Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells

Apoptosis, originally defined by specific morphological changes, is characterised biochemically by non-random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50 kbp fragments prior to, concomitantly with, or in the absence of 180 bp integer fragmentation. Incorporation into fragmented DNA of biotin-labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorporation is visualized using avidin-tagged fluorescein isothiocyanate (FITC) (Gorczyca et al.: Cancer Res 53:1945-1951, 1993; Jonker et al.: Cytometry (Suppl 13): Abstr 99A, 1993). Here, we characterise this assay further in three different haemopoietic cell lines. Drug-induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson-encoded protein tyrosine kinase suppressed drug-induced apoptosis, but did not inhibit drug-induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N-methylformamide in HL60 and MOLT-4 cells, including those high molecular weight DNA fragments formed in MOLT-4 cells which were not further cleaved to 180-200 bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples.