Regulation of renal N-nitrosodimethylamine demethylase activity by androgens, progestin, glucocorticoid, and estrogen in BALB/c mice.

Studies from this laboratory have shown that renal N-nitrosodimethylamine demethylase (NDMA demethylase), an enzyme responsible for the metabolism of NDMA, shows age- and sex-related differences and is modulated by testosterone. These results have been positively correlated with NDMA-induced mutagenicity and carcinogenicity studies; however, relatively large doses of testosterone (100 mg/kg or 2 mg/day) had been used. The objective of this study was to determine whether this tissue specific increase in renal NDMA demethylase was evident only with pharmacological doses or could be observed at lower doses of testosterone, and, to determine whether this testosterone-induced increase in renal NDMA demethylase was specific for androgenic hormones. BALB/c mice were given injections s.c. of varying doses of testosterone (0.01 to 1.5 mg/day) in 0.1 ml of peanut oil for 1, 4, 8, or 16 days; control mice received peanut oil alone. A 3-fold increase in renal NDMA demethylase was observed within 24 h with 0.01 mg of testosterone. Dose-dependent increases of 3-, 5-, and 11-fold were observed at 0.01, 0.05, and 0.10 mg of testosterone, respectively. Hepatic NDMA demethylase was not affected. To determine whether this induction was specific for androgenic hormones, several hormones (androgens, estrogen, glucocorticoid, and progestin) were used. A 5-, 7-, and 2-fold stimulation of renal NDMA demethylase activity was observed in female mice with 5 alpha-dihydrotestosterone, synthetic androgen methyltrienolone, and androgen agonist medroxyprogesterone acetate, respectively. The biologically inactive 5 beta-dihydrotestosterone, stereomer of 5 alpha-dihydrotestosterone, had no effect. Medroxyprogesterone, methyltrienolone, testosterone, and 5 alpha-dihydrotestosterone exert their effect by interacting with androgen receptors; 5 beta-dihydrotestosterone does not bind to androgen receptors. Dexamethasone or estradiol treatment resulted in a significant inhibition of renal enzyme activity in male mice. None of the treatments affected hepatic NDMA demethylase activities in male or female mice. This rapid and tissue specific stimulation of renal NDMA demethylase with low doses of testosterone or with other androgen agonists seems to be androgen receptor mediated and indicates a physiological relevance of this phenomenon.

Epithelial-stromal interactions and tumor progression: meeting summary and future directions.

The Epithelial-Stromal Interactions Workshop was organized with the purpose of accelerating progress in understanding the interrelationship between tumor cells and their microenvironment and applying this knowledge to the control of tumor progression. The format of the meeting was the presentation of brief reports that focused on concepts rather than specifics, with extensive discussion periods to identify the issues and barriers hindering progress in this area. This report summarizes the findings of this meeting, highlighting the intimate relationship between tumor cells and their environment and addressing the opportunities that manipulation of host-tumor interactions has for therapeutic intervention. Several specific recommendations are made to advance knowledge and progress in this field.

Hormonal regulation of the metabolism of carcinogens in renal tissue of BALB/c mice.

The role of androgens in the regulation of carcinogen metabolism in the renal tissue of BALB/c mice was investigated. Kidney microsomal enzyme preparations from mature and immature animals were used in mutagenic studies using the Ames test. Androgen receptors (cytosolic and nuclear) were also evaluated. The results show that the microsomal enzymes from mature males had greater potential to biotransform dimethylnitrosamine than did the microsomal enzymes from mature females or immature animals. Testosterone treatment of mature females or immature animals resulted in a significant increase in the mutagenic ability of their renal microsomal enzymes. Androgen receptors were detected in kidney cytosols of mature and immature animals (both males and females); however, nuclear androgen receptors were detected only in the mature males. Testosterone treatment resulted in a significant accumulation of nuclear androgen receptors in the kidneys of mature females and immature animals. The relationships among mutagenic activity, androgen receptors, the levels of N-demethylase (an enzyme responsible for conversion of dimethylnitrosamine to its active metabolite), dietary fat, and the carcinogen metabolism are discussed.

Mammalian endogenous peroxidases as cellular markers and as biosynthetic endpoints of hormone-mediated activity: viewpoint from cytochemistry.

Aldehyde-resistant, diaminobenzidine-stained endogenous peroxidases form ideal markers for the biochemical endpoints of hormone stimulation and differentiation of certain mammalian cells and tissues. The lactoperoxidase (LPO)-type of endogenous peroxidases are synthesized by the acinar cells of the salivary, Harderian, lacrimal and mammary glands and are present in their secretions. These LPO-type enzymes, that are inhibited by cyanide and aminotriazole, appear to operate extracellularly as bactericidal agents in milk and in other biological fluids. In the mammary gland, lactoperoxidase is a consistent marker enzyme for differentiated acinar cells engaged in lactogenesis. Myeloperoxidase (MPO)-type endogenous peroxidases are prominent markers for the GERL endomembrane system and differentiated lysosomes in certain cells of the reticuloendothelial system and phagocytes. MPO is prominent within eosinophils, peritoneal macrophages and in Kupffer cells. The MPO-type endogenous peroxidases function primarily within lysosomes as bactericidal agents. Thyroid peroxidase (TPO) is relegated to the cisternae of the granular endoplasmic reticulum and Golgi apparatus, to apical cytoplasmic vesicles and to the luminar cell membrane surface of acinar cells. The enzyme is probably activated at release and functions both in the organification reaction (T leads to To) and in the biosynthesis of thyroxine. Thyroid stimulating hormone (TSH) appears to play a key role in the regulation of TPO levels and activity in the thyroid gland. Certain tissues displaying growth-dependency on estrogen (i.e., uterus, cervix, vagina and the DMBA-induced rat mammary tumor) synthesize and secrete endogenous peroxidase into their lumina. These enzymes serve as important marker proteins of estrogen action, in that they occur distal to the binding of estrogen to its receptor protein. Estrogen antagonists, particularly CI-628 (Parke-Davis) and Nafoxidine (Upjohn) that appear to function through the estrogen receptor mechanism, also induce synthesis of the reproductive tract endogenous peroxidase but inhibit growth of these tissues. Progesterone antagonizes the synthesis of the reproductive tract peroxidases and inhibits growth of the tissues as well, in part, through the reduction of the cytosol estrogen receptor protein. Endogenous peroxidase activity appears to represent a reliable marker for rodent breast cancer tissues displaying dependency for estrogen and is of potential interest as a diagnostic marker protein in human breast cancer. Rat uterine peroxidase (UP) has been investigated by microelectrophoretic techniques. The molecular weight of UP has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergents. The isoelectric point of UP is located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, UP was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000.

Tissue specific regulation of renal N-nitrosodimethylamine-demethylase activity by testosterone in BALB/c mice.

Nitrosodimethylamine (NDMA), like several other nitrosamines, is activated by the enzymes--mixed-function oxidases--present in the tissue microsomal fractions, producing mutagenic and carcinogenic effects. Previous studies in BALB/c mice have shown an age, sex and androgenic regulation of NDMA-induced mutagenicity. The present study was designed to test the correlation between renal NDMA-demethylase activity and previously published reports on NDMA-induced mutagenicity. Renal and hepatic NDMA-demethylases were determined from the microsomal fractions by quantitating formaldehyde. Renal NDMA-demethylase showed the presence of two isozymes, I and II, with Km values of 0.6 +/- 0.2 and 20.2 +/- 6.8 mM respectively. Isozyme I was detected in adult males and first appeared at the onset of puberty; it was absent in adult females and in immature mice. Renal isozyme II was detected in both males and females and was independent of age. Testosterone treatment of adult females resulted in the appearance of renal isozyme I. Castration of adult males caused a dramatic decrease in activity, whereas testosterone administration to such castrates increased activity, of renal isozyme I. Hepatic NDMA-demethylase activities were independent of age, sex or testosterone treatment. In conclusion, these results show an age, sex and tissue specific regulation of renal NDMA activity. Renal and hepatic NDMA-demethylase activities correlated positively with earlier studies on NDMA-induced mutagenesis and carcinogenesis.

Inhibition of experimental metastasis of human breast-carcinoma cells in athymic nude-mice by anti-alpha(5)beta(1) fibronectin receptor integrin antibodies.

We have investigated the role of the human alpha(5) beta(1) fibronectin receptor integrin in experimental metastasis. Treatment of human MDA-MB-231 breast carcinoma cells with monoclonal antibodies specific for alpha(5) or beta(1) integrin subunits prior to injection into the tail veins of 7 to 9 week old athymic nude mice significantly decreased the median number of lung colonies that were formed. In contrast, treatment of the cells with monoclonal antibodies specific for the alpha(2) subunit had no significant effect. In vitro, the anti-alpha(5) and the anti-beta(1) monoclonal antibodies both strongly inhibited breast carcinoma cell adhesion to fibronectin, while only the anti-beta(1) monoclonal antibody inhibited adhesion to laminin. In a Boyden chamber invasion assay, the anti-beta(1) antibody almost completely inhibited invasion of the breast carcinoma cells through an artificial Matrigel basement membrane. The anti-alpha(5) monoclonal antibody inhibited in vitro invasion approximately 30%, only if fibroblast conditioned medium was present as a chemoattractant. Cell migration on fibronectin could be inhibited by both the anti-alpha(5) and the anti-beta(1) monoclonal antibody. These results indicate that the alpha(5) beta(1) integrin fibronectin receptor on MDA-MB-231 human breast carcinoma cells plays an important role in experimental hematogenous metastasis and may function in this process by a combination of mechanisms, including tumor cell attachment to fibronectin and fibronectin-directed extravasation of tumor cells into the target organ.

Phorbol myristate acetate-differentiated THP-1 cells display increased levels of MHC class I and class II mRNA and interferon-gamma-inducible tumoricidal activity.

The protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein induce differentiation of human monocytic leukemia (THP-1) cells along the monocyte/macrophage pathway of development. The differentiated cells express many important macrophage functions including phagocytosis and the secretion of immunomodulatory cytokines. Mezerein-differentiated THP-1 cells secrete interleukin-1 beta as well as a tumor cell growth inhibitory factor whose basal level is increased in response to interferon-gamma. However, tumoricidal, as opposed to tumoristatic, activity of differentiated THP-1 has not been documented. We report herein that PMA-differentiated THP-1 cells (PD/THP-1) contain elevated levels of MHC class I and class II mRNAs even in the absence of activating factors, and kill HT-29 human colon carcinoma cells when stimulated with recombinant human interferon-gamma. These two characteristics are important components of the macrophage phenotype. The results presented in this study extend previous observations on THP-1 cells by demonstrating that PD/THP-1 cells display a critical, immunologically relevant macrophage function, and therefore, enhance the utility of THP-1 as a model for the in vitro study of immunomodulatory drugs and macrophage-mediated cytocidal processes.

Inhibition of growth of subcutaneous xenografts and metastasis of human breast carcinoma by swainsonine: modulation of tumor cell HLA class I antigens and host immune effector mechanisms.

Swainsonine, an indolizidine alkaloid, can decrease the organ colonization potential of metastatic murine tumor cells by augmentation of host immune effector mechanisms. In this report the above findings were extended by the demonstration that systemic administration of swainsonine strongly suppressed the growth of human breast carcinoma subcutaneous xenografts and experimentally induced lung metastases. This inhibition was not due to a direct effect of swainsonine on cell growth. However swainsonine treatment of tumor cells resulted in enhanced expression of HLA Class I antigens, and HLA class I mRNA. Swainsonine was a potent immunodulator as evidenced by the increased (a) cytotoxicity of splenocytes and macrophages, and, (b) proliferative potential of splenocytes and bone marrow cells. These data suggest that swainsonine-induced inhibition of tumor growth and metastases may be mediated via activation of host effector cells and/or alteration of tumor cell antigenicity.

Experimental approaches for the prevention of hematogenous metastasis.

With improvements in surgical procedures and cure rates, the probable course of cancer for the majority of patients is now largely determined by metastasis rather than growth of the primary tumor itself. Thus, metastasis has received increasing attention over the past decade. These studies have led to the identification of several of the molecular events crucial for metastatic dissemination, information which is now being used to design therapeutic strategies to inhibit metastasis formation. Even though the molecular events involved in the dissemination of malignant disease are only partially known, several promising agents are now being tested for their capacity to limit the spread of cancer. A few clinical trials have shown benefit in prolonging survival and disease-free state, particularly when such therapy is employed on an adjuvant basis.

Review of prostatic surgical procedures at a predominantly black hospital: a 22-year study.

This article reports the results of a pathoepidemiological study of a cohort of black men who had prostatic surgery at Howard University Hospital between 1968 and 1989. The median age of patients at diagnosis of prostate cancer increased by 8 years during the study period, indicating either a delay in seeking medical care or a true delay in onset of the disease. The trends of prevalence of biopsies with cancer in various age groups suggested a sharp rise in the incidence of prostate cancer after the age of 70. The significant increase in popularity of needle biopsy during the period 1982 to 1989 after a 7-year plateau is an indication of increased perceived value of this procedure with experience and technical improvement. The prevalence of biopsies with cancer in all successive time periods of the study showed a bimodal distribution with an early hump in the 50- to 54-year-old age group. This hump is analogous to the break in the rising incidence of breast cancer between the ages of 47 and 52 (Clemmesen hook). This similarity could be explained on the basis of hormonal dependence of the two cancers.

Swainsonine: a new antineoplastic immunomodulator.

Swainsonine, an indolizidine alkaloid with immunomodulatory activity, has been found to be effective in inhibiting metastatic dissemination and growth of primary tumors of both murine and human origins. The unique ability of swainsonine to exhibit antimetastatic, anti-proliferative, and immunomodulatory activity imparts this drug a promising future in cancer therapy.

Effect of chronic administration of excess dietary vitamin A and zinc on lipid metabolism in rats.

The effects of chronic feeding of excess dietary vitamin A and zinc on serum cholesterol, liver cholesterol and total liver lipids were studied in rats. Sixty-four Sprague-Dawley female rats were randomly allocated to 4 groups: basal diet: 4848 micrograms of vitamin A and 61 mg of Zn/kg of diet; high Zn (33-fold excess of the requirement) + basal vitamin A; basal Zn + high vitamin A (154-fold excess of the requirement) and high Zn + high vitamin A. After 120 days serum cholesterol, liver cholesterol, total liver lipids, and vitamin A and zinc in the liver and kidney were measured. The mean serum cholesterol (+/- SE) for the 4 groups were: 121 (+/- 12.6), 159 (+/- 6), 133 (+/- 7.6) and 186 (+/- 7.6) mg/dl, respectively. Liver cholesterol and total lipids for the 4 groups were determined as 1.8 (+/- 0.05), 2.2 (+/- 0.09), 4.9 (+/- 0.12) and 4.6 (+/- 0.24) mg/g of tissue and 26.3 (+/- 0.7), 26.0 (+/- 1.1), 38.9 (+/- 0.9) and 40.3 (+/- 1.4) mg/g of tissue, respectively. Liver and kidney vitamin A levels were significantly higher in group C than in group D, suggesting increased vitamin A metabolism in the high dietary zinc + high vitamin A groups. In high dietary zinc groups, a significant elevation of serum cholesterol was observed regardless of the vitamin A levels. Total liver lipids and liver cholesterol were significantly higher in rats fed high vitamin A irrespectively of the zinc levels in the diet.