DNA fragmentation during apoptosis is considered to occur in two stages. Walker et al. (9) reported sequential degradation of genomic DNA, initially to high molecular weight (HMW) DNA fragments of approximately 300 kb, followed by the appearance of 50 kb loop-size chromatin fragments which were detected using pulse-field gel electrophoresis. Oligonucleosomal DNA
fragments that produce the characteristic DNA ladder are released when the 50 kb fragments are further degraded. Certain cells, such as MoIt-4 human T cell leukaemia cells (5) and DU145 human prostate cells (8,10) do not display internucleosomal DNA fragmentation, although these cells die with a typical apoptotic morphology. In these cells, however, HMW DNA fragmentation can be observed, suggesting that HMW DNA cleavage is also an important biochemical marker of apoptosis and that internucleosomal DNA cleavage is dispensable for apoptosis (7,11,12). In general, haematopoietic cells, such as human leukaemia HL60 and Jurkat, are very sensitive to induction of apoptosis and undergo internucleosomal DNA fragmentation upon exposure to various apoptotic stimuli, including inhibitors of topoisomerase I (campto-thecin) or topoisomerase II (etoposide = VP-16) or staurosporine (13-17). On the other hand, epithelial cells (e.g. MCF7 human breast cancer or DU145 prostatic carcinoma cells) are generally more resistant to apoptosis (8,10).
The presence of 5'-phosphate (5'-P) and 3'-hydroxyl (3'-OH) DNA termini in apoptotic cells is an important biochemical feature of apoptotic DNA fragments. Indeed, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) method, which is commonly used to detect apoptotic cells, is based on the presence of 3'-OH termini (18). This method is described in Chapter 2. Another characteristic feature of apoptotic DNA fragmentation is that single-stranded DNA breaks are produced as well as double-strand breaks. Peitsch et al. (19) and Walker et al. (20) found that numerous single-strand breaks are generated in internucleosomal DNA regions during apoptosis. They proposed that DNA single-strand breaks accumulate in the linker regions between nucleosomes, eventually causing double-strand scissions which release oligonucleosomes.
Several nucleases are probably responsible for the DNA fragmentation in apoptosis (Table 1). Among the first reported were DNase I (21, 22) and Nucl8 (23). Nucl8 (cyclophilin) was isolated from apoptotic rat thymocytes
(24). Recently, a 33 kDa DNase I-related nuclease (DNase Y) has been isolated from rat cells. Its gene encodes a 36 kDa protein that is ubiquitously expressed and is activated by proteolytic cleavage to a 33 kDa endonuclease
(25). This nuclease resembles the DNase y, a 34 kDa nuclease that was recently purified from rat thymocytes (26). Interestingly, DNase Y is not transcriptionally regulated in apoptosis (25). An acidic endonuclease, DNase II (27, 28) has also been proposed to be involved, as well as several Ca2+/Mg2+-dependent endonucleases (26, 29-31). However, it should be noted that DNase II produces 5'-OH and 3'-P DNA termini (32), which is not consistent with the characteristics of apoptotic DNA fragmentation. Two other Ca2+/Mg2+-dependent endonucleases have also been isolated from apoptotic rodent cells and another endonuclease was isolated from apoptotic mouse T cell hybridoma (30). It consists of three molecular forms (A, B, and C) with apparent molecular weights of 49, 47, and 45 kDa, respectively (30). In addition, a 97-kDa Ca2+/Mg2+-dependent nuclease producing internucleo-
Table 1. Overview of the apoptotic nucleases
Nuc18 ILCME (not named) (not named) DNase I DNase 7 DNase Y DNase II CAD/DFF40 AN34
Ca2+/Mg2+ Ca2+/Mg2+ Ca2+/Mg2+ Ca2+/Mg2+ Ca2+/Mg2+ Ca2+/Mg2+ Ca2+/Mg2+ (-)
Ion dependency ra2+/Mn2+
97 28 31 34
33 40 40
Mouse T cell hybridoma
Rodent and human cells
Human leukaemia HL60
31 29 22 26 25 28
Reference somal DNA cleavage cells has been reported to be present in untreated rat hepatoma (31).
Very recently, a caspase-activated DNase (CAD) has been reported by S. Nagata and co-workers (33). CAD is a 40 kDa protein that induces internucle-osomal DNA cleavage in isolated nuclei in the presence of Mg2+. CAD activity does not require Ca2+, and CAD forms a complex and co-purifies with an inhibitor of CAD (ICAD) that can be cleaved by caspase-3 during apoptosis (33, 34). CAD and ICAD have been isolated independently by X. Wang and co-workers who showed that DNA fragmentation and chromatin condensation depend on a heterodimeric protein composed of two DNA fragmentation factors of 45 and 40 kDa (DFF45 and DFF40 corresponding to ICAD and CAD, respectively) (35, 36). In our laboratory, we purified a 34 kDa Mg2+-dependent and Ca2+-independent endonuclease from etoposide-treated HL60 cells undergoing apoptosis (37), and designated this 34 kDa Mg2+-dependent endonuclease, AN34 (apoptotic nuclease 34 kDa) (37). AN34 introduces single-and double-strand breaks in purified DNA and internucleosomal DNA cleavage in isolated nuclei (37). AN34-induced DNA breaks terminate with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation (37). Interestingly, our data using caspase inhibitor (zVAD-fmk) suggest that the action of AN34 is also controlled by caspase (37), although AN34 is not inhibited by ICAD (DFF45) (A. Yoshida and Y. Pommier, unpublished data).
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