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Primary myeloma cells rapidly apoptose as soon as they are removed from their bone-marrow environment. A likely explanation is that the tumor environment produces survival factors that may counteract a spontaneous activation of pro-apoptotic program. Additional factors may trigger cell cycling in surviving myeloma cells. Interleukin-6 (IL-6) is a well recognized myeloma cell growth factor produced mainly by the tumor environment. However, myeloma cells themselves may produce low levels of autocrine IL-6. The respective roles of paracrine versus autocrine IL-6 are a matter of debate. We investigated these roles using the XG-6 myeloma cell line whose growth is dependent on addition of exogenous IL-6, despite its weak IL-6 mRNA and protein expression. The apoptosis induced by exogenous IL-6 deprivation was blocked by transferring the Mcl-1 gene coding for an anti-apoptotic protein in XG-6 cells. An XG-6Mcl-1 cell line which can survive and grow without adding IL-6 was obtained. We show that anti-IL-6 or anti-gp130 antibodies abrogated cell cycling whereas they did not affect cell survival. These data indicate that the weak autocrine IL-6 produced by myeloma cells is sufficient to trigger cell cycling whereas their survival requires large exogenous IL-6 concentrations. This important role of autocrine IL-6 has to be considered when evaluating the mechanism of action of myeloma cell growth factors. These factors may actually block an activated pro-apoptotic program, making possible a weak production of autocrine IL-6 to promote cell cycling.

Interleukin-6 (IL-6) is a major survival factor for malignant plasma cells involved in multiple myeloma. Using an RNase protection assay, we looked for gene expression of 10 anti- and proapoptotic Bcl-2-family proteins in 12 IL-6-dependent human myeloma cell lines (HMCL). A high Mcl-1 gene expression was found in all HMCLs and the other genes were variably expressed. Out of the 10 Bcl-2-family members, only the Mcl-1 gene was regulated by IL-6. Upon starvation of IL-6, Mcl-1 gene expression decreased in association with myeloma cell apoptosis and was upregulated after adding IL-6 again in association with myeloma cell survival. A constitutive Mcl-1 expression was induced with an Mcl-1-GFP retrovirus in two IL-6-dependent HMCLs. The Mcl-1 HMCLs have a marked reduced apoptosis upon IL-6 starvation compared to HMCLs transduced with control GFP retrovirus and may grow without adding IL-6. These data emphasize the major role of Mcl-1 antiapoptotic protein in the IL-6-induced survival of human myeloma cells.

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.