Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression.

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.

Interleukin 6 induces monocyte chemoattractant protein-1 expression in myeloma cells.

Interleukin 6 (IL-6) is known to play an important role in the biology of the malignant plasma cells in multiple myeloma. In an effort to better understand IL-6 stimulated myeloma cell growth, we have performed gene expression profiling to identify IL-6 early response genes. Using the KAS-6/1 IL-6-dependent human myeloma cell line, IL-6 stimulation dramatically induced expression of monocyte chemoattractant protein-1 (MCP-1) mRNA. To verify this result, we used reverse transcriptase PCR and RNAse protection assays and demonstrated using both assays that MCP-1 is indeed an IL-6 responsive gene in a variety of IL-6-responsive myeloma cell lines. Moreover, we also demonstrated IL-6 stimulated MCP-1 secretion by the myeloma cell lines as well as by fresh patient tumor cells. Lastly, we present evidence that fresh patient tumor cells express mRNA for the MCP-1 receptor, CCR2, as do myeloma cell lines along with a second MCP-1 receptor, CCR11. Although MM cell chemotaxis in response to MCP-1 was only minimal, we were able to demonstrate that MCP-1 stimulated activation of MAPK. Because of the important role that this chemokine plays in both angiogenesis and bone homeostasis, and the ability of MCP-1 to activate myeloma cells, these results suggest a new mechanism by which IL-6 may contribute to disease pathogenesis.

Inhibition of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and functions both as an apoptosis inhibitor and a regulator of cell division. Survivin overexpression is common in many human tumors and correlates with survival in large cell non-Hodgkin's lymphoma. To evaluate this molecule as a potential therapeutic target in large-cell lymphoma, we evaluated the effect of survivin inhibition both in vitro and in vivo. Using an antisense oligonucleotide (ASO) approach, cell growth was significantly inhibited in the DoHH2, RL and HT lymphoma cell lines. In a lymphoma xenograft model, the development of tumors as well as the growth of established tumors was inhibited in the survivin ASO-treated mice compared to controls. To assess the efficacy of the survivin ASO in combination with other biological agents, we combined the survivin ASO with an anti-CD20 monoclonal antibody, rituximab. The effect of survivin ASO and rituximab in combination was additive in vitro. In vivo, however, suppression of tumor growth with the combination was not significantly superior to controls. We conclude that inhibition of survivin expression is an attractive therapeutic strategy in aggressive non-Hodgkin's lymphomas, and that combining survivin ASO with rituximab may enhance the efficacy of this approach.

Increased expression of extracellular matrix metalloproteinase inducer (CD147) in multiple myeloma: role in regulation of myeloma cell proliferation.

Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.

Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX.

DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.

A role for insulin-like growth factor in the regulation of IL-6-responsive human myeloma cell line growth.

Insulin-like growth factors (IGF-I, IGF-II) have long been recognized as important mitogens in many types of malignancies. Because the role of IGFs in growth control of myeloma cells has not been extensively examined, we have used a panel of IL-6-responsive myeloma cell lines to address this issue. Initial studies demonstrated that IGF-I and IGF-II significantly enhanced DNA synthesis by each of the four cell lines, even when assayed in the absence of IL-6. The specificity of the IGF response was confirmed using an IGF-I receptor Ab, and additional studies demonstrated that IGF responsiveness did not result from induction of autocrine IL-6 expression. When IL-6 responsiveness was assayed, three of four cell lines synthesized DNA in response to IL-6 alone; however, the magnitude of responsiveness was greatly enhanced by addition of IGFs. Similar results were obtained when proliferation and cell cycle progression were analyzed. By contrast, the KP-6 cell line was responsive to IL-6 only when IGF was present. Finally, we analyzed the effects of IGF-I on normal B lymphocytes. IGF, however, did not stimulate B cell DNA synthesis, suggesting that IGF responsiveness may represent a key difference between normal and malignant B cells. In summary, these studies suggest that IGFs may play an important role in multiple myeloma by virtue of their ability to directly stimulate tumor cell growth as well as modulate the magnitude of IL-6-driven growth.