Sequences characteristic of hypoxia-regulated gene transcription

1. Technical Field

The present invention relates to the identification of genes that are differentially expressed in hypoxia and use of the genes and gene products for diagnosis and therapeutic intervention. The invention further relates to identification of polynucleotide sequences, and their gene products, that are differentially expressed in hypoxia and the use of the sequences for diagnosis and probes.

2. Background Art

The level of tissue oxygenation plays an important role in normal development as well as in pathologic processes such as ischemia. Tissue oxygenation plays a significant regulatory/inducer role in both apoptosis and in angiogenesis (Bouck et al, 1996; Bunn et al, 1996; Dor et al, 1997; Carmeliet et al, 1998). Apoptosis (see Duke et al, 1996 for review) and growth arrest occur when cell growth and viability are reduced due to oxygen deprivation (hypoxia). Angiogenesis (i.e. blood vessel growth, vascularization) is stimulated when hypo-oxygenated cells secrete factors which stimulate proliferation and migration of endothelial cells in an attempt to restore oxygen homeostasis (for review see Hanahan et al, 1996).

Hypoxia plays a critical role in the selection of mutations that contribute to more severe tumorogenic phenotypes (Graeber et al, 1996). Identifying activated or inactivated genes and gene products in hypoxia and ischemia is needed.

Ischemic disease pathologies involve a decrease in the blood supply to a bodily organ, tissue or body part generally caused by constriction or obstruction of the blood vessels, as for example retinopathy, myocardial infarction and stroke. Therefore, apoptosis and/or angiogenesis as induced by the ischemic condition are also involved in these disease states. Neoangiogenesis is seen in some forms of retinopathy and in tumor growth. These processes are complex cascades of events controlled by many different genes reacting to the various stresses such as hypoxia.

Stroke is the third leading cause of death and disability in developed countries, affecting more than half a million Americans each year. Stroke is an acute neurologic injury occurring as a result of an insult to the brain, thus interrupting its blood supply. Stroke induces neuronal cell death, which leads to the clinical outcomes of patients\' death or disability ranging from total paralysis to milder dysfunction. Cerebral ischemia is the most common type of stroke, which may lead to irreversible neuronal damage at the core of the ischemic focus, whereas neuronal dysfunction in the penumbra may be reversible. Cells in the penumbra have an estimated time window for survival of up to 6 hours. The ability to intervene as soon as the patient is identified is essential for recovery. It is well established that ischemic tissue damage is multifactorial and involves at least excitotoxicity, reactive oxygen species, and inflammation—all leading to neuronal cell death.

Treatment strategies for stroke are aimed to induce rapid reperfusion and rescue of neurons in the penumbral area. Neuroprotective drugs are constantly being developed in an effort to rescue neurons in the penumbra from dying. However, potential cerebroprotective agents need to counteract all the above-mentioned destructive mechanisms. Therefore, current therapy in stroke focuses primarily on prevention, minimizing subsequent worsening of the infarction, and decreasing edema.

The ability to monitor hypoxia-triggered activation of genes can provide a tool to identify not immediately evident ischemia in a patient. Identification of hypoxia-regulated genes permits the utilization of gene therapy or direct use of gene products, or alternatively inactivation of target genes for therapeutic intervention in treating the diseases and pathologies associated with hypoxia, ischemia and tumor growth.

Induction of p53 in response to hypoxia and DNA damage and its ability to inhibit cell growth in response to common cellular stresses, is a major function associated with its role as a tumor suppressor gene (Lane, 1992). Proteins encoded by p53 target genes have been shown to regulate various processes controlling growth and viability of tumor cells, such as cell cycle progression and programmed cell death. Like p53, the growth arrest and DNA damage (GADD) genes are induced in cells exposed to genotoxic stress. GADD genes were originally identified by subtraction hybridization from a cDNA library constructed from UV-irradiated Chinese hamster ovary cells (Forance et al, 1989). The GADD genes code for a diverse range of proteins with a variety of functions, including the suppression of DNA synthesis (Smith et al, 1994), the inhibition of differentiation (Batchvarova et al, 1995) and the induction of apoptosis (Takekawa et al, 1998). The response to genotoxic stress of some GADD genes is rapid but transient whereas others respond more slowly (Fleming et al, 1998). Other stimuli, such as DNA damage or contact inhibition, also increase gene expression. The regulation of these genes by stress is complex and appears to be mediated by multiple pathways. For example, ionizing radiation induces the transcription of GADD45, which inhibits proliferation and stimulates DNA excision repair, through a p53-dependent mechanism (Hollander et al, 1993). In contrast, UV irradiation increases GADD45 expression in the absence of p53 binding directly to the GADD45 promoter (Zhan et al, 1996). GADD45 mRNA levels are also increased during hypoxia, focal cerebral ischemia, and after exposure of cells to agents which elevate the levels of the glucose-regulated proteins (Price et al, 1992, Schmidt-Kastner et al, 1998). In addition to its ability to inhibit proliferation and stimulate DNA repair, GADD45 can also induce apoptosis when overexpressed in cells in vitro (Takekawa et al, 1998).

Recently, a novel p53 target gene and member of the GADD family, PA26 was identified (Velasco-Miguel et al, 1999). PA26 encodes at least three transcript isoforms, of which two are differentially induced by genotoxic stress in a p53-dependent manner. The function of PA26 is unclear.

The present invention provides purified, isolated and cloned polynucleotides (nucleic acid sequences) associated with hypoxia-regulated activity and having sequences designated as any one of SEQ ID NOs:1-12, or having complementary or allelic variation sequences thereto. The expression of these polynucleotides is modulated when cells are subjected to neurotoxic stress. The present invention includes the polynucleotides of SEQ ID NOs:1-12, as well as the naturally-occurring full-length RNAs and corresponding full-length cDNAs which include any one of these sequences.

The invention is further directed to naturally-occurring polynucleotides having at least 70% identity with any of the polynucleotides which include any one of SEQ ID NOs:1-12, or which are capable of hybridizing under moderately stringent conditions to any of such polynucleotides, and whose expression in naturally-occurring neural cells is modulated when the cells are subjected to hypoxic stress.

The present invention is also directed to fragments having at least 20 nucleotides of any of the polynucleotides of the present invention and to polynucleotide sequences complementary to any of such polynucleotides or fragments.

In a preferred embodiment, the isolated polynucleotide is a strand of a full-length cDNA.

The present invention is further directed to isolated proteins encoded by any such full-length cDNA, as well as variants which have an amino acid sequence having at least 70% identity to such an isolated protein and retain the biological activity thereof, or biologically active fragments of such protein or variant, as well as to salts or functional derivatives of any such protein, variant or biologically active fragment.

The present invention is also directed to antibodies specific to any of the proteins, variants or fragments of the present invention and to any molecule which includes the antigen-binding portion of any such antibody.

The present invention also comprehends antisense DNA of a length sufficient to prevent transcription and/or translation of a gene identified in accordance with the present invention, as well as ribozymes which specifically bind and cleave mRNA sequences identified in accordance with the present invention.

The invention also comprehends methods for screening drugs which up-regulate or down-regulate a gene which is transcribed to an RNA containing a sequence of any of any of the polynucleotides of the present invention.

The present invention is additionally directed to pharmaceutical compositions which include the nucleic acids, proteins or polypeptides in accordance with the present invention, along with pharmaceutically acceptable carriers or excipients.

In addition, the present invention is directed to knockout or transgenic non-human animals, in which a gene identified by the present invention has been introduced or knocked out.