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• W2034678057 abstract "We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis. We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis. Chinese hamster ovary deoxyribonuclease expressed sequence tag green fluorescent protein benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone base pair(s) kilobase kilobase pair(s) caspase-activated DNase inhibitor of caspase-activated DNase. Apoptosis is a form of cell death utilized physiologically to maintain tissue homeostasis, as well as in response to various toxic stimuli, such as cancer chemotherapeutic agents. Apoptosis is characterized by cell shrinkage, membrane blebbing, condensation of the chromatin around the periphery of the nucleus, and DNA fragmentation (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6697) Google Scholar). Many stimuli can induce a cell to undergo apoptosis. Most of these stimuli, such as the Fas ligand or cisplatin, interact with defined primary targets, but the pathways that lead to the subsequent apoptotic death remain to be fully elucidated.Much work has been done to identify various steps in the pathway leading to apoptosis. Many of these discoveries can be attributed to the study of model organisms such as the nematode Caenorhabditis elegans in which the death of specific cells occurs at well defined developmental times. C. elegans proteins such as CED-3 and CED-9 have been found to have mammalian homologs that are implicated in apoptosis (2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2422) Google Scholar). For example, BCL-2 is the human homolog of CED-9, and is well known as a suppresser of apoptosis in both species (3Hengartner M.O. Ellis R.E. Horvitz H.R. Nature. 1992; 356: 494-499Crossref PubMed Scopus (709) Google Scholar). CED-3 is the prototype for a family of cysteine proteases now termed caspases that cleave substrates at the carboxy side of an aspartate residue and function to transduce a signal that leads to the degradation of the chromatin (4Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar).The enzyme responsible for the DNA digestion observed in apoptosis has yet to be conclusively identified. It has frequently been suggested that this endonuclease is Ca2+- and Mg2+-dependent (5Nikonova L.V. Nelipovich P.A. Umansky S.R. Biochim. Biophys. Acta. 1982; 699: 281-289Crossref PubMed Scopus (64) Google Scholar, 6Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 7McConkey D.J. Hartzell P. Duddy S.K. Hakansson H. Orrenius S. Science. 1988; 242: 256-259Crossref PubMed Scopus (431) Google Scholar, 8Peitsch M.C. Polzar B. Tschopp J. Mannherz H.G. Cell Death Differ. 1994; 1: 1-6PubMed Google Scholar, 9Hughes F.M. Cidlowski J.A. Cell Death Differ. 1994; 1: 11-17PubMed Google Scholar). This laboratory initially attempted to purify the Ca2+/Mg2+-dependent endonuclease from CHO1 cells, but no such endonuclease was detected. However, an acid-activated endonuclease was found in these cells. This enzyme was purified and identified as deoxyribonuclease II (DNase II) (10Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (430) Google Scholar). DNase II is active only at low pH, and studies have consistently shown that all cells undergoing apoptosis also undergo intracellular acidification (11Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357PubMed Google Scholar, 12Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 13Reynolds J.E. Li J. Craig R.W. Eastman A. Exp. Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (128) Google Scholar, 14Morana S. Wolf C.M. Li J. Reynolds J.E. Brown M.K. Eastman A. J. Biol. Chem. 1996; 271: 18263-18271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Endonuclease activity was also studied in many different leukemic cell lines, and internucleosomal DNA digestion was stimulated at an acidic pH (15Yanagisawa-Shiota F. Sakagami H. Kuribayashi N. Iida M. Sakagami T. Takeda M. Anticancer Res. 1995; 15: 259-266PubMed Google Scholar). Additional studies show that cells can undergo apoptosis after the chelation of Ca2+ hence obviating an essential role for Ca2+ in DNA digestion (16Barry M.A. Eastman A. Biochem. Biophys. Res. Commun. 1992; 186: 782-789Crossref PubMed Scopus (183) Google Scholar, 17Reynolds J.E. Eastman A. J. Biol. Chem. 1996; 271: 27739-27743Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These studies all suggest that DNase II may be involved in apoptotic DNA digestion.To better study the role of DNase II in the apoptotic process we have cloned its cDNA. To accomplish this, we purified bovine DNase II and obtained the NH2-terminal amino acid sequence, facilitating cloning of the bovine cDNA. We have subsequently cloned the complete human cDNA. Full-length cDNA for the human enzyme was subcloned into an expression vector and found to induce apoptosis when transfected into cells.DISCUSSIONMany different endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis. Originally, the apoptotic DNA degradation was attributed to a Ca2+-activated endonuclease in thymocytes (5Nikonova L.V. Nelipovich P.A. Umansky S.R. Biochim. Biophys. Acta. 1982; 699: 281-289Crossref PubMed Scopus (64) Google Scholar, 6Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 25Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1432) Google Scholar). Others have implicated various Ca2+/Mg2+-dependent endonucleases, including DNase I, Nuc18, and, more recently DNase γ, a DNase I homolog found in rat thymocyte nuclei (8Peitsch M.C. Polzar B. Tschopp J. Mannherz H.G. Cell Death Differ. 1994; 1: 1-6PubMed Google Scholar, 9Hughes F.M. Cidlowski J.A. Cell Death Differ. 1994; 1: 11-17PubMed Google Scholar, 26Gaido M.L. Cidlowski J.A. J. Biol. Chem. 1991; 266: 18580-18585Abstract Full Text PDF PubMed Google Scholar, 27Peitsch M.C. Polzar B. Stephan H. Crompton T. MacDonald H.R. Mannerherz H.G. Tschopp J. EMBO J. 1993; 12: 371-377Crossref PubMed Scopus (543) Google Scholar, 28Polzar B. Peitsch M.C. Loos R. Tschopp J. Mannherz H.G. Eur. J. Cell Biol. 1993; 62: 397-405PubMed Google Scholar, 29Shiokawa D. Ohyama H. Yamada T. Takahashi K. Tanuma S. Eur. J. Biochem. 1994; 226: 23-30Crossref PubMed Scopus (146) Google Scholar, 30Tanuma S. Shiokawa D. Biochem. Biophys. Res. Commun. 1994; 203: 789-797Crossref PubMed Scopus (86) Google Scholar, 31Shiokawa D. Ohyama H. Yamada T. Tanuma S. Biochem. J. 1997; 326: 675-681Crossref PubMed Scopus (57) Google Scholar). However, considerable evidence suggests that Ca2+ is not required for DNA digestion (32Eastman A. Cell Death Differ. 1994; 1: 7-9PubMed Google Scholar). For example, depletion of Ca2+ from cells is an effective means to induce internucleosomal DNA digestion (17Reynolds J.E. Eastman A. J. Biol. Chem. 1996; 271: 27739-27743Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).This laboratory was the first to suggest that DNase II might be an alternate endonuclease involved in apoptosis (10Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (430) Google Scholar). One criticism often made of the role of DNase II in apoptosis is the assumption that it is a lysosomal enzyme and not present in the nuclei, although we originally identified the acidic endonuclease activity in the nuclei of CHO cells. Other groups have also reported DNase II activity in the nucleus (33Vorbrodt A. Busch H. The Cell Nucleus. 3. Academic Press, New York1974: 309-344Crossref Google Scholar, 34Anzai N. Kawabata H. Hirama T. Masutani H. Yoshida Y. Okuma M. Blood. 1995; 86: 917-923Crossref PubMed Google Scholar), while others suggest that an acidic nuclease activity is translocated from the cytosolic compartment to the nucleus during apoptosis (15Yanagisawa-Shiota F. Sakagami H. Kuribayashi N. Iida M. Sakagami T. Takeda M. Anticancer Res. 1995; 15: 259-266PubMed Google Scholar). Therefore DNase II appears to be present in the nucleus and more could be translocated from the cytosol during apoptosis.Other reports have also implicated DNase II in the process of apoptosis. When lens fiber cells differentiate, they lose their nuclei in a process that is very similar to apoptosis. The chromatin condenses and the cells degrade their genomic DNA. A nuclease present in fiber cells was found to be cation-independent, and the fragmented DNA had no 3′-hydroxy termini that would result from a DNase I-type nuclease (36Chaudun E. Arruti C. Courtois Y. Ferrag F. Jeanny J.C. Patel B.N. Skidmore C. Torriglia A. Counis M.F. J. Cell. Physiol. 1994; 158: 354-364Crossref PubMed Scopus (35) Google Scholar). DNase II was found by immunocytochemistry to be localized in the cytoplasm but translocated to the nucleus of the fiber cell before degeneration (35Torriglia A. Chaudun E. Chany-Fournier F. Jeanny J. Courtois Y. Counis M. J. Biol. Chem. 1995; 270: 28579-28585Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). These findings suggest DNase II is the endonuclease responsible for the genomic degradation observed during lens nuclear degeneration.We have observed intracellular acidification in numerous models of apoptosis, which is consistent with the involvement of DNase II (11Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357PubMed Google Scholar, 12Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 13Reynolds J.E. Li J. Craig R.W. Eastman A. Exp. Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (128) Google Scholar, 14Morana S. Wolf C.M. Li J. Reynolds J.E. Brown M.K. Eastman A. J. Biol. Chem. 1996; 271: 18263-18271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, we have shown recently that low intracellular pH is not required for DNA digestion during apoptosis and furthermore that low pH can actually suppress apoptosis by preventing activation of caspases (12Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 37Reynolds J.E. Wolf C.M. Eastman A. Int. J. Oncol. 1997; 11: 1241-1246PubMed Google Scholar). It was therefore surprising that transfection of pDNase II induced apoptosis in cells grown under normal culture conditions, suggesting that this protein has activity at neutral pH. All previous analysis of the catalytic activity of DNase II has been performed with a 31-kDa protein, which we now show is truncated from the 40-kDa full-length form. It is possible that this larger protein has different catalytic requirements, and in particular, it may be active at neutral pH. Unfortunately, attempts so far to produce recombinant forms of this full-length protein have been compromised by the lethality of the constructs.An alternate explanation for the chromatin condensation appearing after transfection with pDNase II is that another endonuclease is activated. The proteolytic cascade of apoptosis mediated by caspases is normally required for DNA digestion; this can be suppressed by the caspase inhibitor zVAD-fmk. In these studies, we showed that zVAD-fmk did not prevent chromatin condensation following transfection with pDNase II, suggesting that other endonucleases were not responsible. Accordingly, it appears that the chromatin condensation is a direct consequence of the action of DNase II.Several caspases have been implicated in apoptosis, and numerous substrates have been reported. The caspase family of proteases cleave their substrates on the carboxy side of an aspartate residue. The coding sequence of DNase II contains a potential caspase cleavage site immediately upstream of the terminal serine in the 31-kDa bovine protein that was originally purified and sequenced. This indicated that the protein was produced as a larger precursor and subsequently modified to the acid-active 31-kDa protein by proteolytic cleavage at an aspartate residue. The cleavage site is ESQD in bovine and KSQD in human. Both of these potential recognition sequences have charged residues in the P4 position. The amino acid Lys at position P4 is not a known caspase cleavage site, but Glu at this position is found in a number of substrates of caspase 3 (38Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar).Lysosomal proteins are commonly cleaved after translocation to the lysosomes. The amino terminus of DNase II contains a stretch of hydrophobic amino acids which, by comparison with other lysosomal proteins, is likely to be a signal sequence. Such sequences are readily cleaved by cathepsins; in this case, the predicted cleavage site is prior to leucine 17 (23Yasuda T. Takeshita H. Iida R. Nakajima T. Hosomi O. Nakashima Y. Kishi K. J. Biol. Chem. 1998; 273: 2610-2616Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, the amino acid sequence around the amino terminus of the 31-kDa fragment is not a consensus sequence for lysosomal cathepsins (39Offermann M.K. Chlebowski J.F. Bond J.S. Biochem. J. 1983; 211: 529-534Crossref PubMed Scopus (18) Google Scholar, 40Imoto T. Okazaki K. Koga H. Yamada H. J. Biochem. (Tokyo). 1987; 101: 575-580Crossref PubMed Scopus (10) Google Scholar, 41van Noort J.M. van der Drift A.C.M. J. Biol. Chem. 1989; 264: 14159-14164Abstract Full Text PDF PubMed Google Scholar). Apart from caspases, the only other protease known to cleave at aspartic acid is a lymphocyte granule protease called granzyme or fragmentin (42Shi L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (419) Google Scholar). It is interesting that an earlier report of DNase II suggested it was a dimer of 35 and 10 kDa, which could represent the two fragments of the protein predicted here (19Liao T.-H. J. Biol. Chem. 1985; 260: 10708-10713Abstract Full Text PDF PubMed Google Scholar). However, the same report suggested both fragments were required for activity. During our purification over the S-Sepharose column, we observed a 10-kDa fragment that separated from DNase II but did not result in loss of DNase II activity (data not shown).The gene for DNase II was originally assigned to chromosome 19 using somatic cell hybrid studies in which mouse cells with a human chromosome 19 displayed higher acidic endonuclease activity (22Brook J.D. Shaw D.J. Meredith L. Bruns G.A.P. Harper P.S. Hum. Genet. 1984; 68: 282-285Crossref PubMed Scopus (61) Google Scholar). The exact localization of this gene has been found to be chromosome 19p13.2. The genomic sequence analysis predicted one different splice junction than observed in the cloned cDNA. This computer prediction is unlikely to be correct, since the ESTs found thus far confirm the cloned cDNA sequence. The human genomic sequence was compared with the genomic sequences of the three C. elegans homologs. There was significant conservation observed between human and the threeC. elegans proteins. The 30–36% identity between DNase II and the three C. elegans homologs is comparable with the 35% identity between caspase 3 and CED-3 (43Fernandes-Alnemri T. Litwack G. Fernandes-Alnemri E.S. J. Biol. Chem. 1994; 269: 30761-30764Abstract Full Text PDF PubMed Google Scholar) and considerably greater than the 23% identity between BCL-2 and CED-9 (44Hengartner M.O. Horvitz H.R. Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1043) Google Scholar). It is also interesting that no significant homology is observed in the yeastSaccharomyces cerevisiae, suggesting some function particularly important to a multicellular organism.The identification of the three C. elegans genes may suggest that DNase II could be a family of enzymes. In addition, there is considerable evolutionary conservation of the gene structure as exemplified by the conservation of two splice junctions in the homologs and the human sequences. A C. elegans nuclease, Nuc-1, was previously shown to have a role in DNA digestion following engulfment of a cell corpse (45Ellis R.E. Jacobson D.M. Horvitz H.R. Genetics. 1991; 129: 79-94Crossref PubMed Google Scholar). However, dying cells in Nuc-1-defective nematodes demonstrated a greater number of DNA breaks in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, presumably because Nuc-1 is only required for secondary destruction of already fragmented DNA. 3R. Horvitz, unpublished observation. This indicates that additional endonucleases must exist to degrade DNA during apoptosis. The genes for the DNase II homologs are all on separate loci from Nuc-1, indicating that they are indeed separate genes and should be examined for a role in the apoptotic process.There has been controversy for many years over which endonuclease is responsible for the DNA digestion that occurs during apoptosis. Very recently, a caspase-activated DNase (CAD) was reported as a new candidate (46Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2795) Google Scholar). This endonuclease requires Mg2+, but not Ca2+, consistent with the discussion above that Ca2+ is not required for apoptotic DNA fragmentation. Most intriguing is the observation that CAD dimerizes with an inhibitor, ICAD, that is cleaved during apoptosis. The human homolog of murine ICAD had already been identified as “DNA fragmentation factor,” a protein whose cleavage was shown to trigger DNA digestion (47Liu X. Zou H. Slughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). Hence, CAD is possibly the best candidate for endonuclease activity during apoptosis, although it is possible that ICAD may also inhibit other endonucleases. DNase II remains a possible alternative as it is found ubiquitously in tissues unlike most other candidates; the only cell line we have found that had no detectable DNase II mRNA is noted for its resistance to apoptotic DNA digestion. DNase II is also highly conserved through evolution suggesting its importance in some physiological process. Since completion of this work, another group has also published the cloning of human DNase II, although their cDNA sequence lacks much of the 3′-untranslated sequence (23Yasuda T. Takeshita H. Iida R. Nakajima T. Hosomi O. Nakashima Y. Kishi K. J. Biol. Chem. 1998; 273: 2610-2616Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The current paper adds the exact chromosomal location, the genomic structure, and homology with other species. The cloning of the cDNA will facilitate studies of its activity as well as establishing its biological functions and localization in cells. The transfection experiments presented here clearly show the potential of this enzyme to induce the DNA degradation observed during apoptosis. Future studies will be aimed to definitively establish any role in this process. Apoptosis is a form of cell death utilized physiologically to maintain tissue homeostasis, as well as in response to various toxic stimuli, such as cancer chemotherapeutic agents. Apoptosis is characterized by cell shrinkage, membrane blebbing, condensation of the chromatin around the periphery of the nucleus, and DNA fragmentation (1Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6697) Google Scholar). Many stimuli can induce a cell to undergo apoptosis. Most of these stimuli, such as the Fas ligand or cisplatin, interact with defined primary targets, but the pathways that lead to the subsequent apoptotic death remain to be fully elucidated. Much work has been done to identify various steps in the pathway leading to apoptosis. Many of these discoveries can be attributed to the study of model organisms such as the nematode Caenorhabditis elegans in which the death of specific cells occurs at well defined developmental times. C. elegans proteins such as CED-3 and CED-9 have been found to have mammalian homologs that are implicated in apoptosis (2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2422) Google Scholar). For example, BCL-2 is the human homolog of CED-9, and is well known as a suppresser of apoptosis in both species (3Hengartner M.O. Ellis R.E. Horvitz H.R. Nature. 1992; 356: 494-499Crossref PubMed Scopus (709) Google Scholar). CED-3 is the prototype for a family of cysteine proteases now termed caspases that cleave substrates at the carboxy side of an aspartate residue and function to transduce a signal that leads to the degradation of the chromatin (4Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar). The enzyme responsible for the DNA digestion observed in apoptosis has yet to be conclusively identified. It has frequently been suggested that this endonuclease is Ca2+- and Mg2+-dependent (5Nikonova L.V. Nelipovich P.A. Umansky S.R. Biochim. Biophys. Acta. 1982; 699: 281-289Crossref PubMed Scopus (64) Google Scholar, 6Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 7McConkey D.J. Hartzell P. Duddy S.K. Hakansson H. Orrenius S. Science. 1988; 242: 256-259Crossref PubMed Scopus (431) Google Scholar, 8Peitsch M.C. Polzar B. Tschopp J. Mannherz H.G. Cell Death Differ. 1994; 1: 1-6PubMed Google Scholar, 9Hughes F.M. Cidlowski J.A. Cell Death Differ. 1994; 1: 11-17PubMed Google Scholar). This laboratory initially attempted to purify the Ca2+/Mg2+-dependent endonuclease from CHO1 cells, but no such endonuclease was detected. However, an acid-activated endonuclease was found in these cells. This enzyme was purified and identified as deoxyribonuclease II (DNase II) (10Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (430) Google Scholar). DNase II is active only at low pH, and studies have consistently shown that all cells undergoing apoptosis also undergo intracellular acidification (11Barry M.A. Reynolds J.E. Eastman A. Cancer Res. 1993; 53: 2349-2357PubMed Google Scholar, 12Li J. Eastman A. J. Biol. Chem. 1995; 270: 3203-3211Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 13Reynolds J.E. Li J. Craig R.W. Eastman A. Exp. Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (128) Google Scholar, 14Morana S. Wolf C.M. Li J. Reynolds J.E. Brown M.K. Eastman A. J. Biol. Chem. 1996; 271: 18263-18271Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Endonuclease activity was also studied in many different leukemic cell lines, and internucleosomal DNA digestion was stimulated at an acidic pH (15Yanagisawa-Shiota F. Sakagami H. Kuribayashi N. Iida M. Sakagami T. Takeda M. Anticancer Res. 1995; 15: 259-266PubMed Google Scholar). Additional studies show that cells can undergo apoptosis after the chelation of Ca2+ hence obviating an essential role for Ca2+ in DNA digestion (16Barry M.A. Eastman A. Biochem. Biophys. Res. Commun. 1992; 186: 782-789Crossref PubMed Scopus (183) Google Scholar, 17Reynolds J.E. Eastman A. J. Biol. Chem. 1996; 271: 27739-27743Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). These studies all suggest that DNase II may be involved in apoptotic DNA digestion. To better study the role of DNase II in the apoptotic process we have cloned its cDNA. To accomplish this, we purified bovine DNase II and obtained the NH2-terminal amino acid sequence, facilitating cloning of the bovine cDNA. We have subsequently cloned the complete human cDNA. Full-length cDNA for the human enzyme was subcloned into an expression vector and found to induce apoptosis when transfected into cells. DISCUSSIONMany different endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis. Originally, the apoptotic DNA degradation was attributed to a Ca2+-activated endonuclease in thymocytes (5Nikonova L.V. Nelipovich P.A. Umansky S.R. Biochim. Biophys. Acta. 1982; 699: 281-289Crossref PubMed Scopus (64) Google Scholar, 6Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 25Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1432) Google Scholar). Others have implicated various Ca2+/Mg2+-dependent endonucleases, including DNase I, Nuc18, and, more recently DNase γ, a DNase I homolog found in rat thymocyte nuclei (8Peitsch M.C. Polzar B. Tschopp J. Mannherz H.G. Cell Death Differ. 1994; 1: 1-6PubMed Google Scholar, 9Hughes F.M. Cidlowski J.A. Cell Death Differ. 1994; 1: 11-17PubMed Google Scholar, 26Gaido M.L. Cidlowski J.A. J. Biol. Chem. 1991; 266: 18580-18585Abstract Full Text PDF PubMed Google Scholar, 27Peitsch M.C. Polzar B. Stephan H. Crompton T. MacDonald H.R. Mannerherz H.G. Tschopp J. EMBO J. 1993; 12: 371-377Crossref PubMed Scopus (543) Google Scholar, 28Polzar B. Peitsch M.C. Loos R. Tschopp J. Mannherz H.G. Eur. J. Cell Biol. 1993; 62: 397-405PubMed Google Scholar, 29Shiokawa D. Ohyama H. Yamada T. Takahashi K. Tanuma S. Eur. J. Biochem. 1994; 226: 23-30Crossref PubMed Scopus (146) Google Scholar, 30Tanuma S. Shiokawa D. Biochem. Biophys. Res. Commun. 1994; 203: 789-797Crossref PubMed Scopus (86) Google Scholar, 31Shiokawa D. Ohyama H. Yamada T. Tanuma S. Biochem. J. 1997; 326: 675-681Crossref PubMed Scopus (57) Google Scholar). However, considerable evidence suggests that Ca2+ is not required for DNA digestion (32Eastman A. Cell Death Differ. 1994; 1: 7-9PubMed Google Scholar). For example, depletion of Ca2+ from cells is an effective means to induce internucleosomal DNA digestion (17Reynolds J.E. Eastman A. J. Biol. Chem. 1996; 271: 27739-27743Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar).This laboratory was the first to suggest that DNase II might be an alternate endonuclease involved in apoptosis (10Barry M.A. Eastman A. Arch. Biochem. Biophys. 1993; 300: 440-450Crossref PubMed Scopus (430) Google Scholar). One criticism often made of the role of DNase II in apoptosis is the assumption that it is a lysosomal enzyme and not present in the nuclei, although we originally identified the acidic endonuclease activity in the nuclei of CHO cells. Other groups have also reported DNase II activity in the nucleus (33Vorbrodt A. Busch H. The Cell Nucleus. 3. Academic Press, New York1974: 309-344Crossref Google Scholar, 34Anzai N. Kawabata H. Hirama T. Masutani H. Yoshida Y. Okuma M. Blood. 1995; 86: 917-923Crossref PubMed Google Scholar), while others suggest that an acidic nuclease activity is translocated from the cytosolic compartment to the nucleus during apoptosis (15Yanagisawa-Shiota F. Sakagami H. Kuribayashi N. Iida M." @default.
• W2034678057 created "2016-06-24" @default.
• W2034678057 creator A5016470161 @default.
• W2034678057 creator A5086245428 @default.
• W2034678057 date "1998-11-01" @default.
• W2034678057 modified "2023-10-13" @default.
• W2034678057 title "The Cloning and Expression of Human Deoxyribonuclease II" @default.
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