Acute effects of Rb in retinoblastoma cells

Abstract

Abstract: : Purpose:Retinoblastomas occur due to mutation of the RB–1 gene and loss of Rb protein. Previous studies displayed variable effects of restoring Rb to retinoblastoma cells, with Rb either having no effect, inhibiting growth in vitro, or inhibiting growth after engraftment in vivo. A potential basis for this variability is that growth of Rb–reconstituted cells prior to analysis may select for Rb–resistant clones. Here, two novel approaches were used to examine the acute effects of Rb in Y79 retinoblastoma cells. Methods:As a first approach, Rb was expressed using a MSCV retroviral vector that co–expresses green fluorescent protein (GFP). Infected cells were detected by flow cytometry and fluorescence microscopy, and analyzed for morphology, growth, and cell cycle stage. As a second approach, Y79 cells were transduced with 1) a vector in which a neo resistance gene and poly(A) signal were flanked by loxP sites and followed by GFP or RB–1 cDNAs; and 2) a Cre recombinase fused to a tamoxifen–responsive estrogen–receptor (Cre–ERT2). Cells were treated with tamoxifen to activate the recombinase, excise the neo–poly(A) cassette, and induce GFP or RB–1. Results:Infection of cells with the MSCV vector or derivatives expressing Rb, Rb^Δ21 (encoding a non–functional protein), or Rb^661W (encoding a low penetrance Rb mutant) resulted in ∼8% of cells becoming GFP(+) at 36 h. By 72 h, cells transduced with wild type but not mutant Rb were enlarged, accumulated in the G1 phase of the cell cycle (with 60% fewer cells in S, G2, and M), and had increased side–scatter consistent with entry into a senescence–like state. Rb–transduced cells were also growth impaired and comprised ∼1–2% of cells after two weeks. However, cells remaining after two weeks grew at a rate similar to vector–transduced controls. Induction of Rb with tamoxifen resulted in cells becoming enlarged, accumulating in G1, and having increased side–scatter; as for Rb retrovirus transduced cells. Rb also increased the proportion of cells expressing senescence–associated ß–galactosidase (SA–ß–gal) from 1.5 to 25%. In control cultures, induction of GFP did not evoke cell enlargement, G1 block, or SA–ß–gal activity. Conclusions:These studies indicate that Rb induces an acute cell cycle block and senescence–associated features in Y79 retinoblastoma cells, followed by emergence of resistant cells. Neither the non–functional Rb^Δ21 nor the low penetrance Rb^661W inhibited cell growth. Because Rb^661W suppresses a subset of retinoblastomas, the results suggest that this mutant may utilize a mechanism that is operative in vivo but not in vitro.

Keywords: retinoblastoma • oncology • pathobiology