J Cancer Res Clin Oncol (2006) 132: 302308

DOI 10.1007/s00432-005-0068-2

O R I GI N A L P A P E R

Joo-Young Kim Hye-Jin Shin Tae-Hyun Kim

Kwan-Ho Cho Kyung-Hwan Shin Bu-Kyoung Kim
Ju-Won Roh Sun Lee Sang-Yoon Park
You-Jin Hwang Inn-Oc Han

Tumor-associated carbonic anhydrases are linked to metastases

in primary cervical cancer
Received: 12 October 2005 / Accepted: 30 November 2005 / Published online: 14 January 2006

Springer-Verlag 2006

Abstract Purpose: Carbonic anhydrase IX (CA9) has

emerged as an important surrogate marker for hypoxia
in solid tumors. CA12 shares a role with CA9 in acidication of micromilieu but it is less strictly regulated by
hypoxia than CA9. In this study, we investigated
expression of CA9 and CA12 mRNA in primary cervical
cancer. We also examined whether CA9 expression can
be an indicator of reoxygenation of tumor by measuring
its mRNA expression during fractionated radiotherapy.
Methods: Tumor tissues were obtained from 59 patients
with uterine cervical cancer who underwent radiotherapy, and a second biopsy was taken after patients had
received either 10 or 20 Gy of radiation. The follow-up
period ranged from 2.4 to 75 months (median=23 months). The ratio of CA9 and b-actin mRNA
expression was determined both pre- and during radiation treatment by RT-PCR. Results: CA9 and CA12
mRNA expression was detected in 62.7 and 88.1% of
tumors (i.e. patients), respectively, and co-expression
was observed in 61% of patients. Multivariate analysis
revealed that CA9 expression was the most signicant
factor associated with metastasis-free survival
(P=0.008, hazard ratio 34.8), whereas CA12 mRNA
expression was linked to a lower risk of metastasis

J.-Y. Kim and H.-J. Shin contributed equally to this work.

J.-Y. Kim H.-J. Shin T.-H. Kim K.-H. Cho K.-H. Shin
B.-K. Kim J.-W. Roh S. Lee S.-Y. Park I.-O. Han (&)
Research Institute, National Cancer Center,
Gyeonggi, Goyang, Korea
E-mail: iohan@inha.ac.kr
Tel.: +82-31-9201724
Fax: +82-31-9200149
Y.-J. Hwang
Division of Biological Science, Gachon Medical School,
Inchon, Korea
I.-O. Han
Department of Physiology and Biophysics, Inha University,
College of Medicine, Incheon, Korea

(P=0.007, hazard ratio of 0.07). Tumor CA9 expression

was not altered following either 10 or 20 Gy of radiotherapy. Conclusion: The strong correlation between
CA9 expression and metastasis suggests that CA9
expression might be an important indicator for identifying patients who require more aggressive systemic
therapy.
Keywords Hypoxia Carbonic anhydrase IX (CA9)
XII (CA12) Metastasis Radiotherapy

Introduction
The presence of hypoxic conditions within solid tumors
is one of the most important factors aecting treatment
outcome. As hypoxia-regulated molecular pathways
have been unveiled in recent years, targeting hypoxia is
increasingly considered a sound and important strategy
in treating cancer. Various experimental and clinical
studies have shown that tumor cells under hypoxic
conditions are resistant to radiation-induced cell killing
compared to cells in well-oxygenated conditions. Early
animal experiments showed that as the reoxygenation
process occurs, the hypoxic proportion of a tumor can
remain constant or even fall after radiation (Hall 2000;
Van Putten and Kallman 1968). In clinical radiotherapy
practice, it has been postulated that many types of tumors can be eradicated by step-wise killing of the
reoxygenated proportion of a tumor using a fractionated
radiotherapy regimen over 6 or more weeks. However,
reoxygenation has not been veried to occur in clinical
tumor samples.
Following sporadic eorts to nd useful hypoxia
markers, a recent study has identied 12 hypoxia-overexpressed genes (HOG) using serial analysis of gene
expression in response to hypoxic stimuli (Lal et al.
2001). Among these genes, the isoenzymes of carbonic
anhydrase (CA) families, CA9 and CA12, are shown as
being induced under hypoxic conditions in many cancer

303

cell lines. CA9 protein is expressed in the perinecrotic

region of solid tumors and co-localizes with pimonidazole staining, a well-known hypoxia indicator (Vordermark and Brown 2003). In addition, CA9 expression
is more specically and rapidly regulated by oxygen
concentration compared to other HOG such as glucose
transporter-1 (GLUT-1) and -3 and vascular endothelial
growth factor (VEGF), the expression of which is
heavily regulated by a number of other signaling pathways (Harris 2002; Lal et al. 2001; Semenza 2003). Although both CA9 and CA12 are downregulated by
VHL, the transcription of which are considered to be
under control of HIF-1a, the expression and the tissue
distribution of CA12 gene are not associated with the
expression of CA9 (Ivanov et al. 1998). Unlike to CA9,
which is usually found in tumor tissues, CA12 is distributed in a variety of normal tissues and its expression
becomes stronger in tumors originating from the same
tissues (Ivanov et al. 2001).
Identifying CA9 expression might be particularly
meaningful in cervical cancer due to the possible link
between CA9 induction and human papilloma virus
(HPV) infection. CA9 was cloned from HeLa cells in
1994 and has been known to be the only tumor-associated member of the CA family, which catalyzes the
carbon dioxide to carbonic acid conversion (Opavsky
et al. 1996). CA9 is subsequently shown to be involved in
cellcell and cellmatrix interactions that aect loss of
contact inhibition and anchorage-independent cell
growth in MN/CA9 gene-transfected C33A and SiHa
cervical cancer cell lines (Lieskovska et al. 1999). In
addition, CA9 is considered as a biomarker of HPV
infection, along with P16 and cyclin E (Keating et al.
2001; Resnick et al. 1996), and these molecules can be
used as novel indicators for screening and diagnosis of
HPV infection.
In the present study, we examined whether expression
of CA9 and CA12 were prognostic indicators for patients with uterine cervical cancer. As a potential measure of reoxygenation, we determined the kinetics of
CA9 expression in individual tumors during fractionated
radiotherapy. The results in this paper demonstrated
that CA9 expression is linked to distant metastasis in
patients with uterine cervical cancer. The data further
suggest that patients with high CA9 expression potentially have a considerable hypoxic tumor burden and are
prone to fail at distant sites and hence should be
administered more aggressive systemic treatments.

Methods and materials

Patients and clinical specimen
Pre-treatment cervical cancer tissues were obtained from
the 59 patients who underwent radiotherapy as their
main treatment modality. Our study was approved by
the Institutional Review Board on the experimental
studies and informed consent was obtained from each

patient or a responsible relative accordingly. All patients

had newly diagnosed, histologically proven squamous
cell carcinoma or adenocarcinoma of the cervix. In these
patients, 12 and 32 patients undertook an additional
biopsy at a cumulative radiation dose of 10 and 20 Gy,
respectively. We used 34 mm width tumor tissue and
observed the frozen cut of each tissue under microscope
before RNA is extracted to minimize the possibility of
normal tissue contamination. This second biopsy was
obtained to detect any change in hypoxia-related genes
during fractionated radiotherapy. Multiple biopsies were
taken avoiding the grossly necrotic portion and the tumor tissues thus obtained were xed in the 4% neutral
formalin and were snap-frozen in liquid nitrogen.
Chemical coagulation was avoided as much as possible
for later biopsy procedures. All tumor sections were H-E
stained and were subsequently examined for the presence of tumor cells by a pathologist.
RNA preparation
About 34 mm length of the tumor tissues were taken
from the biopsy specimens and were kept at 80C in
Trizol 500 ll until it was further processed. For RNA
preparation, samples were chopped and minced, followed by addition of 200 ll of chloroform. The samples
were incubated in ice for 5 min and then centrifuged to
separate the aqueous phase. The upper phase was carefully removed and RNA was precipitated by adding 1 ml
of isopropanol followed by centrifugation. The RNA
pellet thus obtained was rinsed with 70% alcohol in
DEPC water. Finally, the pellet was air-dried and was
then solubilized with RNase-free water. RNA concentration was measured by spectrophotometry with optical
density read at 260 nm.
RT-PCR of the CA9, CA12, and VEGF genes
Total RNA (2 lg) was reverse transcribed for 1 h at
37C in a reaction mixture containing 5 U RNase
(Amersham, Piscataway, NJ, USA), 0.5 mM dNTP
(Boeringer Mannheim, Indianapolis, IN, USA), 2 lM
random hexamer (Stratagene, La Jolla, CA, USA), RT
buer, and 5 U reverse transcriptase (Quiagen). The
rst-strand cDNA synthesis was performed with the
oligo (dT) primed rst-strand synthesis in a total volume
of 10 ll with a reverse transcriptase. The product of the
RT-PCR 1 ll was mixed with 10 buer (MgCl2), 3 ll
of 2.5 mM dNTP, 1 ll of 10 pmol forward and reverse
primer, 1 ll of Taq polymerase, and nally RNase and
DNase-free water 20 ll was mixed to make a total volume of 30 ll. The reaction mixture was subjected to
PCR at 94C 5 min, 94C 30 s, 62C 30 s (57C for CA
12 and 55C for VEGF), 72C 30 s for 30 cycles, and
72C 7 min. The primers were CA9, forward, 5-TAAGCAGCTCCACACCCTCT-3, and reverse, 5TCTCATCTGCACAAGGAACG-3; CA12, forward,

304

5-ATGGCAGGTTCAAGTTCCAC-3, and reverse, 5TCGGAACTCATGTCTCCTCC-3; VEGF, forward,

5-GTGGACATCTTCCAGGAGTA-3 and reverse, 5ATCTGCAAGTACGTTCGTTT-3) and GAPDH,
forward, 5-ATGTACGTAGCCATCCATCCAGGC3, reverse, 5-AGGAAGGAAGGCTGGAAGAG-3.
Analysis of the resulting PCR products on 1% agarose
gels showed single-band amplication products with
expected sizes.

general strategy for model selection involving the hierarchic principle with the likelihood ratio chi-square test.
The correlations of the expression of CA9, CA12, and
VEGF were made by Spearmann rank correlation test.
Pre-treatment and post-treatment CA9 expression was
compared by paired T-test. All statistical analyses were
performed with SAS programs (version 8.01, SAS
Institute Inc., Cary, NC, USA).

Results
Real-time PCR of CA9 from the serially obtained tumor
tissues
CA9 DNA amplication was carried out using CA9
cDNA template obtained from the mRNA produced
from the RT-PCR procedure. Real-time PCR reactions
were set up in a reaction volume of 15 ll using the
TaqMan Universal PCR Master Mix (PE Applied Biosystems, CA, USA). A reporter dye FAM (6-carboxyuorescein) was covalently attached to the 5 end and a
quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine) was incorporated into the 3 end of TaqMan
probe with sequence of TGAACTTCCGA GCGACGCAGCC for CA9. PCR primers of CA9 was 5-ACCTGGTGACTCTC GGCTACAG-3as a forward
primer and 5-TTTGAATGG GCGAGTGATTG-3as a
reverse primer. As an endogenous control, real-time
PCR analysis was performed on the b-actin gene in the
same cDNA samples for relative gene expression quantication. The b-actin gene primers and probe labeled
with VICTM dye and MGB/non-uorescent quencher
were provided by Applied Biosystems. DNA amplication was done in a 96-well reaction plate format in an
ABI PRISM 7900HT Sequence Detection system (PE
Applied Biosystems). Both CA9 and b-actin PCR reactions were carried out in triplicate. Multiple negative
water blanks were included in every analysis. Relative
standard curves were established from the control DNA
extracted from HeLa cells (1 lg cDNA/ll). The control
cDNA was provided as 1 lg/ul and was diluted from
1,000 to 0.01 ng/ll. Two-hundred ng/ll of sample
cDNA was loaded and the reactions were run on ABI
PRISM 7900HT Sequence Detection system programmed to 5-min initial step at 95C followed by 40
cycles of 15 s at 95C and 60 s at 60C. The mean Ct
values and quantities in log linear range were used for
quantication of CA9 gene and control gene.
Statistical method
Overall survival, recurrence-free survival, and metastasis-free survival were analyzed by KaplanMeier method. Univariate analysis was made on the relationship of
CA9, CA12, VEGF, and other individual variables with
respect to the above survival parameters using the logrank test. Multivariate analysis for the same variables
was also undertaken with the Cox model using the

Association between CA9, CA12, and VEGF gene

expression and clinical outcome
The 5-year overall survival, local recurrence-free survival, and metastasis-free survival rates for all patients
were 73.4, 69, and 82%, respectively. Distant metastasis
developed in 14 of 59 patients (23.7%). Using RT-PCR,
biopsy tissues from individual tumors were measured for
CA9, CA12 and VEGF mRNA expression, and were
analyzed in terms of clinical outcomes. CA9 and CA12
mRNA was detected in 63% (37/59) and 88% (52/59) of
tumors (i.e. patients), respectively, and VEGF expression was detected in 58% (54/59) of tumors. CA9
expression was closely correlated to that of CA12 and
VEGF (r=0.75, P<0.001). Analysis of clinical outcomes showed that overall survival and local recurrence
were not linked to mRNA expression of any of the three
genes. Univariate analysis showed tumor size and clinical stage were signicant factors related to overall survival of patients (Table. 1, 2, 3), while multivariate
analysis showed tumor size was the only signicant
factor inuencing local control of the disease (P=0.008,
hazard ratio 3). In addition, multivariate analysis
showed that CA9 mRNA expression was the single most
important factor associated with the occurrence of distant metastasis (P=0.008, hazard ratio of 34.8; Fig. 1a).
In remarkable contrast, CA12 mRNA expression was
associated with less likelihood of metastasis (P=0.007,
hazard ratio of 0.07; Figs. 1b). VEGF mRNA expression was not related to any clinical end-point.
The eect of radiotherapy on CA9 gene expression
Using RT-PCR and real-time quantitative PCR, CA9
mRNA expression in tumor samples was measured
quantitatively both prior to radiotherapy and after
receiving either 10 or 20 Gy radiation (two separate
patient groups). We found that after receiving 10 Gy
treatment there was no dierence in CA9 mRNA levels
in biopsy samples compared to levels in tissue taken
prior to treatment. Although there was considerable
decrease in tumor CA9 mRNA expression following
20 Gy irradiation, this change was similar to that observed in expression of the comparison control gene bactin. The mean ratios of CA9 to b-actin mRNA
expression were 1.18 at pre-treatment and 1.03 at the

305
Table 1 Patients characteristics (N=59)
Characteristics

Number (%)

Age
60
>60
Tumor size
4 cm
<4 cm
Stage
I
IIA
IIB
III
IV
CA IX

23 (39)
36 (61)
30 (49)
29 (51)
6 (10)
16 (27)
25 (42)
6 (10)
6 (10)
22 (37)
37 (63)

+
CAXII

7 (12)
52 (88)

+
VEGF

5 (8.3)
54 (90)

+
CA IX and VEGF
++
Others

33 (56)
26 (34)

completion of 20 Gy fractionated radiation therapy

(P=0.22). The corresponding values for 10 Gy were 1.1
and 1.0, respectively (Fig. 2).

Discussion
The aims of this study were to investigate whether
expression of CA9 and/or CA12 in tumors was a prognostic indicator in primary cervical cancer, and whether

Table 2 Univariate analysis of

clinical outcome with variables

Factors

OS
5-year OS

Age
60
>60
Tumor size
>4 cm
4 cm
Stage
I
IIA
IIB
III
IV
CA IX
+
CA XII

OS overall survival, RFS recurrence-free survival, MFS metastasis-free survival

+
VEGF
+

CA9 could be used as a molecular marker for tissue

reoxygenation. The study undertook a retrospective
analysis of 59 patients with cervical carcinoma and
analyzed CA9, CA12, and VEGF mRNA expression by
RT-PCR in both pre- and post-irradiated tumor biopsy
tissues. Expression of VEGF, a powerful mitogen for
endothelial cell proliferation, is induced by hypoxia and
was used as a comparison. Among a variety of parameters examined, we found that CA9 was the most signicant factor predicting development of distant
metastases. Studies on the clinical implication of CA9
expression in various tumor tissues show a variety of
relationships with dierent clinical end-points (Vordermark and Brown 2003). A study similar to the present
one examined CA9 expression in uterine cervical cancer
and reported results consistent with those reported here
in that positive CA9 immunohistochemical staining is
strongly associated with disease-free and metastasis-free
survival (Loncaster et al. 2001).
The reasons why CA9 expression aects uterine cervical cancer metastasis are currently unknown. Olive et al.
(2001) showed a direct relationship between CA9
expression and the hypoxic status of cells using in vitro
and in vivo experimental system, and that cells with high
CA9 expression are radioresistant. If the high CA9
expression mediates the radioresistance of tumor cells by
a certain mechanism, it might be expected to predict local
recurrence. However, our results suggest that CA9
expression is strongly related to the enhanced metastatic
potential of hypoxic tumors and not to the local control of
these tumors by radiotherapy. It is highly likely that CA9
expression may have an important function in modulating tumor invasion and metastasis. The role of CA9 in
acidifying extracellular matrices can be considered as a
reason why CA9-expressing tumor cells are more meta-

RFS
P value

5-year RFS

MFS
P value

5-year MFS

P value

63
79

0.25

64
70

0.40

76
75

0.76

83
62

0.04

57
80

0.02

60
86

0.15

100
61
84
25
0

0.02

100
82
60
44
0

0.003

100
58
88
83
0

0.03

74
72

0.83

66
70

0.98

90
65

0.07

69
73

0.82

100
63

0.13

57
78

0.20

74
70

0.99

66
71

0.89

82
67

0.40

306
Table 3 Multivariate analysis of clinical outcome with various parameters
Factors

Age ( 60 vs. >60)

Tumor size ( 4 cm vs. >4 cm)
Stage (IIV)
CA IX ( vs. +)
CA XII ( vs. +)
VEGF ( vs. +)

Overall survival

Recurrence-free survival

Metastasis-free survival

P value

Hazard ratio

P value

Hazard ratio

P value

Hazard ratio

0.54
0.18
0.29
0.98
0.47
0.44

0.70
2.45
1.40
1.52
2.84
0.56

0.10
0.08
0.11
0.74
0.99
0.49

0.10
3.14
1.64
0.78
1.00
0.60

0.85
0.25
0.46
0.008
0.007
0.28

0.89
2.31
1.3
34.80
0.07
0.41

static; a low pH microenvironment assists in extracellular

matrix degradation, which increases the likelihood of
blood vessel invasion. Identication of the exact mechanisms underlying metastases associated with CA9-overexpressing cervical cancers awaits further studies.
Intriguingly, the present study showed that CA12 and
CA9 expression had very dierent associations with
metastasis. It has been reported that CA12 expression is
linked to more dierentiated, less invasive status of the
tumor cells of the human breast cancer tissues (Wyko
et al. 2001). Besides, CA12 expression became stronger
with increasing age of mouse embryos, indicating
developmental regulation of this protein (Halmi et al.
2004). Usually, CA12 is distributed in a variety of normal tissues and its expression becomes stronger in tumors originating from those same tissues (Ivanov et al.
2001). It is likely that CA12 has an unknown role in
tumor progression and dierentiation in its own right,
which is not related to hypoxia-associated pathways.
Despite the above evidences, which support our ndings
on the inverse correlation of CA12 expression and tumor
metastasis, our data need to be interpreted with caution
when considering the relatively small number of patients
with CA12 negative tumors in the present study.
Another important aim of this study was to dene the
CA9 mRNA levels within the tumor during radiotherapy. We have observed that CA9 and CA12 mRNA

began to be expressed once oxygen concentrations fell

below 5% O2 in the cell culture system (data not shown).
Five percent oxygen level is often found in normal tissues with low oxygen pressure as well as in intermediately hypoxic tumor tissues. Hence, CA9 mRNA
expression can represent a broad range of hypoxia.
Radiobiological modeling shows that the presence of
cells exposed to the intermediate hypoxia between 0.5
and 20 mmHg O2 can be very important in low dose
(1.82 Gy) fractionated radiotherapy as opposed to a
large single dose of radiation (Wouters and Brown
1997). By using RT-PCR rather than immunohistochemistry for CA expression, we expected to measure its
overall level, rather than the hypoxic subvolume within
the tumors. It has been hypothesized that the hypoxic
fraction of tumors remains constant, or even decreases,
during fractionated radiotherapy, with each radiotherapy fraction killing only the well-oxygenized proportion
of tumor cells (Hall 2000; Van Putten and Kallman
1968). The present study shows that while CA9 expression decreased signicantly during radiotherapy, this
reduction was the same as that for the b-actin control
gene. The reduction in CA9 expression appeared to be
mostly due to the reduced tumor cell burden following
radiation. Comparison between the expression levels in
biopsies taken at two dierent times is not likely to
represent the whole tumor due to inherent limitation

1.0

CA9 (+) 5 Yr 67% (n=21)

0.6

1.0

CA12 (+) 5 Yr 83% (n=52)

0.8

Metastasis-Free Survival

Metastasis-Free Survival

CA9 (-) 5 Yr 82% (n=37)

0.4

0.2

0.8
0.6

CA12 (-) 5 Yr 57% (n=7)

0.4
0.2

0.0

0.0
1

Years
Fig. 1 CA9 and CA12 mRNA expression was examined by RTPCR in pre-treatment tumor specimens of 59 patients and the result
was analyzed with respect to the survival parameters. A Metastasisfree survival of the patients with positive CA9 expression (5-year

Years
rate 67%, n=37) versus negative CA9 expression (5-year rate 82%,
n=21). B Metastasis-free survival of the patients with positive
CA12 expression (5-year rate 83%, n=52) versus negative CA12
expression (5-year rate 57%, n=7)

307
1.20

n=32(p=0.38)
1.15

CA9/ -actin mRNA

Fig. 2 Change in the expression

of CA9 mRNA. A second
biopsy was obtained at 10 or
20 Gy of cumulative radiation
dose in 12 and 32 patients,
respectively, and their CA9
mRNA expression was
examined by RT-PCR. Open
box pre-treatment, shaded box
CA9 mRNA at 10 Gy (Lt) and
20 Gy (Rt), respectively. The
bottom and the top of each box
represent 25 percentile and 75
percentile of the observed CA9/
b-actin mRNA ratio. The gure
and the marks in each box
represent the median value of
each group

1.10

1.05

n=12(p=0.79)

1.18
1.03

1.01

1.00

1.00

0.95

0.90
10Gy
pre-treatment

using human tissue samples. An examination of a

comprehensive set of HOGs might be required to explore the changes in hypoxia levels in human tumors.
Furthermore, analysis of heavily irradiated human tumor specimens can be aected by the number of residual
tumor cells as well as the changes in structure and
integrity of subcellular macromolecules after irradiation.
In our experiments, many biopsies collected following
20 Gy treatment had to be excluded from analysis as
they did not contain enough tumor cells or did not allow
for the extraction of enough RNA of good quality.
In conclusion, we examined the expression of CA9 and
CA12 in human cervical tumors. Measuring CAs mRNA
expressions using RT-PCR was sensitive, reproducible
and simple, and yielded signicant prognostic information. CA9 expression was strongly associated with the
development of distant metastases, while CA12 expression was associated with less such metastases. A larger
study would probably nd that CA9 expression is linked
to overall survival in uterine cervical cancer patients and
may also provide further information regarding the
interesting observation that CA12 expression appears to
be associated with less metastasis in these patients. We
propose that CA9 expression be used to identify patients
who require more intensive systemic therapies due to
their increased likelihood of failure at distant sites.
Acknowledgements This work was supported by the National
Cancer Center Grant 0310150.

References
Hall EJ (2000) Oxygen eect and reoxygenation. In: Radiobiology
for radiologists. Lippincott Williams and Wilkins, New York,
pp 103106
Halmi P, Lehtonen J, Waheed A, Sly WS, Parkkila S (2004)
Expression of hypoxia-inducible, membrane-bound carbonic

20Gy

(Radiation Dose)

during-treatment

anhydrase isozyme XII in mouse tissues. Anat Rec 277A:171

177
Harris AL (2002) Hypoxiaa key regulatory factor in tumour
growth. Nat Rev Cancer 2:3847
Ivanov SV, Kuzmin I, Wei MH, Pack S, Geil L, Johnson BE,
Stanbridge EJ, Lerman MI (1998) Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines
by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci
USA 95:1259612601
Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, Merrill MJ, Proescholdt MA, Oldeld EH,
Lee J, Zavada J, Waheed A, Sly W, Lerman MI, Stanbridge EJ
(2001) Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol
158:905919
Keating JT, Ince T, Crum CP (2001) Surrogate biomarkers of HPV
infection in cervical neoplasia screening and diagnosis. Adv
Anat Pathol 8:8392
Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW, Strausberg RL, Kaanders JH, van der Kogel AJ, Riggins GJ (2001)
Transcriptional response to hypoxia in human tumors. J Natl
Cancer Inst 93:13371343
Lieskovska J, Opavsky R, Zacikova L, Glasova M, Pastorek J,
Pastorekova S (1999) Study of in vitro conditions modulating
expression of MN/CA IX protein in human cell lines derived
from cervical carcinoma. Neoplasma 46:1724
Loncaster JA, Harris AL, Davidson SE, Logue JP, Hunter RD,
Wyco CC, Pastorek J, Ratclie PJ, Stratford IJ, West CM
(2001) 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 61:63946399
Olive PL, Aquino-Parsons C, MacPhail SH, Liao SY, Raleigh JA,
Lerman MI, Stanbridge EJ (2001) Carbonic anhydrase 9 as an
endogenous marker for hypoxic cells in cervical cancer. Cancer
Res 61:89248929
Opavsky R, Pastorekova S, Zelnik V, Gibadulinova A, Stanbridge
EJ, Zavada J, Kettmann R, Pastorek J (1996) Human MN/CA9
gene, a novel member of the carbonic anhydrase family:
structure and exon to protein domain relationships. Genomics
33:480487
Resnick M, Lester S, Tate JE, Sheets EE, Sparks C, Crum CP
(1996) Viral and histopathologic correlates of MN and MIB-1
expression in cervical intraepithelial neoplasia. Hum Pathol
27:234239

308
Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev
Cancer 3:721732
Van Putten LM, Kallman RF (1968) Oxygenation status of a
transplantable tumor during fractionated radiation therapy.
J Natl Cancer Inst 40:441451
Vordermark D, Brown JM (2003) Endogenous markers of tumor
hypoxia predictors of clinical radiation resistance? Strahlenther
Onkol 179:801811
Wouters BG, Brown JM (1997) Cells at intermediate oxygen levels
can be more important than the hypoxic fraction in determining tumor response to fractionated radiotherapy. Radiat
Res 147:541550

Wyko CC, Beasley N, Watson PH, Campo L, Chia SK, English

R, Pastorek J, Sly WS, Ratclie P, Harris AL (2001) Expression
of the hypoxia-inducible and tumor-associated carbonic anhydrases in ductal carcinoma in situ of the breast. Am J Pathol
158:10111019