Fermín R. Bethencourt

Interleukin-1 (IL-1α and IL-1β) and its receptors (IL-1RI, IL-1RII, and IL-1Ra) in prostate carcinoma

The principal components of the interleukin‐1 (IL‐1) family are two secreted factors (IL‐1α and IL‐1β), two transmembrane receptors (IL‐1RI [biologically active] and IL‐1RII [inert receptor]), and a natural antagonist receptor of IL‐1 function (IL‐1Ra). Changes in the expression pattern of these IL‐1 members have been reported to be related to disease progression. The objective of the current study was to evaluate these changes in prostatic tissue by means of immunohistochemistry and Western blot analysis.

Transforming Growth Factor β and its Receptor Types I and II. Comparison in Human Normal Prostate, Benign Prostatic Hyperplasia, and Prostatic Carcinoma

An immunohistochemical and semiquantitative comparative study of transforming growth factor beta 1 (TGF-beta 1) and its receptor types I (TGF-beta RI) and II (TGF-beta RII) was carried out in normal prostates and in the prostates from men with benign prostatic hyperplasia (BPH), and men with prostatic adenocarcinoma. Immunoreaction to TGF-beta 1 was limited to the basal epithelial cells in the normal prostates. Some cells in the connective tissue stroma were also stained. In BPH immunolabelling was also observed in columnar (secretory) cells of the epithelium. In prostatic adenocarcinoma, all epithelial cell types were intensely immunostained. Some stromal cells were also stained. Immunostaining to TGF-beta RI was only present in the basal cells in normal prostates. In BPH, this immunoreaction was found in the whole epithelium and in some stromal cells. In prostatic cancer, the immunostaining pattern for this receptor was similar to that of BPH but more intense in the epithelial cells. Immunoreactivity to TGF-beta RII appeared in some basal cells and some scattered columnar cells of the normal prostate epithelium. In the BPH sections, this pattern was maintained, and a weak immunolabelling was also observed in the stroma. In prostate cancer, all epithelial cells appeared intensely labelled. In the stroma, immunolabelling was similar to that of the BPH specimens. The results of the present study suggest that, in normal prostates, only the basal cells of the epithelium possess both receptor types, and hence can transduce TGF-beta 1 signal intracellularly. The basal cells can also secrete this growth factor which would act as an autocrine inhibitory growth factor for them. In addition, TGF-beta 1 is secreted in some zones by stromal cells, acting then as a paracrine growth factor for basal cells in those areas. In BPH, in addition to the basal cells, some secretory columnar cells also secrete TGF-beta 1 and possess both types of TGF-beta 1 receptors, and thus, both epithelial cell types are susceptible to TGF-beta 1 action. Since both receptor types are also present in some stromal cells, these cells also perform an autocrine secretion, in addition to their paracrine secretion to the epithelial cells. TGF-beta RIIs seem to be more numerous than TGF-beta RIs and this lead us to hypothesize that these incomplete receptors might be a protection against the inhibition caused by TGF-beta 1 action. In prostatic carcinoma all cell types display the same characteristics as in BPH, although both receptor types are found in similar numbers, and thus, the above mentioned protection would not occur.

A lectin histochemistry comparative study in human normal prostate, benign prostatic hyperplasia, and prostatic carcinoma.

The partial oligosaccharide sequences of glycoconjugates and the nature of their glycosidic linkages were investigated in normal human prostate, benign prostatic hyperplasia (BPH) and prostatic carcinoma by means of lectin histochemistry, using light microscopy and Western blot analysis. The labeling pattern of BPH differed from that of normal prostate in having more intense staining with DSA, HPA, UEA-I and AAA, and in showing lesser staining with WGA and SBA. Prostatic carcinoma differed from normal prostates in displaying the more intense labeling with PNA, DSA, SBA, DBA, UEA-I and AAA, and in having lesser labeling with WGA. The main differences in labeling pattern between prostatic carcinoma and BPH were that the latter specimens showed more marked staining with PNA, DSA, DBA, SBA, UEA-I and AAA, and lesser staining with WGA and HPA. The staining patterns of SNA, MAA, ConA, LCA and GNA were similar in all three groups of specimens. For most of the lectins studied, including those showing a similar immunohistochemical staining in the three groups of specimens studied, the Western blot analysis showed differences in the banding pattern among normal, hyperplastic, and carcinomatous prostates. Present results suggest that the glycosylation of proteins was modified in both BPH and prostatic carcinoma. In BPH a strong expression of N-acetylgalactosamine residues occurred, while in prostatic carcinoma an increase of sialic aci, galactose and fucose residues was observed. No changes in mannose residues were detected.

Role of IAPs in prostate cancer progression: immunohistochemical study in normal and pathological (benign hyperplastic, prostatic intraepithelial neoplasia and cancer) human prostate

BackgroundIn this study was investigate IAPs in normal human prostate (NP), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma (PC), and their involvement in apoptosis/proliferation via NF-kB (TNF-α, IL-1) stimulation.MethodsImmunohistochemical and Western blot analyses were performed in 10 samples of normal prostates, 35 samples of BPH, 27 samples diagnosis of PIN (with low-grade PIN or high-grade PIN) and 95 samples of PC (with low, medium or high Gleason grades).ResultsIn NP, cytoplasm of epithelial cells were positive to c-IAP1/2 (80% of samples), c-IAP-2 (60%), ILP (20%), XIAP (20%); negative to NAIP and survivin. In BPH, epithelial cells were immunostained to c-IAP1/2 (57.57%), c-IAP-2 (57.57%), ILP (66.6%), NAIP (60.6%), XIAP (27.27%), survivin (9.1%). Whereas low-grade PIN showed intermediate results between NP and BPH; results in high-grade PIN were similar to those found in PC. In PC, epithelial cells were immunostained to c-IAP1/2, c-IAP-2, ILP, NAIP, XIAP (no Gleason variation) and survivin (increasing with Gleason).ConclusionsIAPs could be involved in prostate disorder (BPH, PIN and PC) development since might be provoke inhibition of apoptosis and subsequently cell proliferation. At the same time, different transduction pathway such as IL-1/NIK/NF-kB or TNF/NF-kB (NIK or p38) also promotes proliferation. Inhibitions of IAPs, IL-1α and TNFα might be a possible target for PC treatment since IAPs are the proteins that inhibited apoptosis (favour proliferation) and IL-1α and TNFα would affect all the transduction pathway involucrate in the activation of transcription factors related to survival or proliferation (NF-kB, Elk-1 or ATF-2).

IL-2, Its Receptors, and bcl-2 and bax Genes in Normal, Hyperplastic and Carcinomatous Human Prostates: Immunohistochemical Comparative Analysis

Abstract The presence of interleukin-2 (IL-2) and its receptors (Rα, Rβ, Rγ), and their relationship with the products of bcl-2 and bax genes was studied in normal prostates, benign prostatic hyperplasia (BPH), and prostatic cancer (PC) by ELISA, Western blot, and immuno-histochemistry. A comparative semiquantitative immunohistochemical study was also performed. For all the antibodies assayed, immunoreactions were found in the epithelium and some stromal cells in the three types of specimens studied. These immunoreactions were much more higher in PC samples than in normal prostates. In BPH, immunoreactions were similar to that of normal prostates (bax), similar to that of PC (IL-2 and its three receptors), or intermediate between that of normal prostates and that of PC (bcl-2). Immunoexpressions of IL-2 and its receptors were found in the epithelial basal cells and some stromal cell of normal prostates and might be related to the control of the proliferation-apoptosis equilibrium. The increased expressions of IL-2 and its receptors in the epithelium of prostates in BPH, associated with increased bcl-2 expression which would account for the decrease in the apoptosis index that has been reported in this disorder. The increased expression of both bcl-2 and bax in PC might be involved in the higher apoptosis index reported in PC specimens. Since IL-2 administration seems to have an anti-tumour effect, the increased expression of this interleukin in BPH and PC could be interpreted as an attempt to hinder cell proliferation which would only be efficient at high doses.

Intermediate filaments in the Sertoli cells of the ageing human testis

Abstract The presence and distribution of intermediate filaments (vimentin, keratin, desmin) was studied in the Sertoli cells of elderly men by means of quantitative immunohistochemical methods. Sertoli cells from young men showed moderate immunogold labelling to vimentin throughout the entire cytoplasm between the cell organelles in tubules showing complete spermatogenesis. Immunogold particles were more numerous in the perinuclear cytoplasm and beneath the plasma membrane in all its faces. The testes from elderly men showed different tubule types; some showed complete spermatogenesis and a normal lamina propria, while others had spermatogenic arrest at different levels (spermatids, spermatocytes, spermatogonia). The immunohistochemical reaction to vimentin in the Sertoli cells of tubules with complete spermatogenesis (type a) was similar to that in the cells of young men. In the Sertoli cells of severely damaged tubules (type b) the immunohistochemical reaction was more intense and immunogold particles extended in similar proportions throughout the whole cytoplasm. When immunolabelling intensity was compared between the three groups of tubules, by counting the number of immunogold particles per square micrometre of cytoplasm, it was found to be significantly higher (P≤0.05) in type b tubules of elderly men than either in tubules of young men or in type a tubules of elderly men. Since the average cell surface of Sertoli cells was similar in all tubule types, these data suggest that an actual vimentin increase occurs in Sertoli cells of germ-cell-depleted tubules. Sertoli cell immunogold labelling to keratin was found neither in young men nor in type a tubules of ageing men, whereas a positive immunohistochemical reaction was observed in the Sertoli cells of type b tubules of elderly men. Immunogold particles were localized mainly in the perinuclear cytoplasm, and beneath the lateral and basal cell surfaces. The observation of vimentin increase and keratin re-expression in ageing Sertoli cells only in germ-cell-depleted tubules suggests that the changes in intermediate filaments are related to the local factors associated with completion of spermatogenesis, causing functional changes in Sertoli cells.

Relationship between IL-6/ERK and NF-κB: a study in normal and pathological human prostate gland

BACKGROUND There is growing evidence that inflammation is a causal factor in cancer, where pro-inflammatory cytokines such as IL-6, IL-1 or TNF-α could induce cellular proliferation by activation of NF-κB. This study focuses on the IL-6/ERK transduction pathway, its relationship with NF-κB, and the consequences of dysregulation in the development of prostate pathologies such as benign prostate hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate cancer (PC). METHODS Immunohistochemical and Western blot analyses for IL-6, gp-130, Raf-1, MEK-1, ERK-1, p-MEK, ERK-2, p-ERK, NF-κB/p-50 and NF-κB/p-65 were carried out in 20 samples of normal prostate glands, 35 samples of BPH, 27 samples with a diagnosis of PIN (low-grade PIN or high-grade PIN), and 95 samples of PC (23 with low, 51 with medium and 21 with high Gleason scores). RESULTS Immunoreaction to IL-6, gp-130, ERK-1, ERK-2, p-ERK and NF-κB/p50 was found in the cytoplasm of epithelial cells in normal prostate samples; p-MEK was found in the nucleus of epithelial cells; but not expression to Raf-1, MEK-1 and NF-κB/p65. In BPH, all of these proteins were immunoexpressed, while there was increased immunoexpression of IL-6, gp-130, p-MEK, ERK-1, ERK-2 and NF-κB/p50 (cytoplasm). In PC, immunoexpression of IL-6 and gp-130 were similar to that found in BPH; while immunoexpression of Raf-1, MEK-1, p-MEK, ERK-1, ERK-2, p-ERK, NF-κB/p50 (nucleus and cytoplasm), and NF-κB/p65 (nucleus and cytoplasm) was higher than in BPH. CONCLUSION Translocation of NF-κB to the nucleus in PC and high-grade PIN could be stimulated by the IL-6/ERK transduction pathway, but might also be stimulated by other transduction pathways, such as TNF-α/NIK, TNF/p38, IL-1/NIK or IL-1/p38. Activation of NF-κB in PC could regulate IL-6 expression. These transduction pathways are also related to activation of other transcription factors such as Elk-1, ATF-2 or c-myc (also involved in cell proliferation and survival). PC is a heterogeneous disease, where multiple transduction pathways might alter the apoptosis/proliferation balance. Significant attention should be give to the combination of novel agents directed towards inactivation of pro-inflammatory cytokines than can disrupt tumour cell growth.

Identification of N- and O-linked Oligosaccharides in the Human Epididymis

The oligosaccharide sequences of glycoconjugates in the human normal epididymis and the nature of linkages were studied with lectin histochemistry. The usual terminal sequences of oligosaccharide side chains in epithelial cell secretions were Neu5Ac2,3Galβ 1,3GalNAc; SO4 Galβ1,3GalNAc; and Galβ1,4GlcNAc, and they were mainly found in O-linked glycoproteins. The lectin pattern of mitochondria-rich cells differed from that of principal cells.

Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors☆☆☆

Caspases are essential initiators and executioners of apoptosis. Changes in their expression may contribute to the development of proliferative disorders such as cancer, by altering the death-proliferation homeostatic balance. The aim of this work was to analyze the expression of a broad panel of caspases at the epithelial level in human prostate tissues to assess possible prostatic disease-related alterations. We comparatively analyzed by immunohistochemistry the expression of pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved caspase-3, cleaved caspase-8, and caspase-7, in normal and pathologic (benign hyperplasic, premalignant [high-grade intraepithelial neoplasia], and cancerous [prostate cancer]) human prostate epithelium. Expression of caspases was correlated with clinicopathologic features, including preoperative prostate-specific antigen levels, Gleason scores, and biochemical progression. Percentage of positive samples for all the analyzed caspases decreased in prostate cancer versus normal prostate epithelium. The values obtained for benign prostatic hyperplasia and high-grade intraepithelial neoplasia more qualitatively resembled those of the prostate cancer group. Our results indicate that caspase expression in prostate malignant cells is reduced in a substantial number of patients and that such an alteration occurs in the premalignant stage. Loss of caspase expression could constitute a useful marker for prostate cancer diagnosis. Therapeutic approaches aimed to recover or enhance caspase expression might be effective against prostate cancer.

Immunocytochemical and quantitative study of the tunica albuginea testis in young and ageing men

Abstract A light and electron microscope immunohistochemical study of the tunica albuginea from both young and elderly men was carried out to determine the distribution of the cells that contain actin, vimentin and/or desmin, and to evaluate the possible variations with ageing by means of quantitative studies. Testicular volume and testicular parenchyma volume decreased significantly with age whereas the tunica albuginea volume remained unchanged. These results agree with the scanty quantitative changes observed in the testicular connective tissue with age, and the notion that age-related changes in testicular volume are principally restricted to the seminiferous tubules. Three connective tissue layers could be distinguished in the tunica albuginea in both young and elderly men. The middle and inner layers increased in width with age while the width of the outer layer decreased. The average width of the tunica albuginea increased significantly with ageing. The tunica albuginea of young men and elderly men presented two types of fusiform cells: (1) fibroblast-like cells, which immunoreacted to actin and vimentin, but not to desmin; and (2) myoid cells, which immunoreacted to actin, vimentin and desmin. In both young men and elderly men, the total number of desmin-positive cells (myoid cells) was significantly lower than that of fibroblasts. However, the total number of desmin-positive cells was significantly increased in ageing men. In young testes, desmin-positive cells were more abundant in the outer layer of the tunica albuginea, whereas in elderly men these cells predominated in the middle layer. The increased desmin immunoexpression in the tunica albuginea of ageing men contrasts with the decrease in desmin immunoreaction in other myoid cells of the testis, the peritubular myoid cells, but only in seminiferous tubules that showed severe germ cell depletion. This suggests that changes in intermediate filament immunoexpression in peritubular cells are focalised, and thus, under local control, whereas changes in the tunica albuginea cells are generalised and possibly related to factors also affecting the connective tissue in other organs