Glucose Catabolism in Cancer Cells

One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than their tissues of origin. Hepatomas exhibiting this phenotype are dependent on the high expression of type II hexokinase, which supplies such tumors with abundant amounts of glucose 6-phosphate, a significant carbon and energy source especially under hypoxic conditions. Here we report that the distal region of the hepatoma type II hexokinase promoter displays consensus motifs for hypoxia-inducible factor (HIF-1) that overlap E-box sequences known to be related in other gene promoters to glucose response. Moreover, we show that subjecting transfected hepatoma cells to hypoxic conditions activates the type II hexokinase promoter almost 3-fold, a value that approaches 7-fold in the presence of glucose. Consistent with these findings is the induction under hypoxic conditions of the HIF-1 protein. Reporter gene analyses with a series of nested deletion mutants of the hepatoma type II hexokinase promoter show that a significant fraction of the total activation observed under hypoxic conditions localizes to the distal region where the overlapping HIF-1/E-box sequences are located. Finally, DNase I footprint analysis with a segment of the promoter containing these elements reveals the binding of several nuclear proteins. In summary, these novel studies identify and characterize a marked glucose-modulated activation response of the type II hexokinase gene to hypoxic conditions within highly glycolytic hepatoma cells, a property that may help assure that such cells exhibit a growth and survival advantage over their parental cells of origin.

One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than their tissues of origin. Hepatomas exhibiting this phenotype are dependent on the high expression of type II hexokinase, which supplies such tumors with abundant amounts of glucose 6-phosphate, a significant carbon and energy source especially under hypoxic conditions. Here we report that the distal region of the hepatoma type II hexokinase promoter displays consensus motifs for hypoxia-inducible factor (HIF-1) that overlap E-box sequences known to be related in other gene promoters to glucose response. Moreover, we show that subjecting transfected hepatoma cells to hypoxic conditions activates the type II hexokinase promoter almost 3-fold, a value that approaches 7-fold in the presence of glucose. Consistent with these findings is the induction under hypoxic conditions of the HIF-1 protein. Reporter gene analyses with a series of nested deletion mutants of the hepatoma type II hexokinase promoter show that a significant fraction of the total activation observed under hypoxic conditions localizes to the distal region where the overlapping HIF-1/E-box sequences are located. Finally, DNase I footprint analysis with a segment of the promoter containing these elements reveals the binding of several nuclear proteins. In summary, these novel studies identify and characterize a marked glucose-modulated activation response of the type II hexokinase gene to hypoxic conditions within highly glycolytic hepatoma cells, a property that may help assure that such cells exhibit a growth and survival advantage over their parental cells of origin.
Numerous studies have demonstrated that highly malignant tumors, i.e. those that are poorly differentiated and grow rapidly, exhibit the capacity to metabolize glucose to lactate at much higher rates than normal cells (reviewed in Refs. [1][2][3][4][5]. This high rate is dependent on elevated levels of hexokinase (6,7), which catalyzes the first step in the glycolytic pathway, thus committing glucose to metabolism as glucose 6-phosphate. In rapidly growing hepatomas, it is type II hexokinase, a form that binds to the outer mitochondrial membrane, that is the predominant overexpressed isozyme (8 -10). In previous reports, we have described the isolation and characterization of the hepatoma type II hexokinase promoter and shown that it contains many apparent response elements (10 -12). The most notable of these elements, from the point of view of glucose metabolism, are those for glucose, insulin, and cAMP, all of which activate the promoter in freshly isolated, transfected hepatoma cells (10 -12). The promoter also contains response elements for the tumor suppressor p53, which, in mutated form, also activates the type II hexokinase promoter (13). Although significant, the activation response obtained for each of these ligands or factors is modest ranging from approximately 1.5-to 3-fold in reporter gene assays where -fold activation is calculated relative to a physiological background containing pyruvate (10,12,13).
Recently, our attention has turned to an investigation of the effects of hypoxic conditions on the hepatoma type II hexokinase promoter as such conditions are likely to be one of the greatest stresses experienced by cancer cells (14,15). Such conditions occur within the tumor interior as a result of rapid cell proliferation that outpaces the growth of oxygen-supplying blood vessels. This slows or incapacitates the oxygen-dependent mitochondrial ATP generating system and forces the glycolytic system to compensate for this loss in ATP production. As type II hexokinase catalyzes the first step of glycolysis in rapidly proliferating hepatomas, it seems reasonable to predict that its promoter is designed to elicit a robust activation response to hypoxic conditions. Significantly, in recent years a number of genes regulated by cellular oxygen concentration have been identified, including the hematopoietic factor erythropoietin, the angiogenesis mediator vascular endothelial growth factor, certain genes of the glycolytic pathway, and isoforms of the glucose transporter at the cell surface (reviewed in Refs. 16 and 17). Central to this regulation is hypoxia-inducible factor-1 (HIF-1), 1 a heterodimeric protein (18) that binds most frequently to the core sequence 5Ј-RCGTG-3Ј (R ϭ purine) (17), but in some promoters also to the modified core sequence 5Ј-RCGTC-3Ј (19). Once bound to a gene's promoter via these binding sites, HIF-1 exerts its effect on gene transcription by mechanisms that remain to be elucidated (17).
With the above thoughts and information serving as a guide, we proceeded to identify HIF-1 binding sites on the hepatoma type II hexokinase promoter and to characterize in some detail the promoter's capacity to be regulated under hypoxic conditions. As predicted above and described in detail below, hypoxic stress significantly activates the promoter, a response that is further accentuated by physiological concentrations of glucose and correlates with the induction of HIF-1 expression.

Materials
The pGL2-Basic luciferase reporter vector, the pSV-␤-galactosidase control vector, and the luciferase assay system were from Promega. The TD-20e luminometer for chemiluminescence measurements and the portable anaerobic chamber were purchased from Turner Designs and Fisher, respectively. The cell porator (electroporator) for transfecting mammalian cells, restriction enzymes, DNA modifying enzymes, and an antibiotic-antimycotic mixture were purchased from Life Technologies, Inc. The glucose-deficient RPMI 1640 tissue culture medium was from Sigma. The Maxi Prep plasmid purification system used to prepare plasmid DNA for transfections was from Qiagen, and the monoclonal antibody against the ␣ subunit of HIF-1 (HIF-1␣) was from Novus Biologicals. Polyvinylidene difluoride membranes were from Bio-Rad, and the ECL chemiluminescence system for detecting the antibody was from Amersham Pharmacia Biotech. [␣-32 P]dATP (3000 Ci/mmol) and [␣-35 S]dATP (1000 Ci/mmol) were from PerkinElmer Life Sciences. A gas mixture of 1% O 2 , 5% CO 2 , and 94% N 2 was obtained from MG Industries. The 4.3-kbp proximal promoter of the type II hexokinase gene derived from AS-30D hepatoma cells was isolated, sequenced, and characterized as described in earlier reports from this laboratory (10 -13). Female Sprague-Dawley rats were obtained from Charles River Laboratories. Their care and use in the experiments described below was approved by and conducted in accordance with the guidelines of the Johns Hopkins University Animal Care and Use Committee. The AS-30D hepatoma cell line, an established model cell line exhibiting a high glycolytic rate (6,7), is routinely maintained and propagated in the laboratory of the corresponding author.

Methods
Tumor Cells-AS-30D hepatoma cells were propagated in the peritoneal cavity of female Sprague-Dawley rats (100 -150 g) and isolated as described previously using glucose-supplemented RPMI 1640 medium, in the absence of serum (8). They were then resuspended at a final concentration of 50 ϫ 10 6 cells ml Ϫ1 for transient transfection studies (10).
Detection of HIF-1-To assess the levels of HIF-1 in AS-30D hepatoma cells, the cells were washed once in Dulbecco's modified phosphate-buffered saline (Ca 2ϩ -and Mg 2ϩ -free). Nuclear extracts were prepared from the washed cells as described below, and analyzed by SDS-PAGE and Western blotting using the HIF-1 antibody from Novus Biologicals, the reliability of which has been documented in recent reports (20,21).
Western Analysis-For Western blotting, samples of nuclear extracts from AS-30D hepatoma cells were subjected to SDS-PAGE on 7.5% gels, which were then transferred onto polyvinylidene difluoride membranes in CAPS buffer (10 mM CAPS, 10% v/v methanol, pH 11). The membranes were probed with an anti-HIF-1␣ monoclonal antibody, according to the manufacturer's instructions, and detected by an ECL chemiluminescence system.
Preparation of Nuclear Extracts-Nuclear extracts were prepared according to the method of Dignam et al. (22) from nuclei isolated from fresh AS-30D hepatoma cells and then stored at Ϫ80°C until use.
Construction of Nested Deletion Mutants for Reporter Gene Analyses-The preparation of the hepatoma type II hexokinase promoterluciferase construct containing the full-length 4.3-kbp promoter for reporter gene analysis has been described in an earlier report from this laboratory (10). To generate reporter gene constructs containing nested deletion mutants, the parent plasmid construct was double digested at two restriction sites of the multiple cloning site of the pGL2 vector. KpnI (which creates an exonuclease III-resistant 3Ј terminus at the multiple cloning site), and MluI (which creates an exonuclease IIIsensitive restriction site within the multiple cloning site adjacent to the 5Ј end of the cloned promoter) were used for the directional deletions into the cloned promoter. Nested deletions were generated at 30°C using exonuclease III and nuclease S1 (23, 24), at a reaction concentration of 150 units of exonuclease III/pmol of susceptible 3Ј ends. Aliquots of plasmid DNA were recovered at 1-min intervals, placed on ice until after addition of EDTA to quench the reaction, extracted with phenol/ chloroform, and then precipitated with ethanol. The plasmids were then recircularized with T4 DNA ligase. Mutants with 1-kilobase deletions in the promoter were selected by agarose gel electrophoresis. After the residual length of the promoter inserts was confirmed by DNA sequencing, they were transformed into Escherichia coli SURE cells for maintenance. These were then transformed into methylation-deficient E. coli SCS 110 cells, and plasmids subsequently prepared from these cells were used for reporter gene analysis.
Transient Transfection of AS-30D Hepatoma Cells and Incubation under Normoxic Conditions-Transient transfection conditions for the full-length hexokinase promoter-luciferase construct and the ␤-galactosidase control vector have been reported previously (10). The same transfection conditions were used in this study for these constructs and also for the constructs containing the nested deletion mutants. Briefly, equimolar concentrations of reporter gene vector DNA (equivalent to 10 g of the 4.3-kbp full-length promoter-luciferase reporter gene construct) were maintained during transfections with the nested deletion mutant constructs (i.e. 8.7, 7.8, 6.8, 5.9, and 5.7 g, respectively, of 3-, 2-, 1-, 0.1-, and 0-kbp promoter containing nested deletion mutant plasmids). The ␤-galactosidase vector (2.5 g) was used as a control in all transfections, where 25 ϫ 10 6 hepatoma cells (in 0.5 ml) were used per transfection. The cells and reporter gene constructs were incubated on ice for 10 min and electroporated at 200 V, 800 microfarads. After an additional 10-min recovery period on ice, the transfected AS-30D hepatoma cells were plated into 10 ml of RPMI 1640 glucose-deficient medium supplemented with 2 mM glutamine, 1 mM pyruvate, 25 mM HEPES (pH 7.4), and an antibiotic-antimycotic mixture. For studies in the presence of glucose, the medium was supplemented with 5 mM glucose. For optimal results under normoxic conditions, incubation of cells was carried out at 37°C in 5% CO 2 and 95% atmospheric air for 24 h. The cells were then subjected to lysis using a cold buffer containing 0.6% Triton X-l00, 0.1 M potassium phosphate, and 1 mM DTT, pH 7.8 (25). Finally, aliquots (10 and 50 l) were used to assay luciferase and ␤-galactosidase activity, respectively.
Incubation of Transfected Cells under Hypoxic Conditions-To expose transfected cells to hypoxia, parallel sets of 100-mm tissue culture dishes containing 25 ϫ 10 6 transfected cells/plate were placed in a portable anaerobic chamber. This was sealed and flushed with an "anaerobic" gas mixture of 1% O 2 , 5% CO 2 , 94% N 2 for 2 min, followed by five successive evacuations (at low vacuum, ϳ1-2 p.s.i.) and repressurization (with the same gas mixture at ϳ1-2 p.s.i.) steps. The chamber was finally flushed again with the anaerobic gas mixture, the evacuation/pressurization ports sealed, and transferred to a 37°C incubator. For optimal results, cells were recovered 12-16 h after transfection, washed once in 10 ml of Dulbecco's modified phosphate-buffered saline (Ca 2ϩ -and Mg 2ϩ -deficient), and lysed in 100 l of cold lysis buffer (0.625% Triton X-100, 0.1 M potassium phosphate, 1 mM DTT, pH 7.8). Aliquots (10 and 50 l) were then assayed for luciferase and ␤-galactosidase activities, respectively.
Assays for Luciferase and ␤-Galactosidase, and Quantification of Data-These enzyme assays were carried out essentially as described by the suppliers of the reporter gene vectors (Promega). Luciferase activity (A) was measured by luminometry using a Turner TD-20e luminometer and recorded as relative light units. DNase I Footprint Analysis-DNase I footprint assays were adapted from methods by Kingston (26) and Christy et al. (27). DNA binding reactions were performed in 35 l of binding buffer (20 mM HEPES, pH 7.4, 5 mM DTT, 1 mM MgCl 2 , 60 mM KCl) with 2 ng (Х2 ϫ 10 5 cpm) of DNA template end-labeled by Klenow fill-in reactions (23,24), 1000 ng of sonicated salmon sperm DNA as competitor DNA, and variable amounts of nuclear extract. Reactions were incubated for 20 min on ice, followed by a 60-s digestion at 25°C with 0.3-5 l of a freshly diluted DNase I solution (0.05 g/l). The digestion reactions were stopped by the addition of 100 l of stop buffer (1% SDS, 20 mM EDTA, 200 mM NaCl, 250 g/ml yeast tRNA). The sample was extracted with phenolchloroform and precipitated with two volumes of ethanol. The DNA pellets were dried and resuspended in loading buffer (99% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol), incubated 5 min at 68°C, and loaded on a 6% polyacrylamide, 8.3 M urea, sequencing gel. The gels were subjected to electrophoresis in 1ϫ Tris borate-EDTA, dried, and then subjected to autoradiography for 1-6 h prior to film development. (28,29)-In an earlier report (10), we presented the sequence of the 4.3-kbp proximal promoter region of the hepatoma type II hexokinase gene, (Gen-Bank TM accession number U19605), identified an E-box (CACGTG) in the distal region (Fig. 1A), and demonstrated using reporter gene analysis that the promoter undergoes a modest activation response (2-3-fold) in the presence of a high concentration of glucose (25 mM). Results presented in Fig. 1B summarize the results of further analyses of the distal region from Ϫ3620 to Ϫ3919 kbp for HIF-1 motifs (5Ј-RCGTG-3Ј or 5Ј-RCGTC-3Ј). Significantly, both visual inspection and the use of a computer algorithm (Signal-Scan; Ref. 30) revealed the presence of one HIF-1 motif (5Ј-RCGTG-3Ј) overlapping the previously identified E-box (CACGTG), and a second HIF-1 motif (5Ј-RCGTC-3Ј) about 50 base pairs upstream overlapping a sequence (CACGTC) with 83% identity to the E-box.

Hypoxic Conditions Activate the Hepatoma Type II Hexokinase Promoter by a Process That Is Enhanced by Physiological
Concentrations of Glucose-The identification of two overlapping E-box/HIF-1 sequences within the distal region of the type II hexokinase promoter raised the possibility that this promoter may be responsive to hypoxic conditions by a process that is modulated by glucose. To test this possibility, AS-30D hepatoma cells freshly isolated from the host animals were transfected with a full-length type II hexokinase promoterluciferase reporter gene construct and exposed in standard medium in separate experiments to three sets of conditions: hypoxia alone (1% O 2 , 5% CO 2 , 94% N 2 ), normoxia alone (5 mM glucose, 5% CO 2 , 95% atmospheric air), and hypoxia plus glucose. As shown in Fig. 1C, hypoxic conditions alone activate the FIG. 1. A, position of the overlapping E-box and HIF-I sequences within the type II hexokinase promoter. The box in blue in the distal 4-kbp region designates a Z-DNA region, whereas IRE designates a putative insulin response element. The positions of the CAAT and TATA boxes together with the cAMP response element (CRE) are shown in orange in the proximal region of the promoter. B, nucleotide sequence of the promoter region that contains the overlapping E-box and HIF-1 sequences. Two HIF-1-related sequences (5Ј-ACGTG-3Ј) and (5Ј-ACGTC-3Ј) both shown in red overlap with one "perfect" E-box with the sequence 5Ј-CACGTG-3Ј and with a second sequence 5Ј-CACGTC-3Ј, which has 83% identity to the first. C, transcriptional activity of the 4.3-kbp type II hexokinase promoter in AS-30D hepatoma cells in response to normoxic and hypoxic conditions. Construction of reporter gene vectors, transfections, and reporter gene assays were carried out exactly as described under "Methods." Hepatoma cells were freshly isolated from tumor-bearing animals. Normoxic and hypoxic conditions to which the cells were subjected are described also under "Methods." Activity is expressed as -fold activation over that of the control (pyruvate, normoxic). Each sample contained 1 mM pyruvate as background substrate. In all cases values are the mean of five different experiments, except where pyruvate was used alone. Here two experiments were carried out. promoter almost 3-fold relative to that observed in the presence of a pyruvate containing "background" medium. However, in the presence of hypoxic conditions plus glucose, the latter of which activates the promoter only about 2-fold, the total activation response approaches 7-fold, a value considerably higher than that expected (approximately 5-fold) had the hypoxia ϩ glucose responses been additive. Moreover, this was a reproducible observation in experiments conducted with AS-30D hepatoma cells from different animals. Thus, these results provide evidence that the hepatoma type II hexokinase promoter is activated by hypoxic conditions in hepatoma cells, and that this response is positively modulated in the presence of a concentration of glucose (5 mM) within the physiological range.

The HIF-1 Protein Is Expressed in the Model Hepatoma Cell Line under Study Where Its Level of Expression Can Be Regulated by Oxygen Concentration-The experiments described
above showing that the AS-30D hepatoma type II hexokinase promoter is activated by hypoxic conditions in cells freshly isolated from the host animal implicate the presence of the HIF-I protein. This was confirmed by subjecting nuclear extracts from these cells first to SDS-PAGE ( Fig. 2A, left lanes 1  and 2) and then to Western blot analysis using an HIF-1␣ subunit antibody (Fig. 2A, right lanes 1 and 2). Subsequent experiments carried out by subjecting AS-30D hepatoma cells in tissue culture to normoxic, hypoxic, or hypoxic plus glucose conditions confirmed that HIF-1␣ subunit levels in these cells are very dependent on oxygen concentration (Fig. 2B). Thus, cells maintained under normoxic conditions expressed much less HIF-1␣ subunit than cells maintained under hypoxic conditions (Fig. 2B, lower panel, compare lanes 1 and 2 with lanes  3 and 4). Glucose was without effect on the protein expression levels of the HIF-1␣ subunit under the same conditions (Fig.  2B, lower panel, compare lane 3 with lane 4). In all of these experiments, the apparent molecular mass of the expressed HIF-1␣ subunit was about l00 kDa: a value that correlates closely to that of the calculated value of 93.3 kDa (GenBank accession no. Y09507).
Taken together with the experiments described above, these results implicate the HIF-1␣ subunit as acting within the distal region of the type II hexokinase promoter and participating, at least in part, in the promoter's activation response to hypoxic conditions. These results also indicate that the role of glucose in enhancing the promoter's activation response to hypoxic conditions (Fig. 1C) is not the result of an effect on HIF-1␣ subunit expression.

A Significant Fraction of the Total Hypoxic Response of the Type II Hexokinase Promoter Can Be Functionally Located to the Distal Region Containing the Overlapping E-box/HIF-1
Sequences-A plasmid constructed for luciferase reporter gene analysis using the full-length promoter (Fig. 3A) formed the basis for the deletion analysis study to functionally locate the primary regions within the promoter that are responsive to hypoxia. As described in detail under "Methods," a series of nested deletion mutants (plasmids B-F) that maintained ϳ1kbp deletions at the 5Ј terminus (Fig. 3B) were selected from the larger pool of deletion mutants generated. During transfection assays with the selected nested deletion mutants, equimolar concentrations of reporter gene vectors equivalent to 10 g of the 4.3-kbp full-length promoter-luciferase reporter gene construct (plasmid A) were maintained together with ␤-galactosidase control vector at concentrations indicated under "Methods." Analysis of these clones by reporter gene analysis under these conditions enabled us to identify the distal 1 kbp of the hepatoma type II hexokinase promoter as a significant contributor to its response to hypoxic conditions in the presence of a physiological concentration (5 mM) of glucose (Fig. 3C). This region of the promoter also contributes in part to the total glucose response. In both cases, the remaining part of the total hypoxic response and the total glucose response appears to localize to the proximal region of the type II hexokinase promoter, as plasmid F lacking the TATA region shows almost negligible responses to hypoxia and glucose in reporter gene analysis.
The Overlapping E-box/HIF-1 Sequences Located in the Distal Region of the Hepatoma Type II Hexokinase Promoter Are Protected from DNase I by Nuclear Extracts-Nuclear extracts prepared from AS-30D hepatoma cells freshly isolated from the host animal were used in DNase I footprint analysis of the distal region of the type II hexokinase promoter containing the two overlapping E-box/HIF-1 sequences. Significantly, as shown in Fig. 4A, these two sequences were found to be protected, indicating that nuclear proteins (potential transcription factors) are present in this hepatoma cell line that bind at these sites. In addition, results of the DNase I footprint analysis revealed that four other sequences near the two overlapping E-box/HIF-1 sequences also bind nuclear factors. Of these, one is a potential Sp1 element (5Ј-GTGCAC-3Ј) that overlaps one of the two overlapping E-box/HIF-1 sequences, whereas two others are identified as potential C/EBP and GATA-1 response elements (Fig. 4B). The fourth protected sequence has no specific identification at present. These data are consistent with the results presented earlier in this report and add additional support for the view that the E-box/HIF-l overlapping sequences located in the distal region of the hepatoma type II hexokinase promoter play a role in this promoter's response to hypoxia and glucose. DISCUSSION Results of experiments reported here provide new insights into the mode of regulation of the gene encoding hepatoma type II hexokinase, the predominant hexokinase isoform expressed in poorly differentiated, rapidly growing hepatomas exhibiting the high glucose catabolic phenotype (6 -10). Specifically, we have shown that the promoter for this enzyme contains in its distal region two overlapping E-box/HIF-1 sequences separated by about 50 base pairs (Fig. 1B) that are known to be associated in other gene promoters (16 -18, 28, 29) with responsiveness to glucose (E-box) or hypoxic conditions (HIF-1). In addition, we have shown that activation of the type II hexokinase gene promoter in transfected hepatoma cells, freshly isolated from the host animal, approaches 7-fold in the presence of hypoxic conditions and a physiological concentration (5 mM) of glucose (Figs. 1C and 3C). Significantly, this is the highest activation response we have observed to date for this promoter. Correlat-ing well with these observations are the following three additional findings reported here. (a) Nuclear extracts from hepatoma cells freshly isolated from the host animal express the HIF-1␣ subunit protein ( Fig. 2A); (b) expression of this protein within these cells can be regulated by oxygen concentration (Fig. 2B); and (c) protein factors within the same nuclear hepatoma extracts bind to the overlapping E-box/HIF-1 sequences located within the distal region of the type II hexokinase promoter (Fig. 4). Finally, results presented here show also that the response of the type II hexokinase gene promoter to both glucose and hypoxic conditions is not confined to its distal region, but involves significantly the proximal region as well (Fig. 3C). Elements in this region remain to be identified and studied in detail. Here we have built on preliminary data reported by us previously (31) and characterized the molecular interactions and their induction under hypoxia in detail. Consistent with this latter finding is the recent result reported by others (32) that, in the A549 human lung cell line, the proximal region of the type II hexokinase promoter also contributes significantly to hexokinase expression under hypoxic conditions.
The apparent synergism between glucose and hypoxic conditions reported here in activating hepatoma type II hexokinase FIG. 3. A, outline of the strategy used for generating ϳ1-kbp nested deletion mutants from the 4.3-kbp type II hexokinase promoter-luciferase reporter gene vector. Restriction sites (SmaI, KpnI, MluI) in the 5Ј region are located on the multicloning site of the parental pGL2basic reporter gene vector. Nested deletions were generated exactly as described under "Methods" by digesting with the indicated enzymes. B, nested deletion mutant series used in reporter gene analysis. The length of the residual promoter regions are indicated on the left under each plasmid construct, and the names (plasmids A-F) of these constructs are indicated on the right. Boxes (green and red) in the distal promoter region designate the location of the overlapping E-box and HIF-1 sequences, whereas the box in the proximal promoter region designates the TATA box. Plasmid F, in which the complete promoter has been removed, represents the control plasmid. C, response of nested deletion mutants to hypoxic conditions plus glucose, and to glucose alone following transfection of AS-30D hepatoma cells. Transfection assays were carried out under normoxic and hypoxic conditions exactly as described under "Methods." Where indicated, 5 mM glucose was present. -Fold activation was calculated as described under "Methods" over a control for each deletion mutant in gene expression has, to our knowledge, not been observed previously for a tumor-associated metabolic gene and deserves further investigation, particularly as it relates to biosynthetic enzymes involved in the rapid production of cell building blocks during tumorigenesis. Thus, for cancer cells to survive within a tumor that is proliferating faster than its vascular system, they must be able to thrive in a hypoxic environment and, in so doing, must not only utilize glucose as the major or sole energy source, but as a major carbon source for biosynthesis as well. Therefore, when faced with such a life or death situation, tumor cells subjected to hypoxic conditions may rely not only on glucose ϩ hypoxic response elements within the promoters of their key glycolytic genes, but in the promoters of their biosynthesis-related genes as well.
The results of studies reported here may be relevant also to tumor metastasis. As indicated in earlier reports (14,15), tumor cells subjected to hypoxic stress may acquire additional mutations in tumor suppressor genes like p53 leading to the development of a clonal population of more aggressive cancer cells with the capacity to metastasize and ultimately kill the host (14,15). If such events are found to contribute significantly to tumor metastasis, it seems likely that the gene encoding type II hexokinase that can be markedly activated by hypoxic conditions in the presence of glucose will be involved.