Background: The “masking effect” is the observation that heat shock masks nuclear DNA damage that alters DNA supercoiling ability. These include DNA strand breaks induced by ionizing radiation or relaxation of MAR DNA-nuclear matrix interactions by oxidative stress. We hypothesize that the masking effect occurs when proteins are relocated to sites of DNA or nuclear matrix damage, resulting in the stabilization of these structures. Our objective for these studies is to determine the mechanism of the masking effect and its role in radiation sensitivity.

Methods: The Halo Assay is a measure of the supercoiling ability of the nuclear DNA. Aliquots of HeLa S3 cells were simultaneously treated with a lysis buffer and increasing concentrations of Propidium Iodine (PI). As the PI concentration increases, the PI rewinds the DNA supercoils in the opposite orientation. Micrographs of the relaxation and the rewinding phases are analyzed with image analysis software.
Chromatin Immunoprecipitation Assay (ChIPs) was performed using a kit supplied by Active Motif. The DNA which co-precipitated with nucleophosmin was PCR amplified using primers specific for several different MAR sequences, exon sequences, and promoter DNA sequences. Other assays include clonogenic survival assays, siRNA knockdown of nucleophosmin, and immunohistochemistry, performed following standard methods.

Results: Following heat shock; nucleophosmin relocalizes to the nucleoplasm and binds to DNA. Chromatin immunoprecipitation experiments show that nucleophosmin has a greater affinity for MAR DNA than the other DNA sequences studied. HeLa cells in which nucleophosmin has been knocked-down do not exhibit the masking effect in Halo Assays, suggesting that nucleophosmin binding MAR DNA plays a role in this effect. Nucleophosmin knocked down in HeLa S3 cells treated with 45ºC, 30 min showed a reduced thermal enhancement ratio (1.72) compared with that (2.22) for cells with normal nucleophosmin levels.

Conclusions: Taken together, these data are consistent with the hypotheses that, following hyperthermia, nucleophosmin binds DNA and facilitates the masking effect which increases radiation sensitivity.