Critical Role of Recombinase (HsRAD51) in Genetic Instability in Multiple Myeloma.

Abstract A number of cancers, including multiple myeloma (MM), show a significant genetic instability that evolves with the progression of disease. The molecular basis for the generation of genetic diversity in cancer cells has therefore emerged as an important focus of investigation. We have previously shown that homologous recombination (HR), required for a variety of normal cellular processes, is dysregulated in multiple myeloma and leads to a progressive accumulation of genetic variation over time. Moreover the inhibition or stimulation of HR activity in MM cells, by altering components of the HR pathway, concordantly affects the acquisition of new genetic changes. Consistent with this, an elevated frequency of cancer is observed in patients whose cells have elevated rate of recombination. HsRAD51 recombinase, is a key molecule in HR pathway and its dysregulated expression can increase in HR activity, leading to increased rate of mutations and development or progression of cancer. Based on these data, we hypothesized that hsRAD51 promoter activity may be a target for carcinogenic agents and ability to measure the hsRAD51 promoter activity may provide a simpler method to assess oncogenic potential of test agents. To this end we constructed a mammalian expression vector containing firefly luciferase gene downstream of hsRAD51 promoter. This plasmid was transfected into normal human plasma cells, multiple myeloma cell lines (ARP, OPM1, U266), and normal human diploid fibroblasts. The cells were allowed to recover for 24 hrs and then treated with agents with carcinogenic or anti-oxidant activities, or control vehicles for 2 hrs; lysed and luciferase activity assessed. RAD51 promoter activity was elevated 7- to 1000-fold in MM cells relative to normal plasma cells. Exposure of MM cells to a carcinogen (nickel chloride) was able to further induce hsRAD51 promoter activity (2-fold) in MM cells with already over-expression of hsRAD51. However, the anti-oxidant tocopherol and cannabinol were able to reduce hsRAD51 promoter activity by 3- and 8-fold respectively, while vitamin C and Selenium had minimal effects. In normal diploid fibroblasts, although the exposure to a non-carcinogen, Sodium azide did not induce hsRAD51 promoter activity, the exposure to agents with known transforming ability, Mitomycin C and Nickel chloride, led to a significant induction of hsRAD51 promoter activity (2.5 and 3.5-fold respectively). Moreover, the ability of these chemicals to induce RAD51 promoter activity covaried with their ability to induce homologous recombination activity as measured by a novel plasmid based assay (r = 0.9). We also demonstrate that Induction of RAD51 and HR or its inhibition using siRNAs targeting hsRAD51 can inhibit acquisition of new mutations in MM cells as measured by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. These preliminary results provides us with a simple and powerful method to measure oncogenic potential of known carcinogens and demonstrate a critical role for RAD51 in ongoing genomic instability in MM and a rationale to target RAD51 to prevent progression.