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PDF file - 114K, Supplemental Figure 5 Cellular distribution of HIF-2alpha under normal oxygen conditions. (a) Representative confocal microscopy images (magnification 400x) showing HIF-2α or Nox4 immunofluorescense, DAPI nuclear staining, or merged images. b) Control (NS) or Nox4 shRNA (KD) cells were imaged following exposure to TEMPOL (0.25 mM), DTT (1mM). c) 786-0 NS cells imaged following transduction with Ad-GFP or Ad-MnSOD. d) 786-0 cells expressing pcDNA-Nox4 imaged with or without treatment with TEMPOL (0.25 mM).

PDF file - 376K, Supplemental Figure 2. Characterization of 786-0 cells stably transfected with the human, full-length Nox4 cDNA. (a) Morphology of 786-0 cells following exogenous expression of Nox4 reveals smaller, denser, more-rounded appearance relative to parental 786-0 cells. (b) Cell viability assay. Equal numbers of untransfected cells or 786-0 Nox4 cells were plated in 96-well plates. After attachment, assay reagent was added, incubated at 37{degree sign}C, and the fluorescent signal read at 590 nm.

PDF file - 224K, Supplemental Figure 1. Quantitative RT-PCR (a) and semi-quantitative RT-PCR (b) for detection of Nox1 in 786-0, RCC4 or LNCaP prostate cancer cells using two published primer pairs(14, 17),. Error bar represents +/- SE.

PDF file - 183K, Supplemental Figure 4. Cytotoxicity was measured by CellTiter-Blue (Promega, Madison, WI). 786-0 NS cells were plated at 1 x 104 cells per well in 96-well plates. After 24 hours, dithiothreitol was added as indicated in 5 replicate wells. 20 �l of the CellTiter-Blue reagent was added to each well and viability quantitated by spectrofluorometer (Spectra Max Plus384, Molecular Devices) and expressed as percentage of viability of media-only controls. Error bars indicate +/- S.E. mean.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

PDF file - 98K, Supplemental Figure 3. Lucigenin chemiluminescence assay for superoxide detection performed as described in materials and methods on a) isolated membrane fractions from parental 786-0, RCC4 and Caki-1 cells or isolated cell fractions from 786-0 (b) or RCC4 (c) cells. RLU: relative light units.

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PDF file - 446K, Supplemental Figure 6 Immunohistochemistry for Nox4 and HIF-2alpha in 786-0 NS and KD xenograft explants confirms re-expression of Nox4 in KD tumors with corresponding elevated HIF-2alpha expression. Magnification 200X.

OBJECTIVE/BACKGROUND: Approximately 400,000 adolescents give birth in the US annually. Although one-half experience depressive symptoms, less than 25% comply with referrals for depression evaluation and treatment. The current study tested the effectiveness of an internet based depression intervention on seeking depression treatment. METHODS: Based upon the Theory of Planned Behavior (TPB), the intervention included vignettes, questions and answers, and resources. Before the intervention, immediately after the intervention, and two-weeks later the adolescent mothers (n=151) answered questions related to TPB variables and depression treatment. Data were compared to adolescent mothers (n=138) in the control group. Data were collected in community organizations or home visits for the control group. Adolescent mothers in the intervention group answered questions and completed the intervention from a computer of their choice. RESULTS: The adolescents were primarily African American (89.2%); less than high school educated (51.7%); had given birth in last year (97.1%); with a mean age 18.2 years. The intervention led to significant changes in attitude, perceived control, intention to seek mental health treatment, and actually seeking depression treatment. CONCLUSIONS/DISCUSSION: Untreated postpartum depression dramatically impacts a mother’s relationships with her child, her functioning at work and school, health care seeking behaviors, mothering skills, and her development as well as the development of her child. An Internet based depression intervention is an inexpensive method to increase rates of depression treatment.

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. Cancer Res; 74(13); 3501–11. ©2014 AACR.

Background The excision repair cross complementing (ERCC1) gene product plays a vital role in the nucleotide excision repair and DNA interstrand crosslink repair pathways, which protect the genome from mutations and chromosomal aberrations, respectively. Genetic deletion of Ercc1 in the mouse causes dramatically accelerated aging. We examined the effect of Ercc1 deletion in the development of prostate cancer in a prostate recapitulation model as Ercc1 deficient mice die within four weeks of birth.

The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes. Unlike other tumor types, prostate tumors express high levels of EGR1 relative to normal tissues. However, the mechanism of EGR1 regulation in prostate tumor cells is unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using RT-PCR to quantify EGR1 transcripts, we found that EGF induced EGR1 expression in a dose- and time-dependent manner and the ERK pathway inhibitor, PD98059, abrogated the EGF-mediated EGR1 response in LNCaP and PC3 cells. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (-771 to -245 bp) containing 3 potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment in PC3 cells. Overexpression of Elk-1 was sufficient to activate the EGF-responsive region of EGR1 promoter in PC3 cells and, similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity. Taken together, these results demonstrate for the first time that EGR1 expression in PC3 cells is mediated through an EGF-ERK-Elk-1 signaling cascade.

Background The prostate gland represents a multifaceted system in which prostate epithelia and stroma have distinct physiological roles. To understand the interaction between stroma and glandular epithelia, it is essential to delineate the gene expression profiles of these two tissue types in prostate cancer. Most studies have compared tumor and normal samples by performing global expression analysis using a mixture of cell populations. This report presents the first study of prostate tumor tissue that examines patterns of differential expression between specific cell types using laser capture microdissection (LCM). Methods LCM was used to isolate distinct cell-type populations and identify their gene expression differences using oligonucleotide microarrays. Ten differentially expressed genes were then analyzed in paired tumor and non-neoplastic prostate tissues by quantitative real-time PCR. Expression patterns of the transcription factors, WT1 and EGR1, were further compared in established prostate cell lines. WT1 protein expression was also examined in prostate tissue microarrays using immunohistochemistry. Results The two-step method of laser capture and microarray analysis identified nearly 500 genes whose expression levels were significantly different in prostate epithelial versus stromal tissues. Several genes expressed in epithelial cells (WT1, GATA2, and FGFR-3) were more highly expressed in neoplastic than in non-neoplastic tissues; conversely several genes expressed in stromal cells (CCL5, CXCL13, IGF-1, FGF-2, and IGFBP3) were more highly expressed in non-neoplastic than in neoplastic tissues. Notably, EGR1 was also differentially expressed between epithelial and stromal tissues. Expression of WT1 and EGR1 in cell lines was consistent with these patterns of differential expression. Importantly, WT1 protein expression was demonstrated in tumor tissues and was absent in normal and benign tissues. Conclusions The prostate represents a complex mix of cell types and there is a need to analyze distinct cell populations to better understand their potential interactions. In the present study, LCM and microarray analysis were used to identify novel gene expression patterns in prostate cell populations, including identification of WT1 expression in epithelial cells. The relevance of WT1 expression in prostate cancer was confirmed by analysis of tumor tissue and cell lines, suggesting a potential role for WT1 in prostate tumorigenesis.

Treatment for head and neck cancer precipitates a myriad of distressing symptoms. Patients may be isolated both physically and socially and may lack the self-efficacy to report problems and participate as partners in their care. The goal of this project was to design a telehealth intervention to address such isolation, develop patient self-efficacy, and improve symptom management during the treatment experience. Participatory action research and a review of the literature were used to develop electronically administered symptom management algorithms addressing all major symptoms experienced by patients undergoing treatment for head and neck cancers. Daily questions and related messages were then programmed into an easy-to-use telehealth messaging device, the Health Buddy(R). Clinician and patient acceptance, feasibility, and technology issues were measured. Using participatory action research is an effective means for developing electronic algorithms acceptable to both clinicians and patients. The use of a simple tele-messaging device as an adjunct to symptom management is feasible, affordable, and acceptable to patients. This telehealth intervention provides support and education to patients undergoing treatment for head and neck cancers.

Prior research on instructional media effects suggested that an audiographic approach to World Wide Web based courses would optimize educational effectiveness along with cost effectiveness, although with a possible loss of teacher immediacy that could adversely affect student attitudes. An introductory telecommunication course was converted to an audiographic Web telecourse in which students listened to pre-recorded audio classroom interactions while viewing a detailed course outline and illustrative sites over the World Wide Web. Forty-nine subjects were recruited from a live lecture class and randomly assigned to either the experimental (Web course) group or a control group that took the class in a traditional lecture section. Analysis of covariance (ANCOVA) showed that the experimental group had test scores and student attitude and teacher immediacy ratings equal to those of the control group after controlling for student gender, class level, grade point average and attendance. Open-ended interviews were also conducted to assess qualitative dimensions of student satisfaction. The results supported the audiographic telecourse model as a potentially cost-effective approach to distributing courses over the Web. New directions in research on instructional media effects and teacher immediacy were formulated from an analysis of the unique characteristics of the World Wide Web as an instructional medium.

The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes resulting in diverse responses such as cell growth, proliferation, and apoptosis. Unlike other tumor types, in prostate tumor tissues EGR1 expression is elevated relative to normal tissues. However, the mechanism(s) of regulation of EGR1 in prostate tumor cells are unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using real-time PCR to quantify EGR1 transcripts, EGF induced EGR1 expression levels in a dose and time-dependent manner; and kinase inhibitors, PD98059 and wortmannin, abrogated the EGF-mediated EGR1 response. Analysis of the EGR1 promoter using reporter constructs identified an EGF-responsive region in the proximal promoter containing three potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that EGF enhanced Elk-1 binding at the SRE sites of the EGR1 promoter in PC3 cells, and Elk-1 also bound in the absence of stimulus. Overexpression of Elk-1 enhanced activation of the EGF-responsive region indicating that Elk-1 is sufficient to activate EGR1 in PC3 cells. Similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity indicating that Elk-1 is necessary for EGF-induced EGR1 transcription in both PC3 and LNCaP cells. Taken together, these results demonstrate that EGR1 expression in prostate cancer cells is mediated through an EGF-ERK-Elk-1 signaling cascade. Since elevated EGF and EGF receptor are associated with prostate cancer progression, these results may explain elevated EGR1 in high-grade prostate tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4013. doi:10.1158/1538-7445.AM2011-4013