Inhibitor of programmed cell death

Details Inhibitor of programmed cell death Inhibitor of programmed cell death

2000-02-24

2003-05-27

Huff, Sheela (Department: 1642)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

Reexamination Certificate

active

06570002

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a novel inhibitor of programmed cell death, the protein Aven, and to the use of these sequences in the diagnosis, prevention, and treatment of diseases associated with decreased or increased apoptosis.
BACKGROUND OF THE INVENTION
Normal development, growth, and homeostasis in multi-cellular organisms require a careful balance between the production and destruction of cells in tissues throughout the body. Cell division is a carefully coordinated process with numerous checkpoints and control mechanisms. These mechanisms are designed to regulate DNA replication and to prevent inappropriate or excessive proliferation. In contrast, apoptosis is the genetically controlled process by which unneeded or damaged cells can be eliminated without causing the tissue destruction and inflammatory responses that are often associated with acute injury and necrosis.
The term “apoptosis” was first used to describe the morphological changes that characterize cells undergoing programmed cell death. Apoptotic cells have a shrunken appearance with an altered membrane lipid content and highly condensed nuclei. Apoptotic cells are rapidly phagocytosed by neighboring cells or macrophages without leaking their potentially damaging contents into the surrounding tissue or triggering an inflammatory response.
The processes and mechanisms regulating apoptosis are highly conserved throughout the phylogenetic tree, and much of our current knowledge about apoptosis is derived from studies of the nematode
Caenorhabditis elegans
and the fruit fly
Drosophila melanogaster
. Aberrations in apoptosis regulation have recently been recognized as significant factors in the pathogenesis of human disease. For example, inappropriate cell survival can cause or contribute to many diseases such as cancer, autoimmune diseases, and inflammatory diseases. In contrast, increased apoptosis can cause immunodeficiency diseases such as AIDS, neurodegenerative disorders, and myelodysplastic syndromes.
A variety of ligands and their cellular receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown. Despite the identification of genes necessary for cell death and the ability to regulate apoptosis by known genes, the essential biochemical events in apoptotic death remain largely unknown.
The consistency of the morphologic and biochemical patterns defined as apoptosis within different cell types and species, during normal development and as a response to external stimuli arc consistent with a common cause of cellular mortality. The thesis is supported by the concept of an endogenous program responsible for cells death and the presence of gene products which are positive and negative regulators of apoptosis. The best studied negative regulator of apoptosis is the Bcl-2 proto-oncogene product. It provides the strongest evidence for a sharedmammalian pathway of death by its ability to block a wide variety of cell death models.
The Bcl-2 proto-oncogene is rather unique among cellular genes in its ability to block apoptotic deaths in multiple contexts. Overexpression of Bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms. Induction of apoptosis by diverse stimuli, such as radiation, hyperthermia, growth factor withdrawal, glucocorticoids and multiple classes of chemotherapeutic agents is inhibited by Bcl-2 in vitro models. These effects are proportional to the level of Bcl-2 expression. Additionally, the endogenous pattern of Bcl-2 expression is indicative of a role in the regulation of cell survival in vivo. The Bcl-2 protein seems likely to function as an antagonist of a central mechanism operative in cell death.
The protein encoded by the Bcl-2 proto-oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems. The proto-oncogene Bcl-2 was isolated and characterized as a result of its frequent translocation adjacent to the immunoglobulin heavy chain enhancer in the t(14;18) chromosome translocation present in more than 80% of human follicular lymphomas. These neoplasias are characterized by an accumulation of mature resting B cells presumed to result from a block of apoptosis which would normally cause turnover of these cells. Transgenic mice expressing Bcl-2 under the control of the E&mgr; enhancer similarly develop follicular lymphomas.
The Bcl-2 protein is a 26 kDa membrane-associated cytoplasmic protein. Unlike many other proto-onocogene products, the Bcl-2 protein apparently functions, at least in part, by enhancing the survival of hematopoietic cells of T and B origins rather than by directly promoting proliferation of these cell types. The capacity of Bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody. Upregulation of Bcl-2 expression also inhibits apoptosis of EBV-infected B-cell lines. The expression of Bcl-2 has also been shown to block apoptosis resulting from expression of the positive cell growth regulatory proto-oncogene, c-myc, in the absence of serum or growth factors.
Within vertebrates, Bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor. Other proteins which interact with and/or are structurally related to the Bcl-2 gene product have also been identified, such as, for example, Bcl-x
L
and Bcl-x
S
. The family of Bcl-2 related proteins also includes the nematode protein CED-9 and two DNA virus proteins, LMW5-HL and BHRF-1 of the Epstein Barr Virus. Thus, a family of Bcl-2 like genes exists and evidence indicates that they participate in regulating cell death.
The family of Bcl-2 related proteins has been noted to have homology that is principally, but not exclusively, clustered within two conserved regions entitled Bcl-homology 1 and 2 (BH1 and BH2). This includes Bax, Bcl-X
L
, Mcl-1 and Al, and several open reading frames in DNA viruses including BHRF-1 of Epstein-Barr virus and LMW5-HL of African swine fever virus.
It has been discovered that Bcl-2 also associates in vivo with a 21 kDa protein partner called Bax. Bax shows extensive amino acid homology with Bcl-2 and forms homodimers with itself and heterodimers with Bcl-2 in vivo. Bax is encoded by 6 exons and demonstrates a complex pattern of alternative RNA splicing that predicts a 21 KDa membrane (&agr;) and three forms (&bgr;, &ggr;, and &ohgr;)) of cytosolic protein. Bcl-2 and Bax have biochemical functions that are yet to be delineated but may also modulate cell death/survival through heterodimerization. When Bax predominates and a substantial percentage of Bax is present as homomultimers (e.g., homodimers) and/or free (unbound) Bax monomer or other activated form or complex, programmed cell death is accelerated and the death repressor activity of Bcl-2 is countered. In many cell types when in excess, Bax counters the ability of Bcl-2 to repress cell death. It was unexpected to find that Bax shares extensive homology with Bcl-2, especially within two highly conserved domains. These domains are also the most highly conserved regions of human, mouse, and chicken Bcl-2. These domains are also conserved in an open reading frame BHRF-1 within Epstein-Barr virus and Mcl-1, a gene recently isolated from a myeloid leukemia cell line following induction with phorbol ester.
The Bcl-x gene was identified by low-stringency hybridization using a Bcl-2 polynucleotide probe and encodes two proteins, Bcl-x
L
and Bcl-x
S
, via alternative RNA splicing. The Bcl-x
L
cDNA encodes a polypeptide of 233 amino acids with similar

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