CHROMATIN PROBES FOR DISTINGUISHING MALIGNANT CELLS

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The objective of this study is to develop and apply cytochemical and biophysical probes or techniques applicable to flow cytometry and cell-sorting instruments for analysis of nuclear and cytoplasmic structure that can distinguish between normal proliferating, normal differentiating, premalignant, and malignant cells, as well as characterize in detail various metabolic phases of the cell cycle. By providing new, objective, and quantitative criteria, the probes also could have an application in the classification of tumors and prognostic value in monitoring responsiveness of tumor cells to therapy. The probes are developed to discriminate between proliferating and quiescent tumor cells, which is of potential value in planning chemotherapy protocols. Using a variety of fluorochromes, the following cell features are measured: (1)\total DNA, RNA, and protein content as well as RNA/DNA and RNA/protein ratios of whole cells and isolated nuclei; (2)\membrane potential of mitochondria; (3)\polysomal versus ribosomal RNA; (4)\ratio of double-stranded to single-stranded RNA; and (5)\accessibility of DNA in situ to intercalating probes of various sizes and affinities as well as to exogenous nucleases. In addition, structure of nuclear chromatin is analyzed using combinations of dyes and exploiting the energy transfer phenomenon, studying depolarization of DNA-bound probes, presence and accessibility of SH groups, sensitivity to steroid hormones, and DNA in situ sensitivity to denaturation. More basic studies on chromatin probes include investigations on molecular interactions between natural nucleic acids, homoribo-\or deoxyribo-polymers, and such dyes as acridine orange or pyronin Y. Different tissue culture cell lines, cycling, quiescent and differentiating cell systems, human leukemias, lymphomas, solid tumors, and clinical cytology specimens are used. (3)