Proliferation of U937 cells was increased by growth in 231-CM. U937 cells were grown in the absence (control) or presence of different concentrations of 231-CM for 3, 5 and 7 da. Proliferation was assessed both by MTT (A) and growth curve (B) assays. Bars represent absorbance (at 570 nm), which is proportional to cell proliferation (A) and growth curve represents number of living cells (B). Data represent mean ± S.D. of three independent experiments; *, p = 0.05 (as compared to the control at each time interval).

CTSB activity in U937 cells was increased by growth in 231-CM. U937 cells were grown in the absence (control) or presence of different concentrations of 231-CM for 3, 5 and 7 da (treated). CTSB activity in cell lysates (A) and in media (B) was measured against Z-Arg-Arg-NHMec and is expressed as pmol/min/μg DNA. Activity in the media was measured after pepsin activation of proCTSB as described in Materials and Methods. Data represent mean ± S.D. of at least three independent experiments; *, p = 0.05 (as compared to the control at each time interval).

Gelatinase activity and expression by U937 cells was increased in a time- and dose-dependent manner by growth in 231-CM. (A) Representative zymogram from three independent experiments depicting gelatinolytic activity of proMMP-2 (72 kDa), MMP-2 (62 kDa) and proMMP-9 (92 kDa) in U937 cells. Samples were loaded equally according to protein content and separated on 10% SDS-PAGE containing 1% gelatin (w/v). White bands indicate regions in which gelatin has been hydrolyzed with lanes 5 and 6 being control lanes for gelatinolytic activity of recombinant proMMP-9 and -2, respectively. Lane 1 represents overnight serum-free conditioned media of untreated U937 cells (control); lanes 2, 3 and 4 represent serum-free media of U937 cells grown with different concentrations of 231-CM (1 in 6, 1 in 4 and 1 in 3, respectively) for 3, 5 and 7 da. (B and C) Dose-dependent increase in secretion of total (active and inactive) MMP-2 (ng/ml) and active MMP-9 (ng/ml), respectively, from U937 cells after 7 da of growth with 231-CM as compared to the control U937 grown in complete RPMI media. Data represent results of at least 3 independent experiments and are presented as mean ± S.D.; *, p = 0.05 and **, p = 0.001.

Increased secretion of IL-6 and IGFBP-1 from U937 cells grown in 231-CM. (A) Representative cytokine antibody array of media conditioned by untreated U937 cells (control). (B) Representative cytokine antibody array of media conditioned by U937 cells grown in 231-CM (1 in 4 dilution) for 7 da prior to incubating overnight in serum-free medium. Increases in IGFBP-1 (lane 6 spot 10); osteoprotegrin (lane 8 spot 2) and NAP-2 (lane 7 spot 10); and a decrease in RANTES (lane 4 spot 2) and I-309, (lane 2 spot 1) were observed.

IL-6-NAb reduced 231-CM induced increases in CTSB expression, secretion and activity by U937 cells. U937 cells were grown for 5 da in the absence (control) or presence of 231-CM or 231-CM plus 4 μg/ml IL-6 NAb. Cells were washed and incubated in serum-free medium overnight prior to collecting cell lysates and media. (A) Representative immunoblot of cell lysates and overnight serum-free conditioned media for CTSB. CTSB activity in cell lysates (B) and in media (C) was measured against Z-Arg-Arg-NHMec and is expressed as pmol/min/μg DNA. Specific activity of CTSB is shown as a percentage of that measured in U937 cells treated with 231- CM. Activity in the media was measured after pepsin activation of proCTSB as described in Materials and Methods. Data represent mean ± S.D. of at least three independent experiments; *, p = 0.05; ** p = 0.01.