Two 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 detect 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. Experimental design; Spontaneous apoptous cell death was detected in plastic embedded tumor biopsies from patients with non-Hodgkin's lymphoma (NHL), head and neck squamous cell carcinomas (HNSCC), and breast cancer 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 etoposide (VP16) was also studied in HL60 cells by in situ methods and by agarose gel electrophoresis.

RESULTS

Interestingly, whereas NH1 and HNSCC biopsies showed comparable levels of detectability with the two techniques, the breast tissues be it neoplastic, normal or metastatic, revealed apoptosis detectable only by TUNEL and not by ISEL. Similarly in HL60 cells, the percentage of apoptotic cells or apoptotic index (AI) determined by TUNEL was significantly higher than that determined by ISEL. A double labelling of these HL60 cells for ISEL and TUNEL also revealed a higher proportion of cells labeled positively for TUNEL as compared to those labeled for ISEL. Agarose gel electrophoresis revealed characteristic DNA laddering only at 35 microM 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 differentiated 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 multiples) but have differently staggered ends. These fragments may be 3' recessed, 5' recessed or blunt ended. While TUNEL can label all three types, ISEL labels only those with 3' recessed ends.

CONCLUSIONS

Thus our data show that the DNA fragments formed during spontaneous apoptosis in breast tissues and preferentially during VP16 induced apoptosis in HL60 cells are either 5' recessed or blunt ended, being distinctly different from 3' recessed fragments seen in NHL and HNSCC or with a lesser frequency in VP 16 treated HL60 cells. Specific fragmentation patterns could be a result of activation of different endonucleases which as indicated by our data could be tissue specific and may be differentially activated by different chemotherapeutic agents. Therefore, screening for the presence of specific endonucleases in different tissues and for agents specifically activating them would have major clinical implications.