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From the Laboratory of Immunobiology,*
the Intramural
Research Support Program,
Science Applications
International Corporation, Frederick, Maryland; the Molecular Aspects
of Drug Design Section,§
National Cancer
Institute, Frederick Cancer Research and Development Center, Frederick,
Maryland; the Surgical Neurology Branch,¶
National
Institute of Neurological Disorders and Stroke, National Institutes of
Health, Bethesda, Maryland; the Department of Microbiology and
Molecular Genetics,
College of Medicine,
University of California at Irvine, Irvine, California; the St. Louis
University School of Medicine,**
St. Louis,
Missouri; and the Academy of Sciences,||
Prague,
Czech Republic
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
HIF2-.8,16-18
Both HIF- subunits are pVHL-binding
proteins that are targeted by pVHL, presumably via the VBC complex, for
degradation in normoxic but not hypoxic conditions.8
Therefore, pVHL is responsible for control of the hypoxia-driven
changes in gene expression in tumors, thereby initiating and augmenting
the second stage of tumor development. In VHL-defective tumors,
curiously enough, the two fundamental stages of tumor development occur
either simultaneously or in reverse, first triggering the
hypoxia-cellular response, followed by proliferation of transformed
cells, consistent with the angiogenic phenotype of tumors seen in the
VHL syndrome. Previously, we discovered that in VHL patients the CA9
and CA12 genes are overexpressed in tumors because of the absence of
pVHL, and argued that these CA enzymes might be involved in sensing and
maintaining the acidic tumor microenvironment.10
We have
now extended these observations by analyzing the expression of CA IX
and CA XII in a large sample of cancer cell lines and fresh or archival
tumor specimens. Here we show that either one or both genes are
overexpressed in many tumor types, suggesting that this is a common
feature of cancer cells, consistent with the fundamental role of VHL in
tumor development. We believe that overexpression of these enzymes
contributes to the acidic tumor microenvironment and helps the cancer
cells to grow and metastasize. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used 45 mRNA samples isolated from cancer cell lines provided by the Developmental Therapeutics Program, Frederick Cancer Research and Development Center, National Cancer Institute. Additional mRNA samples from central nervous system (CNS) tumor cell lines U105MG, U251MG, U373MG, and G4; leukemia cell lines U937 and HL60; the renal cell carcinoma cell line UM-RC-29; and the monocyte cell line THP-1 were kindly provided by Dr. Teizo Yoshimura (NCI-Frederick, Frederick, MD). Non-small cell carcinoma (H1373, H1264, H1693, H1944, H838, H1299, H157, H1466, H460, H727, and H28), small-cell carcinoma cell lines (H1184, H2081, H2227, H1086, H841, H69, H1820, H660, H1769, H446, H1238, H748, and H2552), cancer cell lines SCC-35, MCF-7-adr, SQ-20B, 510-HPV-18, and Scid (5020) were kindly provided by Dr. Bruce Johnson (NCI, Bethesda, MD). The human glioblastoma cell line, U87, was purchased from ATCC (Manassas, VA). RNA samples from other cancer cell lines were available on the Clontech MTN blot no. 7757-1, and samples from normal tissues on MTE Array no. 7775--1 blot (Clontech, Palo Alto, CA).
Molecular Techniques
Total RNA and mRNA isolation from cell lines and tumor tissues was done using commercially available kits (Invitrogen, Carlsbad, CA; Life Technologies, Inc., Grand Island, New York). Electrophoresis in formaldehyde gels and Northern blot analysis were performed according to published procedures.10,19 Quantification of Northern hybridization signals was done as described previously10 or using a VE-1000 Video Camera System (Dage-MTI, Inc., Michigan City, IN) and NIH Image Version 1.6.1 software.
Tissue Specimens
The normal adult tissues from all organ sites and the corresponding benign and/or malignant neoplastic tissues were obtained from routine pathology specimens at St. Joseph Hospital (Orange, CA). These organs include the brain (cerebrum, cerebellum, ventricle, pons, pituitary gland), eyes, nose, throat, upper and lower respiratory system, heart, upper and lower gastrointestinal system (esophagus, stomach, small and large intestine), pancreas, liver, biliary system including gallbladder, female and male urogenital system (kidney, ureter, bladder, testicle, cervix, uterus, fallopian tube, ovary), adrenal gland, thyroid, parathyroid, salivary gland, spleen, bone, muscle, cartilage, skin, and the body cavity. All tissue samples were processed within 6 hours of surgical resection and fixed in 10% neutral-buffered formalin or snap-frozen. The formalin-fixed tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for light microscopic examination.
Immunohistochemical Studies
The mouse monoclonal antibody (MN75) used to detect the MN/CA IX
protein and the rabbit polyclonal antibody to CA XII protein have been
described previously.20,21
Immunohistochemical staining of
tissue sections with anti-CA IX and anti-CA XII antibodies was done
using a peroxidase technique with microwave pretreatment, as described
previously.22
Known positive and negative tissue specimens
were included in each run. For CA IX immunostaining, the primary
antibody was used at a 1:10,000 dilution and the CA XII at a 1:500
dilution. The immunohistochemical results were semiquantitative based
on the percentage of the positive cells seen in a total field of a
single section. The pattern of staining was scored as diffuse when
40% of the cells stained and focal when <40% of the cells stained.
A negative score was given to tissue sections that had no evidence of
specific immunostaining.
pH Determination, Glucose Uptake, and Hypoxia Conditions in Cell Culture
Renal clear-cell carcinoma cell line 786-0 and its derivative, expressing the wtVHL transgene, were described earlier.10 They were grown to confluence in Dulbecco’s modified Eagle’s medium (cat. no. 11965-084; Life Technologies, Inc.) with 10% bovine fetal serum (Sigma, St. Louis, MO). Three independent experiments on pH and glucose measurements in the media were done using a Hanna Instruments microcomputer pH meter (model HI931000) and Infinity Glucose Reagent (Sigma Diagnostics, cat. no. 18-20). U87 cell culture and maintenance of hypoxia were performed as previously described.19
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using MTE array blots containing 68 different mRNAs from a variety
of normal adult tissues, we observed that the CA12 gene was highly
expressed in kidney, colon and rectum, esophagus, brain, and the
pancreas, whereas the mammary gland, bladder, uterus, trachea, and
aorta showed low levels of expression. In the brain, only the corpus
striatum [caudate nucleus (E2) and putamen (H2)] produced strong
hybridization signals with the CA12 probe (Figure 1)
. Moderate expression of CA9 was
observed in a limited number of tissues that included stomach and, to
an even lesser extent, heart, liver, pancreas, and salivary gland
(Figure 1)
. These data were consistent with hybridization results
obtained with the MTN blots. In addition, low signals were also
observed in brain and placenta (data not shown). We detected no
significant differences in the distribution of CA12 expression in a
variety of fetal tissues compared to normal adult tissues (Figure 1
,
column 11; and data not shown). In the case of CA9, however, fetal lung
and muscle demonstrated high levels of expression whereas adult lung
and muscle were negative (data not shown).
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and Table 2
, and an example of a Northern blot is
given in Figure 2
. Among 87 RNA samples,
representing tumors of the head and neck, lung, kidney, cervix, ovary,
prostate, breast, colon, skin, and several leukemias, 50 (57%)
expressed either one or both genes at variable intensity, whereas
37 were negative (Table 1
and Figure 2
). Non-small cell lung cancer,
colon and renal cell carcinoma cell lines, and tumors of the CNS showed
the highest proportion of specimens expressing CA9 or CA12 (68, 78, 89,
and 78%, respectively). Fourteen of the 18 tumors (78%) of the CNS
and spinal cord showed expression of the genes (Table 2)
. Among these
tumors 11 expressed both genes and three expressed either CA9 or CA12.
It was previously shown and extensively documented in this study that
expression of CA IX protein may serve as a diagnostic biomarker for
clear cell renal cell carcinoma22-24
and colorectal
tumors,25,26
whereas CA XII has been identified as a
biomarker for non-small-cell lung cancer (10; US patent no. 5,589,579).
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High levels of CA IX expression were consistently observed in the basal cells in and near the infundibulum and medulla of the hair follicle, mesothelial cells, and coelomic epithelium of the body cavities. In the visceral organs, high levels of CA IX expression in the epithelium were identified but limited to rete ovarii, rete testis, ductular efferens, bile ducts, pancreatic ducts, and gallbladder. In the gastrointestinal tract, diffuse CA IX immunoreactivity was observed in the gastric mucosa, ductal cells of Brunner’s glands, and crypt cells of the duodenum, jejunum, and, to a lesser degree, in the terminal ileum and appendix. In the peripheral and central nervous systems, CA IX expression was limited to the ventricular lining cells and the choroid plexus. Interestingly, mesodermal cells of the amniotic/chorionic plate of the placenta and cartilaginous tissues from joint spaces also showed variable degrees of CA IX protein expression.
CA XII was variably expressed in mesothelial cells and the coelomic
epithelium of the body cavity. In general, high levels of CA XII
expression were found in certain tissues that were CA IX-negative.
These tissues were the distal convoluted tubules and the intercalated
cells of the collecting duct of the kidney, sweat glands of the skin,
the epithelium of the breast, some of the proliferative endometrial
glands, and seminal vesicles. Low levels of CA XII expression were also
found focally in ductal cells and mucous cells of the salivary glands
and submucosal glands of the upper respiratory system, epithelial cells
of Schneider’s membrane of the nose, and acinar cells of the pancreas.
Limited numbers of positive epithelial cells were also found in the
prostate, vas deferens, and transitional mucosa of the renal pelvis. In
the gastrointestinal tract, CA XII expression was observed, but was
limited to the surface glands of the large intestine only. Very weak
immunoreactivity of CA XII was also found in the gastric glands. In the
peripheral and central nervous system, the CA XII immunoreactivity was
restricted to the posterior lobe of the pituitary glands, remnant of
Rathke’s pouch, the choroid plexus and limited numbers of ganglion
cells in the cortex. In the placenta, limited numbers of
syncytiotrophoblasts were immunoreactive. The only normal tissues
co-expressing CA IX and CA XII were mesothelial cells, ductular
efferens, and, to a lesser degree, the choroid plexus and gastric
glands. A summary of the distribution of expression in normal tissues
is given in Table 3
, and selected normal
tissues with high expression of CA IX and/or CA XII are illustrated in
Figure 3
.
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We surveyed a large number of benign and malignant human tissues
by immunostaining with specific antibodies. The results are summarized
in Table 4
and Figures 3 to 5
. With a few
exceptions, a wide variety of tumors examined showed plasma membrane,
or both plasma membrane and cytoplasmic immunoreactivity for CA IX and
CA XII. The frequency of co-expression, the distribution of staining
(focal or diffuse), and the intensity of staining varied considerably
even within a single tumor type. For example, CA IX protein was
expressed in 99% of cervical carcinomas tested
(n = 77), and 68% of these tumors showed
diffuse and strong staining, whereas CA XII was expressed in only 33%
of cases tested (n = 24) and the positive
staining was focal and weak. When a comparison of the distribution of
CA IX and CA XII expression in normal tissues (Table 3)
and the
corresponding neoplastic tissues (Table 4)
was made, the expression of
CA IX and CA XII could be grouped into two categories as
follows.
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Examples of co-expression of CA IX/CA XII in normal and neoplastic
tissues are mesothelium/mesotheliomas and choroid plexus/choroid plexus
tumors (Figure 3, E– L)
. Expression of CA XII was seen in ductal
breast epithelium and ductal breast carcinoma (Figure 3, C and D)
, and
expression of CA IX was observed in the biliary duct and carcinomas
derived from the epithelium of the biliary tract (data not shown).
Ectopic Expression of CA IX/CA XII
In general, most tumors ectopically expressed these proteins. The
ectopic or enhanced expression may have clinical implications,
especially in areas of tumor diagnosis and tumor immunotherapy, when
applied to specific organ sites. The best examples were the expression
of CA IX/CA XII proteins in gliomas (Figure 4, C–E)
, and CA IX in cervical
intraepithelial neoplasia and carcinomas,22
and in renal
cell carcinomas.23
In all of these tumors CA IX
immunoreactivity was not seen in the corresponding normal or reactive
tissues, whereas diffuse immunoreactivity was present in the tumors.
Association of CA IX/CA XII Expression with Tumor Type and Degree of Differentiation
Within a given organ, in particular breast, kidney, and brain, CA
IX and CA XII expression was closely related to a specific cell type
and a given degree of differentiation. High levels of CA XII expression
were primarily observed in low-grade ductal carcinoma and lobular
carcinoma of the breast, and low-grade glioma of the brain (Figure 3D
and Figure 4C
). In contrast, strong CA IX immunoreactivity was limited
to high-grade glioma, including glioblastoma multiforme (Figure 4, D and E)
and high-grade ductal breast carcinoma with necrosis (Figure 4F)
.
Expression of CA IX and CA XII Are Diagnostic for Renal Cell Carcinomas
CA IX is not expressed in normal adult kidney (Figure 5A)
, whereas CA XII is expressed in the
distal convoluted tubules and the intercalated cells of the collecting
duct (Figure 5B)
. We had originally shown that ectopic CA IX protein
expression is diagnostic for renal cell clear-cell carcinomas (Figure 5C)
.23
We now show that CA XII is also expressed in these
tumors (Figure 5D)
. Furthermore, CA XII is also expressed in
chromophobe cell carcinomas (Figure 5F)
and oncocytic tumors (Figure 5H)
, whereas CA IX is not expressed in either type of tumor (Figure 5, E and G)
. Co-expression of CA IX and CA XII is also seen in papillary
type tumors and collecting duct carcinomas (data not shown). Thus, the
combination of CA IX and CA XII immunostaining essentially diagnoses
all forms of renal cell cancers.
Induced or Enhanced Expression of CA IX and CA XII in Association with Hypoxia
We observed a strong association between induced or enhanced
expression of CA IX and CA XII proteins and tumor necrosis and hypoxia.
High levels of CA IX protein were expressed by those neoplastic cells
located in and adjacent to the necrotic foci, irrespective of whether
the tumor cells in the absence of necrosis were CA IX immunoreactive or
not. A similar phenomenon was also observed in cells expressing CA XII
in areas adjacent to necrosis, but was less predictable compared to CA
IX expression. The most predominant examples were induced expression of
CA IX protein in hypoxic/necrotic regions of melanoma, and enhanced
expression in necrotic/hypoxic regions of meningiomas, glioblastoma
multiforme, anaplastic ependymoma, and comedo type of breast
carcinomas. The latter three tumors are shown in Figure 4; D, E, and F
.
CA IX was predominantly expressed in the areas of necrosis and hypoxia.
The tumor cells that were more distal to the necrotic/hypoxic areas
showed little or no immunoreactivity.
Effect of Hypoxia on CA 9 and CA12 mRNA Expression in a Glioblastoma Cell Line
To directly examine the effect of hypoxia on the expression of CA9
and CA12 mRNA we analyzed an established glioblastoma cell line, U87,
expressing a normal VHL message (M Merrill and M Proescholdt,
unpublished observations). The cells were grown under normoxic
conditions and then maintained under hypoxic conditions for 6 and 12
hours as previously described.19
Northern blot
hybridization (Figure 6)
revealed that 6
hours of hypoxia resulted in approximately fourfold and 1.6-fold
up-regulation of CA9 and CA12 expression, respectively, while 12 hours
of hypoxia induced expression of both genes 
11-fold to 13-fold,
compared to control.
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We next investigated the correlation of CA9 and CA12 mRNA
expression with the dynamics of extracellular pH changes under cell
culture conditions. Acidification of the bicarbonate-buffered media at
the late confluent stage was much more pronounced in 786-0 cells
(lacking wtVHL), as compared to the same cell line expressing wtVHL
transgenes (
pHe, 0.4). Thus, the increased acidification clearly
correlates with the up-regulation of the CA9 and CA12 mRNA expression
(Figure 7)
. Elevated uptake of glucose at
this stage in the 786-0 cells (15% increase compared to the wtVHL
expressing 786-0, data not shown) suggests that altered glucose
uptake and metabolism may be responsible, at least partially, for the
media acidification.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, indicating
tumor-specific activation of these genes. Other investigators, in less
extensive screenings, have reported expression of CA IX or CA XII in
some common tumors.27,28
To our knowledge this is the
first extensive comparative analysis of the combined expression of
these genes and proteins. Cumulatively, the data presented here and
published before by us10,22,23
and
others21,27,28
firmly establish high expression of
CA9/CA12 genes in a large number of common human malignancies,
suggesting an important role for them in tumor development.
Tumor growth involves complex interactions between cells and their
unique microenvironment, that is characterized by low acidic pHe and
altered hydrostatic and oxygen pressures.6,7
Tight control
of pH homeostasis in tumors is achieved by using proton extrusion
mechanisms that include plasma membrane proton pumps,29,30
proton channels/proton wires,31
sodium/proton
exchangers,32
and monocarboxylic acid
transporters.33
Some mechanisms involved in proton
extrusion and maintenance of pH homeostasis in and around tumor cells
are depicted in Figure 8
. The extremely
efficient cell surface CAs34,35
may play an important role
in controlling the levels of protons and bicarbonate in the immediate
vicinity of the tumor cells by sensing pH and tipping the proton
balance across the cell membrane. The hypothetical pH sensing
function(s) of CA enzymes may be associated with histidine residues in
their catalytic center outside the cells as well as with internal
histidines in the cytoplasmic domains. The fact that CA IX and CA XII
proteins are localized normally on differentiated cells specialized in
acid/base homeostasis (ie, intercalated cells and collecting ducts of
the kidney, gastrointestinal gland cells, and so forth), supports the
proposed role for them in maintaining extracellular acidity in tumors.
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that glioblastoma cells expressing a normal
VHL message when maintained for 12 hours under hypoxic conditions
overexpress the CA9/CA12 genes, indicating a role for hypoxia in this
induction. It is established that in hypoxic cells the proteolytic
function of pVHL is abrogated, resulting in the overexpression of the
HIF1- protein and other VHL target genes.8,18
The
transcriptional trans-activator HIF1 controls the expression
of several dozen target genes including those involved in energy
metabolism (glucose transporters, glycolytic enzymes) and angiogenesis
(VEGF and its receptors), and is considered a master regulator gene
that orchestrates physiological responses to acute and chronic
hypoxia.1,12-14
Thus, loss of pVHL function(s) results in
the stabilization of HIF and transactivation of its target genes.
Therefore, presence or absence of the VHL protein seems to be
responsible for fundamental metabolic changes in tumor cells. The
ultimate consequences of these changes in gene expression in the
absence of a functional VHL protein (because of mutation, epigenetic
silencing, or localized hypoxic conditions) are compromised tumor
microenvironment, a powerful, albeit faulty, disorganized process of
angiogenesis, increased glycolysis, and induction of expression of
transmembrane CA. The frequent, widespread overexpression of the
CA9/CA12 genes in human tumors has several important implications in
the clinical setting. Implications for Tumor Growth and Spread
On the basis of the above considerations, it would seem that tumor microenvironmental acidity could play a predominant promoting role in tumor growth and metastasis and also could underlie resistance to radiotherapy, chemotherapy, and other nonsurgical treatments.6,7 The extracellular matrix metalloproteinases, which are activated by acidic pH, are involved in remodeling stromal and tumor cell surface proteins, thereby promoting tumor cell motility, and contributing to tumor growth and metastasis.37 However, synthesis of matrix metalloproteinases may have opposite effects on tumor angiogenesis, promoting extracellular matrix degradation and new blood vessel formation on the one hand37 and, on the other, blocking angiogenesis by producing angiostatin, a cleavage product of plasminogen.2 Most tumors have been shown to exhibit high vascular permeability and high interstitial fluid pressure.6,7 These may result from activation, as a consequence of the acidic tumor microenvironment, of trans-membrane water channel proteins (aquaporins) that are widely distributed in tumors.38 Aquaporins are known to play a major role in trans-cellular and trans-epithelial water movements in many normal tissues.39,40 The expression of CA IX/CA XII in cystic tumors is an indication that their activity in promoting acidity might facilitate the activity of aquaporins expressed on tumor and endothelial cells, leading to high interstitial fluid pressure and high vascular permeability in tumors.6,7
Implications for Diagnosis
The fact that CA IX/CA XII are expressed in many tumor types may
lead one to conclude that they have limited utility in cancer
diagnosis. However, for selected cancers, they may prove to be powerful
diagnostic biomarkers. We have already shown the utility of CA IX as a
biomarker for cervical dysplasia and carcinoma.22,41
Furthermore, we have recently shown its utility in identifying those
patients who receive a Pap smear diagnosis of "atypical glandular
cells of undetermined significance" and harbor a significant lesion
in their cervix.42
We and others have also shown that
expression of CA IX is an excellent diagnostic biomarker for renal cell
carcinoma.23,24,43
The studies described here, in which we
show that CA XII is expressed in chromophobe tumors, whereas CA IX is
not (Figure 5)
, suggests that the combined detection of CA IX and CA
XII expression will identify all renal cell carcinomas. This also has
important implications for therapy.44-47
Other
investigators have identified the expression of CA IX in
esophageal48
and lung carcinomas49
Finally,
our studies here also suggest that CA XII and/or CA IX may have utility
in diagnosis of brain tumors.
Implications for Tumor Treatment
The CA IX/CA XII enzymes ectopically expressed on the surface of cancer cells provide an excellent target for tumor treatment modalities, including development of highly specific humanized antibodies, antibodies armed with toxins,46 and specific inhibitors of these enzymes.50-52 Antibodies to CA IX have also been developed in Europe and are being successfully used to treat renal cancer.43,47 Dr. Neal Bunder is developing humanized antibodies at the Memorial Sloan-Kettering Cancer Center (personal communication). In addition, antibodies to CA XII were recently patented for treatment of lung cancer (US patent no. 5,589,579). On the other hand, highly specific inhibitors of CA IX/CA XII enzymes tailored to their catalytic centers, could be also used to treat cancer and should be developed. Two of us (AW and WS), with others,50 have shown that acetazolamide, a potent inhibitor of CAs, suppressed invasion of renal cancer cells in vitro. It took Professor T. Maren and the Merck Company 20 years to develop such inhibitors for topical treatment of glaucoma.51,52 Establishing the crystal structures of CA IX and CA XII should take less time and will result in designing more effective specific drugs to manipulate tumor pH, and possibly provide a mechanism for inhibiting tumor growth and tumor spread.
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Acknowledgements |
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Footnotes |
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Supported in part by National Cancer Institute, National Institutes of Health, contract no. NO1-CO-56000 and CA19401 from the National Cancer Institute.
S. I. and S.-Y. L contributed equally to this work.
Accepted for publication November 17, 2000.
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 A. M. Niemela, P. Hynninen, J.-P. Mecklin, T. Kuopio, A. Kokko, L. Aaltonen, A.-K. Parkkila, S. Pastorekova, J. Pastorek, A. Waheed, et al. Carbonic Anhydrase IX Is Highly Expressed in Hereditary Nonpolyposis Colorectal Cancer Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1760 - 1766. [Abstract] [Full Text] [PDF]
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 C. C. Wendler, S. Amatya, C. McClaskey, S. Ghatpande, B. B. Fredholm, and S. A. Rivkees A1 adenosine receptors play an essential role in protecting the embryo against hypoxia PNAS, June 5, 2007; 104(23): 9697 - 9702. [Abstract] [Full Text] [PDF]
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 R. J. Scheibe, G. Gros, S. Parkkila, A. Waheed, J. H. Grubb, G. N. Shah, W. S. Sly, and P. Wetzel Expression of Membrane-bound Carbonic Anhydrases IV, IX, and XIV in the Mouse Heart J. Histochem. Cytochem., December 1, 2006; 54(12): 1379 - 1391. [Abstract] [Full Text] [PDF]
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 S. Kaluz, M. Kaluzova, and E. J. Stanbridge Proteasomal Inhibition Attenuates Transcriptional Activity of Hypoxia-Inducible Factor 1 (HIF-1) via Specific Effect on the HIF-1{alpha} C-Terminal Activation Domain Mol. Cell. Biol., August 1, 2006; 26(15): 5895 - 5907. [Abstract] [Full Text] [PDF]
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 P. Schoffski, H. Dumez, P. Clement, A. Hoeben, H. Prenen, P. Wolter, S. Joniau, T. Roskams, and H. Van Poppel Emerging role of tyrosine kinase inhibitors in the treatment of advanced renal cell cancer: a review Ann. Onc., August 1, 2006; 17(8): 1185 - 1196. [Abstract] [Full Text] [PDF]
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 S. Grosch, T. J. Maier, S. Schiffmann, and G. Geisslinger Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst, June 7, 2006; 98(11): 736 - 747. [Abstract] [Full Text] [PDF]
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F. Dayan, D. Roux, M. C. Brahimi-Horn, J. Pouyssegur, and N. M. Mazure The Oxygen Sensor Factor-Inhibiting Hypoxia-Inducible Factor-1 Controls Expression of Distinct Genes through the Bifunctional Transcriptional Character of Hypoxia-Inducible Factor-1{alpha}. Cancer Res., April 1, 2006; 66(7): 3688 - 3698. [Abstract] [Full Text] [PDF]
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 P. Pan, M. Leppilampi, S. Pastorekova, J. Pastorek, A. Waheed, W. S. Sly, and S. Parkkila Carbonic anhydrase gene expression in CA II-deficient (Car2-/-) and CA IX-deficient (Car9-/-) mice J. Physiol., March 1, 2006; 571(2): 319 - 327. [Abstract] [Full Text] [PDF]
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 R. Cairns, I. Papandreou, and N. Denko Overcoming Physiologic Barriers to Cancer Treatment by Molecularly Targeting the Tumor Microenvironment Mol. Cancer Res., February 1, 2006; 4(2): 61 - 70. [Abstract] [Full Text] [PDF]
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J. A. Haapasalo, K. M. Nordfors, M. Hilvo, I. J. Rantala, Y. Soini, A.-K. Parkkila, S. Pastorekova, J. Pastorek, S. M. Parkkila, and H. K. Haapasalo Expression of Carbonic Anhydrase IX in Astrocytic Tumors Predicts Poor Prognosis Clin. Cancer Res., January 15, 2006; 12(2): 473 - 477. [Abstract] [Full Text] [PDF]
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A. Mayer, M. Hockel, and P. Vaupel Carbonic Anhydrase IX Expression and Tumor Oxygenation Status Do Not Correlate at the Microregional Level in Locally Advanced Cancers of the Uterine Cervix Clin. Cancer Res., October 15, 2005; 11(20): 7220 - 7225. [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 Oncology, October 1, 2005; 7(4): 465 - 475. [Abstract] [PDF]
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 A Ivanova, S-Y Liao, M I Lerman, S Ivanov, and E J Stanbridge STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker J. Med. Genet., July 1, 2005; 42(7): 565 - 576. [Abstract] [Full Text] [PDF]
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M. C. Panelli, E. Wang, and F. M. Marincola The Pathway to Biomarker Discovery: Carbonic Anhydrase IX and the Prediction of Immune Responsiveness Clin. Cancer Res., May 15, 2005; 11(10): 3601 - 3603. [Full Text] [PDF]
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M. Atkins, M. Regan, D. McDermott, J. Mier, E. Stanbridge, A. Youmans, P. Febbo, M. Upton, M. Lechpammer, and S. Signoretti Carbonic Anhydrase IX Expression Predicts Outcome of Interleukin 2 Therapy for Renal Cancer Clin. Cancer Res., May 15, 2005; 11(10): 3714 - 3721. [Abstract] [Full Text] [PDF]
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I. J. Hoogsteen, H. A.M. Marres, K. I.E.M. Wijffels, P. F.J.W. Rijken, J. P.W. Peters, F. J.A. van den Hoogen, E. Oosterwijk, A. J. van der Kogel, and J. H.A.M. Kaanders Colocalization of Carbonic Anhydrase 9 Expression and Cell Proliferation in Human Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., January 1, 2005; 11(1): 97 - 106. [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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A. Bhattacharya, K. Toth, R. Mazurchuk, J. A. Spernyak, H. K. Slocum, L. Pendyala, R. Azrak, S. Cao, F. A. Durrani, and Y. M. Rustum Lack of Microvessels in Well-Differentiated Regions of Human Head and Neck Squamous Cell Carcinoma A253 Associated with Functional Magnetic Resonance Imaging Detectable Hypoxia, Limited Drug Delivery, and Resistance to Irinotecan Therapy Clin. Cancer Res., December 1, 2004; 10(23): 8005 - 8017. [Abstract] [Full Text] [PDF]
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M. Hilvo, M. Rafajova, S. Pastorekova, J. Pastorek, and S. Parkkila Expression of Carbonic Anhydrase IX in Mouse Tissues J. Histochem. Cytochem., October 1, 2004; 52(10): 1313 - 1322. [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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J. S. Lam, A. S. Belldegrun, and R. A. Figlin Tissue Array-Based Predictions of Pathobiology, Prognosis, and Response to Treatment for Renal Cell Carcinoma Therapy Clin. Cancer Res., September 15, 2004; 10(18): 6304S - 6309S. [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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D. Hedley, M. Pintilie, J. Woo, A. Morrison, D. Birle, A. Fyles, M. Milosevic, and R. Hill Carbonic Anhydrase IX Expression, Hypoxia, and Prognosis in Patients with Uterine Cervical Carcinomas Clin. Cancer Res., November 15, 2003; 9(15): 5666 - 5674. [Abstract] [Full Text] [PDF]
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 P. Maxwell HIF-1: An Oxygen Response System with Special Relevance to the Kidney J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2712 - 2722. [Full Text] [PDF]
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A. J. Pantuck, G. Zeng, A. S. Belldegrun, and R. A. Figlin Pathobiology, Prognosis, and Targeted Therapy for Renal Cell Carcinoma: Exploiting the Hypoxia-Induced Pathway Clin. Cancer Res., October 15, 2003; 9(13): 4641 - 4652. [Abstract] [Full Text] [PDF]
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A. Zisman, A. J. Pantuck, M. H. T. Bui, J. W. Said, R. R. Caliliw, N. Rao, P. Shintaku, F. Berger, S. S. Gambhir, and A. S. Belldegrun LABAZ1: A Metastatic Tumor Model for Renal Cell Carcinoma Expressing the Carbonic Anhydrase Type 9 Tumor Antigen Cancer Res., August 15, 2003; 63(16): 4952 - 4959. [Abstract] [Full Text] [PDF]
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J. M. Hernandez, M. H. T. Bui, K.-r. Han, H. Mukouyama, D. G. Freitas, D. Nguyen, R. Caliliw, P. I. Shintaku, S. H. Paik, C.-L. Tso, et al. Novel Kidney Cancer Immunotherapy Based on the Granulocyte- Macrophage Colony-stimulating Factor and Carbonic Anhydrase IX Fusion Gene Clin. Cancer Res., May 1, 2003; 9(5): 1906 - 1916. [Abstract] [Full Text] [PDF]
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S-Y Liao, S Ivanov, A Ivanova, S Ghosh, M A Cote, K Keefe, M Coca-Prados, E J Stanbridge, and M I Lerman Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma J. Med. Genet., April 1, 2003; 40(4): 257 - 261. [Abstract] [Full Text] [PDF]
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S. Kaluz, M. Kaluzova, and E. J. Stanbridge Expression of the Hypoxia Marker Carbonic Anhydrase IX Is Critically Dependent on SP1 Activity. Identification of a Novel Type of Hypoxia-responsive Enhancer Cancer Res., March 1, 2003; 63(5): 917 - 922. [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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 D. Bodmer, W. van den Hurk, J. J. M. van Groningen, M. J. Eleveld, G. J. M. Martens, M. A. J. Weterman, and A. Geurts van Kessel Understanding familial and non-familial renal cell cancer Hum. Mol. Genet., October 1, 2002; 11(20): 2489 - 2498. [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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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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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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S. Gruvberger, M. Ringner, Y. Chen, S. Panavally, L. H. Saal, A. Borg, M. Ferno, C. Peterson, and P. S. Meltzer Estrogen Receptor Status in Breast Cancer Is Associated with Remarkably Distinct Gene Expression Patterns Cancer Res., August 1, 2001; 61(16): 5979 - 5984. [Abstract] [Full Text] [PDF]
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A. V. Ivanova, S. V. Ivanov, A. Danilkovitch-Miagkova, and M. I. Lerman Regulation of STRA13 by the von Hippel-Lindau Tumor Suppressor Protein, Hypoxia, and the UBC9/Ubiquitin Proteasome Degradation Pathway J. Biol. Chem., April 27, 2001; 276(18): 15306 - 15315. [Abstract] [Full Text] [PDF]
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