Patent Publication Number: US-2003228568-A1

Title: Inducible phosphofructokinase and the warburg effect

Description:
CROSS REFERENCE TO RELATED APPLICATION  
     [0001] This patent application is a continuation-in-part from U.S. patent application Ser. No. 08/961,578 filed Oct. 31, 1997. 
    
    
     
       TECHNICAL FIELD OF THE INVENTION  
       [0002] The present invention provides a novel phosphofructokinase isozyme (iPFK-2) that is preferentially transcribed and translated in tumor cells. The discovery of this isozyme, together with its function, led to the discovery of its use as a diagnostic target, as a drug screening target, and antisense compounds that inhibit its translation in cellular cytosol as an anti-tumor treatment.  
       BACKGROUND OF THE INVENTION  
       [0003] The glycolytic pathway is a fundamental anaerobic pathway for sugar metabolism in eukaryotic cells. Glycolysis has a dual role, to degrade sugars to generate energy (ATP) and to provide building blocks for synthetic reactions. The rate of conversion of glucose into pyruvate is regulated to meet these two major cellular needs. In glycolysis, the enzymes hexokinase, phosphofructokinase and pyruvate kinase catalyze irreversible reactions and are regulated enzymes for control points in glycolysis. The enzymes are regulated by reversible binding of allosteric effectors, by covalent modification and by transcriptional control to meet changing metabolic needs. Of the three control enzymes, phosphofructokinase is the most important control point in mammalian glycolysis.  
       [0004] In 1930, Warburg pointed out that tumors have a high rate of anaerobic glycolysis and that they do not show a decreased glycolytic rate at relatively high O 2  concentrations. This loss of regulatory control (i.e., the Pasteur effect) has come to be called the Warburg effect. Supplying tumor cells with glucose results in an inhibition of oxygen consumption, which magnifies the dependence on glucose for energy. Other cellular types do not normally show this effect since they maintain respiration from other substrates even in the presence of glucose. The question of why rapidly growing tumors have a marked tendency to convert the glycolytically-generated pyruvate to lactic acid in the cytosol instead of transporting into the mitochondria for total oxidation has puzzled biochemists for years. The physiologic consequence of this altered metabolic behavior are clear. Tumor tissue generates a high degree of metabolic inefficiency in the host, through an enhanced operation of energy-wasting processes, such as the Cori cycle between the tumor and the liver. As a result of the high glycolytic rate, a large amount of pyruvate is generated, together with an increase in the cytosolic NADH/NAD+ ratio, which favors the reduction of pyruvate to lactate through the action of lactate dehydrogenase. This is also supported by the low mitochondrial content of tumor cells which hampers the possibility of dissipating NADH through the action of the electron transfer chain and the low levels of NADH-shuttle systems found in a great number of tumors. The tumor cell becomes a lactate exporter in a similar way to some muscular fibers in anoxic situations. Although the precise role of the enhanced Cori cycle in tumor-bearing states is not fully determined, it adds inefficiency to the host in a way that, instead of ATP formation of 36-38 molecules during the complete oxidation of glucose to CO 2 , a net loss of 4 ATPs can be expected when two three-carbon molecules are converted to one molecule of glucose.  
       [0005] A distinctive metabolic environment of cancer-bearing individuals has been described (Argilés and Azcón-Bieto,  Mol. Cell. Biochem.  81:3-17, 1988). Tumor invasion upon a host has been metabolically characterized by a reduction of the metabolic efficiency of the host, muscular protein depletion, increased gluconeogenesis, and uncoupling of oxidative phosphorylation. The net result is an energy imbalance leading to cachexia and eventual starvation.  
       SUMMARY OF THE INVENTION  
       [0006] The present invention provides a cancer malignancy diagnostic assay comprising obtaining a sample of a body fluid or tissue (including, for instance, a sample of tumor tissue, performing a sequence identity assay to look for the presence of iPFK-2 specific sequences (SEQ ID NO.: 11). Preferably, the sequence identity assay is selected from the group consisting of PCR (polymerase chain reaction) assays, ELISA immunologic assays, hybridization assays, and combinations thereof. The present invention further provides an anticancer, anti-inflammatory and cachexia pharmaceutical composition comprising a specific antisense oligonucleotide to the inventive isolated iPFK-2 sequence and a pharmaceutically acceptable oligonucleotide carrier. Preferably, the antisense oligonucleotide is a 15-50 base oligonucleotide incorporating an oligonucleotide sequence selected from the group consisting of: 5′-CCAACGGCATCTTCGCGGCT-3′ [SEQ ID NO: 2], 5′-GTCAGTTCCAACGGCATCTT-3′ [SEQ ID NO: 4], and combinations thereof. The present invention further provides a therapeutic agent screening assay to screen for compounds having anti-tumor activity, comprising: (a) obtaining recombinant iPFK-2 having activity that forms fructose 2,6-bisphosphate from fructose 6-phosphate substrate; (b) adding candidate drug at various concentrations or no-drug control vehicle; and (c) assaying for fructose 2,6-bisphosphate as a measure of enzymatic activity. Preferably, the product assay is conducted by means of an enzymatic assay.  
       [0007] The present invention further provides a recombinant iPFK-2 polypeptide expressed by the cDNA sequence provided in SEQ ID NO. 11. The use of the iPFK-2 polypeptide, with known antibody techniques, including known monoclonal antibody techniques, further provides antibodiesy that specifically bind to iPFK-2. Preferably, such antibodies are monoclonal antibodies. 
     
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
     [0008]FIG. 1 shows the predicted amino acid sequence and alignment of the novel iPFK-2 cDNA with PFK-2 sequences deduced from a human placental (Sakai, Kato, Fukusawa et al.,  J. Biochem.  119:506-511, 1996) and a human liver (Lange and Pilkis,  Nuc. Acids Res.  18:3652, 1990) cDNA clone. Boxed residues indicate identity.  
     [0009] FIGS.  2 A-C shows that LPS induces peripheral blood monocytes to rapidly express iPFK-2 mRNA and protein. FIG. 2A shows a RT-PCR analysis. FIG. 2B shows a Northern blot analysis. FIG. 2C shows a Western blot analysis, wherein the anti-iPFK-2 antiserum used in the right panel was pre-absorbed with the iPFK-2-specific peptide against which the antiserum was raised.  
     [0010] FIGS.  3 A-B shows iPFK-2 mRNA expression by human cancer cell lines. FIG. 3A shows a Northern blot analysis of various human cancer cell lines. FIG. 3B shows a RT-PCR analysis of K-562 cells for β-actin, iPFK-2 and human liver PFK-2.  
     [0011] FIGS.  4 A-C shows that an iPFK-2 antagonist antisense oligonucleotide inhibited IPFK-2-specific K562 cell proliferation in vitro. Specifically, FIG. 4A shows a Western blot analysis of the iPFK2 antagonist activity of the antisense oligonucleotide. FIG. 4B shows a fructose-2,6-bisphosphate assay of the iPFK2 antagonist activity of the antisense oligonucleotide(AS) versus the sense (S) sequence. FIG. 4C shows a 5-phosphoribosyl 1-pyrophosphate assay and a K562 cell proliferation assay of the iPFK2 antagonist activity of the antisense oligonucleotide(AS) versus the sense (S) sequence.  
     [0012]FIG. 5 shows in vivo data providing evidence of iPFK-2 antagonist activity of the antisense oligonucleotides and further showing anti-cancer therapeutic activity of iPFK-2 antagonists.  
     [0013]FIG. 6 shows that in vivo endotoxemia induces mouse iPFK-2 mRNA expression in spleen and muscle tissue. 10 week-old BALB/c mice (19-20 gm) were injected i.p. with LPS (12.5 mg/kg) or saline as control. After 6 and 24 hours mice were euthanized by CO 2  asphyxiation and the brain, liver, lower extremity muscles, and spleen were removed by dissection. Total RNA extraction and Northern blot analysis were performed using a mouse iPFK-2-specific cDNA probe (amplified from mouse peritoneal macrophage CDNA by 30 cycle RT-PCR using the following human iPFK-2-specific primers: 5′-TGAGGCAGACGTGTCGGTTC-3′ [SEQ ID NO.: 5], 5′-CAGCAGCTCCAGGAAAGTGT-3′ [SEQ ID NO.: 6]. These in vivo data show that LPS induced iPFK-2 mRNA expression in mice in brain, liver, muscle and spleen tissues.  
     [0014]FIG. 7 shows that iPFK-2 is overexpressed in PBMCs (peripheral blood mononuclear cells) of HIV+ individuals. Total RNA was isolated from 3 uninfected individuals (lanes 1-3) and 5 HIV+ individuals (lanes 4-8) and analyzed by RT-PCR with β-Actin-specific primers (5′-TAAGGAGAAGCTGTGCTACG-3′ [SEQ ID NO.: 7], 5′-ATCTCCTTCTGCATCCTGTC-3′ [SEQ ID NO.: 8], 19 cycles) and iPFK-2-specific primers (5′-ATTGGTCTGGCAACTGCAAA-3′ [SEQ ID NO.: 9], 5′-GGAGCCTCCTATGTGTGACT-3′ [SEQ ID NO.: 10], 23 cycles).  
     [0015]FIG. 8 shows a postulated metabolic scheme for the metabolic role of iPFK-2, particularly in rapidly dividing cancer cells where there is a buildup of lactate from anaerobic metabolism and production of nucleotides to support rapid cell division.  
     [0016]FIG. 9 shows the results of iPFK-2 antisense oligonucleotides inhibiting the proliferation of T cell tumor line MOLT-4. Two different iPFK-2 antisense oligonucleotides were effective and exhibited pharmacologic anti-tumor activity in this predictive assay. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0017] The present invention provides a cancer malignancy diagnostic assay comprising obtaining a sample of a body or tumor fluid or tissue, and performing a sequence identity assay to look for the presence of iPFK-2 specific sequences. Preferably, the sequence identity assay is selected from the group consisting of PCR (polymerase chain reaction) assays, ELISA immunologic assays, hybridization assays, and combinations thereof. The present invention further provides an anticancer pharmaceutical composition comprising an antisense oligonucleotide specific to the inventive isolated PFK-2 sequence and a pharmaceutically acceptable oligonucleotide carrier. Preferably, the antisense oligonucleotide is selected from a 15-50 base oligonucleotide incorporating an oligonucleotide sequence selected from the group consisting of): 5′-CCAACGGCATCTTCGCGGCT-3′ [SEQ ID NO: 2], 5′-GTCAGTTCCAACGGCATCTT-3′ [SEQ ID NO: 4], and combinations thereof.  
     [0018] The present invention further provides a recombinant iPFK-2 polypeptide expressed by the cDNA sequence provided in SEQ ID NO. 11. The use of the iPFK-2 polypeptide, with known antibody techniques, including known monoclonal antibody techniques, further provides an antibody that specifically binds to iPFK-2. Preferably, this antibody is a monoclonal antibody.  
     [0019] The present invention further provides an isolated CDNA sequence encoding an inducible human phosphofructokinase-2 (iPFK-2) enzyme. The cDNA sequence is listed as SEQ ID NO 11. SEQ ID NO. 11 provides a bolded start and stop codon of the coding region. Further, there are underlined base pairs at the C terminal region of the coding region that provide additional amino acids not found in any other PFK-2 isotypes. The inventive iPFK-2 cDNA sequence is useful for producing recombinant iPFK-2 polypeptide, for designing antisense oligonucleotides, and for transfecting cells (both prokaryotic and eukaryotic) to produce recombinant iPFK-2 and fragments thereof. The recombinant iPFK-2 polypeptide, having PFK-2 enzymatic activity, is useful for screening for inhibitors having therapeutic activity as anticancer agents specifically against the inventive inducible iPFK-2 isoform. Anti-cancer therapeutic activity can be attributable to iPFK-2 inhibitors because a novel, AU-rich early response gene is required for leukemia growth. This gene appears to be the inducible iPFK-2 gene, the gene product of which is most prevalent in tumor cells.  
     [0020] The present invention further provides an isolated cDNA sequence encoding an inducible human phosphofructokinase-2 (iPFK-2) isozyme. The examples below detail the efforts that led to the isolation, purification and expression of this isozyme. The isolated isozyme sequence was found to be preferentially expressed in tumor cells and lead to increased glycolytic activity.  
     [0021] The invention is based upon the identification and understanding of a novel gene for PFK-2/FBPase (6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-biphosphophatase (FBPase)) or “iPFK-2” that is induced by pro-inflammatory stimuli and which is distinguished from other similar genes encoding PFK-type enzymes by the presence of multiple copies of an AUUUA mRNA instability motif in its 3′-untranslated end. This AU-rich element is characteristic of mRNAs encoding several inflammatory cytokines (e.g., TNFα, IL-1, IFN-γ, and GM-CSF) and oncoproteins (e.g., c-Fos, c-Myc, and c-Sis) (Greenberg and Belasco, in  Control of Messenger RNA Stability,  Belasco and Brawerman eds., pp. 199-218, Academic Press, New York, 1993). Data presented herein shows that iPFK-2 is expressed constitutively in several human cancer cell lines and was found to be essential for tumor cell growth in vivo. Inhibiting the level of iPFK-2 protein expression (through the use of antisense antagonists) decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), an important precursor for purine and pyrimidine biosynthesis. Accordingly, iPFK-2 is an important regulatory isoenzyme that appears to be essential for tumor growth, whose antagonists have important anti-cancer therapeutic activity, and provides an explanation for long-standing observations concerning the apparent coupling of glycolysis and cancer cell proliferation.  
     [0022] The mRNAs of several cytokines and proto-oncogenes that are members of early response gene families have been noted to contain the sequence motif AUUUA in their 3′ untranslated region (3′UTR). This AU-rich element confers instability to the mRNA molecule and plays a role in regulating its physiologic half life (Caput et al.,  Proc. Natl. Acad. Sci. USA  83:1670-1674, 1986; and Shaw et al.,  Cell  46: 659-667, 1986). An expressed sequence tag (EST) database was searched for cDNA sequences containing conserved AUUUA sequence motifs. One AU-rich EST, unrelated to previously described genes, was identified and the complete cDNA was cloned and sequenced. The DNA sequence of this novel gene was found to share 29% identity with human liver PFK-2 (FIG. 1), which does not contain AU-rich elements. The predicted amino acid sequence showed 69% identity and extensive conservative substitutions (FIG. 1) (Lange and Pilkis,  Nucl. Acids Res.  18:3652, 1990).  
     [0023] The expression of many proto-oncogenes and cytokines bearing the AUUUA motif increases in cells as a consequence of mitogenic or pro-inflammatory stimulation. Thus, only very low levels of iPFK-2 expression were detected by Northern blotting of normal human tissues (i.e., heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen and lymph node). Northern analysis of human monocytes stimulated with lipopolysaccharide (LPS), by contrast, showed that the expression of this novel iPFK-2 gene was rapidly induced (FIG. 2A), hence the term “iPFK-2.” The induction and increase in the level of iPFK-2 expression was similar to that which was observed for the cytokine IL-1β (which also contains AU-rich elements) (FIG. 2B). The expression of the liver (constitutive) isoform of PFK-2 was unaffected by LPS stimulation. Induction of iPFK-2 mRNA was accompanied by a corresponding increase in immunoreactive iPFK-2 protein, as measured by Western blotting analysis utilizing a specific anti-iPFK-2 antibody (FIG. 2C). These data demonstrate that iPFK-2, like other genes with AU-rich motifs in their 3′UTR, is induced in primary human monocytes upon pro-inflammatory activation in vitro. In a separate experiment, iPFK-2 expression in peripheral blood leukocytes of 5 HIV-infected patients was examined. In each case, the level of iPFK-2 mRNA was higher than that observed in control, uninfected individuals (n=3). These data suggest that iPFK-2 is induced upon leukocyte activation in vivo.  
     [0024] An increase in the level of stable expression of proto-oncogenes with AU-rich 3′UTR&#39;s is a characteristic feature of many transformed cells and can be directly oncogenic (Lee et al.,  Mol. Cell. Biol.  8:5521-5527, 1988; Rabbitts et al.,  EMBO J.  4:3727-3733, 1985; and Piechaczyk et al.,  Cell  42:589-597, 1985). Eight human tumor cell lines were examined for iPFK-2 mRNA by Northern blotting and high levels of expression were found (FIG. 3A). The intensities of iPFK-2 hybridization signals were comparable to iPFK-2 signals observed in the RNA obtained from LPS-stimulated primary human monocytes (FIG. 2B). Closer examination of the K562 chronic myelogenous leukemia cell line showed that the expression of iPFK-2 was much higher than that of the hepatic PFK-2 isoform (FIG. 3B). These data suggest that iPFK-2 expression is important in regulating the glycolytic pathway during tumor cell growth.  
     [0025] K562 leukemia cells were transfected with iPFK-2-specific anti-sense oligonucleotides. Both iPFK-2 protein and F2,6BP levels were significantly decreased when compared to cells transfected with oligonucleotide controls (FIG. 4A). These data indicate that the kinase activity of iPFK-2 contributes significantly to intracellular F2,6BP levels. The enhanced glycolytic flux in transformed cells facilitates the biosynthesis of 5-phosphoribosyl pyrophosphate (PRPP), a critical precursor for purine and pyrimidine biosynthesis (Eifenbrody et al.,  Trends Pharmacol. Sci.  1:24-245, 1980). Inhibition of iPFK-2 was found to significantly decrease PRPP levels in K562 cells and this decrease was associated with a corresponding decrease in K562 DNA synthesis and cell proliferation (FIG. 4B). A similar level of inhibition of DNA synthesis was observed after the transfection of iPFK-2 anti-sense oligonucleotides into HL-60, MOLT-4, SW480, G361, and KG1A cell lines. These observations indicate that iPFK-2 catalyzed F2-6BP production may enhance glycolytic flux (through stimuloation of PFK-1) and permit increased channeling of glucose metabolism in the direction of PRPP synthesis.  
     [0026] The role of iPFK-2 in tumorigenesis in vivo was examined by administering iPFK-2-specific antisense oligonucleotides to K562 tumor-bearing nude mice. Within 2 days of treatment, tumors for the iPFK-2 antisense-treated mice were significantly smaller than tumors from the iPFK-2 sense oligonucleotide or PBS-treated mice (FIG. 4C).  
     [0027] Procedures and reagents have been developed to test for iPFK-2 inhibitors using rapid in vitro assays suitable for high throughput screening of compounds or libraries of compounds. By homology to known PFK-2 functional domains, the iPFK-2 polypeptide comprises two distinct domains: the amino-terminal portion of the peptide comprises the kinase domain (responsible for phosphorylating fructose 6-phosphate to yield fructose 2,6-bisphosphate) and the carboxy-terminal portion comprises the phosphatase domain (responsible for hydolyzing fructose 2,6-bisphosphate to yield fructose 6-phosphate).  
     [0028] Expression of iPFK2  
     [0029] The complete iPFK2 open reading frame was cloned by screening an EST cDNA library using an iPFK-2 specific probe, and then cloned into the pT7T3D-Pac plasmid (Pharmacia). The sequence of this insert was confirmed as correct by restriction digestion and sequencing. Insert-carrying vector was used to transform bacteria, and positive colonies were detected by performing restriction analysis and sequencing. Finally, the insert was excised and re-cloned into the pET11b expression vector (Novagen) and expressed following the general procedure described below.  
     [0030] Expression of iPFK2 Kinase Domain  
     [0031] The iPFK-2 kinase domain was obtained by PCR cloning. Briefly, a cDNA pool, obtained from reverse transcription of total mRNA of LPS-stimulated human monocytes was used as template for PCR amplification. Two specific primers, containing NdeI and BamHI restriction sites as a 5′ extension, were custom synthesized: P1, 5′-ACATATGCCGTTGGAACTGACGCAGAGC-3′ [SEQ ID NO. 27], P2, 5′-TGGATCCTCACAGGTAGATGGTACGCGGCT-3′ [SEQ ID NO. 28]. The amplified product was found to be at the predicted size and to correspond to the nucleotide sequence of the iPFK-2 kinase domain (corresponding to the positions 47-797 of the iPFK-2 open reading frame). This DNA amplification product was purified with a GENECLEAN DNA purification kit (BIO IO) and then cloned both into the pT7Blue cloning t-vector (Novagen) and into the pET14b expression vector (Novagen), which contains a histidine tag. Insert-carrying clones were detected by PCR screening, and the insert sequence was checked by sequencing. A single pET14b insert-positive colony was inoculated into 2 ml of LB broth and incubated at 37° C. for 3 hours. The bacterial culture was then transferred to a larger flask, containing 100 ml of LB broth, and incubated at 37° C. Once the OD 600  reached 0.7, the culture was induced by adding IPTG (1 mM), and allowed to grow under shaking overnight at 25° C. Finally, the culture was centrifuged, the supernatant was aspirated and the pellet was frozen to −70° C. These reserved cells were resuspended in lysis buffer (B-PER, Pierce), centrifuged and the supernatant was stored for later use as soluble proteins at −20° C. The kinase domain peptide was purified by using a histidine tag purification kit (Novagen) and tested for its enzymatic activity as described below. This procedure can be used to produce histidine-tagged polypeptides for the kinase domain or the phosphatase domain.  
     [0032] Assay for iPFK2 Kinase Activity  
     [0033] Briefly, the proceure of Sakata et al. ( J. Biol. Chem.  266: 15764-15770, 1991) was followed. The enzymatic activity of iPFK-2 peptide or the iPFK-2 kinase domain (from natural or recombinant sources) is conveniently assayed by measuring production of the metabolite Fru 2,6BP from Fru 6-P and ATP. One unit of activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of metabolite per minute. The reaction is conveniently carried out at 30° C. in a final volume of 200 μl, containing 100 mM Tris-HCl, pH 7.5, 2 mM DTT, 0.1 mM EDTA, 5 mM ATP, 1 mM F6-P, 5 mM potassium phosphate, and 10 mM MgCl 2 . At timed intervals, 20 μl-aliquots are transferred into 180 μl of 50 mM NaOH, and the diluted solutions are heated to 90° C. to stop the reaction. A suitable aliquot of the heated solutions is assayed for F2,6-BP as described below. Test agents are conveniently evaluated as inhibitors of iPFK-2 kinase activity by inclusion in the above incubation, followed by assay for the production of F2,6BP; control experiments are used to determine that test agents do not themselves interfere with the subsequent fructose 2,6-bisphosphate assay.  
     [0034] Fructose 2,6-Bisphosphate Assay  
     [0035] Briefly, the procedure in Van Schaftingen et al. ( Eur. J. Biochem.  129:191-195, 1982) was followed. The F2,6BP assay is conveniently carried out in a 96-well format, at a final volume of 300 μl. In each well, 30 μl enzyme solution (4.5 U/ml aldolase, 17 U/ml glycerol-3-P-dehydrogenase, 50 U/ml triose isomerase, 0.1 U/ml fructose bisphosphate kinase 1-pyrophosphate dependent, in 0.2% BSA) are added to 150 μl of buffer solution (100 mM Tris/acetate buffer, 4 mM magnesium acetate, 100 mM fructose-6-phosphate, in the presence of 0.3 mM NADH). Samples (or standard) (105 μl) are then dispensed into each well, mixing by pipetting. This reaction mixture is incubated at RT for 5 min and then 15 μl of 10 mM pyrophosphate are simultaneously added to each sample and control well, mixing twice by pipetting. The reaction leads to an oxidation of NADH, which is monitored by reading the absorbance of the samples at 1 min intervals for 10 min at 340 nm, yielding OD/min values. The rate of change in absorbance per unit time is a hyperbolic function of the concentration of F2,6BP present in the sample.  
     [0036] Experimental values are determined by interpolation of an appropriate standard curve of fructose 2,6 bisphosphate.  
     EXAMPLE 1  
     [0037] This example illustrates the initial cloning of the iPFK-2 sequence. An expressed sequence tag (EST) containing an AU-rich element was identified in the dbEST database at the National Center for Biotechnology Information by performing a TBLASTN search using the query sequence ATTTATTTATTTA [SEQ ID NO.: 12]. AU-rich EST (GenBank ID F00287) had been obtained from a Homo sapiens skeletal muscle cDNA library and was unrelated to previously identified sequences. 5′- and 3′-rapid amplification of complementary DNA ends (RACE) was performed using a Human Skeletal Muscle Marathon cDNA-ready RACE kit (Clontech Laboratories, Inc., Palo Alto, Calif.). Gene-specific oligonucleotides used for sequential 5′-directed RACE include 5′-ATTGGTCTGGCAACTGCAAA-3′ [SEQ ID NO.: 19], 5′-GATTGTACCATACCTGAAGCACAGCCTC-3′ [SEQ ID NO.: 13], 5′-TCTCCTGCCGCTCCAGCTCCATGATCAC-3′ [SEQ ID NO.: 14], and 5′-GTCAGCTTCTTGGAGATGTAGGTCTTGC-3′ [SEQ ID NO.: 15]. Gene-specific oligonucleotides used for 3′-directed RACE include 5′-TTGGTTTGGGAGCCTCCTATGTGTGACT-3′ [SEQ ID NO.: 16] and 5′-TTGGCGTCTACTGATTCCTCCAACTCTC-3′ [SEQ ID NO.: 17]. DNA amplification products were purified with a QIAEX DNA gel extraction kit (Qiagen, Germany) and then cloned into the pT7Blue T-vector (Novagen, Madison, Wis.). For each amplification product, five recombinant clones were isolated and the DNA inserts were sequenced bidirectionally using the Taq DyeDeoxy Terminator Cycle sequencing kit and an ABI Model 373A DNA sequencer (Applied Biosystems, Foster City, Calif.). The entire predicted amino acid sequence of human iPFK-2 is presented in FIG. 1, which shows a comparison against related sequences.  
     EXAMPLE 2  
     [0038] This example illustrates that LPS induces peripheral blood monocytes to rapidly express iPFK-2 mRNA and protein. PBMCs were isolated by density gradient centrifugation of whole blood through Ficoll (Ficoll-Paque, endotoxin-tested; Pharmacia, Piscataway, N.J.) and then cultured in 6-well plates (2×10 cells/ml/well RPMI with 10% fetal bovine serum, Hyclone Labs, Logan, Utah). Nonadherent cells were removed by changing the media after 24 hours and the remaining, adherent monocytes were incubated alone as control or in the presence of 1 mg/ml LPS ( E. coli  0111:B4; Sigma Chemical Co., St. Louis, Mo.). After incubation for 1.5, 3, 6, 12, or 24 hours, cells were lifted, collected by centrifugation at 300 g for 10 min, and immediately analyzed. Total cellular RNA was isolated by a modified guanidinium isothiocyanate method (RNAzol, Cinna Biotecx, Friendswood, Tex.). For RT-PCR analysis, CDNA was prepared from 1.0 mg of total RNA using 0.25 ng of oligo-(dT) and Superscript II following the manufacturer&#39;s protocol (Gibco/BRL, Grand Island, N.Y.). Two μl aliquots of cDNA then were amplified by PCR in a Perkin-Elmer model 9600 thermal cycler using the primers listed below and the following cycling program: denaturation for 15 sec at 95° C., annealing for 20 sec at 55° C., and extension for 30 sec at 72° C. for the indicated cycles with a final extension for 5 min at 72° C. The following human mRNA primers were custom synthesized: P-Actin, 5′-TAAGGAGAAGCTGTGCTACG-3′ [SEQ ID NO.: 7],5′-ATCTCCTTCTGCATCCTGTC-3′ [SEQ ID NO.: 8]; IL-1β, 5′-CTGTACCTGTCCTGCGTGTT-3′ [SEQ ID NO.: 18], 5′-AGCTCTCTTTAGGAAGACAC-3′ [SEQ ID NO.: 19]; iPFK-2, 5′-ATTGGTCTGGCAACTGCAAA-3′ [SEQ ID NO.: 9], 5′-GGAGCCTCCTATGTGTGACT-3′ [SEQ ID NO.: 10]; Liver PFK-2, 5′-GAAGTCAAACTGAATGTGTC-3′ [SEQ ID NO.: 20],5′-CCTCTTGTAGGCAGTAAGTC-3′ [SEQ ID NO.: 21] (and 5′-AGGCAGTAAGTCTTTATTCG-3′ [SEQ ID NO.: 22],5′-AAGTCAAACTGCCTGTGTCC-3′ [SEQ ID NO.: 23], data not shown) (Gibco/BRL). For Northern blot analysis, RNA (7.5 μg) was electrophoresed through 1.5% agarose-formaldehyde gels, transferred onto nylon membranes (Schleicher &amp; Schuell), and hybridized sequentially with cDNA probes for human iPFK-2 and β-Actin. Probes were produced by PCR using primers described above and then labeled with  32 P by the random-priming method (Megaprime kit, Amersham). Autoradiography was performed at room temperature for 2-6 hr using DuPont Reflection films and intensifying screens. For Western blot analysis, cells were lysed in 2× Laemle sample buffer for 5 min at 95° C. and total cellular proteins were resolved by electrophoresis through 18% SDS polyacrylamide gels under reducing conditions and transferred onto nitrocellulose membranes (Schleicher &amp; Schuell). Membranes were incubated with rabbit polyclonal anti-human iPFK-2 serum (produced by immunization of rabbits with an iPFK-2 carboxy terminal peptide ([NH 2 ]-GQPLLGQACLT-[COOH]) [SEQ ID NO.24] that was conjugated to KLH. This peptide comprises a unique region of iPFK-2 (amino acids 505-515) that differs from the corresponding portion of placental PFK-2 by a 5 amino acid deletion and 8 amino acid mismatches. Immunoreactive iPFK-2 (M r =59 kD) then was visualized by developing membranes with a donkey peroxidase-conjugated anti-rabbit IgG antibody (1:8,000) followed by reaction with ECL reagents (Amersham International, Buckinghamshire, England) (see FIG. 2).  
     EXAMPLE 3  
     [0039] This example illustrates iPFK-2 mRNA expression by human cancer cell lines. A Northern blot, containing 2 μg of polyA RNA per lane from 8 different human cell lines (Clontech Labs), was hybridized sequentially with cDNA probes for GADPH (Clontech Labs) and iPFK-2 as in example 2 above. The cell lines were: promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K562, lymphoblastic leukemia MOLT-4, Burkitt&#39;s lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. For RT-PCR analysis, two μl aliquots of K562 CDNA were amplified by PCR for the indicated cycles in a Perkin-Elmer model 9600 thermal cycler using β-Actin-, iPFK-2, or liver PFK-2-specific primers (primer sequences listed in example 2 above). These data (shown in FIG. 3) show that iPFK-2 is expressed by a large variety of human cancer cell lines and is a likely tumor marker enzyme sequence that can be used to measure the progress of cancer treatment, to initially identify cells as cancerous or to identify a patient as tumor-bearing.  
     EXAMPLE 4  
     [0040] This example illustrates that iPFK-2-specific anti-sense oligonucleotides inhibit K562 cell proliferation in vitro. K562 cells (ATCC) in exponential growth phase were cultured in triplicate in 96-well plates (5×10 3  cells/well) in RPMI (Gibco/BRL) supplemented with 10% FBS. Cells were incubated with PBS as control or transfected by the lipofectin method (Gibco/BRL) for 20 hours with the following phosphorothioate oligonucleotides: S-iPFK-2 (A) (sense, position 35-55): 5′-AGCCGCGAAGATGCCGTTGG-3′ [SEQ ID NO.: 1]; AS-iPFK-2 (A) (anti-sense, position 35-55): 5′-CCAACGGCATCTTCGCGGCT-3′ [SEQ ID NO.: 2]; S-IPFK-2 (B) (sense, position 42-62): 5′-AAGATGCCGTTGGAACTGAC-3′ [SEQ ID NO.: 3]; AS-iPFK-2 (B) (anti-sense, position 42-62): 5′-GTCAGTTCCAACGGCATCTT-3′ [SEQ ID NO.: 4]. Western blot analysis was performed as provided in example 2. Total cellular fructose-2,6-bisphosphate and 5-phosphoribosyl 1-pyrophosphate were measured using the methods described in Van Schaftingen,  Methods. Enz. Anal.  6:335-341, 1984 and Sant et al.,  J. Biol Biochem.  16:11038-11045, 1992, respectively. K562 proliferative activity was measured by the incorporation of [ 3 H]thymidine (4 μCi/ml) (DuPont, Boston, Mass.) into DNA over the last 14 hours of incubation/transfection as quantified by liquid scintillation counting. Data in FIG. 4 are expressed as the mean±SD (n=3). Statistical significance was assessed by two sample T-tests (assuming unequal variances) (*, p&lt;0.05) (Taetle et al.,  Cancer Trmt. Reports  71:297-304, 1987). FIG. 4 shows that a group of antisense oligonucleotides have iPFK-2 antagonist activity and will likely exhibit significant anti-cancer therapeutic activity in view of the widespread prevalence of iPFK-2 and the Warburg effect known for tumor tissue.  
     EXAMPLE 5  
     [0041] This example illustrates that iPFK-2-specific anti-sense oligonucleotides inhibit K-562 tumor growth in vivo. K562 tumor-bearing mice were implanted for the indicated days (FIG. 5) with micro-osmotic pumps containing PBS, (o); S-iPFK-2 (B), (□); or AS-iPFK-2 (B), (▪). K562 cells were collected from exponential growth phase culture in RPMI medium supplemented with 10% FBS and then washed twice and resuspended in PBS (1×10 7  cells/ml). Groups of 5 female BALB/c nude mice (20 gm) (Harlan Labs) were injected s.c. with 0.10 ml of the K562 suspension (1×10 6  cells). The tumors were allowed to grow for 7 days to a mean weight of 0.4 gm before treatment was begun. Alzet micro-osmotic pumps (Alza Corporation, Palo Alto, Calif.) loaded with 0.1 ml of PBS or the phosphorothioate oligonucleotides S-iPFK-2 (B) or AS-iPFK-2 (B) (3.0 mM in PBS, see example 4 for sequences) were implanted s.c. into the tumor-bearing mice. Tumor size after 0, 1, 2, 3, and 4 days was determined with Vernier calipers according the following formula: weight (mg)=(width, mm 2 ×(length, mm 2 ) (Taetle et al.,  Cancer Trmt. Reports  71:297-304, 1987). FIG. 5 shows that the antisense oligonucleotides that exhibited iPFK-2 antagonist activity also demonstrate anti-cancer therapeutic activity. Therefore, iPFK-2 antagonists are useful for treating cancers.  
     EXAMPLE 6  
     [0042] This example illustrates that endotoxemia induces mouse iPFK-2 mRNA expression in spleen and muscle. 10 week-old BALB/c mice (19-20 gm) were injected i.p. with LPS (12.5 mg/kg) or saline as control. After 6 and 24 hours mice were euthanized by CO 2  asphyxiation and the brain, liver, lower extremity muscles, and spleen were removed by dissection. Total RNA extraction and Northern blot analysis were performed as above using a mouse iPFK-2-specific cDNA probe (amplified from mouse peritoneal macrophage cDNA by 30 cycle RT-PCR using the following human iPFK-2-specific primers: 5′-TGAGGCAGACGTGTCGGTTC-3′ [SEQ ID NO.: 25], 5′-CAGCAGCTCCAGGAAAGTGT-3′ [SEQ ID NO.: 26]).  
     [0043] The results are presented in FIG. 6 and show that LPS induced iPFK-2 mRNA expression in mouse tissues. These data illustrate the predictive pharmacologic importance of iPFK-2 as a therapeutic marker for inflammatory conditions.  
     EXAMPLE 7  
     [0044] This example illustrates that iPFK-2 is overexpressed in peripheral blood mononuclear cells (PBMCs) of HIV+individuals. Total RNA was isolated from 3 uninfected individuals (lanes 1-3) and 5 HIV+individuals (lanes 4-8) and analyzed by RT-PCR with β-Actin-specific primers (5′-TAAGGAGAAGCTGTGCTACG-3′ [SEQ ID NO.: 7], 5′-ATCTCCTTCTGCATCCTGTC-3′ [SEQ ID NO.: 8], 19 cycles) and iPFK-2-specific primers (5′-ATTGGTCTGGCAACTGCAAA-3′ [SEQ ID NO.: 9], 5′-GGAGCCTCCTATGTGTGACT-3′ [SEQ ID NO.: 10], 23 cycles).  
     [0045] The results are presented in FIG. 7, and show that iPFK-2 is over-expressed in PBMCs from HIV+ individuals.  
     EXAMPLE 8  
     [0046] This example illustrates anti-tumor therapeutic activity of iPFK-2 antagonists. Test compounds, such as the potential iPFK-2 antagonist 2,5-anhydro-D-mannitol are for inhibition of iPFK-2 enzymatic (i.e., kinase) activity using recombinant iPFK-2 polypeptide as provided herein. 2,5-anhydro-D-mannitol, or other test compounds may then be further tested in an in vitro assay of anti-tumor therapeutic activity that correlates iPFK-2 inhibition of iPFK-2 kinase enzymatic activity with therapeutic anti-tumor pharmacologic activity. K562 tumor cells (1×10 4  cells grown in RPMI supplemented with 10% FBS) are exposed to different concentrations of 2,5-anhydro-D-mannitol or other test compounds to control sugar (glucose) for 12 hours. A cell proliferation assay, for instance, measuring tritiated thymidine uptake, is then used to estimate tumor cell proliferation. Thus, this procedure helps to determine if a test compound is an iPFK-2 antagonist, wherein an antagonist is an agent that acts on iPFK-2 to decrease F2,6 P and/or increase F6P in the assay.  
     [0047] A further tumor cell proliferation assay was also conducted, as described in example 4, with two different iPFK-2 antisense oligonuclotides using the T cell tumor cell line MOLT-4. As shown in FIG. 9, both antisense oligonucleotides inhibited tumor cell proliferation and exhibited anti-tumor therapeutic activity in this predictive in vitro assay.  
    
     
       
         1 
         
           
             31  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               hiPFK-2 antisense  
             
           
            1 

agccgcgaag atgccgttgg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               hi-PFK-2 antisense  
             
           
            2 

ccaacggcat cttcgcggct                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               hi-PFK-2 antisense  
             
           
            3 

aagatgccgt tggaactgac                                                 20 

 
           
             4  
             20  
             DNA  
             Artificial Sequence  
             
               hi-PFK-2 antisense  
             
           
            4 

gtcagttcca acggcatctt                                                 20 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            5 

tgaggcagac gtgtcggttc                                                 20 

 
           
             6  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            6 

cagcagctcc aggaaagtgt                                                 20 

 
           
             7  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            7 

taaggagaag ctgtgctacg                                                 20 

 
           
             8  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            8 

atctccttct gcatcctgtc                                                 20 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            9 

attggtctgg caactgcaaa                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            10 

ggagcctcct atgtgtgact                                                 20 

 
           
             11  
             4220  
             DNA  
             Artificial Sequence  
             
               human iPFK-2  
             
           
            11 

caggtcgagc ggcagggcct ggtggcgaga gcgcagccgc gaagatgccg ttggaactga     60 

cgcagagccg agtgcagaag atctgggtgc ccgtggacca caggccctcg ttgcccagat    120 

cctgtgggcc aaagctgacc aactccccca ccgtcatcgt catggtgggc ctccccgccc    180 

ggggcaagac ctacatctcc aagaagctga ctcgctacct caactggatt ggcgtcccca    240 

caaaagtgtt caacgtcggg gagtatcgcc gggaggctgt gaagcagtac agctcctaca    300 

acttcttccg ccccgacaat gaggaagcca tgaaagtccg gaagcaatgt gccctagctg    360 

ccttgagaga tgtcaaaagc tacctggcga aagaaggggg acaaattgcg gttttcgatg    420 

ccaccaatac tactagagag aggagacaca tgatcctcca ttttggcaaa gaaaatgact    480 

ttaaggcgtt tttcatcgag tcggtgtgcg acgaccctac agttgtggcc tccaatatca    540 

tggaagttaa aatctccagc ccggattaca aagactgcaa ctcggcagaa gccatggacg    600 

acttcatgaa gaggatcagt tgctatgaag ccagctacca gcccctcgac cccgacaaat    660 

gcgacaggga cttgtcgctg atcaaggtga ttgacgtggg ccggaggttc ctggtgaacc    720 

gggtgcagga ccacatccag agccgcatcg tgtactacct gatgaacatc cacgtgcagc    780 

cgcgtaccat ctacctgtgc cggcacggcg agaacgagca caacctccag ggccgcatcg    840 

ggggcgactc aggcctgtcc agccggggca agaagtttgc cagtgctctg agcaagttcg    900 

tggaggagca gaacctgaag gacctgcgcg tgtggaccag ccagctgaag agcaccatcc    960 

agacggccga ggcgctgcgg ctgccctacg agcagtggaa ggcgctcaat gagatcgacg   1020 

cgggcgtctg tgaggagctg acctacgagg agatcaggga cacctaccct gaggagtatg   1080 

cgctgcggga gcaggacaag tactattacc gctaccccac cggggagtcc taccaggacc   1140 

tggtccagcg cttggagcca gtgatcatgg agctggagcg gcaggagaat gtgctggtca   1200 

tctgccacca ggccgtcctg cgctgcctgc ttgcctactt cctggataag agtgcagagg   1260 

agatgcccta cctgaaatgc cctcttcaca ccgtcctgaa actgacgcct gtcgcttatg   1320 

gctgccgtgt ggaatccatc tacctgaacg tggagtccgt ctgcacacac cgggagaggt   1380 

cagaggatgc aaagaaggga cctaacccgc tcatgagacg caatagtgtc accccgctag   1440 

ccagccccga acccaccaaa aagcctcgca tcaacagctt tgaggagcat gtggcctcca   1500 

cctcggccgc cctgcccagc tgcctgcccc cggaggtgcc cacgcagctg cctggacaac   1560 

ctttgctagg gcaagcctgt ctaacatgaa aggttcccgg agcagcgctg actcctccag   1620 

gaaacactga ggcagacgtg tcggttccat tccatttcca tttctgcagc ttagcttgtg   1680 

tcctgccctc cgcccgaggc aaaacgtatc ctgaggactt cttccggaga gggtggggtg   1740 

gagcagcggg ggagccttgg ccgaagagaa ccatgcttgg caccgtctgt gtcccctcgg   1800 

ccgctggaca ccagaaagcc acgtgggtcc ctggcgccct gcctttagcc gtggggcccc   1860 

cacctccact ctctgggttt cctaggaatg tccagcctcg gagaccttca caaagccttg   1920 

ggagggtgat gagtgctggt cctgacaaga ggccgctggg gacactgtgc tgttttgttt   1980 

cgtttctgtg atctcccggc acgtttggag ctgggaagac cacactggtg gcagaatcct   2040 

aaaattaaag gaggcaggct cctagttgct gaaagttaag gaatgtgtaa aacctccacg   2100 

tgactgtttg gtgcatcttg acctgggaag acgcctcatg ggaacgaact tggacaggtg   2160 

ttgggttgag gcctcttctg caggaagtcc ctgagctgag acgcaagttg gctgggtggt   2220 

ccgcaccctg gctctcctgc aggtccacac accttccagg cctgtggcct gcctccaaag   2280 

atgtgcaagg gcaggctggc tgcacgggga gagggaagta ttttgccgaa atatgagaac   2340 

tggggcctcc tgctcccagg gagctccagg gcccctctct cctcccacct ggacttgggg   2400 

ggaactgaga aacactttcc tggagctgct ggcttttgca cttttttgat ggcagaagtg   2460 

tgacctgaga gtcccacctt ctcttcagga acgtagatgt tggggtgtct tgccctgggg   2520 

ggcttggaac ctctgaaggt ggggagcgga acacctggca tccttcccca gcacttgcat   2580 

taccgtccct gctcttccca ggtggggaca gtggcccaag caaggcctca ctcgcagcca   2640 

cttcttcaag agctgcctgc acactgtctt ggagcatctg ccttgtgcct ggcactctgc   2700 

cggtgccttg ggaaggtcgg aagagtggac tttgtcctgg ccttcccttc atggcgtcta   2760 

tgacactttt gtggtgatgg aaagcatggg acctgtcgtc tcagcctgtt ggtttctcct   2820 

cattgcctca aaccctgggg taggtgggac ggggggtctc gtgcccagat gaaaccattt   2880 

ggaaactcgg cagcagagtt tgtccaaatg acccttttca ggatgtctca aagcttgtgc   2940 

caaaggtcac ttttctttcc tgccttctgc tgtgagccct gagatcctcc tcccagctca   3000 

agggacaggt cctgggtgag ggtgggagat ttagacacct gaaactgggc gtggagagaa   3060 

gagccgttgc tgtttgtttt ttgggaagag cttttaaaga atgcatgttt ttttcctggt   3120 

tggaattgag taggaactga ggctgtgctt caggtatggt acaatcaagt gggggatttt   3180 

catgctgaac cattcaagcc ctccccgccc gttgcaccca ctttggctgg cgtctgctgg   3240 

agaggatgtc tctgtccgca ttcccgtgca gctccaggct cgcgcagttt tctctctccc   3300 

cctggatgtt gagtctcatc agaatatgtg ggtagggggt ggacgtgcac gggtgcatga   3360 

ttgtgcttaa cttggttgta tttttcgatt tgacatggaa ggcctgttgc tttgctcttg   3420 

agaatagttt ctcgtgtccc cctcgcaggc ctcattcttt gaacatcgac tctgaagttt   3480 

gatacagata ggggcttgat agctgtggtc cccctctccc ctctgactac ctaaaatcaa   3540 

tacctaaata cagaagcctt ggtctaacac gggactttta gtttgcgaag ggcctagata   3600 

gggagagagg taacatgaat ctggacaggg agggagatac tatagaaagg agaacactgc   3660 

ctactttgca agccagtgac ctgccttttg aggggacatt ggacgggggc cgggggcggg   3720 

ggttgggttt gagctacagt catgaacttt tggcgtctac tgattcctcc aactctccac   3780 

cccacaaaat aacggggacc aatattttta actttgccta tttgtttttg ggtgagtttc   3840 

ccccctcctt attctgtcct gagaccacgg gcaaagctct tcattttgag agagaagaaa   3900 

aactgtttgg aaccacacca atgatatttt tctttgtaat acttgaaatt tattttttta   3960 

ttattttgat agcagatgtg ctatttattt atttaatatg tataaggagc ctaaacaata   4020 

gaaagctgta gagattgggt ttcattgtta attggtttgg gagcctccta tgtgtgactt   4080 

atgacttctc tgtgttctgt gtatttgtct gaattaatga cctgggatat aaagctatgc   4140 

tagctttcaa acaggagatg cctttcagaa atttgtatat tttgcagttg ccagaccaat   4200 

aaaatacctg gttgaaatac                                               4220 

 
           
             12  
             13  
             DNA  
             Artificial Sequence  
             
               query sequence  
             
           
            12 

atttatttat tta                                                        13 

 
           
             13  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            13 

gattgtacca tacctgaagc acagcctc                                        28 

 
           
             14  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            14 

tctcctgccg ctccagctcc atgatcac                                        28 

 
           
             15  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            15 

gtcagcttct tggagatgta ggtcttgc                                        28 

 
           
             16  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            16 

ttggtttggg agcctcctat gtgtgact                                        28 

 
           
             17  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            17 

ttggcgtcta ctgattcctc caactctc                                        28 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            18 

ctgtacctgt cctgcgtgtt                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            19 

agctctcttt aggaagacac                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            20 

gaagtcaaac tgaatgtgtc                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            21 

cctcttgtag gcagtaagtc                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            22 

aggcagtaag tctttattcg                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            23 

aagtcaaact gcctgtgtcc                                                 20 

 
           
             24  
             11  
             PRT  
             Artificial Sequence  
             
               polypeptide  
             
           
            24 

Gly Gln Pro Leu Leu Gly Gln Ala Cys Leu Thr 
 1               5                  10 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            25 

tgaggcagac gtgtcggttc                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            26 

cagcagctcc aggaaagtgt                                                 20 

 
           
             27  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            27 

acatatgccg ttggaactga cgcagagc                                        28 

 
           
             28  
             30  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            28 

tggatcctca caggtagatg gtacgcggct                                      30 

 
           
             29  
             514  
             PRT  
             Artificial Sequence  
             
               iPFK-2  
             
           
            29 

Met Pro Leu Glu Leu Thr Gln Ser Arg Val Gln Lys Ile Trp Val Pro 
 1               5                  10                  15 

Val Asp His Arg Pro Ser Leu Pro Arg Ser Cys Gly Pro Lys Leu Thr 
            20                  25                  30 

Asn Ser Pro Thr Val Ile Val Met Val Gly Leu Pro Ala Arg Gly Lys 
        35                  40                  45 

Thr Tyr Ile Ser Lys Lys Leu Thr Arg Tyr Leu Asn Trp Ile Gly Val 
    50                  55                  60 

Pro Thr Lys Val Phe Asn Val Gly Glu Tyr Arg Arg Glu Ala Val Lys 
65                  70                  75                  80 

Gln Tyr Ser Ser Tyr Asn Phe Phe Arg Pro Asp Asn Glu Glu Ala Met 
                85                  90                  95 

Lys Val Arg Lys Gln Cys Ala Leu Ala Ala Leu Arg Asp Val Lys Ser 
            100                 105                 110 

Tyr Leu Ala Lys Glu Gly Gly Gln Ile Ala Val Phe Asp Ala Thr Asn 
        115                 120                 125 

Thr Thr Arg Glu Arg Arg His Met Ile Leu His Phe Gly Lys Glu Asn 
    130                 135                 140 

Asp Phe Lys Ala Phe Phe Ile Glu Ser Val Cys Asp Asp Pro Thr Val 
145                 150                 155                 160 

Val Ala Ser Asn Ile Met Glu Val Lys Ile Ser Ser Pro Asp Tyr Lys 
                165                 170                 175 

Asp Cys Asn Ser Ala Glu Ala Met Asp Asp Phe Met Lys Arg Ile Ser 
            180                 185                 190 

Cys Tyr Glu Ala Ser Tyr Gln Pro Leu Asp Pro Asp Lys Cys Asp Arg 
        195                 200                 205 

Asp Leu Ser Leu Ile Lys Val Ile Asp Val Gly Arg Arg Phe Leu Val 
    210                 215                 220 

Asn Arg Val Gln Asp His Ile Gln Ser Arg Ile Val Tyr Tyr Leu Met 
225                 230                 235                 240 

Asn Ile His Val Gln Pro Arg Thr Ile Tyr Leu Cys Arg His Gly Glu 
                245                 250                 255 

Asn Glu His Asn Leu Gln Gly Arg Ile Gly Gly Asp Ser Gly Leu Ser 
            260                 265                 270 

Ser Arg Gly Lys Lys Phe Ala Ser Ala Leu Ser Lys Phe Val Glu Glu 
        275                 280                 285 

Gln Asn Leu Lys Asp Leu Arg Val Trp Thr Ser Gln Leu Lys Ser Thr 
    290                 295                 300 

Ile Gln Thr Ala Glu Ala Leu Arg Leu Pro Tyr Glu Gln Trp Lys Ala 
305                 310                 315                 320 

Leu Asn Glu Ile Asp Ala Gly Val Cys Glu Glu Leu Thr Tyr Glu Glu 
                325                 330                 335 

Ile Arg Asp Thr Tyr Pro Glu Glu Tyr Ala Leu Arg Glu Gln Asp Lys 
            340                 345                 350 

Tyr Tyr Tyr Arg Tyr Pro Thr Gly Glu Ser Tyr Gln Asp Leu Val Gln 
        355                 360                 365 

Arg Leu Glu Pro Val Ile Met Glu Leu Glu Arg Gln Glu Asn Val Leu 
    370                 375                 380 

Val Ile Cys His Gln Ala Val Leu Arg Cys Leu Leu Ala Tyr Phe Leu 
385                 390                 395                 400 

Asp Lys Ser Ala Glu Glu Met Pro Tyr Leu Lys Cys Pro Leu His Thr 
                405                 410                 415 

Val Leu Lys Leu Thr Pro Val Ala Tyr Gly Cys Arg Val Glu Ser Ile 
            420                 425                 430 

Tyr Leu Asn Val Glu Ser Val Cys Thr His Arg Glu Arg Ser Glu Asp 
        435                 440                 445 

Ala Lys Lys Gly Pro Asn Pro Leu Met Arg Arg Asn Ser Val Thr Pro 
    450                 455                 460 

Leu Ala Ser Pro Glu Pro Thr Lys Lys Pro Arg Ile Asn Ser Phe Glu 
465                 470                 475                 480 

Glu His Val Ala Ser Thr Ser Ala Ala Leu Pro Ser Cys Leu Pro Pro 
                485                 490                 495 

Glu Val Pro Thr Gln Leu Pro Gly Gln Pro Leu Leu Gly Gln Ala Cys 
            500                 505                 510 

Leu Thr 

 
           
             30  
             520  
             PRT  
             Artificial Sequence  
             
               Placental PFK-2  
             
           
            30 

Met Pro Leu Glu Leu Thr Gln Ser Arg Val Gln Lys Ile Trp Val Pro 
 1               5                  10                  15 

Val Asp His Arg Pro Ser Leu Pro Arg Ser Cys Gly Pro Lys Leu Thr 
            20                  25                  30 

Asn Ser Pro Thr Val Ile Val Met Val Gly Leu Pro Ala Arg Gly Lys 
        35                  40                  45 

Thr Tyr Ile Ser Lys Lys Leu Thr Arg Tyr Leu Asn Trp Ile Gly Val 
    50                  55                  60 

Pro Thr Lys Val Phe Asn Val Gly Glu Tyr Arg Arg Glu Ala Val Lys 
65                  70                  75                  80 

Gln Tyr Ser Ser Tyr Asn Phe Phe Arg Pro Asp Asn Glu Glu Ala Met 
                85                  90                  95 

Lys Val Arg Lys Gln Cys Ala Leu Ala Ala Leu Arg Asp Val Lys Ser 
            100                 105                 110 

Tyr Leu Ala Lys Glu Gly Gly Gln Ile Ala Val Phe Asp Ala Thr Asn 
        115                 120                 125 

Thr Thr Arg Glu Arg Arg His Met Ile Leu His Phe Gly Lys Glu Asn 
    130                 135                 140 

Asp Phe Lys Ala Phe Phe Ile Glu Ser Val Cys Asp Asp Pro Thr Val 
145                 150                 155                 160 

Val Ala Ser Asn Ile Met Glu Val Lys Ile Ser Ser Pro Asp Tyr Lys 
                165                 170                 175 

Asp Cys Asn Ser Ala Glu Ala Met Asp Asp Phe Met Lys Arg Ile Ser 
            180                 185                 190 

Cys Tyr Glu Ala Ser Tyr Gln Pro Leu Asp Pro Asp Lys Cys Asp Arg 
        195                 200                 205 

Asp Leu Ser Leu Ile Lys Val Ile Asp Val Gly Arg Arg Phe Leu Val 
    210                 215                 220 

Asn Arg Val Gln Asp His Ile Gln Ser Arg Ile Val Tyr Tyr Leu Met 
225                 230                 235                 240 

Asn Ile His Val Gln Pro Arg Thr Ile Tyr Leu Cys Arg His Gly Glu 
                245                 250                 255 

Asn Glu His Asn Leu Gln Gly Arg Ile Gly Gly Asp Ser Gly Leu Ser 
            260                 265                 270 

Ser Arg Gly Lys Lys Phe Ala Ser Ala Leu Ser Lys Phe Val Glu Glu 
        275                 280                 285 

Gln Asn Leu Lys Asp Leu Arg Val Trp Thr Ser Gln Leu Lys Ser Thr 
    290                 295                 300 

Ile Gln Thr Ala Glu Ala Leu Arg Leu Pro Tyr Glu Gln Trp Lys Ala 
305                 310                 315                 320 

Leu Asn Glu Ile Asp Ala Gly Val Cys Glu Glu Leu Thr Tyr Glu Glu 
                325                 330                 335 

Ile Arg Asp Thr Tyr Pro Glu Glu Tyr Ala Leu Arg Glu Gln Asp Lys 
            340                 345                 350 

Tyr Tyr Tyr Arg Tyr Pro Thr Gly Glu Ser Tyr Gln Asp Leu Val Gln 
        355                 360                 365 

Arg Leu Glu Pro Val Ile Met Glu Leu Glu Arg Gln Glu Asn Val Leu 
    370                 375                 380 

Val Ile Cys His Gln Ala Val Leu Arg Cys Leu Leu Ala Tyr Phe Leu 
385                 390                 395                 400 

Asp Lys Ser Ala Glu Glu Met Pro Tyr Leu Lys Cys Pro Leu His Thr 
                405                 410                 415 

Val Leu Lys Leu Thr Pro Val Ala Tyr Gly Cys Arg Val Glu Ser Ile 
            420                 425                 430 

Tyr Leu Asn Val Glu Ser Val Cys Thr His Arg Glu Arg Ser Glu Asp 
        435                 440                 445 

Ala Lys Lys Gly Pro Asn Pro Leu Met Arg Arg Asn Ser Val Thr Pro 
    450                 455                 460 

Leu Ala Ser Pro Glu Pro Thr Lys Lys Pro Arg Ile Asn Ser Phe Glu 
465                 470                 475                 480 

Glu His Val Ala Ser Thr Ser Ala Ala Leu Pro Ser Cys Leu Pro Pro 
                485                 490                 495 

Glu Val Pro Thr Gln Leu Pro Gly Gln Asn Met Lys Gly Ser Arg Ser 
            500                 505                 510 

Ser Ala Asp Ser Ser Arg Lys His 
        515                 520 

 
           
             31  
             471  
             PRT  
             Artificial Sequence  
             
               Liver PFK-2  
             
           
            31 

Met Ser Pro Glu Met Gly Glu Leu Thr Gln Thr Arg Leu Gln Lys Ile 
 1               5                  10                  15 

Trp Ile Pro His Ser Ser Gly Ser Ser Arg Leu Gln Arg Arg Arg Gly 
            20                  25                  30 

Ser Ser Ile Pro Gln Phe Thr Asn Ser Pro Thr Met Val Ile Met Val 
        35                  40                  45 

Gly Leu Pro Ala Arg Gly Lys Thr Tyr Ile Ser Thr Lys Leu Thr Arg 
    50                  55                  60 

Tyr Leu Asn Trp Ile Gly Thr Pro Thr Lys Val Phe Asn Leu Gly Gln 
65                  70                  75                  80 

Tyr Arg Arg Glu Ala Val Ser Tyr Lys Asn Tyr Glu Phe Phe Leu Pro 
                85                  90                  95 

Asp Asn Val Glu Ala Leu Gln Ile Arg Lys Gln Cys Ala Leu Ala Ala 
            100                 105                 110 

Leu Lys Asp Val His Asn Tyr Leu Ser His Glu Glu Gly His Val Ala 
        115                 120                 125 

Val Phe Asp Ala Thr Asn Thr Thr Arg Glu Arg Arg Ser Leu Ile Leu 
    130                 135                 140 

Gln Phe Ala Lys Glu His Gly Tyr Lys Val Phe Phe Ile Glu Ser Ile 
145                 150                 155                 160 

Cys Asn Asp Pro Gly Ile Ile Ala Glu Asn Ile Arg Gln Val Lys Leu 
                165                 170                 175 

Gly Ser Pro Asp Tyr Ile Asp Cys Asp Arg Glu Lys Val Leu Glu Asp 
            180                 185                 190 

Phe Leu Lys Arg Ile Glu Cys Tyr Glu Val Asn Tyr Gln Pro Leu Asp 
        195                 200                 205 

Glu Glu Leu Asp Ser His Leu Ser Tyr Ile Lys Ile Phe Asp Val Gly 
    210                 215                 220 

Thr Arg Tyr Met Val Asn Arg Val Gln Asp His Ile Gln Ser Arg Thr 
225                 230                 235                 240 

Val Tyr Tyr Leu Met Asn Ile His Val Thr Pro Arg Ser Ile Tyr Leu 
                245                 250                 255 

Cys Arg His Gly Glu Ser Glu Leu Asn Ile Arg Gly Arg Ile Gly Gly 
            260                 265                 270 

Asp Ser Gly Leu Ser Val Arg Gly Lys Gln Tyr Ala Tyr Ala Leu Ala 
        275                 280                 285 

Asn Phe Ile Gln Ser Gln Gly Ile Ser Ser Leu Lys Val Trp Thr Ser 
    290                 295                 300 

Arg Met Lys Arg Thr Ile Gln Thr Ala Glu Ala Leu Gly Val Pro Tyr 
305                 310                 315                 320 

Glu Gln Trp Lys Ala Leu Asn Glu Ile Asp Ala Gly Val Cys Glu Glu 
                325                 330                 335 

Met Thr Tyr Glu Glu Ile Gln Glu His Tyr Pro Glu Glu Pro Ala Leu 
            340                 345                 350 

Arg Asp Gln Asp Lys Tyr Arg Tyr Arg Tyr Pro Lys Gly Glu Ser Tyr 
        355                 360                 365 

Glu Asp Leu Val Gln Arg Leu Glu Pro Val Ile Met Glu Leu Glu Arg 
    370                 375                 380 

Gln Glu Asn Val Leu Val Ile Cys His Gln Ala Val Val Arg Cys Leu 
385                 390                 395                 400 

Leu Ala Tyr Phe Leu Asp Lys Ser Ser Asp Glu Leu Pro Tyr Leu Lys 
                405                 410                 415 

Cys Pro Leu His Thr Val Leu Lys Leu Thr Pro Val Ala Tyr Gly Cys 
            420                 425                 430 

Lys Val Glu Ser Ile Tyr Leu Asn Val Glu Ala Val Asn Thr His Arg 
        435                 440                 445 

Glu Lys Pro Glu Asn Val Asp Ile Thr Arg Glu Pro Glu Glu Ala Leu 
    450                 455                 460 

Asp Thr Val Pro Ala His Tyr 
465                 470