Identifying intercellular signaling genes expressed in malignant plasma cells by using complementary DNA arrays

Abstract

In multiple myeloma (MM), the growth of primary plasma cells depends not only on interleukin-6 (IL-6), but also on additional unidentified signals delivered by the bone marrow environment. Using Atlas complementary DNA (cDNA) arrays comprising 268 genes coding for intercellular signaling molecules, this study identified genes that are overexpressed in myeloma cells compared to autologous B-lymphoblastoid cell lines. These genes encode the oncogenic Tyro3 tyrosine kinase receptor, the heparin-binding epidermal growth factor-like growth factor (HB-EGF) that is an epithelial autocrine tumor growth factor, the thrombin receptor (TR) that is linked to HB-EGF and syndecan-1 processing and to cell invasion, chemokine receptors CCR1 and CCR2, the Wnt pathway actor Frizzled-related protein (FRZB), and the Notch receptor ligand Jagged 2. These data, obtained with the Atlas cDNA array, were confirmed by reverse transcriptase-polymerase chain reaction or protein analysis or both. Furthermore, Tyro3, HB-EGF, TR, and FRZB gene expression was documented in purified primary malignant plasma cells from patients with plasma cell leukemia or MM. HB-EGF and FRZB were poorly expressed in purified polyclonal plasma cells. Finally, HB-EGF was proved to be an essential autocrine growth factor for the XG-1 myeloma cells. This study shows the po- IntroductionTumor development involves multiple mechanisms that lead to an increase in the survival of tumor cells and a deregulation of the cell cycle. Specifically, cell-to-cell growth signaling is likely to operate between tumor cells and their neighboring stromal cells. 1 In multiple myeloma (MM), the tight interactions between malignant plasma cells and their medullar environment exemplify this oncogenic process. As recently reported, myeloma cells produce the angiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), which may promote the neovascularization of the bone marrow observed as the disease activity increases. [2][3][4] Malignant plasma cells express adhesion molecules such as VLA4 and CD44, whose interactions with stromal cells and the extracellular matrix sustain MM cell survival. [5][6][7] Partly by producing interleukin-1 (IL-1), MM cells induce bone marrow stromal cells to synthesize IL-6, a major cytokine in MM biology. [8][9][10][11] However, purified MM cells cannot be rescued, even by IL-6, and require the presence of stromal cells for in vitro survival. [12][13][14][15] Thus, in the chronic phase of the disease, some molecules produced by stromal cells are critical, combined with IL-6, to promoting MM cell survival and proliferation. Growth factors able to bind heparan sulfate, such as FGFs, could be candidate molecules. Indeed, translocations involving the FGF receptor 3 are found in MM cells from patients with active disease and can confer an increase in the response of MM cells to IL-6 in vitro. 16,17 Moreover, MM cells are the only cells in the bone marrow that express syndecan-1, a heparan sulfate proteoglycan that i...

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3

“…Previous reports have demonstrated that CCR2 is expressed on normal, mature B cells (Frade et al, 1997). Very recently, transcripts for this receptor were also identified in myeloma plasma cells, as demonstrated by cDNA arrays (De Vos et al, 2001). Our data show that CCR2 is also expressed in both, normal and malignant plasma cells, at the protein level.…”

Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma

“…In the current experiments, cells were cultured at a low concentration to limit the biological activity of autocrine loops involving EGF-ligands. Indeed, we have previously shown that autocrine HB-EGF contributes to the IL-6-induced growth of some IL-6-dependent HMCLs cultured at a high cell density (De Vos et al, 2001;Wang et al, 2002;Mahtouk et al, 2004). Compiling our previous data about ErbB expression in HMCLs with those of EGF-ligand expression, we provide here a comprehensive description of autocrine loops in HMCLs.…”

“…Growth factors (1 mg) were added at the beginning of the culture in six culture wells per group. At the end of the culture, cells were pulsed with tritiated thymidine (Amersham Pharmacia Biotech, Orsay, France) for 12 h, harvested and counted as reported previously (De Vos et al, 2001). …”

Bone morphogenic protein 6: a member of a novel class of prognostic factors expressed by normal and malignant plasma cells inhibiting proliferation and angiogenesis

“…Proliferation of HMCLs was investigated as previously published (De Vos et al, 2001). Cells were cultured in RPMI-1640 containing 10% fetal calf serum and graded concentrations (0.00128, 0.0064, 0.032, 0.16, 0.8 and 4 mg/ml) of BMP6.…”