The brain 3ß-HSD up-regulation in response to deteriorating effects of background emotional stress: an animal model of multiple sclerosis.

The brain 3ß-hydroxysteroid dehydrogenase (3ß-HSD), is the enzyme that catalyzes the biosynthesis of a neuroprotective factor, progesterone. The regulation of 3ß-HSD in response to stress exposure in the cuprizone-induced model of Multiple Sclerosis was investigated and the reaction related to the demyelination extremity. 32 female Wistar rats divided into four groups (i.e., control group (Cont), non-stress cuprizone treated (N-CPZ), physical stress- cuprizone treated (P-CPZ) and emotional stress- cuprizone treated (E-CPZ). A witness foot-shock model used to induce background stress for 5 days. An elevated-plus maze applied to validate the stress induction. Followed by 6 weeks of cuprizone treatment, the Y-maze test performed to confirm brain demyelination. 3ß-HSD gene expression as an indicator of progesterone synthesis examined. At the behavioral level, both stressed groups reflected more impaired spatial memory compared to the N-CPZ group (p < 0.01), with more severe results in the E-CPZ group (p < 0.01). The results of mRNA expression of 3ß-HSD illustrated significant elevation in all cuprizone treated groups (p < 0.001) with a higher up-regulation (p < 0.001) in the E-CPZ group. Background stress -particularly emotional type- exacerbates the demyelination caused by cuprizone treatment. The brain up-regulates the 3ß-HSD gene expression as a protective response relative to the myelin degradation extent.

Conservation of Steroidogenic Effect of the Low-Molecular-Weight Agonist of Luteinizing Hormone Receptor in the Course of Its Long-Term Administration to Male Rats.

Abstract-It was shown that the thienopyrimidine derivative TP03, a low-molecular-weight agonist of the luteinizing hormone receptor (LHR), during the treatment of male rats for 7 days steadily increased the production of testosterone (T), whose elevated level was retained for 7 days, and increased the expression of the gene for LHR, which indicates the maintenance of the sensitivity of Leydig cells to gonadotropins. At the same time, the steroidogenic effect of human chorionic gonadotropin (hCG), which significantly increased the T level on the first day of administration, was further weakened, which was accompanied by a decrease in the expression of the gene for LHR in the testes, indicating the development of resistance of Leydig cells to hCG. Along with this, in the case of hCG administration, a compensatory increase in the expression of genes of the steroidogenic enzymes, such as cytochrome P450scc and dehydrogenase 3ß-HSD, was shown in the testes, while in the case of TP03 administration this effect was absent.

The endocannabinoid 2-arachidonoylglycerol dysregulates the synthesis of proteins by the human syncytiotrophoblast.

In recent years, endocannabinoids emerged as new players in various reproductive events. Recently, we demonstrated the involvement of 2-arachidonoylglycerol (2-AG) in human cytotrophoblast apoptosis and syncytialization. However, 2-AG impact in hormone production by the syncytiotrophoblast (hST) was never studied. In this work, we demonstrate that 2-AG activates cannabinoid (CB) receptors, exerting an inhibitory action on cyclic AMP/protein kinase A (cAMP/PKA) and mitogen-activated protein kinase (MAPK) p38 pathways, and enhancing ERK 1/2 phosphorylation. Furthermore, 2-AG affects the synthesis of human chorionic gonadotropin (hCG), leptin, aromatase, 3-ß-hydroxysteroid dehydrogenase (3-ß-HSD), and placental protein 13 (PP13). These 2-AG effects are mediated by the activation of CB receptors, in a mechanism that may involve p38, ERK 1/2 and cAMP/PKA pathways, which participate in the regulation of placental proteins expression. To our knowledge, this is the first study that associates the endocannabinoid signalling and endocrine placental function, shedding light on a role for 2-AG in the complex network of molecules that orchestrate the production of placental proteins essential for the gestational success.

AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia.

PR-104, a phosphate ester of the nitrogen mustard prodrug PR-104A, has shown evidence of efficacy in adult leukemia clinical trials. Originally designed to target hypoxic cells, PR-104A is independently activated by aldo-keto-reductase 1C3 (AKR1C3). The aim of this study was to test whether AKR1C3 is a predictive biomarker of in vivo PR-104 sensitivity. In a panel of 7 patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts, PR-104 showed significantly greater efficacy against T-lineage ALL (T-ALL) than B-cell-precursor ALL (BCP-ALL) xenografts. Single-agent PR-104 was more efficacious against T-ALL xenografts compared with a combination regimen of vincristine, dexamethasone, and l-asparaginase. Expression of AKR1C3 was significantly higher in T-ALL xenografts compared with BCP-ALL, and correlated with PR-104/PR-104A sensitivity in vivo and in vitro. Overexpression of AKR1C3 in a resistant BCP-ALL xenograft resulted in dramatic sensitization to PR-104 in vivo. Testing leukemic blasts from 11 patients confirmed that T-ALL cells were more sensitive than BCP-ALL to PR-104A in vitro, and that sensitivity correlated with AKR1C3 expression. Collectively, these results indicate that PR-104 shows promise as a novel therapy for relapsed/refractory T-ALL, and that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from such treatment in prospective clinical trials.

Arsenic activates the expression of 3ß-HSD in mouse Leydig cells through repression of histone H3K9 methylation.

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3ß-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3ß-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3ß-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3ß-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells.

[Abnormal expression of genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer].

Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.

Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3ß-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Pregnane glycosides interfere with steroidogenic enzymes to down-regulate corticosteroid production in human adrenocortical H295R cells.

A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na(+) /K(+) ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11ß-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21ß-hydroxylase, but not 3ß-hydroxysteroid dehydrogenase/isomerase.

Retinol dehydrogenase 10 but not retinol/sterol dehydrogenase(s) regulates the expression of retinoic acid-responsive genes in human transgenic skin raft culture.

Retinoic acid is essential for skin growth and differentiation, and its concentration in skin is controlled tightly. In humans, four different members of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins were proposed to catalyze the rate-limiting step in the biosynthesis of retinoic acid (the oxidation of retinol to retinaldehyde). Epidermis contains at least three of these enzymes, but their relative importance for retinoic acid biosynthesis and regulation of gene expression during growth and differentiation of epidermis is not known. Here, we investigated the effect of the four human SDRs on retinoic acid biosynthesis, and their impact on growth and differentiation of keratinocytes using organotypic skin raft culture model of human epidermis. The results of this study demonstrate that ectopic expression of retinol dehydrogenase 10 (RDH10, SDR16C4) in skin rafts dramatically increases proliferation and inhibits differentiation of keratinocytes, consistent with the increased steady-state levels of retinoic acid and activation of retinoic acid-inducible genes in RDH10 rafts. In contrast, SDRs with dual retinol/sterol substrate specificity, namely retinol dehydrogenase 4 (RoDH4, SDR9C8), RoDH-like 3α-hydroxysteroid dehydrogenase (RL-HSD, SDR9C6), and RDH-like SDR (RDHL, SDR9C4) do not affect the expression of retinoic acid-inducible genes but alter the expression levels of several components of extracellular matrix. These results reveal essential differences in the metabolic contribution of RDH10 versus retinol/sterol dehydrogenases to retinoic acid biosynthesis and provide the first evidence that non-retinoid metabolic products of retinol/sterol dehydrogenases affect gene expression in human epidermis.

Inner mitochondrial translocase Tim50 interacts with 3ß-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis.

In the adrenals, testes, and ovaries, 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3ßHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3ßHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3ßHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3ßHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3ßHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3ßHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3ßHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3ßHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3ßHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3ßHSD2 expression and activity, representing a new role for translocases in steroidogenesis.

Transactivation activity of human papillomavirus type 16 E6*I on aldo-keto reductase genes enhances chemoresistance in cervical cancer cells.

The oncogenic E6 proteins produced by high-risk human papillomaviruses (HPVs) are invariably expressed in cervical carcinomas and are multifunctional proteins capable of affecting host-cell proliferation by binding and deregulating key host molecules such as p53. High-risk HPVs, including HPV16, have the unique ability to splice the E6 viral transcript, resulting in the production of a truncated E6 protein known as E6*I whose precise biological function is unclear. This study explored the changes in gene expression of the cervical cancer C33A cell line stably expressing HPV16 E6*I (16E6*I) and observed the upregulation of ten genes. Two of these genes were aldo-keto reductases (AKR1Cs), AKR1C1 and AKR1C3, which have been implicated in drug resistance. The results demonstrated that expression of 16E6*I, but not full-length E6, specifically increased AKR1C1 transcript levels although it did not alter AKR1C2 transcript levels. HPV16 E7 alone also had the ability to cause a moderate increase in AKR1C3 at both mRNA and protein levels. Site-directed mutagenesis of 16E6*I revealed that transactivation activity was abolished in R8A, R10A and T17A 16E6*I mutants without altering their intracellular localization patterns. Loss of transactivation activity of the 16E6*I mutants resulted in a significant loss of AKR1C expression and a decrease in drug resistance. Analysis of the AKR1C1 promoter revealed that, unlike the E6 protein, 16E6*I does not mediate transactivation activity solely through Sp1-binding sites. Taken together, it was concluded that 16E6*I has a novel function in upregulating expression of AKR1C and, in concert with E7, has implications for drug treatment in HPV-mediated cervical cancer.

Identification and characterization of the LysR-type transcriptional regulator HsdR for steroid-inducible expression of the 3α-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400-47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-type C. testosteroni after testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3α-HSD/CR expression in C. testosteroni.

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization.

BACKGROUND: Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. METHODS: To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. RESULTS: Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the "hit list" was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. CONCLUSIONS: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug's properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.

Aggressive interactions rapidly increase androgen synthesis in the brain during the non-breeding season.

In male song sparrows (Melospiza melodia), territorial challenges during the breeding season can rapidly increase circulating levels of testosterone (T). During the non-breeding season, male song sparrows are highly aggressive, but the gonads are regressed and plasma T levels are non-detectable and unaffected by territorial challenges. The pro-hormone dehydroepiandrosterone (DHEA) is elevated in song sparrow plasma and brain during the non-breeding season and may be locally converted to sex steroids in the brain to regulate aggression. The enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) converts DHEA to androstenedione (AE) using the cofactor NAD(+), and this is a critical rate-limiting step. We predicted that brain 3beta-HSD activity varies seasonally and is rapidly modulated by aggressive challenges. In the first study, brain 3beta-HSD activity was highest in the non-breeding season in specific regions. In the second study, a simulated territorial challenge rapidly increased aggressive behavior in non-breeding song sparrows. Brain 3beta-HSD activity, when measured without exogenous NAD(+), increased by approximately 250 to 500% in telencephalic regions of challenged subjects. When brain 3beta-HSD activity was measured with exogenous NAD(+), these effects of territorial challenges were not observed. These data suggest that territorial challenges rapidly increase endogenous NAD(+) levels or increase 3beta-HSD activity specifically within a NAD-rich subcellular compartment. Together, these two studies suggest a shift from systemic to local sex steroid signaling in the non-breeding season. Local steroid signaling produces high spatial and temporal specificity of steroid signals and avoids the costs of high systemic T levels during the non-breeding season.

The expression of the nuclear receptors NR5A1 and NR5A2 and transcription factor GATA6 correlates with steroidogenic gene expression in the bovine corpus luteum.

The corpus luteum (CL) is the major site of progesterone (P4) production during the luteal phase of the estrous cycle in cattle. To better understand the molecular mechanisms underlying P4 production, we compared the mRNA and protein expression profiles of key components of the steroidogenic pathway (StAR, CYP11A, and 3beta-HSD) during the bovine CL luteal phase with that of several transcription factors (NR5A1, NR5A2, GATA4, GATA6) known for their roles in the control of steroidogenic gene expression. In the bovine CL, StAR, CYP11A, and 3beta-HSD mRNA and protein levels remained constant at the mid and late luteal phases but markedly declined at the regressed luteal stage. NR5A1 and NR5A2 exhibited a similar pattern with a significant decrease in expression at the regressed luteal stage. Both GATA4 and GATA6 mRNA and proteins could be detected in bovine CL; GATA6 levels, however, were generally higher. Although GATA4 expression did not change during the luteal phase, GATA6 showed a marked decrease at the regressed luteal stage, like NR5A1, NR5A2, and the other steroidogenic markers. Thus, we suggest that NR5A1, NR5A2, and GATA6, but not GATA4, contribute to the transcriptional regulation of steroidogenic gene expression, and hence P4 production, in the bovine CL. Furthermore, we have demonstrated the association of NR5A1 and NR5A2 with the bovine StAR promoter in the mid-luteal CL using chromatin immunoprecipitation, suggesting that these factors have definitive roles in the regulation of StAR gene transcription in vivo.

Childhood adrenocortical tumor: A clinical and immunohistochemical study of 13 cases.

The aim of the study was to investigate the molecular mechanisms in childhood adrenocortical tumors (ACTs), which is still unclear.A total of 9 girls and 4 boys with ACTs were enrolled. Relevant clinical features were obtained from records. Immunohistochemistry of vimentin, chromogranin A, S100, synaptophysin, cytokeratin (CK), type 2 3ß-hydroxysteroid dehydrogenase (3ßHSD), cytochrome P45017α, p53, p21, p27, cyclin D1, Ki-67, insulin growth facter-2 (IGF-2), and ß-catenin were undertaken for 13 tumors and 3 adjacent normal tissues. TP53 mutations in exon 2-11 were analyzed for 6 tumors and 3 blood samples.Virilization was the most common presentation (8/13, 61.5%). Immunohistochemically, p53 was positive in 8 of 13 ACTs and none in controls while p21 was positive in 12 of 13 ACTs and none in controls (P = .0036). Ki-67 was positive in 10 of 13 ACTs, but not in normal tissues (P = .0089). Although the expression of p27, cyclin D1, IGF-2 and ß-catenin were similar between the ACTs and controls, ß-catenin was noted in nuclear of 3 ACTs but not in controls. The difference of type 2 3ßHSD and P450c17α was not significant (P > .05, respectively). Four variants of TP53 were identified in the 6 tumors. C215G variant was found in 5 of 6 while A701G and G743A variants were found in 1 case, respectively. A novel C680G variant was also noted in 1 case. It was notable that C215G variant was found in the blood mononuclear cell of 3 patients.In conclusion, p53 variant and p21 overexpression, and abnormal ß-catenin distribution may be involved in the etiology and mechanism of childhood ACTs.

Gonadotrophin-releasing hormone-I and -II stimulate steroidogenesis in prepubertal murine Leydig cells in vitro.

AIM: To study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis. METHODS: Purified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined. RESULTS: GnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05). CONCLUSION: GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.

Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer.

Androgen receptor (AR) plays a central role in prostate cancer, and most patients respond to androgen deprivation therapies, but they invariably relapse with a more aggressive prostate cancer that has been termed hormone refractory or androgen independent. To identify proteins that mediate this tumor progression, gene expression in 33 androgen-independent prostate cancer bone marrow metastases versus 22 laser capture-microdissected primary prostate cancers was compared using Affymetrix oligonucleotide microarrays. Multiple genes associated with aggressive behavior were increased in the androgen-independent metastatic tumors (MMP9, CKS2, LRRC15, WNT5A, EZH2, E2F3, SDC1, SKP2, and BIRC5), whereas a candidate tumor suppressor gene (KLF6) was decreased. Consistent with castrate androgen levels, androgen-regulated genes were reduced 2- to 3-fold in the androgen-independent tumors. Nonetheless, they were still major transcripts in these tumors, indicating that there was partial reactivation of AR transcriptional activity. This was associated with increased expression of AR (5.8-fold) and multiple genes mediating androgen metabolism (HSD3B2, AKR1C3, SRD5A1, AKR1C2, AKR1C1, and UGT2B15). The increase in aldo-keto reductase family 1, member C3 (AKR1C3), the prostatic enzyme that reduces adrenal androstenedione to testosterone, was confirmed by real-time reverse transcription-PCR and immunohistochemistry. These results indicate that enhanced intracellular conversion of adrenal androgens to testosterone and dihydrotestosterone is a mechanism by which prostate cancer cells adapt to androgen deprivation and suggest new therapeutic targets.

The effects of cryptorchidism on the regulation of steroidogenesis and gap junctional communication in equine testes.

INTRODUCTION: Evidence collected over the years has demonstrated that cryptorchidism is associated with a defect in spermatogenesis and, as a consequence, with either reduced fertility or infertility. However, the effect of cryptorchidism on Leydig cell function is less clear. The aim of our study therefore was to investigate the regulation of steroid hormone biosynthesis and, additionally, intercellular communication in the cryptorchid equine testes. MATERIAL AND METHODS: Testes of mature bilaterally cryptorchid horse and healthy stallions were used for this study. The expression of luteinising hormone receptor (LHR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), aromatase and connexin43 (Cx43) was detected by means of immunohistochemistry. Testosterone and oestradiol levels were measured in testicular homogenates using appropriate radioimmunoassays. RESULTS: In the testes of both normal and cryptorchid stallions, immunostaining for LHR, 3beta-HSD and aromatase was confined to the Leydig cells. In the cryptorchid horse, the intensity of the staining for LHR and 3beta-HSD was weaker, whereas the staining for aromatase was clearly stronger than that of the normal stallion. Radioimmunological analysis revealed disturbance of the androgen-oestrogen balance in the cryptorchid testes. Additionally, in both the seminiferous tubules and interstitial tissue of the cryptorchid a clear reduction of the Cx43 signal was observed. CONCLUSIONS: Decreased expression of LHR and 3beta-HSD and increased expression of aromatase in the cryptorchid testes suggest that hormonal imbalance was caused both by reduced testosterone synthesis and by increased androgen aromatisation. Impaired expression of Cx43 in the seminiferous tubules as well as in the interstitial tissue of the cryptorchid horse indicates that cryptorchidism affects intercellular communication in the testes.