Figure 4

IGFBP7 inhibits myeloma cell proliferation. MM cell lines (A) and primary MM cells (B) were treated with PBS/BSA 0.1% or recombinant human IGFBP7 (rhIGFBP7) at the indicated concentrations for 96 hours. Primary cells were treated with IGFBP7 at 10 μg/ml. Viable cell numbers were evaluated as outlined in the methods section. (C) Proliferation of MM cells was assessed by BrdU assay. HMCLs were treated for 72 hours with PBS/BSA 0.1% or rhIGFBP7 at the indicated concentrations. BrdU was added for the last 19 hours of incubation. Proliferation was assessed by measuring absorbance at 450 nm. (D) Expression of p21 was analysed by quantitative PCR after 24 hour treatment with PBS/BSA 0.1% or rhIGFBP7 at 80 μg/ml. (E) Apoptosis of HMCLs was determined by Annexin V and 7-AAD staining after a 72 hour incubation period in the presence of PBS/BSA 0.1% or rhIGFBP7 at the indicated concentrations. Graphs represent the mean (% positive cells on FACS) of three independent experiments performed in duplicates. (F) KMS-12-BM and MM.1S were cultured in Syn-H serum free medium in the presence of rhIGFBP7, IGF-1 and/or insulin at the indicated concentrations for 96 hours before viable cell numbers were determined. (A-D, F) Values represent the mean ± SD of three independent experiments performed in triplicates and mean ± SD of triplicate experiments for primary MM cells. Asterisks indicate statistical significance compared with the control (* P < 0.05, ** P < 0.01, *** P < 0.001).