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D. Cobrinik, A. Tewari, C. Cheng, S. Rafii, D.H. Abramson, T.C. Lee; Improved Rb Transduction and Growth of Retinoblastoma Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2829.
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Retinoblastomas form due to the loss of Rb in early retinal cells. One way to assess how Rb suppresses retinoblastoma is to examine the effects of restoring Rb. However, this approach has been hindered by difficulties in attaining efficient Rb transduction, and by difficulties in culturing primary retinoblastoma cells in a manner that resembles their growth in vivo. This study sought to improve Rb transduction, to identify genes that are regulated by the ectopic Rb, and to culture primary retinoblastoma cells under conditions that emulate the in vivo tumor environment.
Rb and various Rb mutants were introduced into Y79 cells, using either an MSCV–GFP retroviral vector or a novel lentiviral vector that expresses Rb and GFP from a bidirectional promoter. Transduced Y79 cells were purified by sorting, and their RNAs examined by probing Affymetrix U133 Plus arrays and by quantitative RT–PCR. Primary retinoblastoma cells from two tumors were plated either in the absence of stroma or over stroma that was derived from a prior retinoblastoma explant, and were cultured under ambient hyperoxic (18% O2) or normoxic (3% O2) conditions.
Concentrated retroviral Rb vectors infected up to 40% of Y79 cells, whereas unconcentrated lentiviral Rb vectors infected up to 79% of cells. Retroviral and lentiviral transduction of Rb, but not the non–functional RbΔ21 or the low penetrance Rb661W, dramatically suppressed proliferation. Microarray and RT–PCR analyses identified Rb–regulated genes that are implicated in the cellular stress response and in photoreceptor differentiation. Tumor–derived stroma and normoxia improved survival of primary retinoblastoma cells, and in combination supported robust proliferation.
Lentiviral vectors efficiently transduce retinoblastoma cells and may thereby facilitate biochemical analysis of Rb function. Rb regulates stress–response and differentiation–related genes that might contribute to the suppression of retinoblastoma. Tumor–derived stroma and normoxia substantially improve primary retinoblastoma cell survival and proliferation, and provide a physiologically relevant in vitro system with which to examine Rb tumor suppressor function.
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