Physiopathological role of the enzymatic complex 5α-reductase and 3α/ß-hydroxysteroid oxidoreductase in the generation of progesterone and testosterone neuroactive metabolites.

The enzymatic complex 5α-reductase (5α-R) and 3α/3ß-hydroxysteroid oxidoreductase (HSOR) is expressed in the nervous system, where it transforms progesterone (PROG) and testosterone (T) into neuroactive metabolites. These metabolites regulate myelination, brain maturation, neurotransmission, reproductive behavior and the stress response. The expression of 5α-R and 3α-HSOR and the levels of PROG and T reduced metabolites show regional and sex differences in the nervous system and are affected by changing physiological conditions as well as by neurodegenerative and psychiatric disorders. A decrease in their nervous tissue levels may negatively impact the course and outcome of some pathological events. However, in other pathological conditions their increased levels may have a negative impact. Thus, the use of synthetic analogues of these steroids or 5α-R modulation have been proposed as therapeutic approaches for several nervous system pathologies. However, further research is needed to fully understand the consequences of these manipulations, in particular with 5α-R inhibitors.

Cellular and functional characterization of buffalo (Bubalus bubalis) corpus luteum during the estrous cycle and pregnancy.

In the present paper, cellular composition of buffalo corpus luteum (CL) with its functional characterization based on 3ß-HSD and progesterone secretory ability at different stages of estrous cycle and pregnancy was studied. Buffalo uteri along with ovaries bearing CL were collected from the local slaughter house. These were classified into different stages of estrous cycle (Stage I, II, III and IV) and pregnancy (Stage I, II and III) based on morphological appearance of CL, surface follicles on the ovary and crown rump length of conceptus. Luteal cell population, progesterone content and steroidogenic properties were studied by dispersion of luteal cells using collagenase type I enzyme, RIA and 3ß-HSD activity, respectively. Large luteal cells (LLC) appeared as polyhedral or spherical in shape with a centrally placed large round nucleus and an abundance of cytoplasmic lipid droplets. However, small luteal cells (SLC) appeared to be spindle shaped with an eccentrically placed irregular nucleus and there was paucity of cytoplasmic lipid droplets. The size of SLC (range 12-23µm) and LLC (range 25-55µm) increased (P<0.01) with the advancement of stage of estrous cycle and pregnancy. The mean progesterone concentration per gram and per CL increased (P<0.01) from Stage I to III of estrous cycle with maximum concentration at Stage III of estrous cycle and pregnancy. The progesterone concentration decreased at Stage IV (day 17-20) of estrous cycle coinciding with CL regression. Total luteal cell number (LLC and SLC) also increased (P<0.01) from Stage I to III of estrous cycle and decreased (P<0.05), thereafter, at Stage IV indicating degeneration of luteal cells and regression of the CL. Total luteal cell population during pregnancy also increased (P<0.01) from Stage I to II and thereafter decreased (P>0.05) indicating cessation of mitosis. Increased (P<0.05) large luteal cell numbers from Stage I to III of estrous cycle and pregnancy coincided with the increased progesterone secretion and 3ß-HSD activity of CL. Thus, proportionate increases of large compared with small luteal cells were primarily responsible for increased progesterone secretion during the advanced stages of the estrous cycle and pregnancy. Total luteal cells and progesterone content per CL during the mid-luteal stage in buffalo as observed in the present study seem to be less than with cattle suggesting inherent luteal deficiency.

The bioreductive prodrug PR-104A is activated under aerobic conditions by human aldo-keto reductase 1C3.

PR-104, currently in phase II clinical trials, is a phosphate ester pre-prodrug which is converted in vivo to its cognate alcohol, PR-104A, a prodrug designed to exploit tumor hypoxia. Bioactivation occurs via one-electron reduction to DNA crosslinking metabolites in the absence of oxygen. However, certain tumor cell lines activate PR-104A in the presence of oxygen, suggesting the existence of an aerobic nitroreductase. Microarray analysis identified a cluster of five aldo-keto reductase (AKR) family members whose expressions correlated with aerobic metabolism of PR-104A. Plasmid-based expression of candidate genes identified aldo-keto reductase 1C3 as a novel nitroreductase. AKR1C3 protein was detected by Western blot in 7 of 23 cell lines and correlated with oxic PR-104A metabolism, an activity which could be partially suppressed by Nrf2 RNAi knockdown (or induced by Keap1 RNAi), indicating regulation by the ARE pathway. AKR1C3 was unable to sensitize cells to 10 other bioreductive prodrugs and was associated with single-agent PR-104 activity across a panel of 9 human tumor xenograft models. Overexpression in two AKR1C3-negative tumor xenograft models strongly enhanced PR-104 antitumor activity. A population level survey of AKR1C3 expression in 2,490 individual cases across 19 cancer types using tissue microarrays revealed marked upregulation of AKR1C3 in a subset including hepatocellular, bladder, renal, gastric, and non-small cell lung carcinoma. A survey of normal tissue AKR1C3 expression suggests the potential for tumor-selective PR-104A activation by this mechanism. These findings have significant implications for the clinical development of PR-104.

Type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) contributes to testosterone production in the adrenal reticularis.

CONTEXT: The human adrenal gland produces small amounts of testosterone that are increased under pathological conditions. However, the mechanisms through which the adrenal gland produces testosterone are poorly defined. OBJECTIVE: Our objective was to define the role of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in human adrenal production of testosterone. DESIGN AND METHODS: Adrenal vein sampling was used to confirm ACTH stimulation of adrenal testosterone production. Adrenal expression of AKR1C3 was studied using microarray, quantitative real-time RT-PCR, and immunohistochemical analyses. AKR1C3 knockdown was accomplished in cultured adrenal cells (H295R) using small interfering RNA, followed by measurement of testosterone production. RESULTS: Acute ACTH administration significantly increased adrenal vein testosterone levels. Examination of the enzymes required for the conversion of androstenedione to testosterone using microarray analysis, quantitative real-time RT-PCR, and immunohistochemistry demonstrated that AKR1C3 was present in the adrenal gland and predominantly expressed in the zona reticularis. Decreasing adrenal cell expression of AKR1C3 mRNA and protein inhibited testosterone production in the H295R adrenal cell line. CONCLUSIONS: The human adrenal gland directly secretes small, but significant, amounts of testosterone that increases in diseases of androgen excess. AKR1C3 is expressed in the human adrenal gland, with higher levels in the zona reticularis than in the zona fasciculata. AKR1C3, through its ability to convert androstenedione to testosterone, is likely responsible for adrenal testosterone production.

The developmental increase in adrenocortical 17,20-lyase activity (biochemical adrenarche) is driven primarily by increasing cytochrome b5 in neonatal rhesus macaques.

Adrenarche is thought to be experienced only by humans and some Old World primates despite observed regression of an adrenal fetal zone and establishment of a functional zona reticularis (ZR) in other species like rhesus macaques. Adrenal differentiation remains poorly defined biochemically in nonhuman primates. The present studies defined ZR development in the neonatal rhesus by examining androgen synthetic capacity and factors affecting it in rhesus and marmoset adrenals. Western immunoblots examined expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5 (b5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD), among other key enzymes. 17,20-lyase activity was quantified in adrenal microsomes, as was the contribution of b5 to 17,20-lyase activity in microsomes and cell transfection experiments with rhesus and marmoset P450c17. Expression of b5 increased from birth to 3 months, and was positively correlated with age and 17,20-lyase activity in the rhesus. Recombinant b5 addition stimulated 17,20-lyase activity to an extent inversely proportional to endogenous levels in adrenal microsomes. Although 3betaHSD expression also increased with age, P450c17, 21-hydroxylase cytochrome P450, and the redox partner, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase, did not; nor did recombinant cytochrome P450 oxidoreductase augment 17,20-lyase activity. Cotransfection with b5 induced a dose-dependent increase in dehydroepiandrosterone synthesis by both nonhuman primate P450c17 enzymes. We conclude that the increase in 17,20-lyase activity characteristic of an adrenarche in rhesus macaques is driven primarily by increased b5 expression, without the need for a decrease in 3betaHSD, as suggested from human studies. The rhesus macaque is a relevant and accessible model for human ZR development and adrenal function.

Inactivation of the anticancer drugs doxorubicin and oracin by aldo-keto reductase (AKR) 1C3.

Resistance towards anticancer drugs is a general problem upon chemotherapy. Among the mechanisms of resistance, metabolic inactivation by carbonyl reduction is a major cause of chemotherapy failure that applies to drugs bearing a carbonyl moiety. Oracin is a promising anticancer drug which is presently in phase II clinical trials. Pharmacokinetic studies have revealed that oracin undergoes metabolic inactivation by carbonyl reduction. In the present study, we provide evidence that AKR1C3, a member of the aldo-keto reductase (AKR) superfamily, catalyzes the inactivation of oracin. Moreover, AKR1C3 does also mediate C13 carbonyl reduction of doxorubicin to its inactive hydroxy metabolite doxorubicinol. Doxorubicinol, however, has also been considered responsible for the cardiomyopathy observed upon doxorubicin chemotherapy. Since AKR1C3 is overexpressed in hormone-dependent malignancies like prostate and breast cancer, coadministration of AKR1C3 inhibitors might enhance the chemotherapeutic efficacy of oracin and doxorubicin, and simultaneously reduce the risk of cardiomyopathy upon doxorubicin treatment.

Analysis of HSD3B7 knockout mice reveals that a 3alpha-hydroxyl stereochemistry is required for bile acid function.

Primary bile acids are synthesized from cholesterol in the liver and thereafter are secreted into the bile and small intestine. Gut flora modify primary bile acids to produce secondary bile acids leading to a chemically diverse bile acid pool that is circulated between the small intestine and liver. A majority of primary and secondary bile acids in higher vertebrates have a 3alpha-hydroxyl group. Here, we characterize a line of knockout mice that cannot epimerize the 3beta-hydroxyl group of cholesterol and as a consequence synthesize a bile acid pool in which 3beta-hydroxylated bile acids predominate. This alteration causes death in 90% of newborn mice and decreases the absorption of dietary cholesterol in surviving adults. Negative feedback regulation of bile acid synthesis mediated by the farnesoid X receptor (FXR) is disrupted in the mutant mice. We conclude that the correct stereochemistry of a single hydroxyl group at carbon 3 in bile acids is required to maintain their physiologic and regulatory functions in mammals.

Neurosteroids and the songbird model system.

The brain is now widely recognized as having the capacity to make steroids, neurosteroidogenesis. Although many functions are known for steroids that might be made in the brain, the evolution of and natural biological functions for these neurosteroids are not fully understood. In songbirds, neurosteroids may function in the development of neural circuits controlling song and may also participate in the activation of some steroid-dependent behaviors during the non-breeding season. In addition to neuroanatomical and behavioral evidence, we have physiological, molecular, and biochemical evidence for the expression and activity of steroidogenic enzymes in the brains of developing and adult songbirds. We review the evidence published so far for songbird neurosteroidogenesis and discuss why we believe songbird species are excellent models for the study of brain steroid synthesis and action.

Aldo-keto reductase (AKR) 1C3: role in prostate disease and the development of specific inhibitors.

Human aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20-ketosteroid reductase with a preference for the conversion of Delta4-androstene-3,17-dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9alpha,11beta-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARgamma). Activation of PPARgamma is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9alpha,11beta-PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9alpha,11beta-PGF2.

Tibolone metabolism in human liver is catalyzed by 3alpha/3beta-hydroxysteroid dehydrogenase activities of the four isoforms of the aldo-keto reductase (AKR)1C subfamily.

Tibolone [[7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one] is used to treat climacteric symptoms and prevent osteoporosis. It exerts tissue-selective effects via site-specific metabolism into 3alpha- and 3beta-hydroxymetabolites and a Delta4-isomer. Recombinant human cytosolic aldo-keto reductases 1C1 and 1C2 (AKR1C1 and AKR1C2) produce 3beta-hydroxytibolone, and the liver-specific AKR1C4 produces predominantly 3alpha-hydroxytibolone. These observations may account for the appearance of 3beta-hydroxytibolone in target tissues and 3alpha-hydroxytibolone in the circulation. Using liver autopsy samples (which express AKR1C1-AKR1C4), tibolone was reduced via 3alpha- and 3beta-hydroxysteroid dehydrogenase (HSD) activity. 3beta-Hydroxytibolone was exclusively formed in the cytosol and was inhibited by the AKR1C2-specific inhibitor 5beta-cholanic acid-3alpha, 7alpha-diol. The cytosolic formation of 3alpha-hydroxytibolone was inhibited by an AKR1C4-selective inhibitor, phenolphthalein. The ratio of these stereoisomers was 4:1 in favor of 3beta-hydroxytibolone. In HepG2 cell cytosol and intact cells (which do not express AKR1C4), tibolone was exclusively reduced to 3beta-hydroxytibolone and was blocked by the AKR1C1-AKR1C3 inhibitor flufenamic acid. In primary hepatocytes (which express AKR1C1-AKR1C4), time-dependent reduction of tibolone into 3beta- and 3alpha-hydroxytibolone was observed again in a 4:1 ratio. 3beta-HSD activity was inhibited by both 5beta-cholanic acid-3alpha,7alpha-diol and flufenamic acid, implicating a role for AKR1C2 and AKR1C1. By contrast, the formation of 3alpha-hydroxytibolone was exclusively inhibited by phenolphthalein implicating AKR1C4 in this reaction. 3beta- and 3alpha-Hydroxytibolone were rapidly metabolized into polar metabolites (>85%). The formation of minor amounts of tibolone was also observed followed by AKR1C-catalyzed epimerization. The low hepatic formation of 3alpha-hydroxytibolone suggests that AKR1C4 is not the primary source of this metabolite and instead it maybe formed by an intestinal or enterobacterial 3alpha-HSD.

Mitochondrial 3 beta-hydroxysteroid dehydrogenase (HSD) is essential for the synthesis of progesterone by corpora lutea: an hypothesis.

In mouse ovaries, the enzyme 3 beta-hydroxysteroid dehydrogenase (HSD) is distributed between microsomes and mitochondria. Throughout the follicular phase of the estrous cycle, the HSD activity in microsomes is predominant; whereas, after LH stimulation, HSD activity during the luteal phase is highest in the mitochondria. The current study examined whether or not LH stimulation always results in an increase in mitochondrial HSD activity. This was accomplished by measuring the HSD activity in microsomal and mitochondrial fractions from ovaries of pregnant mice. These animals have two peaks of LH during gestation, and one peak of LH after parturition. It was found that mitochondrial HSD activity was highest after each peak of LH. It is proposed that mitochondrial HSD is essential for the synthesis of high levels of progesterone. The increase in HSD activity in mitochondria after LH stimulation occurs because: 1) LH initiates the simultaneous synthesis of HSD and the cholesterol side-chain cleavage enzyme (P450scc); and, 2) HSD and P450scc bind together to form a complex, which becomes inserted into the inner membrane of the mitochondria. High levels of progesterone are synthesized by mitochondrial HSD because: 1) the requisite NAD+ cofactor for progesterone synthesis is provided directly by the mitochondria, rather than indirectly via the rate limiting malate-aspartate shuttle; and, 2) the end-product inhibition of P450scc by pregnenolone is eliminated because pregnenolone is converted to progesterone.

[Disorder of androgen biosynthesis].

Disorders of androgen biosynthesis are a relatively rare cause of sexual ambiguity in 46XY genetic males. The biosynthesis of androgen requires the steroidogenic acute regulatory protein (StAR) and the steroidogenic enzymes P450scc, 3 beta HSDII, P450c17, 17 beta HSDIII, and 5 alpha-reductase. Deficiencies have been reported in these enzymes, leading to male pseudohermaphroditism. Here we describe three enzymes 3 beta HSDII, P450c17, and 17 beta HSDIII, their roles and mechanisms of action special reference to diagnosis and treatment for the patients with deficiencies of these enzymes.

Steroid hormone synthesis by vaccinia virus suppresses the inflammatory response to infection.

The 3beta-hydroxysteroid dehydrogenase (3beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Vaccinia virus (VV) also synthesizes steroid hormones with a 3beta-HSD enzyme (v3beta-HSD) encoded by gene A44L. Here we examined the effects of v3beta-HSD in VV disease using wild-type (vA44L), deletion (vDeltaA44L), and revertant (vA44L-rev) viruses in a murine intranasal model. Loss of A44L was associated with an attenuated phenotype. Early (days 1-3) after infection with vDeltaA44L or control viruses the only difference observed between groups was the reduced corticosterone level in lungs and plasma of vDeltaA44L-infected animals. Other parameters examined (body weight, signs of illness, temperature, virus titres, the pulmonary inflammatory infiltrate, and interferon [IFN]-gamma levels) were indistinguishable between groups. Subsequently, vDeltaA44L-infected animals had reduced weight loss and signs of illness, and displayed a vigorous pulmonary inflammatory response. This was characterized by rapid recruitment of CD4+ and CD8+ lymphocytes, enhanced IFN-gamma production and augmented cytotoxic T lymphocyte activity. These data suggest that steroid production by v3beta-HSD contributes to virus virulence by inhibiting an effective inflammatory response to infection.

Effect of salt acclimatization on 3 beta-hydroxysteroid dehydrogenase/isomerase activity in the interrenal of Bufo arenarum.

In amphibians, aldosterone (Aldo) is particularly important in the regulation of Na(+) exchange by skin and urinary bladder. In previous works we studied a key enzyme in Aldo biosynthesis, the 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta HSD/I), in the interrenals of Bufo arenarum. In those works a dual localization of the 3 beta HSD/I in both microsomes and mitochondria was described. The mitochondrial, but not the microsomal, enzyme prefers the immediate Aldo precursor, 3 beta-analogue of aldosterone, as substrate. In this order, the enzyme 3 beta HSD/I would be not only a key enzyme for the synthesis of Aldo but additionally, due to its microsomal and mitochondrial localization, a possible target for the regulation of Aldo biosynthesis. With this rationale in mind, we have used in vivo and in vitro approaches to study Aldo regulation. In the present investigation the levels of Aldo were determined in plasma of winter (W) and summer (S) toads subjected to different saline concentrations (0.125 and 0.15 M) or kept on wet land. Saline hyperosmotically treated toads had significantly lower levels than isoosmotically treated toads. These results are consistent with the response in mammals, in which salt loading provokes a reduction in Aldo secretion. In W toads, plasmatic corticosterone (B) concentration was higher than Aldo concentration, whereas in S toads, B/Aldo ratio approached unity. The reduction of Aldo levels after saline dehydration was due to a decline in its biosynthesis. K(m) and V(max) values for 3 beta HSD/I were calculated for mitochondrial and microsomal fractions obtained from animals acclimated to 0.15 M NaCl or kept on land. As previously described, V(max) differs between W and S toads. However, only mitochondrial V(max) changed as a consequence of saline adaptation, suggesting that the mitochondrial enzyme could be involved in the regulation of Aldo biosynthesis.

Control of human luteal steroidogenesis.

The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.

3alpha-Hydroxysteroid dehydrogenase in animal and human tissues.

This review analyzes data on the biological role of 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) in animal and human tissues and describes its main characteristics, mechanism of action, and regulation of activity. Based on published data, a scheme for the actions of androgen, progestin, and glucocorticoids involving the participation of 3alpha-HSD is proposed. According to this scheme, in the mechanism of steroid action 3alpha-HSD not only regulates the concentration of the main effector androgen, 5alpha-dihydrotestosterone, in target cells, but also switches androgen, progestin, and glucocorticosteroid genomic activity to non-genomic activity.

Placental lactogen not growth hormone and prolactin regulates secretion of progesterone in vitro by the 40-45 day ovine corpus luteum of pregnancy.

The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL.

A new insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) deficiency is a rare form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene, causing varying degrees of salt-loss in both sexes and incomplete masculinization of the external genitalia in genetic males. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing and 23 missense mutations) have been identified in the HSD3B2 gene. Results from functional charaterization studies of the mutant proteins agrees with the prediction that no functional type II 3beta-HSD isoenzyme is expressed in the adrenals and gonads of the patients with the severe salt-losing form, whereas the nonsalt-losing form causes an incomplete loss in enzymatic activity, thereby leaving sufficient enzymatic activity to prevent salt loss. Recent studies have highlighted the fact that various mutations appear to have a drastic effect upon the stability of the protein, therefore providing molecular evidence of a new mechanism involved in classical 3beta-HSD deficiency. Finally, the functional characterization of the missense mutations known to be involved in this autosomal recessive disorder provides valuable information concerning the structure-function relationships of the 3beta-HSD enzyme superfamily.

GEN GEN: the genomic genetic analysis of androgen-metabolic genes and prostate cancer as a paradigm for the dissection of complex phenotypes.

Prostate cancer will be diagnosed in about 179,300 men in the US in 1999 alone. Some 37,000 individuals die of this disease annually. Prostate cancer is characterized by a substantial racial/ethnic variation in risk: highest in African-American men, lowest in Asian men and intermediate in Caucasian and Latino men. We set out to investigate as our central hypothesis that genetic variants of genes involved in androgen metabolism by themselves and in combination significantly contribute to prostate cancer progression and its racial/ethnic variation. Specifically, we examined the hypothesis that DNA sequence (allelic) variations in the type II (or prostatic) steroid 5alpha-reductase (SRD5A2) gene contribute substantially to the risk and progression of prostate cancer particularly across racial/ethnic lines. The "candidate gene", SRD5A2, was chosen because the reaction product [i.e. dihydrotestosterone (DHT)] of the enzyme encoded by this gene modulates directly cell division in the prostate. DHT binds to the androgen receptor (AR) and the DHT-AR complex leads to the transactivation of a variety of genes which ultimately modulates cell division in the prostate. Epidemiologic evidence suggests that variation in DHT levels play an important role in risk of prostate cancer. Thus, steroid 5alpha-reductase activity encoded by SRD5A2 variant alleles may be important in regulating intraprostatic DHT steady state levels by controlling its biosynthesis. A second candidate gene, the type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) gene, encodes the enzyme that initiates the metabolic inactivation of testosterone (T) to DHT. We have identified allelic variants in this gene as well. Here I review our strategy for identifying candidate genes for prostate cancer, a multifactorial disease. I summarize the significant findings, particularly of allelic variants in the HSD3B2 and SRD5A2 genes and discuss how they by themselves, in combination and through interactions with the environment may play a role in prostate cancer predisposition and its progression. Our approach, a multidisciplinary genomic genetic (GEN GEN) attack on the problem, may be useful in the analysis of other complex phenotypes as well.