Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6 by Hideaki Ishikawa, Naohiro Tsuyama, Saeid Abroun, Shangqin Liu, Fu-Jun Li, Osamu Taniguchi, and Michio M. Kawano Blood Volume 99(6):2172-2178 March 15, 2002 ©2002 by American Society of Hematology

CD45+ myeloma cell lines proliferate in response to IL-6 CD45+ myeloma cell lines proliferate in response to IL-6.Flow cytometry of the indicated human myeloma cell lines stained for CD45 (A) or CD126 (IL-6Rα) (C) is shown. CD45+ myeloma cell lines proliferate in response to IL-6.Flow cytometry of the indicated human myeloma cell lines stained for CD45 (A) or CD126 (IL-6Rα) (C) is shown. (B) Proliferation of human myeloma cell lines treated with (right bars) or without (left bars) IL-6 (2 ng/mL) was analyzed by BrdU incorporation, shown by mean values and SDs obtained from 3 independent experiments using triplicate samples. The CD45− or CD45+ U266 cells were isolated by a cell sorter from parental U266 cell line. Hideaki Ishikawa et al. Blood 2002;99:2172-2178 ©2002 by American Society of Hematology

STAT3 and MEK1/2-ERK1/2 are activated in response to IL-6 in CD45− myeloma cell lines.(A,B) Activation of STAT3 in myeloma cell lines stimulated with IL-6 for 0, 10, 30, or 90 minutes was determined by Western blot analysis (WB) using antibodies specific fo... STAT3 and MEK1/2-ERK1/2 are activated in response to IL-6 in CD45− myeloma cell lines.(A,B) Activation of STAT3 in myeloma cell lines stimulated with IL-6 for 0, 10, 30, or 90 minutes was determined by Western blot analysis (WB) using antibodies specific for phosphorylated STAT3 at the tyrosine residue 705 (STAT3-P). The activation of the Ras-ERK pathway was assessed by ERK1/2 kinase assay (KA) using Elk-1 as a substrate (A) and by Western blot analysis using the specific antibodies for the phosphorylated MEK1/2 at the serine residues 217 and 221 (MEK1/2-P) (B) for U266 cells (A) and for other myeloma cell lines (B), respectively. (C) Activation of SAPK/JNK and p38 MAPK was analyzed using phosphorylated SAPK/JNK at threonine 183 and tyrosine 185 (SAPK/JNK-P) and phosphorylated p38 MAPK at threonine 180 and tyrosine 182 (p38 MAPK-P) antibodies, respectively. (D) Proliferation of CD45+ U266 cells assessed by BrdU incorporation was suppressed by a MEK1 inhibitor, PD98059 (20 μM). Values of BrdU incorporation are indicated by means and SDs that were obtained from 3 independent experiments using triplicate samples. Hideaki Ishikawa et al. Blood 2002;99:2172-2178 ©2002 by American Society of Hematology

src family PTKs are activated in CD45+ but not CD45− myeloma cell lines.(A) Western blot analysis shows the expression of Lyn (p53 and p56), Fyn, Blk, SHP-1, and SHP-2 in the myeloma cell lines indicated. src family PTKs are activated in CD45+ but not CD45− myeloma cell lines.(A) Western blot analysis shows the expression of Lyn (p53 and p56), Fyn, Blk, SHP-1, and SHP-2 in the myeloma cell lines indicated. (B) The kinase activity of Lyn or Fyn in myeloma cell lines stimulated with (+) or without (−) IL-6 was investigated by kinase assay using enolase as an exogenous substrate. (C) The immunoprecipitation (IP) and Western blot analysis with the indicated antibodies were used to examine the interaction of Lyn or Fyn kinases with CD45 or gp130 molecules in myeloma cell lines treated with (+) or without (−) IL-6. Hideaki Ishikawa et al. Blood 2002;99:2172-2178 ©2002 by American Society of Hematology

Lyn PTK is required for proliferation of CD45+ U266 cells enhanced by IL-6.(A) BrdU incorporation was used to determine the DNA synthesis of CD45+ U266 cells treated with Lyn-specific sense (S, left 2 bars) or antisense (AS, right 2 bars) oligodeoxynucleoti... Lyn PTK is required for proliferation of CD45+ U266 cells enhanced by IL-6.(A) BrdU incorporation was used to determine the DNA synthesis of CD45+ U266 cells treated with Lyn-specific sense (S, left 2 bars) or antisense (AS, right 2 bars) oligodeoxynucleotides (10 μM) in the presence (the second and fourth bars) or absence (the first and third bars) of IL-6. The results obtained from the experiments using 2 different oligodeoxynucleotides 1 and 2 are indicated on the left and right, respectively. (B) The PTK inhibitors PP2 (300 ng/mL, middle) and herbimycin A (1 μg/mL, right) reduce the BrdU incorporation of CD45+ U266 cells by IL-6 (right bars; left bars are without IL-6). Control DMSO is shown on the left. (C) Western blot analysis shows the effect of antisense (AS) or sense (S) oligodeoxynucleotides specific for Lyn on the expression of Lyn, STAT3, and MEK1/2 in CD45+ U266 cells. Western blot analysis is also used to examine the phosphorylation of STAT3 (Tyr705) and MEK1/2 (Ser217/221) in CD45+ U266 cells treated with either Lyn-specific antisense or sense oligodeoxynucleotides in the presence of IL-6. (D) Western blot analysis shows the phosphorylation and expression of STAT3 and MEK1/2 in CD45+ U266 cells treated with PP2 or herbimycin A prior to IL-6 stimulation. Values of BrdU incorporation are indicated by the means and SDs resulting from 3 independent experiments using triplicate samples (A,B). Hideaki Ishikawa et al. Blood 2002;99:2172-2178 ©2002 by American Society of Hematology