Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique.
Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase.
Ehemann
Volker
V
Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220-221, 69120 Heidelberg, Germany. volker_ehemann@med.uni-heidelberg.de
Sykora
Jaromir
J
Vera-Delgado
Jorge
J
Lange
Adelheid
A
Otto
Herwart F
HF
D017209:Apoptosis; D001943:Breast Neoplasms / Q000473:pathology*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D020287:In Situ Nick-End Labeling; D008214:Lymphocytes / Q000378:metabolism
D004247:DNA