IMMUNOLOCALIZATION OF THE RETINOBLASTOMA PROTEIN IN THE HUMAN EYE ANDIN RETINOBLASTOMA

Purpose. To determine whether, by employing recent advances in immunoc ytochemical technique, it is possible to identify reliably the product of the retinoblastoma (RB) susceptibility gene, p110(RBl), in formali n-fixed, paraffin-embedded eyes with commercially available primary an tibodies. If so, the authors sought to determine the distribution of p 110(RBl) in normal human eyes and retinoblastomas in hopes of better u nderstanding its function. Methods. Four antibodies to p110(RBl) were tested on normal human and monkey eyes, as well as on six human retino blastomas. The human tissue was formalin-fixed and paraffin-embedded. Free antigen was used for an absorbed control. The monkey eye had been injected with tritiated (H-3) thymidine 24 hours before enucleation. Results. Three of the four antibodies had acceptable reactivity (a pol yclonal against the carboxyl-terminal epitope and two monoclonals agai nst epitopes near the amino-terminus). Staining was confined to nuclea ted cells of the normal eyes and was strongest in the cycling cells of the lenticular and corneal epithelia. Somewhat weaker reactivity was seen in those corneal epithelial cells in S phase as determined by aut oradiography for H-3-thymidine. Of the six retinoblastomas, three had strong nuclear and cytoplasmic staining and one showed weaker staining in the tumor cells than in the adjacent vascular endothelial cells. T wo of the tumors had positive cytoplasmic and negative nuclear stainin g with an amino-terminal antibody but were completely negative for car boxyl-terminal p110(RBl) reactivity. Conclusions. Using appropriate im munocytochemical techniques, p110(RBl) can be identified in paraffin-e mbedded tissues with commercially available antibodies. The observed s taining pattern in retinoblastoma suggests that RBl transcripts are co mmonly produced in the tumor cells and that they are sometimes, but no t always, capable of nuclear binding. Thus, nuclear binding by the RBI gene product per se is not sufficient to prevent tumor growth, nor do es it indicate the presence of a normal transcript.