HR activity in myeloma cells is induced by nickel chloride and inhibited by siRNAs targeting recombinases. (A) Loss of RAD51 protein expression by siRNAs. Immunostaining using monoclonal antibody to HsRAD51 of ARP cells transfected with (I) control siRNA and (II) HsRAD51-specific siRNAs. (B) Loss of HR activity by siRNAs. HR activity was assessed using the plasmid substrate after the transfection of ARP cells with siRNA directed at HsRAD51 and RAD51B. Recombination frequency is presented as number of recombination events per million transfected plasmid copies. ARP control, MM cells transfected with control siRNAs; ARP 51/51B SiRNA, MM cells transfected with siRNAs directed at RAD51 ands RAD51B. (C) Effect of nickel on promoter regulation of HsRAD51. HsRAD51 promoter was cloned upstream of a luciferase gene in a mammalian expression vector. The resulting construct HsRAD51P-LUC was transfected into normal diploid fibroblasts and the cells were then exposed to nickel chloride (0.3 mg/mL). After a 2-hour exposure, cells were lysed and luciferase activity was assayed with a Luciferase Assay Kit (Clontech). Lanes show luciferase activity in water (lane 1), in ARP cells transfected with RAD51P-Luc (lane 2), and in ARP cells transfected with RAD51P-Luc and exposed to nickel chloride (lane 3). (D) Induction of HsRAD51 and related genes by nickel chloride in ARP cells. ARP myeloma cells, untreated (ARP) or treated with nickel chloride (0.3 mg/mL) for 24 hours (ARP-Ni) were harvested, total RNA was isolated, and processed for gene expression analysis using U133 arrays (Affymetrix). Gene expression is shown by intensity of red color. Color scale shows fold change of gene expression in treated cells relative to untreated ARP cells. (E) HR activity in untreated ARP cells (ARP) or ARP cells exposed to nickel chloride (ARP Nickel; 0.3 mg/mL) for 5 days.