Deoxyribonuclease II proteins and cDNAS

Details Deoxyribonuclease II proteins and cDNAS Deoxyribonuclease II proteins and cDNAS

1999-03-23

2001-02-06

Schwartzman, Robert A. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C536S023100, C536S024500, C435S006120, C435S091100, C435S325000

Reexamination Certificate

active

06184034

ABSTRACT:

BACKGROUND OF THE INVENTION
Controlled cell death is critical for the life of a human; too much cell death can lead to diseases such a neurodegeneration and autoimmune deficiency syndrome (AIDS) while too little cell death can lead to cancer or autoimmune diseases. Recent studies have defined the pathway of cell death as “apoptosis” and have identified some of the biochemical steps involved.
Apoptosis is a homeostatic mechanism involved in the controlled death of obsolete cells during metamorphosis, differentiation, cell turnover, and hormone mediated deletion of thymocytes (Wyllie et al.
Int. Rev. Cytol.
1980 68:251-306). Apoptosis has also been identified as the mechanism of cell killing during growth factor withdrawal (Rodriguez-Tarduchy et al.
EMBO J.
1990 9:2997-3002; McConkey et al.
J. Biol. Chem.
1990 265:3009-3011), T-cell deletion, treatment with many cytotoxic agents (Cohen, J. J. and Duke, R. C.
J. Immunol.
1984 132:38-42; Barry et al.
Biochem. Pharmacol.
1990 40:2353-2362; Kaufmann, S. H.
Cancer Res.
1989 49:5870-5878; and McConkey et al.
Science
1988 242:256-259), and following hypothermia (Barry et al.
Biochem. Pharmacol.
1990 40:2353-2362; Lennon et al.
Biochem. Soc. Trans.
1990 18:343-345; Takano et al.
J. Pathol.
1991 163:329-336).
Central to the mechanism of apoptosis is internucleosomal DNA digestion by endogenous endonucleases. Mammalian cells contain a variety of endonucleases which could be involved in internucleosomal DNA digestion. However, it has been postulated that the primary endonuclease involved in apoptosis is a Ca
2+
/Mg
2+
-dependent endonuclease. Several Ca
2+
/Mg
2+
-dependent endonucleases have been identified, one of which is deoxyribonuclease I (DNase I), (Peitsch et al.
EMBO J.
1993 12:371).
Recent experiments, however, indicate that DNase I may not be the primary endonuclease involved in apoptosis. It has been found that many cells do not contain this endonuclease. The role of DNase I, or any other Ca
2+
/Mg
2+
-dependent endonuclease is further unlikely, as often no increase, or only a minor increase, in Ca
2+
levels occurs in apoptotic cells (Eastman, A.
Cell Death and Differentiation
1994 1:7-9).
An alternate endonuclease that is active below pH 7.0 and has no apparent requirement for Ca
2+
or Mg
2+
has been detected (Barry, M. A. and Eastman, A.
J. Natl Cancer Inst.
1990 82:749). This alternate endonuclease has been identified as deoxyribonuclease II (DNase II; Barry, M. A. and Eastman, A.
Archives of Biochem and Biophys.
1993 300(1):440-450). It is believed that this enzyme is involved in the internucleosomal digestion or fragmentation of DNA which is one of the early steps in the pathway of apoptosis. Another report that has implicated DNase II in cell death involves lens fiber cell differentiation, a process where the cells lose their nuclei in a manner similar to apoptosis (Torriglia, A. et al. 1995
J. Biol. Chem.
270:28579-28585). In this process, the chromatin condenses and the cells degrade their genomic DNA. DNase II was found by immunocytochemistry to be localized in the cytoplasm but translocated to the nucleus of the fiber cell before degeneration. These findings implicate DNase II as the endonuclease responsible for genomic degradation observed during lens nuclear degeneration, and further support a role for this enzyme in mechanisms of cell death.
DNase II has now been isolated and purified and the amino acid sequence determined. Further, the DNA sequences for both the human and bovine proteins have now been cloned.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a cDNA encoding deoxyribonuclease II enzyme.
Another object of the present invention is to provide an isolated, purified deoxyribonuclease II enzyme.
Yet another object of the present invention is to provide antibodies against deoxyribonuclease II which can be used in diagnosing cells at stages in the apoptotic pathway.
Yet another object of the present invention is to provide antisense agents targeted to a cDNA or corresponding mRNA encoding deoxyribonuclease II which can be used to reduce levels of this enzyme.
Yet another object of the present invention is a method for identifying agents that inhibit deoxyribonuclease II activity comprising treating cells with a test agent, transfecting cells with deoxyribonuclease II, maintaining said transfected cells in culture, and monitoring apoptosis in treated and untreated cells to determine whether the test agent modulates apoptosis.
Yet another object of the present invention is a method of inducing apoptosis in selected cells comprising transfecting cells with a vector expressing the deoxyribonuclease II cDNA so that apoptosis is induced.
Yet another object of the present invention is to provide a method of digesting DNA released from dying cells comprising contacting said cells with an effective amount of an isolated, purified deoxyribonuclease II protein so that DNA is digested.
DETAILED DESCRIPTION OF THE INVENTION
The existence of DNase II as a protein of lysosomal origin that is involved in cellular digestion of foreign DNA has been known for many years. Recently, this enzyme has been linked with the DNA fragmentation that occurs at an early stage in apoptosis.
The bovine and human forms of the DNase II protein have now been isolated and purified and the amino acid sequences of these proteins determined. cDNAs encoding the bovine and human form of this protein have also been cloned and characterized. The complete amino acid and nucleotide sequence of human DNase II are provided in SEQ ID NO: 3 and SEQ ID NO: 1, respectively. This cloning was performed by first purifying the bovine protein, sequencing a stretch of amino acids, as depicted in SEQ ID NO: 4, and using molecular biology techniques well known to those of skill in the art to isolate a portion of the bovine cDNA sequence, as depicted in SEQ ID NO: 2. Nine hundred and twenty-seven bases of the bovine cDNA sequence were obtained. The predicted translation of the sequence gave 276 amino acids of which 21 amino acids were upstream of the initial serine obtained by protein sequencing. No initiator methionine codon was found in the 21 amino acids upstream. However, the codon for an aspartate residue was found to be present immediately upstream of the amino terminal serine thus indicating that DNase II is produced as a larger protein which is post-translationally modified at this aspartate residue to produce the acid-activated 31 kDa protein. The potential active site of porcine DNase II had been previously purified and sequenced and consisted of an octomer, ATEDHSKW (SEQ ID NO: 5) (Liao, T. -H. 1985
J. Biol. Chem.
260:10708-10713). The cDNA coding for this octomer is found in the bovine sequence and comprises amino acids 184-191.
This bovine sequence was then used to isolate the human sequence. The bovine cDNA sequence was compared to sequences in the GenBank database and was found to be homologous to three overlapping human ESTs. The human ESTs was used to design primers to amplify human cDNA from a U937 cell line cDNA library. Additional upstream human cDNA sequence was obtained from 4 separate clones that had significant homology to bovine sequence. A primer was designed using sequence that was 5′, Hfor3, and used in a PCR with the Revcon1 primer to amplify a 357 bp fragment of DNase II to be used as a probe for screening of Northern Blots and human cDNA libraries.
Total RNA from 5 human cell lines was analyzed by Northern Blotting. A message of approximately 2 kb was detected in the human myeloid cell line ML-1, and the human breast carcinoma lines MDA-231, T47D, and MCF7. However, no signal was detected from the epithelial carcinoma HeLa cell RNA, a cell line that does not readily undergo DNA laddering during apoptosis.
A lambda Zap HepG2 cDNA library, an ML-1 cDNA library, and a lambda Unizap human macrophage cDNA library were all screened with this 357 bp probe. No full length clones were isolated from the HepG2 and ML-1 libraries. However

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