Effect of human myeloma cells on osteoblast differentiation. Confluent PreOBs obtained from healthy BMMNCs were cocultured with HMCLs (XG-6, RPMI-8226, OPM2, and XG-1) in the presence or absence of a transwell insert or with healthy BMMNCs or the EBV-positive cell line B95.8 for 24 to 48 hours. After 24 hours mRNA was extracted from either PreOBs alone, in the control condition and transwell coculture condition, or from pooled cells in cell-to-cell contact condition. (A) Alkaline phosphatase (ALP), collagen I, and osteocalcin (OC) mRNA expression was evaluated by RT-PCR. (B) Collagen I protein was assessed by Western blot analysis after 48-hour coculture. Figures are representative of 3 independent experiments. (C) OC levels were measured by ELISA assay in the 48 hours conditioned medium of cell-to-cell contact cocultures. Graphs and bars represent the mean OC levels plus SD of 3 independent experiments performed in duplicate. ^*P = .01; ^**P = .001. (D) Alkaline phosphatase (ALP) levels were detected by a fluorimetric method on cell lysates of 48-hour coculture. Graphs and bars represent the mean % minus SD versus the control condition (PreOB alone). ^*P < .05. (BMMNC: bone marrow mononuclear cells; EBV: EBV-positive cell line B95.8)