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The lack of an effective medical treatment for adrenocortical carcinoma (ACC) has prompted the search for better treatment protocols for ACC neoplasms. Sorafenib, a tyrosine kinase inhibitor has exhibited effectiveness in the treatment of different human tumors. Therefore, the aim of this study was to understand the mechanism through which sorafenib acts on ACC, especially since treatment with sorafenib alone is sometimes unable to induce a long-lasting antiproliferative effect in this tumor type. The effects of sorafenib were tested on the ACC cell line H295R by evaluating cell viability, apoptosis and VEGF receptor signaling which was assessed by analyzing VE-cadherin and β-catenin complex formation. We also tested sorafenib on an in vitro 3D cell culture model using the same cell line. Apoptosis was observed after sorafenib treatment, and coimmunoprecipitation data suggested that the drug prevents formation VEGFR-VE-cadherin and β-catenin proteins complex. These results were confirmed both by ultrastructural analysis and by a 3D model where we observed a disaggregation of spheres into single cells, which is a crucial event that represents the first step of metastasis. Our findings suggest that although sorafenib induces apoptotic cell death a small portion of cells survive the treatment and have characteristics of a malignancy. Based on our data we recommend against the use of sorafenib in patients with ACC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cerquetti, Bucci, Raffa, Amendola, Maggio, Lardo, Petrangeli, Torrisi, Toscano, Pugliese and Stigliano.)

Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression ( p < 0.0001) compared to MTT and IR given alone ( p < 0.05). The MTT results confirm its antisteroidogenic activity ( p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death., Competing Interests: The authors have nothing to disclose.

Background: A lack of balance between energy intake and expenditure due to overeating or reduced physical activity does not seem to explain entirely the obesity epidemic we are facing, and further factors are therefore being evaluated. Nickel (Ni) is a ubiquitous heavy metal implied in several health conditions. Regarding this, the European Food Safety Authority has recently released an alert on the possible deleterious effects of dietary Ni on human health given the current levels of Ni dietary intake in some countries. Pre-clinical studies have also suggested its role as an endocrine disruptor and have linked its exposure to energy metabolism and glucose homeostasis dysregulation. Ni allergy is common in the general population, but preliminary data suggest it being even more widespread among overweight patients., Objectives: The aim of this study has been to evaluate the presence of Ni allergy and its association with the metabolic and endocrine profile in overweight and obese individuals., Methods: We have evaluated 1128 consecutive overweight and obese outpatients. 784 were suspected of being allergic to Ni and 666 were assessed for it. Presence of Ni allergy and correlation with body mass index (BMI), body composition, metabolic parameters and hormonal levels were evaluated., Results: We report that Ni allergy is more frequent in presence of weight excess and is associated with worse metabolic parameters and impaired Growth Hormone secretion., Conclusions: We confirm that Ni allergy is more common in obese patients, and we report for the first time its association with worse metabolic parameters and impaired function of the GH-IGF1 axis in human subjects., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Paraganglioma (PGL) is a rare neuroendocrine tumor. Currently, the malignancy is defined as the presence of metastatic spread at presentation or during follow-up. Several gene mutations are listed in the pathogenesis of PGL, among which succinate dehydrogenase (SDHX), particularly the SDHB isoform, is the main gene involved in malignancy. A 55-year-old male without evidence of catecholamine secretion had surgery for PGL of the urinary bladder. After 1 year, he showed a relapse of disease and demonstrated malignant PGL without evidence of catecholamine secretion with a germline heterozygous mutation of succinate dehydrogenase B (SDHB). After failure of a second surgery for relapse, he started medical treatment with sunitinib daily but discontinued due to serious side effects. Cyclophosphamide, vincristine, and dacarbazine (CVD) chemotherapeutic regimen stopped the disease progression for 7 months., Conclusion: Malignant PGL is a very rare tumor, and SDHB mutations must be always considered in molecular diagnosis because they represent a critical event in the progression of the oncological disease. Currently, there are few therapeutic protocols, and it is often difficult, as this case demonstrates, to decide on a treatment option according to a reasoned set of choices.

The sirtuin family comprises seven NAD + -dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins' levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins' expression and clinical and molecular parameters were also analyzed. We found that sirtuin1-6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1-6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins' expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins' levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral adipose-derived stem cells of obese patients.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with an incidence ranging from 0.7 to 2.0 cases/million people per year. Hypercortisolism represents the most common clinical presentation in many patients although, less frequently, some ACC secreting androgens and estrogens are even more pathognomonic compared to cortisol secretion. Currently, radical surgery, when feasible, is still the only curative therapy. Mitotane, an adrenolytic drug, is used in the adjuvant setting and in combination with chemotherapy drugs in metastatic disease. The use of radiotherapy remains controversial, being indicated only in selected cases. New targeted therapies, such as insulin growth factor-1 (IGF-1), mammalian-target of rapamycin (m-TOR), vascular endothelial growth factor (VEGF) inhibitors and others, have recently been investigated with disappointing clinical results. The partial effectiveness of current treatments mandates the need for new therapeutic strategies against this tumor.

Our aim was to systematically evaluate the benefits of degarelix as antagonist versus agonists of gonadotropin-releasing hormones (GnRH) for the treatment of advanced prostate cancer (PC). This comparison was performed either in terms of biochemical or oncological or safety profiles. To this end we, carried out a systematic review and meta-analysis of the literature.We selected only studies directly and prospectively analyzing the two treatments in the same population (randomized phase III studies). We followed the Preferred Reporting Items for Systematic Reviews and meta-analyses process for reporting studies.After we eliminated studies according to the exclusion criteria, 9 publications were considered relevant to this review. These articles described 5 clinical trials that were eligible for inclusion. The follow-up duration in all trials did not exceed 364 days. This meta-analysis and review comprised a total of 1719 men, 1061 randomized to degarelix versus 658 to GnRH agonists treatment for advanced PC. Oncological results were evaluated only in 1 trial (CS21:408 cases) and they were not the primary endpoints of the study. Treatment emerging adverse events were reported in 61.4% and 58.8% of patients in the degarelix and GnRH agonists group, respectively (odds ratio, OR = 1.17; 95% confidence interval, 95% CI: 0.78-1.77, P > 0.1). Treatment related severe cardiovascular side effects were reported (trial CS21-30-35) in 1.6% and 3.6% of patients in the degarelix and GnRH agonists group, respectively (OR = 0.55, 95% CI: 0.26-1.14, P > 0.1).Our analysis evidences relevant limitations in particular for the comparative evaluation of the efficacy and the oncological results related to degarelix., Competing Interests: There are no conflicts of interest for all authors, related to this meta-analysis study.

Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese versus non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity., (© 2015 Wiley Periodicals, Inc.)

Tumors are complex tissues in which transformed cells communicate with the surrounding microenvironment and evolve traits promoting their own survival and malignancy. Hypoxia and inflammation are constant characteristics of prostate tumor microenvironment influencing both cancer stem cells and differentiated tumor cells. HIFs and NF-kB are the key regulators of the transcriptional response to hypoxic and inflammatory stresses, respectively, and a crosstalk between HIFs and NF-kB pathways has been widely documented. Similarly, androgen and estrogen signaling, that play important roles in the growth and function of normal prostate gland, when deregulated, have a significant part in the acquisition of hallmarks of malignant diseases. Moreover, androgen and estrogen receptors have been shown to intersect with the HIF/NF-kB signaling in prostate cancer. Aim of this review is to present the current knowledge regarding the crucial role, in prostate cancer progression, of a molecular network linking hypoxia, pro-inflammatory response and steroid receptors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression.

HIF1α and NFkB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression, and resistance to chemotherapy. In fact, HIF1α and NFkB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic reprograming, inflammatory reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7, and some TLRs, are activated by HIF1α; and that, in turn, alarmin receptors strongly activate NFkB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these molecules are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors with reduction of metastasis formation and, more importantly, with a net increase in survival. This review highlights the central role of HIF1α activated in hypoxic regions of the tumor, of NFkB activation and proinflammatory gene expression in transformed cells to understand their progression toward malignancy. Different molecules and strategies to inhibit these transcription factors will be reviewed. Finally, the central role of a new class of deacetylases called Sirtuins in regulating HIF1α and NFkB activity will be outlined.

Obesity and metabolic comorbidities represent increasing health problems. Endocrine disrupting compounds (EDCs) are exogenous agents that change endocrine function and cause adverse health effects. Most EDCs are synthetic chemicals; some are natural food components as phytoestrogens. People are exposed to complex mixtures of chemicals throughout their lives. EDCs impact hormone-dependent metabolic systems and brain function. Laboratory and human studies provide compelling evidence that human chemical contamination can play a role in obesity epidemic. Chemical exposures may increase the risk of obesity by altering the differentiation of adipocytes. EDCs can alter methylation patterns and normal epigenetic programming in cells. Oxidative stress may be induced by many of these chemicals, and accumulating evidence indicates that it plays important roles in the etiology of chronic diseases. The individual sensitivity to chemicals is variable, depending on environment and ability to metabolize hazardous chemicals. A number of genes, especially those representing antioxidant and detoxification pathways, have potential application as biomarkers of risk assessment. The potential health effects of combined exposures make the risk assessment process more complex compared to the assessment of single chemicals. Techniques and methods need to be further developed to fill data gaps and increase the knowledge on harmful exposure combinations.

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.

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.

In the present report, we investigated the action of retinoic acid (RA) on the transactivation of the epidermal growth factor receptor (EGFR) gene promoter. In a previous study, we showed that the estrogen receptor (ER) α activated by 17β-estradiol (E₂) increased EGFR expression by enhancing the binding of the transcription factor Sp1 to the EGFR minimal promoter in HeLa cells. Here, we demonstrate that ligand-activated RA receptor (RAR) α inhibited EGFR transactivation by competing with Sp1 for binding to the same promoter fragment in the same cell model. When RARα and ERα were coexpressed, the inhibitory effect of RA on transactivation of the EGFR promoter counteracted the enhancement induced by E₂-activated ERα and became more pronounced in the presence of ligand-free ERα. In the MCF7 breast cancer cell line, which endogenously expresses RARα and ERα, RA exerted anti-proliferative effects in the presence of ligand-free ERα. Moreover, interplay between the pathways mediated by the two receptors was observed, as RA counteracted E₂-induced cell proliferation. Our results suggest that the interference with the activity of Sp1 on the EGFR promoter could be related to the observed RA-mediated growth suppression of breast cancer cells., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Background: Recent studies have underlined the role of tumor cells in the endogenous synthesis of pro-inflammatory molecules. We tested whether malignant progression in prostate cancer was associated with the activation of a phenotype typical of the innate immune system., Methods: The expression of a set of molecules involved in tissue inflammation and repair was measured by real-time PCR and Western blot analysis in prostate samples in the absence or slight presence of a detectable leukocyte infiltrate. Whole tumor and non-tumor samples were analyzed in addition to laser-capture microdissected tumor and host epithelium. Receptor for advanced glycation end products, purine receptor, inducible enzymes cyclooxygenase-2 and nitric oxide synthase-2, pentraxin-3 and growth-survival factor receptors such as epithelial growth factor and estrogen alpha and beta receptors were all studied., Results: A global survey approach showed an up-regulation in tumor samples of all of the studied genes, with the exception of ERbeta. A laser-capture microdissection approach highlighted over-expression of pro-inflammatory molecules in each tumor sample examined. Nuclear translocation of nuclear factor-kB subunit p65 was observed in tumor tissues., Conclusions: These data support the evidence that molecules typical of the innate immune system, similar to that of activated leukocytes, are produced by prostate epithelial cells and that their expression is up-regulated in malignant cells. We suggest that the observed pro-inflammatory and repair process activation may represent an important molecular mechanism in the progression of prostate cancer.

In previous studies on HeLa cells we demonstrated estrogen-responsiveness of the epidermal growth factor receptor (EGFR) gene, as 17beta-estradiol (E(2)) and selective estrogen receptor modulators (SERMs) genistein (G), daidzein (D), and 4-hydroxytamoxifen (4OH-T) modulated its transcription in a ligand- and estrogen receptor (ER) isoform-specific way. This study describes further investigations into the role of ERs in mediating the effects induced by E(2) and SERMs on EGFR expression, and the relationship between the actions of ERs and EGFR in U2OS osteosarcoma cells stably expressing ERalpha or ERbeta. Cell number and DNA content determination revealed that E(2), G, and D inhibited proliferation and cell cycle progression and promoted apoptosis in both cell lines. In parallel, changes in cell morphology typical of osteoblast maturation were observed via optical microscopy. Consistently, quantitative PCR and Western blot analysis showed an up-regulation of markers of osteoblast differentiation and bone repair, and a decrease in EGFR expression. The transfection of specific antisense (AS) oligonucleotides strengthened our hypothesis that EGFR reduction caused changes in the proliferation/differentiation pattern comparable to those induced by ER ligands. The link between the ER and EGFR pathways was confirmed by treatment with 4OH-T, which decreased the EGFR level and produced differentiation effects via ERalpha, but induced both EGFR expression and proliferation effects via ERbeta. In conclusion, we show that also in U2OS cells, E(2) and SERMs are able to modulate the expression of the EGFR gene and can affect events strictly controlled by its signaling pathway, such as the maturation of osteoblasts.

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., ((c) 2008 Wiley-Liss, Inc.)

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.

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.

In this study, we evaluate cell death, proliferation and repair in left ventricular endomyocardial biopsies from 20 patients with active lymphocytic myocarditis worsening or recovering from cardiac dysfunction after 6-months immunosuppression. Apoptosis and necrosis were assessed by in situ ligation of hairpin probes, proliferation by Ki67 and MCM5 labelling of myocytes, repair by electron microscopy, morphometric study of percent myofibrillar area and real-time polymerase chain reaction of alpha-and beta-Myosin Heavy Chain (MHC). Apoptosis and necrosis decreased in post- vs pretreatment biopsies by 85 and 62%, respectively in responders, while increased by 42 and 46% in nonresponders. Ki67 and MCM5-positive myocytes were higher vs controls at baseline and increased by 43 and 38% at follow-up in responders and by 75 and 63% in nonresponders. Myofibrillar area reduced in pretreatment samples, increased by 33% at follow-up in responders, correlated with percent enhancement of ejection fraction and was associated with increased alpha-MHC expression and alpha/beta-MHC ratio. In follow-up biopsies of nonresponders, myofibrillar area diminished by 36% and correlated with percent decrease of ejection fraction. Our results suggest that recovery of cardiac function in myocarditis responding to immunosuppression is associated with inhibition of cell death, activation of cell proliferation and with newly synthesized contractile material.

Purpose: FSH, identified as an endogenous product of the prostate, is a glycoprotein with proliferative activity. Increasing evidence of autocrine/paracrine activities of gonadotropins at extragonadal sites led us to investigate the gene expression and cellular localization of FSH-R in normal and diseased human prostates., Materials and Methods: Prostate specimens, including normal gland, BPH, PCa and human androgen refractory (PC3) and androgen dependent (LNCaP) prostate cancer cell lines (European Collection of Cell Cultures, Salisbury, United Kingdom), were analyzed for FSH-R expression by semiquantitative and real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. We also evaluated cyclic adenosine monophosphate production by cultured PC3 and LNCaP stimulated with human FSH., Results: Little FSH-R expression was seen in 9 of 13 normal and 8 of 15 BPH specimens. Of 30 PCa samples 21 were FSH-R positive with generally higher expression compared to normal prostate and BPH samples. Real-time reverse transcriptase-polymerase chain reaction of matched normal/tumor pairs confirmed higher FSH-R mRNA expression in PCa. PC3 cells expressed FSH-R, while LNCaP cells were FSH-R negative. FSH-R protein was mainly localized in the glandular epithelium and in some stromal cells in normal prostate, BPH and PCa specimens. PC3 cells expressed FSH-R protein and their treatment with FSH induced a significant increase in cyclic adenosine monophosphate production., Conclusions: These results indicate that a subset of PCa expresses FSH-R mRNA and protein at levels higher than those of normal and hyperplastic tissues that express FSH-R. This suggests that FSH might contribute to some cases of PCa via a receptor mediated mechanism.

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.

Through the analysis of the transient expression of the luciferase reporter gene in HeLa cells, an evaluation has been made of the transcriptional activity of oestrogens and of selective oestrogen receptor (ER) modulators (SERMs), mediated by the alpha and beta isoforms of the ER, on the epidermal growth factor receptor gene promoter. Oestrogen-activated ERbeta presents a lower transcriptional activity compared with ERalpha, probably due to structural differences in the AF-1 regions of the receptors. Also SERMs induce different responses depending on the receptor isoform bound. Indeed, the phyto-oestrogens, genistein and daidzein, act as weak agonists of the oestrogenic activity via ERalpha, but as full agonists when bound to ERbeta. The synthetic SERM 4OH-tamoxifen, on the other hand, displays an opposite behaviour since it exerts a full agonist action through ERalpha, but acts as a full antagonist via ERbeta. As we have previously shown for ERalpha, an ERbeta/Sp1 functional synergism has also been highlighted, by means of gel mobility shift assays. Moreover, our results show that the sensitivity of target tissues to oestrogens and SERMs can be affected by coexpression of ERs, depending on the formation of appropriate levels of homo- and heterodimers, thus providing a useful approach to predict the effects of hormonal treatment.

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.

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.

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.

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.

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.

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., (Copyright 1999 John Wiley & Sons, Ltd.)

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.

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.

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 (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.

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.

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.

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.

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.

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.

Human benign prostatic hyperplasia (BPH) samples were analyzed to evaluate the presence of immunoreactive epidermal growth factor (irEGF) and EGF receptor (EGFR). In all BPH samples examined both peptide and its receptor were present. Scatchard analysis of binding data of [125I]EGF showed two classes of binding sites with high and low affinity. Intratissular irEGF concentrations showed a significant inverse linear correlation with EGFR levels. Two groups of samples can be identified: the first showing high irEGF concentrations and low levels of EGF binding sites; the second low irEGF and high concentrations of EGFR. The simultaneous presence of EGF and its receptor in BPH samples indicates that this growth factor may act in an autocrine/paracrine manner in human prostatic tissue. The inverse relationship between EGF and the two sites of EGFR lead one to hypothesize that EGF itself could play a central role in determining receptor cell surface availability.

Six human pituitary adenoma cultures, characterized for estrogen and progesterone (Pg) receptors, were treated with 17 beta-estradiol (17 beta-E2), Pg and tamoxifen (TAM) at different concentrations, alone and in combination, for 2, 4 and 8 days. Cell proliferation data showed in most cases a stimulating effect of 17 beta-E2 and an inhibitory effect of Pg on cell growth, which appeared to be correlated with specific receptor expression, but independent of pituitary cell hormone content. A marked inhibitory effect of TAM on cell proliferation was present in all cases, but, on the contrary, was independent of estrogen receptor expression.

The immunosuppressant hormone dexamethasone (Dex) interferes with T cell-specific signals activating the enhancer sequences directing interleukin 2 (IL-2) transcription. We report that the Dex-dependent downregulation of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and calcium ionophore-induced activity of the IL-2 enhancer are mediated by glucocorticoid receptor (GR) via a process that requires intact NH2- and COOH-terminal and DNA-binding domains. Functional analysis of chloramphenicol acetyltransferase (CAT) vectors containing internal deletions of the -317 to +47 bp IL-2 enhancer showed that the GR-responsive elements mapped to regions containing nuclear factor of activated T cells protein (NFAT) (-279 to -263 bp) and AP-1 (-160 to -150 bp) motifs. The AP-1 motif binds TPA and calcium ionophore-induced nuclear factor(s) containing fos protein. TPA and calcium ionophore-induced transcriptional activation of homo-oligomers of the NFAT element were not inhibited by Dex, while AP-1 motif concatemers were not stimulated by TPA and calcium ionophore. When combined, NFAT and AP-1 motifs significantly synergized in directing CAT transcription. Such a synergism was impaired by specific mutations affecting the trans-acting factor binding to either NFAT or AP-1 motifs. In spite of the lack of hormone regulation of isolated cis elements, TPA/calcium ionophore-mediated activation of CAT vectors containing a combination of the NFAT and the AP-1 motifs became suppressible by Dex. Our results show that the IL-2-AP-1 motif confers GR sensitivity to a flanking region containing a NFAT element and suggest that synergistic cooperativity between the NFAT and AP-1 sites allows GR to mediate the Dex inhibition of IL-2 gene transcription. Therefore, a Dex-modulated second level of IL-2 enhancer regulation, based on a combinatorial modular interplay, appears to be present.

Benign prostatic hyperplasia (BPH) is a sex steroid dependent disease. Estrogens and androgens can modulate in different mammalian tissues epidermal growth factor (EGF) production and/or secretion. In order to clarify the relationships between estrogen and androgen receptor concentrations and those of immunoreactive EGF (irEGF), we have evaluated these parameters in 14 human BPH samples, by means of a dextran-coated charcoal method and radioimmunoassay, respectively. Cytosolic steroid receptors did not seem to correlate with irEGF. A linear significative relationship was evident between nuclear androgen receptor (ARn) levels and endogenous irEGF but not between nuclear estrogen receptors and irEGF: in ARn negative BPH samples, irEGF levels were lower than in ARn positive ones. Therefore, it is possible that androgens act at prostatic tissue level, through their own receptors, by modulating EGF production and/or secretion.

Retinoic acid (RA) is known to influence the proliferation and differentiation of a wide variety of transformed and developing cells. We found that RA and the specific RA receptor (RAR) ligand Ch55 inhibited the phorbol ester and calcium ionophore-induced expression of the T-cell growth factor interleukin-2 (IL-2) gene. Expression of transiently transfected chloramphenicol acetyltransferase vectors containing the 5'-flanking region of the IL-2 gene was also inhibited by RA. RA-induced down-regulation of the IL-2 enhancer is mediated by RAR, since overexpression of transfected RARs increased RA sensitivity of the IL-2 promoter. Functional analysis of chloramphenicol acetyltransferase vectors containing either internal deletion mutants of the region from -317 to +47 bp of the IL-2 enhancer or multimerized cis-regulatory elements showed that the RA-responsive element in the IL-2 promoter mapped to sequences containing an octamer motif. RAR also inhibited the transcriptional activity of the octamer motif of the immunoglobulin heavy chain enhancer. In spite of the transcriptional inhibition of the IL-2 octamer motif, RA did not decrease the in vitro DNA-binding capability of octamer-1 protein. These results identify a regulatory pathway within the IL-2 promoter which involves the octamer motif and RAR.

We have studied the regulation of the human c-myc proto-oncogene by glucocorticoid hormones in T lymphoblastic leukemic cells. A significant decrease (50%) of the steady state levels of c-myc mRNA was observed as early as 3 h after dexamethasone treatment of CEM-1.3 human lymphoma cells, reaching less than 5% values, with respect to untreated cells, 24 h after hormone administration. Nuclear run-on experiments showed no modifications of the transcriptional rate from the first exon. However, a slight decrease (15%) of the transcript elongation from the first exon/first intron boundary was observed in the dexamethasone-treated cells. Using actinomycin D to block gene transcription, we have observed a significant increase in the rate of c-myc RNA specific decay after dexamethasone treatment. Furthermore, cycloheximide was able to overcome completely the dexamethasone-induced down-regulation of the c-myc gene expression. Our data suggest that dexamethasone is able to inhibit human c-myc gene expression primarily at the post-transcriptional level, through the synthesis of hormone-induced regulatory protein(s) controlling c-myc transcript stability.

In order to evaluate the biochemical modifications induced by hormonal treatments on human prostatic tissue, the intracellular distribution of tissue DHT and AR were investigated in BPH patients untreated and treated (25-30 days before surgery) with the association of cyproterone acetate (CPA), 100 mg p.o./day plus tamoxifen (TAM), 100 mg p.o./day, or with flutamide (FLU) alone, 750 mg p.o./day. Dextran-coated charcoal and exchange assay in the presence of sodium molybdates (0.2 M) were used for AR determination, employing methyltrienolone as radioligand in the presence of triamcinolone acetonide. Endogenous DHT was measured by RIA, after ether extraction and purification on celite microcolumns. The treatment with CPA plus TAM led to a detection of cytosol AR (ARc) in 50% of the specimens, while nuclear AR (ARn) were never measurable. The FLU treatment did not modify the incidence of ARc, while ARn was not detectable. The cytosolic and nuclear compartmentalization of DHT was scarcely affected by the combined CPA plus TAM treatment, while FLU treatment induced a prevalent cytosolic localization of DHT (DHTc = 283.2 +/- 24.6 S.E. and DHTn = 1138.4 +/- 98.7 S.E. pg/mg DNA in untreated patients; DHTc = 350.4 +/- 97.7 S.E. and DHTn = 589.7 +/- 154.4 S.E. pg/mg DNA in CPA plus TAM treated patients; DHTc = 1101.7 +/- 165.7 S.E. and DHTn = 733.0 +/- 93.9 S.E. pg/mg DNA in FLU treated patients). Both medical treatments, therefore, were able to reduce prostatic growth on account of the reduced value of nuclear DHT content.

A case of ovarian arrhenoblastoma in a 14-year-old girl is reported. The patient presented with primary amenorrhea, severe diffuse hirsutism, moderate clitorial enlargement and slight decrease in breast size. Hormonal examinations revealed high plasma testosterone and androstenedione levels, normal plasma prolactin, drhydroepiandrosterone-sulphate, 17-alpha-hydroxyprogesterone, urinary 17 beta oestradiol, oestrone, FSH and LH. Androgen concentrations decreased under dexamethasone suppression test. Following tumor ablation menses occurred spontaneously and normal hormone patterns were observed.

Ten normoprolactinemic and 10 hyperprolactinemic patients, all with polycystic ovary syndrome (PCO), were subjected to prolactin (PRL) stimulatory tests with thyrotropin-releasing hormone (TRH), 200 microgram intravenously, and haloperidol (a dopamine-blocking agent), 1 mg intramuscularly. The results were compared with those of 8 women with idiopathic hyperprolactinemia and 10 normal female volunteers. Distinctive features of PCO were elevated plasma concentrations of luteinizing hormone, estrone, and testosterone in the presence of normal estradiol, whereas in idiopathic hyperprolactinemia estradiol was reduced. Both groups of patients with PCO exhibited responses to TRH and haloperidol significantly higher than the controls (P less than .001), whereas only the hyperprolactinemic PCO patients reacted with an excessive PRL discharge (P less than .001). As expected, the response to both secretagogue agents was blunted in patients with idiopathic hyperprolactinemia. The present report discusses the possible implication of estrogen and the dopaminergic system in the mechanisms leading to hyperprolactinemia and enhanced PRL release in PCO.