REGULATION OF BCL-2 ONCOPROTEIN LEVELS WITH DIFFERENTIATION OF HUMAN NEUROBLASTOMA-CELLS

When established in culture, human neuroblastoma cell lines typically are comprised of heterogeneous cellular subpopulations, including neur oblastic (N-type), substrate-adherent (S-type), and intermediate (I-ty pe) cells that can be distinguished by their characteristic morphologi es and expression of differentiation-associated antigens. Here we exam ined the relative levels of the Bcl-2 oncoprotein in 15 clones derived from four different neuroblastoma cell lines. Among six clones isolat ed from the SK-N-SH line, levels of p26-Bcl-2 correlated with morpholo gy and differentiation markers with the hierarchy of bcl-2 expression being: N-type cells > N/I-type > I-type > S-type. Furthermore, stimula tion of one of the N-type clones, SH-SY5Y, with the phorbol ester, 12- O-tetradecanoylphorbol-13-acetate, induced differentiation toward a mo re neuronal-like phenotype and resulted in a 5- to 10-fold elevation i n the relative levels of Bcl-2 protein. High relative amounts of p26-B cl-2 protein were also found in an N-type clone derived from the SMS-K CN line. In two N-type clones derived from the LA-N-1 line, however, l evels of Bcl-2 protein were only moderately elevated, and in one N-typ e 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 f eature of N-type cells (three of six clones tested). In contrast, all 5 of the S-type clones evaluated contained relatively low levels of Bc l-2 protein, suggesting that these cells (which may represent embryoni c precursors of Schwann, glial, and melanocytic cells) do not typicall y express the bcl-2 gene at high levels. Consistent with this inverse correlation between Bcl-2 protein levels and S-type characteristics, s timulation 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 i n 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 beta -2-microglobulin. Conversely, stimulation of this I-type clone with re tinoic acid resulted in an accumulation of N-type cells (which are tho ught to represent embryonic precursors of sympathetic neurons), decrea sed vimentin and beta-2-microglobulin, increased neurofilament-M, and a marked elevation in p26-Bcl-2. To begin to explore the functional si gnificance of variations in the levels of Bcl-2 protein among neurobla stomas, a cell line with I-type characteristics and relatively low lev els 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 IM R-5-NEO. IMR-5-BCL-2 cells contained almost-equal-to 5-fold higher lev els of Bcl-2 protein than IMR-NEO and were markedly more resistant to killing by several chemotherapeutic drugs as measured in short-term cy totoxicity assays. Taken together, these findings indicate that Bcl-2 protein levels are regulated during the differentiation of at least so me 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.