| It is important to understand how the genetic heterogeneity associated with multiple myeloma may lead to differences in signal transduction, cell cycle, and response to therapy. We used model cell lines to study the effect of mutations in p53 and ras on growth and therapeutic response of myeloma cells. Stable expression of wt p53 in IL-6 producing U266 cells results in suppression of IL-6 expression and subsequent cell cycle arrest and protection from apoptosis induced by doxorubicin and melphalan. Addition of IL-6 resulted in cell cycle progression and increased sensitivity to both doxorubicin and melphalan. ANBL6 is an IL-6 dependent cell line that is sensitive to dexamethasone, doxorubicin, and melphalan. Addition of IL-6 protects the cells from dexamethasone and melphalan-induced apoptosis; however, it enhances apoptosis induced by doxorubicin. We used the ANBL6 cell line transfected with either a N-ras12 or K-ras12 gene to study the effect of activating ras mutations. Both N-ras12 and K-ras12 were able to protect from apoptosis induced by dexamethasone, doxorubicin, or melphalan. It was surprising that both N-ras12 and K-ras12 could protect from doxorubicin-induced apoptosis, but that IL-6 enhanced dox-induced apoptosis. We used an activated Ras interaction assay to study the kinetics of Ras activation induced by IL-6. IL-6 is able to transiently activate both N- and K-ras in the ANBL6 cell line. In addition, increasing concentrations of IL-6 are able to activate increasing levels of both N- and K-ras. The level of Ras activation correlates with protection from dex-induced apoptosis, but does not explain why N- or K-ras12, but not IL-6, is able to inhibit doxorubicin-induced apoptosis. To explore the mechanism of apoptotic protection provided by N-ras12 further, we examined at the apoptotic pathways activated by doxorubicin. Doxorubicin induces apoptosis through the release of cytochrome c from the mitochondria followed by the activation of caspase 9 and caspase 3. Expression of N-ras12 blocks apoptosis downstream of cytochrome c release by inhibiting caspase 9 activity. This is not associated with changes in expression of IAP, Apaf-1, or SMAC. In addition the apoptotic protection appears to be independent of signaling through either MEK1 or PI3K. |
