From the
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.
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.
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 [
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.
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.
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.
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
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-
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.
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
The
activities were determined essentially as described by the supplier of
the reporter vectors (Promega).
-Galactosidase Assays
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).
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.
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.
-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.
/EMBL Data Bank with accession number(s)
U19605.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.