Abstract. Tumor-specific deregulated expression of claudins, integral membrane proteins found in tight junctions (TJs), has indicated a possible role for TJ disruption in cancer progression. The current study demonstrates the marked overexpression of claudin-3 protein in two breast cancer cell lines of metastatic origin (MCF-7 and MDA-MB-415). Immunofluorescence and differential detergent fractionation analyses revealed that, although claudin-3 was primarily localized at cell junctions, it was also detected intracellularly. Similarly, the siRNA-mediated suppression of claudin-3 did not considerably affect its pattern of subcellular distribution relative to mock-transfected cells. However, there appeared to be a preferential loss of claudin-3 signal in the cytoskeletal fraction. Wound-healing assays were conducted to assess the effect of endogenous overexpression versus siRNA-mediated suppression of claudin-3 on cellular motility in MCF-7 cells. Suppression of claudin-3 protein levels resulted in a marked decrease in the rate of cellular motility relative to mock-transfected cells. These findings suggest that overexpression of claudin-3 may be important in disrupting TJ integrity and thus contribute to enhanced cellular motility, a key component of tumor progression. IntroductionThe incidence of breast cancer continues to rise worldwide, particularly in the USA where approximately one in eight females will be diagnosed with breast cancer during her lifetime (1). While early stage breast cancer is readily treatable, patients with metastatic disease pose a greater therapeutic challenge and account for the majority of breast cancer-related mortalities. This is largely due to the paucity of knowledge with regard to the underlying molecular mechanisms associated with malignant progression.Regulated cellular proliferation and differentiation are dependent upon functional tight junctions (TJs), and the loss of TJ integrity may be important in cancer development and progression. Located immediately beneath the apical surface of adjacent endothelial and epithelial cells, TJs form an effective barrier to the diffusion of solutes through the paracellular pathway and exhibit ion-selective permeability in a cell type-dependent manner. In association with adherens junctions, TJs have been shown to establish and maintain epithelial cell polarity by preventing the diffusion of membrane proteins and lipids between the apical and basolateral regions of the plasma membrane. Recent studies also suggest that the TJ plaque proteins, located on the cytoplasmic side of the TJ, are important for the integration of signaling molecules that regulate processes including gene transcription, cellular proliferation, differentiation and morphogenesis. This was reviewed by Turksen and Troy (2).Claudins, the predominant integral membrane proteins that form the backbone of TJs, are required for the assembly, barrier and pore functions of vertebrate TJs (2). Deregulated expression of various claudin proteins has been reported in breast cancer; over...
Although testosterone (T) is essential for the normal completion of spermatogenesis, the exact T-sensitive control points are still unknown. Using staged tissues (premeiotic, PrM; meiotic, M; and postmeiotic, PoM) from zonal testes of the spiny dogfish Squalus acanthias, and standard [3H] steroid binding analysis, we characterized a T-binding component with physiochemical characteristics resembling classical androgen receptors (AR). [3H]T binding was of high affinity (dissociation constant = 4.4 x 10(-9) M), limited capacity (maximum binding, 94 fmol/g tissue) and relatively stable (t1/2 = 4 h at 4 C). The T-binding component was present in both cytosolic and nuclear extracts, adhered to DNA-cellulose, and displayed predicted sedimentation properties of an activated receptor in vivo or in vitro (5.06S). T, 5 alpha-dihydrotestosterone (DHT), and mibolerone (Mib), but not methyltrienelone (R1881), competed well for [3H]T binding; however, progesterone (P) was equivalent to T in its ability to displace tracer. Subsequent analysis of [3H] P binding revealed a P-binding component that was present in nuclear and cytosolic extracts, adhered to DNA, but differed from AR in its inability to bind Mib. Competition studies in which excess radioinert Mib was used to block AR revealed ligand specificity characteristics of progesterone receptors (PR): promegestone (R5020) greater than P greater than deoxycorticosterone, but T, DHT, dexamethasone, and corticosterone were ineffective competitors. Also, a nonlinear Scatchard plot was obtained, suggesting two P-binding activities, which differed in their binding affinities (dissociation constant = 0.88 vs. 5.9 x 10(-9) M) and capacities (66 vs. 210 fmol/g tissue). Conversely, using [3H]Mib to avoid interference from PR, we confirmed that T, DHT, and P were equivalent in their ability to displace ligand from AR. Comparison of tissues by stage of spermatogenesis revealed different distribution patterns for AR (PrM greater than M much greater than PoM) vs. PR (PoM much greater than M = PrM). These data provide definitive evidence for separate testicular T- and P-binding mechanisms and indicate the presence of temporally distinct sets of steroid-regulated genes.
Abstract. Endometrial cancer is the most common female reproductive cancer in the United States and is associated with deregulated tight junction protein expression. Given the highly estrogen-responsive nature of this tissue, we investigated the effects of estrogen and its agonist, 4-OH TAM, on the expression and subcellular localization of the tight junction protein claudin-4 (CLDN-4), in HEC-1A endometrial cancer cells. In untreated HEC-1A cells, we observed dramatic overexpression of claudin-4 protein. In addition, differential detergent extraction analysis indicated that claudin-4 was localized primarily in the membrane but also found in the cytosolic, nuclear and cytoskeletal fractions. Upon exposure of HEC-1A to estradiol (E 2 ), we observed a biphasic effect both on the overall expression of claudin-4 protein and on its cytosolic and cytoskeletal presence as demonstrated by immunoblot analysis. Immunofluorescence analysis also revealed a biphasic effect of E 2 on claudin-4 expression. In contrast, we observed no changes in expression levels nor in the subcellular distribution patterns of claudin-4 in HEC-1A cells treated with different concentrations of 4-OH TAM. The intracellular presence of CLDN-4 coupled with the biphasic effects of E 2 on CLDN-4 expression in the cytoskeleton suggest that this protein may be involved in cell signaling to and from TJs.
Spermatogenesis is a unique developmental sequence dependent on FSH and androgen. Due to the complex organization of the mammalian testis, however, mechanistic details of regulation are largely unknown. Using the dogfish shark (Squalus acanthias) in which there is a cystic mode of spermatogenesis and a topographic separation of different germ cell stages within the testis, we have obtained new information of general relevance on stage-related biochemical and morphological changes and have proposed a model in which steroids serve as parahormonal regulators of the spermatogenic progression. In addition, techniques developed for culturing staged spermatocysts (intact Sertoliigerm cell units) and isolated, staged Sertoli cells demonstrate the usefulness of this model for studying spermatogenic regulation under defined conditions in vitro.
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