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.

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.

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.

Nicotinic Receptors in the Brainstem Ascending Arousal System in SIDS With Analysis of Pre-natal Exposures to Maternal Smoking and Alcohol in High-Risk Populations of the Safe Passage Study.

Pre-natal exposures to nicotine and alcohol are known risk factors for sudden infant death syndrome (SIDS), the leading cause of post-neonatal infant mortality. Here, we present data on nicotinic receptor binding, as determined by 125I-epibatidine receptor autoradiography, in the brainstems of infants dying of SIDS and of other known causes of death collected from the Safe Passage Study, a prospective, multicenter study with clinical sites in Cape Town, South Africa and 5 United States sites, including 2 American Indian Reservations. We examined 15 pons and medulla regions related to cardiovascular control and arousal in infants dying of SIDS (n = 12) and infants dying from known causes (n = 20, 10 pre-discharge from time of birth, 10 post-discharge). Overall, there was a developmental decrease in 125I-epibatidine binding with increasing postconceptional age in 5 medullary sites [raphe obscurus, gigantocellularis, paragigantocellularis, centralis, and dorsal accessory olive (p = 0.0002-0.03)], three of which are nuclei containing serotonin cells. Comparing SIDS with post-discharge known cause of death (post-KCOD) controls, we found significant decreased binding in SIDS in the nucleus pontis oralis (p = 0.02), a critical component of the cholinergic ascending arousal system of the rostral pons (post-KCOD, 12.1 ± 0.9 fmol/mg and SIDS, 9.1 ± 0.78 fmol/mg). In addition, we found an effect of maternal smoking in SIDS (n = 11) combined with post-KCOD controls (n = 8) on the raphe obscurus (p = 0.01), gigantocellularis (p = 0.02), and the paragigantocellularis (p = 0.002), three medullary sites found in this study to have decreased binding with age and found in previous studies to have abnormal indices of serotonin neurotransmission in SIDS infants. At these sites, 125I-epibatidine binding increased with increasing cigarettes per week. We found no effect of maternal drinking on 125I-epibatidine binding at any site measured. Taken together, these data support changes in nicotinic receptor binding related to development, cause of death, and exposure to maternal cigarette smoking. These data present new evidence in a prospective study supporting the roles of developmental factors, as well as adverse exposure on nicotinic receptors, in serotonergic nuclei of the rostral medulla-a finding that highlights the interwoven and complex relationship between acetylcholine (via nicotinic receptors) and serotonergic neurotransmission in the medulla.

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.

Concurrent prenatal drinking and smoking increases risk for SIDS: Safe Passage Study report.

BACKGROUND: Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality. Although the rate has plateaued, any unexpected death of an infant is a family tragedy thus finding causes and contributors to risk remains a major public health concern. The primary objective of this investigation was to determine patterns of drinking and smoking during pregnancy that increase risk of SIDS. METHODS: The Safe Passage Study was a prospective, multi-center, observational study with 10,088 women, 11,892 pregnancies, and 12,029 fetuses, followed to 1-year post delivery. Subjects were from two sites in Cape Town, South Africa and five United States sites, including two American Indian Reservations. Group-based trajectory modeling was utilized to categorize patterns of drinking and smoking exposure during pregnancy. FINDINGS: One-year outcome was ascertained in 94·2% infants, with 28 SIDS (2·43/1000) and 38 known causes of death (3·30/1000). The increase in relative risk for SIDS, adjusted for key demographic and clinical characteristics, was 11·79 (98·3% CI: 2·59-53·7, p < 0·001) in infants whose mothers reported both prenatal drinking and smoking beyond the first trimester, 3.95 (98·3% CI: 0·44-35·83, p = 0·14), for drinking only beyond the first trimester and 4·86 (95% CI: 0·97-24·27, p = 0·02) for smoking only beyond the first trimester as compared to those unexposed or reported quitting early in pregnancy. INTERPRETATION: Infants prenatally exposed to both alcohol and cigarettes continuing beyond the first trimester have a substantially higher risk for SIDS compared to those unexposed, exposed to alcohol or cigarettes alone, or when mother reported quitting early in pregnancy. Given that prenatal drinking and smoking are modifiable risk factors, these results address a major global public health problem. FUNDING: National Institute on Alcohol Abuse and Alcoholism, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute on Deafness and Other Communication Disorders.

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.

County level incidence rates of chronic lymphocytic leukemia are associated with residential radon levels.

AIM: We previously reported that incidence rates for chronic lymphocytic leukemia (CLL) among US states are significantly correlated with levels of residential radon (RR). Because these correlations could be influenced by confounding and/or misclassification among large geographic units, we reinvestigated them using smaller geographic units that better reflect exposure and disease at the individual level. METHODS: We examined the relationships between CLL and RR per county in 478 counties with publicly-available data. RESULTS: After adjustment for ultraviolet radiation, a possible risk factor for CLL, county rates for CLL and RR were significantly correlated among males and females both together and separately (p < 0.0001). CONCLUSION: CLL is significantly associated with RR at the county level.

The expression of keratin 6 is regulated by the activation of the ERK1/2 pathway in arsenite transformed human urothelial cells.

Urothelial cancers have an environmental etiological component, and previous studies from our laboratory have shown that arsenite (As+3) can cause the malignant transformation of the immortalized urothelial cells (UROtsa), leading to the expression of keratin 6 (KRT6). The expression of KRT6 in the parent UROtsa cells can be induced by the addition of epidermal growth factor (EGF). Tumors formed by these transformed cells have focal areas of squamous differentiation that express KRT6. The goal of this study was to investigate the mechanism involved in the upregulation of KRT6 in urothelial cancers and to validate that the As+3-transformed UROtsa cells are a model of urothelial cancer. The results obtained showed that the parent and the As+3-transformed UROtsa cells express EGFR which is phosphorylated with the addition of epidermal growth factor (EGF) resulting in an increased expression of KRT6. Inhibition of the extracellular-signal regulated kinases (ERK1/2) pathway by the addition of the mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 kinase inhibitor U0126 resulted in a decrease in the phosphorylation of ERK1/2 and a reduced expression of KRT6. Immuno-histochemical analysis of the tumors generated by the As+3-transformed isolates expressed EGFR and tumors formed by two of the transformed isolates expressed the phosphorylated form of EGFR. These results show that the expression of KRT6 is regulated at least in part by the ERK1/2 pathway and that the As+3-transformed human urothelial cells have the potential to serve as a valid model to study urothelial carcinomas.

The Stillbirth Classification System for the Safe Passage Study.

Objective Describe the classification system for assigning the cause of stillbirth in the Safe Passage Study, an international, multi-institutional, prospective analysis conducted by the NIAAA/NICHD-funded Prenatal Alcohol in SIDS and Stillbirth (PASS) Research Network. The study mission is to determine the role of prenatal alcohol and/or cigarette smoke exposure in adverse pregnancy outcomes, including stillbirth, in a high-risk cohort of 12,000 maternal/fetal dyads. Methods The PASS Network classification system is based upon 5 "sites of origin" for cause of stillbirth, further subdivided into mechanism subcategories; both are employed to assign an ultimate cause of death. Each PASS stillbirth was assigned a cause of death and status of sporadic versus recurrent. Adjudication involved review of maternal and obstetrical records; fetal autopsy and placental findings; and required complete consensus in each case. Two published classification systems, ie, INCODE and ReCoDe, were used for comparison. Results Causes of stillbirth classified were fetal (26%), placental (53%), external (5%), and undetermined (16%). Nine cases (47%) had placental causes of death due to maternal disorders that carry recurrence risks. There was full agreement for cause of death across the 3 classification systems in 26% of cases and partial agreement among them in 42% of cases. Conclusions The proposed PASS schema employs a user-friendly classification that provides comparable information to previously published systems. Advantages include its simplicity, mechanistic formulations, tight clinicopathologic integration, provision for an undetermined category, and its wide applicability to perinatal mortality review boards with access to information routinely collected during clinicopathologic evaluations.

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, As(3+), 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.

Increased neuron specific enolase expression by urothelial cells exposed to or malignantly transformed by exposure to Cd²âº or As³âº.

Neuron specific enolase (ENO2, γ-enolase) is a biomarker used to help identify neuroendocrine differentiation in tumors. This laboratory has shown that ENO2 might be a biomarker for exposure to cadmium and arsenite. In this study these observations are extended to the urothelial cell, where environmental exposures are strongly linked to urothelial cancer. The UROtsa urothelial cell line and its Cd²âº- and As³âº-transformed counterparts were used as the model. Acute exposure of the UROtsa cells to both As³âº- and Cd²âº-caused significant increases in ENO2 expression. Treatment with the histone deacetlyase inhibitor was also shown to significantly increase the expression of ENO2 mRNA. The expression of ENO2 was significantly elevated in the Cd²âº- and As³âº-transformed UROtsa cells and tumor transplants. In contrast, ENO1, was unaffected by exposure to As³âº or Cd²âº. Immunofluorescence showed ENO2 associated with both the nucleus and cytoplasm and cytoplasmic ENO2 co-localized with ENO1. The findings extend the evidence suggesting a link between As³âº and Cd²âº exposure and neuroendocrine differentiation in tumors. The results suggest that ENO2 might be a biomarker of human exposure to Cd²âº and As³âº that operates through histone modification.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer.

BACKGROUND: ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. RESULTS: It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. CONCLUSION: This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease.

Amyloid precursor protein expression modulates intestine immune phenotype.

Amyloid precursor protein (APP) is widely expressed across many tissue and cell types. Proteolytic processing of the protein gives rise to a plethora of protein fragments with varied biological activities. Although a large amount of data has been generated describing the metabolism of the protein in neurons, its role in regulating the phenotype of other cells remains unclear. Based upon prior work demonstrating that APP regulates the activation phenotype of monocytic lineage cells, we hypothesized that APP can regulate macrophage activation phenotype in tissues other than brain. Ileums of the small intestines from C57BL6/J wild type and APP(-/-) mice were compared as a representative tissue normally associated with abundant macrophage infiltration. APP(-/-) intestines demonstrated diminished CD68 immunoreactivity compared to wild type mice. This correlated with significantly less cyclooxygenase-2 (cox-2), CD68, CD40, CD11c, and ßIII-tubulin protein levels. Peritoneal macrophages from APP(-/-) mice demonstrated decreased in vitro migratory ability compared to wild type cells and diminished basal KC cytokine secretion. Whereas, APP(-/-) intestinal macrophages had an increase in basal KC cytokine secretion compared to wild type cells. Conversely, there was a significant decrease in multiple cytokine levels in APP(-/-) compared to wild type ileums. Finally, APP(-/-) mice demonstrated impaired absorption and increased motility compared to wild type mice. These data demonstrate the APP expression regulates immune cell secretions and phenotype and intestinal function. This data set describes a novel function for this protein or its metabolites that may be relevant not only for Alzheimer's disease but a range of immune-related disorders.

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer.

BACKGROUND: Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As+3 and Cd+2 sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants. RESULTS: It was shown that both As+3 and Cd+2 exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As+3 or Cd+2. Localization studies showed that ENO2 in the MCF-10A cells transformed by As+3 or Cd+2 had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1. CONCLUSION: The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As+3 or Cd+2. The findings also suggest a possible link between As+3 and Cd+2 exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As+3 and Cd+2.

Comparison of expression patterns of keratin 6, 7, 16, 17, and 19 within multiple independent isolates of As(+3)- and Cd (+2)-induced bladder cancer : keratin 6, 7, 16, 17, and 19 in bladder cancer.

This laboratory has generated a series of seven cadmium (Cd(+2))- and six arsenite (As(+3))-transformed urothelial cancer cell lines by exposure of parental UROtsa cells to each agent under similar conditions of exposure. In this study, the seven Cd(+2)-transformed cell lines were characterized for the expression of keratin 6, 16, and 17 while the six As(+3) cell lines were assessed for the expression of keratin 7 and 19. The results showed that the series of Cd(+2)-transformed cell lines and their respective transplants all had expression of keratin 6, 16, and 17 mRNA and protein. The expression of keratin 6, 16, and 17 was also correlated with areas of the urothelial tumor cells that had undergone squamous differentiation. The results also showed that four of the six As(+3)-transformed cell lines had expression of keratin 7 and 19 mRNA and protein and produced subcutaneous tumors with intense focal staining for keratin 7 and 19. The other two As(+3)-transformed cell lines had very low expression of keratin 7 mRNA and protein and produced subcutaneous tumors having no immunoreactivity for keratin 7; although keratin 19 expression was still present. The peritoneal tumors produced by one of these two cell lines regained expression of keratin 7 protein. The present results, coupled with previous studies, indicate that malignant transformation of UROtsa cells by Cd(+2) or As(+3) produce similar patterns of keratin 6, 7, 16, 17, and 19 in the resulting series of cell lines and their respective tumors.

Kindlin-2 expression in arsenite- and cadmium-transformed bladder cancer cell lines and in archival specimens of human bladder cancer.

OBJECTIVE: To confirm a microarray study that suggested that Kindlin-2 might play a role in the development and progression of bladder cancer. There has been no previous examination of Kindlin-2 expression in human bladder cancer. METHODS: A combination of real-time polymerase chain reaction, Western analysis, and immunohistochemistry was used to characterize Kindlin-2 expression in arsenite (As(+3))- and cadmium (Cd(+2))-transformed human cell lines, their tumor transplants in immunocompromised mice, and in archival specimens of human bladder and bladder cancer. RESULTS: The results show that the Kindlin-2 expression patterns in the cell lines were not duplicated in the tumor tissues. However, it was shown that Kindlin-2 was expressed in the stromal element of all the transplanted tumors and archival specimens of human bladder cancer. It was also shown that a small number of high-grade invasive urothelial cancers have focal expression of Kindlin-2 in the tumor cells. CONCLUSION: Kindlin-2 is expressed in the stromal component of most, if not all, human bladder cancers. Kindlin-2 is not expressed in normal urothelium. Kindlin-2 is expressed in a small subset of high-grade invasive bladder cancers and may have potential as a prognostic marker for tumor progression.

Cadmium, environmental exposure, and health outcomes.

We provide an update of the issues surrounding health risk assessment of exposure to cadmium in food. Bioavailability of ingested cadmium has been confirmed in studies of persons with elevated dietary exposure, and the findings have been strengthened by the substantial amounts of cadmium accumulated in kidneys, eyes, and other tissues and organs of environmentally exposed individuals. We hypothesized that such accumulation results from the efficient absorption and systemic transport of cadmium, employing multiple transporters that are used for the body's acquisition of calcium, iron, zinc, and manganese. Adverse effects of cadmium on kidney and bone have been observed in environmentally exposed populations at frequencies higher than those predicted from models of exposure. Population data raise concerns about the validity of the current safe intake level that uses the kidney as the sole target in assessing the health risk from ingested cadmium. The data also question the validity of incorporating the default 5% absorption rate in the threshold-type risk assessment model, known as the provisional tolerable weekly intake (PTWI), to derive a safe intake level for cadmium.

Cadmium, environmental exposure, and health outcomes / O cádmio, a exposição ambiental ao cádmio e as consequências para a saúde

We provide an update of the issues surrounding health risk assessment of exposure to cadmium in food. Bioavailability of ingested cadmium has been confirmed in studies of persons with elevated dietary exposure, and the findings have been strengthened by the substantial amounts of cadmium accumulated in kidneys, eyes, and other tissues and organs of environmentally exposed individuals. We hypothesized that such accumulation results from the efficient absorption and systemic transport of cadmium, employing multiple transporters that are used for the body's acquisition of calcium, iron, zinc, and manganese. Adverse effects of cadmium on kidney and bone have been observed in environmentally exposed populations at frequencies higher than those predicted from models of exposure. Population data raise concerns about the validity of the current safe intake level that uses the kidney as the sole target in assessing the health risk from ingested cadmium. The data also question the validity of incorporating the default 5 percent absorption rate in the threshold-type risk assessment model, known as the provisional tolerable weekly intake (PTWI), to derive a safe intake level for cadmium.

Uma atualização sobre as questões relacionadas à avaliação dos riscos de saúde, oriundos da exposição a cádmio nos alimentos, é apresentada. Em um estudo de indivíduos sujeitos a uma elevada exposição dietária ao cádmio, a biodisponibilidade do cádmio ingerido foi confirmada, e os achados foram reforçados, diante da constatação de quantidades substanciais de cádmio acumulado, seja nos rins, nos olhos, assim como em outros tecidos e órgãos de pessoas ambientalmente expostas. Levantamos a hipótese de que essa acumulação seria o resultado de uma absorção eficiente do cádmio e do seu transporte sistêmico na absorção dos elementos cálcio, ferro, zinco e manganês pelo corpo humano. Os dados populacionais encontrados têm gerado uma preocupação quanto à validade do nível, atualmente aceito, de ingestão cádmica isenta de riscos, os rins constituindo o objeto único desses levantamentos de avaliação dos riscos à saúde, provenientes da ingestão de cádmio. Os dados também nos levam, na inferência do nível de ingestão cádmica isenta de riscos, a questionar a validade de se incorporar no modelo de avaliação de riscos para a determinação do limiar a taxa default de 5 por cento, conhecida como nível tolerável de ingestão proporcionada semanalmente (PTWI).

Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells.

BACKGROUND: Studies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine. RESULTS: The histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1) binding to metal response elements (MRE) elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells. CONCLUSION: The MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the urothelial cells with either Cd+2 or As+3 modified the chromatin of the MT-3 promoter to a bivalent state of promoter readiness. Urinary cytology for MT-3 positive cells would not improve the diagnosis of urothelial cancer, but might have potential as a biomarker for tumor progression.