Contradistinctive growth responses of cultured rat intestinal epithelial cells to epidermal growth factor depending on cell population density

The growth response of cultured rat intestinal epithelial (RIE-1) cells to epidermal growth factor (EGF) depends on the cell population density. EGF stimulated the proliferation of RIE-1 cells in dense cultures, but inhibited the proliferation of cells growing at low population densities. In contrast, insulin enhanced RIE-1 cell growth irrespective of the population density. The tumour promoter 12-0-tetradecanoylphorbol 13-acetate (TPA), like EGF, inhibited the proliferation of low-density RIE-1 cells, but differed from EGF in that it did not stimulate the growth of dense cultures.     

睾丸酮对大鼠前列腺上皮细胞有丝分裂方向的影响

目的探讨丙酸睾丸酮（T）对大鼠前列腺上皮细胞染色体有丝分裂方向的影响及其差异基因表达。方法 SPF级SD雄性大鼠（110~130 g）20只,随机分为2组。深麻醉下对所有大鼠进行去势手术,恢复一周后,给药组大鼠皮下注射T,每只0.5 mg,每日1次,连续30d;对照组大鼠皮下注射橄榄油0.1 mL。取前列腺。通过免疫组化、HE染色观察结果。并做基因芯片检查差异基因表达谱。结果给药组大鼠前列腺发生形态学增生。前列腺腺腔扩张,腺上皮高度明显增高,前列腺增生。且前列腺上皮细胞染色体有丝分裂的方向与基底膜平行,而对照组前列腺上皮细胞有丝分裂的方向与基底膜垂直。AR免疫组化染色后发现给药组的前列腺上皮中,均可见到AR标记的阳性细胞,而对照组均为阴性。基因芯片和RT-PCR结果：促进细胞增殖的基因如雄激素受体相关蛋白（RAN）、TGM4和Wnt通道的WNT2等基因均上调,而抑制细胞增殖的基因如负调控Wnt通道的DKK3和促进细胞凋亡的Fas等基因下调。结论 T注射后改变了前列腺上皮细胞有丝分裂的方向,Wnt和AR信号转导通路参与了细胞增殖和有丝分裂方向改变。     

Influence of castration and testosterone treatment on the vasoactive intestinal peptide receptor/effector system in rat prostatic epithelial cells

The interaction of vasoactive intestinal peptide (VIP) with prostatic epithelial cells was studied after castration and testosterone replacement in pubertal and mature rats. The number of VIP receptors (but not the affinity) decreased 2 days after castration and returned to normal when subsequently treated with testosterone for 4 days. However, the stimulatory effect of VIP upon cyclic AMP accumulation was unaffected by the androgen withdrawal elicited by the surgical procedure. The results suggest the importance of androgens in the biosynthesis of VIP receptors and also in their coupling to adenylate cyclase by affecting the membrane fluidity.     

Beta-carotene uptake and metabolism in human lung bronchial epithelial cultured cells depending on delivery vehicle

Beta-carotene (BC) could have a protective or pro-carcinogenic role in lung cancer, and cell culture systems are important to evaluate it. Nevertheless, the delivery of the hydrophobic BC to cells is difficult. Different vehicles have been used such as liposomes, tetrahydrofuran and serum lipoproteins, but presenting different problems. Water dispersible beadlets containing BC are a good choice and can produce the greatest BC uptake when compared to the above vehicles, but other beadlet components could alter the results. Dimethylsulfoxide (DMSO) could be a good alternative since it has low toxicity and it enhances the penetration of substances across biologic membranes. We aimed to characterize an appropriate model for delivering all-trans-BC to lung cells in culture and knowing its metabolism. All-trans-BC 5 microM was administered to BEAS-2B cells in beadlets or DMSO, and medium and cell samples were taken at different times. The levels of BC and its main isomers and metabolites were determined by HPLC. All-trans-BC reached the same levels in the medium (about 3.5 microM) either when supplied in beadlets or in DMSO, and, with beadlets, 13-cis-BC was also detected. However the amount of all-trans-BC taken up by the cells was the triple when delivered by DMSO. With both vehicles, intracellular all-trans-BC levels reached its maximum after 24 h of treatment, remaining equal after 72 h. The 9-cis and 13-cis isomers of BC, and oxidized metabolites, were also detected in the cells although in smaller proportion than all-trans-BC, especially with DMSO. An LDH assay did not suggest toxicity of beadlets, DMSO or BC itself. In conclusion, DMSO seems the most appropriate vehicle for delivering BC to lung cells in vitro, and we present a model that allows studying the effects of BC and its metabolism in the lung human BEAS-2B cell line.     

Metabolism of testosterone and dihydrotestosterone in cultured rat hepatoma cells.

Steroid metabolism in hepatoma tissue culture (HTC) cells derived from a male rat was investigated. Steroids in ethanol were incubated with the cells for various lengths of time. Volume of ethanol never exceeded 1% of incubation volume. Thin-layer and paper chromatography were used. Incubation was with tritiated steroids. It was demonstrated that testosterone as well as dihydrotestosterone is transformed. The main enzyme activities detected were 5alpha-reduction and 3alpha-, 3beta, and 17beta-hydroxysteroid dehydrogenation. The pattern of metabolism was reproducible and varied with time, substrate concentration, and number of cells incubated. Some steroids interfered with androgen metabolism. 17beta-estradiol, 17-epitestosterone, and progesterone competed for the 17beta-hydroxyprogesterone dehydrogenase. it is concluded that 3beta and 17beta reduction in the HTC cells may be catalyzed by the same enzyme which might differ considerably from the 3beta-hydroxysteroid dehydrogenase assayed in intact liver cells. A hepatoma derived from a female rat also produced considerable amounts of 3beta-derivatives of testosterone.     

Characterization of cultured human prostatic epithelial cells by cluster designation antigen expression

Cultured prostatic epithelial cells have been extensively studied as a model of prostate biology. What is the lineage relationship of the cultured cells to the epithelial cell types in tissue? How different are cultured cells derived from tumor tissue to those derived from benign tissue? Expression of cluster designation (CD) cell surface molecules has been shown to be useful in characterizing cells according to lineage. A CD profile was therefore generated for cultured human prostatic epithelial cells and compared with those previously established for basal and luminal epithelial cells in the prostate. Presence of CD44, CD49b, CD49f, and CD104 and absence of CD57 suggests that cultured cells were derived from basal cells of prostatic tissues. However, expression of certain CD antigens characteristic of luminal epithelial cells was also observed in subpopulations of cultured cells. The pattern of CD antigens in cultured cells reflects a phenotype similar to that of transit-amplifying cells that have been described in the prostate. Several CD antigens were found expressed by both cultured prostatic epithelial and stromal cells, and are probably associated with cell proliferation. The CD profiles of cultured epithelial cell strains derived from normal compared with malignant tissues were notably similar to each other and to that of the prostate cancer cell line PC-3. We conclude that cells in culture retain expression of certain lineage-characteristic CD antigens. Furthermore, CD antigens can define subpopulations of cells with differential gene expression.     

Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues

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Properties of epithelial cells cultured from human carcinomas and nonmalignant tissues

Human epithelial cell cultures were examined for expression of plasminogen activator and fibronectin matrix. All of the cells examined showed ultrastructural evidence suggesting their epithelial origin, including microvilli and specialized junctions. The nonmalignant cells were also negative for endothelial cell markers (ie, they lacked factor VIII antigen, a nonthrombogenic surface and Weibel-Palade bodies). The nonmalignant lines all produced large amounts of plasminogen activator, whereas the tumor-derived lines showed a gradation of activities, ranging from lines having as much activity as the nonmalignant lines to lines having little or no activity above background. For both normal and malignant cells, addition of dexamethesone only slightly decreased the levels of plasminogen activator. By immunofluorescence microscopy, normal bladder and fetal intestine epithelial cells showed fibronectin in a globular and fibrillar matrix. In contrast, normal mammary epithelial cells had a much diminished amount of fibronectin with a punctate distribution.     

Establishment of a SV40-transformed cell line from primary culture of rat dorsolateral prostatic epithelial cells

A permanent cell line of the rat dorsolateral prostate epithelium was established after transformation of primary cultured cells with simian virus 40 (SV40). The established cells had SV40 T-antigen in their nuclei but lacked such typical characteristics of transformed cells as piled-up growth. They grew in a monolayer with an epithelial morphology, were stained with antikeratin antisera, and also retained an ability to form dome-like structure at a confluent state.     

Uptake of testosterone by tissues of human fetal prostatic rudiments

The common and specific uptake of 3H-testosterone (3H-T) by tissues of urogenital sinus (UGS) and bladder (BL) in human 10-12 weeks fetuses was studied. The values of common and specific 3H-T uptake in UGS were significantly higher than those in BL. A high specific uptake of labeled hormone was also detected in UGS mesenchyme separated from epithelium. The authors did not reveal any sexual dimorphism of 3H-T uptake by UGS tissues.     

Effect of nephritogenic antibody on complement regulation in cultured rat glomerular epithelial cells

In passive Heymann nephritis, a rat model of membranous nephropathy, antibody (anti-Fx1A) activates C on the surface of the glomerular epithelial cell (GEC), leading to GEC injury and proteinuria. In this study, we examined C activation by anti-Fx1A in cultured rat GEC. In addition to anti-Fx1A IgG, anti-Fx1A F(ab')2 and Fab' led to GEC injury in the presence of rat or human sera as sources of C. Cytotoxicity was Mg2+ and factor B dependent, but Ca2+ independent, indicating that anti-Fx1A activated the C alternative pathway (AP). Furthermore, in the presence of Mg2+ and factor B, anti-Fx1A enhanced 125I-C3b deposition on GEC in the absence of classical pathway activation. AP C3 and C5 convertases formed on GEC (GEC-C3bBbP) were inactivated over time, probably due to binding of GEC C regulatory proteins. This inactivation was prevented when GEC-C3bBbP were incubated with anti-Fx1A IgG. An antibody raised against cultured GEC that binds to GEC in vitro and in vivo had no effect on C3 and C5 convertases, suggesting that stabilization of C3bBbP is unique to anti-Fx1A. Anti-Fx1A Fab' also stabilized GEC-C3bBbP, indicating that cross-linking of membrane Ag was not required. C3bBbP on E were not affected by anti-Fx1A, excluding direct stabilization of convertases by anti-Fx1A. Therefore, anti-Fx1A inhibits C regulation on GEC, which can account for its ability to activate the AP. This represents a potentially powerful mechanism of producing disease in vivo.     

Shiga toxin-2 enhances heat-shock-induced apoptotic cell death in cultured and primary glial cells

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.     

5'-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells

Cell cultures of adult rat liver produced two distinct morphologic cell types: epithelial cells polygonal in shape and growing in nests of closely apposed cells, and fibroblast cells stellate in shape with little cell-cell contact at low density growth, but aligning in parallel arrays at high density. These two morphologic variants displayed dramatic differences in histochemically demonstrable 5'-nucleotidase activities. Fibroblast cells exhibited great activity throughout the cytoplasm with no concentration of activity in the cell membrane. The lesser activity in epithelial cells was concentrated on the cell membrane. The importance of this finding to the interpretation of data derived from experiments with whole liver homogenates is discussed.     

A proteome map of primary cultured rat Schwann cells

Background

Schwann cells (SCs) are the principal glial cells of the peripheral nervous system with a wide range of biological functions. SCs play a key role in peripheral nerve regeneration and are involved in several hereditary peripheral neuropathies. The objective of this study was to gain new insight into the whole protein composition of SCs.

Results

Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC-MS/MS) was performed to identify the protein expressions in primary cultured SCs of rats. We identified a total of 1,232 proteins, which were categorized into 20 functional classes. We also used quantitative real time RT-PCR and Western blot analysis to validate some of proteomics-identified proteins.

Conclusion

We showed for the first time the proteome map of SCs. Our data could serve as a reference library to provide basic information for understanding SC biology.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat

The paper presents the steroidogenic features of cultured epithelial cells of rat epididymis and their ability to synthesize steroid hormones. The cytoplasm of epididymal epithelial cells accumulated lipid droplets and contained active enzymes of steroidogenesis. Numerous mitochondria with lamellar cristae occurred near lipid droplets. Frequently, mitochondria formed a direct contact with lipid droplets and smooth endoplasmic reticulum. The hormone assay showed that the epididymal epithelial cells cultured without dihydrotestosterone synthesized and released the following steroids: dehydroepiandrosterone (DHEA), testosterone (T) and 17beta-estradiol (E). The levels of DHEA and T were very low. The concentration of E detected in media of cultured epididymal epithelial cells exceeded many times the concentration of E in control media. The cytoplasmic presence of organelles and enzymes that participated in the steroid synthesis indicated their similarity to steroidogenic cells. Epididymal epithelial cells were capable of moderate in vitro synthesis of androgens. It cannot be excluded that steroidogenesis in the cultured epididymal epithelial cells is maintained to sustain 17beta-estradiol synthesis pathways.     

Autoradiographic investigation of estrogen binding in cultured rat ovarian surface epithelial cells

Autoradiographic evidence presented that the cultured rat ovarian surface epithelium exhibits estrogen receptor-like activity. Two autoradiographic techniques were used; one involved live cells that were labeled and freeze-dried, and the other the labeling of ethanol-fixed cells. Autoradiograms were prepared by dipping cells grown on plastic cover slips in liquid nuclear track emulsion. Exposure times were 1 to 4 weeks. Experiments using a pulse-chase technique in live cells and steroid competition tests in fixed and live cells gave evidence for translocation of tritiated estradiol from cytoplasm to nucleus in live cells and for a component of estrogen-specific binding in live and fixed cells. The techniques presented here for the investigation of estrogen receptors in cultured cells require little tissue, are simple, and relatively quick. Reports based on biochemical analyses of tissue homogenates claim the presence of estrogen receptors in human ovarian carcinomas of surface epithelial origin and in rat ovarian surface epithelium. The results of this study add further evidence that the ovarian surface epithelium has estrogen receptor activity and should be considered an estrogen target tissue.