NEOPLASIA Growth Inhibition of a Human Myeloma Cell Line by All-trans Retinoic Acid Is Not Mediated Through Downregulation of Interleukin-6 Receptors but Through Upregulation of p21WAF1

All-trans retinoic acid (ATRA) has previously been shown to inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of interleukin-6 receptor a (IL-6Ra) with IL-6Rb (gp130) unaffected. To formally test this hypothesis, an expression vector designed for constitutive IL-6Ra expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The expression of IL-6Ra was shown by immunofluorescence with anti–IL-6Ra antibody and 125I-IL-6 binding. In five of six transfectant clones, cellular IL-6Ra was 1.5to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6Ra expression in the parental OPM-2 cells, it enhanced its expression in these five transfectants. The clonogenic growth of these transfectants, however, remained strongly inhibited by ATRA. Further analysis, comparing the parental OPM-2 cells and a representative transfectant, clone C5, showed that IL-6 caused rapid tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones. Pretreatment with ATRA greatly reduced IL-6– induced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cellular IL-6Ra. In contrast, IL-6–induced gp130 phosphorylation was not reduced by ATRA pretreatment in C5 cells, indicating that the expressed IL-6Ra was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenous IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21WAF1 expression and cause dephosphorylation of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells. Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the growth inhibitory activity of ATRA is not mediated through cellular IL-6Ra downregulation and is likely to result from a direct upregulation of p21WAF1 and consequent dephosphorylation of pRB. r 1999 by The American Society of Hematology.