The Importance of Bcl-x L in the Survival of Human RPE Cells

PURPOSE. In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x L has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x L blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress. METHODS. Cultured human RPE cells from three different donors were transfected with modified, 2Ј-O-methoxyethoxy Bclx L -mismatched control antisense oligonucleotides (ASOs), Bclx L -specific ASOs, and Bcl-x L splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x L into Bcl-x S , a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x L and Bcl-x S mRNA transcript levels were analyzed using genespecific primers with reverse transcription-polymerase chain reaction. Bcl-x L protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H 2 O 2. Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays. RESULTS. After Bcl-x L -specific ASO and SSO transfections, Bcl-x L mRNA and protein levels were significantly reduced. Bcl-x S levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x L -specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H 2 O 2 , used as a model oxidant, further enhanced cell death induced by Bcl-x L -specific ASO and SSO. CONCLUSIONS. Bcl-x L plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x L expression and/or prevent conversion of Bcl-x L to Bcl-x S may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x L and enhance Bcl-x S may be useful in inhibiting unwanted RPE cell proliferation in PVR. (Invest Ophthalmol Vis Sci. 2007;48:3846 -3853) DOI:10.1167/iovs.06-1145 A ge-related macular degeneration (AMD) is the leading cause of irreversible blindness in adults over the age of 65 in the United States and currently affects more than 1.75 million individuals. 1 The exact etiology of AMD is unknown and is probably multifactorial. However, it has been hypothesized that cumulative oxidative stress throughout life and associated RPE cell injury play an important role. In normal eyes, there is very little RPE cell turnover, and most RPE cells survive for an individual's lifetime. In geographic atrophy, an advanced form of AMD, RPE cells die by apoptotic and nonapoptotic mechanisms. 2,3 RPE cell death is accompanied by underlying choriocapillaris atrophy and overlying retinal thinning, ultimately resulting in decreased visual acuity. In our laboratory, we have examined the effect of nuclear transcription factor (NF)-B inhibition on tumor necrosis factor (TNF)-␣-induced apoptosis in human RPE cells. 7 NF-B is a transcription factor that controls a wide range of genes, including genes that regulate apoptosis. 8 TNF-␣ is a cytokine that regulates a variety of RPE cell activities, including cell attachment, spreading, chemotaxis, migration, and proliferation

Human RPE expression of cell survival factors. Invest Ophthalmol Vis Sci.

PURPOSE. To determine basal and tumor necrosis factor (TNF)-␣-regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-B. METHODS. Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-B or ␤-galactosidase and treated with TNF-␣ for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively. RESULTS. Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1␤-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-␣ upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-␣ in a time-dependent manner, whereas c-IAP1, survivin, Bcl-x L , and TRAF-2 protein levels were not influenced by TNF-␣ treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-␣ treatment. Overexpression of mutant IB blocked TNF-␣-induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-x L proteins were localized diffusely in RPE cytoplasm. CONCLUSIONS. Multiple RPE cell survival factors are expressed by human RPE cells. TNF-␣ regulates expression of some of these factors in an NF-B-dependent manner, whereas others are not influenced by NF-B. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). (Invest Ophthalmol Vis Sci. 2005;46: 1755-176

Improving cancer care for underserved populations in an academic and community practice setting: protocol for a community health worker pilot navigation programme

Introduction Delaying cancer treatment following diagnosis impacts health outcomes, including increasing patient distress and odds of mortality. Interventions to promote timely healthcare engagement may decrease patient-reported stress and improve quality of life. Community health workers (CHWs) represent an enabling resource for reducing delays in attending initial oncology treatment visits. As part of an ongoing programme evaluation coordinated by the Merck Foundation, we will implement a pilot navigation programme comprising CHW-conducted needs assessments for supporting patients and their caregivers. We aim to investigate (1) the programme’s influence on patients’ healthcare utilisation within the period between their first diagnosis and initial treatment visit and (2) the logistic feasibility and acceptability of programme implementation.Methods and analysis We will employ a hybrid implementation design to introduce the CHW navigation programme at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center. CHW team members will use a consecutive sampling approach. Participants will complete the Problem-Checklist, Chronic Illness Distress Scale and the Satisfaction with Life Domains instruments. CHWs will provide tailored guidance by sharing information available on the Johns Hopkins Electronic Resource databases. The investigators will evaluate patients’ time to initial oncology treatment and healthcare utilisation by reviewing electronic medical records at 3 and 6 months postintervention. Bivariate analyses will be completed to evaluate the relationships between receiving the programme and all outcome measures.Ethics and dissemination This study’s protocol was approved by the Johns Hopkins School of Medicine’s institutional review board (IRB00160610). Informed consent will be obtained by phone by the CHW navigator. Dissemination planning is ongoing through regular meetings between members of the investigator team and public members of two community advisory groups. Study plans include collaborating with other experts from the Johns Hopkins Institute for Clinical and Translational Research and the Johns Hopkins Center for Health Equity for ideating dissemination strategies

Registration of ‘Byrd’ Wheat

’Byrd’ (Reg. No. CV-1073, PI 664257) hard red winter wheat (Triticum aestivum L.) was developed by the Colorado Agricultural Experiment Station and released in August 2011 through a marketing agreement with the Colorado Wheat Research Foundation. In addition to researchers at Colorado State University (CSU), USDA-ARS researchers at Manhattan, KS, St. Paul, MN and Pullman, WA participated in its development. Byrd was selected from the cross ‘TAM 112’/CO970547- 7 made in 2002 at Fort Collins, CO. TAM 112 (PI 643143) is a hard red winter wheat cultivar released by Texas A&M University in 2005. CO970547-7 is an experimental line from CSU with the pedigree ‘Ike’ (PI 574488)/‘Halt’ (PI 584505). Byrd was selected as an F3:4 line in July 2006 and assigned experimental line number CO06424. Byrd was released because of its superior grain yield under nonirrigated and irrigated production conditions in eastern Colorado, its resistance to stripe (caused by Puccinia striiformis Westend. f. sp. tritici Eriks.) and stem rust (caused by Puccinia graminis Pers.:Pers f. sp. tritici Eriks. & E. Henn.), and its superior milling and bread-baking quality attributes. The name Byrd was chosen in honor of former CSU wheat breeder and director of the CIMMYT Global Wheat Program, Dr. Byrd C. Curtis

Registration of ‘Denali’ Wheat

‘Denali’ (Reg. No. CV-1075, PI 664256) hard red winter wheat (Triticum aestivum L.) was developed by the Colorado Agricultural Experiment Station and released cooperatively by Colorado State University (CSU) and Kansas State University (KSU) in August 2011 through a marketing agreement with the Colorado Wheat Research Foundation. In addition to researchers at CSU and KSU, USDA-ARS researchers at Manhattan, KS, St. Paul, MN, and Pullman, WA participated in its development. Denali was selected from the cross CO980829/‘TAM 111’ made in 2001 at Fort Collins, CO. CO980829 is an experimental line from CSU with the pedigree ‘Yuma’ (PI 559720)/PI 372129//CO850034/3/4*Yuma/4/NEWS12. TAM 111 (PI 631352) is a hard red winter wheat cultivar released by Texas A&M University in 2002. Denali was selected as an F5:6 line in July 2007 and assigned experimental line number CO050303-2. Denali was released because of its superior grain yield under nonirrigated and irrigated production conditions in eastern Colorado, its grain volume weight, and its resistance to stripe rust (caused by Puccinia striiformis Westend. f. sp. tritici Eriks.)

Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.Multiple funders. See acknowledgments within article for details.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.semcancer.2015.09.00