Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-10-29
2001-05-22
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C435S320100, C435S325000, C435S440000, C530S350000
Reexamination Certificate
active
06235524
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to the nucleic acid sequences (and corresponding translated products) of novel mutant forms of the
Drosophila hid
gene and methods of identifying and sting antagonists of Hid phosphorylation, in particular, compositions modifying MAPK phosphorylation of Hid.
BACKGROUND OF THE INVENTION
Programmed cell death (PCD) is mediated by a process called apoptosis. Although the investigation of cell death is a relatively new field of study, it has become readily apparent that many disease states are manifested due to the aberrant control of programmed cell death. Recent evidence suggest that the failure of cells to undergo apoptotic cell death might be involved in the pathogenesis of a variety of human diseases including cancer, autoimmune diseases and viral infections. The understanding of survival pathways would be critical in disease states where excessive cell numbers, such as in various cancers, are the result of cell survival signals preventing cell death rater than the result of uncontrolled proliferation. It is known that a number of peptide factors including the neurotrophins, Insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF), and epidermal growth factor (EGF) promote cell survival by suppressing the intrinsic cell death program. The growth factors listed above bind to and activate their respective receptor tyrosine kinases (RTK) at the cell surface which, in turn, stimulate the anti-apoptotic activity of the proto-oncogene ras. Ras controls the activity of a number of effector pathways, two of which result in activation of protein kinases known to mediate its anti-apoptotic effect: the mitogen activated protein kinase p42p44 (MAPK) of the ERK-type (extracellular signal-related kinase) via Raf and the Akt kinase via phosphoinositide 3-kinase (PIK-3). Mutational activation of ras oncogenes is associated with about 30% of all human tumors, potentially suppressing the ability of the body to remove the cancerous cell.
The product of the gene hid is responsible for inducing apoptosis in drosophila embryos as well as transfected insect and mammalian cells. When ectopically expressed, Hid induces apoptosis by activating a caspase protease pathway. In the absence of Hid function many cells that should die fail to do so. Unlike other cell death activating genes (e.g. reaper and grim), hid is expressed in many cells that are not destined to undergo apoptosis. This observation suggests that efficient post-translational survival mechanisms operate in these cells to protect them from Hid-induced apoptosis. Activation of the Ras/MAPK pathway in transfected cells has been shown to decrease or inhibit Hid-induced cell death.
The screening of potential therapeutics has been hindered by both a lack of understanding of the physiological basis of cell death and by a dearth of reagents specific for critical points in the cell death signaling pathways. Additionally, much work has focused on the delineation of the death-inducing pathway and reagents that may block it and not on pathways that confer survival signals. What is needed are reagents and methodologies that allow for the identification and testing of agonists and antagonists of cell survival pathways.
SUMMARY OF THE INVENTION
The present invention generally relates to compositions and methods of identifying and testing Hid pathway agonists and antagonists, and in particular, compositions modifying MAPK phosphorylation of Hid. In addition, the invention relates to methods to identify other members of the Hid signal pathway, methods to identify homologs of Hid which are native to other tissue or cell types and methods to generate reagents derived from the invention.
The present invention contemplates employing novel mutant forms of the wild-type
Drosophila hid
gene (SEQ ID NO:1) in these screening methods. In one embodiment, the present invention contemplates replacing nucleic acid of the
Drosophila hid
gene encoding phosphoacceptor residues with nucleic acid encoding non-phosphorylatable amino acids (e.g. alanine). In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence selected from the group consisting of: Hid
Ala3
cDNA having the nucleotide sequence of SEQ ID NO:2; and Hid
Ala5
cDNA having the nucleotide sequence of SEQ ID NO:3. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. The present invention also contemplates RNA transcribed from the above-indicated cDNAs as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein.
The present invention also contemplates transgenic animals comprising the above-indicated DNA (i.e. the “transgene”) or portions thereof. In a particular embodiment, the transgenic animal of the present invention may be generated with the transgene contained in an inducible, tissue specific promotor.
The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays.
The present invention may also be used to identify new constituents of the Hid signaling pathway. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel Hid pathway constituents or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins that could also be used to isolate novel Hid pathway constituents or natural mutations thereof. In yet another embodiment screens maybe conducted using the yeast two-hybrid system.
The present invention may also be used to identify new homologs of Hid or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
The present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expresssion vector, wherein said vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NOS:2 or 3, ii) a second group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the wild-type
Drosophila hid
gene oligonucleotide sequence (SEQ ID NO:1), and iii) at least one compound suspected of having the ability to modulate MARK/Hid pathway activity; b) contacting said first and second groups of cells with said compound; and c) detecting programmed cell death modulation effects of said compound.
The present invention contemplates another method of screening a compound where the compound exerts an apoptotic effect by causing the multimerization of the translated product of the invention. Multimerization of Hid may be required for its activation. Phosphorylation of Hid by MAPK may prevent said multimerization. It is believed that compounds that multimerize Hid can potentially activate nonphosphorlatable Hid thereby initiating apoptosis. Said method comprising a) providing in any order: i) a first group of cells comprising a first recombinant expression vector, wherein said first vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NOS:2 or 3, ii) a second group of cells comprising a second recombinant expression vector, wherein
Agapite Julie
Bergmann Andreas
McCall Kimberly
Steller Hermann
Massachusetts Institute of Technology
Medlen & Carroll LLP
Paras, Jr. Peter
Priebe Scott D.
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