Rb induces a proliferative arrest and curtails Brn-2 expression in retinoblastoma cells

Abstract

Background: Retinoblastoma is caused by loss of the Rb protein in early retinal cells. Although numerous Rb functions have been identified, Rb effects that specifically relate to the suppression of retinoblastoma have not been defined.

Results: In this study, we examined the effects of restoring Rb to Y79 retinoblastoma cells, using novel retroviral and lentiviral vectors that co-express green fluorescent protein (GFP). The lentiviral vector permitted transduction with sufficient efficiency to perform biochemical analyses. Wild type Rb (RbWT) and to a lesser extent the low penetrance mutant Rb661W induced a G0/G1 arrest associated with induction of p27KIP1 and repression of cyclin E1 and cyclin E2. Microarray analyses revealed that in addition to down-regulating E2F-responsive genes, Rb repressed expression of Brn-2 (POU3F2), which is implicated as an important transcriptional regulator in retinal progenitor cells and other neuroendocrine cell types. The repression of Brn-2 was a specific Rb effect, as ectopic p27 induced a G0/G1 block, but enhanced, rather than repressed, Brn-2 expression.

Conclusion: In addition to Rb effects that occur in many cell types, Rb regulates a gene that selectively governs the behavior of late retinal progenitors and related cells.

Figure 1

Effect of retroviral Rb transduction on Y79 proliferation. A. Structure of the MSCV-GFP retroviral vector and Rb derivatives. Rb is expressed from the MSCV LTR, and GFP from a PGK promoter. B. Western analysis of Rb expression 3 days after infection. C, D. Prevalence of GFP+ cells after infection with the indicated vectors, relative to that at 44 h after infection, displayed as a time course (C) or at day 12 for a separate experiment (D). E. Effect of Rb on cell cycle distribution 84 h after infection. F. Effect of Rb on cell size. Cells infected with MSCV-GFP or MSCV-GFP-Rb were identified by GFP fluorescence and stained for Rb (left) or measured for forward scatter (FSC) by flow cytometry (right). For C-E, infections were in triplicate, data points represent averages, and error bars indicate standard deviation.

Figure 2

Long-term culture of Rb-transduced cells selects for low Rb expression. A. Rb expression in Y79 cells 3 days after transduction with MSCV-GFP and Rb derivatives (lanes 1–3), or in puromycin resistant Y79 cells 26 days after transduction with MSCV-Puro and Rb derivatives (lanes 4–6). Lysates from infected cultures were combined with mock-infected cell lysates to normalize the proportion of GFP+ cells. B. Rb expression after transduction with the indicated MSCV-Puro viruses and puromycin selection, at 19 days (lanes 1, 3, and 5) and 26 days (lanes 2, 4, and 6) after infection. For A and B, actin expression is displayed as a loading control. C. RT-PCR analysis of RB1 and GAPDH mRNA in puromycin resistant cells after infection with the indicated viruses, with PCR for 22, 26, and 30 cycles. D. Prevalence of GFP+ cells at 12 days relative to that at 44 h after infection of Y79 cells, or after infection of puromycin-selected Y79 derivatives that were earlier infected with MSCV-Puro or MSCV-Puro-Rb.

Figure 3

Lentiviral transduction of Y79 with BE-GFP-Rb. A. Structure of BE-GFP-Rb^WT. Rb and GFP are expressed from a bidirectional promoter consisting of EF1α enhancer + promoter (light blue) and a minimal CMV promoter (dark blue) that requires a heterologous enhancer for activity [56]. B. Flow cytometric analysis of GFP expression in transduced cultures at 56 h after infection. Numbers indicate the percent GFP+ cells. C. Y79 cell cycle profiles 60 h after mock infection or transduction with the indicated BE-GFP viruses. D. Western analysis of Rb, p27, and α-actin 60 h after mock infection (lane 1) or infection with the indicated BE-GFP viruses (lanes 2–6).

Figure 4

Comparison of Rb and p27 cell cycle effect. A. Y79 cells were mock infected (lane 1) or infected with BE-GFP, BE-GFP-Rb, or BE-GFP-p27 (lanes 2–4); and examined by western blotting for p27 expression, and for actin expression as a loading control. B. Infected cells were stained with propidium iodide and examined for cell cycle position.

Figure 5

Gene expression effects of Rb and p27. cDNA was prepared from cells at 60 h after infection with the BE-GFP vector and A) the indicated Rb derivatives, or B) either Rb^WT or p27 derivatives. The cDNA was subjected to qRT-PCR for the indicated genes. Expression levels were normalized to β-actin and are displayed relative to that of vector-transduced samples. Values indicate averages of duplicate analyses and error bars indicate standard deviation.