Flow cytometric apoptosis assays indicate different types of endonuclease activity in haematopoietic cells and suggest a cautionary approach to their quantitative use.
Two flow cytometric apoptosis assays, the terminal deoxynucleotidyl transferase (TdT) assay and in situ nick translation (ISNT) assay, were assessed for their ability to quantitate drug-induced apoptosis in CLL lymphocytes. In contrast to HL60 cells, biotinylated dUTP could not be effectively incorporated into apoptotic CLL lymphocytes using exogenous TdT. This suggested that CLL lymphocytes possess a different type of endonuclease that cleaves DNA, leaving blunt or 3' recessed DNA breaks, which are poor substrates for TdT. This possibility was tested using lambda exonuclease, which can convert a blunt or 3' recessed DNA break into a 3' overhang. Apoptotic CLL lymphocytes pre-treated with lambda exonuclease demonstrated increased nucleotide incorporation with TdT. Single-strand DNA breaks are also present in apoptotic CLL lymphocytes, as labelled nucleotides could be incorporated using the in situ nick translation assay. This study suggests that the efficiency of tailing reactions may be limited by the nature of the endonuclease activity in certain cell types and that validation with other parameters is an essential prerequisite to their quantitative use.
McKenna
S L
SL
Department of Haematology, University of Wales College of Medicine, Cardiff, United Kingdom. STBI8033@bureau.ucc.ie
Hoy
T
T
Holmes
J A
JA
Whittaker
J A
JA
Jackson
H
H
Padua
R A
RA
D017209:Apoptosis; D002478:Cells, Cultured; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I; D015342:DNA Probes; D003850:Deoxyribonuclease I / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D005434:Flow Cytometry / Q000379:methods*; D018922:HL-60 Cells / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood* / Q000201:enzymology; D007963:Leukocytes, Mononuclear
D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I; D003850:Deoxyribonuclease I