Comparison of two methods of staining apoptotic cells of leukemia cell lines. Terminal deoxynucleotidyl transferase and DNA polymerase I reactions.
We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)
Yamadori
I
I
Department of Pathology, Okayama University Medical School, Shikata-cho, Okayama, Japan.
Yoshino
T
T
Kondo
E
E
Cao
L
L
Akagi
T
T
Matsuo
Y
Y
Minowada
J
J
D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D001549:Benzamides / Q000494:pharmacology; D002467:Cell Nucleus / Q000648:ultrastructure; D004396:Coloring Agents; D003593:Cytoplasm / Q000648:ultrastructure; D053938:DNA Fragmentation; D004256:DNA Polymerase I / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016591:Histocytological Preparation Techniques; D006801:Humans; D020287:In Situ Nick-End Labeling; D007938:Leukemia / Q000201:enzymology* / Q000473:pathology; D009536:Niacinamide / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000494:pharmacology; D019014:fas Receptor / Q000276:immunology
D000911:Antibodies, Monoclonal; D001549:Benzamides; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D019014:fas Receptor; D009536:Niacinamide; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D004256:DNA Polymerase I; D003850:Deoxyribonuclease I; D015032:Zinc