Detection of apoptosis in leukemic and breast cancer cells with monoclonal antibody to single-stranded DNA.
In cultures of leukemic HL-60 and MOLT-4 cells treated with etoposide all nuclei with distinctive morphology of apoptosis (chromatin condensation at the nuclear periphery, nuclear fragmentation) were stained with monoclonal antibody F7-26 specific for single-stranded DNA. DNA in interphase and mitotic cells of control cultures and DNA in necrotic cells of cultures treated with sodium azide did not bind the antibody. In monolayer cultures of breast cancer cell line MDA-468 treated with tamoxifen for 4 hours all cells detached from substratum. These cells were apoptotic by nuclear morphology and stained with F7-26. Subset of cells without visible chromatin condensation but stained with F7-26 was detected among cells still attached to the substratum 1 hour after addition of tamoxifen. Thus, in breast cancer cells reactivity with F7-26 preceded chromatin condensation detected by fluorescence microscopy. Apoptotic cells stained with the antibody and non-apoptotic cells with background fluorescence were completely separated on two-parameter plots generated on a flow cytometer. Linear relation between percentage of apoptotic cells and ELISA reactivity with F7-26 in the cells attached to microtiter plates was demonstrated. These data show that apoptotic response can be measured by ELISA using staining with F7-26. Cells undergoing apoptosis can be detected by the procedure based on thermal denaturation of DNA in situ in the presence of Mg2+ with subsequent staining with the antibody specific for DNA in single-stranded conformation. Correlation between nuclear morphology typical of apoptosis in various cell types demonstrated that staining with monoclonal antibody F7-26 provides specific cytochemical marker for apoptotic cells.
Frankfurt
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Oncology Laboratory, Cedars Medical Center, Miami, FL 33136.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D015458:Leukemia, T-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002843:Chromatin; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D005047:Etoposide; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008274:Magnesium