Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-05-19
2002-03-19
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S350000, C536S023200, C435S320100, C435S252300, C435S006120, C435S325000
Reexamination Certificate
active
06358723
ABSTRACT:
INTRODUCTION
This invention was made in the course of research sponsored by the National Institutes of Health. The U.S. Government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
Controlled cell death is critical for the life of a human; too much cell death can cause the symptoms of cystic fibrosis and also lead to diseases such a neurodegeneration and acquired immune deficiency syndrome (AIDS). In contrast, 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 hyperthermia (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. It was originally 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 (Sorenson et al.
J. Natl. Cancer Inst.
1990 82:749). This alternate endonuclease was identified as deoxyribonuclease II (DNase II; Barry, M. A. and Eastman, A. Archives of
Biochem and Biophys.
1993 300(1):440-450). It was proposed 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.
However, more recent results have implicated yet another endonuclease, referred to CAD or caspase-activated deoxyribonuclease, in apoptosis (Enari, M. et al. 1998 Nature 391:43-50). Thus, it remains to be determined which specific endonuclease is involved in apoptosis.
The enzyme referred to herein as deoxyribonuclease II&agr; (DNA II&agr;) was isolated and purified and the amino acid sequence determined (PCT/US97/18262). The DNA sequences for both the human and bovine proteins of DNase II&agr; have also been cloned (PCT/US97/18262). Use of DNA II&agr; in alleviating the suffering in patients with cystic fibrosis is also disclosed in this PCT application.
In cystic fibrosis, the lungs of patients fill with the remnants of dead cells, and in particular with the DNA from these dead cells. The presence of DNA makes the mucous plugs too viscous to expel. A suggested therapy for these symptoms is the use of DNase I to digest the DNA, thereby permitting expulsion of the mucous plugs. However, this therapy has not been particularly effective due to inactivity of the DNase I enzyme in the presence of actin, also present in the sputum.
It is believed that DNase II enzymes and variations thereof may provide a more effective therapeutic alternative.
Another isoform of the DNase II enzyme, referred to herein as deoxyribonuclease II&bgr; (DNase II&bgr;) has now been identified and the gene and protein sequences for the mouse and human homolog have been determined.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a cDNA encoding deoxyribonuclease II&bgr;.
Another object of the present invention is to provide an isolated, purified deoxyribonuclease II&bgr; enzyme.
Yet another object of the present invention is to provide antibodies against this protein which can be used in diagnosing cells at various 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&bgr;.
Yet another object of the present invention is to provide a method for identifying agents that inhibit DNase II&bgr; activity comprising treating cells with a test agent, transfecting cells with DNase II&bgr;, 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 to provide a method for inducing apoptosis in selected cells comprising transfecting cells with a vector expressing the DNase II&bgr; cDNA so that apoptosis is induced.
Yet another object of the present invention is to provide a method of digesting DNA released from dead cells with an effective amount of an isolated, purified DNase II&bgr; protein so that DNA is digested.
DETAILED DESCRIPTION OF THE INVENTION
The existence of a deoxyribonuclease II (DNase II) enzyme as a protein of lysosomal origin that is involved in cellular digestion of foreign DNA has been known for many years. Recently, a DNase II enzyme has been linked with the DNA fragmentation that occurs at an early stage in apoptosis. The bovine and human forms of this DNase II protein, referred to herein as DNase II&agr; protein have been isolated and purified and the amino acid sequences of these proteins are disclosed in PCT/US97/18262. cDNAs encoding the bovine and human form of DNase II&agr; have also been cloned and characterized in PCT/US97/18262.
An isoform of this enzyme, referred to herein as deoxyribonuclease II&bgr; (DNase II&bgr;) has now been identified.
This full length gene for this isoform was first identified in mice by sequence comparison to expressed sequence tags entered in Genbank database which were similar, but not identical to DNase II&agr;. Oligonucleotide primers were synthesized to obtain the complete DNase II&bgr; mouse gene. The mouse DNase II&bgr; cDNA sequence is depicted as SEQ ID NO:1. The protein sequence of mouse DNase II&bgr; is depicted in SEQ ID NO:2.
Information from the mouse sequence was used to isolate a human homolog of this gene. The human DNase II&bgr; cDNA sequence is depicted as SEQ ID NO:3. The protein sequence of human DNase II&bgr; encoded by the cDNA of SEQ ID NO:3 is depicted in SEQ ID NO:4.
Mouse and rat cDNAs of this homolog of DNase II&agr; have also been disclosed recently by Shiokawa and Tanuma (
Nucleic Acid Res.
1999 27(20):4083-4089 and
Biochemical and Biophysical Research
Eastman Alan Richard
Krieser Ronald Joe
Licata & Tyrrell P.C
Monshipouri M.
Prouty Rebecca E.
Trustees of Dartmouth College
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