Investigations into metabolism, transport and function of sulfonated steroids in the porcine testicular-epididymal compartment

Sulfonated steroids have been traditionally regarded as inactive metabolites destined for excretion, as they are incapable of binding to classical nuclear steroid receptors. However, by the enzyme steroid sulfatase (STS) they may be converted into free steroids, which may be biologically active directly or after a few additional enzymatic reactions. Thus, as sulfonated steroids commonly circulate at relatively high concentrations, they may form an important pool of precursors for the local (intra-tissue) production of active free steroids. This so-called sulfatase pathway has received increased attention over recent years especially with respect to estrogen metabolism in human hormone-dependent breast cancer, where the intratumoral estrogen production from sulfonated precursors obviously has a much higher capacity in comparison to the de novo synthesis via free steroids. This study is composed of two parts of which the first one addresses the secretory patterns of free and sulfonated steroids in vivo, whereas in the second part the expression of STS and of the steroid sulfotransferases SULT1E1 (estrogen specific) and SULT2B1 (specific for beta-hydroxysteroids) was characterized in the testis and in different segments of the epididymis. Other subjects of the second part of this study were hydrolysis of steroid sulfates and the sulfonation of estrone (E1), dehydroepiandrosterone (DHEA) and pregnenolone (P5) in the tissues investigated. Concentrations of androstenedione, testosterone, pregnenolone sulfate (P5S), dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate (E1S)and 17beta-estradiol-3-sulfate were performed in the Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen (head: Prof. Dr. S. Wudy) applying liquid chromatography tandem mass spectrometry (LC-MS-MS). Moreover, 17beta-estradiol (E2) and E1 were measured by inhouse radioimmunoassays to cope with the low concentrations of free estrogens in boars. In order to get new information on the sulfonation of free steroids and the hydrolysis of steroid sulfates in the porcine testicular-epididymal compartment, subcellular fractions were prepared from tissue samples collected from the testis and from defined sites of the epididymis (EH1, EH2: proximal/distal part of epididymal head; EB1-4: epididymal body, from proximal to distal; ET1, ET2: proximal/distal part of epididymal tail) using differential centrifugation. STS and steroid sulfotransferase activities were measured based on the differential distribution of free and sulfonated steroids between an aqueous phase and an organic solvent, tert butyl-methylether. The immunostaining results were shown that SULTs 1E1 and 2B1, immunostaining was especially prominent in superficial epithelial protrusions. Sporadic staining of weaker intensity was also found in the muscular layer and in the vascular endothelium. With WB, a specific band of the expected molecular size (approx. 61 kDa) was found in the testis and all segments of the epididymis. These results show that STS is widely expressed in the porcine testicular-epididymal compartment, indicating a high potential for sulfatase pathways especially in Leydig cells and the epithelial cells of the rete testis and epididymis. The co-expression of STS with SULTs 1E1 and 2B1 in the epididymal epithelium and especially their colocalization in superficial protrusions are very intriguing. In the epididymal duct, apocrine secretion has been described to give rise to the formation of epididymosomes, small vesicles which are considered as vehicles for the transfer of certain molecules to the maturing sperm cells. Other intriguing findings are the virtual absence of a sulfonation of E1, DHEA and P5 in testicular cytosols as well as the absent or questionable detection of SULTs 1E1 and 2B1 in light of the high efflux of various steroid sulfates from the testis. A plausible explanation could be a significant use of sulfonated steroids as precursors/intermediates in porcine testicular steroidogenesis starting from cholesterol sulfate. The concept of a “sulfate pathway” of steroidogenesis would not only provide an explanation for the production of high amounts of steroid sulfates in the virtual absence of relevant steroid sulfotransferase activities but also for the high STS expression in Leydig cells. According to this concept, STS could play a crucial role in the control of the substrate flow through the steroidogenic enzyme cascade by mediating the transition of sulfonated precursors into the pool of free steroids, with the exact subcellular localization being of importance for the step of the enzyme cascade at which this transition(s) may occur. Thus, in order to corroborate this concept investigations into the utilization of sulfonated substrates by steroidogenic enzymes and on the subcellular localization of STS are necessary

Investigations into metabolism, transport and function of sulfonated steroids in the porcine testicular-epididymal compartment

Sulfonated steroids have been traditionally regarded as inactive metabolites destined for excretion, as they are incapable of binding to classical nuclear steroid receptors. However, by the enzyme steroid sulfatase (STS) they may be converted into free steroids, which may be biologically active directly or after a few additional enzymatic reactions. Thus, as sulfonated steroids commonly circulate at relatively high concentrations, they may form an important pool of precursors for the local (intra-tissue) production of active free steroids. This so-called sulfatase pathway has received increased attention over recent years especially with respect to estrogen metabolism in human hormone-dependent breast cancer, where the intratumoral estrogen production from sulfonated precursors obviously has a much higher capacity in comparison to the de novo synthesis via free steroids. This study is composed of two parts of which the first one addresses the secretory patterns of free and sulfonated steroids in vivo, whereas in the second part the expression of STS and of the steroid sulfotransferases SULT1E1 (estrogen specific) and SULT2B1 (specific for beta-hydroxysteroids) was characterized in the testis and in different segments of the epididymis. Other subjects of the second part of this study were hydrolysis of steroid sulfates and the sulfonation of estrone (E1), dehydroepiandrosterone (DHEA) and pregnenolone (P5) in the tissues investigated. Concentrations of androstenedione, testosterone, pregnenolone sulfate (P5S), dehydroepiandrosterone sulfate (DHEAS), estrone-3-sulfate (E1S)and 17beta-estradiol-3-sulfate were performed in the Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus-Liebig-University, Giessen (head: Prof. Dr. S. Wudy) applying liquid chromatography tandem mass spectrometry (LC-MS-MS). Moreover, 17beta-estradiol (E2) and E1 were measured by inhouse radioimmunoassays to cope with the low concentrations of free estrogens in boars. In order to get new information on the sulfonation of free steroids and the hydrolysis of steroid sulfates in the porcine testicular-epididymal compartment, subcellular fractions were prepared from tissue samples collected from the testis and from defined sites of the epididymis (EH1, EH2: proximal/distal part of epididymal head; EB1-4: epididymal body, from proximal to distal; ET1, ET2: proximal/distal part of epididymal tail) using differential centrifugation. STS and steroid sulfotransferase activities were measured based on the differential distribution of free and sulfonated steroids between an aqueous phase and an organic solvent, tert butyl-methylether. The immunostaining results were shown that SULTs 1E1 and 2B1, immunostaining was especially prominent in superficial epithelial protrusions. Sporadic staining of weaker intensity was also found in the muscular layer and in the vascular endothelium. With WB, a specific band of the expected molecular size (approx. 61 kDa) was found in the testis and all segments of the epididymis. These results show that STS is widely expressed in the porcine testicular-epididymal compartment, indicating a high potential for sulfatase pathways especially in Leydig cells and the epithelial cells of the rete testis and epididymis. The co-expression of STS with SULTs 1E1 and 2B1 in the epididymal epithelium and especially their colocalization in superficial protrusions are very intriguing. In the epididymal duct, apocrine secretion has been described to give rise to the formation of epididymosomes, small vesicles which are considered as vehicles for the transfer of certain molecules to the maturing sperm cells. Other intriguing findings are the virtual absence of a sulfonation of E1, DHEA and P5 in testicular cytosols as well as the absent or questionable detection of SULTs 1E1 and 2B1 in light of the high efflux of various steroid sulfates from the testis. A plausible explanation could be a significant use of sulfonated steroids as precursors/intermediates in porcine testicular steroidogenesis starting from cholesterol sulfate. The concept of a “sulfate pathway” of steroidogenesis would not only provide an explanation for the production of high amounts of steroid sulfates in the virtual absence of relevant steroid sulfotransferase activities but also for the high STS expression in Leydig cells. According to this concept, STS could play a crucial role in the control of the substrate flow through the steroidogenic enzyme cascade by mediating the transition of sulfonated precursors into the pool of free steroids, with the exact subcellular localization being of importance for the step of the enzyme cascade at which this transition(s) may occur. Thus, in order to corroborate this concept investigations into the utilization of sulfonated substrates by steroidogenic enzymes and on the subcellular localization of STS are necessary

Clonage et caractérisation des uridine diphospho-glucuronosyltransferases (UGT) effectuant la glucuronidation des hormones stéroïdiennes chez la souris

La sous-famille Uridine diphosphoglucuronosyltransférases 2B (UGT2B) par son activité enzymatique de glucuronoconjugaison participe à l'élimination des stéroïdes. Les études in vitro faites sur les différentes UGT2B humains, notamment UGT2B15, et le génotypage de cette dernière sur différentes populations humaines ont permis de démontrer que cette enzyme pourrait jouer un rôle dans le cancer de la prostate. Représentant une cible thérapeutique potentielle, une compréhension plus exacte de son rôle physiologique ainsi que des études in vivo s'imposent. Actuellement aucun modèle animal n'existe pour faire l'étude in vivo de UGT2B 15. La souris, grâce au génie génétique, représente la meilleure avenue. Jusqu'à maintenant, aucune caractérisation enzymatique du métabolisme des hormones androgènes à partir des Ugt2b murin n'a été faite. Les présents travaux exposent la caractérisation enzymatique de cinq enzymes Ugt2b murins et tentent d'établir un parallèle entre elles et l'enzyme humain UGT2B 15, notamment entre leurs expressions tissulaires et leurs activités de conjugaisons des hormones androgènes. La perspective globale de ces travaux est de déterminer un orthologue sur lequel sera basé la création de souris déficientes

Biosynthesis of estradiol : Cloning and characterization of rodent 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase types 1 and 7

Abstract17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase reactions between 17β-hydroxy and 17-ketosteroids.In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to study the role of rodent type 1 enzyme in ovarian estradiol (E₂) biosynthesis and its enzymatic characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1 enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the reaction from androstenedione to testosterone at least as efficiently as estrone (E₁) to E₂, while human 17HSD/KSR1 clearly preferred the E₁ to E₂ reaction.A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity. A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its ability to convert E₁ to E₂ and it was chronologically named 17HSD/KSR7. Interestingly, it showed 89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP efficiently catalyzed the reaction from E₁ to E₂. The mouse type 7 enzyme was most abundantly expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7.Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle and second half of pregnancy, in parallel with E₂ secretion from the CL. Using in situ hybridization, cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5 post coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of antimesometrial decidua. Between day 8 and 9 p.c. the enzyme was abundant in decidua capsularis of the developing placenta, after which expression moved to the basal zone. On days 12 and 14 p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts, where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new 17HSD/KSR which is involved in the biosynthesis of E₂ in the ovaries. In addition, E₂ produced locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or implantation and placentation.Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 9 of the University Hospital of Oulu, on September 29th, 2000, at 12 noon.Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase reactions between 17β-hydroxy and 17-ketosteroids. In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to study the role of rodent type 1 enzyme in ovarian estradiol (E₂) biosynthesis and its enzymatic characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1 enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the reaction from androstenedione to testosterone at least as efficiently as estrone (E₁) to E₂, while human 17HSD/KSR1 clearly preferred the E₁ to E₂ reaction. A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity. A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its ability to convert E₁ to E₂ and it was chronologically named 17HSD/KSR7. Interestingly, it showed 89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP efficiently catalyzed the reaction from E₁ to E₂. The mouse type 7 enzyme was most abundantly expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7. Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle and second half of pregnancy, in parallel with E₂ secretion from the CL. Using in situ hybridization, cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5 post coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of antimesometrial decidua. Between day 8 and 9 p.c. the enzyme was abundant in decidua capsularis of the developing placenta, after which expression moved to the basal zone. On days 12 and 14 p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts, where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new 17HSD/KSR which is involved in the biosynthesis of E₂ in the ovaries. In addition, E₂ produced locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or implantation and placentation

Characterization of human placental tissue and cell lines for their xenobiotic and steroid disposition potential through quantitative proteomics

The placenta is a vital fetal endocrine organ that plays an important role in gas, nutrient, and hormone shuttling between maternal and fetal sides, but much is unknown about the placental functions due to logistical, ethical, and technical challenges in investigating this critical human organ. In particular, the xenobiotic and steroid disposition pathways of human placenta and their quantitative significance are not well characterized. Understanding the xenobiotic and steroid disposition pathways is key to mitigating the risks associated with xenobiotic exposure during pregnancy and understanding steroidal mechanisms that can be influenced by xenobiotics. While transcriptomics and targeted proteomics studies have provided some information on placental function, limited data are available on quantitative global proteomics of placenta and placental cells lines. Thus, utilizing global quantitative proteomics, we aim to i) characterize archived human placental tissue and ii) elucidate the resemblance of in vitro placental models to the placenta in terms of the steroid and xenobiotic pathways to better understand mechanisms of xenobiotic disposition and toxicity

Identification and characterization of substances interfering with steroid metabolizing enzymes and retinoic acid-related orphan receptor γt activity

Steroid hormones regulate a wide range of physiological processes by activating nuclear receptors, which then act as transcription factors to control the expression of their target genes. Toxicological safety assessments of chemicals to which humans may be exposed also include a section on endocrine assessments, which often focus on direct effects on the nuclear receptors of the sex steroid hormones, namely the androgen and the estrogen receptors. The effects of chemicals on pre receptor control, which includes the biosynthesis of the steroid hormones themselves, as well as on other nuclear receptors such as the immunomodulating retinoic acid related orphan receptor γt (RORγt) activity have been investigated insufficiently so far. This thesis aims to identify and characterize substances that interfere with steroid metabolizing enzymes and RORγt activity. We have addressed this goal in three different projects, which are focusing on different areas of endocrinology. The steroidogenic enzyme 11β hydroxysteroid dehydrogenase type 2 (HSD11B2), is involved in the pre receptor control of the mineralocorticoid receptor (MR) by inactivating 11 hydroxylated glucocorticoids, that can bind and activate the MR. This ensures the receptors specificity for its natural ligand, aldosterone. Inhibition of HSD11B2 can lead to pseudohyperaldosteronism, an effect that has also been described in clinical case studies with patients treated with the azole antifungals itraconazole and posaconazole. In the first project, we assessed species specific susceptibility towards azole fungicide dependent HSD11B2 inhibition. The results of this investigation provided a possible explanation, why this adverse drug effect was missed during clinical trials. The human HSD11B2 homolog was strongly inhibited by both azole fungicides, while the rat enzyme was only moderately inhibited. Mouse and zebrafish homologs were only weakly inhibited. Using predictions based on homology modeling of HSD11B2 and analysis of chimeric enzyme variants, we were able to identify the C terminal region and the amino acid residues at positions 170 and 172 as relevant structural elements that are partly responsible for the species specific differences between the mouse and human homologs. This study highlights that such species specific differences in the inhibition of steroidogenic enzymes by chemicals may be relevant for toxicological investigations and should therefore be considered in future studies. As an extension of this project, we summarized the methods used for the analysis of the enzymatic activity of HSD11B2 together with further assessment possibilities for the in vitro enzyme activity determination of HSD11B2 and 11β hydroxysteroid dehydrogenase type 1 (HSD11B1) in two chapters for the book series 'Methods in Enzymology'. HSD11B1 catalyzes the reverse reaction of HSD11B2, reducing 11 keto glucocorticoids to their active 11β hydroxy forms. Parabens and UV filters are used as additives in body care products and cosmetics to increase their shelf life. These compounds have been measured in various human matrices, including fetal samples, and are known to have antiandrogenic effects by blocking androgen receptor (AR) activity. To date, the influence of these chemicals on the pre receptor control of AR, specifically the androgen biosynthesis, is poorly understood. 3α hydroxysteroid dehydrogenases (3α HSD) are steroidogenic enzymes that can catalyze one of the last two synthesis steps in the backdoor pathway of the most potent androgen, 5α dihydrotestosterone (DHT). Androgen mediated AR activation is required for normal male genitalia formation during embryogenesis. In the second project, we investigated the effects of parabens and UV filters on the enzymatic activity of different 3α HSDs that can produce DHT in the backdoor pathway using a novel, radiometric enzyme activity assay. We have identified several parabens and benzophenone type UV filters as the first inhibitors of the 3α HSD 17β hydroxysteroid dehydrogenase type 6 (HSD17B6) with IC50 values in the mid and high nanomolar range. Said identified inhibitors were analyzed for their structure-activity relationship using a novel HSD17B6 homology model, which highlighted the importance of the 4-hydroxylated phenyl head group present in both substance classes. The addition of a methyl group to the 4-hydroxy group resulted in a loss of inhibitory capacity in 4-methoxylated benzophenones, as it prevented the formation of a hydrogen bond with the amide group of the cofactor nicotinamide adenine dinucleotide (NAD+) in the binding pocket of the homology model. Parabens and UV filters, like many other chemicals, have so far mainly been investigated for their direct effects on the androgen and estrogen receptors. RORγt is involved in immune response regulation and is essential for the differentiation of T helper 17 cells and their expression of pro inflammatory interleukins. Excessive activation of RORγt has been associated with inflammatory and autoimmune diseases such as psoriasis. In the third project, we evaluated the effect of parabens and UV filters on RORγt activity using a previously established tetracycline inducible reporter gene assay in Chinese hamster ovary cells. We identified hexylparaben, benzylparaben and benzophenone 10 as potent RORγt agonists with EC50 values within the higher nanomolar and lower micromolar range. Those RORγt agonists were also able to enhance pro inflammatory cytokine expression in a mouse EL4 T lymphocyte model. Together with structurally similar chemicals which we identified by virtual screening of a cosmetics database, the chemicals identified as RORγt agonists in this study showed additive effects on the receptor activity when assessed as mixtures. Additional experiments are required to determine whether parabens and UV filters can reach the concentrations described in the second and third projects to exert potential antiandrogenic effects in organs expressing HSD17B6, or possibly aggravate existing inflammatory and autoimmune diseases through the additional RORγt activation. As an additional, fourth project, we examined three case studies of Tunisian patients diagnosed with 17β hydroxysteroid dehydrogenase type 3 (HSD17B3) deficiencies for their underlying molecular causes. HSD17B3 is a steroidogenic enzyme that is exclusively expressed in the testes and catalyzes the last enzymatic step of testosterone synthesis. Testosterone, a potent androgen, is essential for normal male sexual development. Pathogenic mutations in the HSD17B3 gene can lead to undervirilization of the male sexual organs due to insufficient testosterone production during embryogenesis, which is why HSD17B3 deficiencies are classified as 46,XY disorders of sexual development. Genetic analysis of the three patients revealed in one patient the first homozygous mutation in the catalytic tetrad of HSD17B3, p.K202M, for which we were able to show a complete loss of function using a radiometric enzyme activity assay. The second patient was a compound heterozygote with a paternally inherited, already characterized, inactive truncation mutation p.C206X and a maternally inherited splice site mutation (c.490 6 T > C) for which we could show by means of a splicing assay that the mutation causes skipping of exon 7 during mRNA splicing which presumably results in a truncated and inactive enzyme. The last patient turned out to have a homozygous p.C206X mutation of HSD17B3. Consanguineous marriages can promote the emergence and establishment of deleterious mutations such as the ones identified in this project. This study highlights the importance of genetic counseling and the sensitization of medical personnel towards HSD17B3 deficiencies. The projects conducted in this thesis address relevant limitations of endocrine studies that are part of safety assessments of chemicals. By assessing species specific inhibition of HSD11B2 by azole fungicides, and identifying parabens and UV filters as modulators of HSD17B6 and RORγt activities, we have created a foundation for further research. The methods and homology models described in the respective projects of this thesis may prove to be valuable tools for future studies

Molecular analysis and physical mapping of the human 3beta-hydroxysteroid dehydrogenase sigma 5/sigma 4 isomerase gene family

3beta-hydroxysteroid dehydrogenase (3beta -HSD) catalyses the conversion of 3beta-hydroxy- Delta5-steroids into the corresponding Delta4-3-ketosteroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex hormones. Two isoforms encoded by two highly homologous, closely linked 3beta-HSD genes (HSD3B1 and 2) are known to be expressed in humans. However, Southern blot analysis and 3B-HSD type II mutation screening suggested that there was more than two HSD3B genes in humans; therefore, two human genomic gammagem11 libraries were screened with 3beta-HSD type I cDNA and bacteriophage clones containing novel 3beta-HSD sequences were identified. Two of the phage clones were characterised and the segments equivalent to HSD3B1 coding regions were sequenced. The 3beta-HSD coding sequence determined from both of these clones contained frameshift mutations resulting in premature stop codons, and it was concluded that these sequences were unprocessed pseudogene members of the 3beta-HSD gene family. The library screens generated 5 new members of the 3beta-HSD gene family and these were mapped by fluorescent in situ hybridisation (FISH) to chromosome 1p13, the same region of the genome as HSD3B1 and 2. Specific oligonucleotide primer pairs were designed for each gene and using PCR the genes were mapped to a set of 3 overlapping yeast artificial chromosomes (YACs) and 9 overlapping bacterial artificial chromosomes (BACs). The gene order was subsequently confirmed using restriction analysis and 3beta-HSD-specific oligonucleotide probes. The orientation of HSD3B1, 2, Psi1 and Psi4 was determined by extensive restiction analysis of the BACs and the positions of the endpoints in three BAG clones. The estimated length of the entire contig is 500kb with the 3beta-HSD gene cluster over a centrally-based 235kb fragment. cDNA selection techniques were established to detect expressed sequences from the region of the 3beta-HSD gene cluster. Three BAG clones from the contig were biotinylated and hybridised to a placental PGR-amplifiable cDNA library. The hybrids produced were isolated using streptavidin coated magnetic beads. Many 3beta-HSD transcripts were detected from these experiments indicating that the selection process was successful, however no previously identified genes were detected that could be localised to the 3beta-HSD locus. Although, several unknown sequences were discovered which may belong to unidentified genes present within or close to the 3beta-HSD gene cluster

Paracrine Regulation of Human Prostate Cancer Cell Growth and Function

This thesis mainly deals with steroid-growth factor interaction in the regulation of growth and steroid metabolism of LNCaP, DU145 and PC3 human prostate cancer cell lines. Growth response to both androgens and oestrogens and to TGFalpha and TGFbeta1 were investigated; in addition, content and status of steroid (AR and ER) and growth factor (EGFR and TGP?R) receptors were assessed using multiple approaches, including ligand binding assay, immunocytochemistry and reverse transcriptase-PCR (RT-PCR). Furthermore, immunofluorescent staining was used to evaluate expression of EGF, TGFalpha and TGFbeta1 and of the 27 kDa heat shock protein (hsp27) as a marker of oestrogen sensitivity. On the other hand, patterns of both testosterone (T) and oestradiol (E2) metabolism were studied using incubation of cultured cells with labelled steroid precursor and reverse phase-HPLC (RP-HPLC) analysis of precursor degradation and formation of metabolic products. Possible influence of both TGFalpha and TGFbeta1 on rates and direction of metabolism of both steroids was also determined. Growth of LNCaP cells was significantly stimulated by physiological concentrations of the two major androgens (T and dihydrotestosterone: 30.2% and 33.8% respectively, P<0.03) and of E2 (65.8%, P=0.009), using stringent culture conditions. Interestingly, increasing concentrations of E2 (0.01-100 nM) induced a significant inhibition of the proliferative activity of PC3 cells (55.2% at 100 nM E2, P<10-6), while neither androgen SUMMARY significantly affected growth of this cell line. In contrast, DU145 cells proved insensitive to the addition of either androgens or oestrogens. Presence of androgen binding sites in prostate tumour cell lines was ascertained using radioligand binding assay and RT-PCR approaches. High affinity AR were detected in both soluble and pellet fractions of LNCaP and DU145 cells, whilst PC3 cells showed only nuclear AR. These results were also supported by PCR system, where ampifiable AR mRNA was found in all three cell lines. Multiple evidence for high affinity sites of oestrogen binding in LNCaP cells was obtained: i) biochemical assay allowed the detection of high affinity, low capacity binding sites in both soluble and nuclear cell fractions; ii) immunocytochemical and immunofluorescent assays showed a consistently intensive staining for both ER and PgR, as well as hsp27; iii) the RT-PCR system documented the presence of normal or a variant ER mRNA in PC3 cells; the latter, which lacks the entire exon 4, has been recently characterised in our laboratories in human mammary carcinoma cells. Presence of ER in PCS cells was also documented by biochemical and cytochemical assays as well as, indirectly, by hsp27 staining. However, the relative estimate of ER expression displayed levels significantly and consistently lower than those found in LNCaP cells. This finding was confirmed using the RT-PCR approach, where transcript levels for both normal and variant ER mRNA were proportionally lower than in LNCaP cells. Conversely, DU145 cells were found to be consistently ER-negative using biochemical and cytochemical assays, hsp27 staining and RT-PCR system. The evidence that the E2-induced growth was completely reversed in LNCaP cells by the addition of the pure antioestrogen ICI-182,780, clearly suggests that E2 acts via its own receptor. The possibility that the inhibitory effect exerted by E2 on growth of PC3 cells could be mediated via an increase of TGFalpha production was also supported by the fact that use of a neutralising antibody raised against TGFbeta1 produced a three-fold increase of cell growth; this effect was almost completely abolished after addition of 100 nM E2. However, Northern blot analysis did not reveal any increase of TGFbeta1 mRNA following E2 administration in this cell line. Growth of PC3 cells was significantly stimulated by TGFalpha (36% at 50 ng/ml, P<0.003) and inhibited by TGFbeta1 (55% at 5 ng/ml, P<10-6) after 48 hours exposure in routine medium. Proliferative activity of DU145 cells was minimally affected by TGFalpha, but significantly inhibited by TGFbeta1 (28% at 5 ng/ml, P<0.009). In contrast, LNCaP cells proved to be poorly sensitive, at least in the short-term, to either growth factor. Radioreceptor assay showed presence of high affinity binding sites for EGF in all three cell lines and for TGFalpha in DU145 and PC3 cells, whilst no detectable site of TGFalpha binding was found in LNCaP cells. DU145 cells displayed the highest EGFR content, LNCaP the lowest; TGFalphaR were expressed in greater amounts in PC3 than in DU145 cells. EGFR binding data were also confirmed using Western blot analysis, the DU145 cells having EGFR expression levels higher than PC3 and LNCaP cells. In addition, immunofluorescent staining revealed high amounts of both EGFR and TGFalpha, and fairly high EGF in DU145 and, to a lesser extent, in LNCaP cells; by contrast, PC3 cells exhibited low levels of both receptor (EGFR) and ligands (EGF, TGFalpha), but appreciable levels of endogenous TGFbeta1. Overall, these results suggest a differential sensitivity to TGFalpha and TGFbeta1 by prostate cancer cells; TGFalpha response seems to be not proportional to the EGF-R content of individual cell lines, while TGFbeta1 response appears to be inversely related to the androgen-sensitivity of cells. (Abstract shortened by ProQuest.)