Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Methylmercury inhibits prolactin release in a cell line of pituitary origin.

Heavy metals, such as methylmercury, are key environmental pollutants that easily reach human beings by bioaccumulation through the food chain. Several reports have demonstrated that endocrine organs, and especially the pituitary gland, are potential targets for mercury accumulation; however, the effects on the regulation of hormonal release are unclear. It has been suggested that serum prolactin could represent a biomarker of heavy metal exposure. The aim of this study was to evaluate the effect of methylmercury on prolactin release and the role of the nitrergic system using prolactin secretory cells (the mammosomatotroph cell line, GH3B6). Exposure to methylmercury (0-100 µM) was cytotoxic in a time- and concentration-dependent manner, with an LC50 higher than described for cells of neuronal origin, suggesting GH3B6 cells have a relative resistance. Methylmercury (at exposures as low as 1 µM for 2 h) also decreased prolactin release. Interestingly, inhibition of nitric oxide synthase by N-nitro-L-arginine completely prevented the decrease in prolactin release without acute neurotoxic effects of methylmercury. These data indicate that the decrease in prolactin production occurs via activation of the nitrergic system and is an early effect of methylmercury in cells of pituitary origin.

The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.

Validation of immunohistochemistry for somatostatin receptor subtype 2A in human somatotropinomas: comparison between quantitative real time RT-PCR and immunohistochemistry.

Somatostatin receptors subtype 2 (SSTR2) expression in somatotropinomas is recognized as a predictor of response to the currently available somatostatin analogs and may be analyzed, mainly, by quantitative RT-PCR or immunohistochemistry (IHC). The former has the advantages of a higher sensitivity and of being quantitative, while the latter, although semi-quantitative, evaluates protein expression and is routinely used in the evaluation of pituitary adenomas. We aimed to evaluate the SSTR2A protein expression in somatotropinomas and to compare it to our previous data regarding mRNA expression, assessed by quantitative real time RTPCR. Thirteen somatotropinomas were analyzed by IHC and the tumors were scored according to percent of immunostained cells: 0 (<25%), 1 (25-50%) and 2 (>50%). SSTR2A immunostaining was present in all but one somatotropinoma, 4 (31%) tumors were classified as score 0, 4 (31%) as score 1, and 5 (38%) as score 2. Median SSTR2 mRNA content was significantly different among the three IHC scores (p=0.036) and was lower in the score 0 than in the score 2 (p=0.016). The finding that there is a positive correlation between RT-PCR and IHC indicates that IHC can be applied in order to assess the SSTR2A content in somatotropinomas.

Estradiol modulates TGF-ß1 expression and its signaling pathway in thyroid stromal cells.

The higher prevalence of thyroid disease in women suggests that estrogen (E2) might be involved in the pathophysiology of thyroid dysfunction. To approach the question of the effect of stromal cells in the modulation of thyroid epithelial cells activity, we established and characterized a homogeneous stromal cell population (TS7 cells) of rat thyroid gland. These fibroblastic cells synthesize the cytoskeleton proteins α-smooth muscle actin and vimentin, produce basement membrane components and express the cytokine transforming growth factor beta 1 (TGF-ß1). Here, we hypothesized that the effects of E2 on follicular thyroid cells are mediated by TGF-ß1 synthesis and secretion by stromal cells (paracrine action). Thus we investigated the effect of E2 on TGF-ß1 synthesis and its signaling pathway in TS7 cells. In addition, we analyzed the role of TGF-ß1 signaling pathway as mediator of TS7-PC CL3 thyroid epithelial cells interactions. We report that TS7 stromal cells expressed α and ß estrogen receptors (ERα and ERß). Further, both isoforms of TGF-ß1 receptors, TGFRI and TGFRII, were also identified in TS7 cells, suggesting that these cells might be a target for this cytokine in vitro. Treatment of TS7 cells with E2 induced both synthesis and secretion of TGF-ß1. This event was followed by phosphorylation of the transcription factor Smad2, a hallmark of TGF-ß1 pathway activation. Co-culture of PC CL3 cells onto TS7 cells monolayers yielded round aggregates of PC CL3 cells surrounded by TS7 cells. TS7 cells induced a decrease in iodide uptake by PC CL3 cells, probably by a mechanism involving TGF-ß1. Moreover, E2 affected synthesis and organization of the extracellular matrix (ECM) components, tenascin C and chondroitin sulfate, in these co-culture cells. Our results point to the TGF-ß1/Smad-2 signaling pathway as a putative target of estrogen actions on thyroid stromal cells and contribute to understanding the interplay between stromal and follicular cells in thyroid physiology.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model.

BACKGROUND: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. OBJECTIVES: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. METHODS AND RESULTS: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. CONCLUSION: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Modulation of extracellular matrix components by metalloproteinases and their tissue inhibitors during degeneration and regeneration of rat sural nerve.

The success of peripheral nervous system regeneration has been associated with changes on the microenvironment, particularly on the extracellular matrix (ECM) components. In the present study we analyzed by indirect immunohistochemistry, electron microscopy and Western blotting, the distribution of ECM components, metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), during Wallerian degeneration (WD) and nerve regeneration (2nd, 7th and 21st days after injury) on crushed rat sural nerves. Our results showed that laminin alpha3-chain and alpha2-chain are over expressed during the early stages of degeneration and regeneration respectively, whereas type IV collagen expression increased progressively after crush. On the other hand, the expression of chondroitin sulfate was down regulated during the early stages of degeneration, returning progressively to normal values during nerve regeneration. The expression of MMP-3 was almost normal immediately after lesion, and then reduced progressively achieving the smallest expression at 21 days after crush; on the contrary, the expression of MMP-7 increased significantly immediately after crush (2nd day) returning to normal values afterwards. TIMP-1 and TIMP-2 were over expressed at the beginning of WD, returning progressively to normal values after that. These results indicate that the modifications of ECM components, which are favorable for nerve regeneration, are correlated with changes on the balance between MMPs and TIMPs.

Goiter and hypothyroidism in two siblings due to impaired Ca(+2)/NAD(P)H-dependent H(2)O(2)-generating activity.

We report herein the study of two siblings (DESM and DSM) with hypothyroidism, goiter, and positive perchlorate discharge tests (50% and 70%) in a family (M) with no history of consanguinity. Thyroid gland histology showed a predominance of hyperactive follicles, with high epithelial cells and variable colloid content. Thyroid peroxidase iodide oxidation (DESM, 1034; DSM, 1064 U/g protein) and albumin iodination (DESM, 16; DSM, 8 nmol I/mg protein) activities were within the normal range. Tg content was normal in both glands compared with that in diffuse toxic goiter (DESM, 28; DSM, 17; diffuse toxic goiter, 19 mg/g tissue), and Tg could be normally iodinated by thyroid peroxidase in vitro (DESM, 3.4; DSM, 4.3; diffuse toxic goiter, 6.3 nmol I/mg Tg). Thyroid cytochrome c reductase activities in these goiters were higher than that in paranodular tissues (DESM, 473; DSM, 567; paranodular tissues, 78 nmol NADP(+)/h/mg protein). However, thyroid NADPH oxidase activities were very low both in the particulate 3,000 x g (DESM, 4.8; DSM, 44; paranodular tissues, 224 nmol H(2)O(2)/h/mg protein) and in the particulate 100,000 x g fractions (DESM, 40; DSM, 47; paranodular tissues, 200 nmol H(2)O(2)/h/mg protein). Thus, a decreased Ca(2+)/NAD(P)H-dependent H(2)O(2) generation is the probable cause of the organification defect in these goiters.

Differential expression of laminin isoform (alpha2), integrins (alpha3beta1 and alpha6beta4) and cytokeratin 20 in H. pylori gastritis.

The expression of laminin-1 chains (beta1 and gamma1), laminin-2 (merosin), integrin receptors to laminin (alpha3beta1 and alpha6beta4) and cytokeratin (CK20) were studied by immunohistochemical methods in gastric biopsies from antrum of 25 patients. H. pylori gastritis was found in 19 cases and intestinal metaplasia (IM) in four from these 19. Another 13 biopsies, all with IM were immunostained to laminin-2. Laminin-1 chains in normal and gastritis areas without IM were expressed as a strong, linear and continuous deposit in the basement membranes of the superficial and glandular epithelium. In metaplastic glands the reactivity to laminin-1 chains was decreased. Merosin was discontinuous when a moderate to accentuated H. pylori glandular colonization was present. Samples with IM were negative to laminin-2. The alpha3beta1 and alpha6beta4 integrins were negative only in IM gastric biopsies. The CK20 immunoreactivity was strong and homogeneous in the cells at the tip and the upper portion of foveolae in normal areas and in gastritis with IM the reactivity to CK 20 was heterogeneous. A differential expression of laminin isoforms is related to inflammation and subsequent IM caused by H. pylori. The alterations of alpha3beta1 and alpha6beta4 parallel both modifications in merosin and CK20 expression in H. pylori chronic gastritis.

Sulfated glycosaminoglycans from ovary of Rhodnius prolixus.

We have characterized sulfated glycosaminoglycans from ovaries of the blood-sucking insect Rhodnius prolixus, and determined parameters of their synthesis and distribution within this organ by biochemical and histochemical procedures. The major sulfated glycosaminoglycan is heparan sulfate while chondroitin 4-sulfate is a minor component. These glycosaminoglycans are concentrated in the ovarian tissue and are not found inside the oocytes. Besides this, we detected the presence of a sulfated compound distinguished from sulfated glycosaminoglycans and possibly derived from sulfated proteins. Conversely to the compartmental location of sulfated glycosaminoglycans, the unidentified sulfated compound is located in the ovarian tissue as well as inside the oocytes. Based on these and other findings, the possible roles of ovarian sulfated glycosaminoglycans on the process of oogenesis in these insects are discussed.

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Effect of reduced temperatures and brefeldin A on prolactin secretion and on subcellular distribution of the secretory product and membrane antigens in GH3 pituitary cells.

The effects of reduced temperatures (20, 15 or 10 degrees C) and brefeldin A (BFA) on prolactin (PRL) secretion in the GH3 rat pituitary cell line have been compared. Both treatments inhibit PRL release to different extents. Ultrastructural immunocytochemistry reveals that, depending on the treatment, PRL is blocked at different steps during its intracellular transit. The temperatures of 20 and 15 degrees C block the PRL transport at one face of the Golgi stacks whereas both the temperature of 10 degrees C and BFA treatment induce an arrest of PRL at the level of the rough endoplasmic reticulum (RER) cisternae. Moreover, exposure to 10 degrees C or BFA induces an accumulation of a specific Golgi membrane antigen in the dilated RER structures. However, although disorganized and no longer definable under BFA treatment, the Golgi apparatus remains visible at 10 degrees C. These two last treatments cause also an increase in the number of partly rough, partly smooth tubular structures tentatively called 'paired cisternae'.

Subcellular distribution of secretogranins I and II in GH3 rat tumoral prolactin (PRL) cells as revealed by electron microscopic immunocytochemistry.

The GH3 rat pituitary cell line which secretes prolactin (PRL) is characterized by the paucity and small size of secretory granules. We looked for the presence, in these cells and in normal PRL cells, of two acidic tyrosine-sulfated proteins which are widely distributed in dense-core secretory granules of endocrine and neuronal cells, secretogranins I and II, using immunofluorescence and electron microscope immunoperoxidase techniques. Both secretogranins were detected in secretory granules of GH3 cells and of normal cells. Moreover, with our pre-embedding approach, secretogranins were localized within some RER cisternae and within all sacules of the Golgi stacks in both PRL cell models. A few small vesicles, large dilated vacuolar or multivesicular structures, and some lysosome-like structures were also immunoreactive. Double localization of secretogranins and PRL performed on GH3 cells by immunofluorescence indicated that all cells contained secretogranins I and II, whereas only 50-70% of the cells contained PRL. Moreover, in the case of hormone treatment known to increase the number of secretory granules, most if not all mature secretory granules were immunoreactive for secretogranins, whereas in certain cells some of the granules were apparently not immunoreactive for PRL. These immunocytochemical observations show that GH3 cells, which under normal conditions form only a small number of secretory granules, produce secretogranins and package them into these granules.