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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.

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.

After a sudden infant death, parents and caregivers need accurate and open communication about why their infant died. Communicating tragic news about a child’s death to families and caregivers is difficult. Shared and consistent terminology is essential for pediatricians, other physicians, and nonphysician clinicians to improve communication with families and among themselves. When families do not have complete information about why their child died, pediatricians will not be able to support them through the process and make appropriate referrals for pediatric specialty and mental health care. Families can only speculate about the cause and may blame themselves or others for the infant’s death. The terminology used to describe infant deaths that occur suddenly and unexpectedly includes an assortment of terms that vary across and among pediatrician, other physician, or nonphysician clinician disciplines. Having consistent terminology is critical to improve the understanding of the etiology, pathophysiology, and epidemiology of these deaths and communicate with families. A lack of consistent terminology also makes it difficult to reliably monitor trends in mortality and hampers the ability to develop effective interventions. This report describes the history of sudden infant death terminology and summarizes the debate over the terminology and the resulting diagnostic shift of these deaths. This information is to assist pediatricians, other physicians, and nonphysician clinicians in caring for families during this difficult time. The importance of consistent terminology is outlined, followed by a summary of progress toward consensus. Recommendations for pediatricians, other physicians, and nonphysician clinicians are proposed.

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.

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.

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

Prenatal alcohol exposure (PAE) has been linked to poor pregnancy outcomes, yet there is no recognized standard for PAE assessment, and the specific effects of quantity, frequency, and timing remain largely unknown. The Safe Passage Study was designed to investigate the role of PAE in a continuum of poor peri- and postnatal outcomes. The objective of this manuscript is to describe the rationale for, and feasibility of, modifications to the traditional Timeline Followback (TLFB) for collecting PAE information in a large cohort of pregnant women. Participants from the Northern Plains region (in the United States) and Cape Town, South Africa, were followed prospectively using a modified 30-day TLFB interview, administered up to five times, to obtain detailed PAE information. Required modifications for our population included capturing information regarding sharing, type/brand, container size, and duration, in order to accurately record the amount of alcohol consumed. PAE status was defined for 99.9% of the 11,892 enrolled pregnancies at least once during pregnancy and for 92% across all trimesters. Of 53,823 drinks reported, 98% had all items necessary for standard drink computation. Sharing was reported for 74% of drinks in Cape Town, South Africa and for 10% in the Northern Plains. Compared to referent values from the traditional TLFB, 74% and 67% of drinks had different alcohol-by-volume and container size, respectively. Furthermore, a statistically significant difference was found between the number of containers reported and the number of standard drinks computed, using information from the modified TLFB. This is the first study of this size to wholly encompass all of these changes into a single measure in order to more accurately calculate daily consumption and assess patterns over time. The methods used to collect PAE information and create alcohol exposure measures likely increased the accuracy of standard drinks reported and could be generalized to other populations.

Precise identification of drinking and smoking patterns during pregnancy is crucial to better understand the risk to the fetus. The purpose of this manuscript is to describe the methodological approach used to define prenatal drinking and smoking trajectories from a large prospective pregnancy cohort, and to describe maternal characteristics associated with different exposure patterns. In the Safe Passage Study, detailed information regarding quantity, frequency, and timing of exposure was self-reported up to four times during pregnancy and at 1 month post-delivery. Exposure trajectories were developed using data from 11,692 pregnancies (9,912 women) where pregnancy outcome was known. Women were from three diverse populations: white (23%) and American Indian (17%) in the Northern Plains, US, and mixed ancestry (59%) in South Africa (other/not specified [1%]). Group-based trajectory modeling was used to identify 5 unique drinking trajectories (1 none/minimal, 2 quitting groups, 2 continuous groups) and 7 smoking trajectories (1 none/minimal, 2 quitting groups, 4 continuous groups). Women with pregnancies assigned to the low- or high-continuous drinking groups were less likely to have completed high school and were more likely to have enrolled in the study in the third trimester, be of mixed ancestry, or be depressed than those assigned to the none/minimal or quit-drinking groups. Results were similar when comparing continuous smokers to none/minimal and quit-smoking groups. Further, women classified as high- or low-continuous drinkers were more likely to smoke at moderate-, high-, and very high-continuous levels, as compared to women classified as non-drinkers and quitters. This is the first study of this size to utilize group-based trajectory modeling to identify unique prenatal drinking and smoking trajectories. These trajectories will be used in future analyses to determine which specific exposure patterns subsequently manifest as poor peri- and postnatal outcomes.

This report details the proceedings and conclusions from the 3rd International Congress on Unexplained Deaths in Infants and Children, held November 26–27, 2018 at the Radcliffe Institute at Harvard University. The Congress was motivated by the increasing rejection of the diagnosis Sudden Infant Death Syndrome (SIDS) in the medical examiner community, leading to falsely depressed reported SIDS rates and undermining the validity and reliability of the diagnosis, which remains a leading cause of infant and child mortality. We describe the diagnostic shift away from SIDS and the practical issues contributing to it. The Congress was attended by major figures and opinion leaders in this area from countries significantly engaged in this problem. Four categories (International Classification of Diseases (ICD)-11 categories of MH11, MH12, MH14, PB00-PB0Z) were recommended for classification, and explicit definitions and guidance were provided for death certifiers. SIDS was reframed as unexplained sudden death in infancy or SIDS/MH11 to emphasize that either term signifies the lack of explanation following a rigorous investigation. A distinct category for children over the age of 1 was recommended (MH12). Definitions and exclusions were provided for the alternative categories of accidental asphyxia and undetermined. As recommended, unexplained sudden death in infancy or SIDS on a death certificate will code a unique, trackable entity, accurately reflecting the inability to determine a definitive explanation, while satisfying surveillance needs and reliable identification for research efforts. The conclusions will be submitted to the World Health Organization for inclusion in the upcoming ICD-11.

e16523 Background: IARC classified arsenic (As) as “carcinogenic to humans”, but despite the health consequences, there is no molecular signature available yet to predict when exposure may lead to the disease development. First aim of this study is to investigate the genetic changes due to the exposure to carcinogenic compounds such as As. Secondly, how accurately we can predict the disease association when exposed to toxic compounds. Methods: The entire analysis was performed in-silico fashion and data was collected from the public resources such as NCBI database. Two Asian population datasets exposed As were used to find significantly differently express genes. In addition, four cancer cell lines with exposure of As compounds were used to identify the association with cancer. The human bladder cancer biopsy datasets were used to develop a risk predictive model. As per the requirements, numerous machine learning (ML) approaches such as random forest, hierarchical clustering, were used to find the classification and association between the samples and outcome. Statistical approaches such as T-Test, and ANOVA, applied to find the differentially expressed genes associated with different conditions and logistic regression models applied to develop risk prediction models. Results: We identified a set of 1183 genes which were common between both the populations and were significantly changed in humans exposed to As. A subset of 157 genes associated with As exposure and involved in cancer progression was selected for risk prediction model development. A set of four genes (NKIRAS2, AKTIP, HLA-DQA1 and TBC1D7) shows the highest prediction ability of primary bladder tumor with AUC 0.96 (95% CI: 0.82- 0.99) and reproducibility of 0.75 (0.34-0.93) when applied on different dataset. Conclusions: This study identified a list of genes and bladder cancer predictive models that would be very helpful to forecast the outcomes of As exposed in humans.

The rate for the sudden infant death syndrome (SIDS) in Cape Town, South Africa, is estimated to be among the highest in the world (3.41/1000 live births). In several of these areas, including those of extreme poverty, only sporadic, nonstandardized infant autopsy, and death scene investigation (DSI) occurred. In this report, we detail a feasibility project comprising 18 autopsied infants with sudden and unexpected death whose causes of death were adjudicated according to the 1991 NICHD definitions (SIDS, n = 7; known cause of death, n = 7; and unclassified, n = 4). We instituted a standardized autopsy and infant DSI through a collaborative effort of local forensic pathology officers and clinical providers. The high standard of forensic investigation met international standards, identified preventable disease, and allowed for incorporation of research. We conclude that an effective infant autopsy and DSI protocol can be established in areas with both high sudden unexpected infant death, and elsewhere. (SUID)/SIDS risk and infrastructure challenges.

The Safe Passage Study is an international, prospective study of approximately 12 000 pregnancies to determine the effects of prenatal alcohol exposure (PAE) upon stillbirth and the sudden infant death syndrome (SIDS). A key objective of the study is to elucidate adverse effects of PAE upon binding to serotonin (5-HT) 1A receptors in brainstem homeostatic networks postulated to be abnormal in unexplained stillbirth and/or SIDS. We undertook a feasibility assessment of 5-HT1A receptor binding using autoradiography in the medulla oblongata (6 nuclei in 27 cases). 5-HT1A binding was compared to a reference dataset from the San Diego medical examiner’s system. There was no adverse effect of postmortem interval ≤100 h. The distribution and quantitated values of 5-HT1A binding in Safe Passage Study cases were essentially identical to those in the reference dataset, and virtually identical between stillbirths and live born fetal cases in grossly non-macerated tissues. The pattern of binding was present at mid-gestation with dramatic changes in binding levels in the medullary 5-HT nuclei over the second half of gestation; there was a plateau at lower levels in the neonatal period and into infancy. This study demonstrates feasibility of 5-HT1A binding analysis in the medulla in the Safe Passage Study.

3534 Background: The epigenetic transcriptional regulator, Kaiso (ZBTB33) has been identified as a member of the C2H2 zinc finger proteins containing a BTB/POZ -zinc finger family of transcription factors that are implicated in development of cancer. Although, our understanding of clinical relevance of subcellular distribution (cytoplasmic/nuclear) Kaiso in the growth and survival of human Breast cancer (BC) is limited. Methods: We examined a cohort of 555 BC patients who underwent surgery for their primary BC in Greenville, NC using AI and SM approach. Results: The sub-classification BC shows, cytoplasmic Kaiso is differentially enriched in ER- BC (p=0.001) compared nuclear Kaiso (p=0.8) and is significantly enriched in the more aggressive classes LumB (p=0.0017), HER2+ (p=0.05) and TNBC (p=6.1e-07) with respect to LumA BC patients. Additionally, the survival analysis of different compartments of Kaiso demonstrates that high cytoplasmic Kaiso (HR = 16.29 (7.6 – 34.8), p = 5.5e−13) is much more predictive of poor survival compared to nuclear Kaiso (HR = 2.83 (2.02 – 3.8), p = 6.1e−11). At gene expression level, ZBTB33 mRNA levels do not correlate with either nuclear (Spearman correlation: -0.03157, p= 0.7267) or cytoplasmic levels (Spearman correlation: -0.03526, p= 0.6962) of Kaiso. Surprisingly, ZBTB33 mRNA abundance is predictive of poor overall BC survival as demonstrated in two independent publicly available BC cohorts Metabric (HR = 2.14 (1.49 − 3.08), p = 2.7e−05) and Gyorffy B et al. (HR = 1.81 (1.55 − 2.12), p = 2.5e−14). Nuclear and cytoplasmic levels of Kaiso do not show significant differences based on race p=0.27 and p=0.1 respectively. Conclusions: Our data suggest subcellular distribution of high Kaiso is associated with poor prognosis of BC survival and subcellular localizations of Kaiso may play differential biological roles in BC prognosis.

Alzheimer's disease (AD) is a neurodegenerative disorder histologically characterized by amyloid-β (Aβ) protein accumulation and activation of associated microglia. Although these features are well described in the central nervous system, the process and consequences of Aβ accumulation in the enteric nervous system have not been extensively studied. We hypothesized that Aβ also may accumulate in the enteric nervous system and lead to immune cell activation and neuronal dysfunction in the digestive tract not unlike that observed in diseased brain. To test this hypothesis, ileums of the small intestine of thirteen month old AβPP/PS1 and C57BL/6 (wild type) mice were collected and analyzed using immunohistochemistry, western blot analysis, cytokine arrays, and ELISA. AβPP/PS1 mice demonstrated no differences in intestinal motility or water absorption but elevated luminal IgA levels compared to wild type mice. They also had increased protein levels of AβPP and the proteolytic enzyme, BACE, corresponding to an increase in Aβ1-40 in the intestinal lysate as well as an increase in both Aβ1-40 and Aβ1-42 in the stool. This correlated with increased protein markers of proinflammatory and immune cell activation. Histologic analysis localized AβPP within enteric neurons but also intestinal epithelial cells with elevated Aβ immunoreactivity in the AβPP/PS1 mice. The presence of AβPP, Aβ, and CD68 immunoreactivity in the intestines of some patients with neuropathologically-confirmed AD are consistent with the findings in this mouse model. These data support the hypothesis that in AD the intestine, much like the brain, may develop proinflammatory and immune changes related to AβPP and Aβ.

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.

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.

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.

Cervical ganglia and nerve root injury with resultant paralysis of the phrenic nerve is proposed as a mechanism of injury in “shaken baby syndrome” or nonaccidental head injury of children. We report autopsy findings of a 22-month-old girl in which cervical ganglia and nerve hemorrhage was present along with the triad of encephalopathy, bilateral retinal hemorrhage, and bilateral subdural, intradural, and subarachnoid hemorrhages. Clear evidence of near immediate respiratory arrest was documented from first responder medical reports, juvenile witness statements, and perpetrator confession. The narrowing of time of injury from the anatomic findings at autopsy allowed investigative efforts to focus on a single individual and cleared several other adults with access to the child. Additional technical notes based on our experience are provided for users of cervical neck dissection. Hemorrhage in the tissues surrounding the vertebral artery raises a potential additional injury site in cases of neck injury.

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.

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 Cd²⁺- and As³⁺-transformed counterparts were used as the model. Acute exposure of the UROtsa cells to both As³⁺- and Cd²⁺-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 Cd²⁺- and As³⁺-transformed UROtsa cells and tumor transplants. In contrast, ENO1, was unaffected by exposure to As³⁺ or Cd²⁺. 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 As³⁺ and Cd²⁺ exposure and neuroendocrine differentiation in tumors. The results suggest that ENO2 might be a biomarker of human exposure to Cd²⁺ and As³⁺ that operates through histone modification.

Writing a scientific manuscript can be broken down into readily achievable defined steps. Authors should first select a topic and perform a literature search. When the topic of the manuscript is selected, authors should define specific topics to discuss in the manuscript, define the authors and authorship order involved and be aware of any regulatory issues with the study. Authors select a target journal for publication and after familiarizing themselves with the journal format and requirements, write the article from the topics defined earlier, incorporating relevant literature references and finalizing conclusions and interpretative findings. This manuscript also provides a general overview of the publication process for potential authors of scientific research papers.

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.

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.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%, conhecida como nível tolerável de ingestão proporcionada semanalmente (PTWI).

Numerous studies showed that renal proximal tubules cells are the cell type critically affected by chronic exposure to cadmium (Cd(2+)). The aim of the present study was to apply global gene expression technology and a human renal epithelial cell culture model (HPT) to determine whether time of exposure to Cd(2+) exerts a major influence on the resulting pattern of global gene expression. HPT cells were exposed to Cd(2+) for a short, 1-d, period of exposure (9, 27, and 45 μM) versus a longer, 13-d, period (4.5, 9, and 27 μM), with the hypothesis being that the stress response of the cells would be more active during the short time of exposure. The results showed that the differential expression of genes was very extensive for HPT cells exposed to Cd(2+) for 1 d, with more than 1848 genes displaying alterations compared to control and with the major categories of genes being involved in stress responses; cell death; checkpoint arrest, DNA repair, and the cell cycle; inflammatory responses; and cell adhesion, motion and differentiation. In contrast, HPT cells exposed to Cd(2+) for 13 d showed 923 genes to be differentially expressed, with a marked reduction in the number of differentially expressed stress response genes and a significant increase in the number of genes involved in development and differentiation. There were 387 differentially expressed genes common to both times of exposure. Data suggest that unless one is actively seeking to study the acute stress response, global gene expression technology should not be applied within an early time course of toxicant exposure.

Context.—Medical examiner cases are increasingly used as tissue donor referral sources to meet the ever-growing need for transplant tissues. The assumption is often made that traumatic and sudden deaths have minimal risk of unsuspected neoplasia. An autopsy of a registered tissue donor with strong preautopsy clinical assessment of a saddle pulmonary embolus revealed unsuspected acute lymphoblastic leukemia, prompting a review of the incidence of unsuspected neoplasia from a regional forensic autopsy practice. Objective.—To determine the incidence of (1) unsuspected neoplasia, (2) clinical concordance of known neoplasia, and (3) potential donor referral in a regional forensic autopsy service. Design.—A retrospective, 5-year review of 412 autopsies from a regional, primarily forensic, autopsy service to determine the incidence of unsuspected neoplasia, clinical concordance of known neoplasia, and the preautopsy assessment of potential donor referral suitability. Results.—Unsuspected neoplasia rate at autopsy was 7% (29 of 412 patients); cancer was the cause of death in 41% (12 of 29 patients) of these individuals. In patients with a history of cancer, the discordance of cancer diagnosis was 44% (4 of 9 patients [11 patients with known cancer, 2 who refused medical evaluation were excluded from the study]). Nearly 60% (17 of 29 patients) of the unsuspected cancer cases had no apparent reason for deferral of tissue procurement before the autopsy examination. Conclusions.—The 7% incidence of unsuspected cancer in a forensic autopsy practice raises concern for the potential introduction of neoplastic tissue in the donor pool. To ensure the safety of this vital resource, mandatory complete autopsies on deceased donors are advocated as well as a tissue-recipient registry to track donor-related neoplasia.

This laboratory has demonstrated that both cadmium and arsenite can directly malignantly transform the UROtsa cell line (Sens et al. 2004). It was also shown that the tumor heterotransplants produced by the Cd2+- and As3+-transformed cells had histologic features consistent with human transitional cell carcinoma of the bladder. The parent UROtsa cell line was developed from a primary cell culture of human urothelium that was immortalized using the SV40 large T-antigen (Petzoldt et al. 1994, 1995). The UROtsa cells proliferate in a simple serum-containing growth medium, retain a normal cytogenetic profile, grow as a contact-inhibited monolayer, and possess an undifferentiated morphology consistent with basal epithelial cells. They are not tumorigenic as judged by their inability to form colonies in soft agar and tumors in nude mice. Our laboratory also adapted the UROtsa cells to grow in a serum-free growth medium (Rossi et al. 2001). Under serum-free conditions, the cells exhibited enhanced differentiation, as evidenced by the presence of a stratified morphology consistent with the structural features associated with the intermediate layers of the urothelium. The cells grown in serum-free medium retained the properties of immortality, contact inhibition, and nontumorigenicity. Total RNA isolated from the parental UROtsa cells and their As3+- and Cd2+-transformed sublines grown in serum-containing growth medium were subjected to microarray analysis employing the U133 Plus 2.0 Affymetric chip. One of the stronger differentially expressed signals between the parental and the As3+-transformed cells was the keratin 6a gene. Our first goal in the present study was to confirm the differential expression of keratin 6a in the parental and transformed UROtsa cell lines at both the mRNA and protein levels. The second goal was to determine the expression of the keratin 6a gene in tumor heterotransplants derived from the As3+- and Cd2+-transformed UROtsa cells. The final aim was to demonstrate that the results from this model system could potentially translate to human disease by determining if keratin 6a was overexpressed in archival specimens of human bladder cancer.

Previous studies from this laboratory have implicated the expression of the third isoform of metallothionein (MT-3) in the maintenance of proximal tubular vectorial active ion transport. It was shown that HK-2 cells have no expression of MT-3 and do not form domes in culture; whereas, the human proximal tubular (HPT) cells and HK-2 cells stably transfected with MT-3 [HK-2(MT-3)] form these structures. In the present study, this association was further explored by determining the effect of MT-3 expression on the expression of the E -, P -, N -, K -, and Ksp-cadherins. It was demonstrated that the HPT cells and HK-2(MT-3) cells had significant elevations in the expression of messenger RNA and protein for the E -, P -, and Ksp-cadherins compared with that of the HK-2 cells transfected with the blank vector [HK-2(blank vector)]. In contrast, the HK-2(blank vector) cells had significantly elevated expression of N- and K-cadherin compared with both the HPT and HK-2(MT-3) cell lines. These patterns of cadherin expression provide strong evidence that MT-3 might be involved in epithelial to mesenchymal transition that is postulated to occur during several disease states and in the mesenchymal to epithelial transition that occurs during normal kidney morphogenesis. A final goal of the study was to determine if Cd(+2) exposure influenced vectorial active transport of the proximal tubular cells and if this might occur through alterations in the expression of MT-3. It was shown that exposure to Cd(+2) eliminated vectorial active transport by the proximal tubular cell lines, but that Cd(+2) exposure did not reduce the expression of the MT-3 protein. The study shows that the level of MT-3 expression in HPT cells influences transepithelial resistance and cadherin expression but does not influence the Cd(+2)-induced loss of vectorial active transport.

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn(+2) (50, 100, or 150 microM) or Cd(+2) (3, 6, or 12 microM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn(+2) toxicity and a lesser but significant effect on Cd(+2) toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn(+2) or Cd(+2) exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.

The present paper describes a rapid method for identification and characterization of human metallothionein (MT) isoforms in complex cell cultures using high-resolution matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF). In the proposed method, the sample preparation of MTs from cultured cells is both simple and fast. It is accomplished by trypsin cleavage of cell proteins into small peptide species, the majority of which are subsequently removed by gel filtration using beads with an exclusion limit of 4000 Da. In contrast to most cell proteins, MTs remain intact (undigested) upon being treated with trypsin, being excluded by the gel beads and thus recovered by low-speed centrifugation. To identify the protein constitutes of the MT preparation, the MT sample is divided into two parts, one for intact protein accurate mass measurement, the other for tryptic digestion followed by MS and MS/MS analyses. In the latter case, the MT proteins are denatured by the addition of EDTA which strips heavy metals from MTs and renders them susceptible to tryptic digestion. The obtained accurate mass with the unique peptide sequences of each MT isoform allows for unambiguous identification of MT isoforms in the prepared mixture. The method has been applied to RWPE-1 cells derived from normal human prostate epithelium. Four MT isoforms, 1E, 1G, 1X, and 2A, have been confidently identified, being primarily acetylated at N-termini. These results are in agreement with the expression of MT mRNAs in RWPE-1 cells determined by real-time reverse-transcription polymerase chain reaction (RT-PCR).

BACKGROUND:Reductions in sudden infant death syndrome (SIDS) are commonly attributed to modifications in infant sleep environments. Approaches to diagnosis in sudden infant death, death scene investigations, the prevalence of intrinsic risk factors for SIDS, and the potential influence of treatment-related factors on infant vulnerability have also changed. Understanding all contributory factors may help reduce residual SIDS rates.METHODS:We analyzed US Mortality Multiple Causes Records for 1983 to 2012 to compare SIDS postneonatal mortality rates with a projection applying non-SIDS mortality changes, using those changes as a proxy measure for alterations in intrinsic risk. Composites of neglect-related, unknown, and circumstantial respiratory diagnoses were measured, as was a cumulative composite of unexplained infant death diagnoses. Cluster analysis with leading causes of postneonatal mortality and SIDS mortality rates for low birth weight infants were also examined.RESULTS:SIDS and non-SIDS postneonatal mortality rates were concordant over time. Important variance was seen 1994 to 1996, coinciding with Back-to-Sleep initiation. Other variance, eliminated in the cumulative composite, appeared related to differences in diagnostic practices. Changes in SIDS rates resembled changes in mortality from congenital malformations, respiratory distress of the newborn, and diseases of the circulatory system. SIDS rates for low birth weight infants followed broader postneonatal trends.CONCLUSIONS:SIDS mortality followed trends in overall postneonatal mortality, including effects of changes in the infant sleep environment and diagnostic classification. Preventing asphyxia risk in the sleep environment must be coupled with efforts to understand intrinsic biological pathways, some potentially associated with other categories of infant and perinatal mortality.

This laboratory has shown that both Cd(+2) and As(+3) can malignantly transform human urothelial cells. The present study examined metal resistance and the mechanism of cell death when the parental and malignantly transformed UROtsa cells were exposed to Cd(+2) and As(+3). It was shown that the malignantly transformed UROtsa cells were more resistant to the toxic effects of both metals. The assessment of the mode of cell death demonstrated that the parental UROtsa cells died by both apoptosis and necrosis when exposed to either metal. It was shown that apoptosis was the more prominent mechanism of cell death, accounting for over 50% of cell death. Apoptotic cell death was determined by the observation of fragmented nuclei using 4',6-diamidino-2-phenylindole staining, the formation of a DNA ladder, and the detection of cleaved caspase-3 and caspase-9 products in the cell lysates. Necrotic cell death was determined by measuring the release of lactate dehydrogenase into the growth medium. It was determined that the extent of apoptosis of the malignantly transformed UROtsa cells was decreased and that the extent of necrosis was increased compared to the parental UROtsa cells. These observations are consistent with in vivo studies which suggest that As(+3) can act as a tumor promoter during the regeneration of the bladder urothelium. The present in vitro studies suggest that As(+3)-induced cytotoxicity could set the stage for tissue repair due to its own inherent toxicity to normal urothelium, and then subsequently act as a tumor promoter during the regeneration process through the stimulation of the regrowth of cells that have gained increased resistance to As(+3).

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For lowgrade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd +2 or As +3 . In contrast, it was shown that the cells transformed by Cd +2 and As +3 that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd +2 and As +3 . Bladder cancer is the first cancer in which industrial exposurewas shown to be the major factor in disease causation. This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer (Sens et al., 2000). A retrospective immunohistochemical analysis of MT-3 expression was performed on a modest sample set of archival paraffinembedded diagnostic specimens composed of benign and cancerous lesions of the bladder. The cells of the normal bladder were shown to have no immunoreactivity for the MT-3 protein. In contrast, all specimens of bladder cancer were immunoreactive for the MT-3 protein and the intensity of staining correlated to tumor grade. An analysis of MT-3 messenger RNA (mRNA) and protein was also performed on samples obtained from surgically removed normal bladder tissue. Immunoblot analysis failed to detect MT-3 protein. The analysis of MT-3 mRNA expression by reverse transcriptase‐ PCR (RT-PCR) at an input of 500 ng total RNA and 40 reaction cycles showed no reaction product. These findings were in agreement with the negative immunostaining found using benign paraffin-embedded bladder tissue and it was concluded that the normal bladder expressed minimal, if any, MT-3 mRNA and protein. The goal of the present study was to confirm and extend these observations to a larger sample set

This laboratory has shown that MT-3 expression determines the choice between apoptotic or necrotic cell death in Cd(+2)-exposed human proximal tubule cells. Human proximal tubule cells that express MT-3 undergo necrosis when exposed to Cd(+2), while cells that have no basal expression of MT-3 undergo apoptotic cell death. It was also shown that cells which express MT-3 were more sensitive to Cd(+2)-induced cell death than those having no basal expression. In the present study, site directed mutagenesis was used to determine if the unique N-terminal sequence of MT-3 was required for these activities regarding toxicity and cell death. The results demonstrated that HK-2 cells stably transfected with MT-3 that had been modified by converting the 2 prolines at amino acid positions 7 and 9 to threonines was no longer active in promoting necrotic cell death at lower levels of Cd(+2) exposure. This was shown in comparison to cells containing the wild type MT-3 sequence and blank vector controls as regards the % of DAPI-stained fragmented nuclei, DNA laddering, LDH release, caspase-9, and caspase-3 activation. This study demonstrates that the unique N-terminal sequence of MT-3 is required to elicit an effect on the mechanism of Cd(+2)-induced death of the proximal tubule cell. This is the identical sequence that has been shown to be responsible for the growth inhibitory activity of MT-3 in the neural system.

This study was designed to define the effect that overexpression of MT-3 would have on a cell culture model of bladder urothelium. Stable and inducible transfection was used to achieve overexpression of the MT-3 gene in the UROtsa cell line. When the UROtsa cells were stably transfected with the MT-3 coding sequence, there was highly elevated expression of MT-3 mRNA, but no MT-3 protein. An inducible vector showed that low basal levels of MT-3 mRNA and protein could be produced, but that induction only increased MT-3 mRNA and not protein. The clones expressing low basal levels of MT-3 protein also had reduced growth rates compared to control cells. Site directed mutagenesis was used to produce an MT-3 coding sequence where the prolines in positions 7 and 9 were converted to threonines. When this altered MT-3 was stably transfected into the UROtsa cells, the cells were able to accumulate the mutated form of the MT-3 protein. These studies show that MT-3 protein expression is inhibited by post-transcriptional control in the urothelial cell. Modifying the MT-3 protein to resemble the MT-1 isoform removes this component of post-transcriptional control and allows accumulation of the mutated MT-3 protein. The altered sequence involved in post-transcriptional control of MT-3 protein expression is the same sequence implicated in the neuronal growth inhibitory activity associated specifically with the MT-3 isoform of the MT gene family.

Studies have shown, using immunohistochemical staining, that the MT-1 and MT-2 proteins (MT-1/2) are overexpressed in a substantial subset of ductal breast cancers, that overexpression occurs early in the disease process, and that this overexpression is indicative of a poor prognosis. Normal ductal breast epithelium fails to immunostain for the MT-1/2 protein, whereas the myoepithelial cells of the ducts stain intensely. There is no information regarding the expression of the mRNAs for the eight active MT-1 and MT-2 genes in normal breast duct epithelium. Microdissection of normal breast samples was used to obtain total RNA from enriched populations of ductal epithelium and myoepithelium. Analysis by reverse-transcription polymerase chain reaction (RT-PCR) demonstrated that the identity of the MT isoform-specific genes expressed (MT-2A and MT-1X) and their relative levels of expression were similar between the myoepithelial and ductal components. These findings indicate that the ductal and myoepithelial components express similar amounts of MT-2A and MT-1X mRNAs, but that they have distinctly different expression of the MT-1/2 protein. Confluent cultures of MCF-10A breast epithelial cells were exposed to Cd(+2) to test for evidence of post-transcriptional regulation of MT-1/2 protein accumulation in ductal epithelium. It was demonstrated that Cd(+2) elicited only a marginal induction of MT-1E, MT-1X, or MT-2A mRNAs, whereas, there was a marked increase in MT-1/2 protein, reaching levels of 6% of total cell protein under conditions of extended exposure. This study suggests that the mechanism underlying the finding of increased MT-1/2 protein expression in ductal breast cancer may involve, to some degree, the post-transcriptional regulation of MT-1/2 protein expression.

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.

BACKGROUND The third isoform of metallothionein (MT-3) is overexpressed in prostate cancers and PIN lesions. The expression of MT-3 is highly variable but appears to correlate to Gleason score. The goal of the present study was to determine the effect of MT-3 overexpression on the growth of the PC-3 prostate cancer cell line. METHODS PC-3 cells were stably transfected with either the MT-3 or MT-1E gene. Cell growth was determined by counting DAPI-stained nuclei, drug resistance by the colony formation assay, MT mRNA expression by reverse transcriptase-polymerase chain reaction, and MT protein by immunoblot. RESULTS PC-3 cells that overexpress the MT-3 gene are growth inhibited compared with either untransfected cells, cells with blank vector, or cells with similar overexpression of the MT-1E gene. Furthermore, increased chemotherapeutic drug resistance occurred in PC-3 clones derived from MT-3– and MT-1E–transfected cells. CONCLUSION The overexpression of MT-3 can influence the growth and chemotherapeutic drug resistance of the PC-3 prostate cancer cell line. Prostate 52: 89–97, 2002. © 2002 Wiley-Liss, Inc.