ZA200101567B - Hypoxia regulated genes. - Google Patents

Description

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HYPOXIA REGULATED GENES

BACKGROUND OF THE INVENTION TECHNICAL FIELD

The present invention relates to the . identification of the polynucleotide sequence of the 2-2-83 gene which was found to be differentially expressed in ; several pathological systems, such as stroke, hypoxic retina and hypoxic regions of tumors.

BACKGROUND ART

The level of tissue oxygenation plays an important role in normal development as well as in. pathologic processes such as ischemia or tumorigenesis”.

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 a

R70) 00/12139 PCT/US99/20393 ’ i oo Bh ~~ 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. —- — — - — : LL _ So ;

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 protein products or products of their activity (i.e., in the case of metabolic enzymes), or alternatively inactivation of target genes function for therapeutic intervention in treating the diseases and pathologies associated with hypoxia, ischemia and tumor growth.

SUMMARY OF THE INVENTION

According to the present invention, there is provided the polynucleotide sequence of a gene whose expression is modulated by hypoxic conditions, a hypoxia response regulating gene. Also provided is a polynucleotide sequence encoding a gene for protein 2-2-83. A pharmaceutical composition for modulating hypoxia and

. . oo A ischemia having an effective amount of a polynucleotide or protein having the nucleic acid sequence according to SEQ.

ID NO. 1 and 2 or a functional analog thereof and a pharmaceutically acceptable carrier is also provided.

There is provided a method of regulating hypoxic associated pathologies and tumorigenesis by administering an effective amount of a polynucleotide, protein, direct or indirect biologically active product of enzymatic activity of the protein, Or inhibitors of direct or indirect biologically active product of enzymatic activity of the protein having a nucleic acid sequence from at least one from the group containing SEQ. ID No.: 1 and 2 and ] functional analogs thereof and a pharmaceutically acceptable carrier.

A method of regulating hypoxic associated pathologies and tumorigenesis by inhibiting the enzymatic activity of the protein encoded by the polynucleotide sequences from at least one from the group containing SEQ. ID No.: 1 and 2 - and functional analogs thereof is provided.

A hypoxia response regulating is further provided. A neuroprotective and neurotrophic agents : comprising a gene product of the polynucleotide sequences of 2-2-83 according to SEQ. ID No.: 1 and 2 and functional analogs thereof is also provided. There is provided a method of diagnosing the presence of a hypoxia associated pathology or tumorigenesis by screening for the reduced expression of the gene encoded by the nucleic acid sequence according to SEQ. ID No.: 1 and 2 and functional analogs : thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be : readily appreciated as the same becomes better understood by reference to the following detailed description when

: a . Ld considered in connection with the accompanying drawings wherein:

Figure 1 shows the full-length cDNA and protein sequence of rat 2-22-83 gene (SEQ. ID No:l);

Figure 2 shows the full length cDNA and protein sequence of human 2-2-83 gene (SEQ. ID No:2) ;

Figure 3 is a graph showing the increased proliferation rate of fresh 2-2-83 expressing C6 clones;

Figure 4 is a graph showing the normalization of proliferation rate of 2-2-83 expressing cé clones following : — 15 passaging in vitro;

Figures 5 A and B are photographs showing the altered more differentiated morphology of freshly infected with

BE2C cultures and pBABE/2-2-83;

Figures 6 A and B are graphs showing the growth curves of two independent polyclonal BE2C cell populations expressing exogenous 2-2-83; A shows the growth curve of

BE2C/2-2-83/pBABE cells; and B shows the growth curve of

BE2C/pcDNA cells;

Figure 7 is a graph showing the cell cycle distribution within freshly infected with pBABE-2-2-83 BE2C cultures;

Figures 8 A and B are graphs showing the sensitivity to chemical hypoxia of 2-2-83-expressing BE2C cells to hypoxia; A shows the sensitivity of 2-2-83/pBABE cells; and

B shows sensitivity of 2-2-83/pcDNA cells;

Q N :

Figure 9 is a graph showing the sensitivity of differentiated BE2C-2-283 cells to 25-hydroxycholesterol;

Figure 10 is a graph showing the sensitivity of non- differentiated BE2C-2-283 cells to 25-hydroxycholesterol;

Figure 11 is a graph showing the sensitivity of passaged 2-2-83 expressing cé clones to 25- hydroxycholesterol ;

Figure 12 is a graph showing the kinetics of C6 tumor growth in nude mice; and

Figure 13 is a graph showing the inhibition of Cé } 15 tumor growth in nude mice by 2-2-83 expression.

DESCRIPTION OF THE INVENTION }

The present invention identifies a polynucleotide (nucleic acid sequence) whose expression is modulated by hypoxic conditions. More specifically, the polynucleotide is known as 2-2-83 with full-length sequence as set forth herein in SEQ. Ip No. 1 and 2, that can be utilized diagnostically in hypoxia and ischemia and that can be used as a target for therapeutic intervention.

The present invention further provides a gene and { gene products (direct or indirect) that can be utilized ; i therapeutically and diagnostically in hypoxia and ischemia : and that can regulate apoptosis, angiogenesis, tumorigenesis and possess neurotrophic and neuroprotective activity. The present invention also provides the initial evidence of - the existence of previously non-identified pathways of stereoidogenesis in mammalian cells.

By regulate or modulate or control, it is meant

’ that the process is either induced or inhibited" “to the — degree necessary to effect a change in the process and the associated disease state in the patient. Whether induction or inhibition is being contemplated will be apparent from the process and disease being treated and will be known to those skilled in the medical arts.

The gene of the present invention is identified for gene therapy. (diagnostics) and therapeutics that have direct causal relationships between a disease and its related pathologies and up- Or down-regulator (responder) genes and biological processes. That is, the present invention is initiated by a physiological relationship between cause and effect.

By gene therapy as used herein refers to the : ee 15 transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease OY condition phenotype. The genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest encodes a suicide gene.

For a review see, in general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997).

Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.

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These genetically reimplanted cells have been shown to express the transfected genetic material in situ.

In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ (Culver, 1998). These genetically altered cells have been shown to express the transfected genetic material in situ.

The gene expression vehicle is «capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the ’ 15 nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5' UTR and/or 3°

UTR of the gene can be replaced by the 5' UTR and/or 3' UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5' UTR and/or 3' UTR of the actual gene to be transferred and only include the specific amino acid coding region.

The expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below. ‘Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. such methods can be found generally described in Sambrook - 7 -

BN pr

» et al., Molecular Cloning: A Laboratory ~Manual, Cold -

Springs Harbor Laboratory, New York (1989, 1992), in

Ausubel et al., Current Protocols in Molecular Biology,

John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor,- MI (1995),

Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors: A Survey of Molecular Cloning Vectors and

Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United

States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and . 5,487,992 for positive-negative selection methods.

IN © 18 © _— introduction of nucleic acids by infection offers several advantages over the other listed ‘methods. Higher efficiency can be obtained due to their infectious nature.

Moreover, Viruses are Very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors Or ligands to alter target specificity through receptor mediated events.

A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector AdenopS53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression 10 cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a - 8 - nN

LY . . mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.

Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.

Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through . the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.

Features that limit expression to particular cell types can also be included. such features include, for . example, promoter and regulatory elements that are specific for the desired cell type.

Tn addition, recombinant viral vectors are useful : for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells.

The result is that a large area becomes rapidly infected, most of which was not jnitially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of - o - g

’ ~~ targeted cells. — a

As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect 5s and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic ; system are to be treated, then a viral vector that is oo specific for blood cells and their precursors, preferably . for the specific type of hematopoietic cell, would be used.

Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily

13 N . available within the art or can be constructed by one skilled in the art using well-known methodology.

The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject.

Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case . of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection.

DNA can also be administered using a gene gun. (Ziao & Brancksman, Nuceic Acids, Res. 24, 2630-2622 i (1996)) . The DNA is precipitated onto the surface of microscopic metal beads. The microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers. For example, the Acal™ Gene Delivery Device manufactured by

Aegacetus, Inc., Middleton, WI, is suitable. Alternatively, nucleic DNA can pass through gkin into the bloodstream simply by spotting the DNA onto skin with chemical or mechanical irritation (see WO 95/05853) .

An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can - 11 -

A x 7 ~ “alleviate the targeting requirement required with other — - forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, thén promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.

The present invention provides a method of ’ regulating angiogenesis, apoptosis, tumorigenesis or other — hypoxia-associated pathologies as well as neurite outgrowth . in a patient in need of such treatment by administering to a patient a therapeutically effective amount of the polynucleotide as encoded by the nucleic acid sequence as identified herein or alternatively by administration of a non-protein product of the gene's activity.

The present invention further provides a method of regulating angiogenesis, apoptosis, tumorigenesis or other hypoxia-associated pathologies as well as neurite outgrowth in a patient in need of such treatment by administering to a patient a therapeutically effective amount of the antagonist of the protein as encoded by the nucleic acid sequence as identified herein or alternatively by administration of the antagonist to non-protein product of the gene's activity, or inactivation of a gene by chemical compound.

The present invention further provides a method of regulating angiogenesis, apoptosis or tumorigenesis in a patient in need of such treatment by administering to a patient a therapeutically effective amount of direct or

. indirect biologically active products of the enzymatic activity of the protein encoded by the identified gene as active ingredients in a pharmaceutically acceptable carrier. These gene products can also be used as neuroprotective or neurotrophic agents.

The protein of the present invention can be produced recombinantly (see generally Marshak et al, 1996 "Strategies for Protein Purification and Characterization.

A laboratory course manual." CSHL Press) and analogues can be due to post-translational processing. The term Analogue as used herein is defined as a nucleic acid sequence or protein which has some differences in their amino acid/nucleotide sequences as compared to the native sequence of SEQ. ID NO. 1. Ordinarily, the analogue will be generally at least 70% homologous over any portion that . is functionally relevant. In more preferred embodiments the homology will be at least 80% and can approach 95% homology to the protein/nucleotide sequence. ;

The amino acid or nucleotide sequence of an analogue can differ from that of the primary sequence when at least one residue is deleted, inserted or substituted, but the protein or nucleic acid molecule remains functional. Differences in glycosylation can provide protein analogues.

Functionally relevant refers to the biological property of the molecule and in this context means an in vivo effector or antigenic function or activity that is directly or indirectly performed by a naturally occurring protein or nucleic acid molecule. Effector functions include but are not limited to include receptor binding, any enzymatic activity or enzyme modulatory activity, any carrier binding activity, any hormonal activity, any activity in promoting or inhibiting adhesion of cells to : extracellular matrix or cell surface molecules, or any structural role as well as having the nucleic acid sequence p - —~gncode functional protein- and --be expressible The —_— antigenic functions essentially mean the possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring protein. Biologically active analogues share an effector function of the native which can, but need not, in addition possess an antigenic function.

The antagonist/regulating agent/active ingredient is dosed and delivered in a pharmaceutically acceptable carrier as described herein. The term antagonist or antagonizing is used in its broadest sense. Antagonism can include any mechanism or treatment which results in inhibition, inactivation, blocking or reduction in gene activity or gene product. It should be noted that the ’ inhibition of a gene or gene product can provide for an — increase in a corresponding function that the gene or gene . product was regulating. The antagonizing step can include blocking cellular receptors for the gene products and can include antisense treatment as discussed herein below. For example, a patient can be in need of inducing apoptosis in tumorogenic cells or angiogenesis in trauma situations where for example a limb must be reattached or in a transplant where revascularization is needed.

The pharmaceutical compositions containing the active ingredients of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the medical arts. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination - 14 -

’ of symptoms and other indicators as are selected as appropriate measures by those skilled in the medical arts.

The pharmaceutical compositions can be combinations of the active ingredients but will include at least one active ingredient.

In the method of the present invention, the pharmaceutical compositions of the present invention can be administered in various ways taking into account the nature of compounds in the pharmaceutical compositions. It should be noted that they can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants . and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intra-arterial, intramuscular, intra-peritoneally, and intra-nasal administration as well as intra-thecal and infusion techniques. Implants of the compounds are also useful. The patient being treated is a warm-blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.

It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses can be single doses or multiple doses over a period of several days, but single doses are preferred.

The doses can be single doses or multiple doses over a period of several days. The treatment generally has a length proportional to the length of the disease process s oo - and drug effectiveness and the patient species being treated.

When administering the compound of the present ~ invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion) . The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, - by the use of a coating such as lecithin, by the a maintenance of the required particle size in the case of dispersion and by the use of surfactants. Non-aqueous vehicles such a cottonseed oil, sesame o0il, olive oil, : soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for . example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds .

Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, : vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems ] useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.

Many other such implants, delivery systems, and modules : 20 are well-known to those skilled in the art.

A pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.

In one embodiment, the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used. The quantity to be administered will vary for the - 17 - db oo ~ patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably will be from 10 pg/kg to 10 mg/kg per day.

The present invention also provides a method of diagnosing the presence of ischemia in a patient including the steps of analyzing a bodily fluid or tissue sample from the patient for the presence or gene product of at least one expressed gene (up-regulated) or their proteins and where ischemia is determined if the up-regulated gene or gene product is ascertained as described herein in the

Example. The bodily fluids can include tears, serum, urine, sweat or other bodily fluid where secreted proteins from the tissue that is undergoing an ischemic event can be localized. Additional methods for identification of the } gene or gene product are immunoassays, such as and ELISA or . radioimmunoassays (RIA), can be used as are known to those : in the art particularly to identify gene products in the : samples. Immunohistochemical staining of tissue samples is also utilized for identification. Available immunoassays are extensively described in the patent and scientific literature. See, for example, United States patents 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521. Further for identification of the gene, in situ hybridization, Southern blotting, single strand conformational polymorphism, restriction endonuclease fingerprinting (REF), PCR amplification and DNA-chip analysis using nucleic acid sequence of the present invention as primers can be used.

The above discussion provides a factual basis for the use of the sequences of the present invention to identify hypoxia-regulated genes and provide diagnostic probes to identify ischemia. The methods used with and the utility of the present invention can be shown by the - 18 - a

7 ‘WO 00/12139 PCT/US99/20393 following non-limiting examples.

MATERIALS AND METHODS

Most of the techniques used in molecular biology are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs can serve as a guideline.

General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described were generally followed as in ; Sambrook et al., Molecular Cloning: A Laboratory Manual,

Cold Springs Harbor Laboratory, New York (1989), and in ) Ausubel et al., Current Protocols in Molecular Biology,

John Wiley and Sons, Baltimore, Maryland (1989) particularly for the Northern Analysis and In Situ analysis - and in Perbal, A Practical Guide to Molecular Cloning, John

Wiley & Sons, New York (1988), and in Watson et al.,

Recombinant DNA, Scientific American Books, New York.

Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications,

Academic Press, San Diego, CA (1990).

Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, were performed as generally described in Sambrook et al., 1989,

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory Press, and methodology as set forth in United

States patents 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference.

Microarray hybridization analysis.

- Preparation of custom hvpoxia-gpecific microarrays

The cell system for gene discovery consisted of the rat glioma cell line C6. The cells were exposed to hypoxia for 4 or 16 hours and the pattern of gene expression was compared to cells grown under normal conditions. DNA microarrays were prepared from clones of subtracted cDNA libraries enriched for sequences differentially regulated by hypoxia.

Subtracted libraries were made from the RNA populations extracted from C6 cells cultured in the following conditions: 1. 16 hours hypoxia vs. normoxia (enrichment for genes upregulated after 16 hours hypoxia). 2. Normal vs. 16 hours hypoxia (enrichment for genes : downregulated after 16 hours of hypoxia). 3. 4 hours hypoxia vs. normal (enrichment for genes . upregulated after 4hours of hypoxia) .

Three enriched libraries from the three. groups above were made by the SSH method using the "PCR select cDNA subtraction kit" from Clontech. From library 1, 1,000 colonies were grown and the plasmids prepared in 96 well format. From libraries 2 and 3, 500 colonies were processed from each. Thus, a total of 2,000 individual plasmids were prepared and used for the fabrication of a

Gene Expression Microarray (GEM). For this, the inserts of each plasmid were amplified by PCR and robotically fabricated on the glass.

Preparation of probes for microarray hybridization

Isolated messenger RNA was labeled with fluorescent dNTP's using a reverse transcription reaction to generate labeled cDNA probes. mRNA is extracted from C6 cells cultured in normoxia conditions and labeled with Cy3- dCTP (Amersham) from C6 cells cultured under hypoxia conditions (either 4 or 16 hours) and labeled with CyS5-AdCTP

= "WO 00/12139 PCT/US99/20393 (Amersham). Two differently labeled cDNA probes were then mixed and hybridized onto microarrays (Schena et al, 1996).

Following hybridization the microarrays were scanned using a laser scanner and the amount of fluorescence of each of the fluorescence dyes was measured for each cDNA clone on the microarray giving an indication of the level of mRNA in each of the original mRNA populations being tested.

Comparison of the fluorescence on each cDNA clone on the microarray between the two different fluorescent dyes is a measure for the differential expression of the indicated genes between the two experimental conditions.

The following probes were made from C6 and A172 . for screening the GEM: i5 1. Normoxia (Cy3 labeled ) + 16 hours hypoxia (CyS labeled) . 2. Normoxia (Cy3 labeled) + 4 hours hypoxia (Cy5 labeled).

The detected sequences are divided into three categories: 1. Novel genes; 2. known genes not known before this publication to be hypoxia regulated; and 3. known genes known to be differentially regulated under hypoxia conditions. Gene 2-2-83 (SEQ. ID. No.:l)was identified as a novel gene fragment whose expression is downregulated by hypoxia.

Utilizing microarray hybridization the sequences set forth herein were identified and «cloned as being differentially expressed under hypoxia conditions (see also

Braren et al, 1997).

In parallel assessment of 2-2-83 (SEQ. ID. No.:1) gene expression by Northern Analysis, the results where found to coincide with those of microarray hybridization analysis. As well in other experiments, the results from in situ hybridization analysis showed a high degree of

) — - correlation with the Northern Analysis. and microarray i analysis.

In Situ Hybridization Analysis:

In situ hybridization analysis was performed to assess the 2-2-83 (SEQ. ID. No.:1) gene expression pattern in normal tissues and in pathological models as described herein.

Disease Models For Tn Situ Hybridization Analysis

Hypoxic Rat Retina:

Hypoxia in retina was created by exposing of new born rat pups to hyperoxia which led to the reduction of blood supply. Upon transfer to normal oxygen conditions, — relative hypoxia is formed. The hypoxic retina was excised, : . fixed, sliced and used for the hybridization with 3°S-dATP : labeled riboprobes.

Solid Tumors:

Cé rat glioma-derived solid tumors were obtained by subcutaneous injection of the suspension of C6 cell into nude mice. Sections of two tumor samples were used in in situ hybridization. One sample represented a solid tumor of about 4X3 mm in size. No significant morphological variations between different tumor regions were observed.

However, at the tumor periphery, there was a region showing elevated expression of VEGF (indicative for hypoxia). The second sample represented a tumor of about 3X1 mm in size, containing a "core" region comprised of "white" thrombus and necrotic masses. This "core" region was surrounded by tumor cells forming the "wall" of varying thickness, from about five to fifteen cell layers. VEGF was found expressed by the closest to the core layer of tumor cells. The most - 22 <

Ht WO 00/12139 PCT/US99/20393 distant cell layers showed no VEGF expression.

Middle Cerebral Artery Occlusion (MCAO) Stroke Model:

The model was implied in the stroke-prone 5S spontaneously hypertensive rat strain. Occlusion was permanent and unilateral, and produced by electro- coagulation of MCA. This led to focal brain ischemia at the ipsilateral side of ©brain cortex leaving the contralateral side intact (control). Experimental animals were sacrificed 4 and 24 hours after the operation. Brains were removed, fixed in formalin, embedded into paraffin and coronal sections were performed for the further use in in situ hybridization with candidate genes-specific

Co riboprobes. VEGF and PGK (phosphoglycerokinase, a glycolitic enzyme upregulated by hypoxia) specific riboprobes were used as positive controls. At 24 hours post operation, a significant up-regulation of VEGF expression was revealed in the brain cortex in the areas adjacent to - the ischemic core region. Heavily labeled (presumably glial) cells could be seen at the ipsilateral to the injury side. In addition, a strong hybridization signal was displayed by few cells at the contralateral side suggesting the stimulation of VEGF response through interhemispheric communication.

Cell Lines:

C6 - Rat glioma cell line. Culture conditions:

DMEM supplemented with 10% FCS, 20 U/ml penicillin, 20 g/ml streptomycin.

BE2C - Differentiated human neuroblastoma cells are a suitable and reliable model for in vitro study of processes that occur in brain of patients suffering from acute and chronic neurodegenerative or hypoxic disorders.

BE2C is a subclone of the SK-N- BE(2) human neuroblastoma

TT Tell line. Unlike the parental cell line, which grows as a - mixed population of adherent and floating cells, BE2C cells are strictly adherent. The cells have a polygonal form and grow as clusters of flattened neuroblasts with numerous short cytoplasmic processes, while a few cells can also have one long neurite. The BE2C cells exhibit moderate levels of tyrosine hydroxylase and dopamine beta hydroxylase activity. They contain neurofilaments and specifically express D2-dopaminergic, alpha2-adrenergic, m2/m4-muscarinic and delta-opioid receptors.

BE2C was modified to express the retroviral ecotropic receptor. This manipulation made the BE2C cells suitable for retroviral gene delivery. BE2C cells are maintained in RPMI 1640 medium supplemented with 10% of heat-inactivated FCS, 2 wM L-glutamine, 1 mM sodium

SI pyruvate, 20 U/ml penicillin, 20 mg/ml streptomycin and 0.5 pg/ml fungizon (Gibco BRL). For neuronal differentiation, cultures of the neuroblastoma cells are exposed to 40 mM of all-trans retinoic acid (RA). After 5-6 days, cells extend neurite processes and show neuronal-like differentiation.

For infection or transfection experiments, confluent non- differentiated BE2C cultures are washed with PBS, detached with Trypsin-EDTA and subcultured to poly-L-lysine-coated plates at low density.

Differentiated and non-differentiated BE2C cells were tested for sensitivity to dopamine, L-Glutamate toxicity and hypoxia (0.5% O,). Cells viability was measured by Neutral Red assay (Biorad). Type of cell death was determined by DAPI staining. Optimal experimental conditions were calibrated.

Pro- and Antiapoptotic Activity Tests In Transient

Transfection Assays: - 24 -

In order to evaluate the potential pro-apoptotic apoptotic of gene 2-2-83, Hela and 293 cells were transiently co-transfected with 4 mg of the 2-2-83 gene plasmid and 2 mg of GFP expressing plasmid. Twenty-four and forty-eight hours post transfection the cells were fixed with 4% formaldehyde and stained with DAPI.

The antiapoptotic properties of the gene were examined in a similar assay by adding 1.2 mg of a pro- apoptotic expression construct (intracellular domain of Fas or RIP death-inducing domain) to the transfection.

The percentage of the apoptotic cells (among the

GFP-expressing cells) in 2 independent experiments was calculated. For further analysis of the cell cycle and the : apoptosis, 10° cells from the transfectants were harvested, resuspended in 1 ml PBS containing 0.05% triton and 50 mg/ml propidium iodide (PI) and subjected to FACS analysis.

Stable Transfection Of C6 Glioma Cells:

C6 cells were stably transfected by pCDNA3 vector either empty or expressing a gene 2-2-83 using a lipofectamine procedure. After three weeks of G418 (1.5 mg/ml) selection, independent clones were isolated. The level of gene expression was measured by Northern blot.

Total RNA samples (10 mg) from the G418 selected clones were separated on formaldehyde gels, transferred to nylon membrane, and hybridized with a 2-2-83-specific probe. C6 samples (3 mg of poly-A RNA) were taken from 16 hours hypoxia treated cells as a positive control.

Stable Transfection Of Human BE2C Cells:

Stable 2-2-83 expressing polyclonal cell populations were obtained by either retroviral transduction with pBABE-Puro-2-2-83 retroviral vectors into BE2C- ecotropic viral receptor expressing cells or by transfection of BE2C cells with pCDNA3-2-2-83 (Fugene 6 reagent -

— -Boehringer). ~~ The - corresponding empty vector served as control in both cases. High titer virus for infection was produced by ecotropic packaging cell line transfected with expression constructs by Ca/phosphate technique.

After either puromycin (pBabe) or G418 (pcDNA3) treatment, stable transfectants were selected as a batch (no single clones were selected). Total RNA was isolated from the cells for Northern blot analysis confirming the expression of 2-2-83.

Measuring The Sensitivity Of 2-2-83 Expressing C6 Clones To

Hypoxia: . : For the assay, the cells were plated at low density onto 96-well plates (5000 cells/well). 24 hours ; later, the cells were exposed to hypoxic conditions (0.4% _ 0,) for 3 days. Viable cells' density was estimated colorimetrically by neutral red assay. Note that there was a strong dependence between response to hypoxia and the density of cells.

Endothelial Cell Proliferation Assay:

Conditioned media from 2-2-83 expressing clones was examined for its ability to induce/inhibit bovine endothelial cell (BAEC) proliferation. For the proliferation assay, the cells were plated at low density onto 96-well plates (500 cells/well). 24 hours later, the culture medium is replaced with 50 ml of DMEM supplemented with 5% calf serum and 50 ml of the test sample (condition medium from Cé cell clones expressing the candidate genes).

For the inhibition assay, bFGF (0.5 ng/ml) was added one hour later. The medium was replaced 72 hours later as described above. The assay was terminated after 6-8 days by fixation with 2.5% formaldehyde and the cell density was estimated colorimetrically by staining with methylene blue.

Tumorogenesis In Nude Mice: 1.5x10° C6 glioma cells from stably expressing 2- 2-83 and pcDNA3-GFP negative control cells, were injected subcutaneously into 4 weeks male nude mice (2 clones of each gene, 3 mice per group). Cell clones were injected individually and as various mixtures with control cells.

Following initial evidence of tumor development, tumor diameters are measured every second day. When individual tumors reach an average diameter of 1.5 cm?, the tumor is operated. The tumors are preserved in freshly prepared 10% buffered formalin fixative for histopathological examination. Tumor vascularization are monitored in tumor sections. Blood vessel endothelial cells are visualized by : incubation with anti-von Willebrand factor. Amounts of apoptotic cells in tumor samples were assessed by TUNEL ) staining of tumor sections. 25-Hydroxycholesterol treatments ]

C6 clones, stably expressing 2-2-83 as well as pcDNA3 clones were examined for their response to 25-

Hydroxycholesterol treatments.

For the assay, the cells were plated at low density onto 24-well plates (20,000 cells/well) or onto 96- well plates (5000 or 10,000 cells/well). Twenty-four hours later the cells were exposed to 25-Hydroxycholesterol at different concentrations (1.25-20 pg/ml) for 3 days. The cell density was estimated by counting the viable cells (for the 24-well plates) or colorimetrically by neutral red assay (for the 96-well plates).

The assay was repeated using C6 clones stably expressing 2-2-83, GFP or pcDNA3 from early passages. For the assay, the cells were plated on low density plates ~ (20,000 on to 96-well plates: 5000 cells/well). Twenty-four : hours later the cells were exposed to 25-Hydroxycholesterol (10 pg/ml) for 1-3 days. The cell density was estimated

- : colorimetrically by neutral red assay.. _ a }

BE2C human neuroblastoma cells, transduced with pBABE- ouro-2-2-83, were also examined for their response to 25- hydroxycholesterol. For the assay, the cells were plated at 5S low density onto 96-well plates precoated with poly-L- lysine (5000 cells/well) and incubated in the presence of retinoic acid for induction of neuronal differentiation.

Five days later the cells were exposed to 25- hydroxycholesterol at different concentrations (0.15-20 ug/ml) for 2 days. The cell survival was estimated by neutral red assay. Three independent experiments were performed.

In addition, non-differentiated BE2C cells stably expressing 2-2-83 were treated with 25-hydroxycholesterol = 15 at concentrations 5 - 40 pg/ml for 72 hours. The initial = cell seeding density was 10,000/well in 96-well plate. : = Literature review on plant homologues of gene 2-2-83 and ' their function.

The Arabidopsis thaliana homologue of gene 2-2-83 is a gene referred as diminuto (DIM) (Takashi et al., 1995), dwarfl (DWFl) (Feldmann, 1991) or CBBl1 (Kauschmann et al., 1996). The dim mutant was initially isolated as slowly growing dwarf. This mutant phenotype could be rescued by the application of brassinolide (plant biologically active end pathway sterol). The comparison of sterol composition of normal and mutant plants has revealed that several biochemical reactions can be affected by dim mutation. In comparison to wt plants, the mutant ones accumulate 24-

Methylenecholesterol and isofucosterol, but contain the significantly reduced amounts of campesterol, sitosterol and end pathway sterols (Klahre et al., 1998). It was demonstrated that DIM activity is necessary for both the isomerization and reduction of 24-methylencholesterol. DIM - 28 -

) is an integral ER transmembrane protein, that is anchored by its N-terminus into the membrane from the cytoplasmic side of membranous compartment (Klahre et al., 1998).

Brassinosteroids.

As well as animals, plants contain steroid compounds that are active at similarly low concentrations as steroid hormones. Animals mainly synthesize cholesterol, ergosterol is the predominant sterol in yeasts, and sitosterol, stigmasterol, and campestrol are the most abundant sterols in plants. In mammalian cells, cholesterol serves as the precursor of steroid hormones, which are characterized by reduced complexity caused by removal of most of the side chain. Plants use campesterol as a precursor for brassinosteroid (BR) biosynthesis and do not . substantially shorten the side chain to form active hormones but rather employ a series of reduction and hydroxylation steps to do so. BRs play an important role in . plant growth and development. Over 60 analogues have been detected in a wide variety of plants in the past 18 years.

Brassinolide is the most biologically active one. It elicits cell elongation/proliferation and shows strong synergistic interactions with auxin and additive interactions with gibberellins. Arabidopsis mutants that accumulate reduced endogenous amounts of BRs or BR- insensitive mutants have very similar phenotype: they grow as dwarfs and their fertility is impaired (for reviews, see

Clouse, 1996; McMorris, 1997). So far, three genes involved in brassinosteroids metabolism have been identified: CPD (Szerkeres et al., 1996), DWF4 (Choe et al., 1998), DET2 (Li et al., 1996) and one gene, BRI1l (receptor kinase), was shown to be involved in BR-mediated signal transduction (Li and Chory, 1997). CPD, DWF4 and DET2 - all encode cytochrome P450-like enzymes. DET2 encoding a close homologue of animal steroid Sa-reductase can also oo -substitute it functionally, working on- testosterone_ and. — progesterone as substrates (Li et al., 1997).

EXAMPLE 1. : 5

Cloning of 2-2-83 cDNA.

On Northern blots comprised from C6 (rat glioma) and

A172 (human glioma) mRNA extracted from cells under hypoxic and normoxic conditions, 2-2-83 was found down regulated after 16 hours of hypoxia. The 2-2-83-specific cDNA probe hybridized to a single mRNA species of 74.0 Kb. Both rat and human orthologs of 2-2-83 cDNA were cloned. Their nucleotide and putative amino acid sequences are shown in

Fig. 1 and Fig.2, respectively. Rat cDNA clone is 3838 bp - 15 long and contains an open reading frame potentially coding hh for a protein of 516 amino acids ( nucl. 24 - 1572). Human

Be cDNA is 4096 bp long and also codes for a 516 amino acid - protein (nucl. 39 - 1587).

EXAMPLE 2.

Bioinformatic analysis.

Protein structure and domain analysis revealed that 2-2-83 has three potential transmembrane domains between amino acids 31 - 51, 137 - 157, and 209 - 229 (SMART). The protein was also predicted to have an uncleavable signal peptide (PSORT). Amino acids 133 - 234 constitute a FAD-binding domain found in several FAD- dependent oxydoreductases (PRODOM) . The search of available sequence databases revealed that human 2-2-83 nucleotide sequence is almost identical to human sequence

D13643 designated as KIAA0018. The putative proteins encoded by rat and human 2-2-83 genes are close homologues of proteins found in several plant species (S71189 from

Arabidopsis thaliana and P93472 from pea), and from C. elegans (017397). However, the putative protein encoded by

KIAAO018 cDNA (Q15392) appears truncated (390 amino acids instead of 516 amino acids encoded by human 2-2-83 gene).

This is due to a frameshift mutation within the KIAA0018 nucleotide sequence which resulted in a deletion of C residue between the positions 1166 - 1167. The overall structure of 2-2-83 protein from different species is similar. However, the second and the third putative transmembrane domains were detected only in mammalian species (probably due to the substitution of Tre/Ser and

Gly residues found in mammalian species to Asn residues in non-mammals within the second putative TM domain, and ‘ substitution of Cys to Gln within the third putative TM domain). The putative non-cleavable signal peptide also } failed to be detected within the non-mammalian species. The

FAD-binding domain is conserved through the evolution of 2- 2-83 protein homologues (Mushegian & Koonin, 1995).

EXAMPLE 3.

Expression pattern of 2-2-83 gene in normal mouse embryonal development.

Expression pattern of gene 2-2-83 in embryogenesis was studied by in situ hybridization on parasagittal sections of mouse embryos at days 12.5, 14.5 and 16.5 postconception (dpc). In most of 2-2-83 expressing cells, intensity of hybridization signal varied from weak to moderate. Only embryonic liver and sebaceous glands can be regarded as sites of strong 2-2-83 gene expression.

Central nervous system. The hybridization signal is widely spread throughout the mouse embryo central nervous system.

— -- The strongest neural expression was found at 12.5 and 14.5 _ ~ dpc stages in the ependymal layer of developing spinal cord and in brain (especially at the ventral side of brain ventricles). By the 16.5 dpc stage, the 2-2-83 expression - 5 disappears from the ependymal lining of central canal of the spinal cord as well as from the lateral and the fourth brain ventricles. However, the expression signal is still found in the third ventricle. Another prominent CNS region of 2-2-83 expression is the mantle layer (gives rise to the gray matter) of the spinal cord where hybridization signal could be seen at 12.5 and 14.5 dpc stages. This signal is preserved in some (but not all) neuroblasts of the ventral horn also at 16.5 dpc stage. A weak hybridization signal can be observed in developing brain cortex and in olfactory : 15 lobes. Neuroblasts of some of medulla oblongata and of _ hypothalamus nuclei (unidentified) display a weak : i hybridization signal at 12.5 and 14.5 dpc stages. These ~ nuclei were absent from the available sections of 16.5 dpc - embryos.

Peripheral nervous system. The peripheral nervous system (spinal ganglia and brain ganglia) as well as the autonomous nervous system (sympathetic ganglia) are 2-2-83- positive at all studied stages.

Non neural ectoderm derivatives.

Teeth. Expression of 2-2-83 could be detected in teeth primordia at all stages studied. At 12.5 dpc, the hybridization signal was also evident in dental lamina, an ectodermal invagination that manifests the earliest stage of tooth formation. At 14.5 and 16.5 dpc, the signal could be seen in ameloblasts, cells destined to produce enamel.

Skin. The 2-2-83 expression in skin could not be detected before the 16.5 dpc stage when a weak signal appeared in the suprabasal cells of epidermis. Interestingly, this signal could be seen only at the ventral side of the body.

At 16.5 dpc, a strong hybridization signal appeared also in the sebaceous glands in the association with developing vibrissae. Simultaneously, a weak hybridization signal also appeared in the external root sheath of vibrissae. Cells of the same type also displayed a weak expression in the hair roots of adult skin.

Heart and vascular system. A weak hybridization signal was detectable in cardiomyocytes at 12.5 and 16.5 dpc. By 16.5 dpc, this signal had disappeared.

Urogenital system. Kidneys and adrenals are present on sections of 12.5 and 16.5 dpc embryos and are absent from } the 14.5 dpc sections due to a cutting plane. At both available stages, a weak hybridization signal is seen in the tubular structures of kidneys and in adrenals.

Seminiferous tubules of developing testes are 2-2-83- positive on 14.5 and 16.5 dpc sections. Unlike the adults’ testis, the expression 2-2-83 pattern in embryo testes appeared uniform.

Skeletal system. The 2-2-83 gene displays a transient expression pattern in developing skeleton at 12.5 and 14.5 dpc. At 12.5 dpc stage, the hybridization signal is prominent in vertebrae primordia where it concentrates over the condensed portion of sclerotome. The signal in chondrocranium (e.g. in primordium of basioccipital bone) is weak and it can be seen in the innermost (i.e. most differentiated) cartilage. These cartilage cells show expression throughout the cartilaginous elements of skeleton also at 14.5 dpc. By 16.5 dpc, the hybridization signal disappears from the skeletal system.

Primitive qut ~~ derivatives. At 12.5 dpc, a weak hybridization signal can be seen in epithelial lining of all primitive gut derivatives present on studied embryo sections: esophagus, trachea, lungs, the pancreatic § primordium and the midgut. The 2-2-83 expression levels in these structures appear to gradually decline at the later developmental stages. Thus, at 14.5 dpc, the hybridization signal is already undetectable in esophagus and trachea. By 16.5 dpc, the signal disappears also from lungs and pancreas. The thymus primordium is present both on 14.5 and 16.5 dpc sections. However, the 2-2-83 expression in thymus is detectable only at 14.5 dpc. Thyroid gland is present only on 16.5 dpc sections when the hybridization signal concentrates in the peripheral part of primordium. As : 15 mentioned above, the 2-2-83 hybridization signal in the — liver is very prominent at -all stages studied, and it is : displayed by the liver parenchymal cells and not by the - hematopoietic cells.

EXAMPLE 4.

Expression pattern of geme 2-22-83 in normal adult rat tissues.

Expression of 2-2-83 was assessed by in situ hybridization to paraffin sections containing multiple adult rat tissues and was found in several types of cells.

Brain. In rat brain, in situ hybridization on sagittal and coronal sections reveals wide expression of this gene throughout the brain structures. Microscopic study shows that the hybridization signal concentrates over at least two cell types, neurons and oligodendrocytes. The intensity of hybridization signal and number of expressing cells vary between brain structures and even between cells within the same structure. In general, cells showing the strongest hybridization signal are concentrated in the posterior parts of brain: pons and medulla oblongata. However, single cells displaying very strong hybridization signal can be seen also in other brain regions, e.g. in midbrain (see below). Both white matter and gray matter within pons and medulla oblongata contain cells showing intensive hybridization signal. Heavily labeled neurons mark the nuclei of reticular formation in the gray matter. Strong expression in oligodendrocytes delineates the fibers of pyramidal tract. Single strong expressing trophic oligodenrocytes are scattered throughout white and gray matter in all brain structures. ’ Distinct layers of cerebellum show different hybridization patterns. No signal was detected in the : molecular layer with the exception of few scattered strongly labeled (presumably neuronal) cells. Most of the

Purkinje cells show hybridization signal of moderate - intensity. Most of oligodendrocytes in the white matter of cerebellum are 2-2-83-negative, but single oligodendrocytes do display very high expression levels. The same irregular pattern of expression can be also observed throughout the oligodendrocytes within the cerebellar nuclei. Most of the neurons in these nuclei show moderate hybridization signal.

Significant variation in the intensity of hybridization signal is observed throughout the midbrain region. Most of neurons show weak to moderate signal while single neurons display very strong expression. These strongly expressing neurons are very prominent in periaqueductal gray matter and in the red nucleus.

Neurons of the cerebral cortex also display a variable hybridization signal intensity, and expression appears to be stronger in ‘the deeper cell layers than in the outer ones. The maximal expression in neurons is observed in the most anterior (orbital) cortex region. Like oo "in other areas of gray matter, here too a very strong - : hybridization signal can be detected in single trophic oligodendrocytes.

In hippocampal neurons, the highest intensity of 2-2-83 expression is in the CA3 nucleus, while- expression in more posterior fields and in the dentate gyrus appears lower.

The pattern of 2-2-83 expression in the forebrain and midbrain regions of thalamus and hypothalamus is similar, and neurons of practically all nuclei display different hybridization signal intensity varying from weak to moderate.

The region of the lowest 2-2-83 expression in brain is presented by striatum. ’ — Skin. The strongest hybridization signal was observed in cells within the basal layer of sebaceous glands. Basal - cells are actually the stem cells which proliferate and give rise to terminally differentiated cells that fill the inner space of the gland. Terminally differentiated cells accumulate lipids within their cytoplasm and undergo apoptotic death that results in release of the fatty secrete.

Viscera. Weak expression of 2-2-83 was detected in the upper layers of urothelium and in surface epithelium of fundic stomach. Much stronger signal was observed in piloric surface epithelium.

Reproductive system. Expression of 2-2-83 was detected in rat testes: in basal cells (apparently spermatogonia) of some seminiferous tubules, probably, because of differential regulation at distinct stages of spermatogenesis. 2-2-83 is expressed in ovaries. The most - 36 -

prominent feature of its expression at this site is the close resemblance of the VEGF expression pattern in corpus luteum (CL): very strong hybridization signal in granulosa cells of postovulatory follicles undergoing luteinization and vascularization, and in young CL. In -mature CL expression of 2-2-83 as well as of VEGF is less prominent.

Another type of cells showing the similar pattern of expression of both genes are theca cells of secondary follicles. Derivatives of theca cells, lutein cells of interstitial glands, display a consistent hybridization signal with 2-2-83 while they are in general VEGF-negative.

EXAMPLE 5. ) Expression pattern of 2-2-83 in disease models. . Hypoxic rat retina. Hypoxia in retina was created by exposing of new born rat pups to hyperoxia which led to the reduction of blood supply (Alon et al., 1995). Upon transfer - to normal oxygen conditions, relative hypoxia is formed. The hypoxic retina was excised, fixed, sliced and used for the hybridization with *°*S-dATP labeled 2-2-83 specific antisense riboprobe. 2-2-83 RNA levels were found downregulated in response to hypoxia.

Solid tumors. C6 rat glioma-derived solid tumors were obtained by subcutaneous injection of the suspension of Cé cell into nude mice. Sections of two tumor samples were used : in in situ hybridization. One sample represented a solid tumor of about 4X3 mm in size. No significant morphological variations between different tumor regions were observed.

However, at the tumor periphery, there was a region showing elevated expression of VEGF (indicative for hypoxia). The

CL second sample represented a tumor of about 3X1 mm in size, containing a "core" region comprised of "white" trombous and necrotic masses. This "core" region was surrounded by tumor oT “cells forming the *wall" of varying thickness, from about : five to fifteen cell layers. VEGF was found to be expressed : by the closest to the core layer of tumor cells. The most distant cell layers showed no VEGF expression. Gene 2-2-83 displayed a uniform expression pattern in the second, necrotic, tumor sample. In the first sample, hybridization signal concentrated mainly at the tumor periphery but was notably absent from the VEGF-positive "cap". Therefore, in

C6 tumor, 2-2-83 also appeared as downregulated by hypoxia.

Middle cerebral artery occlusion (MCAO) stroke model.

The model was implied in the stroke-prone spontaneously hypertensive rat strain. Occlusion was permanent, unilateral, by electrocoagulation of MCA. This led to focal brain ischemia at the ipsilateral side of brain — cortex leaving the contralateral side intact (control). : : Experimental animals were sacrificed 4 hours after the : operation. Brains were removed, fixed in formalin, embedded into paraffin and coronal sections were performed to be used in in situ hybridization with 2-2-83-specific and

PGK (phosphoglycerokinase, glycolitic enzyme, upregulated by hypoxia) specific riboprobes.

In normal brain, 2-2-83 is strongly expressed in neurons of distinct areas of adult rat brain including cerebral cortex (see below). Results of the MCAO experiment revealed that in ischemic regions of cortex, expression of both genes was undetectable apparently due to cell death.

However, in regions adjacent to stroke (prone to delayed apoptosis), the level of 2-2-83 expression was reduced compared to the contralateral normal cortex. Expression of

PGK remained unchanged.

INTERMEDIATE DISCUSSION ON POTENTIAL ROLE OF DIMINUTO (2-2- 83) IN ANIMAL CELLS.

Highly homologous proteins are 1likely to have similar functions. Proteins highly homologous to plant

DIM/DWF1 were also found in mammals (2-2-83 in the present invention) and in C. elegans. C. elegans rely on plant

S sterols for their own sterol synthesis and are able to reduce 24-methylenencholesterol (Lozano et al., 19895).

Therefore, the conservation of Diminuto-like enzyme in this species is explainable. In yeast, no Diminuto homologues were found. Accordingly, the reaction catalyzed by DIM presumably does not occur because the analogous reduction of the corresponding bond in ergosta-5,7,22,24(28)- tetraen-3-0ol to yield ergosterol is known to be catalyzed by an unrelated enzyme. Using labeled precursors, the ’ reaction catalyzed by DIM/DWF1 was not detected in mammalian cells (Nes et al., 1973). Therefore, the reason . for the strict conservation of DIM sequences in animal cell evolution needs further study and explanation. There are three main possible explanations: (1) the reaction ; catalyzed by DIM is probably related only to the degradation of dietary sterols; (2) the reaction that is catalyzed by Diminuto in animals is different from that in plants; (3) the reaction is similar, but the substrate is yet unknown. In general, there are few animal steroids possessing functionalized side chain, e.g. 25-hydroxy vitamin D3 (calcidiol):; 1,25~dihydroxy vitamin D3 (calcitriol), their homologs, cholic acids and 24-oxy- (hydroxy-, epoxi-) steroids. The latter group of steroids emerged only recently in conjunction with biological activity. 24-hydroxy- and epoxysteroids are likely to activate the LXR receptors expressed in liver and brain. 24-oxysterol is highly abundant in brain. However, several reports indicate its potential neurotoxicity. : Tissue and cell-specific pattern of expression of gene 2-2-83 support its involvement in steroidogenesis in animals. Several sites where high levels of 2-2-83 mRNA

TT B “were detected are known as sites where steroids are either - synthesized or stored. Thus, 2-2-83 transcript was found in sebaceous glands where cholesterol compounds are among major constituents. In ovary, regulation of 2-2-83 expression did not correlate with the hypoxic state (judging by VEGF-specific staining), allowing to suggest that it is related to steroidogenesis (estrogen and progesterone synthesis). In brain, expression of 2-2-83 was found mainly in brain stem and in the vicinity of spinal cord, regions rich in myelin, that is also rich in steroids. Finally, expression of 2-2-83 in liver is connected to the synthesis of cholic acid.

In plants, DIM activity is crucial for cell elongation. In animals, cells that are able to elongate (to send long projections)are of neural and glial origin. oe Therefore, the function of gene 2-2-83 is somehow connected : i to neurite and axonal growth. Indeed, in rat brain, 2-2-83 = ‘is expressed in nuclei of brain stem, in reticular - formation and in the stem-spinal cord boundary, all are the regions where neurons have extremely long projections. The fact of 2-2-83 expression in trophic oligodendrocytes points out that some final products of reactions catalyzed by the protein encoded by animal DIM possesses neurotrophic and/or neuroprotective activity. Previously, several natural and synthetic steroids were found to have a neuroprotective activity. In this regard, it was found that downregulation of 2-2-83-specific transcription was present in both in hypoxic retina and in stroke penumbra.

Since steroids synthesized with the aid of animal Diminuto have neuroprotective activity, these compounds can be used as neuroprotective drugs at least in case of above mentioned pathologies.

EXAMPLE 6.

Subcellular localization of 2-2-83 protein in mammalian cells.

Transient transfection of Hela cells with 2-2-83-

GFP fused protein revealed that it is found in ER 24 hours after transfection. However, later it concentrates to granular structures close to plasma membrane. Taking into account the defined ER localization of DIMINUTO in plants, these granular structures are microsomes.

EXAMPLE 7.

Overexpression of 2-2-83 in transient assays neither induces apoptosis nor protects cells from FAS-induced apoptosis. ’ Since downregulation of 2-2-83 expression was observed in hypoxic tissues that contain a lot of apoptotic . cells (hypoxic retina, stroke penumbra, hypoxic regions of glial tumors) the potential association of 2-2-83 expression either with intrinsic apoptotic activity or with : intrinsic ability to protect from apoptosis was assessed.

For this, c¢DNA3-2-2-83 plasmid was transiently : transfected together with pcDNA3-GFP plasmid in Hela and 293 cells. 24 and 48 hours later the cells were fixed and stained with DAPI. No apoptotic effect was observed in the transfected cells. In order to evaluate the potential anti- apoptotic properties of the 2-2-83 protein, FAS-expressing plasmid was included into the co-transfection mixture. No effect opposing FAS-induced apoptosis was observed.

EXAMPLE 8.

) Stably overexpressing ~~ 2=-2-83 €6 glioma and _BE2C- neuroblastoma cells display altered phenotype.

Cell clones expressing 2-2-83 from the pcDNA3 expression vector were obtained by transfection of C6 cells. In BE2C cells, two different polyclonal cell populations stably expressing 2-2-83 either from pcDNA3 vector or from pBABE retroviral vector were obtained. 2-2-83 C6 stable cell clones have some distinct features compared to control: the cells look more flattened, tend to aggregate starting from the very low cell density, send multiple short processes and short time after transfection display higher proliferation rates than control «cells reaching very high density within the aggregates (Fig.3). They also seem to have some adhesion _— problems as they easily detach from plates after mechanical : v insults. Later on, although preserving the initial levels o of exogenous 2-2-83 expression, cells slow down : proliferation to the control rates (Fig.4). 2-2-83 cells kept in culture for long periods (more than a month) proliferate slower than parental cells (see EXAMPLE 9).

BE2C cells freshly infected with pBABE-2-2-83 send longer processes and look much more differentiated than control cells (Fig.5). The proliferation rates were similar to controls (Fig.6). Interestingly, FACS analysis for cell «cycle distribution in BE2C cells freshly transduced with pBABE-2-2-83 revealed that it is distinct from control: relatively more cells had either less than 2n or higher than 2n DNA content, suggesting certain accumulation of apoptotic and proliferating cells in population. Since both processes compensate one another, similarity in growth curves between control and 2-2-83 expressing BE2C cells are explainable. ’ EXAMPLE 9.

Control C6 cells being co-cultivated with "old" 2-2-83 expressing cultures send longer processes.

Since plant 2-2-83 ortholog, Diminuto, is involved in steroid synthesis, and steroids are molecules able to enter and to leave the cell freely, testing was done to determine if the conditioned medium of 2-2-83 expressing cells can influence the parental cells phenotype. For this, equal amounts of cells from either C6-2-2-83 cell clones (kept in culture for more than a month) or from C6-pcDNA3 cell clones (kept in culture for more than a month) were mixed with equal amount of parental C6 cells engineered to express GFP. Cells were plated and observed ) microscopically. First, it was immediately evident under the light microscopy, that while the mixtures of vector . transfected and parental C6 cells grew as homogenous populations of small cells with typical C6 morphology, in . mixed populations containing 2-2-83 expressing cells there were islands of slowly proliferating flattened cells.

Analysis of cells under fluorescent microscopy revealed, that these cells were GFP-negative, hence 2-2-83 expressing. To estimate the relative amount of GFP-positive and negative cells in mixed populations, they were FACS sorted. In control cells mixtures, amount of GFP-positive (parental C6) and negative cells (vector-transfected) appeared equal, while GFP-negative 2-2-83-expressing C6 cells constituted only 20% of mixed cell population though the initial numbers of plated GFP-positive and negative cells were equal. Moreover, observation of cultured cells under fluorescent microscopy demonstrated that parental C6 cells send long processes in the direction of 2-2-83- expressing cells. This was not observed in control plates.

EXAMPLE 10.

0 —-~C6 and BE2C 2-2-83 expressing cells are slightly wore — : resistant to hypoxia-induced cell death than parental cells. 2-2-83-expressing C6 cell clones were subjected to hypoxia (0.5% oxygen) treatment for 3 .days. Two polyclonal BE2C-2-2-83 cell populations were subjected to chemical hypoxia by addition of iron-chelator agent DFO (100uM) to culture media. Both cell types that overexpress 2-2-83 appeared slightly more resistant to hypoxia-induced apoptosis than control cells (Fig. 8).

EXAMPLE 11

Conditioned media from 2-2-83 overexpressing cells in inert in angiogenesis assays. — Since expression of endogenous 2-2-83 is reduced ‘ by hypoxic conditions, the potential involvement of 2-2-83 in inhibition of angiogenesis (as it is potentiated by hypoxia) was assessed. Conditioned medium from C6 clones stably expressing 2-2-83 protein were tested twice for their ability to induce/inhibit endothelial cell proliferation. No effect was observed.

EXAMPLE 12.

C6 and BE2C cells overexpressing 2-2-83 display an altered sensitivity to 25-Hydroxycholesterol-induced cytotoxicity. 24-hydroxy- and epoxysteroids are likely to activate the LXR receptors expressed in liver and brain. 24-oxysterol is highly abundant in brain. Since these compounds resemble to some extent the plant DIMINUTO substrates, it was questioned whether cells expressing exogenous 2-2-83 gene will be more resistant to cytotoxic action of 25-hydroxycholesterol (Sigma) because of its potential conversion by 2-2-83-associated enzymatic - 44 - x activity. For this, C6-2-2-83 cell clones and 2-2-83-BE2C cell populations were treated with 25-hydroxycholesterol as described in Materials and Methods, and cell death was monitored by neutral red staining. The results indicate that freshly infected BE2C cells expressing .2-2-83 and fresh C6 2-2-83 expressing clones are slightly more sensitive to 25-hydroxycholesterol than control cells (Fig.9, 10), while after several passages, C6-2-2-83 clones acquire almost complete resistance to 25- hydroxycholesterol-induced apoptosis (Fig. 11).

EXAMPLE 13 * Introduction of 2-2-83 expression in C6 cell suppresses their tumorigenicity in nude mice.

The objective of the assay was to determine the pattern of tumor growth, vascularisation and survival in nude mice, following injection of transfected C6-2-2-83 cells in order to assess the influence of 2-2-83 gene on apoptosis, proliferation and angiogenesis in in vivo conditions. 1.5x10%6 C6 glioma cells from stably expressing clones of the candidate gene 2-2-83 and pcDNA3 negative control, were injected subcutaneously into 4-6 weeks female nude mice (2 different clones, 4 mice per group, 2 independent experiments). :

Following initial evidence of tumor development, tumor diameters were measured twice a week. Four weeks postinjection, the tumors were operated and weighted.

Tumor growth was detectable and measurable for control C6-pCDNA3 cells implanted in nude mice by 11 days postimplantation, while none of the C6-2283 clones had produced visible tumors (Fig.1l2). One of the 2-2-83 clones (A8) had not produced visible tumors even by 30 days

— ~~ postimplantation in two independent experiments. The second : 2283 clone (B6) had given rise to significantly smaller tumors (Fig. 13).

Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the ? nature of words of description rather than of limitation. i Obviously, many modifications and variations of : the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the described invention, the invention can be practiced otherwise than as specifically described.

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Claims (19)

CLAIMS What Is Claimed Is:

1. A polynucleotide sequence whose expression is modulated by hypoxic conditions.

2. A polynucleotide sequence encoding a gene for protein 2-2-83.

3. The polynucleotide sequence according to claim 2, wherein said sequence is from a mammal.

4. The polynucleotide sequence according to : claim 3, wherein said mammal is a rat.

5. The polynucleotide sequence according to claim 4, wherein said sequence is the nucleic acid sequence according to SEQ. ID No.:1.

6. The polynucleotide sequence according to claim 3, wherein said mammal is a human.

7. The polynucleotide sequence according to claim 6, wherein said sequence is the nucleic acid sequence according to SEQ ID No.:2.

8. A pharmaceutical composition for modulating hypoxia and ischemia comprising an effective amount of a polynucleotide having the nucleic acid sequence according to SEQ ID No.: 1 or 2 and functional analogs thereof and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition for modulating hypoxia and ischemia comprising an effective amount of a protein encoded by the nucleic acid sequence according to SEQ ID No.: 1 or 2 and functional analogs thereof and a pharmaceutically acceptable carrier.

10. A method of regulating hypoxic associated pathologies and tumorigenesis by administering an effective amount of a polynucleotide having a nucleic acid sequence from at least one from the group comprising SEQ. ID No.: 1 and 2 and functional analogs thereof and a pharmaceutically acceptable carrier.

11. A method of regulating hypoxic associated pathologies and tumorigenesis by administering an effective amount of a protein encoded by the polynucleotide sequence from at least one from the group comprising SEQ. ID No.: 1 : and 2 and functional analogs thereof and a pharmaceutically acceptable carrier. a

12. A method of regulating hypoxic associated pathologies and tumorigenesis by administering an effective = amount of a direct or indirect biologically active product B of enzymatic activity of the protein encoded by the polynucleotide sequence from at least one from the group comprising SEQ. ID No.: 1 and 2 and functional analogs thereof and a pharmaceutically acceptable carrier.

13. A method of regulating hypoxic associated pathologies and tumorigenesis by inhibiting the enzymatic activity of the protein encoded by the polynucleotide sequences from at least one from the group comprising SEQ. ID No.: 1 and 2 and functional analogs thereof.

14. A method of regulating hypoxic associated pathologies and tumorigenesis by administering an effective amount of inhibitors of a direct or indirect biologically

- active product of enzymatic activity of the protein encoded — — by the polynucleotide sequence from at least one from the group comprising SEQ. ID No.: 1 and 2 and functional analogs thereof and a pharmaceutically acceptable carrier.

15. A hypoxia response regulating gene.

16. The hypoxia response regulating gene according to claim 14, wherein said gene having the nucleic acid sequence according to SEQ ID No.:1-2.

17. A neuroprotective and neurotrophic agents comprising a gene product of the polynucleotide sequences of 2-2-83 according to SEQ. ID No.: 1 and 2 and functional analogs thereof. :

18. The neuroprotective and neurotrophic agent : according to claim 16, wherein said gene product is a protein or the direct or indirect product of the protein's enzymatic activity.

19. A method of diagnosing the presence of a hypoxia associated pathology or tumorigenesis by screening for the reduced expression of the gene encoded by the nucleic acid sequence according to SEQ. ID No.: 1 and 2 and functional analogs thereof.