Abstract

Malignant plasma cells home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and confined to the BM are not well understood. The G-protein coupled receptor CXCR4 and its chemokine SDF-1 regulate migration in lymphocytes. In this study, we explore the molecular mechanisms involved in migration and adhesion of plasma cells in response to SDF-1. The following inhibitors were used: the Gi protein inhibitor pertussis toxin, PTX (Sigma, Aldrich, MO) 50–100ng/ml for 16 hrs; the PI3K inhibitor LY294002 (EMD Biosciences, CA) 25–50uM for 20 minutes; the mTOR inhibitor rapamycin (LC Laboratories, MA) 20–50nM for 16 hrs; the MEK inhibitor PD098059 (Alexis Biochemicals, CA) 25–50uM for 90 minutes; the p38MAPKinase inhibitor SB203580 (Calbiochem, CA) 10uM for 16 hrs; the PKC isosyme inhibitors (Biomol International LP, PA), PKC beta 5–20uM, PKC epsilon 5–20uM, PKC zeta 5–20uM; and the PLC inhibitors D609 and U73122 (EMD Biosciences, San Diego, CA) 5–20uM for 1 hr. The concentrations and durations of these inhibitors did not induce apoptosis. The transwell migration assay (Costar, Corning, NY) was used with MM cell lines (MM.1S, OPM2 and Kas6/1). Serial concentrations of SDF-1 were used in the lower chambers. The adhesion assay (EMD Biosciences, San Diego, CA) was used with MM cells treated with the inhibitors and in the presence or absence of serial concentrations of SDF-1. Student-t test was used for statistical analysis. SDF-1 induced a bell-shaped migration curve of MM cells, with 10–30nM inducing maximum migration (324% compared to untreated control) while higher doses of SDF-1 (100nM) did not induce migration in all MM cells tested. All inhibitors were tested in the presence or absence of 30nM SDF-1. PTX 50ng/ml significantly inhibited migration to 30% as compared to control in the presence of 30nM SDF-1 (p=0.004). The PI3K inhibitor and MEK inhibitors inhibited migration by 57% and 58% respectively indicating that the PI3K and ERK MAPkinase play a role in MM migration. The combination of the two agents was not additive (59%) indicating signaling through the same pathway. Immunoblotting confirmed that ERK1/2 are downstream of PI3K. The p38MAPkinase inhibitor did not affect migration. All PKC inhibitors induced a dose dependent inhibition of migration as compared to untreated control, PKC-epsilon (75%–33%), PKC-beta (63–30%), PKC-zeta (49–40%) indicating that all isoenzymes play a role in migration of MM cells. The PLC inhibitors inhibited migration in a similar fashion (52–24%). Serial concentrations of SDF-1 increased adhesion in MM cells in a dose dependent fashion with 10nM inducing 180% increase in adhesion, 30nM inducing 296% and 100nM inducing 365% increase in adhesion as compared to control. PTX 50ng/ml in the presence of 30nM SDF-1 inhibited migration to 64% as compared to control. Similar results were observed with LY294002 and PD098059. In summary, we delineate molecular mechanisms that regulate MM migration and adhesion in response to SDF-1. We demonstrate that MM cells migrate in response to SDF-1 through the Gi protein, PI3K, PKC, PLC and ERK MAPkinase pathways, but not through the p38MAPkinase pathway, and that ERK MAPkinase is downstream of PI3K. Studies to delineate the role of these inhibitors in MM homing in vivo are underway so that future clinical trials designed to regulate MM homing and migration can be performed. Supported in part by an ASH Scholar Award and an MMRF grant.

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