BCL-2 INHIBITS CHEMOTHERAPY-INDUCED APOPTOSIS IN NEUROBLASTOMA

bcl-2 is the first member of a new class of protooncogenes the product s of which inhibit programmed cell death (PCD) or apoptosis. We have p reviously determined that Bcl-2 is expressed in a significant percenta ge of untreated primary neuroblastoma (NBL) tumors. In these specimens Bcl-2 expression correlated with other markers of poor prognosis sugg esting a role for Bcl-2 in the malignant behavior of NBL tumor cells. To investigate this possibility, a Bcl-2-negative human NBL cell line (Shep-1) was transfected with a beta expression vector (pSFFVneo-bcl-2 ). Multiple unique clones were isolated which showed variable levels o f Bcl-2 protein by quantitative immunoprecipitation. Vector-transfecte d controls were generated simultaneously. Clones expressing high level s of Bcl-2 were resistant to cisplatin- and etoposide-induced cytotoxi city in a dose-dependent manner. Analysis of propidium iodide-stained nuclei by flow cytometry after cisplatin or etoposide treatment reveal ed marked DNA degradation in vector-transfected controls whereas bcl-2 transfectants showed a dose-dependent inhibition of DNA degradation. Analysis by pulsed-field gel electrophoresis revealed relatively large fragment DNA degradation (similar to 50 kilobases) in the absence of internucleosomal degradation in vector-transfected control cells treat ed with either cisplatin or etoposide. In contrast, Bcl-2-expressing c ells showed significantly less DNA degradation at all time points. The se single gene transfection experiments have revealed that expression of Bcl-2 renders specific NBL cells resistant to chemotherapy-induced PCD and support the hypothesis that Bcl-2 enhances the malignant pheno type of NBL by promoting tumor resistance to chemotherapy agents.