Image analysis-based measurement of DNA supercoiling changes in transformed and nontransformed human cell lines

Abstract

An image analysis system was used to visualize and measure the changes in nucleoid diameter (nuclear matrix core plus extruded DNA loops) which occur when increasing concentrations of propidium iodide are used to titrate the DNA supercoiling response. Parallel core size measurements allow estimates of the changes in apparent DNA loop size. Unlike sedimentation assays, DNA loop size estimates are not influenced by particle mass, require no prior cell labeling, and can be performed on a per cell basis. This technique was used to examine changes in DNA loop characteristics which may occur when cells are transformed or undergo changes in their proliferative state. SV40-transformation of human diploid fibroblast lines resulted in a significant increase in both the nucleoid core and average DNA loop size. Lymphoblast cell lines also had larger nucleoid dimensions than normal lymphocytes. The response of several established human tumor cell lines indicated slightly increased loop but not core sizes as compared to normal human diploid fibroblasts. Changes in proliferative state also resulted in changes in DNA loop characteristics as measured in this assay. Both quiescent fibroblasts and unstimulated lymphocytes appeared to have smaller or more condensed DNA loop structures than their proliferating counterparts. These results demonstrate the utility of this assay in detecting changes in DNA loop structure which occur in association with changes in the proliferative activity of cells in culture.