Antibody-specific detection of CAIX in breast and prostate cancers
Ying Li
a
, Hai Wang
a
, Egbert Oosterwijk
c
, Yamil Selman
b
, Juan C. Mira
b
, Theresa Medrano
b
,
Kathleen T. Shiverick
b
, Susan C. Frost
a,
*
a
Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA
b
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL 32610, USA
c
Urological Research Laboratory, Department of Urology, University Hospital Nijmegen, Nijmegen, The Netherlands
article info
Article history:
Received 9 June 2009
Available online 16 June 2009
Keywords:
CAIX
b-Tubulin
Antibody-specificity
Breast cancer cells
Prostate cancer cells
Hypoxia
abstract
Carbonic anhydrase IX (CAIX) is frequently expressed in human tumors and serves as a marker for
hypoxia. Further, CAIX expression is considered a predictor of poor survival in many, but not all, cancer
types. Herein, we compare the specificity of two CAIX antibodies: the M75, monoclonal antibody which
recognizes an epitope in the N-terminus and a commercially available polyclonal antibody generated
against a C-terminal peptide (NB100-417). Western blot analysis of multiple breast cell lines revealed
that the polyclonal antibody detected both membrane-bound and soluble proteins. The M75 antibody
recognized only the membrane-bound species, which is presumed to be CAIX. These data were confirmed
in an aggressive prostate cell line. We further compared these antibodies in prostate tumors by immuno-
histochemistry. Staining with NB100 was comparable to that of the M75 antibody, but only at high dilu-
tion. Otherwise, cytoplasmic staining was also noted. Two-dimensional gel electrophoresis followed by
mass spectrometric analysis revealed that the cytoplasmic protein detected by NB100 is b-tubulin. This
cross-reactivity could lead to false-positives for CAIX expression in samples where cytosolic proteins are
present.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
Carbonic anhydrase IX (CAIX) is a membrane-bound form of the
carbonic anhydrase (CA) family of zinc metalloenzymes that cata-
lyzes the reversible conversion between carbon dioxide and bicar-
bonate. The CA family members participates in the regulation of
pH, CO
2
and HCO
3
À
transport, and water and electrolyte balance
[1].
Expression of CAIX is associated with tumor cell hypoxia in a
variety of human tumors [2], including breast [3,4] and urologic
cancers [5–7]. CAIX is a membrane glycoprotein in which the cat-
alytic domain, along with a unique N-terminal, proteoglycan do-
main, faces the extracellular milieu [8]. CAIX is upregulated by
hypoxia [9] and its gene is a target of hypoxia-inducible factor-1
(HIF1
a
) [10]. One of the striking features of cancer cells is their
ability to acidify their environment and the orientation of CAIX
suggests that it may serve as one of the mechanisms by which can-
cer cells regulate extracellular pH and induce cytoplasmic alkalin-
ization [11]. Multiple studies have shown that the expression of
CAIX in breast tumors, as well as other solid tumors, is associated
with poor prognosis [3,4,12,13]. Thus, CAIX is being used clinically
as a diagnostic tool which has implications for therapy and patient
outcome. This demands the most careful analysis of CAIX expres-
sion as it may directly impact patient care.
In the 1980’s, Oosterwijk et al. generated a monoclonal anti-
body (G250) against a cell surface protein expressed by renal car-
cinoma cells [6]. Using molecular cloning, this antibody was shown
to bind to CAIX [7]. Later, Pastorekova et al. developed a monoclo-
nal antibody against a 54/58 kDa protein called MN expressed
endogenously in a human mammary tumor cell line [14]. This anti-
body was also shown to target CAIX [15]. The specific epitope for
the G250 antibody is unknown, but it has excellent specificity for
CAIX in immunohistochemical analysis. The M75 (considered the
gold standard for CAIX identification) recognizes the extracellular
proteoglycan-like domain and is useful for western blotting,
immunoprecipitation, and immunohistochemistry. CAIX antibod-
ies are now available commercially. One of the first companies to
offer this product was Novus Biologicals (Littleton, CO). Their poly-
clonal antibody was generated against a peptide in the C-terminus,
a domain which faces the cytoplasmic compartment. R&D Systems
(Minneapolis, MN) also has a number of monoclonal and poly-
clonal antibodies available. In this paper, we compare the specific-
ity of the polyclonal antibody from Novus Biologicals (NB100-417)
with that of the monoclonal antibody, M75. In three different
breast cell lines and a prostate cell line, our data show that
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.06.064
* Corresponding author. Address: Department of Biochemistry and Molecular
Biology, University of Florida, College of Medicine, 1600 SW Archer Rd., Box 100245,
Gainesville, FL 32610, USA. Fax: +1 352 392 2953.
E-mail address: sfrost@ufl.edu (S.C. Frost).
Biochemical and Biophysical Research Communications 386 (2009) 488–492
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