but also enlarges pituitaries with hyperplastic and neoplastic changes, especially in the pars intermedia. The pituitaries of the diethylstilbestrol-implanted animals weigh 90 to 150 mg; those of control animals without diethylstilbestrol pellets weigh 7 to 12 mg. The enlarged pituitaries have 9.7 x 10(-10) M progesterone receptors compared to 0.75 x 10(-10) M in the controls. Castrated male hamsters were hypophysectomized, implanted with diethylstilbestrol pellets, fed laboratory chow ad libitum, and given 5% glucose in water to drink. New pellets were implanted every 3 months, and the animals survived for 12 to 15 months. At autopsy, none of the animals had a tumor. Sixty-two of 65 control castrated males with the same schedule of pellet implantation developed tumors. Hypophysectomized castrated males implanted with diethylstilbestrol pellets were given daily injections of 1 microgram each of follicle-stimulating hormone, luteinizing hormone, and prolactin; or with 0.9% NaCl solution. These animals survived for 12 to 15 months, but none developed kidney tumors. Other castrated males were hypophysectomized and implanted with diethylstilbestrol pellets, and 2 months later tumor tissues were transplanted under the kidney capsule. Eighty days later, no tumors were evident in the kidneys of these animals. Control castrated males were implanted with diethylstilbestrol pellets, and 2 months later tumor tissue was transplanted under the kidney capsule. Between 60 and 85 days later, 13 of the 15 controls had developed renal tumors. The concentrations of follicle-stimulating hormone, luteinizing hormone, and prolactin were measured by radioimmunoassays. The concentrations of circulating follicle-stimulating hormone and luteinizing hormone in animals with diethylstilbestrol implants decreased with time and, by 7 months, were similar to those in hypophysectomized animals. The concentration of prolactin in animals with diethylstilbestrol pellets increased with time and reached twice the value in the control animals without diethylstilbestrol pellets. These studies suggest that some factor secreted by the pituitary may be involved as a promoter or a cocarcinogen in the estrogen induction of kidney tumors.

Ornithine decarboxylase activity and polyamine content of the placenta and decidual tissue in the rat.

Measurements of ornithine decarboxylase (ODC) activity and of the amounts of putrescine, spermidine and spermine in the placenta and uterus of the pregnant rat and in decidual tissue of the pseudo-pregnant rat showed that these wax and wane with the growth rate of the tissues. Human term placenta has essentially no ODC activity, but placentae from 15-week human gestations have substantial amounts of enzyme. The ODC activity of the rat placenta increases 40-fold from day 10 to day 17 of pregnancy, then gradually decreases. The content of the polyamines in the placenta also reaches a peak on day 17. The ODC activity of the endometrium between implantation sites remains low until the end of pregnancy and then increases eight-fold on days 21 and 22. This may represent increased uterine activity in preparation for parturition. The ODC activity and the polyamine content of decidual tissue responds to administered oestradiol with increases that reach a peak within four hours. The ODC activity and polyamine content of decidual tissue decrease after the fifth day of decidualization. Nuclei isolated from decidual tissue respond to the addition of spermine or spermidine with an increase in the rate of RNA synthesis. Spermidine increases the elongation of RNA chains, but does not initiate the synthesis of new RNA chains in decidual nuclei.