CYP19 mRNA Expression Research Articles

225 GENE MUTATION AND ALTERED EXPRESSION OF NBS1 IN HBV ASSOCIATED HEPATOCELLULAR CARCINOMAS

Background and Aims: The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D33) inhibits cell growth and induces cell differentiation and apoptosis in numerous tumors, such as colon, breast and prostate cancers. The role of 1,25-D3 in the development of the hepatocellular carcinoma has not yet been established. The aim of the present study was to examine the gene expression of the vitamin D inactivating CYP24A1 as well as the activating 1-alpha-hydroxylase (CYP27B1) enzyme after 1,25-D3 treatment in hepatocellular carcinoma cell lines (1,25-D). Methods: HepG2 and HUHneo (Dept. of Molecular Virology, Univ. of Heidelberg) cells were treated with 4nM concentrations of 1,25-D3 in Optimem medium and were incubated in 24-well plates in two parallels for treatment duration of 30, 60, 120 minutes, 5, 8, 10, 12, 14, 24, 26, 28 hours. Total RNA was isolated from each sample with Roche High Pure Total RNA Isolation kit. Five hundred ng of total RNA was reverse-transcribed to cDNA. The expression differences of selected genes were analyzed by Taqman probe-based quantitative real-time PCR. Relative quantification was carried out from collected data (threshold cycle numbers) by Applied Biosystems 7500 System SDS software 1.3. Results: CYP24A1 mRNA expression significantly (p < 0.001) increased in response to 1,25-D3 administration in a timedependent manner in both cell lines. In HepG2 cell line the CYP24A1 mRNA expression showed 5300-fold elevation reaching the maximum value at 8 hours. In the HUHneo cells the increase was 152-fold of the baseline level, and the curve reached the maximum at 14 hours. There was no change in CYP27B1 gene activity of the cells treated with 1,25-D3. The activating effect of vitamin D3 on CYP24 mRNA expression was at 28 hours still detectable. Conclusions: Our novel data raise the possibility that the significant increase in the CYP24A1 gene expression of hepatocellular carcinoma cell lines following vitamin D administration may stimulate the inactivation of 1,25-D3, thus “protecting” the tumor cells against the anti-cancer effects of 1,25-D3. This is the first report on the role of vitamin D3 on the CYP24A1 mRNA transcription in hepatocellular carcinoma cells in vitro.

The Human Placenta Expresses CYP17 and Generates AndrogensDe Novo

The human placenta is believed to have insignificant CYP17 expression, rendering it dependent on the maternal and fetal compartments for the necessary androgenic precursors to yield the high levels of estrogens seen in pregnancy. The aim of the study was to analyze whether the human trophoblast is capable of expressing CYP17 and producing androgens de novo. Human trophoblasts from fresh placentas and JEG-3 cells were used for all experiments. CYP17 mRNA analysis was performed via RT-PCR, and protein detection by Western blot and immunohistochemical staining. Steroid products were quantified using RIAs. CYP17 mRNA was expressed in both cell types. CYP17 protein was detected by Western blotting and localized by immunostaining mainly to the cytoplasm of syncytiotrophoblasts. Measurement of 17α-hydroxyprogesterone, androstenedione, and their aromatized products in the media further demonstrated CYP17 expression and activity in the human trophoblast. Baseline levels of CYP17 steroid products were higher in primary cells and significantly increased in the presence of 22-hydroxycholesterol. We have demonstrated CYP17 mRNA and protein expression and activity in human trophoblasts. Considering the precursor concentration, blood flow, and mass of the placenta, we suggest that its contribution of androgens is an important source of estrogen production in pregnancy.

Expression of steroidogenic enzymes during equine testicular development

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3βHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3βHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3βHSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.

Valproic acid augments vitamin D receptor-mediated induction of CYP24 by vitamin D3: A possible cause of valproic acid-induced osteomalacia?

Valproic acid (VPA) is a wide spread anticonvulsant and mood-stabilizing agent, the use of which is associated with hepatotoxicity, bone marrow suppression and osteomalacia. In the current paper we propose a possible mechanism of VPA-induced osteomalacia involving accelerated catabolism of 1α,25(OH) 2–vitamin D3 (VD3) due to increased expression of CYP24. We demonstrate that VPA strongly potentiates CYP24 mRNA expression by VD3 in human hepatocytes (HH) and in human embryonic kidney cells (HEK293). By the method of gene reporter assay we found that VPA increases basal and VD3-inducible activity of CYP24 promoter (pCYP24-luc) in human liver adenocarcinoma (HepG2) and in HEK293 cells in dose-dependent manner. In order to delineate the role of inhibitory effects of VPA on histone deacetylase 1 (HDAC1), we compared the effects of VPA with trichostatin A (TSA) on basal and inducible levels of CYP24 mRNA and pCYP24-luc transactivation. Transactivation of CYP24 promoter by VD3 was enhanced in the presence of both TSA and VPA. In contrast, VD3-inducible expression of CYP24 mRNA was enhanced by VPA but not by TSA, implying that HDAC1 inhibition is not the major reason for VPA effects on CYP24. We examined the effects of VPA on mitogen-activated protein kinases as the important transcriptional regulators of VDR. VPA activated extracellular signal-regulated kinase (ERK) but not c-Jun-N-terminal kinase (JNK) and p38 MAPKs. In conclusion, VPA enhances transcriptional activity of VDR and increases expression of CYP24 mRNA in the presence of VD3 in physiological concentrations. The mechanism involves activation of ERK and partly the inhibition of HDAC1.

Berberine reduces insulin resistance induced by dexamethasone in theca cells in vitro

Theca cells with dexamethasone-induced insulin resistance showed defective glucose uptake and excessive testosterone production, both of which were effectively antagonized by berberine. Therefore, insulin-resistant theca cells may contribute to the pathogenesis of hyperandrogenism in polycystic ovary syndrome.

Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: Targeting the cAMP signalling cascade

Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line (H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 μM 8:2 FTOH for 24 h and productions of progesterone, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dose-dependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly less basal (+ GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total cellular ATP level nor basal (−GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.

P450‐aromatase activity and expression in human testicular tissues with severe spermatogenic failure

There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction.

Endometrial aromatase mRNA as a possible screening tool for advanced endometriosis and adenomyosis

Endometriosis (ENDs) and adenomyosis (ADNs) are estrogen-dependent diseases. The aim of this study was to determine the clinical usefulness of examining endometrial biopsy specimens for aromatase cytochrome P450 (CYP19) as a diagnostic importance for endometriosis and adenomyosis. In general, the RT-PCR analyses of 101 samples revealed increased aromatase mRNA expression in eutopic endometrium in women with endometriosis and adenomyosis compared to healthy controls (p = 0.0002). The highest number of positive cases (93.3%) of CYP19 mRNA expression was detected in women with advanced disease stages. Concrete expression of CYP19 mRNA level in controls was 0.68 compared to patients with ADNs (1.21), ENDsL stage I-II (1.15) and ENDsA stage III-IV (1.65) (p < 0.0001), respectively. The possible influence of increased body mass index on aromatase expression in each group showed in controls an insignificant slight increase of aromatase expression, contrary to cases where this trend was the opposite. The results point to the higher (2.45-fold) difference in aromatase expression in patients with endometriosis stage III-IV compared to controls and provide direct evidence that screening for eutopic endometrial aromatase expression in combination with clinical data could be of discriminative value in the prediction of estrogen-dependent diseases, independent from body mass index.

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

Abstract 4973: C/EBPβ increases expression of the vitamin D3 catabolizing enzyme, CYP24, in non-small cell lung cancer cells

Abstract Introduction: We previously reported that the vitamin D3 catabolizing enzyme CYP24 is over-expressed in non-small cell lung cancer (NSCLC) cells, where it decreases the growth inhibitory effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Studies were conducted to establish the clinical relevance of this finding and the mechanism for CYP24 over-expression in NSCLC cells. Methods: CYP24 mRNA expression was quantified by real-time PCR in patient-matched histologically normal lung tissue and primary lung tumors. To elucidate factors that control CYP24 expression, a panel of NSCLC cell lines that exhibit a 70-fold variation in CYP24 mRNA levels under basal growth conditions was used (relative mRNA expression H292 =1.0; 201T = 7.0; 128.88T = 68; A549 =69). CYP24 gene copy number was assessed by fluorescence in situ hybridization. CYP24 promoter activity was measured following transfection of NSCLC cells with either a wildtype promoter-luciferase reporter plasmid or plasmids harboring CYP24 promoter mutations. Transcription factor binding to CYP24 promoter elements was evaluated by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays. Results: CYP24 was significantly over-expressed in tumors (p = 0.0017), with 53% of NSCLC cases showing a minimum of 5-fold over-expression. CYP24 gene copy number was comparable among the NSCLC cell lines used. When NSCLC cells were transfected with a wildtype CYP24 promoter-luciferase reporter plasmid, significant differences in promoter activity were observed that paralleled endogenous CYP24 transcript levels. ChIP assays revealed that the CCAAT-enhancer binding protein C/EBPβ binds to the endogenous CYP24 promoter under basal growth conditions in 128.88T cells. To establish a role for C/EBP proteins in regulating CYP24 transcription, we introduced mutations into the C/EBP binding site within the CYP24 promoter. The mutations disrupted C/EBP binding and significantly reduced basal promoter activity in 128.88T and A549 cells. EMSAs that employed a consensus C/EBP site as probe demonstrated that C/EBP binding increases with CYP24 promoter activity: nuclear extracts from untreated H292 and 201T cells contained low levels of C/EBP binding activity, whereas 128.88T and A549 extracts contained high levels. Conclusions: CYP24 mRNA is frequently over-expressed in primary lung tumors. A C/EBPβ-mediated transcriptional control mechanism contributes to the increased expression of CYP24 in NSCLC cells. Signaling pathways that increase C/EBPβ expression/activation may contribute to lung cancer by upregulating CYP24 and allowing neoplastic cells to bypass the antiproliferative effects of 1,25(OH)2D3. This work was supported by the University of Pittsburgh Center for Environmental Oncology, the Hillman Foundation, the Pittsburgh Foundation and R01 CA132844. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4973.

Epigenetic mechanisms regulating CYP19 transcription in human breast adipose fibroblasts

Cytochrome aromatase p450, encoded by the gene CYP19, catalyzes the synthesis of estrogens from androgens. In post-menopausal women, adipose becomes the major site for estrogen production, where basal CYP19 transcription is driven by distal promoter I.4. In breast adipose fibroblasts (BAFs), CYP19 expression is elevated in the presence of tumour-derived factors through use of promoters I.3 and II. We show for the first time that DNA methylation contributes to CYP19 regulation in BAFs and breast cell lines. Promoter I.4 and I.3/II-derived mRNA were not dependent on the CpG methylation status within respective promoters. However, inhibition of DNA methylation with 5-aza-2′-deoxycytidine resulted in a significant ∼40-fold induction in CYP19 mRNA expression in BAFs and breast cell lines. These studies uncover a new layer of complexity in the regulation of aromatase where CYP19 appears to be inhibited by DNA methylation and evokes the possibility that disruption to this epigenetic regulation may give rise to an increase in aromatase levels in the breast.

Concentrations of anti-Mullerian hormone in fluid from small human antral follicles show a negative correlation with CYP19 mRNA expression in the corresponding granulosa cells

Ovaries surgically removed for fertility preservation from a total of 24 women served as a source of human small antral follicles, including the follicular fluid (FF) and the corresponding granulosa cells (GC). The FF was used to evaluate the intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-B, estradiol, progesterone, androstenedione and testosterone. In GC mRNA expression of the AMH type II receptor (AMH-r2) was determined and correlated to the mRNA expression of CYP19 (aromatase), FSH-receptor (FSH-r) and LH-receptor (LH-r) and to the hormonal profiles of the corresponding FF. GC and FF from a total of 64 follicles (diameter of 3-9 mm) were evaluated. Concentrations of AMH in FF showed a highly significant inverse correlation with CYP19 mRNA expression in the corresponding GC and with concentrations of estradiol, progesterone and inhibin-B in the FF. However, a small subgroup of follicles exhibited high levels of AMH simultaneously with relative high levels of CYP19 mRNA. In contrast to AMH, mRNA expression of AMH-r2 was significantly positively correlated to the mRNA expression of FSH-r and CYP 19, but failed to correlate to any other measured parameters. These data confirms an intimate correlation between follicular AMH levels, AMH-r2, FSH-r expression and estradiol secretion in the developing human follicle.

Epigenetic Control of Estrogen Synthesis in Human Preadipocytes and Breast Cancer Cells.

Abstract Estrogen plays a significant role in the development and progression of breast cancer (BC). Cytochrome aromatase p450, encoded by the gene CYP19, is the key enzyme catalyzing the synthesis of estrogens from androgens. In post-menopausal women, adipose tissue becomes the major site for estrogen production and an increase in CYP19 expression in mammary adipose is strongly linked to the hormone-dependent progression of BC. Therefore, understanding CYP19 regulation in BC is critical for the development of therapeutic measures. Previous studies of CYP19 regulation in BC have mainly focussed on hormone-induced regulation of transcription via upstream tissue- and promoter-specific regulatory regions. While this is clearly important, it is increasingly evident that epigenetic regulation of expression, such as DNA methylation, is also a common factor in the progression of cancers. The aim of this study was to investigate whether CYP19 expression is under epigenetic regulation and to determine if such mechanisms contribute to the tissue- and promoter-specific expression of CYP19 observed in BC.CYP19 transcripts in cancer-free primary human breast adipose fibroblasts (BAFs) stimulated with cytokines and glucocorticoids (e.g. TNFα and dexamethasone) are derived from the distal promoter 1.4, whereas breast tumour-derived factors (e.g. prostaglandin E2) induce a regulatory switch to proximal promoters 1.3 and II. DNA from BAFs maintained under these two conditions was treated with sodium bisulfite allowing for methylation analysis of CpG dinucleotides. Methylation mapping revealed a stochastic heterogeneous level of methylation among 9 CpG sites (promoter 1.4) and 11 CpG sites (promoters 1.3 and II) in untreated BAFs.No correlation was observed between promoter methylation and promoter activation after response to stimuli. However, treatment of BAFs with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, increased total CYP19 mRNA expression up to 40-fold, primarily via promoter 1.4 thorugh up-regulation of trans-acting factors, suggesting CYP19 is under tonic inhibition by methylation in BAFs. Similar results were also obtained in four BC cell lines. Ongoing work has elucidated trans-acting factors of CYP19 epigenetically silenced in BAFs. These studies uncover a new layer of complexity in the regulation of aromatase expression in BC and may identify new targets for epigenetic therapy, including DNA methylation inhibitors. These findings will translate to BC by determining the methylation state of CYP19 in clinical BC specimens, and by correlation with clinicopathological parameters such as steroid receptor status, grade and stage. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1147.

Regulation of synthesis function of porcine ovarian granulose cells by mitogen-activated protein kinase signaling pathway

To investigate the role of mitogen-activated protein kinase (MAPK) signaling pathway regulating synthesis function of steroid hormone of porcine ovarian granulose cells. Porcine ovarian granulose cells were cultured in medium added with dimethyl sulfoxide (DMSO) at concentration of 50 micromol/L or with MAPK inhibitor PD98059 (PD) for 48 hours. Then, granulose cells in DMSO medium were added with activator of adenylate cyclase (20 micromol/L) or blank agent for next 48 hours incubation, which were defined as observation group 1 and control group 1, similarly, granulose cells in PD medium were also added with activator of adenylate cyclase (20 micromol/L) or blank agent for next 48 hours, which were defined as observation group 2 and control group 2. The level of estradiol (E(2)), progesterone (P) and testosterone (T) were detected by chemoluminescence. The mRNA expression of 17alpha-hydroxylase/17,20-lyase (CYP17) and aromatase P450 (P450 arom) were assessed by RT-PCR. (1) Hormone expression: the level of T, P and E(2) were (29.5 +/- 2.5) nmol/L, (80 +/- 5) nmol/L, (49 +/- 4) pmol/L in control group 1 and (42.3 +/- 3.4) nmol/L, (170 +/- 15) nmol/L, (75 +/- 6) pmol/L in control group 2, which showed significant difference (P < 0.05). In the mean time, the level of T, P and E(2) were (106.2 +/- 7.6) nmol/L, (210 +/- 16) nmol/L, (130 +/- 11) pmol/L in observation group 2 and (47.2 +/- 3.5) nmol/L, (130 +/- 6) nmol/L, (81 +/- 6) pmol/L in observation group 1, which also reach statistical difference (P < 0.05). (2) The expression of enzyme: the expression of CYP17 mRNA was increased by 50% and P450 arom mRNA was decreased by 20% between control group 1 and 2. However, the mRNA expression of P450 arom and CYP17 were upregulated remarkably, especially, the expression of CYP17 mRNA were increased by 125%. MAPK signaling pathway plays an inhibitory role in regulating synthesis of steroid hormone of ovarian granulose cell.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

BackgroundTheca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK).MethodsOvarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells.ResultsAkt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA) does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor) suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca cells.ConclusionThese results indicate that LH stimulates CYP17 mRNA expression and androgen production in theca cells via activation of the PI3K/Akt pathway. The LH-induced Akt phosphorylation and androgen production are modulated by the MAPK signaling in bovine theca cells.

Increased ratio of mRNA expression of the genes CYP17 and CYP11B1 indicates autonomous Cortisol production in adrenocortical tumors

Due to increased use of imaging techniques, adrenal incidentalomas are frequently detected. The majority are non-hyperfunctioning adrenocortical tumors. We have previously shown that expression of the gene CYP17, coding for the enzyme in the cortisol pathway, correlates with cortisol release from adrenocortical tumors in vitro. The aim of this study was to compare clinical data with mRNA expression of CYP17 and CYP11B1 in adrenocortical tumors from patients with and without Cushing's syndrome and to identify adrenal tumors that may cause subclinical Cushing's syndrome. A retrospective study of 34 patients undergoing adrenalectomy due to an adrenal tumor. Clinical data were collected. In the adrenal gland the mRNA expression of the genes CYP17 and CYP11B1 was studied with in situ hybridisation technique. The median ratio of CYP17/CYP11B1 expression in tumors from patients with Cushing's syndrome was significantly higher than the median ratio in the non-hyperfunctioning tumors. Tumors from 2 patients with subclinical Cushing's syndrome had ratios within the upper range for non-hyperfunctioning tumors. The ratio between the expression of the genes CYP17 and CYP11B1 in tumors from patients with Cushing's syndrome is significantly higher than in the non-hyperfunctioning tumors. This indicates that 17alpha-hydroxylase is a major determinant of cortisol overproduction. The patients with subclinical Cushing's syndrome in this study are too few to draw any firm conclusions although the results suggest that subclinical Cushing's syndrome may be identified post-operatively with this method.

Expression of Estrogenicity Genes in a Lineage Cell Culture Model of Human Breast Cancer Progression

The mRNA levels of estrogen receptors (ERα and ERβ), cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1 and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β‐hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1 and catechol‐O‐methyltransferase (COMT) were examined in MCF7 and in MCF10A‐derived cell lines representing a lineage cell culture model for breast cancer progression. Relative to MCF7, MCF10A‐derived cells expressed low levels of ERα but comparable levels of ERβ. In benign parental MCF10A breast epithelial cells, SULT1E1 mRNA was induced in confluent relative to subconfluent cell cultures. Proestrogenic 17βHSD1 mRNA was expressed in all of the MCF10A lineage cell lines while 17βHSD2 mRNA was most abundant in MCF10A cells. CYP1B1, SULT1A1 and SULT2B1 were expressed in MCF7 cells, while STS and COMT were widely detected across cell lines. Treatment with the potent histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1, SULT2B1 and CYP19 mRNA, but suppressed CYP1B1, STS, COMT, 17βHSD1 and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to ER‐positive MCF7 cells, key determinants of breast estrogen metabolism are differentially regulated during neoplastic progression. Supported by ES05823 and ES06636.

Inhibition of Adrenocortical Carcinoma Cell Proliferation by Steroidogenic Factor-1 Inverse Agonists

ABSTRACT Context: Transcription factor steroidogenic factor-1 (SF-1) plays a pivotal role in the control of adrenocortical cell steroidogenesis and proliferation. SF-1 amplification and overexpression are found in most cases of childhood adrenocortical tumors (ACTs). Objective: Our objective was to investigate the effect of SF-1 inverse agonists of the alkyloxyphenol and isoquinolinone classes on the proliferation of human adrenocortical cell lines expressing SF-1 (H295R), in conditions of basal and increased SF-1 expression, or negative for SF-1 expression (SW-13). Main Outcome Measures: Proliferation assays, immunoblots, flow cytometric analyses, steroid hormone assays, and real-time quantitative PCR were used. Results: SF-1 inhibitors of the alkyloxyphenol class displayed a dose-dependent inhibitory effect on both SF-1-positive and -negative ACT cells, whereas SF-1 inverse agonists of the isoquinolinone class selectively inhibited cell proliferation elicited by SF-1 overexpression. These drugs also inhibited stimulated steroid hormone secretion and CYP21 and CYP17 mRNA expression. Conclusion: SF-1 inhibitors may represent a useful tool in the chemotherapy of ACTs.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

생후 발달과정 동안 숫 흰쥐의 Efferent Ductules과 부정소에서 Cytochrome P450 Aromatase(Cyp19) 발현 양상 분석

본 연구는 생후 발달과정에서 숫 흰쥐의 생식기인 Efferent ductules(EDs)과 부정소에서 cytochrome P450 aromatase(Cyp19) 유전자의 발현과 연령별 발현 양상을 알아보는데 목적이 있다. 조직으로부터 추출된 total RNA는 역 전사 반응을 통해 cDNA로 바뀌어진 후 real-time PCR 기법을 사용하여 부위별 그리고 연령별로 Cyp19 유전자 발현을 알아보았다. Cyp19 유전자의 발현은 EDs에서 90일령을 제외한 7일, 14일, 그리고 30일령 등 성숙기 이전의 모든 연령에서 나타났으며, 부정소에서는 7일령을 제외한 모든 연령에서 보여졌다. 특히 부정소에서 Cyp19의 발현은 부정소의 각 부위별로 특이한 양상을 보였다. 본 연구 결과를 통해 남성 생식기의 EDs와 부정소의 각 부분에서 Cyp19 유전자 발현은 연령과 부위에 따라 차별적으로 조절되는 것을 알 수 있었다. The present study was performed to determine expression of cytochrome P450 aromatase(Cyp19) in the efferent ductules(EDs) and the epididymis of male rat reproductive tract at different postnatal ages. Total RNAs isolated were reverse-transcribed, and cDNAs were utilized for real-time PCR analysis. In the EDs, the Cyp19 transcript was expressed at all prepubertal ages with the highest level at 14 days of age, but not at 90 days of age. Expression of Cyp19 mRNA in the epididymis was found at all age groups, except 7 days of age. Distinct expression patterns of Cyp19 transcript were shown in each segment of the epididymis. These results indicate that expression of Cyp19 gene in the excurrent duct of male reproductive tract is differentially regulated in age-dependent and segment-specific manners.