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Department of General and Environmental Physiology, University of Bari, Bari, Italy and
* Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany
1Correspondence: Deutsches Krebsforschungszentrum, Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. E-mail: M.Tommasino{at}DKFZ-Heidelberg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cytoplasmic alkalinization • NHE-1 • HPV16 E7 oncoprotein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
). Several studies support the concept that proliferation and
transformation are associated with an increased intracellular pH (pHi).
Rapid stimulation of the
Na+/H+ exchanger, with
consequent cytoplasmic alkalinization, appears to be a universal
response of quiescent cells to growth-promoting factors
(2)
. Microinjection of activated ras p21 into quiescent
mouse fibroblasts (NIH3T3) results in a transient morphological
transformation, cell proliferation, and a rapid rise in pHi, whereas
microinjection of the unaltered proto-oncogene p21 fails to produce any
response (3)
. In agreement with the ras microinjection
data, long-term expression of oncogenic ras also leads to an
intracellular alkalinization even when bicarbonate-dependent transport
systems are activated by the presence of bicarbonate in the culture
medium (4)
. Moreover,
Na+/H+ exchanger-deficient
cell lines either do not induce tumor formation or show severely
retarded tumor growth when implanted in immuno-deprived mice
(5)
. By an alternative approach, Perona and co-workers
provided additional evidence for the importance of pHi in cellular
transformation. Expression of the gene for the yeast plasma membrane
H+-ATPase in immortalized rodent fibroblasts
leads to a tumorigenic transformation (6)
and a rise in
pHi (7)
. Although these findings demonstrate an
association of cytoplasmic alkalinization with tumorigenic
transformation, it is not clear yet whether the rise of pHi plays a key
role in the induction of cellular events that lead to transformation or
is itself a consequent event of transformation (reviewed in refs
8
, 9
). Furthermore, the causal relationship between the
shift to glycolytic metabolism and the increases in pHi and
proliferation remains unclear.
To better characterize the role and mechanism of the
Na+/H+ exchanger in
transformation-dependent intracellular alkalinization and to understand
whether it is an essential and early event in malignant transformation,
we have developed an experimental cell model using the E7 oncoprotein
of the human papillomavirus type 16 (HPV16). HPV16 E7 protein is able
to induce tumorigenic transformation of mouse fibroblasts and to
cooperate with another viral oncoprotein, E6, to immortalize primary
human keratinocytes (reviewed in ref 10
). These activities
are in part explained by the ability of E7 to interact with the
so-called ‘pocket’ proteins pRb, p107, and p130
(11
12
13)
. The pocket proteins are central regulators of
cell cycle division. They negatively regulate, via direct association,
the activity of several transcription factors, including members of the
E2F family (14)
. Under normal cell cycle regulation,
phosphorylation of pRb, which is mediated by cyclin-dependent kinase
(CDK) activity, leads to the disruption of pRb/E2F inactive complexes.
HPV16 E7 binds the pocket proteins and, analogous to the CDK-mediated
phosphorylation, releases active E2Fs, which in turn positively
regulate transcription of a group of genes encoding proteins essential
for S-phase progression (reviewed in ref 15
). The
association of intracellular alkalinization with E7-driven
transformation or the role of a possible alkalinization in the
E7-dependent events has not yet been determined. Using the NIH3T3 cell
line, in which transformation can be induced and followed by
transcriptional activation of the HPV16 E7 oncogene, we demonstrate
that cytoplasmic alkalinization is an early event in tumorigenic
transformation and is driven by a stimulation of NHE-1 activity via an
increase in the affinity of the NHE-1 proton regulatory site. Most
important, annulment of the E7-induced cytoplasmic alkalinization
prevents the development of the transformed phenotype. Similar data
were obtained in human keratinocytes, the natural host of the virus,
which are immortalized by expression of the HPV16 E6 and E7 oncogenes.
Last, treatment of nude mice with the amiloride analog 5-(N,N-dimethyl)
amiloride (DMA), a specific inhibitor of NHE-1, delays the development
of HPV16-keratinocyte tumors. Together our findings show that
activation of the NHE-1 and resulting cellular alkalinization play a
key role in oncogenic transformation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NIH3T3 cells constitutively expressing E7 genes were generated by
infection with recombinant retroviruses (pBabepuro vector
(16)
expressing HPV16 HA-tagged E7 proteins, wild-type or
mutant. Cells were then selected in presence of 2.5 µg/ml of
puromycin. Two days later, puromycin-resistant colonies were pooled and
used for the experiments.
The 2BN11 cell line was created by infecting NIH3T3 cells with
recombinant retrovirus expressing the HPV16 E7 gene under the control
of a tetracycline repressed promoter (17)
. After selection
in puromycin, the final clone (2BN11) was selected on the basis of
having undetectable basal E7 expression in the presence of tetracycline
and high inducibility as determined by Western blot and reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis.
Partial inhibition of glycolysis was obtained by culturing the cells for 6 h in DMEM medium high glucose (4500 mg/l) supplemented with increasing concentrations of deoxyglucose (0.1–0.5 mg/ml).
RNA preparation and RT-PCR
2BN11 total RNA was prepared using StrataPrep Total RNA Miniprep
Kit (Stratagene, San Diego, Calif.). The first strand cDNA was
synthesized using 1 µg of total RNA and a commercially available kit
(MBI, Hanover, Md.). The E7 and GAPDH fragments were amplified
by PCR using specific primers and 3 µl of each cDNA sample as
template.
Determination of intracellular pH, intrinsic buffering capacity
(ß1), and H+ efflux rates
Cytoplasmic pHi was measured spectrofluorimetrically with the
fluorescent pH-sensitive probe, BCECF, trapped intracellularly in cell
monolayers grown on plastic coverslips. Cells were loaded for 1 h
at room temperature with the acetoxymethyl ester of BCECF (10 µM) in
‘Na+ ringer’. After loading, coverslips with
confluent monolayers were inserted at an angle of 60° in a
fluorometer cuvette designed to permit easy solution change with
multiple perfusion solutions as described previously (18)
.
Fluorescence was monitored in a Shimadzu RF 5000 spectrofluorometer
using alternately 455 nm (pH insensitive) and 490 nm (pH-sensitive) as
excitation wavelengths with a xenon light source (5 nm bandwidth).
Emission was measured at 530 nm (15 nm bandwidth). pHi was calculated
from the fluorescence emission ratio of the two excitation wavelengths
using a standard calibration procedure based on the use of nigericin
(0.5 µM) in high-potassium K clamp ringer (19)
. The
activity of the Na+/H+
exchanger was measured by monitoring pHi recovery after an
intracellular acid load produced with the NH4Cl
prepulse technique (20)
. The rate of
Na+-dependent alkalinization was determined by
linear regression analysis of 15 points taken at 4 s intervals.
The use of CO2/HCO3-free
solutions minimizes the likelihood that
Na+-dependent HCO3
transport was responsible for the observed pHi changes.
The pHi dependence of intracellular buffer capacity was computed by the
NH4 pulse method (21)
. ßi refers
to the ability of intrinsic cellular components, excluding
HCO3-CO2, to buffer changes
in pHi and is defined as the moles of H+ required
to produce a one-unit shift in pHi per liter of intracellular fluid.
The actual activity of the exchanger in terms of proton flux rate
(mM/min) is determined by multiplying the rates in pHi change by the
cells intrinsic buffering capacity (ßi) at the pHi in which the
measurement was being made. The pHi dependence of total net proton
extrusion rate was calculated from the data of pHi recovery curves as
described (22)
.
Equilibration of external and internal pH with tributyltin bromide
The pHi of the cells was clamped by incubation with 5 µM
tributyltin bromide (Aldrich, Milwaukee, Wis.) in DMEM at a medium pH
of either 6.9 or 7.4 with Na-HEPES buffer (23)
. Initial
experiments indicated that the pHex and pHi are rapidly equilibrated in
the presence of tributyltin bromide (data not shown).
Determination of cellular parameters
Proliferation was measured in each sample by determining cell
number as described previously (24)
and by measuring the
incorporation of bromouridine following either the Cell Proliferation
ELISA colorimetric assay or the BrdU Labeling and Detection kit I
according to the manufacturer instructions (Boehringer Mannheim,
Mannheim, Germany). Cells were labeled for 3–6 h with BrdU.
The ability to grow in an anchorage-independent way was measured by the formation of colonies on soft agar. The base layer was prepared with DMEM:0.5% low melting temperature agarose (SeaPlaque, FMC BioProducts, Rockland, Maine). The feed layer was then prepared by resuspending 5000 cells in DMEM:0.25% low melting temperature agarose plus experimental compounds and pouring onto the previously prepared base layer (1/2 of the volume of the lower layer). Culture medium was changed every 2 days and colonies counted after 21 days.
To measure lactate production, cell monolayers were incubated for 10
min in a buffer without glucose (in 110 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 4 mM
Na2P04, 50 mM Na-HEPES, pH
7.4). The cells were then washed twice in the same buffer and
glycolysis was initiated by addition of 20 mM glucose
(23)
. The concentration of lactate released into the
buffer was measured either by an enzyme-linked assay system as
described (25)
or with a colorimetric kit supplied by
Sigma (St. Louis, Mo.).
Determination of cyclin E promoter activity
Activation of the cyclin E promoter by HPV16 E7 was determined
in NIH3T3. Cells were transfected by calcium phosphate precipitation as
described elsewhere (26)
. At 16 h after transfection,
cells were cultured in DMEM containing 0.5% fetal calf serum (FCS) for
24 h in absence or presence of 5 µM DMA. Protein extracts were
prepared and luciferase and ß-galactosidase assays performed. Details
of the construct containing the cyclin E promoter are given elsewhere
(27)
.
Protein extraction and Western blotting
For the detection of HPV16 E7, ß-tubulin, cyclins E and
A, and NHE-1, total cellular extracts were prepared as described in ref
28
, 29
. 100 micrograms of total extract was precipitated
in acetone (9:1/v/v 20 min at -20°C, centrifuged (12,000
g, 10 min) and resuspended in 20 µl of gel loading buffer.
Total cell extracts were fractionated by electrophoresis on a
polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS).
Proteins were transferred onto a Polyscreen PVDF transfer membrane
(DuPont, Wilmington, Del.) in a Trans-Blot semi-dry electrophoretic
transfer cell (Bio-Rad, Hercules, Calif.). Immunocomplexes were
detected with ECL reagent (Amersham, Little Chalfont, U.K.). We used
the following antibodies: anti HA-tag monoclonal antibody (MMS-101R
BabCo, dilution 1:1000), anti HPV16 E7 monoclonal antibody (Triton,
dilution 1:200), anti-human cyclin E antibody (14761C PharMingen;
dilution 1:1000), anti-human cyclin A antibody (H-432 Santa Cruz
Biotechnology; dilution 1:1000), anti-human p34CDK2 antibody (kindly
provided by Dr. M. Pagano, New York University Medical Center, New
York; dilution 1:2000), anti ß-tubulin monoclonal antibody (Tub 2.1
Sigma; dilution 1:1000), anti NHE-1 antibody (kindly provided by Dr.
Pouysségur, CNRS, Nice, France; dilution 1:1000).
Generation of tumors in nude mice and treatment protocol
Swiss athymic CD-1 (nu/nu) nude mice weighting 20–25 g were
injected subcutaneously on the back with HPKIA cells
(107 cells in 100 µl phosphate buffer). Half
the mice were treated every other day by intraperitoneal (i.p.)
injections of 15 µg DMA/gm body weight whereas the other half
received only the vehicle (4% DMSO in sterile water). Tumor length,
width, and height were measured with calipers the third day after
injection and then weekly.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
To determine whether E7-dependent transformation leads to a cytoplasmic
alkalinization, immortalized NIH3T3 cells were infected with
recombinant retroviruses expressing either HPV16 E7 wild-type or a
transformation-deficient mutant (30
, 31)
. The effect of E7
expression on steady-state pHi was then determined by
spectrofluorometry as described in Materials and Methods. As seen in
Fig. 1A
, the wild-type protein elevated resting pHi by 
0.25
units, whereas the transformation negative E7 mutant was not able to
induce alkalinization. Addition of DMA, a specific inhibitor of the
Na+/H+ exchanger isoform 1
(NHE-1), to the culture medium abolished the E7-induced alkalinization.
Western analysis (Fig. 1B
) showed that the inability of the
E7 mutant to alkalinize the cells was not due to different expression
levels and DMA treatment did not decrease the E7 protein expression.
These results are consistent with the reported specificity of only
oncogenic and not proto-oncogenic ras in inducing intracellular
alkalinization in NIH3T3 cells (3)
.
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E7-dependent alkalinization is an early event and drives subsequent
glycolysis
The above experimental system does not permit determination of the
dynamic processes occurring during transformation. To address these
issues, a cell model was constructed in which the transformation of
normal cells could be rapidly induced and early events subsequently
monitored. NIH3T3 cells were infected with a recombinant retrovirus in
which HPV16 E7 gene expression is under the control of a promoter that
is negatively regulated by tetracycline (17)
, clone 2BN11.
As can be seen in Fig. 2
, in 2BN11 cells HPV16 E7 can be detected only after tetracycline
removal. The HPV16 E7 protein was present at lower levels in 2BN11
cells than in CaSki, an HPV16-positive cell line derived from human
cervical cancer (Fig. 2)
. The development of transformed phenotypes
(e.g., increased proliferative rate, anchorage-independent growth,
serum independence, and increased glycolytic metabolism) are under the
strict control of E7 expression in 2BN11 cells (data not shown).
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The effect of E7 expression on steady-state pHi in 2BN11 cells was then
determined. E7-mediated transformation was induced by removing
tetracycline from the culture medium for 24 h and the resting pHi
was determined by spectrofluorometry. In 2BN11 cells, resting pHi
increased with E7-induced transformation from 6.89 ± 0.017 to
7.16 ± 0.019 (n=25, P<0.001). The same
shift in pHi was observed in cells cultured in the presence of
bicarbonate (data not shown). Nontransfected NIH3T3 cells have a
similar resting pHi as 2BN11 cells, which do not express E7
(6.91±0.019, n=5, P>0.5), and addition or
removal of tetracycline had no effect on this value (data not shown).
The alkalinization in transformed 2BN11 cells was blocked by addition
of 5 µM of DMA (6.86±0.012, n=6), whereas 10 µM of the
bicarbonate transporter inhibitor, 4,4' diisothiocynanostilbene-2, 2'
disulfonic acid, had no effect on the pHi (7.19±0.015,
n=5). To confirm that the variation of pHi was due the
inhibition of NHE-1 and not to an unspecific effect of DMA or to the
inhibition of other isoform of NHE, we used another specific inhibitor
of the NHE-1 isoform, HOE 694 (reviewed in refs 1
, 2
).
Similar results were obtained with NHE-1 inhibitors DMA and HOE 694
(data not shown). These data, together with those shown in Fig. 1
,
demonstrate that cellular alkalinization is associated with E7-induced
transformation and is dependent on the
Na+/H+ exchanger.
To determine whether alkalinization is an early event in
transformation, the temporal sequence of the change in resting pHi was
monitored over a time course before and after induction of E7
expression. Monolayers loaded with the pHi-sensitive fluorescent
indicator were first perfused with Na+ ringer
plus 1 µM tetracycline. In these conditions, resting pHi
(6.88±0.014, n=9) remained constant for as long as 5 h
(data not shown). Figure 3A
shows a representative experiment of the time course of
changes in resting pHi in perfused monolayers. After tetracycline
removal, resting pHi remained constant for 90 min and then started a
slow, regular rise that continued until it reached a new constant value
after 1.5 to 2 h, reflecting the new resting pHi (trace a). The
mean time necessary for the initiation of alkalinization after
tetracycline removal was 1.4 ± 0.11 h (n=9) and
was independent of the original perfusion time with tetracycline. The
mean time to accomplish the alkalinization after initiation was
1.8 ± 0.25 h (n=9). Trace b shows that the
addition of DMA at the time of tetracycline removal completely
inhibited the alkalinization. As expected, since DMA inhibition is
fully reversible (reviewed in refs 1
, 2
), its removal
(arrow) resulted in a very rapid alkalinization to the same elevated
resting pHi as in the cells that had not been inhibited (trace a).
Alkalinization was concomitant with activation of E7 transcription
after the removal of tetracycline. RT-PCR analysis showed that the
levels of E7 mRNA rapidly increase when cells are cultured in absence
of tetracycline (Fig. 3B
). However, low levels of mRNA can
also be detected in cells grown in medium containing tetracycline (Fig. 3B
), but they are not sufficient to induce transformation.
Indeed, 2BN11 cells have the same features of the parental cells,
NIH3T3, when cultured in tetracycline-containing medium (data not
shown). This rapid alkalinization is similar to that reported for
microinjection of high concentrations of oncogenic ras (3)
and suggests that the cellular alterations necessary for the
transformation-dependent alkalinization were accumulating during this
time but that alkalinization could not take place due to the block of
the exchanger. As glycolytic metabolism also increases with E7-driven
transformation, it was important to determine the relationship between
the activation of alkalinization and glycolysis. Figure 3C
shows that the transformation-dependent increase in lactate production
(squares) started between 2 and 2.5 h after tetracycline removal
and was inhibited by DMA (triangles). These data show that the shift to
glycolytic metabolism is directly driven by the rise in pHi.
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E7-dependent transformation results in an increase in the affinity
of the intracellular NHE-1 proton regulatory site
As the above data indicate that the
Na+/H+ exchanger is
directly involved in the E7-dependent alkalinization, we next
elucidated the alterations of
Na+/H+ exchanger kinetics
during E7-induced transformation. We first determined the activity of
the exchanger by measuring its rate in returning pHi to the resting
value at two external sodium concentrations after an acid load
(20)
. As illustrated in Fig. 4A
, in the absence of sodium there was no recovery of pHi and
addition of sodium (135 mM) to the cells produced a rapid rise in pHi
(traces a), which was slower in 34 mM sodium (traces b). HPV16
E7-expressing cells (-tet, 24 h) had an increased recovery rate
at both 34 and 135 mM external sodium and returned to a higher resting
pHi value than did the nontransformed (+tet) cells (6.85±0.017
(n=4) vs. 7.10 ± 0.018 (n=8) for +tet and
-tet treatments, respectively). Similar increases in
Na+/H+ exchange-dependent
recovery occurred at both short (3–6 h) and long (24–96 h) periods
after E7 induction (data not shown). The response of intrinsic
buffering capacity (ßi) to transformation was
next determined and used to calculate absolute exchanger activity
(21)
. The average calculated H+
extrusion rate due to
Na+/H+ exchanger activity
significantly increased from 6.33 ± 1.38 in normal cells to
9.45 ± 1.81 and 12.24 ± 1.58 mM H+
min-1 in cells, in which E7 expression was
induced for 3 and 48 h, respectively (n=12,
P<0.001). Western blot analysis showed that the increased
H+ extrusion rate was not due to a change in
NHE-1 protein expression in either short- or long-term E7-induced
transformation (Fig. 4B
). This increase in initial transport
rate without a corresponding increase in transporter protein expression
is indicative of an increased transporter turnover rate
(32)
. To determine whether the increased
H+ extrusion rate was a result of alterations in
the NHE-1 proton regulatory site kinetics, the pHi dependence of the
NHE-1 activity was analyzed (20)
. Figure 4C
shows that 3 h of E7 induction shifted the curve representing the
dependence of exchanger activity (mM H+/min) on
pHi to the right. This is indicative of an increase in the apparent
affinity of the intracellular proton regulatory site of the exchanger
(2)
. Longer periods of E7 expression produced only
slightly more pronounced rightward alkaline shifts (data not shown).
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Role of transformation-dependent alkalinization in driving other
transformed phenotypes
Intracellular pH has been shown to be an important regulator of a
number of cellular processes such as cell shape, proliferation,
apoptosis, and secretion of proteases (1
, 2
, 33
34
35)
.
However, the role of the alkalinization that occurs during
transformation in the induction of anchorage-independent growth and in
serum independence has not been clarified (reviewed in refs 8
, 9
). To characterize the role of early E7-dependent
alkalinization in the subsequent development of these transformed
phenotypes, E7-expressing cells were cultured in the presence or
absence of 5 µM DMA. In Fig. 5
it can be seen that the inhibition of the intracellular alkalinization
by DMA after tetracycline removal was able to decrease the growth rate
to the level of nontransformed cells (Fig. 5A
) and to
suppress the development of serum-independence (Fig. 5B
) and
anchorage-independent growth (Fig. 5C
). The lack of effect
of DMA on basal growth is consistent with the hypothesis that the
Na+/H+ exchanger and the
rise in pHi have a permissive role in the initiation of proliferation
without being involved in steady-state growth. To exclude the
possibility that abrogation of E7-induced transformation was due to a
nonspecific DMA effect, we reduced or annulled the E7-induced
alkalinization by culturing the cells in a growth medium having a pH of
6.9 in the absence or presence of the proton ionophore, tributyltin
bromide (23)
. Lowering the external pH results in a
drastically reduced pHi in 3T3 cells that is resistant to change by
proton or bicarbonate transporters (7)
. Tributylin bromide
functions as an anion exchanger permitting the rapid equalization of
pHi with medium pH and it is preferable to another common ionophore,
nigericin, since it inserts only in the plasma membrane
(23)
. As can be observed in Fig. 6A
, the decrease of external pH to 6.9 was
sufficient to reduce the E7-dependent increase in growth rate in DMEM
containing 10% (Fig. 6A
), whereas clamping pHi to 6.9
further increase this effect. Similarly, the same treatments—decrease
of external pH or clamping pHi to 6.9—negatively influence the
E7-mediated stimulation of serum-independent growth (Fig. 6B
) and lactate production (Fig. 6C
). In
contrast, clamping pHi to 7.4 further increased the levels of the
transformed phenotypes (Fig. 6A
, B
, C
). Together these data
demonstrate that inhibition of alkalinization is sufficient to suppress
the development of the other transformed phenotypes.
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Inhibition of serum-independent growth by DMA does not affect E7
induced cyclin E activation
The ability of HPV16 E7 to drive quiescent cells into S phase is
in part mediated by neutralization of pRb function and consequent
activation of E2F-regulated genes, e.g., cyclin E (15)
.
Since the inhibition of cytoplasmic alkalinization by DMA prevents
E7-expressing cells to enter S phase, it was important to determine
whether variations of intracellular pH influence the ability of HPV16
E7 to activate E2F-driven transcription. For this purpose, NIH3T3 cells
were cotransfected with a construct expressing HPV16 E7 protein
together with a vector containing the cyclin E promoter cloned in front
of the luciferase gene (27)
. As shown in Fig. 7
, the addition DMA to the culture medium had no effect
on the E7-dependent transactivation of the cyclin E promoter. These
data indicate that in the E7-mediated induction of serum-independent
growth, alkalinization must act on a pathway(s) parallel to the
activation of E2F-driven transcription.
|
Alkalinization in HPV16 transformed human primary keratinocytes
drives other transformed phenotypes
To further confirm the role of cytoplasmic alkalinization in
HPV-induced transformation, we extended our study to human primary
keratinocytes, the natural host of the virus. HPKIA cells are primary
human keratinocytes immortalized by transfection of the entire HPV16
genome that become spontaneously transformed at high passage number
(36)
. As shown in Table 1
, the resting pHi was higher in the transformed (late passage)
keratinocytes than in the immortalized (early passage) cells. This
alkalinization was linked to an increased NHE-1 activity and the
addition of DMA reversibly reduced the resting pHi to levels similar to
those for the immortalized cells. The effect of DMA on growth rate and
soft agar colony formation was next assessed (Fig. 8
). The late passage cells had a higher growth rate (Fig. 8A
, B
) and a greater capacity for colony formation in soft agar (Fig. 8C
) than early passage HPKIA. All these parameters were
reduced to levels similar to those in the early passage cells by the
addition of DMA and subsequent reduction of pHi to normal levels (Fig. 8)
. These data are in full agreement with those obtained in the rodent
fibroblast model and underline the conserved nature of the mechanism.
|
|
As the data in NIH3T3 cells indicate that E7-induced, serum-independent
growth requires intracellular alkalinization independently of the
stimulation of E2F-driven transcription, we could predict that the
expression of E2F-regulated genes would not differ between early and
late passage HPKIA cells. Therefore, we determined the endogenous
levels of cyclin E and cyclin A, which are encoded by E2F-regulated
genes, in early and late passage HPKIA cells. As shown in
Fig. 9
, there was no significant difference in the expression of either
protein in the early passage compared to the late passage cells
cultured in the absence or presence of DMA. Similar results were
obtained in the analysis of cyclin E and cyclin A transcripts by
Northern blotting (data not shown). These data confirm that cytoplasmic
alkalinization and activation of E2F-regulated transcription act
independently in driving transformation.
|
Activation of glycolysis does not contribute to the increased
proliferation in transformed HPKIA cells
As in the 2BN11 cells, we observed an increase in glycolytic
metabolism in the transformed HPKIA cells (late passage) that was
reduced to levels measured in the early passage cells by DMA treatment
(data not shown). We next determined whether the increased glycolytic
metabolism plays a role in the stimulated growth rate associated with
transformation in HPKIA cells. Glycolysis can be inhibited by
incubation with deoxyglucose. We observed that addition of 0.1 mg/ml
deoxyglucose to the culture medium (containing 4.5 mg/ml glucose)
decreases glycolysis to the levels of DMA-treated cells (Fig. 10
). In these conditions we observed that the concomitant reduction in
proliferation was only 20% of that occurring when the
alkalinization was blocked with DMA. Since deoxyglucose can induce
apoptosis, most likely due to the ATP deprivation, the percentage of
apoptotic cells was also determined. No significant difference of cell
death was observed when cells were grown in normal medium or at these
concentrations of deoxyglucose (data not shown). On the basis of these
findings we conclude that the pHi-induced stimulation of glycolysis
plays a minor role in the activation of proliferation during
transformation.
|
DMA blocks tumor cell growth in vivo
Finally, the effect of the direct inhibition of NHE-1 activity on
tumor initiation and growth was measured in an in vivo
experimental system. Late passage HPKIA cells were injected
subcutaneously in nude mice. Half of the mice were treated every other
day by i.p. injections of 15 µg DMA/gm body weight whereas the other
half received only the vehicle. Tumor size was measured the third day
after injection and then at weekly intervals. As can be seen in
Fig. 11
, DMA treatment retarded tumor development. These data are consistent
with results from independent in vivo studies in which
tumors were induced with different cancer cell types (37
, 38)
.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The alkalinization is driven
by the activation of NHE-1, as inhibition of NHE-1 activity by its
specific inhibitor, DMA, results in the abrogation of the increased pH.
The stimulation of Na+/H+
exchanger activity occurs via an increase in the affinity of the
intracellular proton regulatory site of the exchanger and is not
dependent on increased
Na+/H+ exchanger
expression. This produces an alkaline shift of the relationship between
the rate of the NHE-1 and pHi that resembles the mechanism involved in
activation of the exchanger by serum, growth factors, and hormones
(2)
.
Most important, our data show that the alkalinization is a key event
for the establishment and maintenance of oncogenic transformation. Upon
expression of the HPV16 E7 oncoprotein, the cells develop a series of
phenotypes characteristic of neoplastic transformation: increased
growth rate, anchorage-independent growth, serum-independent growth,
increased glycolysis, and cytoplasmic alkalinization. Specific
inhibition of NHE-1 activity by DMA prevented the development of the
transformation-associated increase in growth rate, serum independence
of growth, anchorage-independent growth and glycolytic metabolism, and
in vivo tumor development in nude mice. In line with the
reported inhibition by amiloride analogs of the reinitiation of growth
in quiescent cells without affecting steady-state growth
(39)
, DMA inhibited only the component of growth
stimulated by transformation. The abrogation of development of these
transformed phenotypes is not due to an unspecific effect of DMA as
decreasing of pHi at nontransformed levels by acidification of culture
medium or clamping of pHi with the proton ionophore, tributyltin
bromide, was also able to block the development of the transformed
phenotypes.
Our data also demonstrate that alkalinization is not simply a consequence of viral protein expression. In human keratinocytes the expression of HPV16 E6 and E7 genes is not sufficient to induce alkalinization (low passage immortalized HPKIA cells). Since E6 and E7 associate with and inactivate the tumor suppressor proteins p53 and pRb, respectively, it is possible to conclude that alkalinization is not dependent on pathways that are regulated by these two cellular proteins. pRb neutralization by HPV16 E7 leads to activation of the members of the E2F family, which in turn activate the transcription of positive cell cycle regulator genes, e.g., cyclins E and A. Our findings support the idea that the alkalinization-dependent proliferation is not mediated by transcriptional activation of E2F-regulated genes. In transient transfection experiments, we observed that inhibition of NHE-1 by DMA does not alter the ability of E7 to activate the cyclin E promoter. Moreover, the endogenous levels of cyclin E and A do not vary in transformed HPKIA cells (late passage) when cultured in absence or in presence of DMA, even though they have a different intracellular pH. Thus, additional cellular pathways are affected by the increase of pHi to stimulate proliferation.
Cancer cells are characterized by an increased rate of glucose
consumption metabolized predominantly by glycolysis under aerobic
conditions (40
, 41)
. This elevated rate has been
considered to constitute the predominant metabolic component in highly
malignant tumors (42)
, but little is known about its
relative contribution to energy metabolism and growth in cells that
have just undergone transformation. We show that activation of
glycolysis (increased lactate production) is subsequent to and
dependent on E7-induced alkalinization. Furthermore, we have observed
that deoxyglucose treatment decreases the glycolysis to the levels of
transformed HPKIA cells treated with DMA, but had little effect on
cellular growth in comparison with the DMA treatment. Therefore,
glycolysis appears not to play an important role in the increase in
growth rate observed on transformation in HPKIA cells.
In summary, our data demonstrate that alkalinization is an early and essential event in transformation and generates a permissive condition for the establishment and maintenance of transformation-associated phenotypes. The model system for cellular transformation presented in this study should provide a powerful tool for elucidating the signal transduction steps preceding alkalinization and the pH-dependent processes involved in the regulation of proliferation.
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ACKNOWLEDGMENTS |
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Received for publication January 26, 2000.
Revision received May 9, 2000.
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REFERENCES |
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