The primary long term objective of this program is to continue development of new cytochemical, biophysical and molecular probes and techniques to measure content and/or conformation of various constituents of individual cells. These probes (techniques) will be designed for cell analysis by flow cytometry and cell sorting and are expected to have clinical applications in cancer screening, diagnosis, classification and prognosis, in evaluation of drug sensitivity of tumors and in monitoring treatment effects. Specific current aims (projects) of this study are: 1) evaluation of the possible effect of differences in chromatin structure on DNA content estimates in colon and breast tumors; 2) RNA content estimates: a) development of sensitive method(s) of RNA measurement applicable to nuclei isolated from solid tumors; b) studies of mechanisms responsible for differential staining of rRNA in ribosomes vs polyribosomes in situ with pyronin Y, and application of this phenomenon to a new lymphocyte stimulation assay; (3) studies and application of the resonance energy transfer between pyronin Y and rhodamine 123 as a new mitochondrial probe; (4) extension of the method for assay of DNA denaturation in situ to nuclei isolated from colon, breast, lung and bladder tumors; (5) analysis of nuclear proteins selectively released by antitumor drugs having affinity to nucleic acids, and testing antibodies developed against these proteins as a possible probe of drug effects on target cells. The second objective (specific aim or project 6) is to apply the developed probes to studies of cell growth, progression through the mitotic cycle, differentiation and response of cells to different antitumor drugs. The data will be correlated with kinetics of those processes and will provide a more complete description of metabolic changes and cell heterogeneity during the cell cycle. The third objective (specific aim or project 7) is, by using biophysical and biochemical methods, to study molecular interactions between the probes and various cell constituents, predominantly nucleic acids. Because many probes are either antitumor drugs themselves or drug analogs, these studies in addition to being helpful in developing new diagnostic techniques, are expected to reveal information on mechanisms of drug action on cells at the molecular level. Correlating these data with drug studies on whole cells will provide a comprehensive view on intracellular drug targets, mechanisms involved in drug binding, and cytotoxicity as related to cell kinetics or metabolic state. This in turn will help assess the cellular features predicting susceptibility to particular drugs and aid in new drug design.
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