TUNEL assay as a measure of chemotherapy-induced apoptosis.
Chemotherapy induces injury to tumor cells, which subsequently die by a number of processes. One of those processes is apoptosis, and its measurement can be a useful tool to understanding the mechanisms of action of chemotherapy agents, drug resistance, and tumor biology. Cells undergoing apoptosis eventually cleave their DNA with nucleases in a characteristic pattern that leaves free 3'OH ends. This permits the identification of cells undergoing apoptosis by transfer of fluorescence-labeled dUTP to the free 3'OH ends using the enzyme terminal deoxynucleotidyl transferase (TdT) in a process termed terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL). This chapter outlines the concepts governing the measurement of apoptosis following chemotherapy by TUNEL in cells in culture and in tissue biopsied from patients or laboratory animals that received chemotherapy. It lists the reagents, procedures for cell and tissue handling, labeling techniques, caveats, and appropriate controls for labeling 3'OH termini with TdT and quantifying the results.
Wieder
Robert
R
University of Medicine & Dentistry of New Jersey, Division Medical Oncology/Hematology, Newark, USA.
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D002448:Cell Adhesion; D003584:Cytological Techniques; D004247:DNA / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D008856:Microscopy, Fluorescence; D009369:Neoplasms / Q000188:drug therapy / Q000473:pathology*; D010232:Paraffin / Q000737:chemistry
D000970:Antineoplastic Agents; D010232:Paraffin; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D019286:Endopeptidase K