©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucose Catabolism in Cancer Cells
ISOLATION, SEQUENCE, AND ACTIVITY OF THE PROMOTER FOR TYPE II HEXOKINASE (*)

Saroj P. Mathupala , Annette Rempel (§) , Peter L. Pedersen (¶)

From the (1)Laboratory for Molecular and Cellular Bioenergetics, Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

One of the most characteristic phenotypes of rapidly growing cancer cells is their propensity to catabolize glucose at high rates. Type II hexokinase, which is expressed at high levels in such cells and bound to the outer mitochondrial membrane, has been implicated as a major player in this aberrant metabolism. Here we report the isolation and sequence of a 4.3-kilobase pair proximal promoter region of the Type II hexokinase gene from a rapidly growing, highly glycolytic hepatoma cell line (AS-30D). Analysis of the sequence enabled the identification of putative promoter elements, including a TATA box, a CAAT element, several Sp-1 sites, and response elements for glucose, insulin, cAMP, Ap-1, and a number of other factors. Transfection experiments with AS-30D cells showed that promoter activity was enhanced 3.4-, 3.3-, 2.4-, 2.1-, and 1.3-fold, respectively, by glucose, phorbol 12-myristate 13-acetate (a phorbol ester), insulin, cAMP, and glucagon. In transfected hepatocytes, these same agents produced little or no effect. The results emphasize normal versus tumor cell differences in the regulation of Type II hexokinase and indicate that transcription of the Type II tumor gene may occur independent of metabolic state, thus, providing the cancer cell with a selective advantage over its cell of origin.


INTRODUCTION

The ability to maintain an increased rate of glucose utilization and the capacity to sustain high rates of glycolysis under aerobic conditions are the most common biochemical phenotypes of rapidly growing cancer cells(1, 2, 3, 4, 5) . This elevated rate of glucose catabolism is important for highly malignant tumors, which obtain over 50% of their energy, and the anabolic precursors for biosynthetic pathways, via glycolysis(6, 7) . The role of hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), which commits glucose to catabolism in the first step of the glycolytic pathway, has come under increased scrutiny in efforts to understand the molecular basis for the aberrant glycolytic phenotype (7, 8, 9) and has been considered also as a potential target for arresting tumor cell growth(10, 11) .

In comparison to normal cells, the activity of hexokinase is markedly elevated in highly glycolytic, rapidly growing tumors(4, 7, 9, 12) . Two factors are largely responsible for this enhanced activity, one of which involves a propensity for the tumor enzyme to bind to the outer mitochondrial membrane, and the other which involves the enzyme's overproduction. Mitochondrial binding of the tumor enzyme has been studied in depth(3, 4, 6, 7, 8, 9, 12, 13) and has been shown to provide the enzyme with preferential access to mitochondrially generated ATP (9) and to reduce its sensitivity to product inhibition by glucose 6-phosphate(3) , an important regulator of hexokinase in normal cells (3, 13-16). Tyrosine phosphorylation of the tumor enzyme (17) may also play a role in this process but remains to be investigated. Although the mitochondrial bound hexokinase phenotype is also observed for some normal tissues including brain and skeletal muscle(18) , it is much less than that observed in rapidly growing cancer cells.

The overproduction of hexokinase in cancer cells has been given only modest attention, but has nevertheless resulted in the important observation that mRNA levels for this critical key enzyme are also markedly elevated(19, 20, 21) . Therefore, it is of critical importance to elucidate the mechanism of hexokinase transcription in cancer cells in order to identify those factors responsible for its overexpression. In addition, the identification of factors involved in the transcriptional overexpression of hexokinase in tumor cells may also uncover novel signal transduction cascades that are initiated when a cell acquires the cancer phenotype.

To address the issue of enhanced transcriptional regulation of hexokinase in tumor cells, we chose to study the highly glycolytic, rapidly growing hepatoma cell line AS-30D. This cell line has been characterized in detail in this laboratory with respect to its high glycolysis and the role in this process of mitochondrial bound hexokinase(3, 6, 8, 9, 22) . Of the four common hexokinase types (I, II, III, and IV (glucokinase)), it is the Type II, and to a lesser extent, the Type I isozyme that are overexpressed in rapidly growing, highly glycolytic tumors(12, 22) . Therefore, studies reported in this paper utilized a PCR generated probe derived from the Type II hexokinase cDNA(23) , corresponding to the first exon(24) , to screen an AS-30D hepatoma genomic library. As indicated in detail below, the resultant studies led to the cloning and sequencing of the Type II tumor hexokinase promoter, the identification of a number of response elements, and evidence that a number of these elements may play a role in the regulation of tumor Type II hexokinase gene transcription.


EXPERIMENTAL PROCEDURES

Materials

-Fix II phage DNA and Gigapack II Gold packaging extracts were from Stratagene, BA-85 nitrocellulose membranes were from Schleicher and Schuell, [-P]dATP and [-S]dATP were from DuPont NEN. The original source for the Type II hexokinase cDNA clone from rat skeletal muscle was Dr. John E. Wilson, Department of Biochemistry, Michigan State University, East Lansing, MI. Sequenase V. 2.0 and single strand binding protein were from United States Biochemicals. pGL-2 vector series and pSV--galactosidase control vector were from Promega. Chemiluminescence was measured using a TD-20e luminometer (Turner Designs/Promega). Electroporation was carried out in a Cell-Porator electroporator (Life Technologies, Inc.). Other plasmids, restriction enzymes, DNA modifying enzymes, DNA and RNA molecular weight standards were from Life Technologies, Inc. RPMI 1640 (glucose-deficient) tissue culture media, hormones, and growth factor analogs were from Sigma. Plasmid DNA was purified using Nucleobond-AX (Nest Group, Inc.) or Qiagen Maxi-prep (Qiagen, Inc.) plasmid purification columns.

Methods

Tumor Cells

AS-30D hepatoma cells were propagated in female Sprague-Dawley rats as described previously(6, 9) . The hepatoma cells, in ascitic form, were harvested 6-7 days post-transplantation. For genomic DNA isolation (see below), the cells were purified by differential centrifugation in Chance-Hess medium (6) and resuspended in phosphate-buffered saline. For transfection studies, RPMI 1640 medium was used for purification and resuspension.

Isolation of Hepatocytes

Hepatocytes were isolated from female Sprague-Dawley rats (200-250 g) by collagenase perfusion (25) with minor modifications. In brief, after perfusing the liver the hepatocytes were resuspended in 20 ml of RPMI 1640 medium. An equal volume of 90% (v/v) Percoll solution (17 mM NaCl, 5.4 mM KCl, 81.3 mM MgSO, 1 mM phosphate buffer, pH 7.4) (26) was added and mixed. The viable hepatocytes were separated by centrifugation (50 g, 5 min) and washed once in RPMI 1640 medium.

Northern Blot Analysis

Isolation of total RNA from liver, AS-30D cells, brain, and skeletal muscle was performed by phenol-chloroform extraction(27) . Total RNA (20 µg) was size fractionated on a 1.2% agarose formaldehyde gel(28) , transferred to a nylon membrane (Hybond-N, Amersham Corp.) by capillary transfer, and then UV-cross-linked. The gel was stained with ethidium bromide to verify equal RNA sample loading. Expression of Type I hexokinase was studied using an EcoRI/BamHI fragment (0.7 kbp)()of the mouse hepatoma Type I hexokinase cDNA (29) corresponding to the N terminus. For Type II hexokinase a full-length cDNA encoding the rat skeletal muscle Type II enzyme (23) was used. The probes were labeled with [-P]dATP by random priming, and hybridization bands were visualized by autoradiography (-70 °C, 3 days).

Genomic Library Construction

Genomic DNA was isolated from AS-30D hepatoma cells as described previously(28) . Partial digestion of the genomic DNA with Sau3AI to generate 10-20 kbp DNA fragments and partial fill-in of the Sau3AI ends with Klenow fragment to create ends which are incompatible with each other, but are complimentary to XhoI partially filled in ends, were carried out as described previously(30) . Isolation of -Fix II phage DNA by a liquid lysate method and modification of -Fix II phage DNA to generate XhoI half-site arms, and ligation of the half-site -arms to the partially filled-in AS-30D genomic DNA, were carried out also as described previously(30) . The ligated DNA was packaged in vivo using Gigapack II Gold packaging extract according to manufacturer's instructions. The recombinants were screened on duplicate nitrocellulose membranes (132 mm) at a density of 5 10 plaque-forming units/plate.

A 260-bp PCR generated DNA fragment corresponding to the positions -197 to +63 of Type II hexokinase (translation start point referenced as +1) (23) was [-P]dATP radiolabeled by nick translation and used as the probe to screen the library. Hybridizations were performed at 42 °C for 20 h in a solution consisting of 50% formamide, 5 SSPE, 5 Denhardt's solution, 0.1% SDS, 100 µg/ml salmon sperm DNA, and 1 µg of the labeled probe (2.5 10 counts/min in 50 µl). The filters were washed at room temperature in 2 SSC, 0.05% SDS for 2 h, followed by a second wash in 1 SSC, 0.1% SDS at 65 °C for 1 h, and exposed to Kodak X-OMAT-AR films at -80 °C. Six positive -clones were identified by autoradiography and plaque-purified. Liquid lysates were prepared from each and DNA isolated as described previously(30) .

Southern Blot Analysis

DNA (10 µg) from each -clone was digested with XbaI, fractionated on a 0.8% agarose gel, and transferred onto a Zeta-probe (Bio-Rad) nylon membrane in 10 SSC. The membrane was probed using the same 260-bp probe as before. Hybridization was carried out for 20 h at 45 °C in a solution consisting of 45% formamide, 6 SSC, 10 mM EDTA, 5 Denhardt's solution, 0.5% SDS, 0.1 mg/ml salmon sperm DNA, and 1 µg (2.5 10 counts/min in 50 ml) of the radiolabeled probe. The membrane was washed sequentially in 2 SSC, 0.5% SDS for 5 min at room temperature, 2 SSC, 0.1% SDS for 30 min at 37 °C, 0.1 SSC, and 0.5% SDS for 30 min at 65 °C. Hybridized bands were visualized by autoradiography as described before.

Subcloning and Sequencing of Genomic Fragments

XbaI-digested DNA fragments which hybridized to the Type II hexokinase cDNA probe upon Southern blot analysis were isolated from each -clone by preparative agarose gel electrophoresis under the same conditions as described above, followed by electroelution. The individual DNA fragments were subcloned into the XbaI site of plasmid vector pUC 18. The DNA sequence of one of the inserts was determined using the dideoxy chain termination method (31) in the presence of single strand binding protein (United States Biochemicals) using synthetic oligonucleotide primers. Manual and automated methods were used to determine the DNA sequence of the insert in both orientations.

Plasmids for Transient Transfections

The promoterless luciferase plasmid vector pGL2-Basic was used for all promoter studies. An SV40 promoter--galactosidase reporter vector (pSV--galactosidase) was used as an internal control for evaluating the efficiency of transfections in each experiment. An SV40 promoter-luciferase reporter vector (pGL2-Control) was used to evaluate the transcription strength of the tumor Type II hexokinase promoter.

The XbaI-digested DNA fragment described above, which contained the proximal promoter region and the first exon of the AS-30D tumor Type II hexokinase gene, as identified by DNA sequencing, was inserted into the compatible NheI site of the luciferase reporter plasmid pGL2-Basic, upstream of the luciferase cDNA. This construct was sequenced at the sites of ligation using synthetic oligonucleotides to verify orientation and accuracy of ligation. A part of the first exon, including the coding region of tumor Type II hexokinase, was excised from the reporter construct by XhoI digestion followed by religation. For transfections, plasmid DNA was purified using Nucleobond-AX or Qiagen Maxi-prep ion-exchange cartridges.

Transient Transfections

The tumor Type II hexokinase promoter-reporter construct (10 µg) or the pGL2-Control vector (10 µg) was transfected with 2.5 µg of the pSV--galactosidase vector into AS-30D hepatoma cells using 25 10 cells (in 0.5 ml) per transfection. Hepatocytes were transfected with DNA using 20 10 cells (in 0.5 ml) per transfection. Briefly, the cells and plasmid DNA were incubated on ice for 10 min and electroporated at 200 V, 800 microfarad. After 10 more min on ice, the cells were plated into 10 ml of RPMI 1640 glucose-deficient media, pH 7.4, supplemented with an antibiotic-antimycotic mixture (Life Technologies, Inc.), 25 mM HEPES, and 1 mM sodium pyruvate or 1 mM sodium lactate. Based on the transfection study, individual cell samples were further supplemented with 25 mM glucose, 100 nM bovine insulin, 10 µM glucagon, 100 µM dibutyryl cAMP, 100 nM TPA, or combinations thereof. The transfected cells were incubated at 37 °C in 5% CO. Cell extracts were prepared 24-h post-transfection using cold lysis buffer (0.625% Triton X-100, 0.1 M potassium phosphate, and 1 mM dithiothreitol, pH 7.8)(32) . Luciferase activity in the cell lysates was measured as relative light units.

Luciferase and -Galactosidase Assays

The activities were determined essentially as described by the supplier of the reporter vectors (Promega).


RESULTS

Expression of Hexokinase mRNA in the Highly Glycolytic AS-30D Hepatoma Cell Line

To determine the expression levels of Type I and II hexokinase in AS-30D hepatoma cells relative to their expression in normal rat liver, Northern blot analysis was carried out on total RNA (Fig. 1). For Type I hexokinase, an EcoRI/BamHI fragment of the c37 mouse hepatoma Type I hexokinase cDNA (29) was used as the probe, and for Type II hexokinase a full-length cDNA encoding rat skeletal muscle Type II hexokinase (23) was used. Both probes showed specific hybridization bands with rat brain and skeletal muscle RNA (Fig. 1, A and B) used as positive controls for Type I and II hexokinase, respectively. As shown both isozymes could be easily detected in AS-30D cells. However, Type II hexokinase showed a much stronger hybridization signal as compared to the Type I isozyme. In normal rat liver neither hexokinase transcript could be detected.


Figure 1: Expression of hexokinase Type I and II in normal rat liver and AS-30D hepatoma cells. To study hexokinase expression Northern analysis was carried out. For Type I hexokinase, the blot was hybridized with a 0.7-kbp fragment of the c37 tumor Type I hexokinase cDNA (panel A) and for Type II hexokinase, a full-length cDNA of rat skeletal muscle Type II hexokinase was used (panel B) (see ``Experimental Procedures''). Single mRNA species in AS-30D cells (lane 4) were detected similar in size to the hybridization bands obtained with rat brain (lane 1) and skeletal muscle RNA (lane 2) which were used as controls for the Type I and II hexokinase messages, respectively. The Type I and II hexokinase messages were below the detection level in normal rat liver (lane 3). Loading of RNA was estimated by ethidium bromide staining of the gel (panel C).



Isolation of the Tumor Type II Hexokinase Promoter

To identify the cis-transcriptional control elements that regulate the expression of the tumor Type II hexokinase gene, a 5.1-kbp genomic clone containing the proximal promoter region and the first exon of the tumor Type II hexokinase was isolated and mapped. For the isolation, approximately 5 10 plaques were screened from an unamplified AS-30D genomic library packaged at an efficiency of 6.6 10 plaque-forming units/µg. Six plaques, which hybridized to a 260-bp PCR amplified probe corresponding to a part of the Type II skeletal muscle hexokinase first exon, were isolated (Fig. 2A). The recombinant phage, denoted 22-1, 22-2, 22-3, 25-1, 27-1, and 29-1, contained genomic DNA inserts in the size range of 10-20 kbp. Recombinant -DNA prepared from each isolate was completely digested with XbaI. DNA fragments containing sequences corresponding to the first exon of skeletal muscle Type II hexokinase were identified by agarose gel electrophoresis (Fig. 3A) followed by Southern hybridization (Fig. 3B) using the 260-bp PCR product as probe. Specific XbaI digested DNA fragments, in the size range 0.75 kbp (phage 22-1), 5 kbp (phage 22-2 and 29-1), and 9 kbp (phage 22-3, 25-1, and 27-1), were isolated from individual -clones and subcloned into the plasmid vector pUC 18. A pUC 18 subclone containing the approximately 5-kbp DNA insert from the -clone 29-1 was sequenced at the termini using pUC 18 universal primers to test for the presence of DNA similar to the Type II hexokinase first exon, as described below, and selected for further characterization.


Figure 2: Strategy used for AS-30D genomic library construction and isolation of the tumor Type II hexokinase promoter. Sau3AI partially digested AS-30D DNA was ligated to compatible SacI-digested -Fix II arms to generate the genomic library (panel A). From one -clone (29-1) a 5.15-kbp promoter containing DNA segment was isolated, subcloned into plasmid pUC 18, and sequenced (panel B). The 5.15-kbp DNA insert was subcloned into pGL2-Basic, a luciferase reporter vector (panel C) using compatible XbaI, NheI sites. A DNA fragment corresponding to the coding region within the first exon was removed using XhoI, to yield a 4.3-kbp promoter-reporter gene construct. Striped bars indicate luciferase cDNA. Hatched bars indicate AS-30D DNA. Open boxes indicate the multicloning sites (mcs) and DNA of individual vectors +1, transcription start site; cos, -cohesive termini.




Figure 3: Identification of -subclones harboring the tumor Type II hexokinase promoter region. DNA from six positive -clones (lanes 1-6; clones 22-1, 22-2, 22-3, 25-1, 27-1, and 29-1) were digested with XbaI and separated by agarose gel electrophoresis (panel A). DNA fragments similar to Type II hexokinase first exon (white bars) were identified by Southern blot analysis (panel B). Molecular weight markers (-HindIII), in kbp, are shown to the right.



Sequencing and Analysis of the Tumor Type II Hexokinase Promoter

The 5-kbp subclone, denoted 29-1/XbaI, was sequenced in both directions (Fig. 2B) to yield a full-length DNA sequence of 5150 bp (Fig. 4) containing the promoter, the first exon, and part of the first intron. Analysis of the putative first exon and comparison with the published sequences for the first exon of the Type II hexokinase from adipose tissue (24) and skeletal muscle (23) indicated that the corresponding regions within the AS-30D tumor Type II hexokinase are very similar. The 5.1-kbp subclone contained a 257 bp segment of the first intron, a 63 bp coding region of the first exon, a 461 bp untranslated region of the first exon, and a 4369 bp proximal promoter region (Fig. 4).


Figure 4: The complete nucleotide sequence of the 4.3-kbp prox-imal promoter region, the first exon, and the first intron of the AS-30D tumor Type II hexokinase gene. The putative response element motifs are indicated below the nucleotide sequence. Sequence in italics indicate a motif with the potential to form Z-DNA structures. Direct DNA repeats larger than 10 bp are indicated by dotted lines below the sequence and identified by Roman numerals. A TATA box motif (-30) and a CAAT box motif (-85) are highlighted, and the DNA sequence of the first exon is underlined (+1 to +524). The deduced amino acid sequence is indicated below the first exon. Nucleotides +525 to +790 are part of the first intron.



The promoter sequence was analyzed for response elements using available data bases(33, 34) . Putative response elements found within the promoter by computer analysis are indicated in Fig. 4below the DNA sequence. Response elements that are sensitive to two of the main signal transduction cascades, the protein kinase A and protein kinase C pathways, and to insulin, glucagon, and glucose are indicated in Fig. 5. Many DNA direct repeats, ranging from 7 to 36 bp, were found within the promoter. Those which are longer than 10 bp are indicated. Another interesting motif found within the distal part of the promoter was a 31-bp ``T-G'' pyrimidine-purine repeat (Fig. 4).


Figure 5: Organization of the potential response elements for glucose, insulin, glucagon, TPA, and cAMP on the 4.3-kbp AS-30D tumor Type II hexokinase promoter. +1, transcription start site; closed box, mRNA-untranslated region; hatched box, coding region of the first exon; open box, putative response elements that are sensitive to glucose, insulin, glucagon, cAMP, or TPA.



In summary, over 20 putative response elements or regions of potential relevance to the transcriptional regulation of the tumor Type II hexokinase gene were found, including those for well established regulators of carbohydrate metabolism.

Functional Activity of the Tumor Type II Hexokinase Promoter in the Presence of Known Regulators of Carbohydrate Metabolism

To examine the functional activity of the tumor Type II hexokinase promoter in the presence of potential modulators of greatest interest, we placed the 4.3-kbp promoter in the pGL2-Basic reporter vector (Fig. 3C), which is designed to test a promoter's activity by using luciferase as a reporter gene. AS-30D cells were chosen for the transient gene expression study, to ensure the presence of signal transduction cascades and cell-surface receptors characteristic of the parental tumor line. Transient expression of luciferase derived from the promoter-reporter construct was determined after transfection of AS-30D cells, by assaying luciferase activity 24-h post-transfection. Luciferase activity was normalized to the -galactosidase activity derived from the co-transfected internal control plasmid pSV--galactosidase to correct for differences in transfection efficiency. The fold activation of the promoter was based on the activity observed when the transfected cells were maintained in 1 mM pyruvate containing RPMI 1640 medium (control). Under these ``background'' conditions, and in the presence of 10% serum, the tumor Type II hexokinase promoter supported significant levels of transcription in the same range as that of an SV40 promoter-luciferase construct (pGL2-Control) (data not shown).

The relative activity of the tumor Type II hexokinase promoter in driving transcription in the presence of glucose, insulin, or glucagon was then tested. Preliminary studies using a 1 mM lactate or 1 mM pyruvate substrate background indicated that glucose, insulin, and glucagon were capable of directing expression, where the levels of expression observed for each component were similar regardless of the lactate or pyruvate substrate background. Detailed studies using six independent experiments, carried out in a substrate background of 1 mM pyruvate, are shown in Fig. 6. The highest activation of the promoter was observed in the presence of both insulin (100 nM) and glucose (25 mM), with a 4.3-fold increase in activity. Separately, glucose and insulin gave activation levels of 3.4- and 2.4-fold, respectively. Glucagon alone caused a moderate but reproducible activation (1.3-fold) of promoter activity, which increased to 2.4-fold in the presence of glucose. Insulin and glucagon together activated the promoter by 2.8-fold which was 0.4-fold above the transcription enhancement observed in the presence of insulin alone.


Figure 6: Effect of glucose, insulin, glucagon, TPA, and cAMP on the transcriptional activity of the 4.3-kbp tumor Type II hexokinase promoter. Luciferase activity was assayed 24-h post-transfection of the promoter-reporter construct into AS-30D cells, which were maintained under hormonal, metabolite, or intracellular mediator influence as indicated in the figure. Activities are expressed as fold activation over that of a control (1 mM pyruvate). Each of the samples contained 1 mM pyruvate as substrate background. All values represent the mean of six independent experiments. The individual standard deviations (±12 S.D.) for the fold activation are: glucose, 0.44; insulin, 0.25; glucagon, 0.175; glucose + insulin, 0.5; glucose + glucagon, 0.06; TPA, 0.45; and dibutyryl cAMP, 0.7.



Finally, the effect of analogs that activate two of the major signal transduction pathways, namely the protein kinase A and protein kinase C signaling cascades, on the tumor Type II hexokinase promoter was tested using dibutyryl cAMP (100 µM), an analog of cAMP, and TPA (100 nM), an analog of diacylglycerol, respectively. These analogs increased promoter activity by 2.1- and 3.3-fold, respectively.

These findings emphasize the promiscuity of the tumor Type II hexokinase promoter in its activation response to a wide variety of known modulators of carbohydrate metabolism.

Relative Activity of the Tumor Type II Hexokinase Promoter in Hepatocytes and in AS-30D Hepatoma Cells

To test whether the tumor Type II hexokinase promoter was capable of driving transcription in the tumor's parent cell line, the reporter vector was transfected into hepatocytes. The expression was evaluated for glucose, insulin, or glucagon in a substrate background of 1 mM lactate. Parallel experiments were carried out in AS-30D hepatoma cells. In contrast to the highly modulated promoter activities observed in AS-30D cells for glucose, insulin, and glucagon, the promoter showed no significant modulations in activity when placed within hepatocytes (Fig. 7) and tested with the same modulators. In contrast to the increased activity observed for AS-30D cells stimulated with glucagon, in hepatocytes the activity of the promoter decreased by 0.4-fold. However, in hepatocytes and in AS-30D cells, the basal activity of the promoter (in 1 mM lactate), as measured by relative light units for the reporter gene, was comparable in magnitude.


Figure 7: Transcriptional activity of the 4.3-kbp tumor Type II hexokinase promoter in normal or AS-30D hepatoma cells. Luciferase activity was assayed 24-h post-transfection of the promoter-reporter construct into hepatocytes or into AS-30D hepatoma cells. Hormonal or metabolite conditions used, are indicated on the figure; activity is expressed as fold activation over that of a control (1 mM lactate). Each sample contained 1 mM lactate as substrate background. All values represent the mean of two independent experiments.



These results implicate the presence in AS-30D hepatoma cells of one or more transcription factors essential for the expression of the Type II hexokinase gene that are absent in the parental cell line of origin.


DISCUSSION

Although it has been known for over 60 years that one of the most common phenotypes of malignant cells is an elevated glucose catabolic rate(1) , a property linked in large part to a marked elevation in the enzyme hexokinase(3, 4, 7, 22) , the genetic basis underlying these biochemical observations has remained unknown. The novel set of studies reported here are the first to shed light on this important problem. To this end, we have employed the AS-30D hepatoma cell line which grows rapidly, exhibits a high glycolytic rate(3, 6, 22) , and markedly overexpresses a form of hexokinase with a propensity to bind tightly to the outer mitochondrial membrane(3, 8) . Although both hexokinases Type I and II were found to be overexpressed in AS-30D hepatoma cells as compared to normal rat liver, it was the Type II isozyme which was by far the most abundant. Therefore, we have proceeded to isolate and sequence the proximal promoter of the responsible gene and to use reporter-gene analysis to gain insight into the importance of its vast, intriguing features.

Analysis of the 4.3-kbp proximal promoter region of the AS-30D tumor Type II hexokinase revealed both a putative TATA box (AATAA, -30) (35) and a CAAT box(-85), indicating the precise positioning of transcription initiation for the tumor Type II hexokinase mRNA transcript. This is in contrast to the staggered transcription initiation and the lack of either a TATA box or a CAAT box, observed for liver glucokinase(36) , the principal expressed hexokinase isoform in normal liver cells. Putative consensus sites for Ap-2 (GGCAGCCC, -41), a factor inducible by both protein kinase A and protein kinase C pathways(33, 34) , and for ATF-1 (CCACGTC, -70), which is specifically induced by the protein kinase A pathway(33, 34) , were located immediately upstream of the transcription start site. Since both these sites are located in close proximity to the TATA element and the CAAT element, further studies will indicate their importance in transcription enhancement. In addition, Ap-2 sites were the most common and ubiquitous elements within the 4.3-kbp promoter (-3850, -2040, -1965, -1500, -1260, -1110, -665, and -315). Six putative Ap-1 consensus sites (-3469, -2735, -2320, -1955, -1590, and -860) for the complex fos-jun, which is a primary nuclear transducer of the protein kinase C cascade, could be found throughout the 4.3-kbp promoter.

Of the known liver-enriched transcription factors HNF-1, HNF-3, HNF-4, and c/ebp(37, 38) , putative consensus sites could be found for only c/ebp (-4150, -3725, -2550, -1440, -1060, -660, -620, and ;260). However, several putative sites for the factor HNF-5(39) , which usually binds at sites close to the above mentioned liver-specific factors, could be found distributed within the promoter (-4160, -3915, -3330, and -2200). Therefore, the tumor Type II hexokinase promoter may contain additional consensus sites for the hepatic nuclear factors, or for their oncogenic variants, such as vHNF-1, which replaces HNF-1 in de-differentiated cells(33, 34) . Consensus sites for such factors remain to be elucidated by DNA footprinting analysis of the tumor Type II hexokinase promoter.

Regarding known ubiquitous factors that regulate expression of genes coding for glycolytic and gluconeogenic enzymes(38) , namely, sites for ``CCAAT-box'' binding factors, octamer factor, Sp-1, CREB/ATF, Ap-1, b-HLH, and nuclear hormone receptors all of which enhance transcription (38), putative sites could be found for Sp-1 (four sites), CREB/ATF (two sites), Ap-1 (five sites), and for steroids (SRE) (five sites). Also found within the promoter was one putative site(-2955) for factor PPAR, a member of the steroid hormone receptor superfamily, that is thought to play a role in tumor development in liver and in triglyceride and cholesterol homeostasis(40) . As cholesterol biosynthesis in most tumor cells is uncontrolled(41) , it will be interesting to identify possible regulatory interactions between the glycolytic and sterol biosynthetic pathways.

Putative sites for Pea-3, a factor inducible by TPA, epidermal growth factor, and the oncoproteins v-src, v-mox, v-raf, and c-Ha-ras(42) , were identified within the distal (four sites, -3965, -3645, -3625, and -3255) and proximal (two sites, -1415, -1370) regions of the promoter. Within the 4.3-kbp promoter, four putative Sp-1-binding sites were identified (-3290, -2220, -1110, and -55). Since DNA-bound Sp-1 factors self-associate, these sites, placed approximately at 1000 bp intervals within the promoter, may bring together the distal promoter segments for enhancement of transcription. Between base pairs -3811 and -3841, a 31-bp ``GT'' repeat was located. This motif, located in the distal region of the tumor Type II hexokinase promoter, is also found within the proximal promoter region of rat pancreatic -cell glucokinase (a 33-bp tract)(43) , as well as within a human glucokinase gene-associated satellite repeat DNA sequence (a 31-bp tract)(44) . Such repetitive purine-pyrimidine DNA segments have potential to form Z-DNA structures and induce changes in the helicity of adjoining B-DNA.

Within normal liver cells, Type IV hexokinase (glucokinase) is the predominantly expressed isoform(45) , and transcription of this enzyme is enhanced by both glucose and insulin (fed state), and inhibited by glucagon (fasted state)(46) . Significantly, the Type II hexokinase gene, which is markedly overexpressed in AS-30D hepatoma cells, is essentially silent in liver. Therefore, in order to better understand the genetic basis for the enhanced overexpression of the Type II isoform when liver hepatocytes acquire the tumor phenotype, we chose to test the modulation of the tumor Type II hexokinase promoter by glucose, and by insulin and glucagon, the latter representing the main hormones involved in glucose homeostasis. Significantly, both hormones and glucose activated the promoter, with insulin plus glucose providing the maximal response. With the tumor Type II hexokinase promoter, putative sites for glucose response, namely the E-box (CACGTG)(47) , were identified at two positions, one within the promoter(-3765), and the other in the first intron (+535). Only one putative element could be located that was at least partially representative as responsive to insulin (CAAAATAAGA, -4135)(48) . Since the action of glucagon is modulated via cAMP, the CRE/ATF elements within the promoter are the probable sites for action by this hormone.

The enhancement of the tumor Type II hexokinase promoter activity observed with glucose and both hormones, although surprising, is not entirely unexpected. The result may help explain a strategy devised by rapidly growing tumors to maintain glycolysis at an optimal state, regardless of metabolic state (fed or fasting) of neighboring healthy cells, or of the organism itself. The activation effect of glucagon on the tumor Type II hexokinase promoter is mirrored by the effect of dibutyryl cAMP. Again, this contrasts with the transcriptional deactivation observed for glucokinase in normal liver cells, where glucokinase transcription is repressed upon stimulation by cAMP analogs (41), and helps explain why the ratio of hexokinase/glucokinase increases markedly in hepatoma cells(49) . Finally, activation of the promoter by the phorbol ester TPA implicates a possible role of a signal transduction cascade involving protein kinase C.

As the basal activity of the Type II hexokinase promoter reporter construct in hepatocytes was found to be in the same range as that observed in AS-30D hepatoma cells, it is possible that mechanisms such as methylation and chromosomal translocations may contribute toward silencing expression of the Type II hexokinase isoform in liver. However, the failure of this promoter to be activated in hepatocytes by glucose, insulin, or glucagon, all of which modulate the biosynthesis of glucokinase suggests that a different set or level of transcription factors may be involved in normal and transformed hepatocytes for controlling the expression of hexokinase isoforms and therefore the rate of glucose catabolism.

In recent studies reported elsewhere(24) , insulin has been shown to enhance Type II mRNA levels in adipocytes which are known to undergo differentiation in the presence of this hormone. Therefore, in furture studies it will be of interest to compare the promoter regions of the Type II hexokinase of tumor cells described here to that of the Type II enzyme from normal tissues. It seems likely that the Type II form of hexokinase is the form primarily involved in tissues undergoing differentiation and/or rapid cell proliferation.

Using a Type I hexokinase cDNA probe from brain, we previously isolated a hexokinase (cDNA) from another highly glycolytic hepatoma cell line (c37)(29) . This hexokinase form now recognized as Type I is also overexpressed in the AS-30D hepatoma cells used in this study (Fig. 1), but to a much lesser extent than Type II hexokinase. This suggests the possibility that the regulatory regions of the Type I and II hexokinase forms may share some common activating elements. Nevertheless, in those rapidly growing, highly glycolytic cancers studied to date(21, 22, 23, 50, 51, 52, 53, 54, 55) , it is the Type II isozyme that either dominates or increases upon transformation.

In summary, these studies emphasize normal versus tumor cell differences in the regulation of hexokinase genes involved in glucose catabolism. They indicate also that transcription of the Type II tumor gene may occur independent of metabolic state, a property that may give the tumor cell a distinct growth advantage over normal cells.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 32742 (to P. L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U19605.

To whom correspondence should be addressed. Tel.: 410-955-3827; Fax: 410-955-5759.

§
Postdoctoral awardee of the Deutsche Forschungsgemeinschaft.

The abbreviations used are: kbp, kilobase pairs; ATF/CRE, cyclic AMP response element; Ap-1, activator protein-1; C/EBP, CCAAT-enhancer binding protein; HNF, hepatocyte nuclear factor; TPA, phorbol 12-myristate 13-acetate; EtBr, ethidium bromide; SSC, sodium chloride-sodium citrate solution; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Roxanne Ashworth of the Genetic Resources Core Facility at Johns Hopkins University for carrying out automated sequencing of the promoter. We are grateful also to Jackie Seidl for her help in processing the manuscript for publication.


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