WO1997004104A2 - Tumor type ii hexokinase transcription regulatory regions - Google Patents

Abstract

The present invention relates to a tumor promoter involved in the regulation of glucose catabolism in neoplastic tissues. In particular, this promoter region contains numerous response elements that are involved in regulation of transcription of the Type II hexokinase gene in tumor cells. Such elements are of value for diagnostic and therapeutic applications, such as in controlling tumor growth. In addition, the entire promoter region (about 4.3 kbp) or regulatory segments (response elements) contained therein may be used for expression of naturally-occurring or foreign proteins. Such proteins may be derived from mammalian cells and expressed under the control of transcription factors that bind to specific response elements within the tumor Type II hexokinase promoter. Finally, the promoter is useful in gene therapeutic approaches to diseases including diabetes and cancer.

Description

TUMOR TYPE π HEXOKINASE TRANSCRIPTION REGULATORY REGIONS

The invention was made with government support under NIH Grant C A 32742.

The government may have certain rights in this invention.

TECHNICAL FIFT H OF THE INVENTION

The present invention relates to a tumor promoter and transcription regulatory region involved in the regulation of glucose catabolism in neoplastic tissues. In particular, this transcription regulatory region contains numerous response elements that are involved in regulation of transcription of the Type II hexokinase gene in tumor cells. BACKGROUND OF THE INVENTION It has been known for over six decades that one of the most consistent and profound biochemical phenotypes of cancer cells is their increased rate of glucose utilization. In recent years, hexokinase, which catalyzes the first step of the glycolytic pathway and which is highly overexpressed in tumor cells, has been shown to be a major player in this process of glucose catabolism in cancer cells. In comparison to normal cells, rapidly-growing tumors have elevated hexokinase activity levels (Parry,

D. and Pedersen, P., (1983), J. inl. Chem _^ 25S: 10904- 10912). Glycolysis is defined as the metabolism of glucose to yield either lactic acid under anaerobic conditions or pyruvate under aerobic conditions, the latter being further metabolized to carbon dioxide and water. Rapidly-growing cancer cells have the ability to maintain an increased rate of glucose utilization and the capacity to sustain high rates of glycolysis under aerobic conditions (Warburg, O., (1930), Th Metabolism of Tumors, Arnold Constable, London); Weinhouse, S. , (1966), Gann Monogr. , 1:99-115; Bustamante, E. and Pedersen, P., (1977), Pmc Natl. A rad Sri USA , 14:3735-3739; Aisenberg, A. , (1961), In: The. Glycolysis and Respiration of Tumors. Academic Press, London;

Pedersen, P., (1978), Pm Exp Tumnr Re . 22:190-274). This elevated rate of glucose catabolism is important for highly malignant tumors, such as tumors derived from liver, kidney, and brain, which may obtain over 50% of their energy, and the anabolic precursors for biosynthetic pathways, via glycolysis (Nakashima, et_al.,

(1984), Canrer Researrh, 44:5702-5706; Bustamante, eL_al., (1981), J. mnl. Chem , 256:8699-8704).

There are four hexokinase isozymes in mammals designated as Type I-IV. Type IV is also called glucokinase. In general, hexokinases, known as D-hexose 6- phosphotransferases, catalyze the following reaction:

Glucose + MgATP > Glucose-6-Phosphate + MgADP.

Isozymes are multiple forms of a given enzyme that may occur within a single species of organism or within a single cell. Such multiple forms can be detected and separated by gel electrophoresis of cell extracts; as they are coded by different genes, they differ in amino acid composition and thus, in their isoelectric point values (Lehninger, (1975), In: Biochemistry, Worth Publishers, Inc., NY, pg. 244). (Wilson, J.E. , (1985), In: Regulation nf Carhohydrate Metabolism (Breitner, R., ed.) CRC Press I, pgs. 45-86; Rijksen, et al. , (1985), In: Regulation of Carbohydrate Metabolism (Breitner, R., ed.) CRC Press I, pgs. 89-99; Pilkis, et. al., (1994), J. BM. Chem. , 269:21925-21928). Types Mil exhibit very low Kms (0.02-0.13 mM) for glucose (high affinities), are product inhibited by glucose-6-phosphate, (Glu-6-P), and have a molecular mass near 100 kDa. The Type IV isozyme, in contrast to Types I-III, has a high Km (5-8 mM) for glucose, is insensitive to Glu-6-P inhibition, and has a mass near 50kDa.

The distribution of the four isozymes is tissue specific. Type I is found normally in brain and kidneys, Type II is found in skeletal muscle and adipose tissue, Type III is found in low amounts in several tissues, and Type IV is found predominantly in the liver and pancreas. Within normal liver cells, Type IV hexokinase is the predominantly expressed isoform (Printz, et al., (1993), Annn Rev. Nutr. , 13:463-496), and transcription of this enzyme is enhanced by both glucose and insulin (fed state), and inhibited by glucagon (fasted state) (Granner and Pilkis, (1990), J. Biol. Chem., 265:10173-10176). Significantly, the Type II hexokinase gene, which is markedly overexpressed in hepatoma cells, is essentially silent in liver.

In comparison to normal cells, the activity of Type II hexokinase is markedly elevated in highly glycolytic, rapidly growing tumors (Pedersen, P.L., (1978), Prog. Exp. Tumor Res., 22: 190-274; Bustamante, supra; Arora, et al., (1988), J. R Chem. , 263: 14422-14428; Parry, supra). Two factors are known to be largely responsible for this enhanced activity, one of which involves a propensity for the tumor enzyme to bind to the outer mitochondrial membrane, and the other which involves the enzyme's overproduction. Mitochondrial binding of tumor hexokinase to the outer membrane has been intensely studied (Bustamante, supra; Rose, eLal., (1967), J RM. Chem. , 242:1635-1645; Parry and Pedersen, (1983), T Binl Chem , 25S: 10904-

10912). Mitochondrial binding provides the enzyme with preferential access to mitochondrially-generated ATP and increases the activity and stability of the enzyme. (Arora, supra). Mitochondrial membrane binding also reduces the sensitivity to product inhibition by G-6-P, which is an important regulator of hexokinase in normal cells (Bustamante, supra; Rose, supra; Gumaa, eLaL, Rinrhem. Rinphys. Res. Cnmm. ,

26:771-779; Kurokawa, eLal., (1981), Rinrhem Int. , 2:645-650; Inui, eLal., (1979), J. Biochem., 85:1151-1156). The end product of the hexokinase reaction, glucose-6- phosphate (G-6-P), serves not only as a source of ATP via glycolysis but is also a key intermediate in metabolic processes essential for cell growth and proliferation, as shown in Figure 1.

The cDNAs representing each of the four isozymes have been cloned and sequenced (Nishi, et al., (1988), Rinrhem Rinphys Res Cnmm. , 152:937-943; Schwab, eLal. , (1989), Pmr Natl Acad. Sri USA , 86:2563-2567; Thelen, eLal. , (1991), Arch. Rinrhem Rinphys , 286:645-651; Schwab, et al., (1991), Arch. Rinrhe Rinphy. , 285:354-370; Andreone, et al., (1989), J. BM. Chem., 264:363-

369). In addition, hexokinase cDNAs have been cloned and isolated from different tumors (Thelen, supra; Arora, eLal., (1990), J. Rinl Chem , 265:6481-6488).

Using a Type I hexokinase cDNA probe (from brain), the hexokinase isozyme expressed in a mouse hepatoma cell line (c37) has been cloned and characterized (Arora, supra) and shown to be approximately 92% identical to the hexokinase I sequence derived from rat brain (Schwab, et al., (1989), Proc. Natl. Acad. Sci. USA, 86:2563-2567) and human kidney (Nishi, et al. , (1988), Biochem Biophys. Res Comm., 151:937-943). Now recognized as Type I, it is overexpressed in the AS-30D hepatoma cells (Figure 1), but to a much lesser extent than Type II hexokinase. Thus, regulatory regions of the Type I and Type II hexokinase forms may share some common activating elements. Nevertheless, in those rapidly growing, highly glycolytic cancers smdied to date (Shinohara, supra; Nakashima, supra; Thelen, supra; Sato, et al. , (1972), Gann Monograph nn Cancer Res , 11:279-288; Kikuchi, et al. , (1972), Cancer, 30:444-447; Hammond and Balinsky, (1978), Cancer Res , 38:1323-1328; Singh, et al. , (1978), J. Cell Physiol . , 92:285-292; Rose and Warms, (1982), Arch

Biochem. Biophys. , 211:625-634; Rempel, et al., (1994), Binchem Biophys Acta. , 219:660-668), it is the Type II hexokinase isozyme that either dominates or increases upon transformation. Thus, Type I, although elevated somewhat in highly glycolytic tumors, is elevated to a much lesser extent than Type II (Nakashima, et al., (1988), Cancer Res. , 48:913-919; Rempel, et al. , (1994) Biochem. Biophys. Acta., 1219:660-

668; Parry, et al., (1983), J. R Chem. , 258:10904-10912; Kurokawa, et al., (1982), Mnl. Cell. Biochem. , 45: 151-157).

Both Type I and Type II isoforms bind to the outer mitochondrial membrane pore protein, which is known as VDAC (Nakashima, eLal., (1986), Rinrhem. , 25: 1015-1021). This binding markedly reduces the enzymes' sensitivity to product inhibition by Glu-6-P (Bustamante, eL_al., (1977), Pmr Natl. Arad Sri TIS _I 24:3735-3739), provides preferred access to mitochondrially-generated ATP (Arora, eLal., (1988), J. Biol. Chem,., 263:14422-14428), and also provides protection against proteolytic degradation (Rose, eLal., (1982), Arr.h. Rinrhe Rinphys. , 211:625-634). In addition to these properties and the high content of the mmor enzyme (100-fold elevation), Glu-6-P is rapidly produced. Glu-6-P is a key metabolic intermediate serving as a major carbon source for most biosynthetic products essential for cell growth and division, but also results in ATP synthesis during its catabolism to lactic acid (Figure 1). It is estimated that under aerobic conditions more than half the ATP produced in some mmor cells is derived from glycolytic reactions (Aisenberg, A.C ,

(1961), In: Th Glymfysis and Respiration nf Tumnrs, Academic Press, London, pgs. 8-11; Nakashima, eLal., (1984), Cnnrer Re , , 44:5702-5706), in contrast to normal cells where this value is usually less than 10 percent. Under hypoxic (low oxygen) or anaerobic conditions, the already high glycolytic rate may double (Weinhouse, S. , (1972), Canrer Res _t 32:2007-2016), allowing mmor cells to thrive while neighboring normal cells become growth deficient.

The marked overexpression of Type II hexokinase in tumor cells characterized phenotypically by a high glucose catabolic rate is consistent with an enhanced rate of transcription. Elevated mRNA levels for hexokinase have been found in all highly glycolytic tumor lines examined to date (Shinohara, FEBS Lett, supra; Rempel, supra;

Mathupala, eLal, (1995), J. RM. Chem. , 16918-16925).

Mitochondrial-bound hexokinase is also found in some normal tissues, including brain and skeletal muscle (Wilson., J., (1985), In: Regulation nf Carbohydrate Metabolism, Beitner, R, ed., 1:45-85, CRC Press, Inc. , Boca Raton, Florida), but in significantly lower amounts than in rapidly-growing cancer cells. In addition, mRNA is markedly overexpressed in the AS-30D hepatoma cell line relative to normal liver and skeletal muscle. The deduced amino acid sequence for the hepatoma Type II enzyme differs in only 4 amino acids from the skeletal muscle Type II enzyme (Thelen, supra). Moreover, regions of the enzyme predicted to be involved in catalysis and Glu-6-P inhibition are nearly identical to those found in the tumor Type I isozyme (Mathupala, Rempel, and Pedersen, unpublished results). As the Type II hexokinase isozyme of normal and tumor tissues are essentially identical, the contribution of the Type II enzyme to the high glycolytic phenotype of many tumor cells is mainly due to elevated transcription and not to a property of the enzyme itself. Thus, the role of hexokinase, (ATP: D-hexose 6- phosphotransferase; E.C. 2.7.1.1), which commits glucose to catabolism in the first step of the glycolytic pathway, in particular the Type II isoform has come under increased scrutiny in efforts to understand the molecular basis for the aberrant glycolytic phenotype (Bustamante, supra; Nakashima, ei al., (1986), Binchemistry, 25: 1015-1021; Arora, K. and Pedersen, P., (1988), ./. Binl. Chem. , 263: 17422-17428). Significantly, hexokinase is a potential target for arresting tumor cell growth (Floridi,_eLaL, (1981), J. Natl Cancer Institute.

66:497-499; Floridi, eLal., (1981), Cancer Res., 41:4661-4666). The role of hexokinases, particularly Type II and Type IV, in diabetes is now attracting considerable attention as well (Pilkis, supra).

There remains a need in the art for methods and drugs to effectively treat both neoplasia and diabetes.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a DNA fragment useful in the regulation of transcription of a downstream open reading frame.

It is another object of the invention to provide a method of screening for potential drugs useful in the treatment of cancers and diabetes.

It is yet another object of the invention to provide a method of treating cells which are neoplastic.

It is still another object of the invention to provide nucleic acid probes useful in the diagnosis of cancers. It is a further object of the invention to provide methods for diagnosing tumors.

It is yet another object of the invention to provide a vector for regulated expression of desired proteins in a mammalian cell.

It is still another object of the invention to provide a method for increasing glycolysis in cells. These and other objects of the invention are provided by one or more of the embodiments shown below. In one embodiment of the invention an isolated hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame is provided. The fragment comprises at least one of the response elements identified in Figure 11. According to another embodiment of the invention a method of screening for potential drugs which affect with regulated transcription of tumor hexokinase II is provided. The method comprises the steps of: contacting a test substance with a reporter gene fusion comprising an isolated hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure 11 ; and measuring transcription of the reporter gene in the presence of the test substance; identifying a test substance as a potential drug which increases or decreases the transcription of the reporter gene.

In yet another embodiment of the invention a method of treating cells which overexpress hexokinase II is provided. The method comprises the step of: administering to cells which overexpress hexokinase II a gene fusion comprising a toxic gene and a an isolated hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure

11, whereby the toxic gene is expressed in the cells.

According to still another embodiment of the invention a n isolated nucleic acid probe is provided. The probe comprises at least 15 contiguous nucleotides selected from the sequence of SEQ ID NO: 1. In another aspect of the invention a method for diagnosing tumors which overexpress hexokinase is provided. The method comprises the steps of: determining copy number of a hexokinase II gene in a tissue sample suspected of being neoplastic; wherein a determined copy number of greater than two indicates neoplasia. According to another aspect of the invention a method for diagnosing neoplastic tissues is provided. The method comprises the step of: determining whether cells in a tissue sample suspected of being neoplastic contain a hexokinase π gene which is unmethylated, an unmethylated hexokinase II gene indicating neoplasia. In another aspect of the invention a vector for expression of a desired protein in a mammalian cell is provided. The vector comprises: an isolated hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure 11. In another embodiment of the invention a method is provided for increasing glycoslysis in cells. The method comprises the step of: introducing into cells an unmethylated DNA molecule comprising: a hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure 11 ; and

a nucleic acid encoding a hexokinase II, wherein the hexokinase II DNA fragment is covalently and operatively linked to the nucleic acid encoding a hexokinase II. The present invention thus provides the art with methods and reagents for treating diseases such as cancer and diabetes, as well as methods for diagnosing neoplasia. In addition, the method provides methods for screening for new therapeutic agents for such diseases.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic drawing of the basic metabolic pathway for glucose in tumor cells with a high glycolytic phenotype.

Figure 2 shows a Northern blot analysis carried out on total RNA to determine the expression levels of Type I and Type II hexokinase in AS-30D hepatoma cells relative to their expression in normal rat liver. Specifically, for

Type I hexokinase, the blot was hybridized with a 0.7 kbp fragment of the c37 tumor Type I hexokinase cDNA (Panel 2 A) and for Type II hexokinase, a full length cDNA of rat skeletal muscle Type II hexokinase was used (Panel 2B). Single mRNA species in AS-30D cells (lane 4) were detected similar in size to the hybridization bands obtained with rat brain (lane 1) and skeletal muscle RNA

(lane 2), which were used as controls for the Type I and Type II hexokinase messages, respectively. The Type I and Type II hexokinase messages were below the detection level in normal rat liver (lane 3). Loading of RNA was estimated by ethidium bromide staimng of the gel (Panel 2C). Figure 3 outlines the strategy used for AS-30D genomic library construction and isolation of the tumor Type π hexokinase promoter. Figure 3 A shows SauA I partially digested AS-30D DNA was ligated to compatible Sac I digested λ -Fix II arms to generate the genomic library. Figure 3B shows the subcloning of a 5.15 kbp promoter containing DNA fragment into vector pUC18. This 5.15 kbp DNA insert was subcloned into pGL2-Basic, a luciferase reporter vector using compatible Xba I, Nhe I sites (Figure 3C). A DNA fragment corresponding to the coding region within the first exon was removed using Xho I, to yield a 4.3 kbp promoter-reporter gene construct. Striped bars indicate luciferase cDNA. Hatched bars indicate AS-30D DNA. Open boxes indicate the multicloning sites (mes) and DNA of individual vectors + 1, transcription start site; cos, λ- cohesive termini.. Figures 4A and 4B show the identification of λ-subclones containing the tumor Type II hexokinase promoter region. DNA from six positive λ-clones (Lanes 1-6; clones 22-1, 22-2, 22-3, 25-1, 27-1, 29-1) were digested with Xba I and separated by agarose gel electrophoresis, as shown in Figure 4A. DNA fragments similar to skeletal muscle Type II hexokinase first exon (white bars) were identified by Southern blot analysis, as shown in Figure 4B. Molecular weight markers (λ-Hind III), in kbp, are shown to the right.

Figure 5 shows the complete nucleotide sequence of the 4.3 kbp proximal promoter region, the first exon, and part of the first intron of the AS-30D tumor Type II hexokinase gene. The response element motifs are indicated below the nucleotide sequence. Sequence in italics indicate a motif with the potential to form Z-DNA structures. Direct DNA repeats larger than 10 bp are indicated by dotted lines below the sequence, and identified by Roman numerals. A TATA box motif (-30) and a CAAT box motif (-85) are highlighted and the DNA sequence of the first exon is underlined (+ 1 to +524). Nucleotides +525 to +790 are part of the first intron.

Figure 6 shows cDNA and deduced amino acid sequences of the Type π tumor hexokinase from rat hepatoma AS-30D. The differences between the Type II rat skeletal muscle hexokinase and the AS-30D hexokinase at the nucleotide and amino acid levels are highlighted within the sequence. Figure 7 shows cDNA and amino acid sequences of the PCR-generated probe used for isolation of the promoter for Type II tumor hexokinase from the AS-30D genomic library. The DNA region which corresponds to the 260 bp

PCR product is underlined.

Figure 8 outlines the organization of the potential response elements for glucose, insulin, glucagon, TPA, and cAMP on the 4.3 kbp tumor Type II hexokinase promoter ( + 1, transcription start site; closed box, mRNA untranslated region; hatched box, coding region of the first exon; open boxes, response elements that are sensitive to glucose, insulin, glucagon, cAMP or TPA).

Figure 9 shows the effect of glucose, insulin, glucagon, TPA, and cAMP on the transcriptional activity of the tumor type II promoter. Luciferase activity was assayed 24 hours post-transfection of the promoter-reporter construct into

AS-30D cells, which were maintained under hormonal, metabolite, or intracellular mediator influence as indicated in the figure. Activities are expressed as fold activation over that of a control (ImM pyruvate). Each of the samples contained 1 mM pyruvate as substrate background. All values represent the mean of six independent experiments. The individual standard deviations (.+_

SD) for the fold activation are: glucose , 0.44; insulin, 0.25; glucagon, 0175; glucose + insulin, 0.5; glucose + glucagon, 0.06; TPA, 0.45; and dibutyryl cAMP, 0.7.

Figure 10 shows the transcriptional activity of the tumor type II hexokinase promoter in normal or AS-30D hepatoma cells. Luciferase activity was assayed 24 hours post-transfection of the promoter-reporter construct into hepatocytes or into AS-30D hepatoma cells. Hormonal or metabolite conditions used, are indicated on the figure; activity is expressed as fold activation over that of a control (1 mM lactate). Each sample contained 1 mM lactate as substrate background. All values represent the mean of two independent experiments. Figure 11 outlines segments of the nucleotide sequence of the hepatoma

AS-30D Type II hexokinase promoter region shown in Figure 5.

Transcription factors that bind to specific regions within the DNA sequence are indicated. The respective response elements (transcription factor binding sites) may consist of all or part of the sequence shown. An example of how to interpret the information in Figure 11 is set forth below. interpretation

-4369 TCTAGAGCTCGTCGCGGCCGCGGA

(+) LBP-1 S00487

SEQUENCE POSITION = -4369 first sequence reference point

DNA SEQUENCE = TCTAGAGCTCGTCGCGGCCGCGGA

SENSE STRAND = (+)

TRANSCRIPTION FACTOR = LBP-1

REFERENCE # FOR TRANSCRIPTION = SOO487 FACTOR FROM TRANSCRIPTION FACTOR

DATABASE (TFD) AT THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION

For ease of reference, the definitions used in the specification are as indicated in Table 1. TABLE 1. DEFTNTTTONS

Genomic HNA

A DNA fragment derived from cellular chromosomal DNA rather than from messenger RNA, the source for cDNA clones. Genomic and cDNA clones have different sequences due to RNA splicing. Other means of producing genomic DNA can be used. cDNA (complementary DNA)

DNA synthesized from mRNA in test tubes using an enzyme called reverse transcriptase. The cDNA sequence is thus complementary to that of the mRNA. Complementary DNA is sometimes made with radioactive nucleotides and is used as a hybridization probe to detect specific RNA or DNA molecules. Other means of producing cDNA can be used.

Nucleotide

A monomeric unit of deoxyribonucleic acid ("DNA") or ribonucleic acid (" RNA") consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1 ' carbon of the pentose) and that combination of base and sugar is called a nucieoside.

The base characterizes the nucleotide. The four DNA bases are adenine ("A"), guanine

("G"), cytosine ("C"), and thymine ("T"). The four RNA bases are A, G, C, and uracil ("U").

DNA Sequence

A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

Codon A DNA sequence of three nucleotides (a triplet), which encodes, through its template, an amino acid, a translation start signal, or a translation termination signal.

For example, each of the nucleotide triplets TTA, TTG, CTT, CTC, CTA, and CTG encode the amino acid leucine ("Leu"), TAG, TAA, TGA are translation stop signals, and ATG is a translation start signal. Polypeptide

A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids. Transcription

The process of producing RNA from a structural gene. Translation

The process of producing a polypeptide from mRNA. Expression

The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation. PJasmid

Nonchromosomal, double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for neomycin restistance (neo.sup.R) transforms a cell previously sensitive to neomycin into one which is resistant to it. A host cell transformed by a plasmid or vector is called a "tranformant" . Phage or Bacteriophage

Bacterial virus which consists of nucleic acids encapsulated into a protein envelope or coat (" capsid ") .

Cloning Vehicle or Vector

A plasmid, phage DNA or other DNA sequence which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition or restriction sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g. , replication, production of coat proteins, or loss of promoter or binding sites, and which contain a marker suitable for use in the identification of transformed cells, e.g., neomycin resistance or ampicillin resistance. Molecular Cloning

The process of transferring a specific DNA fragment from one organism into another organism via a vector and stably maintaining the foreign DNA molecule in the second organism.

Recomhinant DNA Molecule or Hyhriri DNA A molecule consisting of segments of non-contiguous DNA, which have been joined end-to-end. Expression Control Sequence

A sequence of nucleotides that controls and regulates expression of genes when operatively linked to those genes. Prohe

A DNA or RNA molecule, which is used to locate a complementary RNA or DNA by hybridizing to it. Often a probe is used to identify bacterial colonies or phage plaques that contain cloned genes and to detect specific nucleic acids following separation by gel electrophoresis. Promoter

The DNA sequence to which RNA polymerase specifically binds and at which it initiates RNA synthesis (transcription) of a specific gene. Antisense

A DNA or RNA molecule constructed in an orientation that is complementary to the protein or mRNA coding sequence of the actual gene.

Exogenous, Foreign or Alien DNA

DNA sequences or sequence motifs not naturally present in the host genome. Hypoxia

Reduction of oxygen supply to tissue below physiological levels despite adequate perfusion of the tissue by blood.

Glycolysis

The enzymatic conversion of glucose to the simpler compounds lactate or pyruvate. Breakdown of glucose under aerobic conditions forms pyruvate. Breakdown of glucose under anaerobic conditions forms lactic acid. Tumor

A neoplasm or a new or abnormal tissue growth which is uncontrolled and progressive.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the promoter and adjacent transcriptional regulatory regions for the tumor Type II hexokinase, which were isolated, sequenced, and subjected to reporter-gene analysis. Several features of the promoter are of special interest. The promoter contains response elements inter alia for insulin, glucose, cAMP, and TPA. In particular, insulin, glucose, glucagon, cAMP analogs, and the phorbol ester, TPA, activate the promoter. These findings suggest that signal transduction cascades involving tyrosine kinase, protein kinase A, and protein kinase C are involved in enhancing transcription of the Type II hexokinase gene in highly glycolytic tumor cells. This is in sharp contrast to the parent hepatocytes where both glycolysis and the level of Type II hexokinase isozyme are very low, and the expressed

Type IV isozyme, glucokinase, although transcriptionally activated by insulin, is inhibited by glucagon and cAMP. Thus, during or subsequent to, the hepatocyte — > hepatoma transformation, a genetic switch takes place in order to provide the hepatoma cells with a low Km (glucose) hexokinase that remains highly expressed regardless of metabolic state.

The invention relates particularly to a DNA fragment or regulatory region comprising a Type II hexokinase promoter and flanking sequences. Preferably, this promoter is derived from the genomic DNA of mammalian tumor cells, especially hepatoma cells. Optionally, the Type II hexokinase promoter is followed by all or part of the Type II hexokinase coding region naturally linked to the promoter. Particularly preferred as the first exon. Additionally, the DNA fragment may contain sequences which are required for efficient translation of mRNA. Also, mutant forms of the DNA fragment, which retain promoter or regulatory function, are encompassed by this invention.

A regulatable promoter is a promoter where the rate of RNA polymerase binding and/or initiation is modulated by external stimuli. Stimuli may include glucose, insulin, oxygen, light, heat stress and the like. Inducible, suppressible, and repressible promoters are some types of regulatable promoters. Based upon the characterization of the disclosed promoter and its regulatory sequences, the Type II hexokinase promoter is a regulatable promoter.

For ease of reference, the abbreviations used herein are as indicated in Table 2.

TABLE 2. Abbreviations

kbp kilobase pairs

IRE insulin response element

ATF/CRE cyclic AMP response element

Ap-1 activator protein- 1

C/EBP CCAAT-enhancer binding protein

GIRE glucose response element

HNF hepatocyte nuclear factor

EGF epidermal growth factor

TPA phorbol 12-myristate 13-acetate pfu plaque-forming units

EtBr ethidium bromide

PBS phosphate buffered saline

SSC sodium chloride-sodium citrate solution

Glu-6-P glucose-6-phosphate cDNA complementary deoxyribonucleic acid

DTT dithio threitol

NIDD non-insulin-dependent diabetes

Isolated DNA fragments according to the present invention are genomic DNA fragments which are no longer attached to the chromosome from which they derive. The fragments can be isolated from genomic DNA, or from genomic DNA libraries. or synthesized according to the sequences disclosed herein. The DNA fragments can be isolated from a variety of different mammalian species using known techniques for isolating homologous sequences. The response elements contained within the fragment are highly conserved among species. The response elements are numerous in the approximately 4.3 kbp upstream from the transcription start site of tumor Type II hexokinase. All or less than all of the response elements can be used. In addition to the RNA polymerase binding site, a number of different binding sites for transcription factors are found. These can be identified in any particular sequence by comparison to the known sites (sequences) for the transcription factors. The sites in the sequence of the fragment of SEQ ID NO:l have been found using a computer program for comparing sequences. Such programs can be applied to any particular sequence of a promoter and regulatory region of a mammalian tumor Type II hexokinase. Figure 11 lists the response elements and their locations using nucleotide coordinates. As shown below, many of these response elements observed by inspection of the sequence have been found to be functional. The response elements of the present invention regulate the transcription of a downstream open reading frame. This has been demonstrated using reporter genes, such as luciferase. Other reporter genes, i.e., genes which produce a readily observable or assayable product, can be used as is convenient. Many such genes are known in the art. Other open reading frames which can be covalently joined to the response elements of the present invention include genes encoding toxic products.

These include toxins as well as enzymes which convert innocuous compounds to toxic ones. Well known examples of such toxic genes are ricin and heφes simplex virus thymidine kinase. Any such gene can be used as is appropriate for the particular application. Covalent joining of the transcription regulatory elements of the present invention to open reading frames other than for hexokinase forms recombinant constructs which are termed gene fusions.

Gene fusions according to the present invention can be used in a variety of methods. Gene fusions having a reporter gene attached to a transcriptional regulatory region can be used to screen for potential drugs. Test substances which increase transcription of the reporter gene would be useful for treating diabetes. They could be used to increase the rate of glycolysis in the cells which would increase the absorption of glucose from the bloodstream. Conversely, test substances which decrease transcription of the reporter gene could be used to treat neoplasia, as up-regulation of glycolysis appears to be important for their unregulated growth. Test substances are contacted with the reporter gene fusions, either in an in vitro transcription setting or in an in vivo cellular situation. Transcription can be measured in the presence and absence of the test substance. Any suitable method among the many which are known in the art can be used for measuring transcription. Typical assays utilize labeled nucleotide substrates which are incorporated into transcripts and which can be quantitated. The assays can be performed in the presence of the particular stimuli which affect the transcriptional regulatory factors which bind to the transcription regulatory region of the present invention.

The gene fusions of the present invention can also be used for treating cells which overexpress hexokinase π. Many mmor cells have been found to have the high glycolytic rate phenotype. Thus a treatment method can take advantage of this by using a transcription regulatory region which is very active in the particular cells which one desires to treat, namely that of tumor type II hexokinase. According to one aspect of the invention, humans and mammals are treated when they have been determined to have mmor cells with an increased rate of glucose utilization over normal cells and/or the capacity to sustain high rates of glycolysis under aerobic (solution or physiological fluid saturated with dissolved oxygen at room temperamre (25 degrees Celsius)), hypoxic or under conditions known as hypoxia (low oxygen levels and/or a solution or physiological condition having less saturated conditions as compared to aerobic conditions but not reaching anaerobic conditions), or anerobic conditions (solution or physiological fluid having extremely low [near zero] oxygen levels but not hypoxic levels). Another benefit of using the response elements of the present invention is that they appear to be active in mmor cells but not in normal cells. Thus they provide a means of selectively expressing genes in tumor cells. While not wishing to be bound by any particular theory, one cause of the selective expression may be the presence of mutant forms of p53 in mmor cells. According to the method of the invention, a gene fusion, which comprises a toxic protein regulated by a transcription regulatory region of the present invention, is administered to cells which overexpress hexokinase II. The toxic gene is thus expressed at a high rate and selectively in the tumor cells. Suitable vectors for administering such fusion genes are known in the art and include without limitation, adenoviruses, retroviruses, herpes viruses, and minichromosomes. Delivery methods as are known in the art can be used, including liposomes, infection, inhalation, etc.

Nucleic acid probes of the present invention comprise at least 15 contiguous nucleotides selected from the sequence of SEQ ID NO: 1. Preferably they are selected from the regulatory region comprising nucleotides -4369 to -1. More preferably they are selected from nucleotides number -4369 to -1158. However, other sequences including those of the first exon may be included. Nucleic acid probes may or may not be labeled with a detectable label, including but not limited to a radiolabel, a fluor, and an enzyme. According to another aspect of the invention, the amplification of the hexokinase type II gene in tumor cells can be used as a diagnostic marker of neoplasia and as a prognostic marker of an aggressive type of mmor. Amplification is any gene copy number greater than 2, although copy numbers of greater than 4, 6, 8, and 10 are possible. Determination of copy number can be by any means known in the art, including fluorescence in situ hybridization, quantitative polymerase chain reactions, quantitative Southern blotting. A suitable control sample can be used from a somatic tissue which is observed to be morphologically normal.

Another diagnostic method of the present invention stems from the observation that the type II hexokinase gene is methylated and expression-silent in normal cells but unmethylated and heavily expressed in tumor cells. Thus by determining whether cells in a tissue sample contain an unmethylated or methylated hexokinase II gene one can ascertain neoplasia and/or aggressive mmor behavior. Any test known in the art for determining DNA methylation can be used. For example, restriction endonucleases like Dpnl which only cleave sequences containing methylated adenine or cytosine can be used to distinguish between methylated and unmethylated sequences. Methylation foot printing of the gene can also be used to determine the methylation status of the gene.

Vectors are provided which employ the transcription regulatory fragment of the present invention for expression of a desired protein in a mammalian cell. Because of the multiple means of regulating the mmor type II hexokinase, this system provides a very attractive tool for manipulation of expression levels and conditions. Vectors according to the invention include without limitation viral and plasmid vectors as are known in the art.

According to another aspect of the invention, humans and mammals are treated when they have been determined to have cells with a decreased level or rate of glucose utilization over normal cells and/or an incapacity to sustain high rates of glycolysis under aerobic conditions, such as in non-insulin-dependent diabetes. Even in cases where glucase utilization rates are normal treatment to increase the rate can benefit the patient by removing excess glucose from the blood. Glycolysis in cells is increased by transfecting them with an unmethylated copy of the mmor type II hexokinase gene.

Unmethylated copies can be obtained, inter alia, by passaging the DNA though a non- methylating host. The gene comprises both the regulatory region and the coding region of the hexokinase. By increasing the copy number of the gene, the rate of glycolysis increases, thereby increasing the amount of glucose which is absorbed by the cells from the bloodstream. Particularly suitable cells for such treatment include muscle, liver, and adipose cells.

The disclosed DNA sequence encoding the tumor Type II hexokinase promoter may be synthesized chemically or isolated by one of several approaches well known to one skilled in the art. For example, the complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete sequence (Edge, (1981), Nature, 292:756; Nambair, eLal., (1984), Science, 221: 1299; Jay, et al. , (1984), I Binl Chem _; 259:6311). Isolation methods may include nucleic acid hybridization using appropriate single stranded or double stranded DNA or oligonucleotide probes. Such probes can be constructed synthetically, based upon the DNA or amino acid sequences disclosed herein, or isolated from genomic or cDNA clones also described herein.

The preparation of oligonucleotides and DNA libraries, as well as screening by nucleic acid hybridization, are well known in the art (Sambrook, supra). Standard hybridizing conditions and procedures are known in the art (Southern, E., (1975), L Mol. Biol., 98:503); Sambrook, supra). If the nucleic acid is mRNA, it is contacted with a labeled Type II hexokinase gene probe complementary to the RNA under standard hybridizing procedures. The probe may be DNA, cDNA, or RNA depending upon the nucleic acid extracted and the method of hybridization chosen. The probe may be part of the sequence of the Type II hexokinase gene, including all coding and non-coding regions, a sequence including only the coding or non-coding regions, or any fragment(s) thereof. Preferably, the probe is mmor Type II hexokinase cDNA. The nucleic acid also may be amplified using PCR or RT-PCR prior to contact with the probe.

The construction of a DNA library is well known to the skilled artisan. The library can, for example, consist of a genomic library from a human source.

Preferably, the DNA libraries are constructed of chromosomal DNA. The genomic DNA or cDNA is cloned into a vector suitable for construction of a library.

Once the library is constructed, oligonucleotides or other DNA or RNA molecules may be used to probe the library to identify the segment carrying the sequence encoding the Type II hexokinase promoter in the case of a gene library.

Oligonucleotides can be designed and produced for use as hybridization probes to locate the sequences encoding the promoter. In general, the probes are synthesized chemically, preferably based upon known nucleic sequences, such as the 260 bp probe as shown in Figure 7. Ultimately, the isolated segments of DNA are ligated together so the correct sequence is constructed.

In designing probes, the nucleotide sequences can be selected as to correspond to codons encoding the amino acid sequence. Since the genetic code is redundant, degenerate probes include several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences, which encode a particular amino acid sequence. Thus, it is generally preferred, in selecting a region of the sequence upon which to base degenerate probes, that the region not contain amino acids whose codons are highly degenerate (Lathe, (1985), J. Mnl. Biol. , 181:1-12; Sambrook, supra).

The assembled sequence can be cloned into any suitable vector or replicon and maintained there in a composition which is substantially free of vectors that do not contain the assembled sequence. This provides a reservoir of the assembled sequence, and segments or the entire sequence can be isolated from the reservoir by excision with restriction enzymes or by polymerase chain reaction (PCR) amplification. The polymerase chain reaction is performed by methods and conditions disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195, Sambrook, surpa, and in Perkin Elmer Cetus PCR kit protocols. The DNA polymerase, deoxyribonucleotide triphosphates (dNTPS)

(e.g, dATP, dCTP, dTTP, and dGTP), and amplification buffer (e.g., glycerol, tris- hydrochloric acid, potassium chloride, Tween 20, and magnesium chloride) are commercially available (Perkin Elmer Cetus). The amplification process may be performed for as many cycles as desired. Numerous cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice (Sambrook, supra). The Type U hexokinase promoter and/or gene sense and antisense primers for use in PCR may be selected from primers described herein or others synthesized from the Type II hexokinase promoter and/ or gene. The primers may be produced using a commercially available oligonucleotide synthesizer, such as Applied Biosystems Model 392 DNA/RNA synthesizer. Either the sense or antisense primer may be labeled with a detectable marker by known procedures such as phosphorylation with bacteriophage T4 polynucleotide kinase (Sambrook, supra). Suitable markers include, but are not limited to, fluorescence, enzyme, or radiolabels such as ³²P and biotin. An expression vehicle may be any vector which is capable of transfecting mammalian cells and expressing a desired gene. The gene may encode a therapeutic agent for treating cells in need of such therapy either in vitro or in vivo. Suitable expression vehicles which may be employed include, but are not limited to, eukaryotic vectors, prokaryotic vectors, and viral vectors, such as adenovirus vectors, adeno- associated viral vectors, retroviral vectors (e.g., Moloney Murine Luekemia Virus, vectors derived from retrovriuses such as Rous Sarcoma Virus), herpes virus vectors, DNA-protein complexes, and receptor-mediated vectors. Any such vector may be contained within a liposome. Preferably, the vector of choice includes the mmor Type II hexokinase promoter. Other suitable promoters, however, that may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter, and the human cytomegalovirus (CMV) promoter (Miller, et al., (1989) Biotechniques, 7:980-990).

The construction of vectors containing desired DNA segments linked to appropriate DNA sequences is accomplished by techniques similar to those used to construct the segments. These vectors may be constructed to contain additional DNA segments, such as bacterial origins of replication to make shuttle vectors (for shuttling between prokaryotic hosts and mammalian hosts).

Procedures for construction of mutant sequences are well known in the art. A DNA sequence encoding a mutant form of Type II hexokinase promoter, for example, can be synthesized chemically or prepared from the wild-type sequence by several techniques, e.g. , primer extension, linker insertion, and PCR (Sambrook, supra).

Mutants can be prepared which have deletions, substitutions, and insertions relative to the wild-type sequence. Confirmation of specific mutant sequences can be conducted by sequence analysis and/or assays described herein.

Nucleic acids can be extracted from desired cells by known techniques. The nucleic acids may comprise DNA or RNA, preferably, genomic DNA or mRNA. For example, specific cells can be lysed using proteinase K in the presence of detergents, such as sodium dodecyl sulfate (SDS), NP40, or Tween 20. If the nucleic acid is genomic DNA, it is then extracted using known techniques, such as phenol/chloroform extraction, or other procedures (U.S. Patent Nos. 4,900,677 and 5,047,345). Alternatively, DNA can be isolated using one of the commercially available kits, such as Oncor Genomic DNA isolation kit. RNA can be extracted using various known procedures, such as guanidinium thiocyanate followed by centrifugation in cesium chloride (Sambrook, supra). Specifically, genomic DNA can be isolated from a cancer cell line known as hepatoma AS-30D. A method for culturing cells is described in Example 1 , this and other methods are well known in the art. The DNA can be restricted into smaller-sized fragments of 10 to 20 kbp using the restriction enzyme Sau3 AI. To generate a random population of such fragments for laboratory manipulations, the DNA fragments can be placed in viruses. For this purpose, the DNA fragments can be ligated to viral DNA isolated from the bacteriophage λ-Fix II (Stratagene Cloning Systems, La Jolla, CA). A sub-population of the genomic library cana be screened using a DNA probe synthesized using the Type II hexokinase cDNA of rat skeletal muscle (Thelen and Wilson (1991), Arch Rinrhem Rinphys , 286:645- 651), using plaque hybridization and detection (Sambrook, supra). Viral particles that are identified by this technique, as containing the DNA fragments that harbor DNA similar to the DNA probe, can be isolated. DNA within these viral particlesca be released by digestion with Proteinase K and purified by selective precipitation. To isolate and identify the DNA element described in this invention, the DNA can be digested with restriction enzyme Xba I, and the digested DNA fragments can be separated using agarose gel electrophoresis and detected by Southern hybridization, again using a PCR amplified 260 bp probe corresponding to part of the rat skeletal muscle hexokinase Type II cDNA. DNA sequencing of the identified fragments can serve to locate the promoter region, the hexokinase coding regions (exons) and, additionally, the restriction site(s) which may be useful in further processing, for example, for cutting off DNA sequences which are not necessary for promoter function. Depending on the choice of restriction enzyme, the DNA fragments containing the Type II hexokinase promoter and/or transcriptional regulatory regions may also include at the 3' and 5' termini original flanking DNA sequences which do not affect the promoter function and may be used as connecting sequences in the subsequent cloning procedures. If desired, these sequences can be ligated to chemically synthesized DNA linkers, which preferably include the recognition sequence of an appropriate restriction enzyme. This allows a convenient connection of the Type II hexokinase promoter and/or transcriptional regulatory region with foreign polypeptide coding regions. It is also possible to isolate and/or construct a DNA fragment which contains the Type II hexokinase promoter and part or all of the adjacent signal sequence from the Type U hexokinase protein coding region. When ligated to an appropriately cut foreign polypeptide coding region, the resulting hybrid DNA will be expressed in appropriate expression systems to yield desired polypeptides. The polypeptide coding region controlled by the promoter may be derived from genomic DNA or from cDNA prepared via the mRNA route or may be synthesized chemically. The isolated DNA element of 5150 base pairs was placed in a plasmid vector pUC18, for further laboratory manipulations. A vector is a replicon, such as a plasmid, phage or cosmid, into which another DNA segment may be attached so as to bring about the replication of the attached segment. For example, useful vectors may comprise segments of chromosomal DNA, non-chromosomal DNA, such as various known derivatives of SV40 and bacterial plasmids (e.g, plasmids from E. coli including pBR322, pBluescript

(Stratagene), pGEM (Promega), pUC118, pUC119, pUC18, and pUC19), or synthetic DNA sequences, phage DNAs (Ml 3) including derivatives of phage (e.g., NM 989), vectors useful in yeasts, vectors useful in eukaryotic cells, such as vectors useful in animal cells, (e.g., those containing SV40, adenovirus and retrovirus-derived DNA sequences), and vectors derived from combinations of plasmids and phage DNA (such as plasmids which have been modified to employ phage DNA), or other derivatives thereof.

The nucleotide stmcture of a 5150 bp DNA element comprising the mmor type II hexokinase promoter was determined by the Sanger method of dideoxy-mediated chain termination. Alternatively, the nucleic acid may be sequenced using the Maxam-

Gilbert chemical degradation of DNA method (Sambrook, supra), or other procedures known to those skilled in the art. The nucleotide sequence of the promoter region includes the disclosed sequence as disclosed in Figure 5 and conservative variations thereof. In a preferred embodiment, the nuleotides are -4369 to -1, or regulatory fragments contained therein. Regulatory fragments are defined as critical fragments which regulate expression of Type II hexokinase. Figure 6 shows the deduced amino acid sequence for the As-30D hepatoma Type II hexokinase.

The overproduction of hexokinase in cancer cells correlates with markedly elevated mRNA levels. (Johansson, eLal., (1985), Biochem. Biophys. Res. Commun. , 131:608-613; Paggi, eLal. , (1991), Rinrhem Rinphy Re Cnmmur, _J 1 8:648-655;

Shinohara, eLal., FERS Lett. , 291:55-57). Northern blot analysis was carried out on total RNA to determine the expression levels of Type I and Type II mRNA hexokinase in AS-30D hepatoma cells relative to their expression in normal rat liver (Figure 2). The level of Type II hexokinase mRNA may be determined by methods well known in the art, such as Northern blotting, dot and slot hybridization, SI nuclease assay, or ribonuclease protection assays (Sambrook, supra).

In Northern blotting, the RNA is separated by known techniques (Chirgwin, et al., (1979), Biochemistry, 15:5294) and transferred to an activated cellulose, nitrocellulose, or nylon membrane. The mRNA is then hybridized with a radiolabeled DNA or RNA probe followed by autoradiography. The probe may be the full length

Type II hexokinase gene or fragments thereof. Preferably, the probe is the full length Type II hexokinase cDNA. In dot and slot hybridization, the RNA is hybridized to an excess of a radiolabeled Type II hexokinase DNA or RNA probe (Kafatos, et al., (1979), Nucleic Acids Res , 7:1541; Thomas, R, (1980), Prn Natl Aca Sci . , 27:5201; White, eLal., (1982), J. Biol Chem , 252:8569). The amount of the Type

II hexokinase mRNA can then be determined by densitometric tracing of the audioradiograph and comparison to the amount of normal Type II hexokinase mRNA. In SI nuclease assay or ribonuclease protection assay, RNA is hybridized with labeled DNA or RNA probes derived from genomic DNA (Berk, eLal., (1977), Cell, 12:721; Casey, eLal, (1977), Nucleic Acids Res.

Transgenic organisms, such as transgenic mammals, transgenic mice, transgenic fish, etc., may be formed by introducing the nucleic acid molecule of the present invention into their genome, i.e. , a Type II hexokinase gene or any other gene regulated by the disclosed promoter or a mutated Type π hexokinase gene or promoter, or variations thereof. Preferably, the transgenic animal is a mouse. Methods for producing transgenic organisms containing a recombinant nucleic acid molecule are well known in the art (Alberts, et al. , (1989), Molecular Biology of the Cell, 2d. , Garland Publishing Inc., New York, pgs. 267-269; Gasser, eLal. , (1989), Science, 244: 1293-1299; European Patent Application No. 0257472, filed Aug. 13, 1987 by De La Pena, eLal. ; PCT Pub. No. WO 88/02405, filed Oct 1, 1987, by Trulson, eLal.;

PCT Pub. No. WO 87/00551, filed Jul. 16, 1986, by Verma; Wagner, et al., U.S. Patent No. 4,873,191; Rogers, eLal., (1987), Meth in F.n/ymol , 151:253-277; Cocking, et al., (1987), Science, 216:1259-1262; Burton, eLal., U.S. Patent No. 5,416,017). Methods and compositions for regulating the expression of foreign or alien

DNA in a host, such as a mammalian host, are described below. A powerful promoter, such as the tumor Type II hexokinase promoter, is also useful for high levels of protein production. In addition, the promoter, which is regulated by a number of transcription factors, may be manipulated to enhance and/or modify protein production. The compositions of the present invention, i.e., specific sense or antisense sequences, preferably, critical regulatory or response elements or sequences, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents. If appropriate, pharmaceutical compositions may be formulated into preparations including, but not limited to, solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, in the usual ways for their respective route of administration. Methods known in the art can be utilized to prevent release or absorption of the composition until it reaches the target organ or to ensure time- release of the composition. A pharmaceutically-acceptable form should be employed which does not inactivate the compositions of the present invention. In pharmaceutical dosage forms, the compositions may be used alone or in appropriate association, as well as in combination with, other pharmaceutically-active compounds. For example, in applying the method of the present invention for delivery of a nucleic comprising a Type II hexokinase promoter and/or gene-related elements, such delivery may be employed in conjunction with other means of treatment of cancer or diabetes, for example.

Accordingly, the pharmaceutical compositions of the present invention can be delivered via various routes and to various sites in an animal body to achieve a particular effect. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation, or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous intradermal, as well as topical administration.

The composition of the present invention can be provided in unit dosage form, wherein each dosage unit, e.g., a teaspoon, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other pharmaceutically-active agents. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically-acceptable diluent, carrier (e.g. , liquid canier such as a saline solution, a buffer solution, or other physiological aqueous solution), or vehicle, where appropriate. The specifications for the novel unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical compositon in the particular host.

Additionally, the present invention specifically provides a method of transferring nucleic acids to a host, which comprises administering the composition of the present invention using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for the particular application. The "effective amount" of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art. For example, one desired effect might comprise effective nucleic acid transfer to a host cell. Such transfer could be monitored in terms of a therapeutic effect, e.g., alleviation of some symptom associated with the disease being treated, or further evidence of the transfened gene or expression of the gene within the host, e.g, using PCR, Northern or Southern hybridization techniques, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody- mediated detection, or particularized assays, as described in the examples, to detect protein or polypeptide encoded by the transfened nucleic acid, or impacted level or function due to such transfer. These methods described are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan.

Furthermore, the amounts of each active agent included in the compositions employed in the examples described herein, i.e. , add range, provide general guidance of the range of each component to be utilized by the practitioner upon optimizing the method of the present invention for practice either in vitro or in vivo. Moreover, such ranges by no means preclude use of a higher or lower amount of a component, as might be wananted in a particular application. For example, the acmal dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, dmg disposition, and metabolism. Similarly, amounts may vary in vitro applications depending on the particular cell line utilized, e.g. , the ability of the plasmid employed for nucleic acid transfer to replicate in that cell line. Furthermore, the amount of nucleic acid to be added per cell or treatment will likely vary with the length and stability of the nucleic acid, as well as the namre of the sequence, and is particularly a parameter which needs to be determined empirically, and may be altered due to factors not inherent to the method of the present invention, e.g., the cost associated with synthesis. One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular simation. The following examples are to aid in the understanding of the invention, and should not be construed in any way as limiting its scope.

EXAMPLE 1 ; EXPRESSION OF HEXOKTNASE mRNA TN THE HIGHLY GLYCOLYTTC AS-30D HEPATOMA CELL LINE

Northern blot analysis was carried out on total RNA to determine the expression levels of Type I and Type II hexokinase in AS-30D hepatoma cells relative to their expression in normal rat liver (Figure 2). The level of Type II hexokinase mRNA may be determined by methods well known in the art, such as Northern blotting, dot and slot hybridization, SI nuclease assay, or ribonuclease protection assays (Sambrook, supra). In northern blotting, the RNA is separated by known techniques (Chirgwin, et al. , (1979), Biochemistry, 18:5294) and transfened to an activated cellulose, nitrocellulose, or nylon membrane. The mRNA is then hybridized with a radiolabeled DNA or RNA probe followed by autoradiography. The probe may be the full length Type II hexokinase promoter and/or gene or fragment thereof. Preferably, the probe is the full length Type II hexokinase cDNA. In dot and slot hybridization, the RNA is hybridized to an excess of a radiolabeled Type II hexokinase DNA or RNA probe (Kafatos, eLal., (1979), Nucleic Acids Res. , 2:1541; Thomas, R, (1980), Pro Natl Acad. Sci. , 22:5201; White, eLal., (1982), T Biol. Chem. , 257:8569). The amount of the Type II hexokinase mRNA can then be determined by densitometric tracing of the audioradiograph and compared to the amount of normal Type II hexokinase mRNA. In SI nuclease assay or ribonuclease protection assay, the RNA is hybridized with labeled DNA or RNA probes derived from genomic DNA (Berk, eLal. , (1977), Cell, 12:721; Casey, eLal, (1977), Nuclei Acids Res , 4:1539). The products of hybridization are then digested with nuclease SI or RNAase under conditions favoring digestion of single stranded nucleic acids. The amount of the Type II hexokinase fragments can then be measured by electrophoresis and compared to the size of normal Type II hexokinase mRNA fragments. In another embodiment, mutated Type II hexokinase promoter and/or gene may be determined using single stranded conformation polymorphism analysis (Orita, eLal., (1989), Proc. Natl. Acad. Sci. ,

86:2766-2770) and PCR.

Specifically, isolation of total RNA from hepatocytes, AS-30D cells, and skeletal muscle was performed by phenol-chloroform extraction (Chromczynski, eLal., (1987), Anal Rinrhem , 1£2 156-159). Total RNA (20 μg) was size fractionated on a 1.2% agarose formaldehyde gel (Sambrook, eLaL, (1989), In: Molecular Cloning:

A Ifihnrπtnry Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), transfened to a nylon membrane in alkaline conditions by downward capillary blotting (Chromczynski, P. , (1992), Anal . Biochem. , 2M: 134-139), and then UV- crosslinked. The gel was stained with ethidium bromide to verify equal RNA sample loading and transfer. An α-[³³]PdATP labeled probe conesponding to the full length

Type II cDNA hexokinase from skeletal muscle (Thelen and Wilson, (1991), Arch. Biochem. Biophys. , 286: 645-651) was used to detect the Type like mRNA in the blot. For Type I hexokinase, an EcoRI/BamH I fragment of the c37 mouse hepatoma Type I hexokinase cDNA (Arora, et al., (1990), . BM. Chem. , 265:6481-6488) was used as the probe (Figure 7), and for Type II hexokinase a full length cDNA rat skeletal muscle Type II hexokinase (Thelen, supra) was used. Both probes showed specific hybridization bands with rat brain and skeletal muscle RNA (Figures IA and IB) used as positive controls for Type I and Type II hexokinase, respectively. Hybridization bands were visualized by autoradiography on day 5 (-70 degrees Celsius). Both isozymes could be detected easily in AS-30D cells. Type II hexokinase, however, showed a much stronger hybridization signal as compared to the Type I isozyme. Neither hexokinase transcript could be detected in the normal rat liver.

AS-30D hepatoma cells were propagated in female Sprague-Dawley rats (Nakashima, supra; Arora, supra). The hepatoma cells, in ascitic form, were harvested

6 to 7 days post-transplantation. Hepatocytes were isolated from female Sprague- Dawley rats (200-250 g) by collagenase perfusion (Freshney, R., (1987), In: Ciilture of Animal Cells: A Manual nf Basic Technique, 2nd Ed., Wiley-Liss, New York, pgs. 264-265). After perfusing the liver, the hepatocytes were resuspended in 20 ml of RPM-1640 medium. An equal volume of 90% (v/v) Percoll solution (17 mM NaCl,

5.4 mM KCl, 81.3 mM MgSO4, 1 mM phosphate buffer, pH 7.4) (Berry eLal., (1991), I .ah Tech Binchem Mnl Bin! . , 21:24-25, 56-57) was added and mixed. The viable hepatocytes were separated by centrifugation (50 x g, 5 min.) and washed once in RPMI-1640 medium.

EXAMPLE 2: ISOLATION OF THE TITMOR TYPE TT HEXOKTNASE PROMOTER

To identify the cis-transcriptional control elements that regulate the expression of the mmor Type II hexokinase gene, a 5.1 kbp genomic clone containing the proximal promoter region and the first exon of the mmor Type II hexokinase was isolated and mapped. Genomic DNA was isolated from AS-30D hepatoma cells (Sambrook, supra). Partial digestion of the genomic DNA with Say-3AI to generate 10 to 20 kbp DNA fragments and partial fill-in of the Sau-3AI ends with Klenow fragment to create ends which are incompatible with each other, but are complimentary to Xho I partially filled ends (Promega Protocols and Applications Guide, (1991), 2nd

Ed. , Promega Corp. , Madison, WI). Isolation of λ-Fix II phage DNA by a liquid lysate method and modification of λ-Fix II phage DNA to generate Xho I half-site arms, and ligation of the half-site λ-arms to the partially filled-in AS-30D genomic DNA. The ligated DNA was packaged in vivn using Gigapack II Gold packaging extract (Stratagene). The recombinants were screened on duplicate nitrocellulose membranes (132 mm) (Schleicher and Schuell) at a density of 5 x IO⁴ pfu/plate. For the isolation, approximately 5 x 10^s plaques were screened from an unamplified AS- 30D genomic library packaged at an efficiciency of 6.6 x 10° pfu/ug. Six plaques, which hybridized to a 260 bp PCR, as shown in Figure 7, amplified probe corresponding to a part of the Type II skeletal muscle hexokinase first exon, were isolated as shown in Figure 2A. The recombinant phage, denoted 22-1, 22-2, 22-3, 25-1, 27-1, and 29-1, contained genomic DNA inserts in the size range of 10 kbp to 20 kbp. Recombinant λ-DNA prepared from each isolate was completely digested with Xba I. DNA fragments containing sequences conesponding to the first exon of skeletal muscle Type II hexokinase were identified by agarose gel electrophoresis, as shown in Figure 3 A, followed by Southern hybridization, Figure 4B, using the 260 bp PCR product as probe. The 260 bp PCR generated DNA fragment conesponding to the positions -197 to +63 of Type II hexokinase (translation start point referenced as + 1) (Thelen, supra) was [α-³²P]dATP radiolabeled by nick translation and used as the probe to screen the library. Specific Xba I digested DNA fragments, in the size range

0.75 kbp (page 22-10, 5 kbp (phage 22-2 and 29-1), and 9 kbp (phage 22-3, 25-1, and 27-1), were isolated from individual λ-clones and subcloned into the plasmid vector pUC18. A pUC18 subclone containing the approximately 5 kbp DNA insert from the λ-clone 29-1, was sequenced at the termini using pUC 18 universal primers to test for the presence of DNA similar to the Type II hexokinase first exon, as described below in Example 3, and selected for further characterization.

EXAMPLE 3; SEQUENCING AND ANALYSES OF THE TITMOR TYPE TT HEXOKINASE PROMOTER

The 5 kbp subclone, 29-1/Xba I, was sequenced in both directions as shown in Figure 3B to yield a full length DNA sequence of 5150 bp (Figure 5) containing the promoter, the first exon, and part of the first intron. Analysis of the putative first exon and comparison with the published sequences for the first exon of the Type II hexokinase from adipose tissue (Printz, eLaL, (1993), R Chem , 268:5209-5219) and skeletal muscle (Thelen, supra) indicated that the conesponding regions within the AS-30D tumor Type II hexokinase are very similar. The 5J kbp subclone contained a 257 bp segment of the first intron, a 63 bp coding region of the first exon, a 461 bp untranslated region of the first exon, and a 4369 bp proximal promoter region (Figure 5).

The promoter sequence was analyzed for response elements using available databases (Fasisst, eLal., (1992), Nucleic A rids Re . , 20:3-26; Locker, eLal. , (1993), In: Gene Transcription: A Practical Appraach (Hames, B.D., and Higgins, SJ. edsJ, Oxford U. Press, New York, NY. Numerous response elements found within the promoter by computer analysis are indicated in Figure 5 below the DNA sequence and in Figure 11. Response elements that are sensitive to two of the main signal transduction cascades, the protein kinase A and protein kinase C pathways, and to insulin, glucagon, and glucose are indicated in Figure 8. Many DNA direct repeats, ranging from 7 bp to 36 bp, were found within the promoter. Those which are longer than 10 bp are indicated. Another interesting motif found within the distal part of the promoter was a 31 bp 'T-G' pyrimidine-purine repeat (Figure 5).

In summary, numerous response elements or regions of potential relevance to the transcriptional regulation of the mmor Type II hexokinase gene were found, including those for well established regulators of carbohydrate metabolism. EXAMPLE 4: FUNCTIONAL ACTTVTTY OF THE TUMOR TYPE IT

HEXOKINASE PROMOTER TN THE PRESENCE OF KNOWN REGULATORS OF CARBOHYDRATE METABO TSM

The functional activity of the mmor Type II hexokinase promoter in the presence of potential modulators of greatest interest was examined. Using a reporter gene construct consisting of the mmor Type II hexokinase promoter and the luciferase gene, the relative activity of the promoter in driving transcription in the presence of glucose, insulin, glucagon, dibutyryl cAMP, and TPA was tested.

The 4.3 kbp promoter was placed in the pGL2-Basic reporter vector, shown in Figure 4C), which is designed to test a promoter's activity by using a luciferase as a reporter gene. The promoterless luciferase plasmid vector, pGL2-Basic, was used for all promoter studies. An additional promoter-probe vector, such as the promoterless chloramphenicol acetyl transferase (CAT), could be used to test the promoter activity of the Type II hexokinase promoter. An SV-40 promoter-β-galactosidase reporter vector (pSV-β -galactosidase) was used as an internal control for evaluating the efficiciency of transfection in each experiment. An SV-40 promoter-luciferase reporter vector (pGL2-Control) was used to evaluate the transcription strength of the mmor Type II hexokinase promoter. The xba I digested DNA fragment, which contained the proximal promoter region and the first exon of the AS-30D mmor Type II hexokinase gene, identified by DNA sequencing, was inserted into the compatible Nhe I site of the luciferase reporter plasmid pGL2-Basic, upstream of the luciferase cDNA. This construct was sequenced at the sites of ligation using synthetic oligonucleotides to verify orientation and accuracy of ligation. A part of the first exon, including the coding region of mmor Type II hexokinase, was excised from the reporter construct by Xho I digestion followed by religation. The mmor Type II hexokinase promoter- reporter construct (10 μg) was transfected with 2.5 μg of the pSV-β-galactosidase vector into AS-30D hepatoma cells using 25 x 10° cells in 0.5 ml per transfection. Hepatocytes were transfected with DNA using 20 x 10° in 0.5 ml per transfection. Briefly, the cells and plasmid DNA were incubated on ice for 10 minutes and electroporated at 200 volts, 800 μF. After 10 additional minutes on ice, the cells were plated into 10 ml of RPMI-160 glucose-deficient media (pH 7.4) supplemented with an antibiotic-antimycotic mixture, 25 mM Hepes, and 1 mM sodium pyruvate or 1 mM sodium lactate. Based on the transfection study, individual cell samples were further supplemented with 25 mM glucose, 100 mM bovine insulin, 10 μM glucagon, 100 μM dibutyryl cAMP, 100 nM TPA, or combinations thereof. The transfected cells were incubated at 37 degrees in 5 percent carbon dioxide. Cell extracts were prepared 24 hours post-transfection using cold lysis buffer (0.625% Triton X-l 00, 01. M potassium phosphate, and 1 mM DTT, pH 7.8) (Showe eLal., (1992), Nucleic Acids Res , 20:3153-3157). Luciferase activity in the cell lysates was measured as relative light units (RLU) using standard methods (de Wet, eLal., (1987), Mnl Cell Binl , 2:725- 737; Turner TD020e Luminometer (Turner Designs); Promega Luciferase Assay

System Kit).

AS-30D cells were chosen for the transient gene expression smdy, to ensure the presence of signal fransduction cascades and cell-surface receptors characteristic of the parental mmor line. Transient expression of luciferase derived from the promoter- reporter construct was determined after transfection of AS-30D cells, by assaying luciferase activity 24 hours post-infection. Luciferase activity was normalized to the β-galactosidase activity derived from the co-transfected internal control plasmid pSV-β- galactosidase to conect for differences in transfection efficiency. The fold activation of the promoter was based on the activity observed when the transfected cells were maintained in 1 mM pyruvate containing RPMI- 1640 medium (control). Under these

"background" conditions, and in the presence of 10% serum, the mmor Type II hexokinase promoter supported significant levels of transcription comparable to that of an SV 40 promoter (data not shown).

The relative activity of the mmor Type II hexokinase promoter in driving transcription in the presence of glucose, insulin, glucagon was then tested. Preliminary studies using a 1 mM lactate or ImM pyruvate substrate background indicated that glucose, insulin, and glucagon were capable of directing expression, where the levels of expression observed for each component were similar regardless of the lactate or pyruvate substrate background. Detailed studies using six independent experiments, canied out in a substrate background of 1 mM pyruvate, as shown in Figure 9. The highest activation of the promoter was observed in the presence of both insulin (100 nM) and glucose (25 mM), with a 4.3 fold increase in activity. Separately, glucose and insulin gave activation levels of 3.4 and 2.4 fold, respectively. Glucagon alone caused a moderate but reproducible activation (1.3 fold) for promoter activity, which increased to 2.4 fold in the presence of glucose. Insulin and glucagon together activated the promoter by 2.8 fold which was 0.4 fold above the transcription enhancement observed in the presence of insulin alone.

The effect of analogs that activate two of the major signal transduction pathways, namely the protein kinase A and protein kinase C signaling cascades, on the mmor Type II hexokinase promoter was tested using dibutyryl cAMP (100 μM), and analog of cAMP, and TPA (100 nM), an analog of diacylglycerol, respectively. These analogs increased promoter activity by 2J fold and 3.3 fold, respectively. These findings emphasize the promiscuity of the mmor Type II hexokinase promoter in its activation response to a wide variety of known modulators of carbohydrate metabolism. Thus, in hepatocytes, where insulin, glucose, and glucagon are all known to regulate the expression of Type IV hexokinase, these same agents produced little or no effect on the activity of the transfected Type II mmor hexokinase promoter. These results implicate the presence of one or more unique transcription factors essential for the activation of the Type II hexokinase promoter, and therefore, the overexpression of the Type II enzyme in hepatoma cells. The significance of these findings emphasize that normal versus tumor cell differences in the regulation of hexokinase genes involved in glucose catabolism, and indicate that transcription of the Type II mmor gene may occur independent of metabolic state. Thereby providing the cancer cell with a selective advantage over its cell of origin in the production of the key metabolic precursor Glu- 6-P. EXAMPLE S. RELATIVE ACTTVITY OF TUMOR TYPE II HEXOKINASE

PROMOTER TN HEPATOCYTES AND TN A .30D HEPATOMA CELLS

The reporter vector was transfected into hepatocytes in order to test whether the tumor Type II hexokinase promoter was capable of driving transcription in the tumor's parent cell line. The expression was evaluated for glucose, insulin, or glucagon in a substrate background of 1 mM lactate. Parallel experiments were canied out in AS- 30D hepatoma cells. In contrast to the highly modulated promoter activities observed in AS-30D cells for glucose, insulin, and glucagon, the promoter showed no significant modulations in activity when placed within hepatocytes (Figure 10) and tested with the same modulators. In hepatocytes and in AS-30D cells, the basal activity of the promoter (in 1 mM lactate), as measured by relative light units for the reporter gene, were comparable in magnimde. These results implicate the presence in AS-30D hepatoma cells of one or more transcription factors essential for the expression of the Type II hexokinase gene that are absent in the parental cell line of origin. EXAMPLE 6: RESPONSE ELEMENTS

Analysis of the 4.3 kbp proximal promoter region of the AS-30D mmor Type II hexokinase revealed both a putative TATA box (AATAA, -30) (Breathnach, eLaL, (1981), Ann Rev Rinrhem , 50:349-383) and a CAAT box (-85), indicating the precise positioning of transcription initiation for the mmor Type II hexokinase mRNA transcript. This is in contrast to the staggered transcription initiation, and the lack of either a TATA box or a CAAT box, observed for liver glucokinase (Magnuson, eLal. , (1989), Prnc Natl Aca Sci USA, 86:4838-4832), the principal expressed hexokinase isoform in normal liver cells. Interestingly, response elements for glucose, insulin, glucagon, cAMP, and the phorbol ester TPA were identified for the Type II mmor hexokinase promoter.

Putative consensus sites for Ap-2 (GGCAGCCC, -41), a factor inducible by both protein kinase A and protein kinase C pathways (Faisst, supra; Locker, supra), and for ATF-1 (CCACGTC, -70), which is specifically induced by the protein kinase A pathway, were located immediately upstream of the transcription start site. Since both these sites are located in close proximity to the TATA element and the CAAT element, further studies will indicate their importance in transcription enhancement.

In addition, Ap-2 sites were the most common and ubiquitous elements within the 4.3 kbp promoter (-3850, -2040, -1965, -1500, -1260, -1110, -665, -315). Six putative Ap-1 consensus sites (-3469, -2735, -2320, -1955, -1590, -860) for the complex fos- jun, which is a primary nuclear transducer of the protein kinase C cascade, could be found throughout the 4.3 kbp promoter.

Of the known liver-enriched transcription factors HNF-1, HNF-3, HNF-4, and c/ebp (Lai and Darnell, (1991), TIBS, 16:427-430; Lemaigre and Rousseau, (1994), Biochem. J., pgs. 1-14), putative consensus sites could be found for only c/ebp (-4150, -3725, -2550, -1440, -1060, -660, -620, -260). However, several putative sites for the factor HNF-5 (Grange, eLaL, (1991), Nucleic Add. Res , 19:131-139), which usually binds at sites in close proximity to the response elements for the above-mentioned liver- specific factors, could be found distributed within the promoter (-4160, -3915, -3330, -2200). Therefore, the mmor Type II hexokinase promoter may contain additional consensus sites for the hepatic nuclear factors, or for their oncogenic variants, such as vHNF-1, which replaces HNF-1 in de-differentiated cells (Faisst, supra; Locker, supra). HIF-1 protein (hypoxia-inducible factor) was recently discovered (Semenza, eLal., (1995), J. Binl Chem , 269:23757-23763; Wang and Semenza, (1995), .1, Biol. CherrL, 220: 1230-1237). A response element for HIF-1 was found. As a result, low oxygen concentrations are likely to result also in an activation response via the HIF-1 protein (hypoxia-inducible factor). HIF-1 and glucose response elements are essentially identical. As a result, signal transduction pathways involving glucose, tyrosine kinase, protein kinase A, and protein kinase C are implicated in the transcriptional regulation of mmor Type II hexokinase. Consensus sites for such factors remain to be elucidated by DNA footprinting analysis of the Tumor Type II hexokinase promoter.

Regarding known ubiquitous factors that regulate expression of genes coding for glycolytic and gluconeogenic enzymes (Lemaigre, supra), namely sites for 'CCAAT-box' binding factors, Ocatmer factor, Sp-1, CREB/ATF, Ap-1, b-HLH, and nuclear hormone receptors all of which enhance transcription, putative sites could be found for Sp-1 (4 sites), CREB/ATF (2 sites), Ap-1 (5 sites), and for steroids (SRE)

(5 sites). Also found within the promoter was one putative site (-2955) for factor PPAR, a member of the steroid hormone receptor superfamily, that is thought to play a role in tumor development in liver and in triglyceride and cholesterol homeostasis (Issemann and Green, (1990), Namre, 142:645-650). Three response elements for the factor p53, a well-known tumor suppessor gene product, were identified in the proximal and distal region of the promoter (-4240, -4195, -1610).

Sites for Pea-3, a factor inducible by TPA, EGF, and the oncoproteins v-src, v-mox, v-raf, and c-Ha-ras (Gutman and Wasylyk, (1990), EMBO, 9:2241-2246) were identified within the distal (4 sites, -3965, -3645, -3625, -3255) and proximal (2 sites,

-1415, -1370) regions of the promoter. Within the 4.3 kbp promoter, four Sp-1 binding sites were identified (-3290, -2220, -1110, -55). Since DNA bound Sp-1 factors self-associate, these sites, placed approximately at 1000 bp intervals within the promoter, may bring together the distal promoter segments for enhancement of transcription. Between base pairs of -3811 and -3841, a 31 bp 'GT' repeat was located. This motif, located in the distal region of the mmor Type II hexokinase promoter, is also found within the proximal promoter region of rat pancreatic beta-cell glucokinase (a 33 bp tract) (Magnuson and Shelton, (1989), T Biol Chem , 264: 15936-15942), as well as within a human glucokinase gene associated satellite repeat DNA sequence (a 31 bp tract) (Tanizawa, et al., (1992), Mnl. F.ndncrinnl . ,

6: 1070-1081). Such repetitive purine-pyrimidine DNA segments have potential to form Z-DNA strucmres, and induce changes in the helicity of adjoining B-DNA.

EXAMPLE 7: INTRODUCTION OF AN ANTTSENSF MOLECULE TO REDUCE HEXOKTNASE ACTIVITY:

It may be desirable to inhibit the expression of the Type II hexokinase promoter and/or gene because of its association with cancer. Various approaches may be taken to reduce the hexokinase activity in mmor cells. One approach involves the introduction of an RNA molecule (antisense polynucleotide). Antisense technology involves the juxtaposition of the targeted gene in a reverse orientation behind a suitable promoter, such that an antisense RNA molecule is produced. This antisense construct is then transfected into the engineered cell and, upon its expression, the engineered cell produces a RNA molecule that will bind to, and prevent the processing/ translation of RNA produced by the targeted gene, in this case the hexokinase Type II gene. In order to generate antisense molecules with exact sequence identity to the homologue of hexokinase JJ being expressed by the mmor cells, the hexokinase variant present in the AS-30D hepatoma cell line will be converted to cDNA by reverse transcribing the mRNA and amplification of the DNA product (Hushes, eLal., (1991), J. Biol. Chem., 266:4521-4530). The oligonucleotides used for amplification will be based upon the published sequence of the rat skeletal muscle hexokinase II (Thelen, supra). The oligonucleotides include restriction enzyme recognition sequences at their 5' ends to facilitate directional cloning of the amplified cDNA into the selected vector in an antisense orientation. Because the vector contains both the transcription termination and polyadenylation signal sequences downstream of the cloning cassette, processing of the antisense transcripts should proceed normally. An antisense molecule can be made synthetically in reverse orientation to the sequence provided in Figure 5 or variations of such specific response elements of the sequence. Alternatively, expression constructs may be used which comprise a promoter operably linked to at least 20 nucleotides of the antisense strand of Type II hexokinase cDNA. The expression construct directs the synthesis in a cell of a RNA molecule which is complementary to Type II hexokinase mRNA.

EXAMPLE ϋ: DRUG SCRFFNTN

The described sequences or variations thereof can be used to screen substances for potential therapeutic agents for disease states, such as cancer and NIDD. A substance which decreases the activity of the Type II hexokinase promoter or production of the gene, is a potential therapeutic agent for cancer treatment. One which increases activity is a potential drug for treating diabetes. Means used to determine amounts of activity are well known in the art, including, but not limited to, radioactive components, antibodies, etc. For example, use of Type II hexokinase promoter elements driving expression of any reporter gene permits identification of pharmacologic agents capable of depressing or increasing the function of the promoter. Detection of loss of expression of Type II hexokinase or any marker gene by in situ hybridization (Baldino, eLal., (1989), Methods in Enzymol . , 168:761-777; Emson, et al. , (1989), Methods in F.nzymnl , 168:753-61; Harper, eLal., (1987), Methods in

EnzyxnoL, 151:539-551; Angerer, eLal., (1987), Methnds in F.nzymnl. , 152:649-661; Wilcox, eLal., (1986), Methnds in F.nzymnl , 124:510-533) experiments can identify agents which have the potential to supress the function of the Type II hexokinase promoter. One prefened method for detecting mRNA associated with expression of the cross-reactive protein is in situ hybridization to tissue sections, preferably from mmors.

EXAMPLE 9: THFRAPEUTTC

The present invention includes methods to treat humans and animals determined to have cells which have an increased rate of glucose utilization over normal cells and/or the capacity to sustain high rates of glycolysis under aerobic (solution or physiological fluid samrated with dissolved oxygen at room temperamre (25 degrees Celsius)), hypoxic or under conditions known as hypoxia (low oxygen levels and/or a solution or physiological condition having less samrated conditions as compared to aerobic conditions but not reachning anerobic conditions), or anerobic conditions

(solution or physiological fluid having extremely low [near zero] oxygen levels but not hypoxic levels), such as cancer cells. Gene therapy for cancer involves use of hexokinase II transcriptional regulatory regions to drive expression of a toxic gene in mmor cells to kill the mmor cells or inhibit their growth. Another method of treatment included in the invention is to treat animals determined to have a decreased level or rate of glucose utilization over normal cells and/or an incapacity to sustain high rates of glycolysis under aerobic conditions, such as NIDD. In addition, even normal rates of glucose utilization can be elevated according to the invention in order to lower blood glucose levels. Individuals who have NIDD may have a defect (mutation) in the insulin receptor, the glucose transporter, or the Type II hexokinase. Gene therapy for NIDD involves introducing to the cell the Type II hexokinase gene with its promoter in an unmethylated form so that it will be expressed.

EXAMPLE 10: HYPOXTA TNDUCIBTLTTY

Within the distal region of the regulatory region of mmor hexokinase II, a potential hypoxia inducible factor (HIF-1) binding motif (CACGTGCT) is present at nucleotides -3765 to -3758. To determine whether the promoter was responsive to hypoxic conditions, we used a promoter-luciferase reporter gene construct, in transient transfection experiments where the mmor cells were maintained in an environment of

1 % oxygen. Reporter gene analysis done 24 hrs post-transfection, indicated activation of the type II hexokinase promoter by hypoxia. A 4- to 7-fold activation of the promoter was observed under different substrate backgrounds of pyruvate (1 mM to 10 mM) and glucose (5 mM to 25 mM). This study suggests the involvement of oxygen partial pressure as another step in regulating the transcriptional control of the Type II hexokinase gene, in controlling the glycolytic flux of cancer cells at the first and committed step in glucose catabolism. This is consistent with the observations that most malignant mmor cells survive and proliferate in an oxygen depleted, hypoxic environment. In addition, it is consistentrapidly growing, highly malignant tumor cells, a high rate of glycolysis is maintained inespective of the in vivo oxygen stress.

EXAMPLE 11. RELATIONSHIP BETWEEN THE TYPE TT HEXOKINASE GENE AND PS3 PROTETN OF TTTMOR CELLS

In order to elucidate the basis for high expression of the p53 protein in the experimental rat hepatoma cell line AS-30D, preliminary studies were completed in cloning, and sequencing the p53 cDNA. The p53 protein under smdy was found to be mutated at two positions: 103 (Gly-Ser) and 256 (Glu-Gly). "In vivo" overexpression of the p53 protein was then canied out in AS-30D mmor cells co¬ transfected with the Type II hexokinase promoter-luciferase reporter gene construct. The results show that the promoter for the Type II hexokinase gene is positively regulated by the p53 protein. This indicates also that the p53 elements identified on the 4.3 kbp proximal promoter of the Type II hexokinase gene by computer analysis are in fact functionally active.

The cloned p53 cDNA was co-expressed with the Type II hexokinase promoter- luciferase reporter gene construct. A cytomegaloviral (CMV) promoter- vector was used to drive expression of cloned p53 in the tumor cells. Transfected cells were maintained in RPMI-1640 medium (serum-less) containing 1 mM pyruvate. The cells were lysed 20 hrs post-transfection, and luciferase activity measured. The CMV vector lacking a cDNA insert was used in parallel co-transfection experiments as a negative control. As a separate control for the p53 response elements on the hexokinase promoter, a 108 bp minimal promoter construct of the hexokinase gene (denoted Mut C) which contained the TATA element, an ATF and an AP2 element, and the CAAT element, was used. When experiments were repeated in the presence of serum, the induction levels observed for the full length hexokinase promoter were of similar magnimde to that observed under serum-starved conditions, demonstrating the high expression of p53 via the CMV promoter in co-expressed cells. The activation seen for the Mut C construct in the presence of serum is possibly due to activation of the AP2 and ATF elements by the PKA and PKC pathways upon serum induction.

EXAMPLE 12: AMPLIFICATION OF THE GENE ENCODING TYPE TI

HEXOKINASE TN CANCER CELT .S

We have demonstrated by Southern blot analysis and fluorescence in situ hybridization (FISH) that in the rapidly growing rat AS-30D hepatoma cell line, enhanced hexokinase activity is associated with at least a 5-fold amplification of the type II gene relative to normal hepatocytes. The amplified genes are located chromosomally, comprise the whole gene, and most likely are at the site of the resident gene. No ranangement of the gene could be detected. Therefore, overexpression of hexokinase type II in these cells is based, at least in part, on a stable gene amplification. Cells and Cell Culture. Clone 9 (CRL 1439), a rat hepatocyte cell line, was obtained from the American Type Culmre Collection and grown in RPMI 1640 mediun. AS-30D hepatoma cells were grown in the peritoneal cavity of female Sprague-Dawley rats, harvested and purified as described previously. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion.

Hexokinase Assay. Hexokinase activity was determined spectrophotometrically on whole cell lysates using a glucose 6-phosphate dehydrogenase coupled assay. Activity is expressed as milliunits (mU) defined as the formation of one nmol NADPH per min. Southern-blot analysis. High molecular weight DNA was isolated from AS-30D hepatoma cells and hepatocytes as described. DNA (30 μg) was digested with the indicated restriction enzymes. To avoid technical problems resulting from incomplete hydrolysis, digestions were repeated several times with an excess of restriction enzymes. The digested DNA was fractionated on a 1 % agarose gel and transfened to nylon membranes (Amersham). Probe labelling, hybridization, and detection were performed with the Fluorescein Gene Images System (Amersham) according to the manufacmrer's instructions. Either the full-length cDNA or a 260 bp fragment conesponding to the position -197 to +63 of rat skeletal muscle hexokinase Type II were used as probes. Fluorescence in situ hybridization. The pUC18 plasmid containing the 3.6 kb cDNA clone of the rat hexokinase (HKII) gene was nick-translated with biotin-14 dATP

(BRL, Gaithersburg, MD), with 25% incorporation as determined by tritium tracer incoφoration. Slides with chromosome spreads were made from AS-30D hepatoma cells and clone 9 (normal control), harvested by standard cytogenetic techniques. Fluorescence in situ hybridization was performed as described with modifications. Probe mix (2X SSCP, 50% formamide, 10% dextran sulfate, 5 ng/(μl biotinylated probe, and 20 (μg/μl salmon sperm DNA) was denatured at 70 °C for 5 min, quickly chilled on ice, placed on slides and hybridized at 37°C overnight. Slides were washed in 50% formamide/2XSSC at 43 °C for 20 minutes, and 2 changes of 2XSSC at 37°C for 5 min each. Biotinylated probe was detected with FITC-avidin and amplified with biotinylated anti-avidin, using reagents from "in situ hybridization kit" (Oncor Inc.,

Gaithersburg, MD), following manufacturer's instructions.

Preliminary Southern-blot analysis using digested genomic DNA from hepatocytes and AS-30D hepatoma cells revealed that the hexokinase Type II probe hybridized with much greater intensity to the hepatoma DNA than to the hepatocyte DNA. To estimate the differences in hybridization intensities we performed a dilution experiment. The hybridization signals with different amounts of EcoR I/Xba I digested AS-30D hepatoma genomic DNA were compared to the signal obtained with 30 μg of DNA isolated from hepatocytes (Fig. 1). The blots were probed with two different probes specific for the hexokinase Type II gene (Fig. IA and IB). The intensities of the resulting bands indicate that 3-6 μg hepatoma DNA were equivalent to 30 μg hepatocyte DNA. From this experiment we estimated that AS-30D hepatoma cells contain approximately 5-10 fold more copies of the hexokinase Type II gene than normal hepatocytes. In addition it is clear from Fig. IA that the signal intensities of all Type II hexokinase related bands obtained with AS-30D hepatoma DNA are the same. This indicates that the amplification extends to the whole coding region of the hexokinase gene. Moreover, when the membranes were probed again with DNA fragments specific for the 5' -flanking region of the hexokinase gene similar results were obtained (data not shown). Thus, the amplified unit in AS-30D hepatoma cells also includes the promotor region of the hexokinase Type II gene. Densitometric quantification of autoradiograms made from different Southern-blots confirmed the data obtained in the dilution experiment, and a factor of approximately 5 was calculated for the amplification. Additional support for the hexokinase Type II gene amplification in AS-30D hepatoma cells came from experiments searching for the hexokinase Type II promotor region in these cells and in hepatocytes. Thus, 6 positive plaques were obtained when 5 x 105 plaques were screened from an AS-30D hepatoma genomic library, whereas only 2 positives were found in 2.5 x 106 plaques of a normal liver library. Taking into consideration that the liver library had been prior amplified the estimated factor for amplification is near 6, in accordance with results from Southern-blot analysis. Instability of the genome is a well-known phenomenon of transformed cells and amplification is a frequently observed mechanism for the overexpression of oncogenes including N-myc and the epidermal growth factor receptor gene. It is well known that a strong relationship frequently exists between a gene that is amplified and cell growth. The amplification of the hexokinase Type II gene is consistent with this relationship as the role of this critical metabolic enzyme is to provide cells with both energy and precursors for nucleotide and lipid biosynthesis. In a recent report, we provided evidence that increased expression of one or more transcription factors is involved in the elevated production of hexokinase Type II in AS-30D hepatoma cells. Work presented here suggests that amplification of the gene for the same enzyme may play a role as well.

Southern-blot analysis displayed some faint restriction fragments with the hepatocyte DNA which were not observed in the AS-30D hepatoma DNA. As the restriction enzymes used, EcoR I and Xba I, are both sensitive to methylation of their recognition sequence, this raises the possibility that methylation differences exist within the hexokinase Type II gene in normal hepatocytes and AS-30D hepatoma cells.

Several studies reviewed in have demonstrated that DNA methylation plays a role in gene regulation. Therefore, methylation could be involved in differential expression of hexokinase Type II in normal and mmor cells. Further experiments to test this hypothesis are in progress. For some oncogenes it is well known that amplification is accompanied by recombination and rearrangement of the gene locus. To look for structural differences in the hexokinase Type II gene locus in normal and AS-30D hepatoma cells restriction fragment length polymorphism (RFLP) analysis was canied out. To circumvent problems due to methylation differences of normal and mmor DNA, methylation insensitive restriction enzymes (Rsa I, Nde I, Hinc III) were used. For each enzyme the same restriction fragment pattern is observed in both hepatocyte and AS-30D hepatoma DNA. Thus, no macroscopic reanangement of the hexokinase gene is seen at this level of resolution. Also, this result renders it unlikely that a translocation of the hexokinase gene locus has occured in AS-30D hepatoma cells. Therefore, the amplification described above appears to occur at the site of the resident gene, and the possibility that the hexokinase Type II gene in AS-30D hepatoma cells has come under the control of different regulatory sequences through translocation seems remote.

To obtain additional support for the amplification and localization of the hexokinase Type II gene, in situ hybridization experiments were performed. Because primary hepatocytes divide very rarely and rapidly dedifferentiate, we used clone 9

(CRL 1439), a non-tiimorigenic, normal liver cell line as a control for in situ hybridization. These cells exhibit no detectable hexokinase activity in contrast to AS-30D hepatoma cells where the activity is 762 mU/mg. The liver homogenate, which in addition to hepatocytes contains other cell types, exhibits a low but detectable hexokinase activity. In situ hybridizations using the hexokinase Type II cDNA as probe revealed that in AS-30D hepatoma cells a signal could be readily detected in every metaphase (20/20) and interphase cell. Occasional (4/20) tetraploid cells which were observed in the AS-30D hepatoma cell population showed a hybridization signal on two chromosomes indicating that the gene was amplified before the chromosomes were duplicated. The single positive chromosome seen in the AS-30D sample most likely represents the amplification site on one chromosome homolog only, but the loss of the other homologous chromosome cannot be ruled out. In contrast, in clone 9, no interphase signals were seen and only 1 of 20 metaphase cells showed a faint specific signal. Because the probe used for in situ experiments was rather small (3.6 kbp), genes with a low copy number cannot be easily detected with this size probe. This confirms again that the copy number of the hexokinase Type II gene is much more abundant in AS-30D hepatoma cells than in control cells. Although FISH does not allow exact quantitation of the amplification, it is consistent with at least a 5-fold increase in copy number. Moreover, the amplified sequence was localized to a single chromosome in AS-30D hepatoma cells suggesting that the amplification is present on only one of the two homologous chromosomes, a finding not uncommon for amplified genes (20). Chromosomally localized gene amplification represents one of the more stable forms of amplified genes. Stable retention of amplified genes and their passage to daughter progeny are ensured only when such genes are integrated within a chromosome. Unstable amplified genes which are very common in transformed cells are characteristically associated with extrachromosomal elements called double minutes. However, double minutes were never observed in our studies of AS-30 hepatoma cells. In summary, results reported here provide for the first time evidence that a hexokinase gene (Type II) is amplified in a mmor cell line exhibiting a high glucose catabolic phenotype. This amplification is stable, not associated with a rearrangement of the hexokinase gene locus, and probably occurs at the site of the resident gene.

The invention has been described with reference to the presently prefened embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. All references and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: PEDERSEN, PETER L. MATHUPALA, SAROJ P.

REMPEL, ANNETTE

(ii) TITLE OF INVENTION: TUMOR TYPEII HEXOKINASE TRANSCRIPTION REGULATORY REGIONS

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: BANNER & ALLEGRETTI, LTD. (B) STREET: 1001 G STREET, ELEVENTH FLOOR

(C) CITY: WASHINGTON DC

(E) COUNTRY: USA

(F) ZIP: 20001

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/001,199

(B) FILING DATE: 14-JUL-1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: KAGAN, SARAH A.

(B) REGISTRATION NUMBER: 32,141

(C) REFERENCE/DOCKET NUMBER: 1107.57886

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-508-9100

(B) TELEFAX: 202-508-9299

(2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5150 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus

( i) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TCTAGAGCTC GCGGCCGCGA GCTCTAATAC GACTCACTAT AGGGCGTCGA CTCGATCCAA 60

CTGGCCTAGA ACTCACAGCC ATCCTCTTGC CTCTACCTAT GGAGTGTGGA GATTAAAGGC 120

ATGTTCTACC ATGTCTTAAT TTTAAAATAC CTATGGAGTG TGGAGATTAA AGGCATGTTC 180

TACCATGTCT TAATTTTAAA ATAGGAATAT TTGTGGATTG AGGTCTTGAG CAAAATAAGA 240

TTTTTCCCAA GAGAGTTTCC TGAAGCCTAA GTAGACTCAG GTCCTTCTCA TGCAGGGCCA 300

ATCTAGGGCC AGGAGCAGGA CCAACTGGTG TGAAATCAGA AAGATGGTAC TCATAGCTAT 360

TAGTCCATCT CTGGTTGACA CTCCCAGACT CCCCTACATC TCAAGACACA GACATACGTG 420

GCTTTTTATG AATCCATTTT TCTGGTCTGT ATTATTTGTT CTGTGTGTTA ATTTTATGTC 480

TTAAACTAAA CAGAAATCCT TTAAGGAAAG AACACCCCGC CCCTCTCCTG TGTGTGTGTG 540

TGTGTGTGTG TGTGTGTGTA CACGTCTGTG TGAGTATCTC GCACCCTGTA AAGGGCTTAA 600

TAAACACGTG CTGATTGATT CCCCTCCTGG AATGTGAATG TGGACTGCCA ATCTGCCAGT 660

CTACAATGTG TGTGCCTGTA TGTGCTCATG GGGAGAGAGA GGGAGAAATA AAATAGACTC 720 TAAGGAAGAA TCTTGAGGAC AGGAAAGTCA GAGCTACACA CCTCACTTTT GAGTGGGTAG 780

CTGTCCCCTG ATTTGACACA TACAGATGGG TTAGGGGATA TCACTGTACT CACTCCAGCC 840

ACCTCCCAGG GTTACTGGGA ACTCTGTGAG AGATCATCCC ATAAAGTACC CTGTGAACAT 900

GAGTTAGTCC TCATAAAGTG GGACCAGAAA AGAGAATGGA GAATGGAGCT GAAGTGTGTG 960

TGCAAGTAAG TGTGTGTGAG ATCCAGCTAA TTGGACTCAG CTGATGGAGT GCTTGCCTAG 1020

CACGCATGAA TCCTCATGTT TGCCTCTGAT CGCAAGACCT GAAAAAAAAA AAAAATAGGC 1080

GAGGTAGACA GTGCCTGTAA CCTCAGCGCT GAGGAAGTGG AGGCCGGAGG ATGGGAAGCT 1140

CAAGACTGTC CTTGGTTGCA TGTTTAGTTA GAGGCCATCT TGGGCCACAT GATCCTGTCC 1200

CAAAATAAAC AAAGGAATAC AATTAGTCCA TAGGGAGGAG ATCATAGTTG ACCTGACCCC 1260

ACTGATTTTG ATCTTAGTTG TCTGAGGGAA ATTATTTTAT ATACTGATTT AACTATCGGT 1320

TTTTTAAGTG TCTCAAAATG TTTTTATTTC ATGTACACCC TTATTGGGTG TGCATATGCA 1380

TGTAGGTACA CACATGCCAT GGTAAGTGTG TAAAGGTCAG AAGTCAATTT TCTTGAGTTG 1440

ATTCTCTCCT GTGACCACAT GGGTCCTAGG GTTCACCTCA AGTAGTCGGG GTTCAGCGAC 1500

AAGCGCCTTT ACCCACCGAG CCACCTTGCC AGCACCCGAA GTGTTTCCAG AAAGGTCTTT 1560

TTTTTTTTCT CTTTGCTGCT TACTTTTAAC CTATGCCATC AATTCTGCCT CAGACTTCTG 1620

AACACCTAAA GCCTTAATCA GCCTCTGTGC CTCACCCTTG TCTCACTCCA GCCTTTATCT 1680

TATCTGGGAG TTCCTGTCTC TTCTCCTTCA GGCCGGGTCC TTTCCTCCCA TTCATGTGGA 1740

GAGCAGCTTT TGTCCTACAA AAGCTTTAAG CATCTCAGAG TCTGTGTCAG AAAGAGAGGA 1800

GCTGGCTTAT GAGGCTGTTG CAATTGGGTG AAAGACACTG GTGAACTGTG AGGCAGACCA 1860

ATGGGAAGGG TTTGAGAACT AATATAGAAA ATGAAAGTCT CTCCTTTGTG TCGTATAATC 1920

ATATGTGACA TCACTAAATC ATCTACTAAC TTACACAATA AATACCTACA TGGTGCCTAC 1980 CATGTGATAG AGCGCCCCCA CACGAGGTAC TGCAGATAGA AGGGAATGAT ATAGACGCAG 2040

ATGCTTATTC AGACAGGTAG GACAGAATGG ATATAACACT TAGAAAAGGA CCCGGGTGTG 2100

GTGGCACGGT GGCACATACC TTAGATCCCA CCACCCGGGT GTTGGGGCTG AGGCAGATGA 2160

CTTTTGTTTG TTTTTTTGTT TTCAGTTTTG CTTTTTTTCA AGACAGGGTC TCTCCGTGCA 2220

GCACTGCCTG TCCTGGAACT CGCTTTGCTG GCCACGTTTG TGGCCTTGAA CTCACAAAGT 2280

GCTGGTGCCT GGCTGTAAAA TTAATTTCTC TCTCCCTCTC TCCCCCCCTC CCCCACCTCT 2340

CTCGCTACTT GCTTGGTAGA CCAGACTGGC TTCGAACTCA GAGATTTGCC TGCGTTTGCC 2400

GCCCAAGGGC TGTGATTAAA GGTATGTGCC ACCATGTCCA GCCTTAAAAA TTACTTCTAA 2460

TAGTCATTCT TAGGAGTTTG GATTTTATTT GAAGATAAGA AAACAATAAT GGTTTTAAGA 2520

CTCTTCCCCC CCAAAAAGAC AGTTTGGTAT ATATCTATCA ATCAATCTAA TCTTATCTCC 2580

TGCCTGCCTG CGTATCTATC TATCTATCTA TCTATCTATC TATCTATCTA TCTATCCATC 2640

CATCCAAGGT CTCATGCTTA CCAAGTTGGG CTTGAACTCC TGACTCTTCT GTCTCCACCT 2700

CTGGAGCACT GGGACTACAA ATTTGTGCCA CCCCACAAAG CACTGGCTTG TATTTTAAAC 2760

AAGTCTCTTT AGCTCTTGAG TAAGAGGGTT CATGGTGGTC AAACTAGAGG TAGCTAAAAA 2820

TGGCAGCTAA GTGACATTAC ACGGACTCGG GTGGAGTCAT CGATGGCCTG GCCATGAGGG 2880

TCTGGCCTTT TTGATTTGCA GTTAGACTAA CTGCTCCCCG ATGGAGTGGA TAGTTGTAAG 2940

AGCAGGTGGC AGGAAGACAC CATGGATGGT GATGTCATTT GTGGAGACAA CTGGTAAAGG 3000

AAAAAAAAAA CCTAAGAAGT TCAGCTTAGT ACATGTTAAG TGTGAGGTGC TAGTCACCTC 3060

TGCTGGGATG ACAAACCATA GCTGGCTAAG AAAGTTAGAA GCCCTGGGGG AAAGTTCTTG 3120

CTTTGTAGAT TAGGTTTGTG AGTACCTGTG TGCAGATGGT GTAACTGCTG TCGACAGTGC 3180

TGGGAGGAAT CGCCCATCGA GGGAATAGAT GAACCACACC AGAGAACATG GTAGAAGCGG 3240 CCCAGCAGAG CAACGTGGGC TGGGGTGTAC TTCAGTCGGC AGAGTGCTTT GATCTCCAGT 3300

AGTGGCCCAT GCCACTCCAG AGGTGGGGAG AGAGCTTGGG GAGCGAGACT GTTTGGAAAT 3360

GGTAGGCCCT GTCTTCTTTC CATGTAACTT TCCAACTCCC AGGTTTCCCA TTCTCCACCA 3420

GCAACAACCT CATGCCATTT GAGGTGCTAC TTCAATATCG CTGGCGTCTA CTCATCTATG 3480

TGAACTTAAG AGTCTACTCA TCTATGTGAC TAAGAGTCTG GTGTCAGGCG TGAGCTGAGG 3540

TAGAGGTGGG CTCTTCTCAG CCTCTATAAA CCAATTCACA CCACTTGAGC CAAGCAGTTA 3600

CACATGCACT TTCTCCTCCC GCCTATCAGT CCTAGCTCCT GACAAGGTTT CTCTCCAGCC 3660

TTTTACTTTC CTGGCTTCAA GAAAGGCGGG ATAATATACC AGGGTGGGGA GATTGCATTT 3720

CAGAGTGAGA CGTGTTCTGT CTTCACCTAC CACTTGTTGG CGATGTGACC TTGGGCAAAG 3780

CTCATTAACA GCACAGTGCC TAGTTCCCTA ATTTGTAAAA CATATGCTAT AGGTGTGACG 3840

ATTACGAAGG GCTGACTTTT GTAATGGCTT TGCTTCAGGG ATCTGCAGAC TCGTTGAGCC 3900

ACAATTAGGA TGAGAATCAA GGTGCTTCAG ACTTGTGACA GGGCACTGGC GGCCCCTCAC 3960

ATGATCCTCA GATACCAGAT TGTGGCGTGT GCTGCTAGGA TCACTTGTCT TTCCAGTCTC 4020

CCAACATCTC TTGGGTCCGT GATCACGCGC CCCCCACCCG AAGCCCAGCC TGACGCGGCG 4080

GTGGCTCATG CGCCCTGGAG TCCCGGGCTC TAGCCACGGA ACACACGTCC CAACTCTGGC 4140

GCCCGGCTCC GCCCCTAGCC TCGGGCGCGT CTCTCCCGCC GCCTGCTTGG GTGCTGGAGC 4200

AGCCGCGCCC GCGGGCTCTG GGCGCTGATT GGCTGTGGAC TGCGGGCGGG CAGCCGGAGA 4260

GCGTACACAC CCTCTTCCCG CAGCCAATGA GCGCGCCCAC GTCACTGTCT TGGGCGGCCC 4320

AAAGAGCCGG CAGCCCCTCA ATAAGCCACA TTGTTGCACC AACTCCAGTG CTAGAGTCTC 4380

AGGACACCAC AGGCTACACG GAGTTATCCC GCTTAGGAGA CCCGAAGGCA GGAGCATCAC 4440

TCCAGTGACT CTGATAAGGT GCGATCGCCC GAGAGGAACA GAACTGTCAT TTTTGCGAAG 4500 TTGAGCCTTA CGGATCCCGT GGGCGAAGTT AGCGACGGGA CGCTGAGCAA CTAGACCGGA 4560

CGGCAGGAGT GAGACTTAGG TGCCTTCTAG TAGTTGTGAC TTAAAAAAAA AAAAAAAAGG 4620

AAAAGAAAAA AGGAGGAAAA CCTGTTTCTG GAAACGCGAG GCCCTCAGCT GGTGAGCCAT 4680

CGTGGTTAAG CTTCTTTGTG TGGCTCCTGG AGTCTCCGAT CCCAGCCGGA CACCCGGGCC 4740

TGGTTTCAAA GCGGTCGAAC TGCTCTGCCC GCTCCACCGG TAGCGCTCGA GCCTCGGTTT 4800

CTCTACTCGA CCCCGACTCG CCGCAGCAGG ATGATCGCCT CGCATATGAT CGCCTGCTTA 4860

TTCACGGAGC TCAACCAAAA CCAAGTGCAG AAGGTAAGTC GGCACGGGCG GGAGCTGCTG 4920

GCTCGCTTCG GACCAAGTTG CGTGCTCTCC GGGAATCTGG AGCACGCAGA GGACCTGCTT 980

CCTCCTCCGG GGCTGGGGAC GTGGAACCAG TCTGAGTAGC TGGGAAAGTC CTGAGCGCCA 50 0

GAAACCACGT CTGCTAGGCA CCCTCGTGGC CCGGCCGCGC ATCACCGATA CTCCCACTTT 5100

CCCGGGATCC GCGAGCATCC TCCCCACCCT TAAAGCCCCT AATTTCTAGA 5150

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2771 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Rattus rattus

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 18..2771 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CGACTCGCCG CAGCAGG ATG ATC GCC TCG CAT ATG ATC GCC TGC TTA TTC 50

Met Ile Ala Ser His Met Ile Ala Cys Leu Phe 1 5 10

ACG GAG CTC AAC CAA AAC CAA GTG CAG AAG GTT GAC CAA TTT CTC TAC 98 Thr Glu Leu Asn Gin Asn Gin Val Gin Lys Val Asp Gin Phe Leu Tyr 15 20 25

CAC ATG CGT CTC TCA GAT GAG ACC CTT CTG GAG ATT TCT AGG CGG TTC 146 His Met Arg Leu Ser Asp Glu Thr Leu Leu Glu Ile Ser Arg Arg Phe 30 35 40

CGG AAG GAG ATG GAG AAA GGG CTA GGA GCT ACC ACG CAC CCT ACA GCA 194 Arg Lys Glu Met Glu Lys Gly Leu Gly Ala Thr Thr His Pro Thr Ala 45 50 55

GCT GTG AAA ATG TTG CCT ACC TTT GTG AGG TCA ACT CCG GAT GGG ACA 242 Ala Val Lys Met Leu Pro Thr Phe Val Arg Ser Thr Pro Asp Gly Thr 60 65 70 75

GAA CAT GGG GAG TTC CTG GCT CTG GAT CTT GGA GGA ACC AAC TTC CGT 290 Glu His Gly Glu Phe Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg 80 85 90

GTG CTC CGA GTA AGG GTG ACG GAC AAT GGC CTC CAG AGA GTG GAG ATG 338 Val Leu Arg Val Arg Val Thr Asp Asn Gly Leu Gin Arg Val Glu Met 95 100 105

GAG AAC CAG ATC TAC GCC ATC CTT GAG GAC ATC ATG CGG GGC AGT GGA 386 Glu Asn Gin Ile Tyr Ala Ile Leu Glu Asp Ile Met Arg Gly Ser Gly 110 115 120

ACC CAG CTG TTT GAC CAC ATC GCC GAA TGC CTG GCC AAC TTC ATG GAC 434 Thr Gin Leu Phe Asp His Ile Ala Glu Cys Leu Ala Asn Phe Met Asp 125 130 135

AAG CTA CAA ATC AAA GAG AAG AAG CTC CCT CTG GGT TTC ACC TTC TCG 482 Lys Leu Gin Ile Lys Glu Lys Lys Leu Pro Leu Gly Phe Thr Phe Ser 140 145 150 155 TTC CCC TGC CAC CAG ACA AAA CTG GAT GAG AGT TTT TTG GTC TCG TGG 530 Phe Pro Cys His Gin Thr Lys Leu Asp Glu Ser Phe Leu Val Ser Trp 160 165 170

ACT AAG GGG TTC AAG TCC AGT GGC GTG GAA GGC AGA GAT GTG GTG GAC 578 Thr Lys Gly Phe Lys Ser Ser Gly Val Glu Gly Arg Asp Val Val Asp

175 180 185

CTG ATC CGG AAG GTT ATC CAG CGC AGA GGG GAC TTT GAC ATT GAC ATT 626 Leu Ile Arg Lys Val Ile Gin Arg Arg Gly Asp Phe Asp Ile Asp Ile 190 195 200

GTG GCC GTG GTG AAT GAC ACA GTT GGG ACC ATG ATG ACT TGT GGC TAT 674 Val Ala Val Val Asn Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr 205 210 215

GAT GAT CAG AAC TGC GAG ATT GGT CTC ATT GTG GGC ACT GGC AGC AAC 722 Asp Asp Gin Asn Cys Glu Ile Gly Leu Ile Val Gly Thr Gly Ser Asn 220 225 230 235

GCC TGC TAC ATG GAG GAA ATG CGT CAT ATT GAC ATG GTG GAG GGA GAT 770 Ala Cys Tyr Met Glu Glu Met Arg His Ile Asp Met Val Glu Gly Asp 240 245 250

GAG GGG CGC ATG TGC ATC AAC ATG GAG TGG GGA GCC TTT GGG GAC GAC 818 Glu Gly Arg Met Cys Ile Asn Met Glu Trp Gly Ala Phe Gly Asp Asp

255 260 265

GGT ACA CTC AAT GAC ATC CGA ACC GAG TTT GAC CGA GAG ATC GAC ATG 866 Gly Thr Leu Asn Asp Ile Arg Thr Glu Phe Asp Arg Glu Ile Asp Met 270 275 280

GGC TCG CTG AAC CCT GGG AAG CAG CTG TTT GAG AAG ATG ATT AGC GGG 914 Gly Ser Leu Asn Pro Gly Lys Gin Leu Phe Glu Lys Met Ile Ser Gly 285 290 295

ATG TAC ATG GGG GAG CTG GTC AGG CTC ATC CTG GTG AAG ATG GCC AAG 962 Met Tyr Met Gly Glu Leu Val Arg Leu Ile Leu Val Lys Met Ala Lys 300 305 310 315

GCA GAG CTG TTG TTC CAA GGG AAA CTC AGC CCA GAA CTC CTT ACC ACT 1010 Ala Glu Leu Leu Phe Gin Gly Lys Leu Ser Pro Glu Leu Leu Thr Thr 320 325 330

GGC TCC TTC GAG ACC AAA GAT GTC TCG GAT ATT GAA GAG GAT AAG GAT 1058 Gly Ser Phe Glu Thr Lys Asp Val Ser Asp Ile Glu Glu Asp Lys Asp 335 340 345

GGA ATC GAG AAG GCC TAC CAA ATC CTG ATG CGC CTG GGT CTG AAT CCA 1106 Gly Ile Glu Lys Ala Tyr Gin Ile Leu Met Arg Leu Gly Leu Asn Pro 350 355 360

TTG CAG GAG GAT TGT GTG GCC ACG CAC CGA ATC TGC CAG ATT GTG TCC 1154 Leu Gin Glu Asp Cys Val Ala Thr His Arg Ile Cys Gin Ile Val Ser 365 370 375

ACG CGC TCG GCC AGT CTG TGC GCA GCC ACC CTG GCC GCG GTG CTG TGG 1202 Thr Arg Ser Ala Ser Leu Cys Ala Ala Thr Leu Ala Ala Val Leu Trp 380 385 390 395

CGA ATC AAA GAG AAC AAG GGC GAG GAG CGA CTT CGC TCC ACC ATC GGT 1250 Arg Ile Lys Glu Asn Lys Gly Glu Glu Arg Leu Arg Ser Thr Ile Gly 400 405 410

GTC GAT GGC TCC GTC TAC AAG AAA CAT CCC CAT TTT GCC AAG CGT CTC 1298 Val Asp Gly Ser Val Tyr Lys Lys His Pro His Phe Ala Lys Arg Leu 415 420 425

CAT AAG GCA GTG AGG AGG CTG GTG CCC GAC TGT GAT GTC CGC TTC CTC 1346 His Lys Ala Val Arg Arg Leu Val Pro Asp Cys Asp Val Arg Phe Leu 430 435 440

CGC TCT GAG GAT GGC AGC GGC AAG GGG GCT GCT ATG GTG ACG GCG GTG 1394 Arg Ser Glu Asp Gly Ser Gly Lys Gly Ala Ala Met Val Thr Ala Val 445 450 455

GCT TAC CGT CTG GCT GAC CAA CAC CGG GCC CGC CAG AAG ACC CTG GAG 1442 Ala Tyr Arg Leu Ala Asp Gin His Arg Ala Arg Gin Lys Thr Leu Glu 460 465 470 475

TCT CTG AAG CTG AGC CAC GAG CAG CTT CTG GAG GTT AAG AGA AGA ATG 1490 Ser Leu Lys Leu Ser His Glu Gin Leu Leu Glu Val Lys Arg Arg Met 480 485 490 AAG GTG GAA ATG GAG CAG GGT CTG AGC AAG GAG ACG CAT GCG GTC GCC 1538 Lys Val Glu Met Glu Gin Gly Leu Ser Lys Glu Thr His Ala Val Ala 495 500 505

CCT GTG AAG ATG CTG CCC ACT TAC GTG TGT GCC ACT CCA GAT GGC ACA 1586 Pro Val Lys Met Leu Pro Thr Tyr Val Cys Ala Thr Pro Asp Gly Thr 510 515 520

GAG AAA GGA GAC TTC TTG GCC TTG GAT CTT GGA GGA ACA AAC TTC CGG 1634 Glu Lys Gly Asp Phe Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg 525 530 535

GTC CTG CTG GTG CGT GTG CGT AAT GGC AAG CGG AGG GGC GTG GAG ATG 1682 Val Leu Leu Val Arg Val Arg Asn Gly Lys Arg Arg Gly Val Glu Met 540 545 550 555

CAT AAC AAG ATC TAC TCC ATC CCA CAG GAG GTT ATG CAT GGC ACT GGG 1730 His Asn Lys Ile Tyr Ser Ile Pro Gin Glu Val Met His Gly Thr Gly 560 565 570

GAA GAG CTC TTC GAC CAC ATT GTC CAG TGC ATT GCG GAC TTC CTG GAG 1778 Glu Glu Leu Phe Asp His Ile Val Gin Cys Ile Ala Asp Phe Leu Glu 575 580 585

TAC ATG GGC ATG AAG GGC GTG TCC CTG CCT TTG GGT TTC ACA TTC TCC 1826 Tyr Met Gly Met Lys Gly Val Ser Leu Pro Leu Gly Phe Thr Phe Ser 590 595 600

TTC CCT TGC CAG CAG AAC AGC CTA GAC CAG AGC ATC CTC CTC AAG TGG 1874 Phe Pro Cys Gin Gin Asn Ser Leu Asp Gin Ser Ile Leu Leu Lys Trp 605 610 615

ACA AAG GGA TTC AAG GCA TCT GGC TGC GAG GGT GAG GAT GTG GTC ACC 1922 Thr Lys Gly Phe Lys Ala Ser Gly Cys Glu Gly Glu Asp Val Val Thr 620 625 630 635

TTG CTG AAG GAA GCG ATT CAC CGG CGA GAG GAG TTT GAC CTG GAT GTG 1970 Leu Leu Lys Glu Ala Ile His Arg Arg Glu Glu Phe Asp Leu Asp Val 640 645 650 GTT GCC GTG GTG AAT GAC ACA GTT GGG ACT ATG ATG ACT TGT GGC TAC 2018 Val Ala Val Val Asn Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr 655 660 665

GAA GAC CCT CAC TGT GAA GTT GGC CTC ATT GTT GGC ACC GGA AGC AAC 2066 Glu Asp Pro His Cys Glu Val Gly Leu Ile Val Gly Thr Gly Ser Asn 670 675 680

GCC TGC TAC ATG GAA GAG ATG CGT AAT GTG GAG CTG GTG GAC GGA GAG 2114 Ala Cys Tyr Met Glu Glu Met Arg Asn Val Glu Leu Val Asp Gly Glu 685 690 695

GAG GGA CGG ATG TGT GTC AAC ATG GAG TGG GGA GCA TTT GGG GAC AAT 2162 Glu Gly Arg Met Cys Val Asn Met Glu Trp Gly Ala Phe Gly Asp Asn 700 705 710 715

GGC TGC CTG GAT GAC TTG CGG ACC GTG TTT GAT GTT GCT GTG GAT GAG 2210 Gly Cys Leu Asp Asp Leu Arg Thr Val Phe Asp Val Ala Val Asp Glu 720 725 730

CTT TCT CTC AAC CCT GGC AAA CAG AGG TTC GAG AAG ATG ATC AGC GGC 2258 Leu Ser Leu Asn Pro Gly Lys Gin Arg Phe Glu Lys Met Ile Ser Gly 735 740 745

ATG TAC TTG GGA GAG ATT GTG CGC AAC ATT CTC ATC GAT TTC ACG AAG 2306 Met Tyr Leu Gly Glu Ile Val Arg Asn Ile Leu Ile Asp Phe Thr Lys 750 755 760

CGG GGG CTG CTC TTC CGA GGC CGC ATC TCA GAG CGC CTC AAG ACA AGG 2354 Arg Gly Leu Leu Phe Arg Gly Arg Ile Ser Glu Arg Leu Lys Thr Arg 765 770 775

GGA ATC TCT GAA ACT AAG TTC CTG TCT CAG ATA GAG AGC GAC TGC CTA 2402 Gly Ile Ser Glu Thr Lys Phe Leu Ser Gin Ile Glu Ser Asp Cys Leu 780 785 790 795

GCC CTG CTA CAG GTT CGT GCC ATC CTG CGC CAC CTA GGG CTG GAG AGC 2450 Ala Leu Leu Gin Val Arg Ala Ile Leu Arg His Leu Gly Leu Glu Ser 800 805 810 ACG TGC GAT GAC AGC ATC ATC GTG AAG GAG GTG TGC ACT GTG GTT GCC 2498 Thr Cys Asp Asp Ser Ile Ile Val Lys Glu Val Cys Thr Val Val Ala 815 820 825

CGG CGC GCT GCA CAG CTC TGT GGC GCA GGC ATG GCC GCC GTA GTG GAC 2546 Arg Arg Ala Ala Gin Leu Cys Gly Ala Gly Met Ala Ala Val Val Asp 830 835 840

AAG ATA AGA GAG AAC CGT GGG CTG GAC AAC CCC AAA GTG ACA GTG GGC 2594 Lys Ile Arg Glu Asn Arg Gly Leu Asp Asn Pro Lys Val Thr Val Gly 845 850 855

GTG GAC GGG ACT CTG TAT AAG CTT CAT CCT CAC TTT GCC AAG GTC ATG 2642 Val Asp Gly Thr Leu Tyr Lys Leu His Pro His Phe Ala Lys Val Met 860 865 870 875

CAT GAG ACG GTG AGA GAT CTG GCT CCG AAA TGT GAC GTG TCC TTC CTG 2690 His Glu Thr Val Arg Asp Leu Ala Pro Lys Cys Asp Val Ser Phe Leu 880 885 890

GAA TCC GAG GAC GGC AGT GGG AAG GGA GCA GCT CTC ATC ACT GCC GTG 2738 Glu Ser Glu Asp Gly Ser Gly Lys Gly Ala Ala Leu Ile Thr Ala Val 895 900 905

GCC TGC CGC ATC CGG GAG GCT GGG CAG AGA TA 2771 Ala Cys Arg Ile Arg Glu Ala Gly Gin Arg 910 915

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 917 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ile Ala Ser His Met Ile Ala Cys Leu Phe Thr Glu Leu Asn Gin 1 5 10 15 Asn Gin Val Gin Lys Val Asp Gin Phe Leu Tyr His Met Arg Leu Ser 20 25 30

Asp Glu Thr Leu Leu Glu Ile Ser Arg Arg Phe Arg Lys Glu Met Glu 35 40 45

Lys Gly Leu Gly Ala Thr Thr His Pro Thr Ala Ala Val Lys Met Leu 3 50 55 60

Pro Thr Phe Val Arg Ser Thr Pro Asp Gly Thr Glu His Gly Glu Phe 65 70 75 80

Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Val Arg 85 90 95

Val Thr Asp Asn Gly Leu Gin Arg Val Glu Met Glu Asn Gin Ile Tyr

100 105 110

Ala Ile Leu Glu Asp Ile Met Arg Gly Ser Gly Thr Gin Leu Phe Asp 115 120 125

His Ile Ala Glu Cys Leu Ala Asn Phe Met Asp Lys Leu Gin Ile Lys 130 135 140

Glu Lys Lys Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys His Gin 145 150 155 160

Thr Lys Leu Asp Glu Ser Phe Leu Val Ser Trp Thr Lys Gly Phe Lys 165 170 175

Ser Ser Gly Val Glu Gly Arg Asp Val Val Asp Leu Ile Arg Lys Val

180 185 190

Ile Gin Arg Arg Gly Asp Phe Asp Ile Asp Ile Val Ala Val Val Asn 195 200 205

Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr Asp Asp Gin Asn Cys 210 215 220

Glu Ile Gly Leu Ile Val Gly Thr Gly Ser Asn Ala Cys Tyr Met Glu 225 230 235 240 Glu Met Arg His Ile Asp Met Val Glu Gly Asp Glu Gly Arg Met Cys 245 250 255

Ile Asn Met Glu Trp Gly Ala Phe Gly Asp Asp Gly Thr Leu Asn Asp 260 265 270

Ile Arg Thr Glu Phe Asp Arg Glu Ile Asp Met Gly Ser Leu Asn Pro 275 280 285

Gly Lys Gin Leu Phe Glu Lys Met Ile Ser Gly Met Tyr Met Gly Glu 290 295 300

Leu Val Arg Leu Ile Leu Val Lys Met Ala Lys Ala Glu Leu Leu Phe 305 310 315 320

Gin Gly Lys Leu Ser Pro Glu Leu Leu Thr Thr Gly Ser Phe Glu Thr

325 330 335

Lys Asp Val Ser Asp Ile Glu Glu Asp Lys Asp Gly Ile Glu Lys Ala 340 345 350

Tyr Gin Ile Leu Met Arg Leu Gly Leu Asn Pro Leu Gin Glu Asp Cys 355 360 365

Val Ala Thr His Arg Ile Cys Gin Ile Val Ser Thr Arg Ser Ala Ser 370 375 380

Leu Cys Ala Ala Thr Leu Ala Ala Val Leu Trp Arg Ile Lys Glu Asn 385 390 395 400

Lys Gly Glu Glu Arg Leu Arg Ser Thr Ile Gly Val Asp Gly Ser Val

405 410 415

Tyr Lys Lys His Pro His Phe Ala Lys Arg Leu His Lys Ala Val Arg 420 425 430

Arg Leu Val Pro Asp Cys Asp Val Arg Phe Leu Arg Ser Glu Asp Gly 435 440 445

Ser Gly Lys Gly Ala Ala Met Val Thr Ala Val Ala Tyr Arg Leu Ala 450 455 460 Asp Gin His Arg Ala Arg Gin Lys Thr Leu Glu Ser Leu Lys Leu Ser 465 470 475 480

His Glu Gin Leu Leu Glu Val Lys Arg Arg Met Lys Val Glu Met Glu 485 490 495

Gin Gly Leu Ser Lys Glu Thr His Ala Val Ala Pro Val Lys Met Leu 500 505 510

Pro Thr Tyr Val Cys Ala Thr Pro Asp Gly Thr Glu Lys Gly Asp Phe 515 520 525

Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val Arg 530 535 540

Val Arg Asn Gly Lys Arg Arg Gly Val Glu Met His Asn Lys Ile Tyr 545 550 555 560

Ser Ile Pro Gin Glu Val Met His Gly Thr Gly Glu Glu Leu Phe Asp 565 570 575

His Ile Val Gin Cys Ile Ala Asp Phe Leu Glu Tyr Met Gly Met Lys 580 585 590

Gly Val Ser Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys Gin Gin 595 600 605

Asn Ser Leu Asp Gin Ser Ile Leu Leu Lys Trp Thr Lys Gly Phe Lys 610 615 620

Ala Ser Gly Cys Glu Gly Glu Asp Val Val Thr Leu Leu Lys Glu Ala 625 630 635 640

Ile His Arg Arg Glu Glu Phe Asp Leu Asp Val Val Ala Val Val Asn 645 650 655

Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr Glu Asp Pro His Cys 660 665 670

Glu Val Gly Leu Ile Val Gly Thr Gly Ser Asn Ala Cys Tyr Met Glu 675 680 685 Glu Met Arg Asn Val Glu Leu Val Asp Gly Glu Glu Gly Arg Met Cys

690 695 700

Val Asn Met Glu Trp Gly Ala Phe Gly Asp Asn Gly Cys Leu Asp Asp 705 710 715 720

Leu Arg Thr Val Phe Asp Val Ala Val Asp Glu Leu Ser Leu Asn Pro 725 730 735

Gly Lys Gin Arg Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu 740 745 750

Ile Val Arg Asn Ile Leu Ile Asp Phe Thr Lys Arg Gly Leu Leu Phe 755 760 765

Arg Gly Arg Ile Ser Glu Arg Leu Lys Thr Arg Gly Ile Ser Glu Thr 770 775 780

Lys Phe Leu Ser Gin Ile Glu Ser Asp Cys Leu Ala Leu Leu Gin Val 785 790 795 800

Arg Ala Ile Leu Arg His Leu Gly Leu Glu Ser Thr Cys Asp Asp Ser 805 810 815

Ile Ile Val Lys Glu Val Cys Thr Val Val Ala Arg Arg Ala Ala Gin 820 825 830

Leu Cys Gly Ala Gly Met Ala Ala Val Val Asp Lys Ile Arg Glu Asn 835 840 845

Arg Gly Leu Asp Asn Pro Lys Val Thr Val Gly Val Asp Gly Thr Leu 850 855 860

Tyr Lys Leu His Pro His Phe Ala Lys Val Met His Glu Thr Val Arg 865 870 875 880

Asp Leu Ala Pro Lys Cys Asp Val Ser Phe Leu Glu Ser Glu Asp Gly 885 890 895 Ser Gly Lys Gly Ala Ala Leu Ile Thr Ala Val Ala Cys Arg Ile Arg 900 905 910

Glu Ala Gly Gin Arg 915

Claims

We claim:

1. An isolated hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure 11.

2. The fragment of claim 1 which is responsive to hypoxia.

3. The fragment of claim 1 which is responsive to p53.

4. The fragment of claim 1 which is responsive to glucose.

5. The fragment of claim 1 which is responsive to insulin.

6. The fragment of claim 1 which is responsive to AP-1.

7. The fragment of claim 1 which is responsive to AP-2.

8. The fragment of claim 1 which is responsive to ATF/CRE.

9. The fragment of claim 1 which has the sequence shown in SEQ ID NOJ

(Figure 5).

10. The fragment of claim 1 which is covalently joined to a reporter gene.

11. The fragment of claim 1 which is covalently joined to a toxic gene.

12. A method of screening for potential drugs which affect regulated transcription of mmor hexokinase II, the method comprising the steps of: contacting a test substance with the reporter gene fusion of claim 10; and measuring transcription of the reporter gene in the presence of the test substance; wherein a potential drug is identified when a test substance increases or decreases the transcription of the reporter gene.

13. The method of claim 12 wherein the transcription is performed in the presence of a transcription factor which binds to the response element.

14. A method of treating cells which overexpress hexokinase II comprising the step of: administering the gene fusion of claim 11 to cells which overexpress hexokinase II, whereby the toxic gene is expressed in the cells.

15. An isolated nucleic acid probe comprising at least 15 contiguous nucleotides selected from the sequence of SEQ ID NO: 1.

16. The isolated nucleic acid probe of claim 15 wherein the probe is selected from the nucleotides number -4369 to -1158.

17. The isolated nucleic acid probe of claim 15 which is detectably labeled.

18. A method for diagnosing mmors which overexpress hexokinase, comprising the steps of: determimng copy number of a hexokinase II gene in a tissue sample suspected of being neoplastic; wherein a determined copy number of greater than two indicates neoplasia.

19. The method of claim 18 wherein a copy number of greater than ten indicates neoplasia.

20. The method of claim 18 wherein copy number is determined by hybridization to a nucleic acid probe comprising at least 15 contiguous nucleotides seleted from the sequence of SEQ ID NO: 1 wherein the probe is detectably labeled.

21. The method of claim 18 wherein copy number is determined by comparing hybridization of a hexokinase II gene probe with a normal tissue to hybridization of the probe with the tissue sample suspected of being neoplastic.

22. The method of claim 18 wherein copy number is determined by a quantitative polymerase chain reaction.

23. The method of claim 18 wherein copy number is determined by fluorescence in situ hybridization (FISH) analysis.

24. A method for diagnosing neoplastic tissues, comprising the step of: determining whether cells in a tissue sample suspected of being neoplastic contain a hexokinase II gene which is unmethylated, an unmethylated hexokinase II gene indicating neoplasia.

25. The method of claim 24 wherein the step of determining whether the hexokinase II gene is unmethylated is performed by use of restriction endonucleases which are methylation sensitive.

26. A vector for expression of a desired protein in a mammalian cell, comprising: an isolated hexokinase II DNA fragment according to claim 1.

27. The vector of claim 26 further comprising: a gene encoding the desired protein, wherein the gene is covalently linked to the DNA fragment and the DNA fragment regulates the expression of the desired gene in a mammalian cell.

28. The vector of claim 27 wherein the DNA fragment is upstream from the desired gene.

29. A method for increasing glycolysis in cells, comprising the step of: introducing into cells an unmethylated DNA molecule comprising: a hexokinase II DNA fragment capable of regulating transcription of a downstream open reading frame, wherein the fragment comprises at least one of the response elements identified in Figure 11 ; and

a nucleic acid encoding a hexokinase II, wherein the hexokinase II DNA fragment is covalently and operatively linked to the nucleic acid encoding a hexokinase II.

30. The method of claim 29 wherein the cells are liver cells.

31. The method of claim 29 wherein the cells are muscle cells.

32. The method of claim 29 wherein the cells are adipose cells.