WO1998036774A1 - Method of treating a lactate dehydrogenase-a (ldh-a)-associated disorder - Google Patents

Abstract

Methods of inhibiting proliferation of cells associated with a cell proliferative disorder by contacting the cells with a glucose antimetabolite where the cells have reduced levels of a member of the anti-apoptotic gene family, such as Bcl-2 are provided. Methods for identifying compounds which inhibit such cells are also disclosed.

Description

METHOD OF TREATING A LACTATE DEHYDROGENASE-A (LDH-AV

ASSOCIATED DISORDER

Field of the Invention

The present invention relates generally to cellular metabolism and more specifically to methods of treating disorders associated with elevated levels of LDH-A and low to basal levels of anti-apoptotic proteins such as bcl-2.

Background of the Invention

Alterations of the glycolytic pathway including elevation of lactate dehydrogenase-A activity are thought to be hallmarks of cancer cells, which unlike normal cells are able to produce lactate aerobically, a phenomenon known as the Warburg effect (Bodansky, 1975; Goldman et al., 1964; Pedersen, 1978; acker and Spector, 1981; Warburg, 1930; Warburg, 1956). The LDH-A gene is a delayed early serum response, epidermal growth factor, cAMP and phorbol ester-inducible gene whose role in neoplasia remains unestablished despite its widespread use as a prognostic tumor marker (Chung et al., 1995; Huang and Jungmann, 1995; Matrisian et al, 1985; Short et al., 1994). Lactate dehydrogenase is a tetrameric enzyme with five isoforms composing of combinations of two subunits, LDH-A and LDH-B. The LDH-A subunit converts pyruvate to lactate under anaerobic conditions in normal cells. The other isoenzyme, LDH-B, kinetically favors the conversion of lactate to pyruvate and is found at high levels in aerobic tissues such as the heart. Hereditary LDH-A subunit deficiency causes early postimplantation embryonic lethality in homozygotic mice (Merkle et al, 1992). Human LDH*A deficiency presents clinically as an exertional myopathy, which is associated with a severe inability of excercised muscles to produce lactic acid (Kanno et al., 1980). In addition to its role in intermediary metabolism, the LDH-A isozyme may be functionally involved in the transcriptional modulation of gene expression and/or DNA replication, since its tyrosine phosphorylated form localizes to the cell nucleus, and has been found to be a single-stranded DNA binding protein with DNA helix-destabilizing activity (Cooper et al., 1983; Grosse et al., 1986; Sharief et al, 1986; Williams et al.. 1985; Zhong and Howard, 1990).

c-myc, by contrast, is an early serum response gene whose deregulated expression is the molecular signature of Burkitt's lymphomas and is frequently found in various commonly occuring solid tumors (Cole, 1986; Dalla-Favera et al., 1982; Dang and Lee, 1995). The c-Myc protein participates in the regulation of cell proliferation, differentiation and apoptosis induced by serum deprivation (Eilers et al., 1989, 1991 ; Evan et al. 1992; Harrington et al., 1994; Hermeking and Eick, 1994; Packham and Cleveland. 1995; Wagner et al., 1994; White, 1996). c-Myc is a basic-helix-loop-helix/leucine zipper (bHLH/Z) transcription factor that heterodimerizes with another protein termed Max via the HLH/Z domain to bind a DNA consensus core sequence, CACGTG or E-box (Blackwood et al., 1992; Evan and Littlewood, 1993; Meichle et al., 1992; Prendergast and Ziff, 1992). Although c-Myc provides the transregulatory function for the heterodimer to activate through E-boxes or suppress gene transcription through initiator elements, its targets have not been comprehensively characterized and are only beginning to emerge from various studies (Ayer et al., 1995; Bello-Fernandez et al.. 1993; Galaktionov et al., 1996; Gaubatz et al., 1995; Grandori et al, 1996; Gu et al., 1993; Jones et al, 1996; Kretzner et al., 1992; Lee et al., 1996; Li et al., 1994; Miltenberger et al., 1995; Philipp et al., 1994; Schuldiner et al., 1996).

Since LDH-A is important for glycolysis and growth arrest as a response to nutrient deprivation is evolutionarily conserved (Parde.e et al., 1978), we asked whether glucose, a critical nutrient for all organisms, may play a role in cell proliferation and in particular whether glucose deprivation alters the phenotype of c-Myc transformed cells. Similar to the effects of glucose withdrawal that cause yeast to arrest before the Gl START point (Gillies et al., 1981), we observe that glucose deprivation arrests non-transformed cells in Gl . In contrast, glucose deprivation induces extensive apoptosis of c-Myc transformed fibroblasts, lymphoblastoid or lung carcinoma cells. Overexpression of LDH-A alone is sufficient to induce apoptosis of Ratla cells deprived of glucose but not those deprived of serum, suggesting c-Myc induction of LDH-A expression causes the transformed cells to undergo apoptotic cell death in the absence of glucose. This observation also suggests that the apoptotic program induced by serum withdrawal is distinguishable from that stimulated by glucose deprivation. Our studies, thus, add novel insights into the coupling of glucose metabolism and the cell cycle and to our intellectual armamentarium on cancer therapeutics.

Summary of the Invention

The characteristic ability of cancer cells to overproduce lactic acid aerobically was recognized by Warburg about seven decades ago, although its molecular basis has been elusive. The lactate dehydrogenase-A (LDH-A) gene, whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc responsive gene. Stably transfected Rat la fibroblasts that overexpress LDH-A alone or those transformed by c-Myc overproduce lactate, suggesting that overexpression of LDH-A is sufficient to induce the Warburg effect. LDH-A overexpression is required for c-Myc-mediated transformation, since lowering its expression reduces soft agar clonogenicity of c-Myc-transformed Ratla fibroblasts, c- -Myc-transformed human lymphoblastoid and Burkitt's lymphoma cells. To assess whether deregulated LDH-A expression alters cell cycle regulation by nutrients, we determined the effects of glucose withdrawal on cell growth and survival. Glucose deprivation or treatment with the glucose antimetabolite, 2-deoxyglucose. causes non-transformed cells to arrest in the Gl phase of the cell cycle, whereas c-Myc transformed fibroblasts, lymphoblastoid or lung carcinoma cells undergo extensive apoptosis that is blocked by Bcl-2. Overexpression of LDH-A alone in Ratla fibroblasts is sufficient to induce apoptosis with glucose deprivation, but not with serum withdrawal, suggesting that LDH-A mediates the apoptotic effect of c-Myc when glycolysis is blocked. Our studies have linked c-Myc to the induction of LDH-A whose expression is sufficient for the Warburg effect and sensitizes cells to a unique apoptotic pathway induced by glucose deprivation. The inventors sought to identify c-Myc target genes using representational difference analysis (RDA) and found that LDH-A behaves as a direct c-Myc -responsive gene that may be involved in Myc-mediated cell transformation (Hubank and Schatz. 1994; Lisitsyn et al., 1993). In studies reported here, we observed that overexpression of LDH-A alone is sufficient to increase production of lactate indicating that increased LDH-A may be responsible for the Warburg effect in tumors.

In a first embodiment, the invention provides a method for ameliorating a cell proliferative disorder associated with elevated levels of LDH-A and levels of at least one anti-apoptotic protein that are the same or less than a normal cell not having the disorder in a subject, the method comprising contacting cells of the subject with a therapeutically effective amount of a compound which inhibits LDH-A activity. The anti-apoptotic protein illustrated in the present invention is bcl-2 and levels of bcl-2 are typically less than those levels found in a normal cell (e.g., a non-cancerous cell). Preferably the subject is a human. The contacting can be in vivo or ex vivo, for example in bone marrow cells. Preferably, the cells are transformed with an oncogene, such as the myc, ras, fos, SV40 large T antigen and adenovirus El A oncogenes. The inhibitor may be an antibody, such as a monoclonal or polyclonal antibody. Alternatively, the inhibitor can be an LDH-A antisense nucleic acid. The antisense nucleic acid may, for example, be complementary to a region in the LDH-A promoter. The region of the LDH-A promoter may contain an oncogene protein binding site.

In another embodiment, the invention provides a method for ameliorating a cell proliferative disorder in a subject wherein the cells have levels of at least one anti- apoptotic protein that are the same or less than a normal cell not having the disorder and have elevated levels of lactic acid, the method comprising contacting cells of the subject with a therapeutically effective amount of a compound which affects glycolysis. The anti-apoptotic protein is preferably bcl2 and the cells are preferably associated with oncogenic transformation. The compound that affects glycolysis is preferably an inhibitor of glycolysis such as a glucose anti-metabolite. An exemplary glucose antimetabolite is 2-deoxyglucose (2-DG).

In another embodiment, the invention provides a method for identifying a compound which modulates cell proliferation in cells that produces elevated levels of LDH-A and levels of at least one anti-apoptotic protein that are the same or less than a normal cell not having the disorder comprising incubating components comprising the compound and said cell, wherein the incubating is carried out under conditions sufficient to allow the compound to interact with said cells; and measuring the effect of the compound on proliferation of the cell. The compound can be, for example, an LDH-A antisense nucleic acid, as described above, or a glucose antimetabolite, such as 2-deoxyglucose (2- DG). As a further step in the method, the effect of the compound on glycolysis can be measured.

The invention also provides a cell line for screening glucose anti-metabolite compounds characterized by having levels of at least one anti-apoptotic protein that are the same or less than a normal cell; and having elevated levels of LDH-A. Preferably, the anti- apoptotic protein is bcl2.

In yet another embodiment, the invention provides an antisense nucleic acid sequence that binds to LDH-A encoding nucleic acid. The antisense may bind to the LDH-A promoter region.

Brief Description of the Drawings

Figure 1 : LDH-A is a direct c-Myc target gene. (A) RNase protection assay showing elevated LDH-A expression (320 bp protected band) in non-adherent Ratla-Myc (Rla-Myc) versus Ratla (Rla) cells. A rat vimentin probe (220 bp protected band) was used as an internal control. (B) Nuclear run-on assays with isolated nuclei from non-adherent cells demonstrate an increased transcriptional rate of LDH-A in Rat la-Myc as compared to Ratla cells. Vimentin served as a control. (C) Northern blots showing LDH-A expression in a cell line expressing a Myc-estrogen receptor fusion protein on exposure to cycloheximide (CHX) alone (top panel) or exposure to both CHX and hydroxytamoxifen (4-HOTM) (middle panel) for the indicated times. The bottom panel shows control vimentin expression on the same blot as the middle panel.

Figure 2: Deregulated c-Myc expression elevates LDH-A mR A and activity in lymphoid cells. (A) Relative LDH enzymatic activities (a.u. = arbitrary units) for non-c-Myc transformed CB33 lymphoblastoid cells (lane 1), c-Myc transformed CB33 cells (lane 2), Ramos (lane 3), ST486 (lane 4) and DW6 (lane 5) Burkitt's lymphoma cell lines are shown. (B) The corresponding c-Myc protein levels determined by immunoblot analysis for each cell line are shown. (C) Northern blot showing LDH-A mRNA (1.7 Kb) levels in the indicated cell lines (bottom of figure). The lower band corresponds to LDH-B mRNA (1.4 Kb) that cross-react with randomly primed rat LDH-A radiolabelled probes. Fifteen micrograms of total RNA were loaded per lane, and an ethidium bromide stained gel with the 18S ribosomal RNA band is shown as control for sample loading. (D) Bcl-2 protein levels by immunoblot analysis are shown for each cell line. Note that the ST486 cell line (lane 4) displayed large amounts of Bcl-2 protein as compared with CB33 lymphoblastoid (lanes 1 and 2) and the other Burkitt's cells (lane 3 and 5). Each lane was loaded with the same amount of total lysate protein.

Figure 3: c-Myc is able to transactivate the rat LDH-A promoter in an E-box dependent manner in NTH 3T3 cells. The top diagram depicts the wild-type LDH-A promoter with two E-boxes (El and E2) and the corresponding mutations of the E-boxes, mEl and mE2. The graph shows wild-type and mutant LDH-A promoter-reporter responses to empty vector (RSV) or a c-Myc (RSVMyc) expression vector. The responses of the wild-type LDH-A promoter to a Myc mutant lacking the helix-loop-helix region (RSVMycDHLH) the transcription factor USF (RSVUSF) are also shown; in both cases there were no activation of the double E-box mutant reporter. Empty vector, Myc or USF expression plasmids were lipofected at 50 ng/60 mm dish. Data are averages of four to eight experiments with standard errors shown.

Figure 4: Reduction of LDH-A expression in c-Myc transformed cells inhibits anchorage-independent growth. (A) LDH-A enzymatic activities (arbitrary units (a.u.); mean of four experiments with standard error shown) of pooled Ratla/C (Rla/C) puromycin-resistant control cells, pooled Ratla-LDH-A (Rla-LDH-A) overexpressing LDH-A, pooled Ratla-Myc/C (Rla-Myc/C) puromycin-resistant control cells or pooled Ratla-Myc AS-LDH-A (Rla-Myc AS -LDH-A) with antisense LDH-A expression are shown. (B) Immunoblot showing human c-Myc protein levels in pool cell lines corresponding to those indicated in panel A. Expression of antisense LDH-A did not affect ectopic human c-Myc protein levels (compare third and fourth lanes). Extracts from equivalent number of cells were electrophoresced and immunoblotted with the 9E10 anti-human c-Myc antibody. CRM = cross reacting material. (C) The graph shows adherent growth rates of the four pooled cell lines indicated in panel A. The ordinate shows cell number per 60 mm dish (average of triplicate experiments with standard errors shown) at the indicated times. The shading of the bars corresponds to that shown in panel A. (D) Anchorage-independent growth assay of Rat 1 a-Myc/C cells and Rat la-Myc AS-LDH-A (photomicrograph) demonstrates a reduction in colony formation (graph) associated with decreased LDH-A levels. Data are averages of two experiments with standard error shown. (E) RNase protection assay demonstrating the expression of LDH-A antisense transcript (AS-LDH-A; 320 bp protected fragment using the LDH-A sense probe); vimentin was used as an internal control. (F) Comparative anaerobic to aerobic growth rates of cell lines described in panel A. Cells were grown adherently on 100 mm plastic dishes in a hypoxic chamber (described in the Methods section) or in regular oxgenated conditions for 48 h, and cell numbers (average of triplicate experiments with standard errors shown) were determined after trypsinization.

Figure 5 : (A) Antisense LDH-A expression reduced clonogenicity of c-Myc transformed lymphoblastoid cells, CB33-Myc. Control empty vector transfected CB33-Myc cells were subjected to soft agar clonogenic assays. Representative colonies formed (cloning efficiency of 2 x 10-4) are shown in the upper four panels. With the antisense LDH-A expressing pooled clones, CB33Myc AS-LDH-A, there was a four-fold reduction in cloning efficiency and a reduction in colony sizes. Representative microscropic fields are shown in the lower four panels. (B) Antisense LDH-A expression reduced clonogenicity of the DW6 Burkitt's lymphoma cell line. Pooled stably transfected G418-resistant DW6 cells were cultured in soft agar. The left panel represents a composite photograph of petri dish halves containing empty vector (left half) or antisense LDH-A vector (right half) transfected DW6 cells. The right panel is a higher magnification showing soft agar colonies of the same cells shown in the left panel. (C) Growth rates of the indicated cell lines were determined by Coulter counting cells grown in suspension cultures. The ordinate shows cell numbers per 60 mm plate (average of triplicates with standard error shown) at the indicated times.

Figure 6: Cell cycle distribution and BrdU incoporation characteristics of Ratla, R- atla-LDH-A, Ratla-Myc and Ratla-Myc-Bcl-2 cells. Two dimensional flow cytometric distributions of DNA content stained by propidium iodide (abscissa) and BrdU labeling (ordinate) are shown for each cell line cultured for 24 hours in the presence (upper panels) or absence (lower panels) of glucose. Distributions of nuclei in each compartment (Gl, S or G2/M) were estimated via deconvolution of propidium iodide staining profiles and are shown at the bottom of each panel. The figures in parentheses (lower panels) for the S phase indicate the percentage of BrdU labeled cells. The percentage of cells in S phase and those labeled with BrdU were equal for the upper panels.

Figure 7: Glucose deprivation induced apoptotic cell death of c-Myc or LDH-A overexpressing cells. Ratla, Ratla-LDH-A, Ratla-Myc and Ratla*Myc±Bcl-2 cells were cultured for 24 hours in the presence (upper panels) or absence (lower panels) of glucose. Cells were harvested, stained with propidium iodide, and DNA strand breaks were labeled with biotin^dUTP with TdT as described in the methods section. DNA content determined by propidium iodide staining is shown on the abscissa and DNA strand breaks content is shown on the ordinate. Note that only the glucose deprived Rat la* LDH-A and Ratla-Myc cells display significant numbers of cells with DNA strand breaks. Bcl-2 coexpression with c-Myc (Rat la* Myc* Bel* 2) greatly reduces apoptotic cell death with glucose deprivation. The numbers below each panel indicate the percentage of cells that were apoptotic and labeled with biotin-dUTP.

DETAILED DESCRIPTION OF THE INVENTION

LDH-A is a target of c-Myc

To identify potential c-Myc target genes, we used Ratla fibroblasts that only require ectopic c-Myc expression to be transformed (Barrett et al., 1995; Small et al. 1987; Stone et al., 1987). In particular, Ratla cells transformed by c-Myc display anchorage-independent growth, whereas nontransformed cells require adherence for cell proliferation. To identify genes that are regulated by c-Myc and contribute to the anchorage independent growth phenotype of Ratla-Myc cells, we synthesized cDNAs from non-adherent Ratla-Myc and Ratla cells and used cDNA RDA to identify differentially expressed genes (Hubank and Schatz, 1994; Lisitsyn et al, 1993). Among 21 differentially expressed genes (B. Lewis et al., unpublished results), LDH-A proved to be one that is highly differentially expressed, displaying a 6.7(+ 0.7)-fold elevated expression in Myc transformed Ratla cells (Figure 1A). Nuclear run-on experiments (Figure IB) demonstrated an enhanced transcriptional rate of LDH-A in Ratla-Myc cells as compared to Ratla fibroblasts. To determine whether LDH-A gene might be transcriptionally activated by Myc, we used a previously described Ratla cell line expressing a Myc-estrogen-receptor (Myc-ER) fusion protein that is activated by the addition of hydroxytamoxifen to the growth medium (Eilers et al, 1989; Grandori et al., 1996). Activation of Myc-ER by hydroxytamoxifen in confluent cells causes induction of LDH-A, which is not inhibited by the protein synthesis inhibitor cycloheximide (Figure IC). No stimulation of LDH-A expression was observed with cycloheximide alone (Figure IC). These observations suggest that induction of LDH-A expression by Myc is direct and does not require new protein synthesis. We studied lymphoid cells and also observed a correlation between c-Myc overexpression and the levels of LDH-A mRNA and activity (Figure 2). Nontransformed CB33 lymphoblastoid cells, those transformed by c-Myc (CB33-Myc) and three Burkitt's lymphoma cell lines with c-myc chromosomal translocations were studied. The c-Myc transformed lymphoblastoid and Burkitt's lymphoma cell lines all have elevated c-Myc protein levels which is associated with elevations of LDH-A mRNA (1.7 Kb) and enzymatic levels compared to the non-transformed lymphoblastoid cells. The lower band in Figure 2, panel C corresponds to LDH-B mRNA (1.4 Kb) that cross-hybridized with randomly primed rat LDH-A radiolabelled probes (Sharief et al, 1994). Note that our LDH enzyme assay does not distinguish between LDH-A and LDH-B; therefore, LDH-B activity may contribute to background activity seen in Figure 2, panel A. While elevated c-Myc protein levels are correlated with elevated LDH-A mRNA and activity, note that the ST486 cell line have a very high level of c-Myc protein but a relatively lower amount of LDH-A mRNA. This observation suggest that other factors influence the magnitude of elevation of LDH-A mRNA by c-Myc. The correlation between Myc and LDH-A enzyme activity was also observed in the small cell lung carcinoma NCI H209 cell line (H209-Myc) that was stably transfected to overexpress c-Myc (Barr et al., 1991). Similar to the CB33 cells, the H209 cells grow in suspension in large cellular aggregates. This aggregation is abolished by overexpression of c-Myc, which also elevates LDH activity by 30% in the lung H209-Myc cells. Our observations indicate that c-Myc increases the expression of LDH-A in different cell lines and augments LDH-A mRNA in Rat 1 a fibroblasts at the transcriptional level.

The rat LDH-A promoter contains two consensus Myc/Max binding sites or E-boxes, CACGTG, that are also conserved in mouse and man suggesting that c-Myc may be able to regulate the transcription of LDH-A through these E-boxes (Fukasawa and Li, 1987; Short et al., 1994; Takano and Li, 1990). Transient transfection experiments with a c-Myc expression vector demonstrated an E-box-dependent transactivation of the LDH-A promoter-luciferase reporter gene (Figure 3). Mutation of either or both E-boxes (Figure 3) abrogated Myc-dependent transactivation. The expression vector producing a c-Myc mutant lacking the helix-loop-helix domain was unable to activate the LDH-A promoter (Figure 3) suggesting that dimerization with Max is required for transactivation. An expression vector for the transcription factor USF (Gregor et al., 1990), which also binds CACGTG, was also able to stimulate the LDH-A promoter although only half as efficiently as the c-Myc expression vector (Figure 3). In vitro electrophoretic mobility shift DNA-protein binding assays demonstrated the ability of recombinant Myc/Max proteins to bind the LDH-A promoter E-boxes (data not shown). These observations taken together with the time course of serum induction of c-myc followed by LDH-A expression in fibroblasts (Matrisian et al, 1985) and the direct induction of LDH-A expression by the Myc-ER fusion protein suggest that LDH-A is a direct Myc-responsive target gene.

Increased expression of LDH-A induces the Warburg effect

To determine whether LDH-A overexpression is sufficient to increase lactate production aerobically (the Warburg effect) or transformation, we created pooled stably transfected Ratla cells that constitutively express rat LDH-A. When subjected to soft agar anchorage-independent growth assay, the Ratla-LDH-A cells were unable to proliferate as well as Ratla-Myc cells in suspension indicating that increased LDH-A expression is insufficient to induce full transformation. Moreover, LDH-A in unable to cooperate with activated Ras in transforming primary rat embryo cells, indicating that its expression is insufficient to replace the activity of c-Myc. In contrast, we observed that both c-Myc transformed and LDH-A ectopically expressing Ratla cells produce more lactate than the control stably transfected Ratla cells (Table 1). These observations suggest that the Warburg effect induced in fibroblasts by c-Myc is largely due to the deregulated expression of LDH* A. Necessity of elevated LDH-A for c-Myc induced soft agar growth of fibroblasts and lymphoid cells

We sought to determine whether elevated LDH-A expression is necessary for c-Myc mediated anchorage-independent growth by constructing Ratla-Myc cells and c-Myc transformed lymphoblastoid cells expressing antisense LDH-A (Lombardi et al.. 1987). Reduction of LDH activity in Ratla-Myc cells by antisense expression (Figure 4A and 4D), which did not alter ectopically expressed Myc protein levels (Figure 4B), dramatically decreased soft agar clonogenicity of Ratla-Myc cells in soft agar (Figure 4C) (14). Nonetheless, the growth rates of Ratla-Myc and those expressing antisense LDH-A (Ratla-Myc AS-LDH-A) were virtually the same when they were seeded on plastic dishes indicating that reduced LDH-A activity did not inhibit adherent growth (Figure 4E). In contrast to antisense LDH-A expression, stable transfection of an antisense prothymosin a (Frangou-Lazaridis et al., 1988) expression vector with the same SV40 promoter reduced prothymosin a levels but did not reduce anchorage independent growth of Ratla-Myc cells (C. V. Dang and S. L. Berger, unpublished observation). Prothymosin a is a c-Myc target gene implicated in cell proliferation (Eilers et al., 1991). These results indicate that LDH-A is necessary for c-Myc mediated anchorage-independent growth of Rat la cells, although LDH-A overexpression alone is insufficient to induce the extent of growth in soft agar characteristic of c-Myc transformed Rat la cells.

We hypothesized that the anaerobic conditions within an expanding soft agar colony of cells competing for nutrients and oxygen may select against cells with low LDH-A levels and are thus inefficient in anaerobic glycolysis. To test this hypothesis, we subjected adherent Ratla/C, Ratla-LDH-A, Ratla-Myc and Ratla-Myc- AS -LDH-A cells to hypoxia (Figure 4F). Cells were cultured adherent to plastic dishes with HEPES-buffered medium (pH 7.55) in a hypoxic chamber with 0% oxygen as previously described (Wang et al., 1995). After 48 hours of oxygen deprivation, the rate of cell proliferation was determined by counting trypsinized cells. Both Ratla-Myc and Ratla-LDH-A cells continued to grow in hypoxic conditions as compared to the control Ratla/C cells. Intriguingly, the Ratla-Myc- AS -LDH-A cells had significantly reduced growth rates when deprived of oxygen. These observations support our hypothesis as well as provide a plausible biological basis for differences in the growth properties of these cells in soft agar.

In addition to the effects seen in fibroblasts, reduction of LDH-A expression also inhibited soft agar colony formation of human lymphoid cells transformed by c-Myc. EBV-immortalized human lymphoblastoid CB33 cells can be transformed by c-Myc in vitro, resulting in the ability to form soft agar colonies (Lombardi et al., 1987). We constructed c-Myc transformed lymphoblastoid cells that express antisense LDH-A. The mock transfected c-Myc transformed lymphoblastoid cells form colonies in soft agar as previously reported (Lombardi et al., 1987), whereas cells transfected with antisense LDH-A showed a four-fold reduction in soft agar cloning efficiency (Figure 5A). Antisense LDH-A expression was also achieved in two of three Burkitt's lymphoma cell lines resulting in the reduction of LDH activity. The DW6 Burkitt's lymphoma cell line displayed a very high soft agar cloning efficiency that was reduced more than 100-fold with antisense LDH-A expression (Figure 5B). Both the DW6 and c-Myc transformed lymphoblastoid CB33 cells that express antisense LDH-A display normal growth characteristics in standard suspension culture conditions (Figure 5C). The Ramos Burkitt's cell line displayed a much lower cloning efficiency, but its clonogenicity was 40-fold decreased with antisense LDH-A expression (data not shown). These results indicate that elevated LDH-A levels associated with overexpression of c-Myc is necessary for neoplastic transformation as measured by soft agar clonogenic assays.

We studied the effect of antisense LDH-A expression on activated Ras-mediated transformation of Ratla cells to determine whether reduction of LDH-A affects other oncogene-induced transformation. Expression of activated Ras causes focus formation of Ratla cells (778 + 36 foci/100 mm dish; n = 3) that was reduced 3.6-fold by coexpression of antisense LDH-A (214 + 22 foci/100 mm dish; n = 3). We further determined the potential toxicity of antisense LDH-A expression in a colony suppression assay to determine the contribution of toxicity to the suppression of Ras-mediated transformation. In this assay, puromycin-resistant colonies were counted after co-transfection of the puromycin-resistance marker gene with the empty expression vector or antisense LDH-A expression vector. The anti-sense LDH-A expression vector reduced puromycin-resistant colony formation by 40% as compared to control empty expression vector. These observations suggest that antisense-LDH-A expression only slightly reduces (1.5-fold) the transforming potential of Ras. These effects, however, are quite different from the phenotypes observed with antisense LDH-A expression in stably transfected Myc-transformed cells. In the case of the Myc-transformed cells, the difference in phenotypes is profoundly different when cells are grown in soft agar.

Glucose deprivation induces apoptosis of c-Myc transformed cells Since LDH-A is intimately linked to glucose metabolism and its expression is enforced by c-Myc, we determined whether glucose deprivation would alter the phenotype of c-Myc transformed cells. Glucose deprivation of non-transformed Ratla cells causes a reduction in BrdU incorporation, an enrichment in Gl phase cells and reduction in S-phase and G2/M cells (Figure 6). Only 67% of the Ratla-Myc cells with S-phase DNA content incorporated BrdU indicating that these cells either arrested or died in S-phase. Microscopically, 20% of the c-Myc transformed cells, but not cells coexpressing Bcl-2 with Myc, were round, retractile and floating after 24 h of glucose withdrawal. In contrast to the Ratla-Myc cells, the Ratla-Myc-Bcl-2 cells show a cell cycle profile that appears similar to the control Ratla cells with reduction in the S-phase population. Virtually all S-phase Ratla-Myc-Bcl-2 cells, however, incorporate BrdU (Figure 6). We therefore determined the extent of apoptotic cell death using the terminal deoxynucleotidyl transferase (TdT) end-labelling flow cytometric assay. As shown in Figure 7, the untransformed Rat la cells displayed minimal apoptotic cell death with glucose withdrawal. In contrast, 19% of the c-Myc transformed cells have undergone apoptosis and were TdT positive after 24 h of glucose deprivation. The apoptotic cells have DNA content spreading from the Gl into the S-phase pool as well as some in the G2 pool. Coexpression of Bcl-2 completely blocked glucose deprivation induced apoptosis of c-Myc transformed cells. These observations uncover a novel glucose-dependent apoptotic pathway that is activated by c-Myc overexpression and is inhibited by coexpression of Bcl-2.

We employed the inhibitor, 2-deoxyglucose, to determine whether inhibition of glycolysis is also able to induce apoptosis of c-Myc transformed cells. 2-Deoxyglucose is thought to inhibit glycolysis via competitive inhibition after it is phosphorylated by hexokinase (Kaplan et al., 1990). Table 2 shows that 2-DG differentially induces apoptosis of Ratla-Myc cells versus nontransformed Rat la cells and CB33 lymphoblastoid cells transformed by c-Myc as compared to the nontransformed cells. Bcl-2 also blocks the ability of 2-DG to induce apoptosis of c-Myc transformed fibroblasts.

We also studied three Burkitt's lymphoma cell lines and found that only the Ramos and DW6 cell lines, but not ST486, displayed apoptosis with exposure to 2-DG, although all three cell lines overexpress c-Myc and have elevated LDH levels (Table 2). Both Ramos and ST486 cells have mutant p53 (Gaidano et al, 1991), yet the Ramos cell line underwent apoptosis with 2-DG suggesting that wild-type p53 is unnecessary for 2-DG-induced apoptosis. The Bcl-2 level in the ST486 is highly elevated as compared to lymphoblastoid cells or the DW6 and Ramos Burkitt's lymphoma cell lines suggesting that Bcl-2 may cause ST486 to resist 2-DG induced apoptosis (Figure 2). These observations substantiate the connection between c-Myc expression and LDH*A levels. They further suggest that wild-type p53 is not required, whereas Bcl-2 is able to block glucose-deprivation induced apoptosis.

Overexpression of LDH-A sensitizes rat fibroblasts to glucose deprivation, but not serum deprivation, induced apoptosis To determine whether enforced expression of LDH-A by c-Myc plays a role in glucose deprivation induced apoptosis, we studied pooled stably transfected Ratla cells (Ratla-LDH-A) that overexpress LDH-A. Intriguingly, overexpression of LDH-A increases the population of Rat la cells in S-phase in the presence of glucose (Figure 6). Withdrawal of glucose, however, was associated with the dramatic reduction in BrdU positive cells in S-phase. LDH-A ectopically expressing Ratla cells, similar to c-Myc overexpressing cells, displayed significant apoptotic cell death with glucose withdrawal (Figure 7). Unlike the Ratla-Myc cells that displayed 21 percent apoptotic cell death after 24 h of serum withdrawal, Ratla-LDH-A cells growth arrested did not display significantly increased (5.7 %) apoptotic cell death. These observations indicate that induction of apoptosis by glucose deprivation and serum deprivation are distinctly different pathways and that LDH-A links c-Myc to glucose-dependent apoptosis.

DISCUSSION c-Myc regulates the expression of LDH-A

Our results indicate that c-Myc is able to directly increase the expression of LDH-A at the transcriptional level and transactivate the LDH-A promoter in an E-box dependent manner. In fibroblasts, serum stimulates c-myc expression at 2 to 3 hours followed by LDH-A expression at 4 to 6 hours (Matrisian et al., 1985). This time course of induction is consistent with LDH*A being a target of c-Myc. Using cells expressing a Myc-estrogen receptor fusion, we observed the induction of LDH-A expression by hydroxytamoxifen. This induction is not inhibited by the protein synthesis inhibitor cycloheximide. suggesting that the induction of LDH-A by Myc is direct. We also observed a correlation between c-Myc overexpression and elevated LDH levels in c-Myc transformed fibroblasts, lymphoblastoid and lung cells as well as in Burkitt's lymphoma cell lines. In addition, inducible c-Myc expression was previously reported to elevate LDH-A expression in two different fibroblast cell lines (Tavtigian et al., 1994). These observations strongly support the hypothesis that LDH-A is a direct target of c-Myc; however, it is possible that other E-box (CACGTG) binding transcription factors, such as USF (Gregor et al., 1990), TFE-3 (Beckmann et al., 1990) or HIF-1 (Semenza, personal communication; Wang et al., 1995), may also regulate the expression of LDH-A under other physiological circumstances. In fact, our results suggest that USF is also able to transactivate the LDH-A promoter, albeit with decreased efficiency. Since LDH-A expression is altered in response to various stimuli (Chung et al, 1995: Huang and Jungmann, 1995; Matrisian et al., 1985; Short et al, 1994), it is likely that it is regulated by different transcription factors. Our results demonstrate that LDH-A expression is elevated in c-Myc transformed cells and suggest that LDH-A is a direct target of c-Myc.

Is LDH-A responsible for the Warburg effect?

Seven decades ago, Warburg studied glycolysis in a variety of human and animal tumors and found that there was a trend toward an increased rate of glycolysis in tumor cells, resulting in the excessive production of lactic acid from glucose (Warburg. 1930; Warburg, 1956). This phenomenon known as the Warburg effect was a subject of intense investigation, controversy and intrigue, yet the molecular basis of the Warburg effect has remained unclear (Racker and Spector, 1981; Racker and Spector, 1981). Our findings are instructive when the Warburg effect and previous links between elevated LDH-A levels and human cancers are taken into consideration (Bodansky, 1975; Bredin et al., 1975; Carda-Abella et al., 1982; Csako et al., 1982; Goldman et al., 1964; Kawamoto, 1994; Li et al., 1988; Nevin and Mulholland, 1988; Nishikawa et al, 1991; Schneider et al., 1980; Sharief et al., 1994; Tanaka et al., 1984; Vergnon et al., 1984; Woollams et al., 1976). In particular, an elevated LDH level is an independent predictor of poor clinical outcome in Burkitt's lymphoma, in which activation of the c-myc gene by chromosomal translocations is a sine qua non (Csako et al., 1982; Dalla-Favera et al., 1982: Magrath et al.. 1980; Schneider et al., 1980). Our results indicate that c-Myc is able to activate the expression of LDH-A, increase lactate production and perhaps accounts for the elevation of LDH-A levels in various forms of commonly occurring human cancers. An elevation of lactate production in a transgenic mouse model that overexpresses c-Myc in the liver, without development of liver tumor, further supports the induction of the Warburg effect by c-Myc (Valera et al., 1995). Moreover, we observe that ectopic LDH-A expression is sufficient to induce the Warburg effect in fibroblasts without conferring the full transformed phenotype of anchorage-independent growth. Necessity of LDH-A induction for Myc-mediated transformation R e d u c e d expression of LDH-A in our model systems is able to block Myc-mediated soft agar colony formation, suggesting that elevated LDH-A expression in human cancers may be necessary for their neoplastic phenotype. Our observations are instructive when the growth properties of Myc-transformed Ratla or lymphoblastoid cells expressing antisense LDH-A in soft agar are compared with the same cells in the absence of suspension in agar. The growth rates of these Myc-transformed cells are virtually the same when they were grown without suspension in agar, suggesting that LDH-A overexpression is required for soft agar colony formation but not for cell viability. The biochemical basis for this difference remains to be established; however, it is conceivable that a threshold level of glycolysis and production of lactate is necessary for soft agar colony formation. We hypothesize that the anaerobic conditions within an expanding soft agar colony may select against cells with low LDH-A levels and are inefficient in anaerobic glycolysis. This hypothesis is supported by our observations that LDH-A expression correlated with the ability of Ratl a-LDH-A and Ratl a-Myc, but not Ratl a or Ratl a-Myc -AS-LDH-A, to proliferate when adherent cells were cultured in hypoxic conditions. Our observation on the effect of hypoxia on stably transfected Ratla cells constitutively expressing c-Myc contrasts with the previously reported hypoxia-induced apoptosis of cells expressing a Myc-estrogen receptor fusion protein exposed to tamoxifen (Graeber et al., 1996). In fact, we have also observed hypoxia-induced cell death with Ratla cells expressing the Myc-ER fusion protein upon exposure to hydroxytamoxifen. This effect is enhanced when the growth medium is not buffered in hypoxic chambers to physiological pH. The key differences between the constitutive and inducible Myc expressing Ratla systems are as yet unresolved. Our hypothesis, however, is supported by the finding that the early embryonic lethality of homozygous LDH-A deficient mice is probably due to the postimplantation anaerobic conditions (Ellington, 1987) that exist before formation of the chorioallantoic placenta (Merkle et al., 1992). Glucose deprivation induced apoptosis

Our observation that glucose is required for nontransformed cells to progress through the Gl-S boundary has been previously observed with 3T3 fibroblasts (Holley and Kiernan, 1974). Furthermore, Saccharomyces cerevisiae growth arrest prior to the START point when deprived of glucose (Gillies et al., 1981). Although the oxygenated yeast cells were arrested by glucose deprivation, the starved yeast cells were able to synthesize ATP suggesting that the signal for cell cycle progression is created during the catabolism of glucose and not strictly by the energy supply. The observation that the temperature sensitive mutant cdcl9 is a mutated form of the yeast pyruvate kinase gene further indicates that glycolysis is tightly linked to cell cycle control (Aon et al.. 1995). Depletion of ATP per se through mitochondrial uncoupling also arrest mammalian cells in Gl, and in G2 with extensive ATP depletion (Sweet and Singh, 1995). This observation along with our observation of a residual G2-M population of non-transformed Ratla cells after glucose deprivation suggest that a glucose-dependent restriction of passage through G2-M may also exist. Our results taken together with previous studies suggest that there exists an evolutionarily conserved glucose-dependent cell cycle checkpoint that appears to overlap with START in yeast and the restriction point in mammalian cells (Pardee, 1974).

Intriguingly, c-Myc overexpression in glucose starved fibroblasts or lymphoblastoid cells resulted in extensive apoptosis. This apoptotic pathway is suppressed by Bcl-2 in c-Myc transformed Ratla cells. We examined the effect of 2-deoxyglucose on three Burkitt's lymphoma cell lines and found that the Ramos and DW6, but not the ST486, cell lines underwent apoptosis; although all three cell lines overexpress c-Myc and have elevated LDH-A levels. Since the Ramos and the ST486 cell lines both contain mutant p53 (Gaidano et al., 1991), it appears that the glucose-dependent apoptotic pathway is independent of wild-type p53 activity. In contrast, wild type p53 appears to be required for c-Myc induced apoptosis with serum deprivation (Evan et al., 1992; Hermeking and Eick, 1994; Wagner et al, 1994). The high Bcl-2 protein level in the ST486 Burkitt's cell line and its low levels in the Ramos and DW6 cell lines suggest that Bcl-2 is a critical determinant of Myc-dependent glucose deprivation induced apoptosis.

Studies performed almost four decades ago indicate that infusions of 2-deoxyglucose into cancer patients were well tolerated (Landau et al., 1958). In leukemic patients, the white cell count fell during the 24-hour period following a single 2-deoxyglucose infusion and glycolysis was lowered in the leukemic cells. With the available modern molecular probes, cancer cells may be characterized with regard to their molecular characteristics including Bcl-2/Bcl-XL status and it is conceivable that this historic antimetabolite may be effective in activating apoptosis in certain neoplasms with high LDH-A levels.

LDH-A overexpression induces apoptosis with glucose deprivation

Our results indicate that overexpression of LDH-A in rat fibroblast is sufficient to sensitize cells to glucose deprivation induced apoptosis. This observation supports the hypothesis that LDH-A is a downstream target of c-Myc that mediates this unique apoptotic phenotype. The connection between LDH-A overexpression, glucose deprivation and the common pathway leading to apoptotic cell death remains to be elucidated. We speculate that constitutive generation of NAD+ and lactate by LDH-A and the decrease in the regeneration of NADH by inhibition of glycolysis contribute to the oxidative stress on the cells, which then triggers the final death pathway (Hockenbery et al. 1993).

In summary, we surmise that the oncogenic transcription factor c-Myc acts pleiotropically to transform cells by upregulating components of the cell cycle machinery (Galaktionov et al., 1996; Hoang et al, 1994; Jansen-Durr et al, 1993; Philipp et al., 1994), stimulating the production of biosynthetic enzymes such as ornithine decarboxylase to prepare cells for S-phase entry (Bello-Fernandez et al., 1993; Grandori et al.. 1996; Miltenberger et al., 1995; Schuldiner et al., 1996), and activating the expression of metabolic enzymes such as LDH-A to ensure an adequate supply of energy or signals for cell proliferation (Bodansky, 1975; Goldman et al, 1964). This view readily explains why the retroviral v-myc oncogene (Bishop, 1978) is sufficient to acutely cause avian tumors and why alterations of c-myc gene expression are frequently observed in human cancers (Csako et al., 1982; Dalla-Favera et al, 1982; Magrath et al., 1980; Schneider et al., 1980).

Compounds that affect LDH-A activity also include peptides, peptidomimetics, polypeptides, chemical compounds and biologic agents. LDH-A activity can be assayed using methodology as described in the present Examples. Incubating includes conditions which allow contact between the test compound and LDH-A. Contacting includes in solution and in solid phase, or in a cell. The test compound may optionally be a combinatorial library for screening a plurality of compounds. Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, etal, Proc. Natl Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al, Science, 241:1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al, Science, 242:229-237, 1988).

When a cell proliferative disorder is associated with the over-expression of LDH-A, a therapeutic approach which directly interferes with the translation of LDH-A messages into protein is possible. For example, antisense nucleic acid or ribozymes could be used to bind to the LDH-A mRNA or to cleave it. ^■ Antisense RNA or DNA molecules bind specifically with a targeted gene's RNA message, interrupting the expression of that gene's protein product. The antisense binds to the messenger RNA forming a double stranded molecule which cannot be translated by the cell. Antisense oligonucleotides of about 15-25 nucleotides are preferred since they are easily synthesized and have an inhibitory effect just like antisense RNA molecules. In addition, chemically reactive groups, such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can be attached to an antisense oligonucleotide, causing cleavage of the RNA at the site of hybridization. These and other uses of antisense methods to inhibit the in vitro translation of genes are well known. in the art (Marcus-Sakura, ^«α/., Biochem., 172:289, 1988).

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40. 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target prothymosin al producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172:289. 1988).

Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher. et al, Antisense Res. and Dev., H3V227, 1991; Helene, C, Anticancer Drug Design. 6(6):569Λ99\).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single- stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J.Amer.Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated. There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while "hammerhead" -type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.

These and other uses of antisense methods to inhibit the in vivo translation of genes are well known in the art (e.g., De Mesmaeker, et al, 1995. Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr. Opin. Struct. Biol. 5:343-355 Gewirtz, A.M., et al, 1996b. Facilitating delivery of antisense oligodeoxynucleotides Helping antisense deliver on its promise; Proc. Natl. Acad. Sci. U.S.A. 93:3161-3163 Stein, CA. A discussion of G-tetrads 1996. Exploiting the potential of antisense: beyond phosphorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323).

Delivery of antisense, triplex agents, ribozymes, competitive inhibitors and the like can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Various viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a polynucleotide sequence of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include but are not limited to Ψ2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for antisense polynucleotides a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al, Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylchohne, phosphatidylserine, phosphatidyletha- nolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylchohne, dipalmitoylphosphatidylcholine and distearoylphos- phatidylcholine.

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific. cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. In general, the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells. A ligand may be any compound of interest which will bind to another compound, such as a receptor.

The therapeutic agents useful in the method of the invention can be administered parenterally by injection or by gradual perfusion over time. Administration may be intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol. polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants. chelating agents and inert gases and the like.

Antibodies to LDH-A peptide or fragments could be valuable as diagnostic and therapeutic tools to aid in the detection of diseases in which LDH-A is a pathological factor. Therapeutically, antibodies or fragments of the antibody molecule could also be used to neutralize the biological activity of LDH-A in diseases where LDH-A is involved in the overgrowth of tissue. Such antibodies can recognize an epitope of LDH-A, or fragments thereof, suitable for antibody recognition and neutralization of LDH-A activity. As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen, such as a LDH-A peptide, to which the paratope of an antibody, such as an LDH-A-specific antibody, binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.

Preparation of an antibody requires a substantially purified moiety that can provide an antigenic determinant. The term "substantially pure" as used herein refers to LDH-A, or variants thereof, which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Substantially purified or "isolated" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. One skilled in the art can isolate Tal using standard techniques for protein purification. The substantially pure peptide will yield a single major band on a non-reducing polyacrylamide gel. The purity of the LDH-A peptide can also be determined by amino-terminal amino acid sequence analysis. Tal peptide includes functional fragments of the peptide, as long as the activity of LDH-A remains. Smaller peptides containing the biological activity of LDH-A are included in the invention. As used in the present invention, the term "antibody" includes, in addition to conventional antibodies, such protein fragments that have the ability to recognize specifically and bind the LDH-A protein or variants thereof. Regions of the gene that differ at the protein level are well defined. A protein can be raised by expression of the wt gene or of the variants, or, preferably, fractions therefore. For example, the nucleic acid sequence can be cloned into expression vectors. According to this embodiment, the sequence of interest can first be obtained by employing PCR, as described above, or from a synthetic gene construction with overlapping and ligated synthetic oligonucleotides. Another alternative would involve synthesis of a short peptide. All those methodologies are well known to one skilled in the art. See, for example, Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes 1 and 2 (1987), with supplements, and Maniatis et al, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory.

The genetic sequence discussed above then is expressed in any known, commercially available systems. Vectors for subcloning the sequence of interest, and subsequent expression into bacterial, yeast, baculovirus, insect, or tissue culture are well known to one skilled in the art. The subcloning process could, according to one embodiment, produce a fused protein with a short N- or C-terminal extension to facilitate subsequent purifications on columns or by use of antibodies. Alternatively, the protein of interest is purified by standard protein purification protocols. See for example PROTEIN PURIFICATION - PRINCIPLES AND PRACTICE, Springer Varlag publ., New- York; and PROTEIN BIOTECHNOLOGY, Humana Press, Totowa, NJ.

The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al, Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1 -5 (Humana Press 1992); Coligan et al. , Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992), which are hereby incorporated by reference. The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al, sections 2.5.1-2.6.7; and Harlow et al, ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988), which are hereby incorporated by reference. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatogra- phy with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan et al, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al, Purification oflmmunoglobulin G (IgG), in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (Humana Press 1992). Methods of in vitro and in vivo multiplication of monoclonal antibodies are well-known to those skilled in the art. Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages. Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies. Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an airlift reactor, in a continuous stirrer reactor, or in immobilized or entrapped cell culture. Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal. The invention provides a method for detecting Tal, or variants thereof, which includes contacting an anti-Tal antibody with a cell or protein and detecting binding to the antibody. An antibody which binds to Tal peptide is labeled with a compound which allows detection of binding to Tal . There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the antibody, or will be able to ascertain such, using routine experimentation. For purposes of the invention, an antibody specific for LDH-A peptide may be used to detect the level of LDH-A in biological fluids and tissues. Any specimen containing a detectable amount of antigen can be used.

The antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

The antibodies of the invention can be bound to many different carriers and used to detect the presence of an antigen comprising the peptide of the invention. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases. natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.

Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, puridoxal, and fluorescein, which can react with specific antihapten antibodies.

The invention includes antibodies immunoreactive with Tal peptide or functional fragments thereof. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al, Nature, 256:495, 1975). The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')₂, Fv and SCA fragments which are capable of binding an epitopic determinant on Tal .

(1) An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

(2) An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner. (3) An (Fab')₂ fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')₂ fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

(5) A single chain antibody ("SCA") is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.

Alternatively, a therapeutically or diagnostically useful anti-Tal antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Natl Acad. Sci. USA 86: 3833 (1989), which is hereby incorporate din its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al, Nature 321 : 522 (1986); Riechmann et al, Nature 332: 323 (1988); Verhoeyen et al, Science 239: 1534 (1988); Carter et al, Proc. Nat 'I Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al, J. Immunol 150: 2844 (1993), which are hereby incorporated by reference.

Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al, Ann. Rev. Immunol 12: 433 (1994), which are hereby incorporated by reference. Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).

In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be sued to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994); Lonberg et al, Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6: 579 (1994), which are hereby incorporated by reference.

As is mentioned above, antigens that can be used in producing LDH-A- specific antibodies include LDH-1 peptides or LDH-A peptide fragments. The polypeptide or peptide used to immunize an animal can be obtained by standard recombinant, chemical synthetic, or purification methods. As is well known in the art, in order to increase immunogenicity, an antigen can be conjugated to a carrier protein. Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). In addition to such carriers, well known adjuvants can be administered with the antigen to facilitate induction of a strong immune response. EXAMPLES

Description of Plasmids

The rat LDH-A sense expression vector was constructed by ligating an EcoRI/Bglll 1.6 kb LDH-A cDNA fragment from pLDH-2 (Matrisian et al., 1985) into the corresponding sites of pSG5, an SV40 promoter driven expression vector (Strategene; La Jolla, CA). Antisense pSG5 vector was constructed by three piece ligation of the same LDH-A cDNA fragment with pSG5 Ndel/Bglll and pSG5 Ndel/EcoRI fragments. The rat prothymosin α cDNA (kindly provided by Dr. P. Szabo, Cornell University, NY) was inserted in the antisense direction into the EcoRI site of pSG5 (Frangou-Lazaridis et al., 1988).

An EBV ori-based episomal antisense LDH-A expression vector was constructed by a three piece ligation of an LDH-A cDNA Hindlll/NotI fragment (from pBSKS shuttle vector) with pHEBoCMVneo Sstl/Hindlll and pHEBoCMVneo Sstl/NotI fragments (partially digested with SstI). This episomal vector was used in lymphoid cells (Lombardi et al., 1987).

The rat LDH-A subunit promoter fragment (-1173/+25 bp; GenBank/EMBL data Bank, accession no. U05674) was as described previously (Short et al., 1994). A Xbal-restricted 642 bp promoter fragment was subcloned into a luciferase reporter, pGL2Luc (Promega; Madison, WI), to generated pGLDH637Luc. The two Myc/Max type E-boxes, CACGTG, in the LDH-A promoter were mutated to CCCGGG by PCR-assisted methods. The 5' E-box was mutated using a pair of primers: 5'-TTGGGGTGTCGCAGCACCCGGGGAGCCACTCTTGCAGG; 5-

'-TCTAGACGCAGAGCAGCACG. The mutated PCR amplified fragment was then used in a PCR reaction as the 3 '-primer with another 5'-primer: 5'-CTGCTATGGCGGATAGACC. The final PCR product was subcloned into the TA cloning system (Invitrogen Co., San Diego, CA) and the mutated Aatll/Nsil promoter fragment was subcloned back into the promoter reporter construct, pGLDH637Luc. The 3' E-box was similarly mutated using a pair of primers: 5- '-CTGCTATGGCGGATAGACC; 5'-TGCGGGAACCCCCGGGTAGGCTGGGCCG.

The double E-box mutant was made by combining the single-E-box mutants through one of two BssHII restriction sites located in between the two E-boxes and a flanking EcoRI site. The mutated promoters were confirmed by DNA sequence analysis.

Representational Difference Analysis.

Rat la or Ratla-Myc cell lines (2 x 10⁶ cells/150mm plates) were plated on 150mm plates coated with a layer of 0.7% agarose in DMEM. Cells were grown for 48 hours. RNA was isolated by guanidium thiocyanate lysis followed by cesium chloride centrifugation.

Poly A+ mRNAs were selected on an oligo dT column. cDNA was then synthesized using a kit (Promega; Madison, WI). cDNAs were then digested with either Dpn II or

Sau 3A and subjected to RDA as previously described (Hubank and Schatz, 1994;

Lisitsyn et al., 1993). RDA selected amplicons were subcloned directly into PCR 2.1 via

TA cloning (Invitrogen, San Diego, CA).

RNA analyses and nuclear run-on assays. Total RNA from Ratl a and Ratl a-Myc cells was isolated by guanidium thiocyanate lysis followed by cesium chloride centrifugation. 15 μg of total RNA was used in RNase protection assay with the RPA II kit (Ambion Inc., Austin, TX) according to the manufacturer's protocol. Rat vimentin mRNA levels were independent of Myc expression as determined by Northern blot analysis; therefore vimentin was used as an internal control. Northern blot analyses were performed as described (Hoang et al., 1994). Nuclear run-on assays were performed as described (Groudine et al., 1981).

Luciferase Assay

Luciferase activity was measured using the luciferase assay system (Promega, Madison, WI). Data were normalized for total protein as measured by the method of Bradford (Bradford, 1976). Immunoblotting.

Cells were lysed in 10%> SDS, heated to 95°C for 5 min, sheared through 26G needles, and supematants were collected after centrifugation at 16,000 g for 5 min. Protein concentrations were measured with the BCA Protein Assay reagent (Pierce Co., Rockford, IL). Equal volumes of lysates and 2X Laemmli buffer were mixed (Laemmli, 1970), and equivalent amounts of total proteins were resolved by SDS/ 10% PAGE and subjected to immunoblot analysis. c-Myc and Bcl-2 were detected using a monoclonal mouse anti-Myc antibody 9E10 (1 :1000 dilution) (Evan et al., 1985) and polyclonal anti-human Bcl-2 antibody (1 : 1000 dilution; Pharmingen, San Diego, CA), respectively. The blots were incubated with secondary goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated antibody (1 :10,000 dilution; Bio-Rad Laboratories, Rockville Center, NY), and reactive polypeptides were detected by the enhanced chemoluminescence system (Amersham Corp., Arlington Heights, IL).

LDH Enzyme Assay and Glucose-Lactate Measurements Cells were collected and sheared through a 26G needle in a hypotonic buffer (15mM KC1, lOmM Tris-HCl, 1.5 mM MgC12, and 6 mM mercaptoethanol at pH 7.4) (Nakamura et al., 1984). The cell lysates were centrifuged at 16,000g for 5 min and the supematants were collected for LDH enzyme assays. LDH enzyme assay kit (Sigma Co., St. Louis, MO) was used according to manufacturer's instructions to measure LDH-A enzyme activity. Glucose and lactate levels in culture media were measured with a YSI model 2300 Stat Glucose/Lactate Analyzer (Yellow Springs, OH).

Cell Culture and Transfection

Rat fibroblast cells were cultured in a humidified atmosphere of 5% CO₂ in air at 37°C using Dulbeccops modified eagle medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (GIBCO-BRL, Gaithersburg, MD) and antibiotics. Human lymphoid cells were similarly cultured in Iscoveps modified Dulbecco ps medium (IMDM). Human lung carcinoma H209 cells were cultured in RPMI 1640 medium. For glucose deprivation studies, Ratla cells were washed two times with phosphate buffered saline and then cultured with DMEM without glucose or pyruvate (GIBCO-BRL, Gaithersburg, MD) containing dialyzed 10% fetal bovine serum (GIBCO-BRL, Gaithersburg, MD) supplemented with sodium pyruvate.

Ratla fibroblasts were transfected with pSG5-LDH-A sense or antisense and a puromycin resistance marker plasmid (pBABE puro) using Lipofectin (GIBCO BRL, Gaithersburg, MD) as described (Hoang et al., 1994). Pooled transfected Ratla cells were selected with 0.75 μg/ml puromycin (Sigma Chemical Co., St. Louis, MO). Lymphoid cells were transfected with Lipofectin (BRL, Gaithersburg, MD) and selected in 800 μg/ml G418.

A Ratla cell line expressing the c-Myc-estrogen receptor fusion protein (gift of J. M. Bishop; UCSF) was passaged in DMEM with 10% fetal calf serum and cultured at 80% confluency in DMEM without phenol red ^• (BRL-GIBCO; Gaithersburg, MD) and charcoal-treated fetal bovine serum (10%) v/v; Hyclone, Logan, UT) for 48 h prior to induction of Myc. To induce c-Myc activity, Myc-ER cells grown to confluency were exposed to 0.25 mM 4-hydroxytamoxifen (Research Biochemical International; Natrick, MA) for the indicated times as previously described (Grandori et al., 1996). To block protein synthesis these cells were exposed to 10 mM cycloheximide (Sigma Chemical Co., St. Louis, MO) 30 minutes before addition of hydroxytamoxifen.

Hypoxic treatment of cells was performed as previously described (Wang et al., 1995). Cells were cultured in DMEM (6 ml/100 mm dish) with 25 mM HEPES (pH 7.55) with 10% fetal calf serum for 48 h in hypoxic chambers. The chambers were sealed and gassed with 0% oxygen, 5 % carbon dioxide and 95% nitrogen gas. Measurements of glucose comsumption and lactate production of Ratla cells yielded a molar ratio of 2 lactate molecules produced per glucose molecule consumed, suggesting that effective hypoxia was achieved. Growth rates of lymphoid cells and fibroblasts were determined by plating 5 x 104 and 1 x 104 cells, respectively, per 60 mm dish. At the indicated time points, cells from triplicate dishes were counted in a Coulter counter. Fibroblasts were trypsinized prior to Coulter counting.

Soft agar clonogenic, Ras-transformation, and colony suppression assay

The soft agar anchorage-independent growth assay was performed as previously described (Hoang et al., 1994). 5 X 105 Ratla fibroblasts were mixed with 0.4% agarose and poured onto a bed of 0.1% agarose in 100-mm dish. Both top and bottom agarose were prepared in DMEM with 10% FBS. For lymphoid cells, soft agar assays were performed with 5 X 105 cells in 0.3% top agarose with IMDM as described previously (Lombardi et al., 1987).

Ratla cells were lipofected with pEJ-ras (2 μg/100 mm dish) encoding activated H-ras and either the empty pSG5 expression plasmid (4 μg/100 mm dish) or the LDH-A antisense expression plasmid pAS-LDH-A (4 mg/100 mm dish). Transformed foci were determined by photography at 2 weeks after transfection. For colony suppression assay, either the empty pSG5 expression plasmid (4 μg/100 mm dish) or the LDH-A antisense expression plasmid pAS-LDH-A (4 μg/100 mm dish) was lipofected with the pBABE-Puro (0.2 μg/100 mm dish) puromycin-resistance marker plasmid. Puromycin-resistant colonies were counted in triplicate experiments, 7 days after the beginning of drug selection.

Apoptosis assay and BrdU labeling

The cell cycle distribution and the fraction of actively proliferating cells were determined by two dimensional flow cytometry. Time dependent data were obtained from cells grown to a half confluent monolayer in culture flasks. Following incubation for 30 min with bromo-deoxyuridine (BrdU, lOμM), the cells were washed, fixed in 70% ethanol at -20°C, digested with pepsin (0.4 mg/ml in 0.1 N HC1) for 30 min, incubated in 2N HC1 for 20 min at room temperature (Schutte et al., 1987), then stained with a fluorescein isothiocyanate (FΙTC)-labeled anti-BrdU antibody (Becton-Dickinson Immunocvtometry Systems, San Jose, CA). The nuclei were subsequently stained for total DNA with propidium iodide (PI). PI and FITC fluorescence, as well as forward light scattering, were detected using a Coulter EPICS 752 flow cytometer equipped with MDADS 11 software. V 1.0. Cell cycle distribution profiles were determined with a curve fitting program Elite v.3.0 (Coulter Co., Hialeah, FL).

DNA fragmentation characteristic of apoptosis was quantified using two dimensional flow cytometry (Gorczyca et al., 1993). Cells were fixed in 1% formaldehyde followed by 70%) methanol, washed, and incubated at 37oC with the deoxynucleotide analog biotin-16-dUTP plus terminal deoxynucleotidyl transferase (TdT) (Boehringer Mannheim, Indianapolis, IN). Cells were then treated with a FITC-conjugated avidin (Boehringer Mannheim, Indianapolis, IN), followed by PI staining and analyzed by flow cytometry as described above.

Table 1 : Lactate production to glucose consumption molar ratios for Ratla cell lines

Ratla Ratla-Myc Ratla-LDH

1.28 1.44 1.68

1.28 1.48 1.62 1.28 1.44 1.58

1.06 1.48 1.56

1.23 + 0.11 1.46 + 0.02 1.61 + 0.05

Values represent the moles of lactate produced per mole of glucose consumed for various cell lines after 24 h incubation with fresh medium. Values from four separate experi- ments and the mean with standard deviations are shown for each cell line.

Table 2. 2-Deoxyglucose induction of apoptosis in c-Myc overexpressing cells

Percent apoptotic cell death

Cell line control 2-deoxyglucose (10 mM)

Ratla 0.2 4.6 Ratla-Myc 0.8 21.9

Ratla-Myc-Bcl-2 0.4 0.3

CB33 (lmphoblastoid) 1.6 8.0

CB33-Myc 5.7 57.4

Ramos (Burkitt's) 0.3 23.3 DW6 (Burkitt's) 1.0 7.6

STE486 (Burkitt's) 3.9 0.2

H209 (lung) 0.2 3.0

H209-Myc 1.4 153

Each cell line was cultured in their respective medium with 10 mM deoxyglucose for 1 day before they were harveted for flow cytometric analysis for apoptosis.

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Claims

WE CLAIM:

1. A method for ameliorating a cell proliferative disorder associated with elevated levels of LDH-A and levels of at least one anti-apoptotic protein that are the same or less than a normal cell not having the disorder in a subject, the method comprising contacting cells of the subject with a therapeutically effective amount of a compound which inhibits LDH-A activity.

2. The method of claim 1 , wherein the anti-apoptotic protein is bcl2.

3. The method of claim 1, wherein the cell proliferative disorder includes overgrowth of cells.

4. The method of claim 1, wherein the cell proliferative disorder is associated with oncogenic transformation.

5. The method of claim 1, wherein the oncogene is selected from the group consisting of myc, ras, fos, SV40 large T antigen and adenovirus El A.

6. The method of claim 1 , wherein the inhibitor is antibody.

7. The method of claim 6, wherein the antibody is monoclonal.

8. The method of claim 6, wherein the antibody is polyclonal.

9. The method of claim 1 , wherein the inhibitor is an LDH-A antisense nucleic acid.

10. The method of claim 9, wherein the antisense nucleic acid is complementary to a region in the LDH-A promoter.

11. The method of claim 10, wherein the region of the LDH-A promoter contains an oncogene protein binding site.

12. The method of claim 11 , wherein the oncogene is myc.

13. The method of claim 1 , wherein the contacting is in vivo.

14. The method of claim 1 , wherein the contacting is ex vivo.

15. The method of claim 14, wherein the cells are bone marrow cells.

16. A method for ameliorating a cell proliferative disorder in a subject wherein the cells have levels of at least one anti-apoptotic protein that are the same or less than a normal cell not having the disorder and have elevated levels of lactic acid, the method comprising contacting cells of the subject with a therapeutically effective amount of a compound which affects glycolysis.

17. The method of claim 16, wherein the anti-apoptotic protein is bcl2.

18. The method of claim 16, wherein the cell proliferative disorder includes overgrowth of cells.

19. The method of claim 16, wherein the cell proliferative disorder is associated with oncogenic transformation.

20. The method of claim 19, wherein the oncogene is selected from the group consisting of myc, ras, fos, SV40 large T antigen and adenovirus El A.

21. The method of claim 16, wherein the compound is a inhibitor of glycolysis.

22. The method of claim 21 , wherein the compound is a glucose anti -metabolite.

23. The method of claim 22, wherein the compound is 2-deoxyglucose (2-DG).

24. A method for identifying a compound which modulates cell proliferation in cells that produces elevated levels of LDH-A and levels of at least one anti- apoptotic protein that are the same or less than a normal cell not having the disorder comprising:

(a) incubating components comprising the compound and said cell, wherein the incubating is carried out under conditions sufficient to allow the compound to interact with said cells; and

(b) measuring the effect of the compound on proliferation of the cell.

25. The method of claim 24, wherein the compound is an LDH-A antisense nucleic acid.

26. The method of claim 25, wherein the antisense nucleic acid is complementary to a region in the LDH-A promoter.

27. The method of claim 26, wherein the region of the LDH-A promoter contains an oncogene protein binding site.

28. The method of claim 24, wherein the compound is a glucose antimetabolite.

29. The method of claim 28, wherein the compound is 2-deoxyglucose (2-DG).

30. The method of claim 24, further comprising measuring the effect of the compound on glycolysis.

31. The method of claim 24, wherein the anti-apoptotic protein is bcl2.

32. A cell line for screening glucose antimetabolite compounds characterized by: a) having levels of at least one anti-apoptotic protein that are the same or less than a normal cell; and b) having elevated levels of LDH-A.

33. The cell line of claim 32, wherein the anti-apoptotic protein isbcl2.

34. The cell line of claim 32, wherein the cell is transformed with myc-oncogene.

35. An antisense nucleic acid sequence that binds to LDH-A encoding nucleic acid.

36. An antisense nucleic acid sequence that binds to LDH-A promoter region.