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Regulation of Bcl-2 Oncoprotein Levels with Differentiation of Human Neuroblastoma Cells¹

La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037 [M. H., S. K., S. T., D. C-H.]; Laboratory of Cellular and Biochemical Genetics, Memorial Sloan-Kettering Cancer Center, New York 10021 [B. A. S., J. L. B.]; and Department of Biological Sciences, Fordham University, Bronx, New York 10458 [R. A. R.]

When established in culture, human neuroblastoma cell lines typically are comprised of heterogeneous cellular subpopulations, including neuroblastic (N-type), substrate-adherent (S-type), and intermediate (I-type) cells that can be distinguished by their characteristic morphologies and expression of differentiation-associated antigens. Here we examined the relative levels of the Bcl-2 oncoprotein in 15 clones derived from four different neuroblastoma cell lines. Among six clones isolated from the SK-N-SH line, levels of p26-Bcl-2 correlated with morphology and differentiation markers with the hierarchy of bcl-2 expression being: N-type cells > N/I-type > I-type > S-type. Furthermore, stimulation of one of the N-type clones, SH-SY5Y, with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induced differentiation toward a more neuronal-like phenotype and resulted in a 5- to 10-fold elevation in the relative levels of Bcl-2 protein. High relative amounts of p26-Bcl-2 protein were also found in an N-type clone derived from the SMS-KCN line. In two N-type clones derived from the LA-N-1 line, however, levels of Bcl-2 protein were only moderately elevated, and in one N-type clone from the SK-N-BE(2) line the levels of Bcl-2 protein were low. Thus, high relative levels of Bcl-2 oncoprotein are not a universal feature of N-type cells (three of six clones tested). In contrast, all 5 of the S-type clones evaluated contained relatively low levels of Bcl-2 protein, suggesting that these cells (which may represent embryonic precursors of Schwann, glial, and melanocytic cells) do not typically express the bcl-2 gene at high levels. Consistent with this inverse correlation between Bcl-2 protein levels and S-type characteristics, stimulation of an I-type clone derived from the SK-N-BE(2) line with 5-bromodeoxyuridine was accompanied by an accumulation of S-type cells in these cultures, decreased Bcl-2 protein, diminutions in the neuronal markers neurofilament-M and neuron-specific enolase, and an increase in the relative levels of the S-type marker proteins vimentin and ß-2-microglobulin. Conversely, stimulation of this I-type clone with retinoic acid resulted in an accumulation of N-type cells (which are thought to represent embryonic precursors of sympathetic neurons), decreased vimentin and ß-2-microglobulin, increased neurofilament-M, and a marked elevation in p26-Bcl-2. To begin to explore the functional significance of variations in the levels of Bcl-2 protein among neuroblastomas, a cell line with I-type characteristics and relatively low levels of p26-Bcl-2 (IMR-5) was stably infected with either a control or Bcl-2-encoding retrovirus, thus producing the lines IMR-5-BCL-2 and IMR-5-NEO. IMR-5-BCL-2 cells contained 5-fold higher levels of Bcl-2 protein than IMR-NEO and were markedly more resistant to killing by several chemotherapeutic drugs as measured in short-term cytotoxicity assays. Taken together, these findings indicate that Bcl-2 protein levels are regulated during the differentiation of at least some neuroblastoma cell clones in culture, thus raising the possibility of pharmacologically altering bcl-2 gene expression in neuroblastomas in vivo and thereby modulating sensitivity to chemotherapeutic drugs.

¹ This work was supported by NIH Grants CA-47956, CA-08748, and CA-41520, by Grant DHP-32B from the American Cancer Society, and by a Scholar Award to J. C. R. from the Leukemia Society of America.

² Current address: Mie University School of Medicine, Department of Pediatrics, Mie 514, Japan.

³ Current address: Hopitaux de Paris, Service d'Anatomie, Paris 92118, France.

⁴ To whom requests for reprints should be addressed, at La Jolla Cancer Research Foundation, Cancer Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037.

Received 6/10/93. Accepted 8/ 6/93.

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