engleski

Bone Morphogenetic Protein-4 and -6 Are Over-Expressed in the Multiple Myeloma Bone Marrow Samples

Multiple myeloma (MM) is a B-lymphocyte neoplasia characterized by the slow proliferation of plasma-cells in bone marrow (BM) and bone destruction. Although several bone morphogenetic proteins (BMP) have been shown to elicit apoptosis of myeloma cells in vitro, we proposed that BMPs may have other non-apoptotic and pro-survival effects. BM was obtained from MM patients (n = 25) and control subjects (n = 14) after the informed consent and approval of the Ethical Board. Selected BM MM samples were magnetically separated for CD138 positive cells. Primary MM samples and myeloma lines (NCI H-929 and Thiell) were cultured for 48 hours and treated by recombinant human (rh)BMP-2 or -6 (500 ng/mL for both) with or without BMP inhibitor rhNoggin. cDNA was extracted from mononuclear BM cells or cell lines, amplified by quantitative PCR using TaqMan assays for BMPs, corresponding receptors and downstream regulatory molecules, and normalized to GPDH. For detection of apoptotic and dead cells, we used flow cytometric annexin V/propidium iodide staining. ELISA assays for p53, Bax and Bcl-2 were performed on cultured cell lysates. Expression of BMP-4, -6 and Alk-2 was significantly higher in MM BM samples compared with control BM (around 10-fold for BMP-4, 35-fold for BMP-6, 2-fold for Alk-2), with significant positive correlation of BMP-6 expression and plasma-cell percentage in MM samples (p<0.01). Upon in vitro BMP-2 or -6 treatment, insignificant changes in the percentage of death/apoptotic cells were observed paralleled by down-regulation of pro-apoptotic Bax and p21 (up to 2-fold and 5-fold respectively), and up-regulation of inhibitors of differentiation ID-1 and -2 (up to 200-fold and 5-fold respectively). Changes induced by BMPs were blocked by the addition of Noggin. ELISA confirmed the involvement of p53 pathway and decrease in the expression of Bax. Selected CD138 positive myeloma cells showed relatively higher endogenous expression of BMPs/receptors compared with corresponding full BM samples and CD138 negative population, providing a basis for autocrine action. We found that BMP-4 and -6 are over-expressed in MM BM samples and selected CD138 positive myeloma cells, indicating the role of BMPs as proliferative and survival autocrine factors in MM. Our study opened the possibility to further test clinical application of BMP inhibitors in MM treatment.

myeloma; BMPs; cell survival

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