Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Lipido-sterolic extract of Serenoa repens (LSESr, Permixon) treatment affects human prostate cancer cell membrane organization.

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.

Expression of Fas and Fas ligand in human testicular germ cell tumours.

In the present study, we analysed the expression of Fas ligand (FasL) and its cognate receptor Fas in 14 seminomatous testicular germ cell tumours (TGCT) and six normal testicular tissues obtained following orchiectomy. Tissue samples have been processed to prepare either total RNA or protein extracts or fixed and embedded in paraffin for immunohistochemistry (IHC) experiments. Quantitative RT-PCR experiments demonstrated in TGCT a significant (p < 0.01) increase of the FasL mRNA expression of 21.1 +/- 5.4 fold, with respect to normal tissues. On the contrary, in the same cancer tissues, the levels of Fas mRNA were significantly (p < 0.01) reduced to 0.27 +/- 0.06 fold. These observations were confirmed in western blot experiments showing a significant increase of FasL and a concomitant decrease of Fas proteins in testicular cancer tissues, with respect to normal testis. Moreover, IHC experiments showed a strong FasL immuno-reactivity in six out of eight TGCT samples analysed, while Fas immuno-positivity was found in cancer cells of only two TGCT tissues. In addition, in all tumour samples, infiltrating lymphocytes were Fas positive. However, no correlation could be observed between Fas or FasL mRNA variations and clinical parameters such as patient's age, TNM stage or tumour size. We also compared the serum levels of soluble FasL (sFasL) of 15 patients affected by seminomatous TGCT, of four patients with non-seminomatous TGCT and six age-matched healthy males. No significant differences in sFasL serum level could be identified. In conclusion, our data demonstrated that the majority of seminomas are characterized by an increased expression of FasL and a concomitant reduction of Fas, with respect to human normal testis, and that sFasL serum level is not a tumour marker for patients affected by TGCT.

Cancer stem cells in prostate adenocarcinoma: a target for new anticancer strategies.

Prostate cancer (PC) is major common malignancy in males in most industrialized Western countries, where it is the most commonly diagnosed cancer affecting men after middle age (>50 years). Over 90% of PC patients with incurable disease respond to primary treatment, which consists of intervention to lower serum testosterone. However, the duration of response is short (12-33 months) and in almost all patients, is followed by the emergence of a phenotype resistant to androgen deprivation in therapy (known as hormone or androgen-resistant PC). Considerable research efforts have been directed towards the identification of markers associated with the initiation and progression of PC, yet there is little consensus about the target cell within prostate epithelium that is susceptible to malignant transformation. Stem cells may represent a major target for mutations leading to cancer as their longevity assures continued presence during the long latency between carcinogenic agents exposure and cancer development. Therefore in order to allow the development of more effective treatment strategies for PC, a better understanding of the molecular changes that underlie cancer stem cells is required.

Expression of epidermal growth factor receptor in neoplastic pituitary cells: evidence for a role in corticotropinoma cells.

The oncogenic effects of epidermal growth factor (EGF) have long been established. EGF receptor (EGFr) is overexpressed in many types of tumors and constitutes a target for cancer treatment. The pituitary gland is a target of EGF action and it is very likely that EGFr plays a role in pituitary tumor formation and progression. However, there is a controversy in the literature concerning EGFr expression in the different types of pituitary adenomas. In the present study we investigated the expression pattern of the wild type EGFr (EGFrWT) and the constitutively active variant III (EGFrvIII) at the mRNA and protein levels in a large series of pituitary tumors. EGFrWT was found in a high percentage of hormone-secreting tumors, but only in a small fraction of non-functioning pituitary adenomas, while no expression of the EGFrvIII could be detected by nested RT-PCR in any tumor. Among the hormone-secreting adenomas, the highest incidence of EGFr expression was found in Cushing's pituitary adenomas. Furthermore, immunohistochemistry for the phosphorylated EGFr revealed the presence of activated EGFr in most Cushing's adenomas, compared with most pituitary adenomas. Taking into account that downregulation of p27/Kip1 plays a significant role in corticotrope tumorigenesis and that EGFr mitogenic signaling results in decreased p27/Kip1, we searched for a correlation between EGFr expression and p27/Kip1 levels in corticotropinomas. Low p27/Kip1 immunoreactivity was observed in corticotropinomas expressing EGFr. On the other hand, somatotropinomas expressing EGFr had high p27/Kip1 immunoreactivity. These data suggest a corticotrope-specific phenomenon and indicate that EGFr may have a role in the unbalanced growth of corticotrope tumoral cells.

Effects of long-term treatment with the anti-androgen bicalutamide on human testis: an ultrastructural and morphometric study.

AIMS: To assess the effects of more than 4 years' treatment with the anti-androgen bicalutamide on human testis by clinical, ultrastructural and morphometric analysis. METHODS AND RESULTS: Two patients (aged 74 and 69 years) with prostate cancer were treated for more than 4 years with bicalutamide 50 mg daily. Clinical characterization and follow-up included luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and prostate-specific antigen (PSA) measurements and clinical response of the tumours. Due to progression of the disease, patients underwent surgical orchidectomy as a further androgen withdrawal therapy. Testis biopsies were studied by light and electron microscopy and analysed by morphometry. Control samples were obtained from the normal testis of two patients with testicular cancer who underwent orchidectomy. Clinical follow-up showed a good response in the control of tumour growth and serum PSA decreased to < 4 ng/mL; concentrations of serum LH, FSH and testosterone were within the normal range. Testicular morphology of treated patients was unexpectedly well preserved; the organization of seminiferous tubules was normal with all the germ line elements and mature spermatozoa present. In some areas, a net increase of peritubular connective tissue was evident which may be a consequence of the age of the patients. CONCLUSIONS: Long-term bicalutamide (50 mg) treatment appears to have very little impact on testis ultrastructure and sperm maturation, while it is effective in the control of androgen-dependent prostatic tumours.

Transforming growth factor beta 1 and androgen receptors in prostate neoplasia.

OBJECTIVE: To investigate the interplay between transforming growth factor (TGF) beta 1, androgen receptors and stromal-epithelial interactions in benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma areas of prostate neoplasia. STUDY DESIGN: In this immunohistochemical study we investigated staining patterns and then determined the correlation between TGF-beta 1 expression and androgen receptor status in the epithelium and stroma of 60 paraffin-embedded tissues from radical prostatectomies. RESULTS: Staining patterns differed in the epithelium and stroma of tumor and peritumor prostatic tissue. TGF-beta 1 immunostaining (H-scores) in the epithelium and stroma increased significantly from BPH to PIN and from BPH to prostate carcinoma in the epithelium (P < .05), whereas androgen receptor (AR) immunoreactivity significantly (P < .05) increased from BPH to PIN to prostatic carcinoma in epithelium and stroma. TGF-beta 1 did not correlate with histologic grade of differentiation, whereas AR proteins were more strongly expressed in Gleason score 5 and 6 than score 7 tumors (P < .05). Nonlinear regression showed a significant correlation (P < .01) between TGF-beta 1 and AR expression only in the stromal compartment of PIN. CONCLUSION: These findings argue in favor of an interaction between TGF-beta 1 and AR in the early stages of prostate carcinogenesis and suggest that TGF-beta 1 plays a central role in stromal-epithelial interactions during the early stages of malignant transformation.

Effects of triptorelin, a gonadotropin-releasing hormone agonist, on the human prostatic cell lines PC3 and LNCaP.

Some analogues of gonadotropin-releasing hormone (GnRH) influence the in vitro proliferation of cultured human cells by complex interactions that are only partially understood. This study explored the effect of Triptorelin, a GnRH agonist, on the LNCaP and PC3 prostatic cell lines, which are, respectively, responsive and unresponsive to androgen stimulation. The toxicity and cell cycle modifications induced by the drug were investigated by FACScan analysis; the effect on cell proliferation in different culture conditions was determined by counting in a Burker chamber; and the expression of binding sites for 125I-Triptorelin was revealed by displacement experiments. PC3 cell growth was completely unaffected by Triptorelin. The drug caused a double stimulatory-inhibitory action on the growth of actively proliferating LNCaP cells, depending upon the dose and environment. A significant inhibitory effect on proliferation, ranging from 25% to 65% compared with controls, was observed at a high dose (10(-4) M) according to the culture conditions; and a proliferative effect (42% compared with controls) was observed at a lower dose (10(-7) M) only in fetal bovine serum-supplemented medium. Displacement experiments revealed the expression of moderately high affinity and low affinity binding sites in LNCaP cells (Kd = 2.6 x 10(-8) and 7.7 x 10(-6) M) but only low affinity binding sites in PC3 cells (Kd = 2.7 x 10(-6) M), which suggests that the expression of binding sites with different affinity could be associated with a biological response to the drug. Proliferation studies in the presence of Cetrorelix, a GnRH antagonist, confirmed the different sensitivity of the 2 cell lines to GnRH analogues and showed that the proliferative effect of Triptorelin on LNCaP cells can be inhibited by the antagonist. Data confirm the cell specificity of Triptorelin's action and the peculiarity of its effects on prostatic cell proliferation in our experimental conditions.

Identification of an estrogen-mediated deoxyribonucleic acid-binding independent transactivation pathway on the epidermal growth factor receptor gene promoter.

To investigate the estrogenic effects on the transcriptional regulation of the epidermal growth factor (EGF) receptor (EGFR) gene, we assayed its promoter ability to direct transcription of the luciferase reporter gene after transfection into HeLa cells. Our studies demonstrated a dose-dependent activation of the EGFR gene transcription by ligand-bound estrogen receptor alpha (ERalpha). This action was retained by the 36-bp core promoter fragment and did not require the receptor DNA binding domain, as demonstrated by analyzing the role of ERalpha deletion mutants on EGFR gene promoter-derived constructs. The 36-bp promoter fragment does not contain an estrogen response element but an imperfect thyroid hormone response element half-site that overlaps the Sp1 binding site. ERalpha does not bind this imperfect thyroid hormone response element half-site but is able to enhance binding of Sp1 to its site, in gel mobility shift assays, suggesting that the mechanism by which the receptor stimulated the transcription involved protein-protein interactions that replaced DNA binding. To explain this action, we propose a model in which induction of the EGFR gene expression by estrogens in HeLa cells is dependent upon the formation of a transcriptionally active ERalpha-Sp1 complex that binds to the GC-rich (Sp1) region of the minimal promoter.

Transforming growth factor-beta expression in prostate neoplasia.

OBJECTIVE: To understand the role of transforming growth factor (TGF) -beta 1, -beta 2 and -beta 3 proteins and TGF-beta type I and II receptors in prostate neoplasia; to determine the correlation between expression of TGF-beta s and their relative receptors in the epithelial and stromal compartments of benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate carcinoma; and to determine whether TGF-beta and TGF-beta receptor expression is associated with the grade of tumor differentiation. STUDY DESIGN: Sixty prostate neoplasms were analyzed by immunohistochemistry using anti-TGF-beta 1, -beta 2, -beta 3, -beta RI and -beta RII antibodies. RESULTS: TGF-beta and TGF-beta receptor immunoreactivity was more strongly expressed in prostate carcinoma than in PIN and BPH, and TGF-beta type I and type II receptors were less strongly expressed than TGF-beta 1-3 proteins. The difference between epithelial and stromal compartments reached significance (P < .05) for all TGF-beta isoforms and related receptors only in BPH, whereas a significant difference was found for TGF-beta protein in all grades of PIN but not for prostate carcinoma tissue. Luminal epithelial cells of BPH and PIN coexpressed all three TGF-beta isoforms and preferentially TGF-beta RII. Conversely, basal epithelial cells stained strongly for TGF-beta 1, -beta 3 and -beta RI but not for TGF-beta 2 and more strongly for TGF-beta RI than -beta RII. Linear regression showed a positive correlation between TGF-beta 1 and -beta 2, between TGF-beta 2 and -beta 3 and between TGF-beta RI and -beta RII proteins in all areas. The epithelium of Gleason score 7 tumors contained significantly higher TGF-beta 2 protein levels than Gleason score 3 and 4, and 5 and 6 tumors (P < .05). CONCLUSION: Stromal and epithelial cells of malignant and nonmalignant prostatic tumors express all three TGF-beta isoforms and their related receptors. These may act as both paracrine and autocrine factors to influence prostate function and the stromal-epithelial cell interaction. TGF-beta and -beta R immunoreactivity noted in basal cells indicates that in BPH and PIN, TGF-beta Rs and signaling pathways remain intact. The overexpression of TGF-beta proteins and underexpression of TGF-beta receptors in prostate cancer could suggest a mechanism for prostate cancer cells to escape the growth inhibitory effect of TGF-beta, thus leading to a more malignant phenotype.

Nitric oxide synthases in normal and benign hyperplastic human prostate: immunohistochemistry and molecular biology.

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The inducible NOS (iNOS or NOS-2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co-expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium.

Transforming growth factor-beta expression in human testicular neoplasms.

OBJECTIVE: To investigate the localization of transforming growth factors (TGF-beta 1, -beta 2 and -beta 3) and their receptors (TGF-beta RI and RII). STUDY DESIGN: The study included 26 paraffin-embedded tissues from human testicular neoplasms: 15 seminomas, 2 embryonal carcinomas, 1 immature teratoma, 4 immature teratomas with embryonal carcinoma, 1 immature teratoma with seminoma, 1 seminoma with embryonal carcinoma and 2 gonadal stromal tumors (Leydig cell tumors). RESULTS: TGF-beta 1 immunoreactivity was cytoplasmic and was expressed in 22 (84.6%), TGF-beta 2 in 20 (77%), TGF-beta 3 in 11 (42.3%), TGF-beta-RI in 21 (80.8%) and TGF-beta-RII in 18 (69.2%) of the 26 neoplasms. The percentage of positive immunostained cells and the intensity of staining were significantly higher in tumor than in peritumor nonneoplastic testis. In the peritumor nonneoplastic testis, Leydig, Sertoli and germ cells coexpressed both the three TGF-beta isoforms and TGF-beta-RI and RII. The myoepithelial cells of the seminiferous tubules showed immunoreactivity for TGF-beta RI and RII but not for TGF-beta s. In tumor testis areas the pattern of TGF-beta and TGF-beta receptor expression and distribution varied according to the histologic type of testicular tumor. Seminomas showed a diffuse pattern of TGF-beta immunoreactivity, whereas immature teratomas had focal and patchy distribution. In teratomas, differentiated structures contained more TGF-beta s than undifferentiated structures.

Epidermal growth factor binding sites in human pituitary macroadenomas.

The number of epidermal growth factor (EGF) binding sites was determined by competitive binding assays in a series of 46 pituitary macroadenomas. A single concentration of 125I-EGF (1 nM) was used for all experiments. In four cases, a displacement curve was obtained by adding increasing concentrations of cold EGF, and Scatchard analysis showed the presence of two classes of EGF binding sites, with Kd1 = 0.62 +/- 0.23 nM and Kd2 = 53.8 +/- 8.2 nM for the high- and low-affinity binding sites respectively. The distribution of EGF binding sites was studied in 42 cases by a single-point assay, in the presence and in the absence of a 100-fold cold EGF excess. A non-parametric distribution of EGF binding sites was observed (median 10.2 fmol/mg membrane protein, range 0.0-332.0). EGF-receptor positivity, defined as EGF binding > or = 10.0 fmol/mg protein, was observed in 23 samples (54.8%), especially in prolactinomas (76.5%, P < 0.05 vs other tumors taken together) and in gonadotrope adenomas (62.5%). EGF binding was higher in invasive than in non-invasive adenomas (median: 12.8 vs 0.0 fmol/mg membrane protein, P = 0.047), and especially in adenomas invading the sphenoid sinus (median 26.7 fmol/mg membrane protein, P = 0.008 vs other adenomas). EGF binding also tended to increase with the grade of supra/extrasellar extension according to Wilson (P = 0.15). Sex steroid receptors (SSRs) were simultaneously determined in both cytosolic and nuclear fractions of 31 pituitary adenomas. Estrogen and progesterone receptors were determined by an enzyme-linked immunoassay and androgen receptors by a competitive binding assay with [3H]methyltrienolone. No correlation could be found between EGF binding and either the gender and gonadal status of the patients, or the expression of SSRs by the adenomas. We conclude that the EGF family of growth factors may play a role in the evolution of a significant subset of human pituitary adenomas, especially in their invasiveness, and that a high EGF binding capacity may represent an additional marker of aggressiveness for these tumors. Sex steroids do not appear to have a significant role in the regulation of EGF binding in vivo in these tumors.

Androgen receptors in ovarian tumors: correlation with oestrogen and progesterone receptors in an immunohistochemical and semiquantitative image analysis study.

The expression and distribution of androgen, estrogen and progesterone receptors was examined by immunohistochemical staining in 31 paraffin-embedded sections from ovarian tumors and the results were assessed by semiquantitative image analysis. Immunohistochemical staining showed heterogeneous patterns of steroid receptor distribution, with mainly nuclear immunoreactivity. Eighty-four percent of benign and malignant ovarian tumors expressed androgen receptors (AR), 74.19% estrogen receptors (ER) and 41.16% progesterone receptors (PR). All benign tumors showed immunoreactivity for the three steroid receptors. Malignant tumors expressed higher AR and ER histochemical scores (H-scores) than PR (82% vs 71% vs 39%). The incidence and expression levels of the steroid receptors varied widely in the different histological types of malignant tumors. Spearman rank analysis showed a positive significant (P < 0.05) correlation between AR- and ER and between ER- and PR-H-scores. In malignant ovarian tumors, neither AR, ER nor PR immunohistochemical scores correlated with tumor FIGO stage. Densitrometric analysis of immunostained steroid receptors is a valid method for assessing the steroid status, because it reduces subjective elements in scoring sections and increases the reliability of results. The high incidence of AR expression confirms the functional role of AR in ovarian tumors and suggests that the determination of AR content in ovarian cancer could have prognostic value.

Cellular receptors for sex steroids in human pituitary adenomas.

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [3H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n = 33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66.7, 60.6 and 81.8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between pre-operative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41.4 vs 15.4%, P < 0.05), whereas a high expression of ERcs and ARcs (> 15 fmol/mg protein) was more common in tumors found in men (34.5 vs 10.3%, P < 0.05 and 54.5 vs 24.0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34.8 +/- 11.3 vs 4.8 +/- 5.1 fmol/mg protein, P = 0.007) and gonadotroph cell adenomas lower ARc values (1.3 +/- 0.8 vs 38.2 +/- 10.6 fmol/mg protein, P = 0.048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (> 90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.

Effects of the lipidosterolic extract of Serenoa repens (Permixon) on human prostatic cell lines.

BACKGROUND: Permixon is a drug used in the treatment of benign prostatic hyperplasia. We studied its androgenic and antiandrogenic effects in the prostatic cell lines LNCaP and PC3, respectively responsive and unresponsive to androgen stimulation. METHODS: We performed FACScan analysis to investigate toxicity, 3H thymidine and 35S methionine incorporation to determine antiproliferative and metabolic effects, electron microscopy to study ultrastructural changes and cotransfection experiments to elucidate the role of wild type androgen receptor. RESULTS: In LNCaP cell line, Permixon induced a double proliferative/differentiative effect, not observed in PC3 cells. In PC3 cells cotransfected with wild-type androgen receptors and CAT reporter genes under the control of a androgen responsive element, the drug inhibited androgen-induced CAT transcription. CONCLUSIONS: Our data indicate a role of the androgen receptor in mediating the effects of Permixon in LNCaP cells. Cotransfection experiments in PC3 cells support a clear antiandrogenic action of the drug.

Gene methylation of oestrogen and epidermal growth factor receptors in neoplastic and perineoplastic breast tissues.

Oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) gene methylation was evaluated in neoplastic and perineoplastic breast tissues from 20 patients. In both tissues, ER gene methylation was inversely correlated with protein levels, while EGFR gene methylation was not. A preferential ER gene hypomethylation was found in neoplastic tissues, suggesting a significant role in neoplastic transformation.

Expression of steroid receptors in acoustic neuroma.

The expression of steroid receptors has been investigated in an attempt to clarify the role of steroid hormones in the proliferation and progression of acoustic neuromas. Specimens of tumours taken during translabyrinthine surgery were tested for cytosolic (c) and nuclear (n) steroid receptors. Oestrogen and progesterone receptor levels were evaluated by enzymatic immuno-assay, while androgen receptor binding levels were detected by dextran-coated charcoal method in a single-step determination. In some cases, the six point Scatchard analysis of cytosolic and nuclear androgen receptor was also performed. Threshold values were: 3 fmol/mg of proteins for cytosolic steroid receptors and 20 fmol/mg DNA for nuclear steroids, which corresponded to approximate median values of cytosolic and nuclear oestrogen and progesterone, respectively. Oestrogen and progesterone appeared to be localized more frequently in the nuclei rather than in the cytosol (70% oestrogen and progesterone positivity in the nuclei; 30% oestrogen, 40% progesterone positivity in the cytosol), while androgen receptors were preferentially localized in the cytosol (80% positivity in the cytosol; 40% positivity in the nuclei). A negative non-linear correlation between cytosolic oestrogen and cytosolic androgen receptors was found. There was a direct linear correlation between cytosolic oestrogen and nuclear oestrogen levels. A strict correlation between nuclear oestrogen and nuclear progesterone incidence was shown. Preliminary analysis of clinical data and biochemical parameters showed that cytosolic progesterone levels inversely correlated with tumour size.

Effects of gonadal steroids on the growth of human pituitary adenomas in vitro.

The effects of testosterone (T), dihydrotestosterone (DHT) and methyltrienolone (R 1881) on cell proliferation of eight human pituitary tumors in culture wre assessed by [3H]thymidine incorporation and compared to those of progesterone (Pg) and 17 beta-estradiol. Receptors for androgens (AR), estrogens (ER) and progesterone (PgR) were characterized. AR had a significant inhibitory effect on all AR-positive tumors, whatever their hormonal content. Inhibitory effects of either T and DHT < R1881 < Pg were observed in tumors co-expressing AR and PgR. The inhibitory effect of R 1881 on a PgR-positive/AR-negative tumor suggested that R 1881 action was partially PgR-mediated. The effects of either T or the nonaromatizable DHT and R 1881 were unrelated to ER expression. We conclude that AR can modulate the growth of human pituitary tumors through direct receptor-mediated intracellular pathways which may be common to various pituitary cell types.

Estrogen receptors: new perspectives in breast cancer management.

The imbalance between proliferative and differentiative estrogenic effect, caused by quantitative and qualitative alteration of the estrogen receptor (ER) expression, may play a determinant role in mammary neoplastic transformation. Our studies demonstrate that ER levels are significantly higher in human mammary neoplastic tissues when compared to perineoplastic tissues and that increased ER expression is associated with ER gene hypomethylation. During progressive multifactorial carcinogenesis, ER overexpression may represent an early step in neoplastic transformation. In fact, high levels of ER represent good markers of differentiation and can predict the likelihood of benefiting from anti-estrogen therapy. Nevertheless, about 35% of ER-positive breast cancers are resistant to endocrine therapy and 10% of ER-negative tumors behave as hormone-sensitive tumors. Recent studies on ER mRNA variants, which naturally occur in human breast tumors, demonstrated mutations, deletions and alternative splicings, yielding deletions of exons 3, 4, 5 and 7. ER variants exhibited altered functions or changed the responsiveness to hormonal therapy. Analysis of these variants could be a useful parameter to better predict tumor responsiveness to anti-estrogen therapy. Recently, a regain of hormonal responsiveness by ER-negative breast cancer cells has been reported following ER gene transfection. However, estradiol treatment inhibits rather than stimulates cell growth as well as the metastatic and invasive potential of the ER gene transduced cells. Transfer of the ER gene may be considered as a new therapeutic approach in the management of hormone-independent breast cancer.