STANDARDIZED MULTIPARAMETRIC CELL CYCLE ANALYSIS

Case Western Reserve University, Cleveland OH

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Abstract

DESCRIPTION: (Adapted from the Applicant's Abstract) Flow cytometry of cellular DNA content is used routinely to evaluate cell proliferation. It is common to couple this parameter assay with immunofluorescence to measure important molecules in a cell cycle specific manner. These molecules include proliferation and phase- specific markers as well as regulatory molecules with cell cycle rate limiting function, e.g., cyclins, c-myc, Rb. Most interesting molecules are intracellular. This cytometric approach to molecular quantification is useful in basic research and in the clinical evaluation of hemotological and solid tumors. In clinical analysis, DNA content provides prognostic information and many two parameter analyses provide more accurate DNA content measurements. Expression levels of regulatory proteins may have prognostic significance, however, it is unlikely that a measurements of a single molecule will be useful, except in rare instances, because of the highly complex and apparently redundant nature of cell regulatory mechanisms. The literature on single markers attests to this. A cytometric approach provides the unique ability to quantify on a single cell basis two or more proteins and DNA content, thus providing correlated gene expression analysis resolved at the cell cycle level, and thus, by inference in a dynamic (time resolved) manner. Current problems with this approach are technological rather than rational. These are (1) multiple markers are required but the multiparametric methodology has not been developed, (2) lack of standardization does not permit direct comparison of data generated across time or between laboratories, (3) the reported levels are relative, and (4) the sensitivity level is known to need improvement but the required lower level has not been established. The ability to solve each of these problems exists but technological development has been slow, largely, because no single unified effort has been made by any one laboratory. The research proposed herein seeks to rectify this by developing (1) multiparameter immunofluorescence cell cycle analysis employing up to three antigens coupled with DNA content, (2) development of methodology to determine the absolute number of molecules rather than relative fluorescence, (3) standardization by creating cell line and/or microbead staining standards, and (4) increasing assay sensitivity through development of improved chemistry, hardware and software.