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 p21(WAF1)

All-trans retinoic acid (ATRA) has previously been shown to inhibit the gro wth of OPM-2 human myeloma cells. The growth inhibition was postulated to r esult from a transcriptional downregulation of interleukin-6 receptor alpha (IL-6R alpha) with IL-6R beta (gp130) unaffected. To formally test this hy pothesis, an expression vector designed for constitutive IL-6R alpha expres sion was constructed and used for transfection of OPM-2 cells. Six stable t ransfectants were cloned. The expression of IL-6R alpha was shown by immuno fluorescence with anti-IL-6R alpha antibody and I-125-IL-6 binding. In five of six transfectant clones, cellular IL-6R alpha was 1.5- to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6R alpha expression in the parental OPM-2 cells, it enhanc ed its expression in these five transfectants. The clonogenic growth of the se transfectants, however, remained strongly inhibited by ATRA. Further ana lysis, 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-i nduced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cell ular IL-6R alpha. In contrast, IL-6-induced gp130 phosphorylation was not r educed by ATRA pretreatment in C5 cells, indicating that the expressed IL-6 R alpha was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenou s IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21(WAF1) 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- 6R alpha downregulation and is likely to result from a direct upregulation of p21(WAF1) and consequent dephosphorylation of pRB. (C) 1999 by The Ameri can Society of Hematology.