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Bcl-X_L Is Expressed in Neuroblastoma Cells and Modulates Chemotherapy-induced Apoptosis¹

Departments of Pediatrics [M. G. D., R. J., V. P. C.) and Pathology [G. N.], University of Michigan, Ann Arbor, Michigan 48109-0684; Division of Hematology-Oncology, Case Western Reserve University, Cleveland, Ohio 44106-4937 [M. J. C.]; and Howard Hughes Medical Center and Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, Illinois 60637 [C. B. T.]

bcl-x is a new member of the bcl-2 gene family and is highly expressed in neural tissues. The present study was designed to determine the expression of the bcl-x gene products in neuroblastoma (NB) and their role in the modulation of chemotherapy-induced apoptosis. Twenty-seven NB cell lines were screened by quantitative immunoprecipitation for Bcl-X_L, Bcl-X_S, and Bcl-2 expression. None of the cell lines expressed Bcl-X_S. Twenty-four of 27 (88%) of the NB cell lines expressed Bcl-X_L and 21 of 27 (78%) were positive for Bcl-2. The level of Bcl-x_L and Bcl-2 expression was variable among the lines analyzed. Bcl-2 expression was restricted to cells of chromaffin lineage, whereas Bcl-x_L was seen in both chromaffin and nonchromaffin lines. To determine whether Bcl-x_L could mediate chemotherapy resistance, a NB cell line expressing negligible levels of Bcl-x_L was transfected with a bcl-x_L expression vector, and unique clones were generated expressing variable levels of Bcl-x_L. Cells were treated either with cisplatinum (CP), 4-hydroperoxy-cyclophosphamide (4-HC), or etoposide (VP-16) to induce apoptosis, and cell viability and DNA degradation were determined. Following treatment with CP or 4-HC, Bcl-x_L-expressing cells showed significantly increased viability as compared to vector-transfected controls (P < 0.005). Flow cytometric analysis of propidium iodide-stained nuclei following CP or 4-HC treatment revealed significantly increased DNA degradation in controls as compared to Bcl-x_L-expressing lines (P < 0.004). DNA analysis by pulsed-field gel electrophoresis revealed high molecular weight (40 kb) DNA degradation in controls, whereas the DNA in cells expressing Bcl-x_L was largely intact. In contrast to CP and 4-HC, results with VP-16 revealed a short-term delay in the onset of apoptosis in Bcl-x_L-expressing cells with no long-term survival advantage. The results of these studies indicate Bcl-x_L is expressed in NB cells and functions in a manner analogous to Bcl-2 by inhibiting chemotherapy-induced apoptosis.

¹ This work was supported by National Institute of Child Health Development Grant CHRC HD2880-3 (to V. P. C.), NIH RO1 CA64556-01 (to G. N.), and American Cancer Society EDT-30 (to M. J. C.). G. N. is supported by NIH Research Career Development Award CA 64421-01. M. G. D. is a University of Michigan Cancer Center Pardee Fellow.

² To whom requests for reprints should be addressed, at Department of Pediatrics, A510D, MSRB 1, Box 0684, 1500 W. Medical Center Dr., University of Michigan, Ann Arbor, MI 48109-0684.

Received 1/25/95. Accepted 4/17/95.

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