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Tumor Biology |
Institute of Molecular Medicine [C. C. W., N. J. P. B., K. J. T., A. L. H.] and the Nuffield Department of Clinical Laboratory Sciences [H. T., K. L. T.], John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; Department of Pathology, University of Manitoba, Winnipeg, Manitoba, R3E OW3 Canada [P. H. W.]; Institute of Virology, Slovak Academy of Sciences, 84246 Bratislava, Slovak Republic [J. P.]; Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, Middlesex HA6 2JR, United Kingdom [A. S., G. D. W.]; Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, United Kingdom [P. H. M., C. W. P., P. J. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genes that are up-regulated by microenvironmental hypoxia through
activation of HIF include glucose transporters, glycolytic enzymes, and
angiogenic growth factors (5
, 10
, 11)
. For some HIF
targets such as VEGF, a clear function in promoting tumor growth is
established (12)
. However, the full range of HIF target
genes has not yet been defined, and identification of additional genes
responding to this pathway is likely to provide further insights into
the consequences of tumor hypoxia and HIF activation. Indirect support
for the importance of microenvironmental activation of HIF has also
been provided by recent demonstrations of constitutive activation of
HIF after inactivation of the VHL tumor suppressor gene
(13)
and amplification of the HIF response by other
oncogenic mutations (14, 15, 16, 17)
. Mutations in VHL cause the
familial syndrome and are also found in the majority of sporadic RCCs
(18)
. The gene product pVHL forms part of a
ubiquitin-ligase complex (19
, 20) that targets HIF-
subunits for oxygen-dependent proteolysis (13
, 21)
. In
VHL-defective cells, HIF- is stabilized constitutively, resulting in
up-regulation of hypoxia-inducible genes such as VEGF
(13)
. Although the pVHL ubiquitin-ligase complex may have
other targets (20)
and other functions of pVHL have been
proposed that may contribute to tumor suppressor effects (22
, 23)
, these recent findings raise important questions as to the
range of genes affected by constitutive HIF activation and the role of
these genes in oncogenesis.
In this respect, one interesting group of genes is the tumor-associated transmembrane CAs CA9 (24, 25, 26, 27) and CA12 (28 , 29) . CAs catalyze the reversible hydration of carbon dioxide to carbonic acid (30) , providing a potential link between metabolism and pH regulation. The membrane-linked isoforms CA9 and CA12 were identified by RNA differential display as genes that are down-regulated by pVHL (29) , although the effect of hypoxia was not examined and the mechanism of regulation was not defined. Interestingly, CA9 can confer a variety of features of the transformed phenotype when transfected into NIH 3T3 cells (24) .
In this study, we demonstrate that in contrast to constitutive up-regulation in pVHL-defective cell lines, both CA9 and CA12 are strongly induced by hypoxia in a broad range of other cell types. The induction of CA9 by hypoxia was striking and has been studied in detail. We show that the CA9 promoter is tightly regulated by a HIF-responsive HRE close to its transcriptional start site, and that the gene product is expressed in a perinecrotic manner in many types of human cancer, overlapping with VEGF mRNA and the hypoxia marker pimonidazole. In keeping with constitutive activation of HIF after inactivation of pVHL, the focal pattern of expression observed in most tumors contrasted with that observed in RCCs, where CA IX was globally up-regulated. Our findings define a new biochemical pathway that is regulated by HIF, suggest that CA IX may be a useful marker for HIF activation either by microenvironmental hypoxia or genetic events such as VHL inactivation, and provide additional insights into mechanisms by which the HIF pathway might mediate effects on tumor metabolism.
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MATERIALS AND METHODS |
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RNA Analysis.
Total RNA was extracted by a modified acid/guanidinium
thiocyanate/phenol/chloroform method (RNAzol B; Cinna/Biotec
Laboratories), dissolved in hybridization buffer (80% formamide, 40
mM PIPES, 400 mM sodium chloride, and 1
mM EDTA, pH 8) and analyzed by RPA. To generate appropriate
riboprobe templates, cDNA fragments of human CA9
(nucleotides 3632–3771, accession number Z54349) and CA12
(nucleotides 301–450, accession number AF037335) were amplified by PCR
and ligated into pSP72 (Promega). DNA templates for generating
32P-labeled RNA probes were linearized for
16 h with BglII and transcribed with SP6 RNA
polymerase. For CA9 and CA12, RPAs were performed
on 30 µg of total RNA, using an internal control assay for U6 small
nuclear RNA as described (13)
.
Construction of Reporter Plasmids.
To generate plasmids p-506 and p-173, sequences of the CA9
gene between -506 and +43 relative to the transcriptional start site
were amplified by PCR from genomic DNA. PCR products were ligated into
pGL3-basic, a promoterless and enhancerless luciferase expression
vector (Promega). To generate plasmids p-36, MUT1, and MUT2,
complementary oligonucleotides with ends corresponding to the 5'
restriction cleavage overhangs of BglII and MluI
were annealed and ligated into
BglII/MluI-digested pGL3-basic. Oligonucleotides
(sense strand) were: p-36 (forward),
5'-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCACGTACAGCCCGTACACACCG-3';
MUT1 (forward),
5'-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCTTTTACAGCCCGTACACACCG-3';
MUT2 (forward),
5'-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCAAGTACAGCCCGTACACACCG-3'.
Nucleotides introduced for cloning are lowercase; mutations are
underlined. All CA9 promoter sequences were confirmed by
dideoxy sequence analysis.
Transient Expression Assays.
Cells at 
70% confluence in 60-mm dishes were transfected with 1
µg of a luciferase reporter construct and 0.4 µg of control
plasmid, pCMV-ßgal (Promega), using FuGENE 6 (Roche Diagnostic)
according to the manufacturer’s instructions. Cells were then
incubated at 20% O2 for 8 h, followed by
20% or 0.1% O2 for 16 h.
Luciferase activity was determined in cell lysates using a commercial
assay system (Promega) and a TD-20e luminometer (Turner Designs).
ßgal activity in cell lysates was measured using
o-nitrophenyl-ß-D-galactopyranoside
as substrate in a 0.1 M phosphate buffer (pH 7.0)
containing 10 mM KCl, 1 mM
MgSO4, and 30 mM
ß-mercaptoethanol. To correct for variable transfection efficiencies
between experimental conditions, the luciferase:ßgal ratio was
determined for each sample. For cotransfection assays, cells also
received 0.1–1 µg each of pCDNA3/HIF-1 or pCDNA3/HIF-2
containing the entire human HIF-1 or HIF-2 open reading frame,
respectively. Transfections were balanced with various amounts of
pCDNA3 (Invitrogen) and pCDNA3/HIF- such that all cells received the
same total quantity of DNA.
Cell Lysis and Immunoblotting.
Whole-cell protein extracts were prepared from tissue culture cells by
10-s homogenization in denaturing conditions as described
(31)
. Whole-cell protein extracts were prepared from
tumors by fine section of frozen tissue and 30-s homogenization in
denaturing conditions identical to tissue culture extracts. For Western
analysis, aliquots were separated by SDS-PAGE and transferred to
Immobilon-P membranes. CA IX was detected using the mouse monoclonal
antihuman CA IX antibody M75 (1:50) as described (32)
.
Horseradish peroxidase-conjugated goat-antimouse immunoglobulin (DAKO;
1:2000) was applied for 1 h at room temperature. ECL Plus
(Amersham Pharmacia) was used for visualization.
Immunohistochemistry.
Formalin-fixed, paraffin-embedded tissue specimens collected by
standard surgical oncology procedures were obtained from the Pathology
Department, John Radcliffe Hospital (Oxford, United Kingdom).
Immunostaining of paraffin sections was performed after dewaxing and
rehydrating 4-µm sections. For CA IX detection, endogenous peroxidase
was blocked with 0.5% hydrogen peroxide in water for 30 min. To block,
10% normal human serum in TBS was applied for 15 min. M75 (see
"Immunoblotting"; 1:50) was applied for 30 min at room temperature.
Secondary polymer from Envision kit (DAKO) was applied for 30 min at
room temperature. For pimonidazole detection, sections were digested
with 0.01% Pronase (Sigma) in PBS for 30 min at 37°C. Endogenous
peroxidase was blocked with 0.1% hydrogen peroxide in water for 30
min. To block, Protein Block (DAKO) was applied for 5 min.
Anti-pimonidazole IgG1 antibody (Natural Pharmacia; 1:100) was applied
for 1 h at room temperature. Biotinylated rabbit antimouse
secondary (DAKO; 1:200) was applied for 1 h at room temperature.
ABC complex horseradish peroxidase conjugate (DAKO) was applied for
1 h at room temperature. Visualization of CA IX and pimonidazole
staining was by diaminobenzidine substrate. Slides were counterstained
with hematoxylin before mounting in Aquamount (BDH). Substitution of
primary antibody with PBS was used as a negative control for both
antibodies.
CA IX and pimonidazole were studied in semiserial tissue sections. The percentage of tumor cells showing positive staining for CA IX or pimonidazole and the extent of overlap between these regions within each tissue section was assessed by light microscopy at low magnification by three observers (C. C. W., P. H. W., and H. T.) and a consensus was determined.
In Situ mRNA Hybridization.
Specific localization of VEGF mRNA was accomplished by in
situ hybridization using an antisense riboprobe. Briefly,
pBluescript (Stratagene) containing 517 consecutive complementary
nucleotides of the VEGF121 transcript (439
consecutive nucleotides of which are complementary to
VEGF165, VEGF189, and
VEGF206) was linearized with EcoRV for
16 h at 37°C. Labeled transcripts were synthesized using T7
(antisense) and SP6 (sense) polymerase in the presence of
[35S]UTP (>800 Ci/mmol; Amersham Pharmacia).
The methods for pretreatment, hybridization, washing, and dipping of
slides in Ilford K5 for autoradiography were as described for
formalin-fixed, paraffin-embedded tissue (33)
. The
presence of hybridizable mRNA in tissue sections was established in
semiserial sections using an antisense ß-actin probe. Hybridizations
using a sense probe were used to control for nonspecific signal.
Autoradiography was at 4°C (two exposures per section for VEGF
visualization at 10 and 18 days), before developing in Kodak D19 and
counterstaining by Giemsa’s method. Sections were examined under
conventional and reflected light-/dark-field conditions.
Pimonidazole Administration.
Patients with squamous or basal cell carcinomas of the skin and
patients with newly diagnosed transitional cell bladder carcinoma were
studied. Signed informed consent was obtained in all cases.
Pimonidazole hydrochloride was selected as the hypoxia marker because
of its high water solubility, chemical stability, efficient tumor
uptake, and low toxicity. Patients received 500
mg/m2 of pimonidazole hydrochloride,
1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole hydrochloride
(Hypoxyprobe) in 100 ml of normal saline i.v. over 20 min. This dose is
25% of the maximum tolerated dose (34)
. Patients with
tumors in skin underwent incisional or Trucut biopsy under local
anesthetic 2–24 h after pimonidazole infusion. Patients with bladder
carcinoma underwent transurethral resection of the tumor under general
anesthetic 2–24 h after pimonidazole infusion. Tissue samples were
immediately placed in 10% neutral buffered formalin, protected from
light, and then processed into formalin blocks.
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RESULTS |
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. We also examined expression in other RCC lines that are
either defective (KRL140, SKRC28, UMRC2, and 786-0) or wild type
(Caki-1) for VHL, and in a stable transfectant of 786-0 re-expressing
wild-type pVHL (WT 8). Representative results from three cell lines are
illustrated in Fig. 1A
. In the VHL-defective cells, both
CA9 and CA12 were constitutively expressed and
unresponsive to changes in oxygen tension. In the wild-type VHL cell
lines, both genes, when expressed, were induced by hypoxia.
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. Because many hypoxia-inducible
genes are up-regulated by treatment of cells with the iron chelator DFO
(35)
, we also tested the effect of DFO and found a similar
induction of both CA9 and CA12 mRNA (Fig. 1C)
.
CA9 Promoter Analysis.
To investigate the unusually tight regulation of CA9 mRNA by
hypoxia, we tested for oxygen-dependent function of the CA9
promoter. In the first set of experiments, we tested luciferase
reporter genes containing 0.5 kb of CA9 5' flanking
sequences (-506 to +43) and a deletion to nucleotide -173 (-173 to
+43) in transiently transfected HeLa cells. Both constructs showed very
low levels of activity in normoxic cells but were induced strongly by
hypoxia (Fig. 2A)
. By contrast, a similar reporter linked to a minimal SV40
promoter showed no induction by hypoxia.
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subunit and cannot form the
HIF-1 transcriptional complex (36)
. In the CHO wild-type
parental subline C4.5, the -173 nucleotide promoter conferred 17-fold
transcriptional induction by hypoxia. In contrast, in the
HIF-1-deficient Ka13 subline, this hypoxic induction was absent
(Fig. 2B)
. Cotransfection of human HIF-1 restored
hypoxia-inducible activity to the CA9 promoter in the Ka13
cells and increased normoxic activity in both C4.5 and Ka13 (Fig. 2B)
. In C4.5 and Ka13 cells at 0.1%
O2, luciferase expression was increased 1.6- and
17-fold, respectively, by cotransfection of human HIF-1. Thus,
hypoxia-inducible activity of the CA9 promoter is completely
dependent on HIF-1 and strongly influenced by the level of HIF-l.
Activity of the CA9 promoter in Ka13 cells could also be
restored by cotransfection of HIF-2, although normoxic activity was
higher and fold induction by hypoxic stimulation was reduced (data not
shown).
Inspection of the CA9 5' flanking sequences revealed a
consensus HRE beginning 3 bp 5' to the transcriptional start site,
orientated on the antisense strand, reading 5'-TACGTGCA-3' (Fig. 2
,
left). To test the importance of this site, we constructed a
CA9 minimal promoter containing this sequence (-36 to +14).
This minimal promoter retained hypoxia-inducible activity in C4.5 cells
but had no inducible activity in Ka13 cells (Fig. 2C)
.
Absolute levels of activity were lower in comparison to the -173
nucleotide promoter construct, being reduced 8 fold, indicating that
although sequences -173 to -36 amplified promoter activity,
responsiveness to hypoxia was conveyed by the minimal sequence
containing the HRE. To confirm the importance of this HRE, two
mutations were made within its core (antisense strand): a 3-bp
substitution from CGT 
AAA (MUT1), and a single substitution of G
T (MUT2; Fig. 2
, left). Both mutations completely
ablated hypoxia-inducible activity, although basal activity was
preserved or slightly increased for MUT1 (Fig. 2C)
.
Regulation of CA IX Protein by Oxygen.
As a first step toward understanding the significance of
hypoxia-inducible expression of CA9 mRNA, the effect of
hypoxia was examined on CA IX protein levels in whole-cell extracts.
Immunoblots of representative cells using anti-CA IX monoclonal
antibody M75 are illustrated in Fig. 3A
. Striking induction of CA IX protein by hypoxia was
observed in multiple cell lines, whereas the VHL-defective RCC4 cells
showed constitutive up-regulation of CA IX protein. Thus, hypoxic
up-regulation of CA9 mRNA is clearly reflected at the
protein level. We next examined the response of CA IX to increasing
degrees of hypoxia (Fig. 3B)
. The level of CA IX hypoxic
induction after 16 h of exposure increased with decreasing oxygen
tensions from 5 to 0.1%. Because the original description of CA IX was
as an antigen induced by culture of cells at high density
(37)
, we also compared the effects of culture at high
density with those of hypoxia. In normoxic cultures of A549 cells, high
density clearly induced CA IX, although the effect was considerably
smaller than that of hypoxia (Fig. 3C)
.
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and no staining in normoxic RCC4/VHL cells (Fig. 4B)
. Then, we compared immunostaining and immunoblots of
tissue extracts from similar regions of tumor and normal tissue in four
sets of paired samples from surgical excisions of head and neck tumors.
By immunostaining, CA IX expression was low or absent in normal tissue
surrounding the tumors but was expressed at significant levels in each
tumor specimen. Results of immunoblot analysis correlated closely with
immunostaining, signals being very low or undetectable in each normal
tissue sample, and correlated with the extent of CA IX immunostaining
in tumor samples (data not shown).
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. Expression was
localized to the cellular membrane. Tracing a line from the necrotic
center to adjacent viable cells revealed a gradient of CA IX
expression, with the highest levels observed in cells closest to or
within necrotic regions (Fig. 4, C–F)
.
Because pVHL inactivation leads to loss of CA9 regulation by
oxygen in cultured cells and is common in clear cell renal carcinoma
but not other renal tumors, we next compared expression patterns in a
second series of 35 clear cell renal tumors and eight papillary renal
tumors. Representative sections from a clear cell and a papillary tumor
are illustrated in Fig. 4, G and H
. Expression
patterns were markedly different. In 33 of 35 clear cell tumors, (both
sporadic and derived from VHL syndrome patients), CA IX was expressed
throughout tumor tissue; strong membrane staining was observed in tumor
cells, regardless of proximity to necrosis or vessels (G).
In contrast, in papillary renal tumors CA IX immunostaining was much
less evident but was observed in tumors containing areas of necrosis,
where, as with the nonrenal tumors, staining was strikingly focal and
perinecrotic (four of eight papillary tumors contained necrosis, and
all four showed focal CA IX positivity; Fig. 4H
). Thus, the
tight regulation of CA9 expression by oxygen observed in
cell culture appeared to be reflected in strikingly focal patterns of
expression around areas of necrosis.
Relationship of CA IX Expression with an Endogenous and an
Administered Hypoxia Marker in Human Tumors.
To compare CA IX expression with potential markers of tumor hypoxia, we
examined expression of VEGF mRNA and activation of the bioreductive
hypoxia marker pimonidazole in relationship to CA IX staining. Serial
sections of a subset of our first series of tumors were analyzed for
VEGF mRNA expression by in situ hybridization, and CA IX
expression was analyzed by immunostaining. Representative views from an
ovarian and head and neck tumor sample are illustrated in Fig. 5
. VEGF mRNA was expressed at varying levels throughout tumor tissue but
was increased greatly in regions adjacent to necrosis. CA IX
immunostaining showed strong overlap but was more tightly limited to
perinecrotic regions.
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, and assessment of pimonidazole and CA IX staining with corresponding
overlap for each tumor biopsy are indicated in Table 1
. In most tumors (16 of 20), pimonidazole staining was more extensive
across tumor sections than CA IX staining, being primarily banded
around necrotic areas (Fig. 6, C and E)
and the
periphery of papillary structures in bladder carcinomas (Fig. 6A)
. Although less extensive, the large majority of CA IX
immunostaining localized within regions of pimonidazole adduct
formation and was also associated with necrosis (Fig. 6, D and F)
or the periphery of papillary structures in bladder
carcinomas (Fig. 6B)
. Some regions containing CA IX were
observed that were slightly farther removed from necrosis than regions
staining positive for pimonidazole (Fig. 6, C and D)
. In 4 of 20 cases, CA IX staining was more extensive than
pimonidazole staining. In these cases, in addition to the
characteristic perinecrotic and peripheral papillary expression, a
proportion of CA IX expression was not obviously associated with such
regions in the plane of the section. Nevertheless, within these four
tumors the pimonidazole-positive regions were consistently localized
within regions of CA IX positivity, again demonstrating the overlap
between these markers. Despite the relationship between pimonidazole
and CA IX at the microscopic level in all tumors, we did not observe an
overall correlation between the percentage of tumor stained for
pimonidazole and CA IX (Table 1)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies of the CA9 promoter demonstrated that sequences close to the transcriptional initiation site were sufficient to convey a hypoxia-inducible response, that this activity was mediated by HIF, and that it was dependent on a consensus HRE lying adjacent to the initiation site. The CA9 promoter contains neither a TATA box nor a consensus initiator sequence at the cap site (38) . The association of this unusual anatomy with tight regulation by hypoxia is therefore of interest and suggests that it may be informative to pursue the mechanism by which HIF interacts with the basal transcriptional machinery operating on this gene. Furthermore, irrespective of the mechanism, the strong inducibility conveyed by the minimal CA9 promoter is unusual and may itself be of utility, for instance in the refinement of gene therapy vectors seeking to target therapeutic gene expression to hypoxic regions of tumors (39 , 40) .
Our findings also raise a number of issues relevant to recently
published analyses of the CA9 promoter that did not examine
the effect of hypoxia: (a) they provide an explanation for
the remarkably low levels of CA9 promoter activity recently
reported under standard culture conditions (41)
, because
promoter activity is so strongly dependent on hypoxia; (b)
they are consistent with the positive activity demonstrated for
sequences -173 to +31 (41)
and show that the
transcriptional effects mediated by these sequences interact with the
HRE in the minimal promoter to amplify the response to hypoxia;
(c) they are consistent with the absence of a DNase I
footprint in the region of the HRE (41)
, because even in
hypoxia it has been shown that HIF-1 binding characteristics are such
that an in vitro footprint is not demonstrated
(42)
; (d) they provide a potential explanation
for the repressive effects of p53 expression on the activity of the
CA9 promoter in some cells (43)
, because it has
been suggested that p53 can interact with the regulation of HIF-1
stability so as to reduce activity of the HIF/HRE complex (15
, 16)
.
In tissue culture, CA9 demonstrated a very marked difference between constitutive expression in VHL-defective RCC cells and strong induction by hypoxia in cells known or presumed to be VHL competent. This provided an opportunity to determine the extent to which these contrasting patterns of regulation in culture were reflected in patterns of expression within native tumors. In our series of renal tumors, we found a striking contrast between generalized expression in clear cell carcinomas, which are usually defective in VHL, and focal perinecrotic expression in papillary renal tumors, which are usually wild type for VHL. Notwithstanding the absence of direct ascertainment of VHL genotype in all of the tumors analyzed, this strongly suggests that effects of VHL status on HIF-dependent, hypoxia-inducible gene expression are reflected in patterns of expression within native tumors. Up-regulation by constitutively active HIF therefore provides an explanation for the utility of CA9 as a marker for clear cell carcinoma. The pattern of diffuse expression in clear cell carcinoma is in agreement with findings of a previous analysis of CA IX expression in which the authors focused on high levels of expression in clear cell carcinoma versus absent expression in a variety of benign lesions and postulated that CA IX expression might be useful as a marker of malignant change (25) . That study also noted focal expression in papillary renal carcinoma, although the authors did not comment on the relation to necrosis. In our studies, we found that the striking localization of focal CA IX expression to zones of necrosis is not just observed in papillary renal carcinoma but also in several series of nonrenal tumors. The pattern is similar to that first described for VEGF mRNA (5) , and we compared directly the pattern of CA IX immunostaining with that of in situ mRNA hybridization for VEGF in several types of tumors. In this work, we used in situ mRNA hybridization for VEGF to localize the site of production, because, in contrast with CA IX, some isoforms of VEGF are secreted. Patterns of expression for CA IX and VEGF mRNA were clearly concordant. However, CA IX expression was more strikingly delimited, being essentially limited to regions surrounding zones of necrosis.
The concordance of hypoxia-inducible versus constitutive patterns of expression in tissue culture with focal perinecrotic versus diffuse patterns of expression in tumors strongly supports the view that the focal perinecrotic pattern of expression is driven by microenvironmental hypoxia. Furthermore, the particularly tight regulation of CA9 by hypoxia suggested that it might be useful as a hypoxia marker. It was, therefore, of interest to compare the pattern of CA IX immunostaining with staining for the hypoxia marker pimonidazole (44, 45, 46) . Our analysis demonstrated clear overlap of the staining patterns, supporting expression of CA IX in hypoxic regions. Previous studies have compared the distribution of immunodetectable pimonidazole adducts with VEGF immunostaining. One study concluded that pimonidazole and VEGF displayed the same pattern of staining on adjacent sections during the angiogenesis associated with a model of liver fibrogenesis (47) , whereas an earlier study emphasized the discrepancies between pimonidazole and VEGF staining, although regions of overlap were demonstrated (48) . Among the explanations considered for the differences between the distribution of VEGF staining and pimonidazole adducts were regulation of VEGF by nonhypoxic stimuli and diffusion of VEGF from hypoxic sites of production. For CA9, basal expression in normoxic cells was lower than we have observed for VEGF, induction by hypoxia was more striking, and the protein was not secreted. Despite this, we also observed differences in pimonidazole and CA IX staining. The substantial regions of overlap presumably reflect regions where tumor hypoxia was of sufficient duration and severity to activate both markers. Regions of nonoverlap could reflect the operation of additional positive or negative influences on expression or activation or different time frames of induction or activation. For instance, pimonidazole adducts are formed over a relatively short period of time and are then long-lived (45 , 46) , whereas we have found that CA IX is a stable protein that, in tissue culture, accumulated over a long period of hypoxia (data not shown). Thus, CA IX induction might only be expected in regions of relatively chronic tumor hypoxia and would reflect a different hypoxic time frame from pimonidazole activation. Correlation of focal CA IX expression with direct measurements of tumor oxygenation and with clinical parameters of outcome will be of interest.
The demonstration that an extracellular CA is up-regulated by microenvironmental tumor hypoxia has potentially important implications for understanding the regulation of tumor pH and the response to hypoxia. It has been widely held that lactate production by glycolysis is a major cause of the acidic extracellular pH of tumors (49) , and indeed glycolytic enzymes are induced by hypoxia (11) , as is lactate production (50) . However, tumors grown from mutant cells with glycolytic defects show a similar extracellular acidosis in the absence of lactate accumulation (51 , 52) , indicating that other mechanisms must be involved. Recently, it has been proposed that extracellular CAs could convert CO2 diffusing from oxygenated areas to carbonic acid and promote the generation of bicarbonate and hydrogen ions (29 , 52) . Bicarbonate might then be exchanged for intracellular chloride, providing a mechanism for maintaining the characteristic extracellular acidosis and intracellular alkalosis that is postulated to promote tumor growth (53) . Thus, it is likely that the hypoxia-inducible behavior of tumor-associated CAs could exert important biological effects through an influence on microenvironmental pH. This could have therapeutic implications because CA inhibitors have been shown to inhibit the invasion of renal cell carcinoma lines in model culture systems (54) and have synergistic effects with other chemotherapeutic agents in animal models (55) . The potential for strong induction by hypoxia will now need to be considered in assessing the diagnostic and therapeutic implications of tumor-associated extracellular CAs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Wellcome Trust
and the Imperial Cancer Research Fund. 
2 To whom requests for reprints should be
addressed, at Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN,
United Kingdom. Phone: (44) 1865-287531; Fax: (44) 1865-287533; E-mail: peter.ratcliffe{at}imm.ox.ac.uk
3 The abbreviations used are: HIF-1,
hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor;
VHL, von Hippel-Lindau; RCC, renal cell carcinoma; CA, carbonic
anhydrase; CA9, carbonic anhydrase 9 gene (including any
genomic sequence and mRNA); CA IX, carbonic anhydrase 9 protein; HRE,
hypoxia response element; RPA, RNase protection assay; ßgal,
ß-galactosidase; DFO, desferrioxamine; CHO, Chinese hamster ovary.
Received 6/13/00. Accepted 10/18/00.
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R. E. Schweppe, T. H. Cheung, and N. G. Ahn Global Gene Expression Analysis of ERK5 and ERK1/2 Signaling Reveals a Role for HIF-1 in ERK5-mediated Responses J. Biol. Chem., July 28, 2006; 281(30): 20993 - 21003. [Abstract] [Full Text] [PDF]
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L. M. Brown, R. L. Cowen, C. Debray, A. Eustace, J. T. Erler, F. C. D. Sheppard, C. A. Parker, I. J. Stratford, and K. J. Williams Reversing Hypoxic Cell Chemoresistance in Vitro Using Genetic and Small Molecule Approaches Targeting Hypoxia Inducible Factor-1 Mol. Pharmacol., February 1, 2006; 69(2): 411 - 418. [Abstract] [Full Text] [PDF]
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N. O. Ibrahim, T. Hahn, C. Franke, D. P. Stiehl, R. Wirthner, R. H. Wenger, and D. M. Katschinski Induction of the Hypoxia-Inducible Factor System by Low Levels of Heat Shock Protein 90 Inhibitors Cancer Res., December 1, 2005; 65(23): 11094 - 11100. [Abstract] [Full Text] [PDF]
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M. A. Proescholdt, C. Mayer, M. Kubitza, T. Schubert, S.-Y. Liao, E. J. Stanbridge, S. Ivanov, E. H. Oldfield, A. Brawanski, and M. J. Merrill Expression of hypoxia-inducible carbonic anhydrases in brain tumors Neuro-oncol, October 1, 2005; 7(4): 465 - 475. [Abstract] [PDF]
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S. Sobhanifar, C. Aquino-Parsons, E. J. Stanbridge, and P. Olive Reduced Expression of Hypoxia-Inducible Factor-1{alpha} in Perinecrotic Regions of Solid Tumors Cancer Res., August 15, 2005; 65(16): 7259 - 7266. [Abstract] [Full Text] [PDF]
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M. A. Titus, C. W. Gregory, O. H. Ford III, M. J. Schell, S. J. Maygarden, and J. L. Mohler Steroid 5{alpha}-Reductase Isozymes I and II in Recurrent Prostate Cancer Clin. Cancer Res., June 15, 2005; 11(12): 4365 - 4371. [Abstract] [Full Text] [PDF]
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A. Soubry, J. van Hengel, E. Parthoens, C. Colpaert, E. Van Marck, D. Waltregny, A. B. Reynolds, and F. van Roy Expression and Nuclear Location of the Transcriptional Repressor Kaiso Is Regulated by the Tumor Microenvironment Cancer Res., March 15, 2005; 65(6): 2224 - 2233. [Abstract] [Full Text] [PDF]
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E. Hervouet, J. Demont, P. Pecina, A. Vojtiskova, J. Houstek, H. Simonnet, and C. Godinot A new role for the von Hippel-Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis Carcinogenesis, March 1, 2005; 26(3): 531 - 539. [Abstract] [Full Text] [PDF]
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S. J. Kim, Z. N. Rabbani, R. T. Vollmer, E.-G. Schreiber, E. Oosterwijk, M. W. Dewhirst, Z. Vujaskovic, and M. J. Kelley Carbonic Anhydrase IX in Early-Stage Non-Small Cell Lung Cancer Clin. Cancer Res., December 1, 2004; 10(23): 7925 - 7933. [Abstract] [Full Text] [PDF]
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U. Berchner-Pfannschmidt, F. Petrat, K. Doege, B. Trinidad, P. Freitag, E. Metzen, H. de Groot, and J. Fandrey Chelation of Cellular Calcium Modulates Hypoxia-inducible Gene Expression through Activation of Hypoxia-inducible Factor-1{alpha} J. Biol. Chem., October 22, 2004; 279(43): 44976 - 44986. [Abstract] [Full Text] [PDF]
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I. P. Stolze, Y.-M. Tian, R. J. Appelhoff, H. Turley, C. C. Wykoff, J. M. Gleadle, and P. J. Ratcliffe Genetic Analysis of the Role of the Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-inducible Factor (HIF) in Regulating HIF Transcriptional Target Genes J. Biol. Chem., October 8, 2004; 279(41): 42719 - 42725. [Abstract] [Full Text] [PDF]
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W. G. Kaelin Jr. The Von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6290S - 6295S. [Abstract] [Full Text] [PDF]
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W. M. Stadler Therapeutic Options for Variant Renal Cancer: A True Orphan Disease Clin. Cancer Res., September 15, 2004; 10(18): 6393S - 6396S. [Abstract] [Full Text] [PDF]
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R. J. Appelhoff, Y.-M. Tian, R. R. Raval, H. Turley, A. L. Harris, C. W. Pugh, P. J. Ratcliffe, and J. M. Gleadle Differential Function of the Prolyl Hydroxylases PHD1, PHD2, and PHD3 in the Regulation of Hypoxia-inducible Factor J. Biol. Chem., September 10, 2004; 279(37): 38458 - 38465. [Abstract] [Full Text] [PDF]
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J. D. Blais, V. Filipenko, M. Bi, H. P. Harding, D. Ron, C. Koumenis, B. G. Wouters, and J. C. Bell Activating Transcription Factor 4 Is Translationally Regulated by Hypoxic Stress Mol. Cell. Biol., September 1, 2004; 24(17): 7469 - 7482. [Abstract] [Full Text] [PDF]
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N. Robertson, C. Potter, and A. L. Harris Role of Carbonic Anhydrase IX in Human Tumor Cell Growth, Survival, and Invasion Cancer Res., September 1, 2004; 64(17): 6160 - 6165. [Abstract] [Full Text] [PDF]
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M. Kaluzova, S. Kaluz, M. I. Lerman, and E. J. Stanbridge DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1{alpha} in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX Mol. Cell. Biol., July 1, 2004; 24(13): 5757 - 5766. [Abstract] [Full Text] [PDF]
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J. T. Erler, C. J. Cawthorne, K. J. Williams, M. Koritzinsky, B. G. Wouters, C. Wilson, C. Miller, C. Demonacos, I. J. Stratford, and C. Dive Hypoxia-Mediated Down-Regulation of Bid and Bax in Tumors Occurs via Hypoxia-Inducible Factor 1-Dependent and -Independent Mechanisms and Contributes to Drug Resistance Mol. Cell. Biol., April 1, 2004; 24(7): 2875 - 2889. [Abstract] [Full Text] [PDF]
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R. A. Cairns and R. P. Hill Acute Hypoxia Enhances Spontaneous Lymph Node Metastasis in an Orthotopic Murine Model of Human Cervical Carcinoma Cancer Res., March 15, 2004; 64(6): 2054 - 2061. [Abstract] [Full Text] [PDF]
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Y. Azuma, S.-C. Chou, R. A. Lininger, B. J. Murphy, M. A. Varia, and J. A. Raleigh Hypoxia and Differentiation in Squamous Cell Carcinomas of the Uterine Cervix: Pimonidazole and Involucrin Clin. Cancer Res., October 15, 2003; 9(13): 4944 - 4952. [Abstract] [Full Text] [PDF]
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 L. A. Sonna, M. L. Cullivan, H. K. Sheldon, R. E. Pratt, and C. M. Lilly Effect of hypoxia on gene expression by human hepatocytes (HepG2) Physiol Genomics, February 6, 2003; 12(3): 195 - 207. [Abstract] [Full Text] [PDF]
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D. E.B. Swinson, J. L. Jones, D. Richardson, C. Wykoff, H. Turley, J. Pastorek, N. Taub, A. L. Harris, and K. J. O'Byrne Carbonic Anhydrase IX Expression, a Novel Surrogate Marker of Tumor Hypoxia, Is Associated With a Poor Prognosis in Non-Small-Cell Lung Cancer J. Clin. Oncol., February 1, 2003; 21(3): 473 - 482. [Abstract] [Full Text] [PDF]
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M. H. T. Bui, D. Seligson, K.-r. Han, A. J. Pantuck, F. J. Dorey, Y. Huang, S. Horvath, B. C. Leibovich, S. Chopra, S.-Y. Liao, et al. Carbonic Anhydrase IX Is an Independent Predictor of Survival in Advanced Renal Clear Cell Carcinoma: Implications for Prognosis and Therapy Clin. Cancer Res., February 1, 2003; 9(2): 802 - 811. [Abstract] [Full Text] [PDF]
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J. H. A. M. Kaanders, K. I. E. M. Wijffels, H. A. M. Marres, A. S. E. Ljungkvist, L. A. M. Pop, F. J. A. van den Hoogen, P. C. M. de Wilde, J. Bussink, J. A. Raleigh, and A. J. van der Kogel Pimonidazole Binding and Tumor Vascularity Predict for Treatment Outcome in Head and Neck Cancer Cancer Res., December 1, 2002; 62(23): 7066 - 7074. [Abstract] [Full Text] [PDF]
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 M. Yao, M. Yoshida, T. Kishida, N. Nakaigawa, M. Baba, K. Kobayashi, T. Miura, M. Moriyama, Y. Nagashima, Y. Nakatani, et al. VHL Tumor Suppressor Gene Alterations Associated With Good Prognosis in Sporadic Clear-Cell Renal Carcinoma J Natl Cancer Inst, October 16, 2002; 94(20): 1569 - 1575. [Abstract] [Full Text] [PDF]
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C. Willam, N. Masson, Y.-M. Tian, S. A. Mahmood, M. I. Wilson, R. Bicknell, K.-U. Eckardt, P. H. Maxwell, P. J. Ratcliffe, and C. W. Pugh Peptide blockade of HIFalpha degradation modulates cellular metabolism and angiogenesis PNAS, August 6, 2002; 99(16): 10423 - 10428. [Abstract] [Full Text] [PDF]
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S. Kaluz, M. Kaluzova, A. Chrastina, P. L. Olive, S. Pastorekova, J. Pastorek, M. I. Lerman, and E. J. Stanbridge Lowered Oxygen Tension Induces Expression of the Hypoxia Marker MN/Carbonic Anhydrase IX in the Absence of Hypoxia-inducible Factor 1{alpha} Stabilization: A Role for Phosphatidylinositol 3'-Kinase Cancer Res., August 1, 2002; 62(15): 4469 - 4477. [Abstract] [Full Text] [PDF]
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 R. H. WENGER Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression FASEB J, August 1, 2002; 16(10): 1151 - 1162. [Abstract] [Full Text] [PDF]
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E. P. Hui, A. T. C. Chan, F. Pezzella, H. Turley, K.-F. To, T. C. W. Poon, B. Zee, F. Mo, P. M. L. Teo, D. P. Huang, et al. Coexpression of Hypoxia-inducible Factors 1{alpha} and 2{alpha}, Carbonic Anhydrase IX, and Vascular Endothelial Growth Factor in Nasopharyngeal Carcinoma and Relationship to Survival Clin. Cancer Res., August 1, 2002; 8(8): 2595 - 2604. [Abstract] [Full Text] [PDF]
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M. Leppilampi, P. Koistinen, E.-R. Savolainen, J. Hannuksela, A.-K. Parkkila, O. Niemela, S. Pastorekova, J. Pastorek, A. Waheed, W. S. Sly, et al. The Expression of Carbonic Anhydrase II in Hematological Malignancies Clin. Cancer Res., July 1, 2002; 8(7): 2240 - 2245. [Abstract] [Full Text] [PDF]
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 M. S. Wiesener, M. Seyfarth, C. Warnecke, J. S. Jurgensen, C. Rosenberger, N. V. Morgan, E. R. Maher, U. Frei, and K.-U. Eckardt Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma Blood, May 15, 2002; 99(10): 3562 - 3565. [Abstract] [Full Text] [PDF]
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C. N. Coleman, J. B. Mitchell, and K. Camphausen Tumor Hypoxia: Chicken, Egg, or a Piece of the Farm? J. Clin. Oncol., February 1, 2002; 20(3): 610 - 615. [Full Text] [PDF]
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P. L. Olive, C. Aquino-Parsons, S. H. MacPhail, S.-Y. Liao, J. A. Raleigh, M. I. Lerman, and E. J. Stanbridge Carbonic Anhydrase 9 as an Endogenous Marker for Hypoxic Cells in Cervical Cancer Cancer Res., December 1, 2001; 61(24): 8924 - 8929. [Abstract] [Full Text] [PDF]
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A. Giatromanolaki, M. I. Koukourakis, E. Sivridis, J. Pastorek, C. C. Wykoff, K. C. Gatter, and A. L. Harris Expression of Hypoxia-inducible Carbonic Anhydrase-9 Relates to Angiogenic Pathways and Independently to Poor Outcome in Non-Small Cell Lung Cancer Cancer Res., November 1, 2001; 61(21): 7992 - 7998. [Abstract] [Full Text] [PDF]
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M. I. Koukourakis, A. Giatromanolaki, E. Sivridis, K. Simopoulos, J. Pastorek, C. C. Wykoff, K. C. Gatter, and A. L. Harris Hypoxia-regulated Carbonic Anhydrase-9 (CA9) Relates to Poor Vascularization and Resistance of Squamous Cell Head and Neck Cancer to Chemoradiotherapy Clin. Cancer Res., November 1, 2001; 7(11): 3399 - 3403. [Abstract] [Full Text] [PDF]
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A. Lal, H. Peters, B. St. Croix, Z. A. Haroon, M. W. Dewhirst, R. L. Strausberg, J. H. A. M. Kaanders, A. J. van der Kogel, and G. J. Riggins Transcriptional Response to Hypoxia in Human Tumors J Natl Cancer Inst, September 5, 2001; 93(17): 1337 - 1343. [Abstract] [Full Text] [PDF]
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J. A. Loncaster, A. L. Harris, S. E. Davidson, J. P. Logue, R. D. Hunter, C. C. Wycoff, J. Pastorek, P. J. Ratcliffe, I. J. Stratford, and C. M. L. West Carbonic Anhydrase (CA IX) Expression, a Potential New Intrinsic Marker of Hypoxia: Correlations with Tumor Oxygen Measurements and Prognosis in Locally Advanced Carcinoma of the Cervix Cancer Res., September 1, 2001; 61(17): 6394 - 6399. [Abstract] [Full Text] [PDF]
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S. K. Chia, C. C. Wykoff, P. H. Watson, C. Han, R. D. Leek, J. Pastorek, K. C. Gatter, P. Ratcliffe, and A. L. Harris Prognostic Significance of a Novel Hypoxia-Regulated Marker, Carbonic Anhydrase IX, in Invasive Breast Carcinoma J. Clin. Oncol., August 15, 2001; 19(16): 3660 - 3668. [Abstract] [Full Text] [PDF]
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N. J. P. Beasley, C. C. Wykoff, P. H. Watson, R. Leek, H. Turley, K. Gatter, J. Pastorek, G. J. Cox, P. Ratcliffe, and A. L. Harris Carbonic Anhydrase IX, an Endogenous Hypoxia Marker, Expression in Head and Neck Squamous Cell Carcinoma and its Relationship to Hypoxia, Necrosis, and Microvessel Density Cancer Res., July 1, 2001; 61(13): 5262 - 5267. [Abstract] [Full Text] [PDF]
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C. C. Wykoff, N. Beasley, P. H. Watson, L. Campo, S. K. Chia, R. English, J. Pastorek, W. S. Sly, P. Ratcliffe, and A. L. Harris Expression of the Hypoxia-Inducible and Tumor-Associated Carbonic Anhydrases in Ductal Carcinoma in Situ of the Breast Am. J. Pathol., March 1, 2001; 158(3): 1011 - 1019. [Abstract] [Full Text] [PDF]
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