The Effect of Retinoic Acid on Arsenite-Transformed Malignant UROtsa Bladder Cancer Cells: In Vitro Model of Basal Muscle-Invasive Bladder Cancer.

Bladder cancer (BC) is the eighth most common cause of cancer death in the United States of America. BC is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Genetically, MIBCs are categorized into the more aggressive basal subtype or less aggressive luminal subtype. All-trans retinoic acid (tretinoin), the ligand for the RAR-RXR retinoic acid receptor, is clinically used as a differentiation therapy in hematological malignancies. This study aims to determine the effects of retinoic acid on arsenite-transformed malignant urothelial cells (UROtsa As), serving as a model for basal muscle-invasive bladder cancer. We treated three independent isolates of arsenite-transformed malignant human urothelial UROtsa cells (UROtsa As) with tretinoin for 48 h. Cell viability, proliferation, and apoptosis were analyzed using crystal violet staining and flow cytometry. mRNA and protein level analyses were performed using RT-qPCR and the Simple Western™ platform, respectively. Tretinoin was found to reduce cell proliferation and urosphere formation, as well as decrease the expression of basal markers (KRT1, KRT5, KRT6, EGFR) and increase the expression of luminal differentiation markers (GATA3, FOXA1). Mechanistically, the antiproliferative effect of tretinoin was attributed to the downregulation of c-myc. Our results suggest that targeting the retinoic acid pathway can diminish the aggressive behavior of basal muscle-invasive urothelial cancer and may enhance patient survival.

Proteasomes Are Critical for Maintenance of CD133+CD24+ Kidney Progenitor Cells.

Kidney progenitor cells, although rare and dispersed, play a key role in the repair of renal tubules after acute kidney damage. However, understanding these cells has been challenging due to the limited access to primary renal tissues and the absence of immortalized cells to model kidney progenitors. Previously, our laboratory utilized the renal proximal tubular epithelial cell line, RPTEC/TERT1, and the flow cytometry technique to sort and establish a kidney progenitor cell model called Human Renal Tubular Precursor TERT (HRTPT) which expresses CD133 and CD24 and exhibits the characteristics of kidney progenitors, such as self-renewal capacity and multi-potential differentiation. In addition, a separate cell line was established, named Human Renal Epithelial Cell 24 TERT (HREC24T), which lacks CD133 expression and shows no progenitor features. To further characterize HRTPT CD133+CD24+ progenitor cells, we performed proteomic profiling which showed high proteasomal expression in HRTPT kidney progenitor cells. RT-qPCR, Western blot, and flow cytometry analysis showed that HRTPT cells possess higher proteasomal expression and activity compared to HREC24T non-progenitor cells. Importantly, inhibition of the proteasomes with bortezomib reduced the expression of progenitor markers and obliterated the potential for self-renewal and differentiation of HRTPT progenitor cells. In conclusion, proteasomes are critical in preserving progenitor markers expression and self-renewal capacity in HRTPT kidney progenitors.

Pevonedistat Inhibits SOX2 Expression and Sphere Formation but Also Drives the Induction of Terminal Differentiation Markers and Apoptosis within Arsenite-Transformed Urothelial Cells.

Urothelial cancer (UC) is a common malignancy and its development is associated with arsenic exposure. Around 25% of diagnosed UC cases are muscle invasive (MIUC) and are frequently associated with squamous differentiation. These patients commonly develop cisplatin (CIS) resistance and have poor prognosis. SOX2 expression is correlated to reduced overall and disease-free survival in UC. SOX2 drives malignant stemness and proliferation in UC cells and is associated with development of CIS resistance. Using quantitative proteomics, we identified that SOX2 was overexpressed in three arsenite (As3+)-transformed UROtsa cell lines. We hypothesized that inhibition of SOX2 would reduce stemness and increase sensitivity to CIS in the As3+-transformed cells. Pevonedistat (PVD) is a neddylation inhibitor and is a potent inhibitor of SOX2. We treated non-transformed parent and As3+-transformed cells with PVD, CIS, or in combination and monitored cell growth, sphere forming abilities, apoptosis, and gene/protein expression. PVD treatment alone caused morphological changes, reduced cell growth, attenuated sphere formation, induced apoptosis, and elevated the expression of terminal differentiation markers. However, the combined treatment of PVD with CIS significantly elevated the expression of terminal differentiation markers and eventually led to more cell death than either solo treatment. Aside from a reduced proliferation rate, these effects were not seen in the parent. Further research is needed to explore the potential use of PVD with CIS as a differentiation therapy or alternative treatment for MIUC tumors that may have become resistant to CIS.

Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results.

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

Arsenite Exposure to Human RPCs (HRTPT) Produces a Reversible Epithelial Mesenchymal Transition (EMT): In-Vitro and In-Silico Study.

The human kidney is known to possess renal progenitor cells (RPCs) that can assist in the repair of acute tubular injury. The RPCs are sparsely located as single cells throughout the kidney. We recently generated an immortalized human renal progenitor cell line (HRTPT) that co-expresses PROM1/CD24 and expresses features expected on RPCs. This included the ability to form nephrospheres, differentiate on the surface of Matrigel, and undergo adipogenic, neurogenic, and osteogenic differentiation. These cells were used in the present study to determine how the cells would respond when exposed to nephrotoxin. Inorganic arsenite (iAs) was chosen as the nephrotoxin since the kidney is susceptible to this toxin and there is evidence of its involvement in renal disease. Gene expression profiles when the cells were exposed to iAs for 3, 8, and 10 passages (subcultured at 1:3 ratio) identified a shift from the control unexposed cells. The cells exposed to iAs for eight passages were then referred with growth media containing no iAs and within two passages the cells returned to an epithelial morphology with strong agreement in differential gene expression between control and cells recovered from iAs exposure. Results show within three serial passages of the cells exposed to iAs there was a shift in morphology from an epithelial to a mesenchymal phenotype. EMT was suggested based on an increase in known mesenchymal markers. We found RPCs can undergo EMT when exposed to a nephrotoxin and undergo MET when the agent is removed from the growth media.

Contamination Assessment and Potential Human Health Risks of Heavy Metals in Urban Soils from Grand Forks, North Dakota, USA.

Heavy metal (HM) pollution of soil is an increasingly serious problem worldwide. The current study assessed the metal levels and ecological and human health risk associated with HMs in Grand Forks urban soils. A total 40 composite surface soil samples were investigated for Mn, Fe, Co, Ni, Cu, Zn, As, Pb, Hg, Cr, Cd and Tl using microwave-assisted HNO3-HCl acid digestion and inductively coupled plasma mass spectrometry (ICP-MS) analysis. The enrichment factor (EF), contamination factor (CF), geoaccumulation index (Igeo), ecological risk and potential ecological risk index were used for ecological risk assessment. The park soils revealed the following decreasing trend for metal levels: Fe > Mn > Zn > Cr > Ni > Cu > Pb > As > Co > Cd > Tl > Hg. Based on mean levels, all the studied HMs except As and Cr were lower than guideline limits set by international agencies. Principal component analysis (PCA) indicated that Mn, Fe, Co, Ni, Cu, Zn, As, Cd, Pb, Cr and Tl may originate from natural sources, while Hg, Pb, As and Cd may come from anthropogenic/mixed sources. The Igeo results showed that the soil was moderately polluted by As and Cd and, based on EF results, As and Cd exhibited significant enrichment. The contamination factor analysis revealed that Zn and Pb showed moderate contamination, Hg exhibited low to moderate contamination and As and Cd showed high contamination in the soil. Comparatively higher risk was noted for children over adults and, overall, As was the major contributor (>50%), followed by Cr (>13%), in the non-carcinogenic risk assessment. Carcinogenic risk assessment revealed that As and Cr pose significant risks to the populations associated with this urban soil. Lastly, this study showed that the soil was moderately contaminated by As, Cd, Pb and Hg and should be regularly monitored for metal contamination.

Chronic Arsenic Exposure Upregulates the Expression of Basal Transcriptional Factors and Increases Invasiveness of the Non-Muscle Invasive Papillary Bladder Cancer Line RT4.

The bladder is a target organ for inorganic arsenic, a carcinogen and common environmental contaminant found in soil and water. Urothelial carcinoma (UC) is the most common type of bladder cancer (BC) that develops into papillary or non-papillary tumors. Papillary tumors are mostly non-muscle invasive (NMIUC), easier treated, and have a better prognosis. Urothelial carcinoma can be molecularly sub-typed as luminal or basal, with papillary tumors generally falling into the luminal category and basal tumors exclusively forming muscle invasive urothelial carcinomas (MIUC). It is unclear why some UCs develop more aggressive basal phenotypes. We hypothesized that chronic arsenic exposure of a papillary luminal bladder cancer would lead to the development of basal characteristics and increase in invasiveness. We treated the human papillary bladder cancer cell line RT4 with 1 µM arsenite (As3+) for twenty passages. Throughout the study, key luminal and basal gene/protein markers in the exposed cells were evaluated and at passage twenty, the cells were injected into athymic mice to evaluate tumor histology and measure protein markers using immunohistochemistry. Our data indicates that chronic As3+- treatment altered cellular morphology and decreased several luminal markers in cell culture. The histology of the tumors generated from the As3+-exposed cells was similar to the parent (non-treated) however, they appeared to be more invasive in the liver and displayed elevated levels of some basal markers. Our study demonstrates that chronic As3+ exposure is able to convert a non-invasive papillary bladder cancer to an invasive form that acquires some basal characteristics.

Protein interactions with metallothionein-3 promote vectorial active transport in human proximal tubular cells.

Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, ß-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq.

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal-derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.

Postbaccalaureate terminal degree and career choices of students who performed undergraduate research.

This study analyzed terminal degree and career choices of students who performed undergraduate research. In one analysis, the study compared terminal degree and career choices between a course-based undergraduate research experience (CURE) and traditional non-course-based undergraduate research experiences at one primarily undergraduate institution (PUI). Students who pursued postbaccalaureate programs chose terminal degrees at levels exceeding 75%, with no significant difference between a CURE experience and a traditional research experience. Analysis of terminal degree and career choices at four PUIs providing traditional research experiences showed a marked difference in the number of students pursuing terminal degrees. Two PUIs showed rates > 75%, whereas students at the other two PUIs pursued terminal degrees <50% of the time. The majority of students not pursuing terminal degrees chose M.S. degrees in education and healthcare. An analysis was also performed among students participating in traditional summer undergraduate research on a research-intensive university (RIU) campus with a medical school. Students were accepted from two programs, an NIH IDeA Network of Biomedical Research Excellence (INBRE) program recruiting students from the RIU and an NSF Research Experiences for Undergraduates (REU) program recruiting undergraduates from rural PUIs and minority-serving institutions, particularly tribal colleges. Analysis showed that >70% of the students who pursued postbaccalaureate programs chose terminal degrees. INBRE undergraduates displayed a marked preference for the M.D. degree (73.9% vs. 17.4%), whereas the REU students chose the Ph.D. degree (75.0% vs. 22.9%). American Indian students were also analyzed separately for career choice and showed an equal preference for the M.D. and Ph.D. degrees when pursuing postbaccalaureate education. Overall, the results provide evidence that undergraduate student research stimulates student careers in areas needed by the nation's citizen stakeholders.

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.

Elevated glucose represses lysosomal and mTOR-related genes in renal epithelial cells composed of progenitor CD133+ cells.

Hyperglycemia is one of the major health concern in many parts of the world. One of the serious complications of high glucose levels is diabetic nephropathy. The preliminary microarray study performed on primary human renal tubular epithelial (hRTE) cells exposed to high glucose levels showed a significant downregulation of mTOR as well as its associated genes as well as lysosomal genes. Based on this preliminary data, the expression of various lysosomal genes as well as mTOR and its associated genes were analyzed in hRTE cells exposed to 5.5, 7.5, 11 and 16 mM glucose. The results validated the microarray analysis, which showed a significant decrease in the mRNA as well as protein expression of the selected genes as the concentration of glucose increased. Co-localization of lysosomal marker, LAMP1 with mTOR showed lower expression of mTOR as the glucose concentration increased, suggesting decrease in mTOR activity. Although the mechanism by which glucose affects the regulation of lysosomal genes is not well known, our results suggest that high levels of glucose may lead to decrease in mTOR expression causing the cells to enter an anabolic state with subsequent downregulation of lysosomal genes.

Meta-analysis of gene expression profiling reveals novel basal gene signatures in MCF-10A cells transformed with cadmium.

Cadmium (Cd2+) is an environmental toxicant and a human carcinogen. Several studies show an association of Cd2+ exposure to the development of breast cancer. Previously, we have transformed the immortalized non-tumorigenic cell line MCF-10A with Cd2+ and have demonstrated that the transformed cells have anchorage independent growth. In a separate study, we showed that transformation of the immortalized urothelial cells with the environmental carcinogen arsenite (As3+) results in an increase in expression of genes associated with the basal subtype of bladder cancer. In this study, we determined if transformation of the MCF-10A cells with Cd2+ would have a similar effect on the expression of basal genes. The results of our study indicate that there is a decrease in expression of genes associated with keratinization and cornification and this gene signature includes the genes associated with the basal subtype of breast cancer. An analysis of human breast cancer databases indicates an increased expression of this gene signature is associated with a positive correlation to patient survival whereas a reduced expression/absence of this gene signature is associated with poor patient survival. Thus, our study suggests that transformation of the MCF-10A cells with Cd2+ produces a decreased basal gene expression profile that correlates to patient outcome.

Activation of PPARγ and inhibition of cell proliferation reduces key proteins associated with the basal subtype of bladder cancer in As3+-transformed UROtsa cells.

Environmental exposure to arsenite (As3+) has a strong association with the development of human urothelial cancer (UC) and is the 5th most common cancer in men and the 12th most common cancer in women. Muscle invasive urothelial cancer (MIUC) are grouped into basal or luminal molecular subtypes based on their gene expression profile. The basal subtype is more aggressive and can be associated with squamous differentiation, characterized by high expression of keratins (KRT1, 5, 6, 14, and 16) and epidermal growth factor receptor (EGFR) within the tumors. The luminal subtype is less aggressive and is predominately characterized by elevated gene expression of peroxisome proliferator-activated receptor- gamma (PPARγ) and forkhead box protein A1 (FOXA1). We have previously shown that As3+-transformed urothelial cells (As-T) exhibit a basal subtype of UC expressing genes associated with squamous differentiation. We hypothesized that the molecular subtype of the As-T cells could be altered by inducing the expression of PPARγ and/or inhibiting the proliferation of the cells. Non-transformed and As-T cells were treated with Troglitazone (TG, PPARG agonist, 10 µM), PD153035 (PD, an EGFR inhibitor, 1 µM) or a combination of TG and PD for 3 days. The results obtained demonstrate that treatment of the As-T cells with TG upregulated the expression of PPARγ and FOXA1 whereas treatment with PD decreased the expression of some of the basal keratins. However, a combined treatment of TG and PD resulted in a consistent decrease of several proteins associated with the basal subtype of bladder cancers (KRT1, KRT14, KRT16, P63, and TFAP2A). Our data suggests that activation of PPARγ while inhibiting cell proliferation facilitates the regulation of genes involved in maintaining the luminal subtype of UC. In vivo animal studies are needed to address the efficacy of using PPARγ agonists and/or proliferation inhibitors to reduce tumor grade/stage of MIUC.

Enrichment of genes associated with squamous differentiation in cancer initiating cells isolated from urothelial cells transformed by the environmental toxicant arsenite.

Arsenic is an environmental toxicant with long-term exposure associated with the development of urothelial carcinomas. Our lab has developed an in-vitro model of urothelial carcinoma by exposing the immortal, but non-tumorigenic bladder cell line, the UROtsa, to arsenite (As3+). These transformed cells form tumors in immune-compromised mice, which resemble urothelial carcinomas with components of the tumor exhibiting squamous differentiation. The goal of the present study was to determine the differences in global gene expression patterns between the As3+-transformed UROtsa cells and the urospheres (spheroids containing putative cancer initiating cells) isolated from these cell lines and to determine if the genes involved in the development of squamous differentiation were enriched in the urospheres. The results obtained in this study show an enrichment of genes such as KRT1, KRT5, KRT6A, KRT6B, KRT6C, KRT14 and KRT16 associated with squamous differentiation, a characteristic feature seen in aggressive basal subtypes of urothelial cell carcinoma (UCC) in the urospheres isolated from As3+-transformed UROtsa cells. In addition, there is increased expression of several of the small proline-rich proteins (SPRR) in the urospheres and overexpression of these genes occur in UCC's displaying squamous differentiation. In conclusion, the cancer initiating cells present in the urospheres are enriched with genes associated with squamous differentiation.

Characterization and determination of cadmium resistance of CD133+/CD24+ and CD133-/CD24+ cells isolated from the immortalized human proximal tubule cell line, RPTEC/TERT1.

Stem/progenitor cells are involved in the regeneration of the renal tubules after damage due to a toxic insult. However, the mechanism involved in the regeneration of the tubules by the stem cells is not well understood due to the lack of immortal cell lines that represent the stem/progenitor cells of the kidney. A previous study from our laboratory has shown that the immortalized cell line RPTEC/TERT1 contains two populations of cells, one co-expressing CD24 and CD133, the other expressing CD24 only. The goal of the present study was to determine if both these populations could be sorted into separate independent cultures and if so, determine their characteristic features and response to the nephrotoxicant cadmium. The results of our study show that both the populations of cells could grow as independent cultures and maintain their phenotype after extended sub-culture. The CD133+/CD24+ co-expressing cells formed multicellular spheroids (nephrospheres), a characteristic feature of stem/progenitor cells, and formed branched tubule-like structures when grown on the surface of matrigel, whereas the CD133-/CD24+ cells were unable to form these structures. The CD133+/CD24+ cells were able to grow and undergo neurogenic, adipogenic, osteogenic, and tubulogenic differentiation, whereas the CD133-/CD24+ cells expressed some of the differentiation markers but were unable to grow in some of the specialized growth media. The CD133+/ CD24+ co-expressing cells had a shorter doubling time compared to the cells that expressed only CD24, and were more resistant to the toxic effects of the heavy metal, cadmium. In conclusion, the isolation and characterization of these two cell populations form the RPTEC/TERT1 cell line will facilitate the development of studies that determine the mechanisms involved in tubular damage and regeneration particularly after a toxic insult.

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.

STEERing an IDeA in Undergraduate Research at a Rural Research Intensive University.

This study documents outcomes, including student career choices, of the North Dakota Institutional Development Award Networks of Biomedical Research Excellence program that provides 10-week, summer undergraduate research experiences at the University of North Dakota School of Medicine and Health Sciences. Program evaluation initiated in 2008 and, to date, 335 students have completed the program. Of the 335, 214 students have successfully completed their bachelor's degree, 102 are still undergraduates, and 19 either did not complete a bachelor's degree or were lost to follow-up. The program was able to track 200 of the 214 students for education and career choices following graduation. Of these 200, 76% continued in postgraduate health-related education; 34.0% and 20.5% are enrolled in or have completed MD or PhD programs, respectively. Other postbaccalaureate pursuits included careers in pharmacy, optometry, dentistry, public health, physical therapy, nurse practitioner, and physician's assistant, accounting for an additional 21.5%. Most students electing to stop formal education at the bachelor's degree also entered fields related to health care or science, technology, engineering, and mathematics (19.5%), with only a small number of the 200 students tracked going into service or industries which lacked an association with the health-care workforce (4.5%). These student outcomes support the concept that participation in summer undergraduate research boosts efforts to populate the pipeline of future researchers and health professionals. It is also an indication that future researchers and health professionals will be able to communicate the value of research in their professional and social associations. The report also discusses best practices and issues in summer undergraduate research for students originating from rural environments.

Human renal tubular cells contain CD24/CD133 progenitor cell populations: Implications for tubular regeneration after toxicant induced damage using cadmium as a model.

The proximal tubules of the kidney are target sites of injury by various toxicants. Cadmium (Cd+2), an environmental nephrotoxicant can cause adverse effects and overt renal damage. To decipher the mechanisms involved in nephrotoxicity, an in vitro model system is required. Mortal cultures of human proximal tubule (HPT) cells have served, as models but are difficult to acquire and do not lend themselves to stable transfection. The immortalized human proximal tubule cell line HK-2, has served as a model but it lacks vectorial active transport and shows signs of lost epithelial features. Recently a new proximal tubule cell line was developed, the RPTEC/TERT1, and the goal of this study was to determine if this cell line could serve as a model to study nephrotoxicity. Global gene expression analysis of this cell line in comparison to the HK-2 and HPT cells showed that the RPTEC/TERT1 cells had gene expression patterns similar to HPT cells when compared to the HK-2 cells. The HPT and the RPTEC/TERT1 cell line had an increased population of stem/progenitor cells co-expressing CD24 and CD133 when compared to the HK-2 cells. The level of expression of cadherins, claudins and occludin molecules was also similar between the RPTEC/TERT1 and the HPT cells. Acute exposure to Cd+2 resulted in necrosis of the RPTEC/TERT1 cells when compared to the HK-2 cells which died by apoptosis. Thus, the RPTEC/TERT1 cells are similar to HPT cells and can serve as a good model system to study mechanisms involved in toxicant induced renal damage.

The unique C- and N-terminal sequences of Metallothionein isoform 3 mediate growth inhibition and Vectorial active transport in MCF-7 cells.

BACKGROUND: The 3rd isoform of the metallothionein (MT3) gene family has been shown to be overexpressed in most ductal breast cancers. A previous study has shown that the stable transfection of MCF-7 cells with the MT3 gene inhibits cell growth. The goal of the present study was to determine the role of the unique C-terminal and N-terminal sequences of MT3 on phenotypic properties and gene expression profiles of MCF-7 cells. METHODS: MCF-7 cells were transfected with various metallothionein gene constructs which contain the insertion or the removal of the unique MT3 C- and N-terminal domains. Global gene expression analysis was performed on the MCF-7 cells containing the various constructs and the expression of the unique C- and N- terminal domains of MT3 was correlated to phenotypic properties of the cells. RESULTS: The results of the present study demonstrate that the C-terminal sequence of MT3, in the absence of the N-terminal sequence, induces dome formation in MCF-7 cells, which in cell cultures is the phenotypic manifestation of a cell's ability to perform vectorial active transport. Global gene expression analysis demonstrated that the increased expression of the GAGE gene family correlated with dome formation. Expression of the C-terminal domain induced GAGE gene expression, whereas the N-terminal domain inhibited GAGE gene expression and that the effect of the N-terminal domain inhibition was dominant over the C-terminal domain of MT3. Transfection with the metallothionein 1E gene increased the expression of GAGE genes. In addition, both the C- and the N-terminal sequences of the MT3 gene had growth inhibitory properties, which correlated to an increased expression of the interferon alpha-inducible protein 6. CONCLUSIONS: Our study shows that the C-terminal domain of MT3 confers dome formation in MCF-7 cells and the presence of this domain induces expression of the GAGE family of genes. The differential effects of MT3 and metallothionein 1E on the expression of GAGE genes suggests unique roles of these genes in the development and progression of breast cancer. The finding that interferon alpha-inducible protein 6 expression is associated with the ability of MT3 to inhibit growth needs further investigation.