Retinoid-binding Proteins in Retinoblastoma Tumors

Abstract

A combination of Western blot, Northern blot, and radiolabeled ligand-binding techniques was used to investigate retinoid-binding proteins in retinoblastoma (RB) cells from fresh tumors and from 19 RB tumor lines cultured in vitro. Using rabbit anti-bovine cellular retinal-binding protein (CRA1BP) antibodies, no CRA1BP could be detected. As determined by [³H]retinol binding, cellular retinol-binding protein was sometimes not detectable but averaged 2.3 ± 2.7 x̄ ± SD, n = 7) pmol [³H]retinol bound/mg protein, similar to adult retina cytosol. Using [³H]retinoic acid as ligand, cellular retinoic acid-binding protein was not detectable in some lines and averaged 1.0 ± 1.2 (x̄ ± SD, n = 7) pmol [³H]retinoic acid bound/mg protein, well below the adult retina cytosol level of 94.4 ± 16.3 (x̄ ± SD, n = 4) pmol [³H]retinoic acid bound/mg. Using rabbit antibovine interstitial retinol-binding protein (IRBP) antibodies, IRBP of the same molecular mass as human IRBP (135,000) was detected in the medium from all cultured RB cells and averaged 75.9 ± 19.2 pmol/10⁸ cells (bovine IRBP immunochemical equivalents). Cytosol levels were less than 1% of the medium. Using a human IRBP complementary DNA probe, levels of IRBP RNA transcripts in 19 RB cell lines were comparable to adult retina. The Y-79 RB cell line was different from the others; the amount of IRBP in the medium was only about 1% of the RB cell lines. Levels of cellular retinol-binding protein were comparable with the other lines, but cellular retinoic acid-binding protein was 9 times more abundant. IRBP RNA transcripts in Y-79 cells were below the limits of detectability but appeared at low levels after induction of differentiation of Y-79 by 10^-6 m retinoic acid. Although this cell line has been in culture longer than the others, it may also have been initiated at an earlier stage of retinal development.