2 IN-SITU LABELING TECHNIQUES REVEAL DIFFERENT PATTERNS OF DNA FRAGMENTATION DURING SPONTANEOUS APOPTOSIS IN-VIVO AND INDUCED APOPTOSIS IN-VITRO

Background: Two new enzymatic reactions were described recently to det ect apoptotic cell death in situ viz. in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase mediated UTP nick end labeling ( TUNEL) of fragmented DNA. A comparative study was conducted to detect in vivo and in vitro apoptotic death using these two techniques. Exper imental design: Spontaneous apoptotic cell death was detected in plast ic embedded tumor biopsies from patients with non-Hodgkin's lymphoma ( NHL), head and neck squamous cell carcinomas (HNSCC), and breast cance r using these two in situ methods. Uninvolved normal tissues adjacent to breast tumors and a lymph node metastasis of breast tumor were also studied. Furthermore, apoptotic death induced by different doses of e toposide (VP16) was also studied in HL60 cells by in situ methods and by agarose gel electrophoresis. Results: Interestingly, whereas NHL an d HNSCC biopsies showed comparable levels of detectability with the tw o techniques, the breast tissues be it neoplastic, normal or metastati c, revealed apoptosis detectable only by TUNEL and not by ISEL. Simila rly in HL60 cells, the percentage of apoptotic cells or apoptotic inde x (AI) determined by TUNEL was significantly higher than that determin ed by ISEL. A double labeling of these HL60 cells for ISEL and TUNEL a lso revealed a higher proportion of cells labeled positively for TUNEL as compared to those labeled for ISEL. Agarose gel electrophoresis re vealed characteristic DNA laddering only at 35 mu M dose of VP 16. No smearing of DNA was found in any group ruling out the necrotic death. In vivo, in one HNSCC specimen apoptosis and necrosis could be differe ntiated by the difference in staining intensity. Both methods stained necrotic chromatin fragments very lightly. The DNA fragments generated during apoptosis could be of unique lengths (ie 180-200 bp or multipl es) but have differently staggered ends. These fragments may be 3' rec essed, 5' recessed or blunt ended. While TUNEL can label all three typ es, ISEL labels only those with 3' recessed ends. Conclusions: Thus ou r data show that the DNA fragments formed during spontaneous apoptosis in breast tissue and preferentially during VP16 induced apoptosis in HL60 cells are either 5' recessed or blunt ended, being distinctly dif ferent from 3' recessed fragments seen in NHL and HNSCC or with a less er frequency in VP 16 treated HL60 cells. Specific fragmentation patte rn could be a result of activation of different endonucleases which as indicated by our data could be tissue specific and may be differentia lly activated by different chemotherapeutic agents. Therefore, screeni ng for the presence of specific endonucleases in different tissues and for agents specifically activating them would have major clinical imp lications.