P53 REGULATES APOPTOSIS IN HUMAN RETINOBLASTOMA

Objectives: To determine whether apoptosis is a significant mode of ce ll death in human retinoblastoma (RE) and if it is regulated by the ex pression of p53.Methods: Apoptosis was analyzed using the criterion of internucleosomal DNA. degradation as determined by agarose gel electr ophoresis of DNA isolated from tumor specimens. Individual cells under going apoptosis were identified using terminal transferase-mediated bi otindUTP nick end labeling (TUNEL) of fragmented DNA. The expression o f p53 and WAF1 (a protein involved in p53-mediated cell cycle arrest) in human RE was determined by immunocytochemical analysis. The functio n of p53 in human RE cell lines was tested by transfecting them with a complementary DNA encoding a temperature-sensitive isoform of murine p53 under the control of a strong viral promoter. Results: DNA from RE tumor specimens showed a strong nucleosomal ladder of DNA fragments t ypical of apoptosis. The TUNEL staining indicated that poorly and mode rately differentiated cells in tumors were undergoing DNA fragmentatio n. Immunoreactivity for p53 was variable. Cells expressing low levels of p53 seemed viable and expressed WAF1. Cells expressing high levels of p53 were found immediately adjacent to cells undergoing apoptosis. Human RE cells in culture that were expressing a murine temperature se nsitive isoform of p53 died at temperatures that allow this protein to assume a wild-type conformation. Conclusions: Apoptotic cell death is prevalent in RE. The close association of p53-immunoreactive cells an d cells undergoing apoptosis in human tumors, and the ability of exoge nous p53 to stimulate cell death in cultured human RB cells, suggests that p53 plays a role in regulating cell death in RE.