Figure 6 CNDAC-induced DNA damage is... | American Society of Hematology

Figure 6

$Figure 6. CNDAC-induced DNA damage is repaired through the homologous recombination pathway. (A-C) Rad51 and its 2 interacting proteins, Xrcc3 and Brca2, are involved in DNA damage response to CNDAC. (A) Exponentially growing HeLa (top) and HCT116 cells (bottom) were incubated with 4μM and 3μM CNDAC for 24 hours and 72 hours, respectively. Cells were harvested at indicated times and subjected to subcellular fractionation. Fractions: cytoplasmic (S1), nuclear soluble (S2), and chromatin binding (P2). Rad51, Orc2, and RhoA or β-tubulin in the indicated fractions were detected by immunoblotting. (B) AA8 (■, wild-type), irs1SF (ο, Xrcc3-deficient), and 1SFwt8 (▴, hXRCC3-complemented) cells were washed into drug-free medium after a 24-hour exposure to CNDAC at a range of concentrations and allowed to form colonies in 6 to 7 days. (C) V79 (■, wild-type) and V-C8 (ο, Brca2-deficient) cells were treated as in panel B. All points are mean ± SD of triplicate plates. (D) IC50 values of CNDAC and mitomycin C from clonogenic assays using paired hamster lines. (E) CNDAC induces higher levels of SCEs after the second S phase. Continuously exposed to bromodeoxyuridine, AA8 cells were treated with 1μM CNDAC for either 15 hours (1 cell cycle) or for 30 hours (2 cycles). Colcemid was added in the final 1.5 hours. Quantitation of SCEs was performed in a minimum of 20 metaphases per sample.$

CNDAC-induced DNA damage is repaired through the homologous recombination pathway. (A-C) Rad51 and its 2 interacting proteins, Xrcc3 and Brca2, are involved in DNA damage response to CNDAC. (A) Exponentially growing HeLa (top) and HCT116 cells (bottom) were incubated with 4μM and 3μM CNDAC for 24 hours and 72 hours, respectively. Cells were harvested at indicated times and subjected to subcellular fractionation. Fractions: cytoplasmic (S1), nuclear soluble (S2), and chromatin binding (P2). Rad51, Orc2, and RhoA or β-tubulin in the indicated fractions were detected by immunoblotting. (B) AA8 (■, wild-type), irs1SF (ο, Xrcc3-deficient), and 1SFwt8 (▴, hXRCC3-complemented) cells were washed into drug-free medium after a 24-hour exposure to CNDAC at a range of concentrations and allowed to form colonies in 6 to 7 days. (C) V79 (■, wild-type) and V-C8 (ο, Brca2-deficient) cells were treated as in panel B. All points are mean ± SD of triplicate plates. (D) IC₅₀ values of CNDAC and mitomycin C from clonogenic assays using paired hamster lines. (E) CNDAC induces higher levels of SCEs after the second S phase. Continuously exposed to bromodeoxyuridine, AA8 cells were treated with 1μM CNDAC for either 15 hours (1 cell cycle) or for 30 hours (2 cycles). Colcemid was added in the final 1.5 hours. Quantitation of SCEs was performed in a minimum of 20 metaphases per sample.

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