81 research outputs found
Metallothionein isoform 3 and proximal tubule vectorial active transport
Metallothionein isoform 3 and proximal tubule vectorial active transport.BackgroundMetallothionein isoform 3 (MT-3) is expressed in the proximal tubule cells of the human kidney. The goal of the present study was to further characterize the basal expression of MT-3 in the proximal tubule and to determine if MT-3 participates in the maintenance of proximal tubule cell function.MethodsExpression of MT-3 mRNA was determined in the intact proximal tubule using microdissection and reverse transcription-polymerase chain reaction (RT-PCR). Basal expression of MT-3 mRNA and protein was determined in cultured human proximal tubule (HPT) cells and an immortalized proximal tubular cell line, HK-2 cells, using RT-PCR and immunoblotting. The MT-3 gene was stably transfected into the HK-2 cell line using the pcDNA3.1/Hygro (+) vector.ResultsMT-3 mRNA was detected in the proximal tubule of the in situ kidney with relative expression in excess to that of the β-actin housekeeping gene. The mortal HPT cells were shown to express both MT-3 mRNA and protein and to form domes, while immortal HK-2 cells were shown to have no expression of MT-3 mRNA and protein nor to form domes. The stable transfection of MT-3 in HK-2 restored MT-3 expression and dome formation to the HK-2 cells.ConclusionsMT-3 mRNA is present in the human proximal tubule, and MT-3 expression is involved in the transport function of a human renal cell line that retains properties of the proximal tubule
Metallothionein Isoform 3 Expression in Human Skin, Related Cancers, and Human Skin Derived Cell Cultures
Human skin is a well known target site of inorganic arsenic with effects ranging from hyperkeratosis to dermal malignancies. The current study characterizes the expression of a protein known to bind inorganic, As3+, metallothionein 3 (MT-3). Expression of this protein was assessed immunohistochemically with a specific MT-3 antibody on human formalin-fixed, paraffin-embedded biopsy specimens in normal skin, squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Assessment in normal skin using nine normal specimens showed moderate to intense MT-3 staining in epidermal karatinocytes with staining extending into the basal cells and moderate to intense staining in melanocytes of nevi. MT-3 immunoexpression was shown to be moderate to intense in 12 of 13 of SCC, low to moderate in 8 of 10 BCC, and moderate to intense in 12 melanoma samples. MT-3 expression in cell culture models (normal human epidermal keratinocytes, normal human melanocytes, and HaCaT cells) showed only trace expression of MT-3, while exposures to the histone deacytalase inhibitor, MS-275, partially restored expression levels. These results indicate that the epidermis of human skin and resulting malignancies express high level of MT-3 and potentially impact on the known association of arsenic exposure and the development of skin disorders and related cancers
Expression of hsp 27, hsp 60, hsc 70, and hsp 70 stress response genes in cultured human urothelial cells (UROtsa) exposed to lethal and sublethal concentrations of sodium arsenite.
The stress response is one mechanism that the bladder urothelium could potentially employ to protect itself from cellular damage after exposure to arsenic and, in so doing, influence the shape of the dose-response curve at low concentrations of exposure to this environmental pollutant. In the present study, we used the cultured human urothelial cell line UROtsa, a model of human urothelium, to determine the expression of heat shock proteins hsp 27, hsp 60, hsc 70, and hsp 70 after acute and extended exposure of the cells to lethal and sublethal levels of sodium arsenite (NaAsO2). Acute exposure was modeled by exposing confluent cultures of UROtsa cells to 100 micro M NaAsO2 for 4 hr followed by a 48-hr recovery period. Extended exposure was modeled by exposing confluent UROtsa cells to 1, 4, and 8 micro M NaAsO2 for 16 days, with the highest concentration producing cell death by 4 days of exposure. The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined by reverse-transcription polymerase chain reaction and Western analysis. Cell viability was determined by the MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The results demonstrated that the expression of hsp 27, hsp 60, and hsc 70 mRNA and protein were not consistently increased by either acute or extended exposure to NaAsO2. In contrast, hsp 70 expression was induced by NaAsO2 after both acute and extended exposure. The degree and duration of the induction of the hsp 70 protein in the extended time course of exposure to NaAsO2 correlated directly with UROtsa cell cytotoxicity. The substantial level of basal expression of hsp 27, hsp 60, and hsc 70 shown previously in human bladder urothelium, coupled with the inducible expression of hsp 70, could provide the human urothelium with a mechanism to withstand and recover from a low level of arsenite exposure
Конфликт культур в повести Н. В. Гоголя «Вий»
В повести Н.В. Гоголя «Вий» рассмотрена утрата нравственных начал жизни и цельности сознания личности, обусловленная конфликтом культур, который проявляется на разных уровнях. В результате столкновения язычества и православия произошла деформация обеих традиционных моделей мира. Это проявляется и в именах бурсаков, и в поведении семинаристов и казаков на хуторе сотника, и в миропонимании и веросознании главного героя повести. Все это делает гибель Хомы Брута неизбежной
The Urothelial Cell Line UROtsa Transformed by Arsenite and Cadmium Display Basal Characteristics Associated with Muscle Invasive Urothelial Cancers
Muscle invasive urothelial carcinomas are divided into various molecular subtypes with basal and luminal subtypes being the prominent ones. The basal muscle-invasive urothelial carcinomas are generally more aggressive at presentation and significantly enriched with squamous features. Our laboratory has developed an in-vitro model of urothelial cancer by transforming the immortalized cell line UROtsa with arsenite (As3+) and cadmium (Cd2+). In this study, we characterized the tumors formed by these transformed cell lines as more basal-like based on their gene expression patterns with increased expression of KRT1, KRT5, KRT6, KRT14, KRT16, KRT17 and CD44. In addition, histological examination of these tumor transplants showed squamous features enriched in basal muscle invasive urothelial carcinomas. The expression of these genes increased in the transformed cell lines as well as in the urospheres, which are putative cancer initiating cells/stem cells derived from the cell lines. There was also increased expression of these genes in the urospheres derived from the parent UROtsa cell line. Thus, our data shows that the As3+ and Cd2+-transformed cell lines and their derived tumor transplants have gene expression profiles similar to the basal subtype of muscle invasive bladder carcinomas with tumors having enriched squamous features. The increased expression of basal markers in the urospheres suggests that stem cells may be involved in the development of squamous differentiation seen in some of the muscle invasive bladder carcinomas
Short and long term gene expression variation and networking in human proximal tubule cells when exposed to cadmium
Transformation of Human Urothelial Cells (UROtsa) by As3+ and Cd2+ Induces the Expression of Keratin 6a
Zinc, Zinc Transporters, and Cadmium Cytotoxicity in a Cell Culture Model of Human Urothelium
We explored the potential role of zinc (Zn) and zinc transporters in protection against cytotoxicity of cadmium (Cd) in a cell culture model of human urothelium, named UROtsa. We used real-time qRT-PCR to quantify transcript levels of 19 Zn transporters of the Zrt-/Irt-like protein (ZIP) and ZnT gene families that were expressed in UROtsa cells and were altered by Cd exposure. Cd as low as 0.1 µM induced expression of ZnT1, known to mediate efflux of Zn and Cd. Loss of cell viability by 57% was seen 24 h after exposure to 2.5 µM Cd. Exposure to 2.5 µM Cd together with 10–50 µM Zn prevented loss of cell viability by 66%. Pretreatment of the UROtsa cells with an inhibitor of glutathione biosynthesis (buthionine sulfoximine) diminished ZnT1 induction by Cd with a resultant increase in sensitivity to Cd cytotoxicity. Conversely, pretreatment of UROtsa cells with an inhibitor of DNA methylation, 5-aza-2’-deoxycytidine (aza-dC) did not change the extent of ZnT1 induction by Cd. The induced expression of ZnT1 that remained impervious in cells treated with aza-dC coincided with resistance to Cd cytotoxicity. Therefore, expression of ZnT1 efflux transporter and Cd toxicity in UROtsa cells could be modulated, in part, by DNA methylation and glutathione biosynthesis. Induced expression of ZnT1 may be a viable mechanistic approach to mitigating cytotoxicity of Cd
- …