Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-07
2002-08-20
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S243000, C435S325000, C435S410000, C536S024100
Reexamination Certificate
active
06436639
ABSTRACT:
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
(Not Applicable)
TECHNICAL FIELD
The present invention relates to regulatory elements that are linked to a gene involved in apoptosis. The invention further relates to methods for identifying agents that modulate expression of a gene involved in apoptosis.
BACKGROUND ART
Apoptosis, or programmed cell death, is a normal physiologic process that leads to individual cell death. This process of programmed cell death is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and infection by human immunodeficiency virus (HIV). Wyllie (1980)
Nature
284:555-556; Kanter et al. (1984)
Biochem. Biophys. Res. Commun.
118:392-399; Duke and Cohen (1986)
Lymphokine Res.
5:289-299; Tomei et al. (1988)
Biochem. Biophys. Res. Commun.
155:324-331; Kruman et al. (1991)
J. Cell. Physiol.
148:267-273; Ameisen and Capron (1991)
Immunology Today
12:102; and Sheppard and Ascher (1992)
J. AIDS
5:143. Agents that modulate the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.
Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Kerr et al. (1992)
FASEB J.
6:2450; and Cohen and Duke (1992)
Ann. Rev. Immunol.
10:267. The blebs, small, membrane-encapsulated spheres that pinch off of the surface of apoptotic cells, may continue to produce superoxide radicals which damage surrounding cell tissue and may be involved in inflammatory processes.
While apoptosis is a normal cellular event, it can also be induced by pathological conditions and a variety of injuries. Apoptosis is involved in a wide variety of conditions including but not limited to, cardiovascular disease; cancer regression; immune disorders, including but not limited to systemic lupus erythematosus; viral diseases; anemia; neurological disorders; diabetes; hair loss; rejection of organ transplants; prostate hypertrophy; obesity; ocular disorders; stress; aging; and gastrointestinal disorders, including but not limited to, diarrhea and dysentery.
In Alzheimer's disease, Parkinson's disease, Huntington's chorea, epilepsy, amyotrophic lateral sclerosis, stroke, ischemic heart disease, spinal cord injury and many viral infections, for example, abnormally high levels of cell death occur. In at least some of these diseases, there is evidence that the excessive cell death occurs through mechanisms consistent with apoptosis. Among these are 1) spinal cord injury, where the severing of axons deprives neurons of neurotrophic factors necessary to sustain cellular viability; 2) stroke, where after an initial phase of necrotic cell death due to ischemia, the rupture of dead cells releases excitatory neurotransmitters such as glutamate and oxygen free radicals that stimulate apoptosis in neighboring healthy neurons; and 3) Human Immunodeficiency Virus (HIV) infection, which induces apoptosis of T-lymphocytes.
In contrast, the level of apoptosis is decreased to abnormal levels in cancer cells, which allows the cancer cells to survive longer than their normal cell counterparts. As a result of the increased number of surviving cancer cells, the mass of a tumor can increase even if the doubling time of the cancer cells does not increase. Furthermore, the high level of expression in a cancer cell of the bcl-2 gene, which is involved in regulating apoptosis and, in some cases, necrotic cell death, renders the cancer cell relatively resistant to chemotherapeutic agents and to radiation therapy.
It is convenient to divide the process of physiological cell death into phases. Vaux and Strasser (1996)
Proc. Natl. Acad. Sci.
93:2239-2244. The earliest phase is the stimulus that provokes the apoptotic response. This may be an external signal delivered through surface receptors or may originate inside the cell from the action of a drug, toxin, or radiation. The next phase includes detection of this signal or metabolic state and transduction of the signal. Signal transduction pathways send this message to the cell death effector machinery. The effector phase is the third part of the cell death mechanism and includes the proteases that are activated during apoptosis, as well as their positive and negative regulators. The fourth phase of cell death is the postmortem phase, in which the cell's chromatin condenses and its DNA is degraded.
The activation or signaling phase of cell death encompasses a great variety of signal transduction pathways that mediate signals from outside the cell, as well as others that originate inside the cell. Two members of the TNF superfamily of receptors, TNFR 1 and CD95, when bound to their respective ligands, TNF-I and CD95L (FasL) can rapidly transduce an apoptotic cell death signal (6-8). Nagata and Golstein (1995)
Science
267:1449-1456; Cleveland and Ihle (1995)
Cell
81:479-482; Schutze-Osthoff (1994)
Trends Cell Biol.
4:421-426. Cell death induced by the CD95/CD95L system is important for the elimination of potentially autoreactive peripheral T cells and contributes to T cell-mediated cytoxicity, whereas the TNFR I/TNF-I system plays a critical role in host defense against microorganisms and their pathogenic factors.
In recent years, a family of proteins has been discovered that controls apoptosis. The prototype of this family is Bcl-2, a protein that inhibits most types of apoptotic cell death and is thought to function by regulating an antioxidant pathway at sites of free radical generation. Hockenbery et al. (1993)
Cell
75:241-251. Together, the Bcl-2 family of proteins are important intracellular modulators of apoptosis and can be divided into two groups based on their effect on apoptosis. Thus, in a general sense, Bcl-2, Bcl-x
L
, Mcl-1, BHRF-1 and E1B19K are cell death inhibitors (anti-apoptotic), while Bak, Bax and Bcl-x
S
accelerate cell death (pro-apoptotic).
Bcl-2 family members are generally localized to the outer mitochondrial membrane, the nuclear membrane and the endoplasmic reticulum, where they associate with membranes by virtue of their C-terminal hydrophobic tail. All members of the family have two highly conserved regions, called BH1 and BH2, that permit specific interactions between two members to form stable dimers. Their mechanism of action is presently unclear; however, it is known that the ratio of anti-apoptotic to pro-apoptotic Bcl-2 family members in a cell is critical to the cell's survival following initiation of an apoptotic signal.
Bak is a member of the Bcl-2 family and is expressed in heart and other tissues. Bak protein is capable of either killing cells, or actively protecting cells from cell death, depending on how this protein interacts with other cellular proteins. Bcl-2 family members are extremely important in determining the fate of a cell following an apoptotic signal, and Bak may be the most important in the major organs such as heart. In the treatment of heart disease, viral infection and cancer, modulation of the expression of genes encoding proteins that control apoptosis is a major focal point.
Interferons (IFN) were originally discovered in the late 1950s as substances produced in animals infected with viruses that could elicit protection against subsequent viral infection. Isaacs and Lindenmann (1957)
Proc. Royal Soc. Lond.
(
Biol.
) 147:258-267. This activity was shown to reside in a group of functionally related polypeptides, IFN&agr;, -&bgr; and -&ggr;, factors which were further discovered to possess a broad range of biological activities in addition to the
Kiefer Michael C.
Ossina Natalya K.
McKelvey Terry
Sheridan & Ross P.C.
Tanox, Inc.
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