Cell death in retinoblastoma: Electron microscopic, immunohistochemical, and DNA fragmentation studies

Apparent cell loss by apoptosis occurs in carcinomatous tissue. To investig ate cell death in retinoblastoma (Rb), ultrastructural examination, ApopTag staining, electrophoresis to detect apoptotic DNA fragmentation, and flow cytometric studies were performed. Immunostaining for the oncogenic product s bcl-2 and p53 was also carried out. Relationships between the proliferati on fraction (PF), apoptotic index (Al), and the distribution of bcl-2 and p 53 were investigated according to the degree of histologic differentiation of Rb. Ultrastructurally, two patterns of cell death were seen. Necrotic ce lls exhibited vacuolation of cytoplasmic organelles with a marked lytic cha nge in the karyoplasm and cytoplasm. In contrast, apoptotic cells were char acterized by crescentic margination of chromatin, condensation of karyoplas m and cytoplasm, and fragmentation of the nucleus. Differentiated Rb had a low Al value (<1%), whereas undifferentiated Rb had a high Al value (>8%). The PF of undifferentiated RE (31%) was significantly higher than that of d ifferentiated RE (14%). Analysis of DNA fragmentation using 3'-end labeling with terminal transferase indicated that undifferentiated Rb has increased DNA cleavage. The distribution of apoptotic bodies within Rb was inversely correlated with the expression of bcl-2. A majority of tumor cells of diff erentiated Rb were negative for p53, whereas 20-40% of tumor cells of undif ferentiated Rb showed a positive reaction far p53. These findings suggest t hat the degree of susceptibility to apoptosis is closely related to PF, is inversely related to the degree of differentiation of Rb, and is protected by oncogene bcl-2.