CHANGES IN THE STRUCTURE OF NUCLEOIDS ISOLATED FROM HEAT-SHOCKED HELA-CELLS

Using a technique to detect changes in DNA supercoiling which allows one to visualize both DNA unwinding and rewinding in presence of the intercalating dye, propidium iodide (PI), we showed that hyperthermic treatment (30 min at 45 degrees C) of HeLa S3 cells alters the response to the intercalating dye. Depending on the treatment conditions, we observed a reduction in the maximum size of the DNA loop that can be measured at the relaxation point (PI concentration 5-7.5 micrograms/ml). Cellular heating also affected all degrees of DNA rewinding (measured as a function of PI concentrations between 10 and 50 micrograms/ml). By 6 h after cellular heating these heat effects had disappeared. This time interval correlated with the time necessary for recovery from a heat-induced increase to normal nuclear and nucleoid protein content. Using gel electrophoresis we showed that the nucleoids (DNA plus nuclear matrix proteins) after heat exposure are enriched in several polypeptides and that there is a specific increase in HSP 72/73. We hypothesize that the altered response to the intercalating dye after cellular heat shock is due to an increase in polypeptides associated with the nuclear matrix thereby altering the DNA-nuclear protein matrix anchor point

Nuclear matrix as a target for hyperthermic killing of cancer cells

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tu mor cell's ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins