Figure 2

Effects of synchronisation and cell cycle arrest on proliferation of rheumatoid arthritis synovial fibroblasts (RASFs). (a) The effect of nocodazol on the cell cycle of early-cultured RASFs. Treatment of higher-proliferating RASFs with nocodazol (2.5 μg/ml, 18 hours) resulted in a marginal increase of RASFs arrested in G₂/M phase (~4 n DNA at G₂/M peak, black line, representative histogram). (b) Quantitative analysis. Values are mean ± standard deviation as a percentage of RASFs treated/untreated with nocodazol obtained from 3 individual patients with RA. (c) Effects of hydroxyurea (HU) on cell cycle of RASFs were illustrated in a representative three-dimensional histogram with the y-axis (time in hours) pointing away from the observer. RASFs treated with HU for 6 hours (time 0 hours) showed an accumulation of RASFs in G₀/G₁ phase. Analysis of cell cycle 18, 24, and 30 hours after HU exposure showed a decrease of RASFs in G₀/G₁ phase with a simultaneous increase of proliferating RASFs in S phase and G₂/M phase, indicating that the cell population remained highly synchronised. Cell cycle analysis after 42 and 48 hours confirmed an increase of RASFs in G₀/G₁ phase, indicating that cell division commenced between 30 and 48 hours. (d) Quantitative analysis as mean ± standard deviation. (e) Early-cultured RASFs became arrested at G₀/G₁ phase after 8 to 10 days of incubation with ITS medium. Subsequent incubation for another 1 or 2 days with complete Dulbecco's modified Eagle's medium (9/1, 9/2 days) resulted in an increase of proliferating RASFs. Bar graphs in frames on right show quantitative analysis. Values are presented as mean ± standard deviation of percentages of RASFs obtained from at least three individual patients with rheumatoid arthritis. Representative three-dimensional histogram. (f) Quantitative analysis.