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Crucial role of phosphatase CD45 in determining signaling and proliferation of human myeloma cells

European Cytokine Network. Volume 18, Number 3, 1-7, September 2007, Research papers

DOI : 10.1684/ecn.2007.0095

Summary

Author(s) : Madeleine Collette , Géraldine Descamps , Catherine Pellat-Deceunynck, Régis Bataille, Martine Amiot , INSERM, U601, Département de Recherche en Cancérologie, LNC Label, Institut de Biologie, 9 quai Moncousu, 44000 Nantes, France.

Summary : In multiple myeloma, a large number of growth factors (IL-6, IGF-1, FGF, HGF and HB-EGF) are involved in promoting myeloma cell growth. In the present study, a serum-free, cytokine-free, collagen-based assay, which does not allow the generation of spontaneous myeloma colonies, was used to identify the clonogenic growth factors for fourteen myeloma cell lines. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 out of 14 myeloma cell lines. Using a pharmacological Erk inhibitor, we show that the Erk/MAPK pathway is involved in IL-6-induced clonogenicity of CD45+, but not CD45- myeloma cell lines. In contrast to IL-6, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some myeloma cell lines, but always CD45-, and less effectively than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from five out of eight CD45- myeloma cell lines. Finally, the capacity of IGF-1 and FGF to stimulate the clonogenicity of CD45- myeloma cells correlates with their ability to stimulate the Erk/MAPK pathway. We conclude that CD45 expression plays a crucial role in determining signaling and proliferation of human myeloma cell responses to IL-6, IGF-1 and other growth factors. The poor outcome of CD45- myeloma patients could be related to the capacity of CD45-myeloma cells to take advantage of multiple growth factors.

Keywords : multiple myeloma, clonogenicity, CD45, IL-6, IGF-1

Pictures

Figure 1 Clonogenicity of CD45- and CD45+ HMCL. HMCL were separated into two groups according to their CD45 phenotype. CD45- HMCL (LP-1, NCI-H929, JIM-3, NAN-1, RPMI-8226, JJN3, L363) and CD45+ HMCL (XG-6, NAN-4, MDN, XG-1, XG-2 and U266). CD45+ HMCL are indicated in bold and italics. For myeloma colony assays, 10³ myeloma cells were seeded per ml of serum-free, collagen-based, semi-solid stemα III medium with or without cytokines and grown for 15 days. Gels were then dried and stained with MGG, and myeloma colony formation (MCF) was scored.

Figure 2 The Erk/MAPK pathway is involved in IL-6-induced clonogenicity of CD45+ HMCL only. Myeloma cells (10³) were seeded per ml of serum-free, collagen-based, semi-solid stemα III medium containing IL-6, with or without inhibitor (U0126) and grown for 15 days. Gels were then dried and stained with MGG, and MCF was scored. Values represent the mean ± SD of three experiments of duplicate cultures.

Figure 3 Analysis of ERK phosphorylation induced by IGF-1 or FGF in HMCL. Eighteen- hour, serum-starved cells were treated or not with IGF-1 for the indicated time (figure 5B) or with IGF-1 or FGF for 30 minutes (figure 5A and 5C). Equivalent amounts of cell lysates were separated by SDS-PAGE, then immunoblotted with anti-phospho-ERK antibodies. Protein loading was controlled with an anti-ERK total [[“total”: OK ?]].

Figure 4 Effect of the combination of IL-6 +IGF-1 on colony formation. Myeloma cells (10³) were seeded per ml of serum-free, collagen –based, semi-solid stemα III medium containing IL-6, IGF-1 or IL-6 + IGF-1 and grown for 15 days. Gels were then dried and stained with MGG, and colony formation was scored. Values represent the mean ± SD of three experiments of duplicate cultures.

Figure 5 Synergistic effect of the combination of IGF-1+HGF on NCI-H929 colony formation. A) Myeloma cells (10³) were seeded per ml of serum-free, collagen-based, semi-solid stemα III medium containing the indicated growth factor and grown for 15 days. Gels were then dried and stained with MGG, and MCF was scored. Values represent the mean ± SD of three experiments of duplicate cultures. B) The synergistic effect of the combination of IGF-1+HGF on colony formation is mediated by a stronger activation of both PI-3K/Akt and Erk/MAPK pathways. Eighteen-hour, serum-starved cells for were treated or not with IGF-1, IL-6, IGF-1+IL-6, HGF or IGF-1+HGF for 30 minutes. Equivalent amounts of cell lysates were separated by SDS-PAGE, then immunoblotted with anti-phospho-ERK or anti-phospho Akt antibodies. Protein loading was controlled with an anti-ERK total [[“total”: OK ?]].