Our laboratory performs research on the molecular pathways involved in early IL-6 signalling and IL-6-mediated plasmacytoma growth, and on the characterization and identification of alterations that lead to independent growth. A major aspect of our studies focuses on the transcriptional regulation of junB, an immediate early gene induced by IL-6 in plasmacytomas. We have documented a novel 222 base pair multi-site enhancer region downstream of the junB coding region that both upregulates the junB promotor (75-fold) and confers inducibility by IL-6 in plasmacytomas. Extensive mutational and DNA-binding analyses reveal a key role for a novel overlapping STAT3/NF-kB/SP-1 site, however, this site alone is unable to drive IL-6-inducible transcription. We are currently characterizing the contribution and interaction of adjacent fragments within this novel enhancer region. Studies with IL-6-dependent and IL-6-independent plasmacytomas indicate that the transition to IL-6-independence is a key step in the malignant progression of murine plasmacytomas. Our results in indicate that the progression to IL-6 independence is mediated by a mechanism other than autocrine production of IL-6. We also found junB transcription to be constitutively active in independent plasmacytoma cells. DNA-binding and transient expression studies incorporating the above 222bp junB enhancer region indicate that the loss of growth factor dependence is associated with constitutive activation of STAT3. Thus, the alterations leading to IL-6-independent growth appear to be """"""""upstream"""""""" of the activation of the STAT3/APRF transcription factor and the subsequent transcriptional activation of junB. We are continuing to evaluate the activation of other molecules that may be responsible for the constitutive activation of STAT3/APRF site. The ability to restore IL-6-dependence to IL-6-independent plasmacytomas by the introduction of normal DNA with normal lymphocytes (eg. classic B-cell hybridomas) suggests that the transition to growth factor independence may involve the inactivation or functional loss of a negative growth regulatory/tumor suppressor gene. Identification of the gene responsible may elucidate the IL-6-independence mechanism.
