FIG. 6.

Overexpression of RAD51 and Bcl-xL and G₂/M delay are exhibited by BCR/ABL-positive primary leukemia cells: role in drug resistance. (A) Downregulation of RAD51 increased sensitivity of CML-BC cells to cisplatin and mitomycin C. CML-BC patient cells were infected with RAD51(AS)-IRES-GFP virus (GFP+AS, •) or with IRES-GFP empty virus (GFP, ▪). Downregulation of RAD51 in GFP-positive cells was determined by immunofluorescence analysis visualizing the levels of endogenous RAD51 (left panel). Drug sensitivity was assessed by the trypan blue exclusion test after 48 h of exposure to the indicated concentrations of cisplatin or mitomycin C. (B) BCR/ABL enhances expression of Bcl-xL in primary bone marrow cells. Murine bone marrow cells were infected with BCR/ABL-IRES-GFP virus (GFP+BCR/ABL) or IRES-GFP (GFP) empty virus. Bcl-xL expression was examined by Western analysis in GFP-positive cells. (C) BCR/ABL causes G₂/M delay essential for drug resistance. GFP+BCR/ABL-positive cells and GFP-positive control cells were treated with 0.375 μg of cisplatin per ml in the presence of IL-3, and cell cycle analysis was performed after 0, 12, 24, and 48 h (left panel). GFP+BCR/ABL cells cultured in the presence of IL-3 were left untreated (1) or treated with 2 mM caffeine (2), 0.375 μg of cisplatin per ml (3), 0.375 μg of cisplatin per ml plus 2 mM caffeine (4), 1.5 μg of cisplatin per ml (5), 1.5 μg of cisplatin per ml plus 2 mM caffeine (6), 0.1 μg of mitomycin C per ml (7), or 0.1 μg of mitomycin C per ml plus 2 mM caffeine (8). Twenty-four hours later, cells were plated in methylcellulose, and colonies were scored after 7 days (right panel). Results are presented as the percentage of colonies in experimental samples in comparison to the untreated control sample 1 (602 ± 37 colonies arose from 10³ untreated GFP+BCR/ABL cells). ∗, P < 0.05 in comparison to the corresponding group not treated with caffeine. Cell cycle analysis of the selected samples (1, 3, and 4) was performed after 24 h of treatment to confirm that caffeine abolished accumulation of the cells in G₂/M. Results represent two experiments.