[Cancer Research 60, 1173-1176, March 1, 2000]
© 2000 American Association for Cancer Research
Carbonyl Reductase: A Novel Metastasis-modulating Function1
Endom Ismail,
Fahd Al-Mulla2,
Shigeki Tsuchida,
Kohji Suto,
Paul Motley,
Paul R. Harrison and
George D. Birnie
The Beatson Institute for Cancer Research, Glasgow G61 1BD, Scotland [E. I., F. A-M., P. R. H., G. D. B.]; Second Departments of Biochemistry [S. T.] and Pathology [K. S.], Hirosaki University School of Medicine, Hirosaki 036-8562, Japan; and Southern General Hospital Trust, Glasgow G51 4TF, Scotland [P. M.]
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ABSTRACT
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To explore reasons for differences in the malignancy of tumors, we have
compared two cell lines derived from a mouse lung adenocarcinoma cell
line that differ 10-fold in their capacity to form lung metastases from
s.c. primary tumors or after i.v. injection. One mRNA encoding carbonyl
reductase was identified at a relatively high abundance in the subline
with low metastatic capacity but was not detectable in the highly
metastatic subline. Transfection of the former subline with a plasmid
construct expressing antisense carbonyl reductase rendered the cells
highly metastatic. Conversely, the capacity of the highly metastatic
cells to metastasize was markedly reduced after transfection with a
construct expressing carbonyl reductase. We also found that human
prostate cancers show loss of carbonyl reductase expression compared
with normal prostate epithelia. These data suggest that carbonyl
reductase has an important function in modifying the metastatic
behavior of malignant tumors.
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Introduction
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It is clear from studies in animals and humans that metastasis is
a complex multistage process orchestrated by a fine interplay between
genetic, epigenetic, and environmental influences (1
, 2) .
However, the reasons why some tumors display a more malignant phenotype
than other similar ones remain obscure. This is particularly so for the
question as to why one tumor is strongly metastatic, whereas another
apparently identical tumor is much less capable of metastasizing. To
identify genetic aberrations that modify the metastatic phenotype
rather than the genes involved in metastasis per se, we have
made use of two cell lines originally derived from a mouse lung
adenocarcinoma (3)
. One of these cell lines, CMT167, has a
markedly greater metastatic capacity than the other, CMT170
(3)
. Because both DNA fingerprinting and in-gel
renaturation (4)
showed the two cell lines to have a
similar genetic composition, we concluded that the difference in the
metastatic propensities of the two is likely to be due to differences
in the expression of a gene or genes. To explore this, we used
differential display (5)
, an unbiased PCR-based
(3)
method that not only detects mRNAs that differ in
abundance between two populations but also allows the corresponding
cDNAs to be cloned and subsequently sequenced. As a result of these
studies, we identified a gene encoding an enzyme (carbonyl reductase)
that had not previously been suspected of any involvement in malignant
progression or the metastatic process. Moreover, we present evidence of
its involvement in at least one type of human cancer, i.e.,
prostatic cancer.
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Materials and Methods
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Cell Culture and Transfection.
CMT167 and CMT170 cells were cultured in DMEM containing 10% FCS.
Full-length cDNA coding for mouse carbonyl reductase 1 (a gift from J.
Wei) was ligated into the pBabe Puro expression vector in both
orientations to generate sense and antisense carbonyl reductase. Ten
µg of the sense coding plasmid were transfected into CMT167 cells,
and 10 µg of antisense coding plasmid were transfected into CMT170
cells using the
N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl
sulfate lipofection protocol (Boehringer Manheim). Transfected cells
were cultured in DMEM with 10% FCS containing 1.5 µg/ml puromycin.
Northern blotting and Western blotting were used to confirm the
expression of the appropriate carbonyl reductase constructs. As
controls, 10 µg of vector only were transfected into CMT167 and
CMT170 cells. For measurement of cell growth rates in vitro,
replica plates of the cells were prepared, and 0.5 mg/ml
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was
added to each plate at the appropriate time point. After 4 h, the
excess medium was removed, 100 µl of DMSO were added to each plate,
and the absorbance was measured at 595 nm.
Differential Display.
Total mRNA was isolated from CMT167 and CMT170 cells as described
previously (6)
. First-strand cDNA synthesis was performed
using the Delta RNA Fingerprinting kit (CLONTECH) according to
manufacturer’s instructions. Differential display was performed
according to the manufacturer’s instructions (CLONTECH). A total of 62
different combinations of arbitrary and oligodeoxythymidylic
acid primers were tested. A combination composed of the P3
arbitrary primer 5'-ATTAACCCTCACTAAATGCTGGTGG-3' and the T8
oligodeoxythymidylic acid primer
5'-CATTATGCTGAGTGATATCTTTTTTTTTGC-3' resulted in a single
differentially expressed band. The band was isolated from the dried
denaturing polyacrylamide gel, reamplified using the same primers,
cloned into pCR 2.1 (Invitrogen),and sequenced using M13 forward and
reverse primers according to manufacturer’s instructions.
Northern and Western Blotting.
For Northern analysis, total mRNA was isolated as described above, and
the products were electrophoresed in a formaldehyde agarose gel and
transferred to Hybond N+ (6)
. To generate the carbonyl
reductase probe for the Northern blot, the band isolated from
differential display and cloned into pCR 2.1 was recut from a bulk
preparation of the plasmid, purified from an agarose gel, and labeled
with [
-32P]dCTP using Ready-To-Go
random-priming kit (Pharmacia). For Western analysis, protein
lysates were prepared from 80% confluent CMT167 and CMT170 cells,
electrophoresed through a 7.5% SDS-polyacrylamide gel, and blotted as
described previously. The filter was probed with rabbit antihuman
carbonyl reductase polyclonal antibody (7)
overnight at
4°C. Membranes were then washed three times with PBS containing 0.1%
Tween 20 and incubated for 1 h with the appropriate
peroxidase-conjugated secondary antibodies. After three washes with
PBS-0.1% Tween 20, bands were detected with the enhanced
chemiluminescence Western blotting system (Amersham) according to the
manufacturer’s instructions and visualized by exposure to Kodak X-OMAT
film for various times.
Immunocytochemistry.
This was performed as described by Suto et al.
(7)
. Sections (5 µm) were cut and mounted on
3-(triethoxysilyl)-propylamine-coated slides (Merck). Immunostaining
was performed using a Biogenex Optimax Plus automated immunostainer.
All incubations were performed at room temperature and were followed by
a wash in Optimax Buffer (Biogenex). Endogenous peroxidase activity was
blocked using 1% aqueous
H202 for 10 min.
Nonspecific staining was blocked using 20% normal goat serum for 20
min. Sections were then incubated for 1 h in a 1:1000 dilution of
primary antihuman carbonyl reductase antibody (7)
,
followed by a 30-min incubation in prediluted biotinylated secondary
antibody (Dako Chemmate detection kit) and finally by a 30-min
incubation in peroxidase-labeled streptavidin (Dako Chemmate detection
kit). The peroxidase label was visualized with diaminobenzidine for 10
min, followed by nuclear staining with Gill’s triple strength hemalum.
All experiments included negative controls in which the primary
antibody was omitted.
Mice.
All animals used in this study were treated according to the Home
Office license standards. C57 B/T syngeneic mice were used in all
experiments. A single cell suspension of 1 x 105 cells in 0.1 ml of DMEM was injected s.c.
into the right flank of mice (for overall metastasis assay), or
1 x 104 cells in 0.1 ml of DMEM
were injected into the tail vein of mice (for the colonization assay).
Primary tumor growth after s.c. injection was monitored regularly, and
once growth was palpable, measurements of primary tumor volume were
made. Animals were sacrificed 20 days after treatment. The weight and
volume of the primary tumors were measured, and the lungs were
subsequently inflated with 1.5 ml of black India ink solution [15%
(v/v) black India ink, 0.5% (v/v) ammonia solution]. The lungs were
dissected out, rinsed briefly in water, and stored in Fekete’s
solution (100 ml of 70% ethanol, 10 ml of 38% formaldehyde solution,
and 5 ml of glacial acetic acid). Lungs were stored for a minimum of
24 h for bleaching and fixation before the lobes of the lungs were
examined, and metastatic deposits were counted and confirmed
histologically as metastasic tumors.
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Results and Discussion
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The derivation of two mouse lung adenocarcinoma cell lines that
are both metastatic but differ significantly in their metastatic
capability has been described previously (3)
. There is an
approximately 10-fold greater number of lung metastases in syngeneic
mice from the highly metastatic CMT167 subline than from the low
metastatic CMT170 subline (Fig. 1, a and b)
when assayed by either s.c. injection,
a measure of the overall metastatic capacity of cells from invasion to
colonization, or i.v. injection, which largely assays the capacity of
the cells to colonize the lungs. Although tumors formed by CMT167 cells
grew faster than those from CMT170 cells (Fig. 1c)
, the
difference in metastatic capacity of the CMT167 cells was not
dependent on their higher growth rate (Fig. 1d)
and remained
significant even when adjusted for the difference in the growth rates
of the primary tumors (data not shown). Interestingly, the difference
in the growth rates of the tumors in vivo was not a
reflection of the growth rates of the sublines in vitro,
which were identical (data not shown). This suggested that the
difference in vivo is not an intrinsic phenomenon of these
cell lines.
Because the two cell lines have a similar genetic composition
(data not shown), it appeared likely that the difference in metastatic
behavior between the two cell lines is due to differences in gene
expression rather than in the genetic make up of the cells. Comparison
of the mRNA populations of the two cell lines by differential display
(5)
with several pairs of primers detected four
differentially expressed mRNAs, the differential expression of which
was subsequently confirmed by Northern blotting. Of these four mRNAs,
one was not detectable in the highly metastatic CMT167 cells but was
present at a relatively high level in CMT170 cells (Fig. 2)
. Subsequent cloning and sequencing of the corresponding cDNA showed a
99.9% and 80% homology to mouse and human carbonyl reductase mRNA,
respectively. To confirm that the differential expression of carbonyl
reductase at the mRNA level is also reflected at the protein level, a
polyclonal antibody raised against the whole
Mr 33,000–34,000 human
carbonyl reductase protein (7)
was used in Western
blotting analysis of cell lysates. The results confirmed the Northern
blot analysis data (Fig. 2)
. Southern blotting experiments showed that
the failure of CMT167 cells to express carbonyl reductase is not due to
deletion of the gene (data not shown). We cannot, however, rule out
mutation of the gene’s promoter as the mechanism.
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Fig. 2. Carbonyl reductase mRNA and protein expression levels in
CMT167 and CMT170 sublines. Left, Northern blot.
Right, SDS-PAGE autoradiograph.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
vinculin are loading controls for Northern and Western blotting,
respectively.
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To evaluate the role of carbonyl reductase in modifying the metastatic
behavior of cells, we transfected CMT170 cells with an expression
vector encoding antisense carbonyl reductase mRNA and analyzed the
behavior of the cells after s.c. and i.v. injection into syngeneic
mice. Expression of antisense carbonyl reductase mRNA and the
subsequent elimination of carbonyl reductase protein from the CMT170
cells (Fig. 3c)
increased the metastatic capacity of the CMT170 cells to
levels similar to those of CMT167 cells (Fig. 3a)
, whereas
transfection with the empty expression vector had no effect.
Conversely, expression of sense carbonyl reductase mRNA in CMT167 cells
(Fig. 3c)
significantly reduced their metastatic capacity to
match that of CMT170 cells (Fig. 3a)
. Interestingly,
transfection of the cell lines with carbonyl reductase sense or
antisense constructs had a lesser effect on the growth rates of the
tumors in vivo (Fig. 3b)
.
Our data suggest that, in this mouse model, carbonyl reductase has a
novel function in modulating the metastatic behavior of cells,
particularly in the latter stages of the process (because of its effect
on i.v. injected cells), and that its silencing by whatever mechanism
permits a greater tumor growth rate and a more aggressive behavior
in vivo. Carbonyl reductase (E.C.1.1.1.184) is one of
several monomeric, NADPH-dependent oxidoreductases with wide
specificity for carbonyl compounds that are generally referred to as
aldoketoreductases (8
, 9)
. It has been extensively studied
in relation to its ability to reduce a great variety of carbonyl
compounds, such as quinones, the antitumor anthracycline antibiotics
daunorubicin and doxorubicin, and 9-ketoprostaglandins
(10, 11, 12)
. The latter property could be directly relevant
to the phenomenon we have described because carbonyl reductase has been
found to be biochemically, immunologically, and functionally identical
to prostaglandin 9-ketoreductase, which oxidizes prostaglandin
E2, F2, and
D2 to their corresponding, biologically inactive,
15-keto metabolites (13
, 14)
. Prostaglandins, especially
prostaglandin E2, play an important role in
modulating tumor growth and metastasis in a variety of human tumors
(15, 16, 17)
. Moreover, prostaglandin E2
has important functions not only in modulating apoptosis in cancer
cells (18)
but also in regulating angiogenesis (19
, 20) , an essential prerequisite for the establishment of viable
metas-tases.
We appreciate that the novel function for carbonyl reductase we have
described here may be restricted to the model system used in this
study, especially because carbonyl reductase has never before been
associated with tumor growth or metastasis. To determine whether loss
of carbonyl reductase expression is a feature of human cancers, we have
screened a limited number of normal and malignant tissues for carbonyl
reductase expression by immunocytochemistry. Carbonyl reductase is not
expressed in some normal tissues, or its expression may be variable, as
in normal lung tissue, for reasons that have yet to be clarified.
However, a series of 20 prostatic adenocarcinomas was found to have a
reduced or total absence of carbonyl reductase expression, irrespective
of stage or grade, compared with normal prostate epithelium (Fig. 4)
. A much larger series of cases will need to be investigated to more
precisely define the clinical features associated with loss of carbonyl
reductase expression. Recent evidence has also shown that carbonyl
reductase expression is significantly reduced in hepatocellular cancer
(7)
. These preliminary results support a more important
role for carbonyl reductase in modifying the behavior of human
malignant tumors than previously suspected.
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Fig. 4. Carbonyl reductase expression is reduced in advanced
prostate cancers. Carbonyl reductase was measured in sections of
(A) normal prostate or (B) advanced
prostate cancer tissue by immunocytochemistry. The normal prostate
section (A) shows staining of the glandular epithelium
(orange), which was not seen in the prostate cancer
section (B). All experiments included negative
controls in which the primary antibody was omitted.
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Our results demonstrate a novel function for carbonyl reductase in
tumor growth and metastasis. We believe that loss of this enzyme by
genetic or epigenetic mechanisms in a metastatic cell renders it more
malignant in vivo than a cancer cell that expresses the
enzyme. Thus, an investigation of the precise mechanisms by which
carbonyl reductase modulates malignant progression, in particular, the
metastatic process, is now indicated.
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ACKNOWLEDGMENTS
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We thank our colleagues at the Beatson Institute for criticism
and encouragement, L. McGarry for densitometric analyses, and L. Hughes
for excellent secretarial assistance.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by grants from the Cancer Research
Campaign (to P. R. H. and G. D. B.). E. I. was supported by a
grant from the Government of Malaysia, and F. A-M. was supported by a
Fellowship from the Department of Pathology, University of Kuwait
Medical School. E. I. and F. A-M. contributed equally to this work. 
2 To whom requests for reprints should be
addressed. Present address: Department of Pathology, Kuwait
University, P. O. Box 24923, Safat 13110, Kuwait. E-mail: fahd{at}al-mulla.org
Received 10/25/99.
Accepted 1/20/00.
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REFERENCES
|
|---|
-
Price J. T., Bonovich M. T., Kohn E. C. The biochemistry of cancer dissemination. Crit. Rev. Biochem. Mol. Biol., 32: 175-253, 1997.[Medline]
-
Woodhouse E. C., Chuaqui R. F., Liotta L. A. General mechanisms of metastasis. Cancer (Phila.), 80: 1529-1537, 1997.[Medline]
-
Layton M. G., Franks L. M. Heterogeneity in a spontaneous mouse lung carcinoma: selection and characterisation of stable metastatic variants. Br. J. Cancer, 49: 415-421, 1984.[Medline]
-
Roninson I. B. Use of in-gel DNA renaturation for detection and cloning of amplified genes. Methods Enzymol., 151: 332-371, 1987.[Medline]
-
Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science (Washington DC), 257: 967-971, 1992.[Abstract/Free Full Text]
-
Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1987.
-
Suto K., Kajihara-Kano H., Yokoyama Y., Hayakiri M., Kimura J., Kumano T., Takahata T., Kudo H., Tsuchida S. Decreased expression of the peroxisomal bifunctional enzyme and carbonyl reductase in human hepatocellular carcinomas. J. Cancer Res. Clin. Oncol., 125: 83-88, 1999.[Medline]
-
Wermuth B., Mader-Heinemann G., Ernst E. Cloning and expression of carbonyl reductase from rat testis. Eur. J. Biochem., 228: 473-479, 1995.[Medline]
-
Jez J. M., Bennett M. J., Schlegel B. P., Lewis M., Penning T. M. Comparative anatomy of the aldo-keto reductase superfamily. Biochem. J., 326: 625-636, 1997.
-
Jarabak J. Polycyclic aromatic hydrocarbon quinone-mediated oxidation reduction cycling catalyzed by a human placental NADPH-linked carbonyl reductase. Arch. Biochem. Biophys., 291: 334-338, 1991.[Medline]
-
Gonzalez B., Akman S., Doroshow J., Rivera H., Kaplan W. D., Forrest G. L. Protection against daunorubicin cytotoxicity by expression of a cloned human carbonyl reductase cDNA in K562 leukemia cells. Cancer Res., 55: 4646-4650, 1995.[Abstract/Free Full Text]
-
Kelner M. J., Estes L., Rutherford M., Uglik S. F., Peitzke J. A. Heterologous expression of carbonyl reductase: demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance. Life Sci., 61: 2317-2322, 1997.[Medline]
-
Schieber A., Frank R. W., Ghisla S. Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase. Eur. J. Biochem., 206: 491-502, 1992.[Medline]
-
Schieber A., Ghisla S. Prostaglandin 9-ketoreductase from pig and human kidney: purification, properties and identity with human carbonyl reductase. Eicosanoids, 5(Suppl.): S37-S38, 1992.
-
Ablin R. J., Shaw M. W. Prostaglandin modulation of prostate tumor growth and metastases. Anticancer Res., 6: 327-328, 1986.[Medline]
-
Fulton A. M., Zhang S. Z., Chong Y. C. Role of the prostaglandin E2 receptor in mammary tumor metastasis. Cancer Res., 51: 2047-2050, 1991.[Abstract/Free Full Text]
-
Young M. R., Okada F., Tada M., Hosokawa M., Kobayashi H. Association of increased tumor cell responsiveness to prostaglandin E2 with more aggressive tumor behavior. Invasion Metastasis, 11: 48-57, 1991.[Medline]
-
Sheng H., Shao J., Morrow J. D., Beauchamp R. D., DuBois R. N. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res., 58: 362-366, 1998.[Abstract/Free Full Text]
-
Ben-Av P., Crofford L. J., Wilder R. L., Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett., 372: 83-87, 1995.[Medline]
-
Chiarugi V., Magnelli L., Gallo O. Cox-2, iNOS and p53 as play-makers of tumor angiogenesis (review). Int. J. Mol. Med., 2: 715-719, 1998.[Medline]
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Top
ABSTRACT
Introduction
) and 17 other mice injected s.c. with CMT170
cells (
). The regression lines show no significant correlation
between tumor volume and the number of metastases for either CMT167 or
CMT170 cells.
) or s.c. (
) injection
(P = 0.0001, Mann-Whitney rank-sum test).
Conversely, transfection of CMT170 with plasmid coding for antisense
carbonyl reductase mRNA (CMT 170 AS) had the opposite
effects (P = 0.0001), whereas
transfection with vector only had no effect on the cell behavior. The
results represent the mean ± SE for three independent
experiments, each of which contained 15 mice for i.v. injections and 15
mice for s.c. injections. b, as in
a, changes in primary tumor volume after s.c.
injection of cells (P = 0.036).
c, densitometric quantification of the carbonyl
reductase Western blot shown on the right.