Claudin-3 Loss of Expression Is a Prognostic Marker in Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) development is the foremost concern after treatment of patients with high risk with locally advanced or metastatic prostate cancer. Androgen receptor (AR) is the main driver of CRPC development, through its interaction with epigenetic modifier genes, placing epigenetics modifications in the forefront of CRPC development. Comparing the DNA methylation and expression profile of androgen-sensitive and -refractory prostate cancer cells, we describe the epigenetic silencing of claudin-3 (CLDN3) in AR positive cells resistant to androgen deprivation (LNCaP-abl). CLDN3 silencing was associated with DNA methylation, loss of histone acetylation and H3K27 methylation, and was re-expressed by the combined treatment with the epigenetic modulators Aza and SAHA. From a functional point of view, CLDN3 loss was associated with increased cellular invasion. Immunohistochemical analysis showed decreased CLDN3 expression in samples from CRPC patients. Interestingly, CLDN3 expression was significantly decreased in samples from patients with high total Gleason score (≥8) and locally advanced tumors. Finally, CLDN3 loss of expression was associated with worse disease-free survival and time to clinical progression. In conclusion, our findings strongly indicate that epigenetic silencing of CLDN3 is a common event in CRPC that could be useful as a molecular marker for the prognosis of prostate cancer patients and to discriminate aggressive from indolent prostate tumors.

Epigenetic Regulation of Gfi1 in Endocrine-Related Cancers: a Role Regulating Tumor Growth.

Prostate and breast cancer constitute the most common cancers among men and women worldwide. The aging population is one of the main risk factors for prostate and breast cancer development and accumulating studies link aging with epigenetic changes. Growth factor independence-1 (Gfi1) is a transcriptional repressor with an important role in human malignancies, including leukemia, colorectal carcinoma, and lung cancer, but its role in prostate and breast cancer is unknown. We have found that Gfi1 epigenetic silencing is a common event in prostate and breast cancer. Gfi1 re-expression in prostate and breast cancer cell lines displaying Gfi1 epigenetic silencing decreases cell proliferation, reduced colony formation density, and tumor growth in nude mice xenografts. In addition, we found that Gfi1 repress alpha 1-anti-trypsin (AAT) and alpha 1-anti-chymotrypsin (ACT) expression, two genes with important functions in cancer development, suggesting that Gfi1 silencing promotes tumor growth by increasing AAT and ACT expression in our system. Finally, Gfi1 epigenetic silencing could be a promising biomarker for prostate cancer progression because it is associated with shorter disease-free survival. In conclusion, our findings strongly indicate that Gfi1 epigenetic silencing in prostate and breast cancer could be a crucial step in the development of these two-well characterized endocrine related tumors.

Oxidative Stress and Lymphocyte Alterations in Chronic Relapsing Experimental Allergic Encephalomyelitis in the Rat Hippocampus and Protective Effects of an Ethanolamine Phosphate Salt.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) exhibits neuropathological and immunological dysfunctions similar to those found in multiple sclerosis (MS) and has been used as an animal model of MS. Inflammatory infiltrates and oxidative stress have been linked to the development of both diseases. Ethanolamine plasmalogen derivates have been shown to be powerful antioxidants and immunomodulators. Therefore, the objective of this study was to analyse inflammatory infiltrates, the state of the oxidative defences and the possible protective effects of calcium, magnesium and phosphate ethanolamine (EAP) in the CR-EAE rat hippocampus. To this aim, we evaluated, by immunohistochemistry, T cell infiltrates, Iba-1+ (a marker of activated microglia) immunoreactivity and TUNEL (+) cells. We also measured the protein levels and activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR). In addition, reduced (GSH) and oxidized (GSSG) glutathione levels, lipid peroxidation and cholesterol as well as desmosterol content were determined. We found an increase in T cell infiltrates and Iba1+ immunoreactivity, lipid peroxidation, SOD, GP and GR activities as well as enhanced cholesterol levels and a decrease in CAT activity, GSH and desmosterol levels in the first and second attack in the CR-EAE rat hippocampus. Pretreatment of CR-EAE rats with EAP led to a delay in the onset of the clinical signs of the disease as well as a decrease in inflammatory infiltrates and alterations of the antioxidant defences in the hippocampus. Altogether, the present results suggest a protective role of EAP in the CR-EAE rat hippocampus.

Distinct and specific roles of AKT1 and AKT2 in androgen-sensitive and androgen-independent prostate cancer cells.

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

Expression of retinoic acid receptors and retinoid X receptors in normal and vitamin A deficient adult rat brain.

The importance of retinoic acid and retinoid X receptors (RARs and RXRs) in the metabolism and functioning of the nervous tissue is well documented, but few data are available about the differences on their distribution in males and females, as well as about the possible changes in a vitamin A deficient state (VAD). Therefore, the aim of this study has been to use immunohistochemistry to determine the cellular localization of RARs (α, ß, γ) and RXR (α, ß, γ) in brain areas in the normal and vitamin A deficient rat, in both males and females. RARα and ß isotypes were detected in practically all the male brain areas whereas immunostaining was weak or absent in the female brain except RARα. RXRγ was absent in the female brain, while it was observed in some regions in the male. RXRß and γ were the most abundant receptors in both sexes, but RXRα were hardly detected in female brain, but were detected more frequently in male. With a vitamin A-free diet, RARs expression was increased in males, but not in females. In the male brain of VAD rats, RXRα expression was increased in some zones and diminished in others. RXRß and γ expression was decreased in the male brain, but increased or was not modified in those areas of the female brain in which it was observed. These findings indicate that the brain management of retinoic acid differs between males and females, also leading to differences in their response to VAD diet in terms of receptor expression.

EGF promotes neuroendocrine-like differentiation of prostate cancer cells in the presence of LY294002 through increased ErbB2 expression independent of the phosphatidylinositol 3-kinase-AKT pathway.

An increased neuroendocrine (NE) cell population in prostate cancer is associated with more aggressive disease and recurrence after androgen-deprivation therapy, although the mechanism responsible is unknown. In this study, we report that the treatment of LNCaP cells with epidermal growth factor (EGF) in the presence of LY294002, an inhibitor of the phosphoinositol 3'-kinase (PI3K)-AKT pathway, induced an increase of levels and activity of ErbB2. Under these conditions, we also observed cell survival and NE differentiation. When we treated with wortmannin, another PI3K inhibitor, or we knocked down PI3K or AKT isoforms in the presence of EGF, ErbB2 up-regulation was not observed, suggesting that the increase of ErbB2 induced by EGF plus LY294002 is not mediated by the PI3K-Akt pathway. Other targets of LY294002 were also discounted. We also show that ErbB2 up-regulation is directly involved in neuroendocine differentiation but not in cell survival as ErbB2 levels increased in parallel with NE differentiation marker levels, whereas ErbB2 knockdown reduced them; other NE differentiation inducers also increased the ErbB2 levels and the immunohistochemical analysis of prostate cancer samples showed colocalization of ErbB2 and chromogranin A. We found that, in LNCaP cells, EGF in combination with LY294002 increased ErbB2 levels by a PI3K/AKT-independent mechanism and that this increase was associated with the acquisition of a NE phenotype. These results suggest that is worth reconsidering ErbB2 as a drug target in prostate cancer and this should be kept in mind when designing new clinical schedules for the treatment of this disease.

Effect of liver growth factor on both testicular regeneration and recovery of spermatogenesis in busulfan-treated mice.

BACKGROUND: Some adult stem cells persist in adult tissue; however, we do not know how to stimulate stem cells in adults to heal injuries. Liver growth factor (LGF) is a biliprotein with hepatic mitogen activity. Its concentration increases markedly in the presence of any type of liver injury, and it shows in vivo therapeutic biological activity at extrahepatic sites. METHODS: We have analyzed the effect of LGF on the replenishment of germinal cells in the testes of mice injected with busulfan, a common cancer drug that also specifically affects germ line stem cells and spermatogonia. We determined the testicular and epididymal weight, spermatozoal concentration in the epididymis and sperm motility, and performed a histological analysis. RESULTS: Intraperitoneal administration of LGF was able to partially restore spermatogenesis, as well as sperm production and motility, in mice sterilized with busulfan. LGF treatment in busulfan-treated animals that have suffered a disruption of spermatogenesis can accelerate the reactivation of this process in most of the tubules, as shown in the histological analysis. CONCLUSIONS: Our results suggest a potential use of LGF in the mobilization of testicular stem cells and in the restoration of spermatogenesis after busulfan-induced damage to the testicular germinal epithelium.

Hormone-sensitive lipase expression and IHC localization in the rat ovary, oviduct, and uterus.

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.

Human pregnane X receptor is expressed in breast carcinomas, potential heterodimers formation between hPXR and RXR-alpha.

BACKGROUND: The human pregnane X receptor (hPXR) is an orphan nuclear receptor that induces transcription of response elements present in steroid-inducible cytochrome P-450 gene promoters. This activation requires the participation of retinoid X receptors (RXRs), needed partners of hPXR to form heterodimers. We have investigated the expression of hPXR and RXRs in normal, premalignant, and malignant breast tissues, in order to determine whether their expression profile in localized infiltrative breast cancer is associated with an increased risk of recurrent disease. METHODS: Breast samples from 99 patients including benign breast diseases, in situ and infiltrative carcinomas were processed for immunohistochemistry and Western-blot analysis. RESULTS: Cancer cells from patients that developed recurrent disease showed a high cytoplasmic location of both hPXR isoforms. Only the infiltrative carcinomas that relapsed before 48 months showed nuclear location of hPXR isoform 2. This location was associated with the nuclear immunoexpression of RXR-alpha. CONCLUSION: Breast cancer cells can express both variants 1 and 2 of hPXR. Infiltrative carcinomas that recurred showed a nuclear location of both hPXR and RXR-alpha; therefore, the overexpression and the subcellular location changes of hPXR could be considered as a potential new prognostic indicator.

Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells.

Glutamate is an excitatory amino acid that serves important functions in mammalian brain development through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/ kainate receptor stimulation. Neural stem cells with self-renewal and multilineage potential are a useful tool to study the signals involved in the regulation of brain development. We have investigated the role played by AMPA/kainate receptors during the differentiation of neural stem cells derived from fetal rat striatum. The application of 1 and 10 microM kainic acid increased significantly the phosphorylation of the cyclic AMP response element binding protein (CREB), raised bromodeoxyuridine incorporation in O4-positive oligodendrocyte precursors, and increased the number of O1-positive cells in the cultures. Increased CREB phosphorylation and proliferation were prevented by the AMPA receptor antagonist 4-4(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM 2206) and by protein kinase A and protein kinase C inhibitors. Cultures treated with 100 microM kainic acid showed decreased proliferation, a lower proportion of O1-positive cells, and apoptosis of O4-positive cells. None of these effects were prevented by SYM 2206, suggesting that kainate receptors take part in these events. We conclude that AMPA receptor stimulation by kainic acid promotes the proliferation of oligodendrocyte precursors derived from neural stem cells through a mechanism that requires the activation of CREB by protein kinase A and C. In the neurons derived from these cells, either AMPA or kainate receptor stimulation produces neuritic growth and larger cell bodies.

Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C.

Neural stem cells (NSC) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into specific neuronal phenotypes. In the present study, we describe the combined effect of basic fibroblast growth factor (bFGF) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) on the differentiation of fetal rat striatal NSC into tyrosine hydroxylase-positive cells. Tyrosine hydroxylase induction was accompanied by the activation of ERK1/ERK2 mitogen-activated protein kinase and was inhibited by the ERK1/ERK2 pathway blocker PD98059, suggesting that ERK activation may be important for this process. In addition, protein kinase C (PKC) was shown to be required for tyrosine hydroxylase protein expression. The inhibition of PKC by staurosporin, as well as its downregulation, decreased the ability of bFGF+dbcAMP to generate tyrosine hydroxylase-positive cells. Moreover, the PKC activator phorbol 12-myristate 13-acetate (PMA) together with bFGF and dbcAMP led to a significant increase in phospho-ERK1/ERK2 levels, and the percentage of beta-tubulin III-positive cells that expressed tyrosine hydroxylase increased by 3.5-fold. PMA also promoted the phosphorylation of the cyclic AMP response element binding protein that might contribute to the increase in tyrosine hydroxylase-positive cells observed in bFGF+dbcAMP+PMA-treated cultures. From these results, we conclude that the manipulation in vitro of NSC from rat fetal striatum with bFGF, cyclic AMP analogs, and PKC activators promotes the generation of tyrosine hydroxylase-positive neurons.

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours.

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.

Normal and pathological human testes express hormone-sensitive lipase and the lipid receptors CLA-1/SR-BI and CD36.

Numerous studies have demonstrated the important role of cholesterol and cholesteryl esters in tumor cell proliferation and progression of cancer. However, few studies have focused on the role of lipid transporters and lipases in cancer development and progression. The present study examined the expression of hormone-sensitive lipase (HSL) and the scavenger receptors CLA-1/SR-BI and CD36 in normal human testis and in nontumor and tumor testicular disorders by immunohistochemistry and Western blotting analysis. In normal young testes, immunoreaction to CLA-1/SR-BI was found in the spermatid acrosomic vesicle and on the surface of Sertoli and Leydig cells. HSL was detected in spermatogonia, the Golgi region of spermatocytes, the nucleus of spermatids, and the cytoplasm of both Sertoli and Leydig cells. Elderly testes and testes with hypospermatogenesis showed a similar staining pattern to that of normal young testes except for CD36, which was expressed in Sertoli cells. Cryptorchid testes demonstrated intense labeling to HSL and weak labeling to SR-BI in Sertoli cells (nucleus and cytoplasm) and Leydig cells (cytoplasm). Seminiferous tubules with intratubular germ cell neoplasia exhibited intense immunolabeling to the 3 lipid receptors in the surface of neoplastic cells and to HSL in the nucleus. In seminoma and spermatocytic seminoma, neoplastic cells labeled to HSL but failed to stain with antilipid receptors; in the seminiferous tubules at the periphery of the tumour, Charcot-Böttcher crystalloids of Sertoli cells were strongly positive to CLA-1. Testes with mature teratoma showed a weak reaction to CD36 and SR-BI in some cells of enteric-type glands, and immature teratoma were exclusively immunolabeled with HSL. Western blotting analysis revealed that multiple bands were immunolabeled, with differences seen between normal and pathological testes. The results of this study indicate that the presence of lipid receptors (CLA-1/SR-BI) and hormone-sensitive lipase in Leydig cells suggests a role of these proteins in steroidogenesis. Also, these proteins seem to be involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in neoplastic germ cell nuclei suggests a still-unknown function of this enzyme, probably related to cell cycle regulation.

Immunohistochemical detection of the retinoid X receptors alpha, beta, and gamma in human prostate.

The aim of this study was to evaluate the presence and distribution of retinoid X receptors (RXRs) alpha, beta, and gamma in normal, hyperplastic (nodular, basal cell, and atrophic hyperplasia), and carcinomatous human prostates in order to elucidate the relationship among these receptors and the onset and development of prostatic adenocarcinoma. RXRalpha and RXRgamma were immunodetected in all samples of normal, nodular, and basal cell hyperplasia, as well as carcinomatous prostates. In atrophic glands, the expression of both receptors was found in 22.5% of samples. Positive immunostaining for RXRbeta was observed in 53.3% of normal prostates, 100% of samples showed basal cell hyperplasia, and were negative in nodular and atrophic hyperplasia. In prostatic adenocarcinoma, only 3 of 25 samples (the 3 diagnosed as well-differentiated) were positive for RXRbeta. Results suggest that diminished RXRbeta expression might be related to prostate cancer progression and because the responsiveness to retinoic acid treatments depends on the expression of different receptors, it is important to study their expression before therapy.

Cellular characterization of epidermal growth factor-expanded free-floating neurospheres.

Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, alpha- and beta-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)