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Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 12596–12601, October 1998
Medical Sciences
Down-regulation of transmembrane carbonic anhydrases
in renal cell carcinoma cell lines by wild-type
von Hippel-Lindau transgenes
(gene targets
y
differential display
y
carcinogenesis)
SERGEY V. IVANOV*†,IGOR KUZMIN*, MING-HUI WEI*, SVETLANA PACK‡,LAURA GEIL*, BRUCE E. JOHNSON§,
ERIC J. STANBRIDGE¶,AND MICHAEL I. LERMAN
i
*Intramural Research Support Program, Science Applications International Corporation Frederick, Laboratory of Immunobiology, and iLaboratory of
Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702; ‡Laboratory of Pathology, National
Cancer Institute, National Institutes of Health, Bethesda, MD 20889; §Medicine Branch at the Navy, National Cancer Institute, National Institutes of Health,
Bethesda, MD 20892; and ¶Department of Microbiology and Molecular Genetics, University of California, College of Medicine, Ir vine, CA 92717
Edited by William S. Sly, St. Louis University School of Medicine, St. Louis, MO, and approved August 12, 1998 (received for review
June 12, 1998)
ABSTRACT To discover genes involved in von Hippel-
Lindau (VHL)-mediated carcinogenesis, we used renal cell
carcinoma cell lines stably transfected with wild-type VHL-
expressing transgenes. Large-scale RNA differential display
technology applied to these cell lines identified several differ-
entially expressed genes, including an alpha carbonic anhy-
drase gene, termed CA12. The deduced protein sequence was
classified as a one-pass transmembrane CA possessing an
apparently intact catalytic domain in the extracellular CA
module. Reintroduced wild-type VHL strongly inhibited the
overexpression of the CA12 gene in the parental renal cell
carcinoma cell lines. Similar results were obtained with CA9,
encoding another transmembrane CA with an intact catalytic
domain. Although both domains of the VHL protein contrib-
ute to regulation of CA12 expression, the elongin binding
domain alone could effectively regulate CA9 expression. We
mapped CA12 and CA9 loci to chromosome bands 15q22 and
17q21.2 respectively, regions prone to amplification in some
human cancers. Additional experiments are needed to define
the role of CA IX and CA XII enzymes in the regulation of pH
in the extracellular microenvironment and its potential im-
pact on cancer cell growth.
Inactivation of the von Hippel-Lindau (VHL) tumor suppres-
sor gene is responsible for hereditary (VHL disease) and most
sporadic renal cell carcinomas (RCCs) of the clear cell type
(1). The product of the gene, pVHL, was predicted to contain
two protein-binding domains (E. V. Kunin, personal commu-
nication). This prediction for the second domain that maps
close to the pVHL carboxyl terminus (residues 156–195) was
confirmed by protein binding studies (2–5). This domain binds
to elongin B and C subunits of the transcription elongation
factor SIII
y
Elongin, which led to a hypothesis of pVHL
involvement in transcription elongation regulation (3–6). The
initial discovery of three pVHL target genes, VEGF,PDGF-B,
and Glut1 (7, 8), stimulated more research into the molecular
mechanisms of pVHL involvement in regulation of gene
expression. A recent study of VEGF down-regulation by wild
type (wt) VHL transgenes (9) showed that pVHL directly
interacted in this system with the ubiquitous transcription
factor Sp1. Thus pVHL has possible effects on initiation of
transcription from Sp1-driven promoters. The other two genes,
PDGF-B and Glut1 (8), and the TGF alpha gene (10) were
down-regulated by pVHL at the posttranscriptional stages,
most likely by reducing mRNA stability (7, 8, 10). RNA
differential display (RDD) technology was used to study
transcription in RCC cell lines expressing wtVHL transgenes
versus parental nontransfected cells to discover target genes
down-regulated by wtVHL. We report here two additional
VHL target genes, encoding carbonic anhydrases (CA) CA12
and CA9 (11), which are strongly suppressed by wtVHL. Both
protein binding domains of pVHL contribute to the regulation
of CA12 expression whereas CA9 was regulated mostly by the
elongin binding domain. High levels of CA12 expression in
adult kidney, pancreas, colon, and prostate suggest an impor-
tant physiological function for this enzyme.
MATERIALS AND METHODS
Molecular Techniques. All molecular manipulations
(screening cDNA libraries, Northern blot analysis, and PCR)
were performed by using standard methods (12).
DNA Sequence Determination. cDNA clones were se-
quenced on an Applied Biosystems 373 DNA sequencer
(Stretch) using Taq Dyedeoxy Terminator Cycle Sequence kits
(Applied Biosystems) with either vector or clone-specific
walking primers.
mRNA Expression Analyses. Northern blot hybridization
was performed with CA12 and CA9 cDNA probes by using
commercial MTN poly(A) RNA blots (CLONTECH) from a
variety of adult human tissues and tumor cell lines and
poly(A)
1
RNA prepared from RCC and lung cancer cell lines.
In addition, the presence of CA12 and CA9 transcripts was
monitored in silico by BLAST homology searches (ref. 13 and
http:
yy
www.ncbi.nlm.nih.gov
y
BLAST
y
) in public expressed
sequence tag (EST) databases (http:
yy
www.ncbi.nlm.nih.
gov
y
dbEST
y
index.html).
Fluorescent in Situ Hybridization. Metaphase spreads de-
rived from BrdUrd-synchronized normal peripheral lympho-
cytes were used as a template. Probes containing CA9 and
CA12 cDNAs were labeled with digoxigenin 11-dUTP by
nick-translation, and hybridization signals were detected with
rhodamine-conjugated antidigoxigenin antibodies (Boehring-
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424y98y9512596-6$2.00y0
PNAS is available online at www.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: CA, carbonic anhydrase (CA genes are denoted by
Arabic numerals and the corresponding isozymes by Roman numer-
als); EST, expressed sequence tag; mut, mutant; RCC, renal cell
carcinoma; RDD, RNA differential display; VHL, von Hippel-Lindau;
wt, wild type.
Data deposition: The CA12 cDNA sequence reported in this paper has
been deposited in the GenBank database (accession no. AF037335).
†To whom reprint requests should be addressed. e-mail: ivanov@
ncifcrf.gov.
12596
er-Mannheim). The conditions of hybridization, detection of
hybridization signals, and digital-image acquisition, process-
ing, and analyses were performed as previously described (14).
Chromosomes were identified by converting 49,6-diamidino-
2-phenylindole (DAPI) banding into G-simulated banding by
using IP Lab Image Software (Scan Analytics, Vienna, VA).
Rehybridization with alpha-satellite centromeric probes (On-
cor) was performed to confirm chromosomal localization.
Sequence Analyses. World Wide Web-based servers were
used to analyze the cDNAs and deduced protein sequences.
Global sequence alignments were done by using BLAST and
advanced BLAST programs as provided by the National Center
for Biotechnology Information (ref. 13; http:
yy
www.ncbi.
nlm.nih.gov
y
BLAST
y
and http:
yy
www.ncbi.nlm.nih.gov
y
cgi-bin
y
BLAST
y
nph-blast?Jform 51) and BLAST2
y
WU
Blast, provided by the European Molecular Biology Labora-
tory (http:
yy
www2.ebi.ac.uk
y
blast2
y
).
Multiple sequence alignments, global and local, were done
by using the CLUSTAL version Wprogram as provided by the
European Molecular Biology Laboratory (http:
yy
dot.imgen.
bcm.tmc.edu:9331
y
seq-search
y
alignment.html), the Baylor
Computing Center (http:
yy
gc.bcm.tmc.edu:8088
y
search-
launcher
y
launcher.html), and the Wisconsin Genetics Com-
puter Group, package 8, program (http:
yy
www.gcg.com
y
).
Protein domains were discovered on the Pfam (http:
yy
www.
sanger.ac.uk
y
Pfam
y
) and membrane topology on the PSORT
servers (http:
yy
www.cbs.dtu.dk
y
services
y
SignalP
y
). Some
protein motifs were found by visually inspecting local align-
ments or by using the protein motifs server (http:
yy
www.
mips.biochem.mpg.de
y
).
VHL-Expressing Constructs. wtVHL expressing minigenes
were constructed with the pRC
y
CMV-Htag-VHL (15) plas-
mid cut with HindIII–XhoI, and the insert was placed into
pCEP4 (Invitrogen). H-tag was removed by partial digestion
with BamHI–HindIII, followed by ligation. Another wtVHL-
expressing construct used in this work was obtained by reverse
transcription–PCR amplification of the wtVHL reading frame
followed by insertion into the pCR 3.1 expression vector as
recommended by the vendor (Invitrogen). Mutant (mut)
pVHL-expressing constructs were created in a similar way by
using naturally occurring mutations. Mut 1 is a 1-bp deletion
creating a frameshift after residue 175; mut 2 is a 4-bp insertion
creating a frameshift after residue 168; mut 3 and mut 4 are
missense mutations, Gly-93–Asp and Tyr-98 –His, respectively.
Products of mut 1 and mut 2 are devoid of the elongin binding
domain; those of mut 3 and mut 4 possess an intact functional
(5) elongin binding domain (Fig. 1).
RCC Cell Lines Overexpressing wtVHL and mutVHLs.
VHL transgenes were overexpressed in two clear cell type
RCC cell lines. The first one, UM-RC-6 (16), contains an
intragenic 9-bp deletion in the remaining single allele of the
VHL gene creating a frameshift after residue 168 (17). The
second one, 786–0 (18), contains a 1-bp deletion creating a
frameshift in the remaining single allele after residue 104 (17).
All transfections were performed by using Lipofectin
(GIBCO
y
BRL) according to the manufacturer’s protocol.
The VHL-positive clones were selected for the presence of
exogenous VHL mRNA and protein by using Northern, South-
ern, and Western blot analyses (data not shown). VHL trans-
gene mRNAs were amplified by reverse transcription–PCR
and verified by sequencing.
Cell Lines Used in RDD. For RDD we used two cell lines:
UM-RC-6 and its wtVHL-transfected derivative harvested at
a late confluent stage of growth. The following cell lines were
used for subsequent Northern analyses: parental UM-RC-6
and 786–0 and the same cells stably transfected with wt and
mut VHLs.
RDD Analysis. RNA isolation for RDD, cDNA production,
recovery and reamplification of differentially expressed bands,
subcloning, and sequencing were performed as described
previously (19). For equal loading of mRNA samples, the
EagleEye Vision system (Stratagene) was used to control
intensity of ethidium bromide-stained lanes on a pilot gel.
Before hybridization, blots were stained with methylene blue
to show equal loading of the RNA samples. Quantification of
hybridization signals was done by using the PhosphorImager
system (Molecular Dynamics).
RESULTS
Strategy and Experimental Design to Isolate Genes In-
volved in VHL-Mediated Carcinogenesis. The changes in gene
expression in RCC devoid of functional pVHL after introduc-
tion of wtVHL were studied by using differential display. This
straightforward strategy was used to identify genes whose
expression was up-regulated or down-regulated after introduc-
tion of wtVHL-expressing minigenes. The theoretical frame-
work underlying the RDD technology largely depends on
comparing mRNA concentrations at physiological conditions
that maintain steady-state levels of gene expression. The
expression of the various VHL constructs was not inf luenced
by the growth conditions: it was similar in late logarithmic and
confluent cells (data not shown). Therefore, we ran the RDD
at a late confluent stage of cell growth and verified differential
expression by Northern blot analyses in both growing and
resting cells. Fig. 1 presents the domain structure of pVHL and
the structure of mutVHL transgenes used for transfections.
Identification and Analysis of Genes Differentially Ex-
pressed by RCC Cells Overexpressing wtVHL Transgenes. We
compared steady-state RNA concentrations by RDD in late
confluent UM-RC-6 cells (three independent isolates) versus
the same cells stably transfected with a wtVHL minigene
driven by the cytomegalovirus promoter. Eighty arbitrary
13-mer primers in combination with three anchor primers to
FIG. 1. Schematic representation of the domain structure of wt and
naturally occurring mut pVHLs. The three exons are indicated by
different shadowing; the putative protein binding domains are repre-
sented by ovals, the filled one is the elongin binding domain; numbers
above the pictures are amino acid residues, arrows indicate residues
involved in producing mut pVHLs. Mut 1 is a 1-bp deletion creating
a frameshift after residue 175; mut 2 is a 4-bp insertion creating a
frameshift after residue 168; mut 3 and mut 4 are missense mutations,
Gly-93–Asp and Tyr-98–His, respectively. Mut 1 and mut 2 are devoid
of the elongin binding domain; mut 3 and mut 4 possess an intact
functional elongin binding domain.
Medical Sciences: Ivanov et al.Proc. Natl. Acad. Sci. USA 95 (1998) 12597
complete 240 sets of differential display reactions were de-
signed to scan at least 20–25% of the total gene complement
expressed in these cells. Fifty-two bands were found to be
differentially expressed in a reproducible fashion. All were
PCR-reamplified, cloned, sequenced, and used for Northern
blot analyses to confirm that they indeed represented differ-
entially expressed RNAs. Thus far, five have been confirmed
to be down-regulated by wtVHL from two to more than 30
times (data not shown). Sequence analyses revealed matches in
the databases and identified three specific clones as corre-
sponding to known human genes, namely, NOTCH 2 (ref. 20,
GenBank U77493), DEC1 (ref. 21, GenBank AB004066), and
an alpha CA; two clones corresponded to ESTs (GenBank
D86978 and AA165698) of unknown genes.
The alpha CA family (E.C. 4.2.1.1., carbonic dehydratase)
(22, 23) is comprised of 12 isozymes, including the CA XII
identified and cloned in this work. Here we report in detail on
CA XII, and also include data on regulation of another
structurally related transmembrane CA, CA IX (11) that is
down-regulated by VHL (see below). First, their expression at
different stages of cell growth was analyzed in another RCC
cell line, 786–0, which is also devoid of a functional pVHL
(17). Second, the effect of wtVHL and distinct mutVHL
minigenes on the expression of CA12 and CA9 in both RCC
cell lines was ascertained (Figs. 2 and 3). Two classes of
naturally occurring VHL mutations (17, 24) were tested. Mut
1 and mut 2 minigenes encoded truncated proteins devoid of
the elongin binding domain (Fig. 1), whereas mut 3 and mut
4 apparently encoded full-sized proteins with missense muta-
tions in the first putative binding domain and an intact (Fig. 1)
and functional (5) elongin binding domain. Mut and wt VHL
proteins are highly expressed in transfected UM-RC-6 (data
not shown) and 786–0 cells (3–5). The VEGF gene, repeatedly
reported as a VHL gene target in 786–0 cells (7–9), was
included in these analyses as a control. The results (Figs. 2 and
3) clearly show that the expression pattern of the CA9 and
CA12 genes is different in the analyzed RCC lines and
dramatically depends on the growth stage. The CA12 gene is
highly expressed in both cell lines, whereas the CA9 and VEGF
is genes are highly expressed in the 786– 0 cells but not
expressed at all in the UM-RC-6 line. The expression of all
three genes is much higher in a late confluent stage compared
with late logarithmic growth. The impact of the VHL trans-
genes, both wt and mut ones, on CA9,CA12, and VEGF gene
expression is very striking and indeed informative (Figs. 2 and
3). The wtVHL completely suppresses the expression of CA9
and CA12 genes in both confluent and growing 786–0 cells.
VEGF is completely suppressed in confluent 786–0 cells, but
only partially suppressed in growing 786–0 cells. wtVHL
effectively (.80%) suppresses the high level of CA12 expres-
sion in late confluent UM-RC-6 cells but not in growing cells
(Fig. 2). The mutVHL transgenes differentially affect the
expression of CA12,CA9, and VEGF genes (Figs. 2 and 3). Mut
1 and mut 2 transgenes, which lack the elongin binding domain,
slightly suppress CA12 expression but have no effect or even
slightly up-regulate the expression of CA9 and VEGF genes. In
contrast, mut 3 and mut 4 transgenes, which express an intact
functional elongin binding domain, suppress the expression of
all three genes to varying degrees in conf luent cells (Fig. 3). In
growing cells, these mutant VHL proteins completely suppress
FIG. 2. Northern analysis of target genes in growing (Log) and late
confluent (Con) UM-RC-6 cells transfected with wt or mut 1 pVHL.
Quantification of hybridization signals was done by using the Phos-
phorImager system (Molecular Dynamics). Before hybridization the
blots were stained with methylene blue (Lower) to show approx imately
equal loading of the RNA samples (see also Materials and Methods).
Numbers on the right represent size markers in kb.
FIG. 3. Northern analysis of target genes in growing (Log) and late
confluent (Con) 786– 0 cells transfected with wt or mut 2, mut 3, and
mut 4 pVHL-containing plasmids. Quantification of hybridization
signals was done by using the PhosphorImager system (Molecular
Dynamics). Before hybridization the blots were stained with methyl-
ene blue (Lower) to show approximately equal loading of the RNA
samples (see also Materials and Methods). Numbers on the right
represent size markers in kb.
12598 Medical Sciences: Ivanov et al.Proc. Natl. Acad. Sci. USA 95 (1998)
CA9, show no effect on CA12, and only slightly suppress VEGF
gene expression. These observations suggest that the CA12
gene discovered on the RDD screen and the previously cloned
CA9 (11) gene are regulated by VHL. Both domains of the wt
pVHL are required to down-regulate the expression of the
CA12 gene. Naturally occurring mutants that possess an intact
functional elongin binding domain alone could effectively
(.90%) down-regulate the expression of the CA9 gene (Fig.
3). We next explored the possibility that VHL could regulate
the expression of other members of the alpha CA gene family.
wtVHL transgenes did not affect the expression of CA1,CA2,
CA4, and CA5 in the UM-RC-6 and 786–0 RCC cell lines (data
not shown).
Cloning and Characterization of CA12, a Member of the
Alpha CA Gene Family. The differentially expressed cDNA
clone no. 9 was used as a probe to screen a human lung cDNA
library (no. 1024–018, GIBCO
y
BRL). As a result, several
cDNA clones were obtained and then sequenced. Sequence
analysis showed that all of these clones represented partial
cDNA sequences of the same gene. The biggest insert, 2,771 bp
in length, contained a 1,062-bp ORF (354 amino acids), a
115-bp 59untranslated region (UTR), and a 1,591-bp 39UTR,
which contained the original differentially expressed cloned
cDNA sequence 9. After this work was completed, Tureci et al.
(25) reported the cloning of the CA12 cDNA, which contained
108 bp less in the 59UTR. We used a number of bioinformatics
web-based servers (see Materials and Methods) to analyze the
cDNA and the deduced protein sequences. Analysis of the
cDNA showed high degrees of homology to members of the
alpha CA gene family and established CA12 as a member of
this family (data not shown). Analysis of the predicted protein
sequence (354 residues) revealed a signal peptide (cleavage
site between residues 24 and 25) followed by an alpha CA
domain (residues 28–289), which has 55% similarity with the
consensus sequence, followed by a transmembrane peptide
(residues 308–324) and a short cytoplasmic domain (residues
325–354). This protein is a one-pass transmembrane CA with
extracellular amino and intracellular carboxy termini. The CA
domain of CA12 is closely related to the CA domain of the CA6
and CA9 genes. The cytoplasmic domain of CA XII is 50%
identical to the cytoplasmic domain of the CA9 gene product,
which is also a transmembrane CA protein (Fig. 4). Phyloge-
netic reconstruction of alpha CAs (data not shown) indicates
that CA XII, CA IX, and CA VI are more closely related to
each other than to other alpha CAs and group together,
representing a rather early branch in the evolution of the alpha
CA gene family (22). The short cytoplasmic domains of these
two proteins contain several conserved residues including a
threonine, a motif with one tyrosine, GVXYXPA, and one or
two histidine residues (Fig. 4). Both proteins possess all three
zinc-binding histidines obligatory for the catalytic activity in
their CA modules (Fig. 4) (22, 23). The CA IX protein was
predicted (22) and reported (26) to be an active CA enzyme.
Tureci et al. (25) now have shown that expressed CA XII
protein is an active CA isozyme. We next analyzed the
expression patterns of the CA12 gene by Northern blot hy-
bridization to human adult tissues, some tumor cell lines, and
by in silico monitoring public EST databases. Northern blot
hybridization was performed with commercial multiple tissue
poly(A)
1
RNA blots (CLONTECH). CA12 is highly expressed
in colon, kidney, and prostate, moderately in pancreas, ovary,
and testis as 4.3-kb mRNA (Fig. 5A). Very low expression
could be observed in lung and brain on longer exposure (data
not shown). The tumor cell lines tested (Fig. 5B) showed A549
cells (adenocarcinoma of the lung) had very high level of
expression, HeLa S3, and colorectal (SW480) cells moderate
expression, lymphoma low expression, and no expression was
detected in melanoma and several leukemias. The very high
expression in the A549 cells prompted us to analyze the
expression of CA12 in other small cell and nonsmall cell lung
carcinoma cell lines. No expression was detected in 14 small
cell lung cancer and high to moderate expression in four of 10
tested nonsmall cell lung carcinoma cell lines (Fig. 5B). The 15
CA12 ESTs were distributed in the cDNA libraries as follows:
5
y
15 colon, 2
y
15 kidney, 2
y
15 pancreatic islets, 2
y
15 ovarian
carcinoma, and 1
y
15 in fetal brain, fetal heart, uterus, and
endometrial carcinoma. These combined results suggest that
CA12 is expressed in a restricted assortment of normal and
tumor tissues. CA9 is far more restricted then CA12 in its tissue
distribution. It is not expressed in normal kidney tissue (27),
and the only normal tissue that expresses significant amounts
is the gastric mucosa (ref. 27 and S. Y. Liao and E.J.S.,
FIG. 4. Global amino acid alignment of CA XII and CA IX proteins
obtained by using the CLUSTALW alignment tool as provided by the
ExPasy server (http:
yy
expasy.hcuge.ch
y
sprot
y
scnpsite.html). The
overall identity is 35.8%, and the identical residues are marked by p
below the sequence. The three essential Zn-liganded histidine residues
are marked by Zn symbols above them. The short cytoplasmic domains
are boxed with histidine, threonine, serine, and tyrosine residues
underlined. The ideograms depicting the domain structure of the genes
are positioned above the alignment. SP, signal peptide; CA, carbonic
anhydrase domain; TM, transmembrane peptide; CTD, cytoplasmic
domain; PG, proteoglycan domain.
FIG. 5. Northern blot analysis of CA12 expression in normal tissues
(A) and tumor cell lines (B) using the cDNA as hybridization probe.
(A) The RNA filters are from CLONTECH (nos. 7759 and 7760) and
contain 2
m
g of poly(A)
1
mRNA per tissue indicated: lanes 1, heart;
2, brain; 3, spleen; 4, thymus; 5, prostate; 6, testis; 7, ovary; 8, small
intestine; 9, colon; 10, peripheral blood leucocytes. (B) The RNA filter
is from CLONTECH (no. 775) and contains 2
m
g of poly(A)
1
mRNA
per cell line indicated: lanes 1, promyelocytic leukemia, HL-60; 2,
HeLa cells S3; 3, chronic myelogenous leukemia, K-562; 4, lympho-
blastic leukemia, MOLT-4; 5, Burkitt’s lymphoma, Raji; 6, colorectal
adenocarcinoma, SW 480; 7, lung adenocarcinoma, A549; 8, mela-
noma, G361, and poly(A)
1
RNA from human nonsmall cell lung
carcinomas (as indicated: 9, NCI H_1373; 10, NCI H_1264; 11, NCI
H_1693; 12, NCI H_1944; 13, NCI H_838; 14, NCI H_1299; 15, NCI
H_157; 16, NCI H_1466; 17, NCI H_460; 18, NCI H_727).
Medical Sciences: Ivanov et al.Proc. Natl. Acad. Sci. USA 95 (1998) 12599
unpublished observations) and gut enterocytes (28). The chro-
mosomal map positions of the CA9 and CA12 genes were
determined by fluorescence in situ hybridization using the
corresponding cDNAs as probes. The CA9 locus was localized
to 17q21.2–21.3 (Fig. 6A) and the CA12 to 15q22 (Fig. 6B).
These bands are subject to amplifications in quite a number of
human cancers that include prostate, lung, kidney, ovarian,
breast, neuroblastoma, and head and neck tumors (29).
DISCUSSION
Analysis of VHL tumor phenotypes led to the prediction of
several VHL target genes that were verified in the 786–0 cell
system (7–9). Our interest in identifying more VHL gene
targets was because at least some of these genes could mediate
downstream events in VHL and
y
or general carcinogenesis.
Therefore, a more global understanding of VHL-mediated
changes in gene expression might direct us to new applications
for diagnostic and therapeutic intervention in the VHL-caused
tumors. Our study used the RDD technology applied to RCC
cells overexpressing wtVHL transgenes to discover VHL gene
targets. RDD analysis of 20–25% of all transcribed genes in
RCC cells has allowed us to identify five additional VHL gene
targets, suggesting that the total number of potential targets
would not exceed 20–25 genes. RDD analysis with a complete
set of primers should reveal most, if not all, of the yet-to-be-
discovered VHL potential target genes. The 10 known VHL
targets (six in this work and four reported previously, refs. 7–9)
include only one protooncogene the NOTCH2 gene (30, 31).
The physiological functions of the numerous alpha CA
enzymes (22, 23) are based on their ability to catalyze the
reversible hydration of CO
2
, producing carbonic acid that
subsequently decomposes to HCO
3
2
and H
3
O
1
, leading to a
decrease in pH. There is considerable evidence that the
glycosylphosphatidylinositol-anchored plasma membrane CA
IV enzyme exerts an effect on several ionic channels, which
could result in shifting protons and HCO
3
2
across the mem-
brane, leading to a rise in cytoplasmic and decrease in extra-
cellular pH (23, 32). The one-span transmembrane CA IX and
CA XII enzymes, which were predicted and shown to be
catalitically active (22, 25, 26), could serve a similar function.
Their short, cytoplasmic domains containing histidine, threo-
nine, serine, and tyrosine residues could function as sensors of
cytoplasmic pH and elicit cellular responses to certain cues.
The enhanced expression of CA IX (33, 34) and CA XII (35,
36) isozymes in renal and other cancers may add to our
understanding of cancer development and may well explain
evidence accumulated since the time of O. Warburg (37), that
the extracellular pH of human tumors is on average more
acidic than that of normal tissues (38). We argue that these CA
isozymes acidify the immediate extracellular milieu surround-
ing the cancer cells and thus create a microenvironment
conducive to tumor growth and spread. It has been shown that
acidic pH enhances invasive behavior of tumor cells in vitro
(39). The altered control of extracellular and intracellular pH
caused by overexpression of the CA9 and CA12 genes could
represent a pathway in the development of at least a fraction
of human tumors. Cancer cells could use different mechanisms
to maintain high levels of CA9 and CA12 expression such as
inactivation of the VHL tumor suppressor gene and
y
or am-
plification of the corresponding loci (29). We, therefore,
suggest that in addition to being used as potential diagnostic
biomarkers (33, 34, 36, 40) these enzymes should be consid-
ered as targets for novel therapeutic applications.
The molecular mechanisms by which pVHL modulates the
expression of target genes is not well understood. The original
hypothesis based on the discovery of elongin B binding by
pVHL assumed that VHL could negatively regulate transcrip-
tion elongation of target genes by inhibiting the elongin
y
SIII
function (6). Although this expectation was confirmed in vitro
(2, 3), there is no compelling evidence to date that pVHL can
exert the same effect in vivo. The accumulated evidence
suggests that pVHL reduces the stability of mRNA (7, 8)
and
y
or the initiation of transcription (9). Our own experi-
ments demonstrate distinct domains of pVHL contribute
unequally to the modulation of expression of different target
genes (CA9,CA12, and VEGF). pVHL may operate on dif-
ferent levels of gene regulation depending on the particular
gene and the stage of cell growth.
FIG. 6. Subchromosomal localization of the human CA9 and CA12 genes. Metaphase after fluorescence in situ hybridization showing location
of the (A)CA9 gene on the long arm of chromosome 17q21.2–21.3 (arrow) and (B)CA12 gene on the long arm of chromosome 15q22 (arrow).
(Insets) Position of both loci, CA9 and CA12,on49,6-diamidino-2-phenylindole (DAPI)-banded human chromosomes, 17q21.2–21.3 and 15q22,
respectively (arrows).
12600 Medical Sciences: Ivanov et al.Proc. Natl. Acad. Sci. USA 95 (1998)
We thank William Kaelin (Dana Farber Cancer Institute, Boston)
for kindly providing the 786 –0 RCC cell line and the pRC
y
CMV-
Htag-VHL expressing plasmid and David Hewett-Emmett (University
of Texas Health Science Center, Houston) for helpful discussions on
the phylogenetics of carbonic anhydrases. This project has been funded
in whole or in part with federal funds from the National Cancer
Institute, National Institutes of Health, under Contract No. NO1-CO-
56000. It also was supported, in part, by a grant (CA19401) from the
National Cancer Institute (to E.J.S.).
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