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Carcinogenesis |
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Departments of Medicine and Pathology, Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GT11 cDNA expression library derived
from HepG2 HCC cells was screened and the FB-50 mAb
produced against the FOCUS cell line was found to recognize an epitope
on a protein encoded by an AAH (HAAH) cDNA. The HAAH enzyme has
subsequently been found to be up-regulated in several different human
transformed cell lines and tumor tissues as compared with the adjacent
human tissue counterpart. More important was the finding that the
overexpressed enzyme in different human malignant tissues was
catalytically active (2)
.
The HAAH is a protein belonging to the 
-ketoglutarate-dependent
dioxygenase family of prolyl and lysyl hydroxylases, which play a key
role in collagen biosynthesis. This molecule hydroxylates aspartic acid
or asparagine residues in certain EGF-like domains of several proteins
in the presence of ferrous iron. These EGF-like domains contain
conserved motifs that form repetitive sequences in diverse proteins,
such as clotting factors, extracellular matrix proteins, low-density
lipoprotein receptor, Notch homologues or Notch ligand homologues
(3, 4, 5)
. It is believed that EGF-like sequences play an
important role in protein-protein interactions, as shown by mutations
in EGF-like domains of fibrillin that cause Marfan’s syndrome or
factor IX, which produces hemophilia B (6
, 7)
.
In this study, HAAH gene expression was examined in proliferating bile ducts because our previous findings demonstrated that this gene was overexpressed in 100% of human cholangiocarcinomas. We also determined whether overexpression of HAAH in NIH-3T3 cells led to the generation of the malignant phenotype, as measured by the formation of transformed foci, growth in soft agar as an index of anchorage independent growth, and tumor formation in nude mice. Furthermore, we explored the role of enzymatic activity in the induction of transformed phenotype by using a cDNA construct with a mutation in the catalytic site that abolished hydroxylase activity, as described previously (8) . Taken together, our results are consistent with the hypothesis that overexpression of the HAAH gene is associated with malignant transformation of bile ducts.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Constructs.
The murine full-length AAH construct (pNH376) and the site-directed
mutation construct (pNH376-H660) with abolished catalytic activity were
cloned into the eukaryotic expression vector pcDNA3 (Invitrogen Corp.,
San Diego, CA), the full-length HAAH was cloned into prokaryotic
expression vector pBC-SK+ (Stratagene, La Jolla, CA), and
these constructs were a generous gift of Dr. Joseph Dinchuk (DuPont
Pharmaceuticals, Wilmington, DE). The full-length human AAH construct
has been described previously (2)
, and it was subcloned
into the EcoRI site of the pcDNA3 vector. The protein
sequence homology between murine and human AAH is 80%. The pLNCX-UP1
construct that encodes v-src has been described previously
(9)
.
Animal Model of Bile Duct Proliferation.
Rats were divided into nine separate groups of three animals each
except for group 9, which contained five rats. Group 1 served as the
nonsurgical and group 2 as sham-operated surgical controls. The
remaining groups underwent common bile duct ligation to induce
intrahepatic bile duct proliferation and were evaluated at 6, 12, 24,
and 48 h and 4, 8, and 16 days, as shown in Table 1
. Animals were asphyxiated with CO2, and liver samples were
taken from left lateral and median lobes, fixed in 2%
paraformaldehyde, and embedded in paraffin. Liver samples (5 µm) were
cut and stained with H&E to evaluate intrahepatic bile duct
proliferation. Immunohistochemistry was performed with polyclonal
anti-HAAH antibodies that cross-react with the rat protein to determine
levels of protein expression.
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Immunohistochemistry.
Liver tissue sections (5 µm) were deparaffinized in xylene and
rehydrated in graded alcohol. Endogenous peroxidase activity was
quenched by a 30-min treatment with 0.6% H2O2
in 60% methanol. Endogenous biotin was masked by incubation with
avidin-biotin blocking solutions (Vector Laboratories, Burlingame, CA).
The FB-50 mAb (for PSC samples) and polyclonal anti-AAH-hydroxylase
antibodies (for rat liver samples) were added to slides in a humidified
chamber at 4°C overnight. Immunohistochemical staining was performed
using the avidin-biotin horseradish peroxidase complex (ABC) method
using Vectastain kits with diaminobenzidine as the chromogen, according
to the manufacturer’s instructions (Vector Laboratories, Inc.). Tissue
sections were counterstained with hematoxylin, followed by dehydration
in ethanol. Sections were examined by light microscopy for bile duct
proliferation and AAH protein expression. Paraffin sections of
cholangiocarcinoma and placenta served as positive control
(2)
, and hepatosteatosis samples served as a negative
control. To control for antibody binding specificity, adjacent sections
were immunostained with the primary antibody omitted or using
nonrelevant antibody to Dengue virus (10)
. As a positive
control for tissue immunoreactivity, adjacent sections of all specimens
were immunostained with monoclonal antibody to glyceraldehyde
3-phosphate dehydrogenase.
Western Blot Analysis.
Cell lysates were prepared in RIPA buffer containing protease
inhibitors as described previously (2)
. The total amount
of protein in the lysates was determined by Bio-Rad colorimetric assay
(Bio-Rad, Hercules, CA), followed by 10% SDS-PAGE and subsequent
transfer to polyvinylidene difluoride membranes, and subjected to
Western blot analysis using FB-50, HBOH2, and anti-Erk-1 (used as an
internal control for protein loading) as primary antibodies and sheep
antimouse and donkey antirabbit antisera labeled with horseradish
peroxidase as secondary antibodies, as described (11)
.
Antibody binding was detected with enhanced chemiluminescence reagents
(SuperSignal; Pierce Chemical Co., Rockford, IL) and film
autoradiography. The levels of immunoreactivity were measured by volume
densitometry using NIH Image software.
Enzymatic Activity Assay.
AAH activity was measured in cell lysates using the first EGF-like
domain of bovine protein S as substrate, where 14C-labeled
-ketogluterate hydroxylates the domain, releasing
14C-containing CO2, as described previously
(4
, 12
, 13)
. Incubations were carried out at 37°C for 30
min in a final volume of 40 µl containing 48 µg of crude cell
extract protein and 75 µM EGF substrate.
Cell Transfection Studies.
The NIH-3T3 cells were cultured in DMEM (Mediatech, Washington, DC)
supplemented with 10% heat-inactivated FCS (Sigma Chemical Co., St.
Louis, MO), 1% L-glutamine, 1% nonessential amino acids,
and 1% penicillin-streptomycin (Life Technologies, Inc., Grand Island,
NY). Subconfluent NIH-3T3 cells (3 x 105
cells/60-mm dish) were transfected with 10 µg of one of the following
plasmids: (a) nonrecombinant pcDNA3 vector (Invitrogen
Corp., San Diego, CA) as a negative control; (b)
pNH376-H660, the murine AAH cDNA was mutated at histidine position 660
to lysine in the catalytic domain and cloned into the pcDNA3 vector
driven by a cytomegalovirus promoter; (c) pNH376, the
wild-type murine AAH cDNA cloned into the pcDNA3 vector; (d)
pCDHH, wild-type human AAH cDNA cloned into the pcDNA3 vector; or
(e) pLNCX-UP1, a cDNA that encodes v-Src oncogene
(positive control). Cells were transfected using the calcium phosphate
transfection kit according to the manufacturer’s instructions (5
Prime-3 Prime, Inc., Boulder, CO). Comparison of cellular transfection
efficiency was assessed with the various constructs. For this
procedure, confluent plates obtained 48 h after transfection were
split and reseeded into 12 separate 6-cm dishes, and 6 of them were
made to grow in the presence of 400 µg/ml G-418 (Life Technologies,
Inc.)-containing medium. The number of G-418-resistant foci was
determined at 14 days after transfection and used to correct for any
variability in transfection efficiency.
Transformation Assay.
The NIH-3T3 cells were transfected with the various constructs and
allowed to reach confluence after 48 h as described above. Each
6-cm dish was split and seeded into 12 different 6-cm dishes. While six
of them were made to grow in the presence of G-418 to detect
transfection efficiency, the other six were grown in complete medium
without G-418 and with a medium change every fourth day. The number of
transformed foci were counted in these plates without G-418 and
expressed as transformed foci/µg of transfected DNA. Transfection
efficiency was corrected for as described above.
Anchorage-independent Cell Growth Assay.
A limiting dilution technique (0.15 cell/well of a flat-bottomed
96-well-plate) was performed on transfectants grown in G-418 to isolate
cell clones with different levels of HAAH activity, as measured by
Western blot analysis and enzymatic assay of hydroxylase activity.
Cloned cell lines (1.0 x 104 cells) were
suspended in complete medium containing 0.4% low-melting agarose
(SeaPlaque GTG Agarose; FMC Bioproducts, Rockland, ME) and laid over a
bottom agar mixture consisting of complete medium with 0.53%
low-melting agarose. Each clone was assayed in triplicate. The clones
were seeded under these conditions, and 10 days later the size
(positive growth >0.1 mm in diameter) and number of foci were
determined.
Tumorigenicity in Nude Mice.
The same clones, as assessed in the anchorage-independent growth assay,
were injected into nude mice and observed for tumor formation.
Tumorigenicity was evaluated using 10 animals in each of four groups
(Charles River Laboratory, Wilmington, MA) as described previously
(14)
. Group 1 received 1 x 107
cells stably transfected with mock DNA, groups 2–4 received
1 x 107 cells of clones stably transfected
with pNH376 and expressing various levels of murine AAH protein. Nude
mice were kept under pathogen-free conditions in the animal facility of
the Massachusetts General Hospital. Thirty days after tumor cell
inoculation, the animals were sacrificed using isofluorane (Aerrane,
Anaquest, NJ)-containing chambers, and the tumors were carefully
removed and the weights were determined.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Immunohistochemical staining failed
to detect the presence of AAH in proliferating bile ducts at any time,
as shown by the representative example depicted in Fig. 1, E and F
. Analysis of AAH expression in bile ducts derived from sham surgical
controls was also negative, whereas all samples exhibited positive
immunoreactivity with antibodies to glyceraldehyde 3-phosphate
dehydrogenase (data not shown). Thus, bile duct proliferation was not
associated with increased HAAH expression in this animal model system.
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. Adjacent sections immunostained with the FB-50 mAb had
no detectable HAAH immunoreactivity in proliferating bile ducts (Fig. 1D)
. In contrast, sections of cholangiocarcinoma that were
immunostained simultaneously using the same antibody and detection
reagents manifested intense levels of HAAH immunoreactivity in nearly
all tumor cells (Fig. 1A)
as described previously, whereas
adjacent sections of the cholangiocarcinomas exhibited a negative
immunostaining reaction with mAb to Dengue virus (Fig. 1B)
.
Therefore, these findings suggest that HAAH expression was more
associated with malignant transformation rather than cellular
proliferation of intrahepatic bile ducts.
HAAH-associated Transformation of NIH-3T3 Cells.
The transforming capability of the murine AAH and
HAAH genes, as well as the murine AAH mutant construct
without enzymatic activity, were compared with mock DNA (negative
control) and v-Src-transfected NIH-3T3 cells (positive control). This
assay was repeated five times. The transforming capability of murine
AAH was found to be two to three times that of vector DNA control, as
shown in Fig. 2C
. The transforming capacity of the human
gene was greater than that observed with the murine AAH (32 ± 1.5 versus 13 ± 2.6 transformed
foci, respectively). The murine AAH- and HAAH-transfected cells formed
large foci (Fig. 2B)
, resembling those of v-Src-transfected fibroblasts, as compared with the
occasional much smaller foci observed in cells transfected with vector
DNA that displayed the contact inhibition of fibroblast cell lines
(Fig. 2A)
. More importantly, parallel experiments performed
using the mutant pNH376-H660 construct without enzymatic activity
revealed no transforming activity, as shown in Fig. 2, A and C
. This finding suggests that the enzymatic activity may be
responsible, in part, for the transforming activity exhibited by the
HAAH gene.
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. This level of AAH was similar to that found in the foci generated
during the colony formation assay by transient transfection of the cDNA
(data not shown). Protein loading was equivalent in all lanes, as shown
by immunoblotting of the same membranes with an anti-Erk-1 monoclonal
antibody (data not shown). These experiments were repeated three times
with similar results. We confirmed that the increased protein
expression was associated with increased enzymatic activity, as shown
in Fig. 4A
. The capability of these clones to exhibit anchorage-independent cell
growth in soft agar is presented in Fig. 4B
. All three
clones with increased AAH gene expression demonstrated
anchorage-independent cell growth compared with the mock
DNA-transfected clone.
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depicts a representative comparison of the tumor size exhibited by
clone 18, as compared with a mock DNA-transfected clone. Clones 7, 16,
and 18 were highly transformed in this assay and produced large tumors
with a mean weight of 2.5, 0.9, and 1.5 g, respectively (Fig. 5B)
. Taken together, these observations suggest that
overexpression of AAH was associated with induction and/or maintenance
of the malignant phenotype in vivo.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether AAH expression was associated with malignancy
rather than increased cell turnover, we studied two models of bile duct
proliferation. In the animal model, ligation of the common bile duct
induced extensive intrahepatic bile duct proliferation, yet there was
no evidence of AAH gene expression under these experimental
conditions, as shown in Table 1
and Fig. 1, E and F
. Similarly, HAAH gene expression was assessed
in a human disease model associated with bile duct proliferation
because PSC is an autoimmune liver disease associated with destruction
as well as proliferation of the intra- and extrahepatic bile ducts. It
is also noteworthy that PSC is considered to be a premalignant disease,
and a significant proportion of affected individuals will eventually
develop cholangiocarcinoma (15)
. However, we found no
evidence for increased HAAH gene expression in the presence
of extensive bile duct proliferation, as shown in Fig. 1, C and D
.
Having established that HAAH protein levels were elevated in
cholangiocarcinoma (Fig. 1, A and B)
and not in
normal or proliferating bile ducts, it became of interest to directly
assess a role of the enzyme in the generation of a malignant phenotype.
To do this, we transfected the HAAH gene into NIH-3T3 cells
and studied cellular changes, such as increased formation of
transformed foci, colony growth in soft agar, and tumor formation in
nude mice associated with malignant transformation (14)
.
The full-length murine AAH and HAAH genes were
cloned into expression constructs and transiently transfected into
NIH-3T3 cells. We observed an increased number of transformed foci in
cells transfected with both the murine AAH and
HAAH genes as compared with mock DNA-transfected controls.
The increased number of transformed foci, after controlling for
transfection efficiency, was not as high compared with v-Src
gene-transfected cells used as a positive control. It is of interest
that the transforming activity of the HAAH cDNA appeared to
be greater than the murine counterpart, perhaps because endogenous
cellular mechanisms required to regulate HAAH expression and activity
are somewhat species specific. More importantly, the enzymatic activity
of the AAH gene was important because a mutant construct
that abolished the catalytic site had no transforming properties under
these experimental conditions. Next, we established several stably
transfected and cloned NIH-3T3 cell lines with a modest increase in AAH
protein levels and enzymatic activity. Such cell lines were placed in
soft agar to examine anchorage-independent cell growth as another
property of the malignant phenotype. We found that all cell lines grew
in soft agar compared with mock DNA-transfected control. The
correlation between the cellular level of AAH gene
expression and the number and size of colonies formed was not perfect,
but there was a general trend toward more numerous and larger colonies
with higher levels of AAH. It then became of interest to determine
whether these three of these cloned cell lines formed tumors in nude
mice. All three cell lines with increased AAH expression were
oncogenic, as shown by the development of large tumors (Fig. 5)
as
another well-known characteristic of the transformed phenotype
(14)
.
The only known function previously attributed to this
Mr 85,000 protein is hydroxylation of EGF-like
domains in a variety of proteins (3, 4, 5)
. Thus, we
determined whether the cellular changes induced by overexpression of
AAH were related to the enzymatic function. A site-directed mutation
was introduced into the gene that changed the ferrous iron binding site
from histidine to lysine at the 660th position, abolishing hydroxylase
activity of the murine AAH as described previously (8)
.
The pNH376-H660 construct had no transformation activity, as shown in
Fig. 2
, indicating that cellular changes of the malignant phenotype
induced by overexpression may depend, in part, on the biological
activity of the protein. The molecular mechanisms of how HAAH
participates in cellular transformation are unknown. Such EGF-like
domains are found as conserved motifs of several different proteins,
some of which are transforming, such as Notch (16, 17, 18)
.
It will be important in the future to develop assays reflecting the state of Notch protein hydroxylation in the context of overexpression of AAH because EGF-like domains may be involved in receptor-ligand interactions. Indeed, point mutations affecting aspartic acid or asparagine residues in EGF-like domains that are the targets for ß-hydroxylation by AAH reduce calcium binding and therefore could influence protein-protein interactions and thereby influence activation of downstream signal transduction pathways (19) .
The data presented herein provide substantial evidence that high-level HAAH expression is linked to malignant transformation. We demonstrated that overexpression of the AAH cDNAs in NIH-3T3 cells induces a transformed phenotype manifested by increased numbers of transformed foci, anchorage-independent growth, and tumorigenesis in nude mice. In addition, intact AAH enzyme was found to be required for AAH-associated transformation. Of particular interest were the findings that only modest increases in AAH expression and enzyme activity were required for cellular transformation. Small differences in the levels of an extracellular signaling molecule can specify cell fate during development and possibly carcinogenesis. Threshold responses are often determined at the level of transcription, and previous studies have demonstrated that HAAH has been up-regulated at the transcriptional level (2 , 20) . These results suggest that increased AAH gene expression and enzyme activity contribute to the generation or maintenance of the transformed phenotype (1) and are not strictly related to cellular proliferation. Finally, there is very little available data concerning genes that are up-regulated with malignant transformation of bile duct epithelium, and HAAH immunoreactivity is detectable on tumor cell surface membranes (2) . Assay of HAAH protein levels in either biological fluids such as bile or cells obtained by fine-needle aspiration deserves further attention as a possible diagnostic marker of human cholangiocarcinoma in future.
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FOOTNOTES |
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1 Supported in part by NIH Grants CA-35711 and
AA-02666. 
2 To whom requests for reprints should be
addressed, at The Liver Research Center, Rhode Island Hospital and
Brown University School of Medicine, 55 Claverick Street, Providence,
RI 02903. Phone: (401) 444-2795; Fax: (401) 444-2939; Email: Jack_Wands_MD{at}Brown.edu
3 The abbreviations used are: HCC, hepatocellular
carcinoma; HAAH, human aspartyl (asparaginyl) ß-hydroxylase; EGF,
epidermal growth factor; mAb, monoclonal antibody; PSC, primary
sclerosing cholangitis.
Received 7/28/99. Accepted 1/ 5/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ketoglutarate-dependent dioxygenases. Proc. Natl. Acad. Sci. USA, 91: 7227-7231, 1994.This article has been cited by other articles:
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  S. P. Ho, M. S. Scully, C. M. Krauthauser, E. J. Wexler, M. D. Stow, J. E. Dinchuk, J. S. Kerr, and P. A. Friedman Antisense Oligonucleotides Selectively Regulate Aspartyl beta -Hydroxylase and Its Truncated Protein Isoform in Vitro but Distribute Poorly into A549 Tumors in Vivo J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 795 - 803. [Abstract] [Full Text] [PDF]
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 W. G. Kaelin Jr. How oxygen makes its presence felt Genes & Dev., June 15, 2002; 16(12): 1441 - 1445. [Full Text] [PDF]
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 J. E. Dinchuk, R. J. Focht, J. A. Kelley, N. L. Henderson, N. I. Zolotarjova, R. Wynn, N. T. Neff, J. Link, R. M. Huber, T. C. Burn, et al. Absence of Post-translational Aspartyl beta -Hydroxylation of Epidermal Growth Factor Domains in Mice Leads to Developmental Defects and an Increased Incidence of Intestinal Neoplasia J. Biol. Chem., April 5, 2002; 277(15): 12970 - 12977. [Abstract] [Full Text] [PDF]
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J. E. Dinchuk, N. L. Henderson, T. C. Burn, R. Huber, S. P. Ho, J. Link, K. T O'Neil, R. J. Focht, M. S. Scully, J. M. Hollis, et al. Aspartyl beta -Hydroxylase (Asph) and an Evolutionarily Conserved Isoform of Asph Missing the Catalytic Domain Share Exons with Junctin J. Biol. Chem., December 8, 2000; 275(50): 39543 - 39554. [Abstract] [Full Text] [PDF]
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