Flow cytometric analysis of nuclear matrix proteins: method and potential applications.
The nuclear matrix (NM) is an important structural component of the nucleus that participates in the regulation of several diverse metabolic processes. Immunometric assays have shown that alterations in NM-associated functions and morphological characteristics may occur as a result of changes in NM composition. Recent evidence suggests that detection of quantitative or qualitative changes in nuclear matrix protein (NMP) composition may be useful in the diagnosis of cancer and as a reliable indicator of cell death. We have developed an in situ flow cytometric technique for the simultaneous detection of specific NMPs and DNA content in fixed, permeabilized cells. Illustrative results from two different applications of these methods involving two different cell lines (human melanoma and promyelocytic leukemia) are presented, including: 1) measurements of NM breakdown in necrotic and apoptotic cells after treatment with the cytotoxic agents camptothecin, etoposide, or hyperthermia; and 2) detection of changes in NMP content immediately after heat shock. We demonstrate that the technique is useful for the identification of cell-cycle specificity of NM breakdown and allows correlations to be made between the kinetics of DNA fragmentation and NMP solubilization. Furthermore, our studies indicate that flow cytometric detection of changes in NM composition may be useful for identifying different modes and temporal patterns of cell death. We discuss other potential applications of the technique and advantages over standard biochemical assays.
Dynlacht
J R
JR
Department of Radiological Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Henthorn
J
J
O'Nan
C
C
Dunn
S T
ST
Story
M D
MD
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D002453:Cell Cycle / Q000276:immunology; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006358:Hot Temperature; D009687:Nuclear Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D009687:Nuclear Proteins; D004247:DNA