From the Departamento de Bioquímica y
Biología Molecular, Facultad de Medicina, Instituto
Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo
and ¶ Servicio de Anatomía Patológica, Hospital
Clínico-IDIBAPS, 08036 Barcelona, Spain
Received for publication, January 19, 2001, and in revised form, February 27, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have identified and cloned a human fetal lung
cDNA encoding a new protein of the ADAM-TS family (a
disintegrin and metalloproteinase domain, with thrombospondin type-1 modules)
that has been called ADAM-TS12. This protein exhibits a domain
organization similar to the remaining family members including a
propeptide and metalloproteinase-like, disintegrin-like, and
cysteine-rich domains. However, the number and organization of the TS
repeats is unique with respect to other human ADAM-TSs. A total of
eight TS-1 repeats arranged in three groups are present in this novel
ADAM-TS. Analysis of intracellular processing of ADAM-TS12 revealed
that it is synthesized as a precursor molecule that is first activated
by cleavage of the prodomain in a furin-mediated process and
subsequently processed into two fragments of different size: a 120-kDa
N-terminal proteolytically active fragment containing the
metalloproteinase and disintegrin domains, and a 83-kDa C-terminal
fragment containing most of the TS-1 repeats. Somatic cell hybrid and
radiation hybrid mapping experiments showed that the human ADAM-TS12
gene maps to 5q35, a location that differs from all ADAM genes mapped
to date. Northern blot analysis of RNAs from human adult and fetal
tissues demonstrated that ADAM-TS12 transcripts are only detected at
significant levels in fetal lung but not in any other analyzed tissues.
In addition, ADAM-TS12 transcripts were detected in gastric carcinomas
and in tumor cell lines from diverse sources, being induced by
transforming growth factor- Cell-cell and cell-extracellular matrix interactions are essential
for the development and maintenance of an organism. Likewise, proteolysis of the extracellular matrix is of vital importance for a
series of tissue-remodeling processes occurring during both normal and
pathological conditions, such as tissue morphogenesis, wound healing,
inflammation, or tumor cell invasion and metastasis. These events are
mediated by a variety of cell surface adhesion proteins and proteases,
with different structural and functional characteristics (1). Among
them, a group of recently described proteins called ADAMs
(a disintegrin and
metalloproteinase domain) have raised considerable interest
because of their potential ability to perform both functions, adhesion
and proteolysis (2, 3). ADAMs were first associated with reproductive
processes like spermatogenesis and heterotypic sperm-egg binding and
fusion (4). However, over the last few years, the spectrum of
functional roles for ADAMs has considerably expanded to processes such
as myogenesis (5), osteoblast differentiation (6), and host defense
(7). Furthermore, some ADAM family members, including TACE
(tumor necrosis factor- The structural and functional complexity of the ADAM family of cellular
disintegrins has considerably grown after the finding of a series of
new members characterized by the presence of thrombospondin repeats in
their amino acid sequence. The first member of this subfamily,
ADAM-TS1, was identified as a consequence of its association with the development of cancer cachexia as well as with various inflammatory processes (16). Subsequently, the cloning of the cDNA
for procollagen I amino-proteinase, whose deficiency cause Ehlers-Danlos syndrome type VIIC in humans, revealed a significant degree of structural similarities with ADAM-TS1, and it was called ADAM-TS2 (17). ADAM-TS4 and ADAM-TS5/TS11, other members of this
subfamily of disintegrins containing thrombospondin motifs, have been
characterized as aggrecanases responsible for the degradation of
cartilage aggrecan in arthritic diseases (18, 19). ADAM-TS4 has been
found to be responsible for brevican cleavage in glioma cells, a
proteolytic cleavage that has been proposed to be critical in mediating
the invasiveness of these tumors (20). On the other hand, ADAM-TS8
(also called METH-2) and ADAM-TS1 have been characterized as proteins
with angio-inhibitory activity (21). Finally, other family members
identified in human tissues such as ADAM-TS3, ADAM-TS5, ADAM-TS6,
ADAM-TS7, and ADAM-TS9 have been only characterized at the structural
level, and their putative functional significance is still unclear (22,
23).
These recent findings have stimulated the search for new ADAMs
potentially associated with some of the conditions involving cell-cell
interactions or extracellular matrix degradation taking place during
both normal or pathological conditions (1-3). In this work, we have
examined the possibility that additional yet uncharacterized ADAMs
could be produced by human tissues, with the finding of a novel family
member, belonging to the ADAM-TS subfamily that we have called
ADAM-TS12. We describe the molecular cloning and complete nucleotide
sequence of a cDNA coding for this protein. We also report an
analysis of the intracellular processing and enzymatic activity of
ADAM-TS12. Finally, we describe the chromosomal location of the
ADAM-TS12 gene and analyze its expression and regulation in normal and
tumor tissues.
Materials--
A human fetal lung cDNA library constructed
in Isolation of a cDNA Clone for ADAM-TS12 from a Human
Fetal Lung cDNA Library--
A search of the GenBankTM
data base of human ESTs1 for
sequences with homology to members of the ADAM family led us to
identify a sequence (AI039653; WashU-Merck EST Project) derived from a
fetal lung cDNA clone and showing significant similarity with sequences of previously described ADAMs. To obtain this DNA fragment, we performed PCR amplification of a human fetal lung cDNA
(CLONTECH) with two specific primers
5'-CAACCCAGGAGGACATGTGA and 5'-TTCTCACGAGGAGAAGGACC, derived from the
AI039653 sequence. The PCR reaction was carried out in a GeneAmp 2400 PCR system from PerkinElmer Life Sciences for 40 cycles of denaturation
(94 °C, 15 s), annealing (64 °C, 20 s), and extension
(68 °C, 30 s). The 390-bp PCR product amplified from human
fetal lung cDNA was cloned, and its identity was confirmed by
nucleotide sequencing using the kit DR terminator Taq FS and the automatic DNA sequencer ABI-PRISM 310 (PerkinElmer Life Sciences). This cDNA was then excised from the vector, radiolabeled, and used
to screen the same fetal lung cDNA library according to standard procedures.
3'-Extension of Isolated cDNAs--
The 3'-ends of cloned
cDNAs were extended by successive cycles of rapid amplification of
cDNA ends (RACE) using RNA from human fetal lung and the
MarathonTM cDNA amplification kit
(CLONTECH), essentially as described by the
manufacturer. Each cycle of RACE allowed the extension of ~100-200
bp of cDNA toward the 3'-end. Finally, the full-length cDNA was
obtained by PCR amplification using the Expand Long PCR kit (Roche
Molecular Biochemicals). The PCR reactions were performed for 35 cycles
of denaturation (15 s at 94 °C), annealing (15 s at 64 °C), and
extension (3 min at 68 °C), with primers 5'-ATGCCATGTGCCCAGAGGAGCT and 5'-GGGCTTAGAGTTCTTTTGAC. Following gel purification, the
amplification product was cloned and sequenced.
Chromosomal Mapping--
DNA from a panel of
monochromosomal somatic cell hybrids containing a single human
chromosome in a mouse or hamster cell line background was PCR screened
for the presence of the genomic sequence flanked by the primers:
5'-TCCAGTCCGAGTAGATGCCAGTG and 5'-GTGCCACTGAGATGGCAGAGGGG. Amplification conditions were as follows: 35 cycles of denaturation (94 °C, 15 s), annealing (65 °C, 15 s), and extension
(68 °C, 2 min) using the Expand Long PCR kit. Radiation hybrid
mapping was carried out using the Genebridge 4 panel (Human Genome
Mapping Resource Center, Cambridgeshire, UK). DNA samples from this
panel (25 ng) were PCR screened for the presence of the genomic
sequences flanked by the primers 5'-CTTGAGCTCAGGGAGCTCATTCAT and
5'-GGGGAGGCTCTGATTTCTC AGCAA. Amplification conditions were as follows:
35 cycles of denaturation (94 °C, 15 s), annealing (68 °C,
15 s), and extension (72 °C, 1 min). PCR results were converted
to a vector of 93 0 values (no amplification), 1 values
(amplification), and 2 values (blanks and uncertainties) and
submitted to the mapping server of the Whitehead Institute/MIT Center
for Genome Research, with a minimum LOD (log of the odds) score
of 15.
Cell Culture--
Human cancer cells from different sources were
routinely maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO2.
Cells were subcultured weekly by incubation at 37 °C for 2 min with
0.0125% trypsin in 0.002% EDTA, followed by the addition of complete
medium, washing, and resuspension in fresh medium. For most
experiments, ~5 × 105 cells/well were plated out in
100-mm dishes, transferred to serum-free Dulbecco's modified Eagle's
medium for 24 h, and then exposed to the different cytokines and
growth factors at the concentrations and for the times indicated.
Extracellular matrix remaining on the dishes was extracted with Laemmli
sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 5%
mercaptoethanol, and 10% glycerol) and analyzed by SDS-polyacrylamide
gel electrophoresis.
Northern Blot Analysis--
Nylon filters containing 2 µg of
poly(A)+ RNA of a wide variety of human tissues were
prehybridized at 42 °C for 3 h in 50% formamide, 5× SSPE (1×
SSPE = 150 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7.4), 10×
Denhardt's solution, 2% SDS, and 100 µg/ml of denatured herring
sperm DNA and then hybridized with a radiolabeled ADAM-TS12-specific
1.8-kb-long probe, corresponding to the 5'-region of the cDNA.
Hybridization was performed for 20 h under the same conditions.
Filters were washed with 0.1× SSC, 0.1% SDS for 2 h at 50 °C
and exposed to autoradiography. RNA integrity and equal loading was
assessed by hybridization with an actin probe.
Reverse Transcription-PCR Amplification--
To assay the
presence of ADAM-TS12 in human tumor specimens, total RNA was isolated
from malignant tumors by guanidium thiocyanate-phenol-chloroform extraction and used for cDNA synthesis with the RNA PCR kit from PerkinElmer Life Sciences. After reverse transcription using 1 µg of
total RNA and random hexamers as primers according to the instructions
of the manufacturer, the whole mixture was used for PCR with two
ADAM-TS12-specific oligonucleotides (5'-AAGCATGCTCGGCGACATGCG and
5'-ACTGCGAATCCGCACTCCACC), as described above. The PCR products were
analyzed in 1.5% agarose gels. cDNA quality was verified by
performing control reactions with primers derived from the sequence of
actin. Negative controls were also performed in all cases by omitting
the template or reverse transcriptase.
Site-directed Mutagenesis--
The H465Q and E466A mutations in
the metalloproteinase domain of ADAM-TS12 were carried out by PCR-based
methods. An oligonucleotide containing a MumI sequence and
two mutations
5'-TTCACAATTGCCCAAGCGCTAGGACACAGC (with A and C indicating
changes T to A and A to C in the original sequence) and a second
oligonucleotide (5'-TTGCAGAGCCTCTCTCTGCGCTC) were first used to PCR
amplify a DNA fragment with the following conditions: 94 °C for 2 min (1 cycle) and 94 °C for 0.1 s, 60 °C for 0.1 s, and
68 °C for 30 s (20 cycles). The PCR product of the expected
size was digested with MumI and BstEII and cloned in pcDNA3. The presence of the mutations in the plasmid
(pcDNA3-ADAM-TS12-MUT) was confirmed by nucleotide sequencing.
Construction of Eukaryotic Expression Vectors and Western
Blot Analysis--
A full-length cDNA encoding ADAM-TS12 was PCR
amplified with oligonucleotides 5'-ATGCCATGTGCCCAGAGGAGCT and
5'-GGGCTTAGAGTTCTTTTGAC and cloned in the EcoRV site of a
modified pcDNA3 vector containing a 24-bp linker coding for the
hemagglutinin (HA) epitope of human influenza virus. Thus, the
resulting ADAM- TS12 protein was HA-tagged at the C terminus.
Similarly, two oligonucleotides (5'-GTCACTTGACTACAAGGACGACGATGACAAGGG and 5'-AACTGATGTTCCTGCTGCTACT GTTCCCCAGTG) were used to introduce the
FLAG epitope at the BstEII site at position 1642 of the
ADAM-TS12 cDNA. COS-7, HT-1080, or LoVo cells were transfected with
1 µg of plasmids pcDNA3-ADAM-TS12-HA, pcDNA3-ADAM- TS12-FLAG,
pcDNA3-ADAM-TS12-MUT, or pcDNA3 alone, using LipofectAMINE
reagent (Life Technologies, Inc.) according to the manufacturer's
instructions. Transfected cells were used for preparing protein
extracts, which were then analyzed by Western blot. Blots were
visualized by ECL according to the manufacturer's instructions
(Amersham Pharmacia Biotech).
In Vitro Transcription and Translation--
One microgram of
pcDNA3 containing the full-length ADAM-TS12 cDNA was
transcribed and translated using the coupled reticulocyte TNT T7 kit
(Promega) in the presence of [35S]methionine (Amersham
Pharmacia Biotech), following the manufacturer's instructions.
Parallel experiments were conducted using empty pcDNA3 as control.
Protein translation products were analyzed by SDS-polyacrylamide gel
electrophoresis followed by overnight autoradiography.
Enzymatic Assays--
The proteolytic activity of ADAM-TS12 was
determined using the Identification and Characterization of ADAM-TS12--
To identify
putative novel members of the ADAM family expressed in human tissues,
we used the BLAST algorithm to scan the GenBankTM data base
of ESTs looking for sequences with significant similarity to previously
described family members. This analysis allowed us to identify a 469-bp
EST that, after conceptual translation, generated an open reading frame
with significant amino acid sequence similarity to sequences found in
disintegrins purified from snake venom. A cDNA containing part of
this EST was obtained by PCR amplification of total
Further structural analysis of the identified amino acid sequence
showed a significant similarity with other human ADAMs, the maximum
percentage of identities being with members of the ADAM-TS subfamily
(57% with ADAM-TS7). An alignment of the deduced amino acid sequence
confirmed that this protein possesses all characteristic domains of the
ADAM-TS family members including signal sequence, propeptide, and
metalloproteinase-like, disintegrin-like and cysteine-rich domains, as
well as a series of TS-1 repeats (Fig. 1). However, the organization of
the TS-1 repeats of the identified sequence is unique to this protein
(Fig. 2). Thus, all previously described
human family members exhibit a first TS repeat 52 amino acids in length
that is separated from a C-terminal TS module through a cysteine-rich
domain and a spacer sequence. This second module is composed of a
single TS repeat in ADAM-TS5, ADAM-TS6, ADAM-TS7, and ADAM-TS8, two TS
repeats in ADAM-TS1, and three TS repeats in ADAM-TS2, ADAM-TS3, and
ADAM-TS9, whereas ADAM-TS4 lacks any additional repeat. The fetal lung
ADAM-TS described herein contains three TS repeats in this second TS
module, but, in addition, an extra C-terminal TS module can be
identified in its sequence. This third TS module is separated from the
second one by a spacer sequence (spacer-2) 319 amino acids long, and it
is formed by four TS repeats. This latter domain is followed by a
C-terminal extension rich in cysteine residues that shows similarities
with the C-terminal region of other ADAM-TSs. The additional domains
present in ADAM-TS12 are the cause of the large size of this novel
human disintegrin when compared with all remaining family members.
The alignment of the amino acid sequence deduced for ADAM-TS12 with
that of other human ADAM-TSs also revealed the presence of additional
structural hallmarks of the ADAM-TS family members (Figs. 1 and 2).
Thus, the putative proregion contains two conserved Cys residues (at
positions 139 and 160) as well as an additional one located in the
sequence PTCGLKD (positions 206-212)
that resembles the Cys switch motif
(PRCGXPD) present in the matrix
metalloproteinases and involved in maintaining enzyme latency. The
prodomain ends in a dibasic motif that could correspond to a furin
activation sequence for generation of the mature enzyme. The catalytic
domain includes the consensus sequence HEXXHXXGXXHD (at positions 392-403) involved in
the coordination of the catalytic zinc atom at the active site of
metalloproteinases and bearing the Asp residue that distinguishes
reprolysins from matrix metalloproteinases. This domain also contains
the eight cysteine residues present in the catalytic region of all
ADAM-TS family members as well as a conserved methionine residue
located 16 amino acids C-terminal to the zinc-binding site that
contributes to form the Met turn structure present in both reprolysins
and matrix metalloproteinases. Furthermore, the disintegrin-like domain is very similar in size (79 residues) to those found in most ADAM-TSs and contains the eight cysteines present in this region of ADAM-TS proteins with the exception of ADAM-TS6. Finally, the cysteine-rich domain shows a high percentage of sequence identity with the equivalent domain in other ADAM-TSs (59.6% with ADAM-TS7), including the 10 conserved cysteine residues present in all of them. Taking together all
of these structural comparisons, it seems that the newly identified
human protein is a member of the ADAM-TS family of disintegrins, albeit
with a more complex organization of thrombospondin repeats. Following
the nomenclature system for cellular disintegrins, the
officially approved name for this enzyme is ADAM-TS12.
Chromosomal Mapping of the Human ADAM-TS12 Gene--
To determine
the chromosomal location of the human ADAM-TS12 gene, we used a
PCR-based strategy to screen a panel of somatic cell hybrid lines
containing a single human chromosome in a rodent background. The
sequence-tagged site specific for the ADAM-TS12 gene was generated by
using two specific oligonucleotides whose sequence was derived from a
noncoding sequence flanking the second exon of the gene and from a
coding sequence of this same exon. As can be seen in Fig.
3A, positive amplification
results were only obtained in the hybrid containing the autosome number
5. Southern blot analysis confirmed that the amplified band
corresponded to ADAM-TS12 (Fig. 3A). Because no
amplification products of the expected size were observed in the
hybrids containing the remaining human chromosomes, we can conclude
that the ADAM-TS12 gene maps to chromosome 5. To further determine the
chromosomal location of the human gene encoding ADAM-TS12, we used the
same sequence-tagged site oligonucleotides to PCR screen a panel of
radiation hybrids containing human chromosome fragments in a rodent
background. Computer analysis of positive amplification results
indicated that the ADAM-TS12 gene is located in chromosome 5q35 at
54.47 cR from marker WI-6737 (Fig. 3B and data not shown).
Interestingly, ADAM-TS2 and ADAM-TS6 also lie on human chromosome 5, although they are not necessarily clustered. Thus, ADAM-TS2 is located at 5q23, and ADAM-TS6 is located at an undefined locus at this chromosome. However, all of the remaining ADAM-TS genes are dispersed in the human genome. Thus, ADAM-TS1 and ADAM-TS5/TS11 are linked on
21q21-q22, ADAM-TS3 maps to 4q21, ADAM-TS4 maps to 1q31, ADAM-TS8 (METH-2) maps to 11q25, ADAM-TS9 maps to 3p14, and ADAM-TS7 maps to an undefined locus at chromosome 15 (22, 23).
Intracellular Processing and Enzymatic Activity of
ADAM-TS12--
Members of the ADAM family of metalloproteinases are
synthesized as precursor molecules that are subjected to proteolytic processing-mediated events to generate the final active molecules. ADAM-TS12 contains putative Cys switch and furin cleavage motifs that
could be involved in the activation mechanism of this enzyme by removal
of the inhibitory prodomain. To analyze the intracellular processing of
ADAM-TS12, we first prepared a pcDNA3 expression vector
(ADAM-TS12-HA) containing the full-length cDNA for ADAM-TS12 with
an HA epitope at the 3'-terminal end. Western blot analysis of COS-7
cell extracts transiently transfected with this construct revealed the
presence of a band of about 83 kDa, immunoreactive against anti-HA and
absent in cells transfected with an empty vector (Fig.
4A). This size is considerably
lower than that of 175 kDa, which was derived for ADAM-TS12 after
in vitro transcription and translation experiments (Fig.
4B). According to these results, it seems that ADAM-TS12 is
extensively processed at the N-terminal end leading to the removal of a
considerable part of this region. Amino acid sequence analysis of
ADAM-TS12 allowed us to estimate that the putative processing site to
generate a protein of about 83 kDa would be necessarily located after
the cysteine-rich domain of the protein and around its first TS-1
domain. To further analyze this question, we prepared an additional
construct (pcDNA3-ADAM-TS12-FLAG) also containing the HA epitope at the
C-terminal end but including a FLAG epitope at the beginning of the
first TS-1 domain of ADAM-TS12 (position 548 of the protein). After
transfection of COS-7 cells with this double-labeled construct followed
by Western blot analysis, we first confirmed that the size of the
HA-immunoreactive band was identical (83 kDa) to that observed in the
single labeled construct (Fig. 4A). However, when the blot
was incubated with the anti-FLAG antiserum, we detected a major band of
about 120 kDa that was only present in the double-labeled ADAM-TS12
construct. A minor band of about 150 kDa was also detected in the same
cell extract (Fig. 4A). This product could correspond to an
incompletely processed protein. Similar processing events were obtained
when transfections were performed using HT-1080 instead of COS-7 cells (data not shown), indicating that the observed maturation events are
not exclusive of a single cell line. According to these results, we can
conclude that upon synthesis, ADAM-TS12 is subjected to an
intracellular maturation process leading to the generation of a
fragment containing the N-terminal region of the molecule including the
metalloproteinase, disintegrin-like, Cys-rich, and TS-1 domains and a
C-terminal fragment containing the spacer-2 and the four additional
TS-1 domains characteristic of ADAM-TS12.
To address the possibility that this proteolytic cleavage could be an
autocatalytic process mediated by the metalloproteinase domain of
ADAM-TS12, we next prepared an expression vector containing two point
mutations in the cDNA for this protein (pcDNA3-ADAM-TS12-MUT). These mutations would lead to the production of a protein with changes
in His and Glu residues essential for the catalytic activity of
metalloproteinases. However, after transfection of COS-7 cells with
this mutant construct and subsequent Western blot analysis, we did not
observe any difference in the 83-kDa anti-HA immunoreactive band (Fig.
4A). Therefore, we can conclude that the proteolytic event
leading to ADAM-TS12 processing is mediated by a cellular protease
distinct from ADAM-TS12 itself. A preliminary analysis of the ability
of a series of proteinase inhibitors to block this processing event
revealed that only BB-94 was able to partially inhibit the ADAM-TS12
maturation, suggesting that the protease involved in this step is a
metalloprotease (data not shown). Finally, we tried to evaluate the
location of the two fragments generated after ADAM-TS12 processing.
Western blot analysis of conditioned medium from COS-7 cells
transiently transfected with the different constructs (HA, FLAG, MUT)
failed to detect any HA- or FLAG-immunoreactive bands (Fig.
4A and not shown, respectively), indicating that none of the
ADAM-TS12-derived fragments was secreted into the cell culture medium.
By contrast, extracellular matrix preparations from COS-7 cells
transfected with these constructs and analyzed by Western blot with
anti-HA antiserum revealed in all cases the presence of the 83-kDa
immunoreactive band previously detected in total cell extracts (Fig.
4A). Similarly, when the same blot was hybridized with an
anti-FLAG antibody, a 120-kDa immunoreactive band corresponding to the
N-terminal region of the processed ADAM-TS12 was detected in the
construct containing this epitope (Fig. 4A). According to
these results, we can conclude that both fragments generated during
ADAM-TS12 maturation are anchored to the extracellular matrix.
We next tried to determine whether furin, a widely expressed proprotein
convertase, was involved in the first step of the ADAM-TS12 maturation.
To do that, we examined the processing of this protein in
furin-deficient LoVo cells. As shown in Fig. 4C, these cells
were unable to efficiently process ADAM-TS12, indicating that furin was
actually involved in the release of the ADAM-TS12 prodomain. Finally,
we evaluated the possibility that ADAM-TS12 could be an active
metalloprotease. To this purpose, we used the ADAM-TS12 Expression and Regulation in Human Tissues--
To
examine the expression of ADAM-TS12 in human tissues, Northern blots
containing poly(A)+ RNAs prepared from a variety of adult
tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testis, ovary, small intestine,
colon, and leukocytes) and fetal tissues (brain, lung, liver, and
kidney) were hybridized with a 1.8-kb cDNA specific for ADAM-TS12.
As shown in Fig. 5A, a
transcript of about 8 kb was exclusively detected in fetal lung. The
predominant expression of ADAM-TS12 in human fetal tissues suggests
that this novel enzyme could participate in some of the tissue
remodeling processes taking place in this tissue during physiological
conditions. In an effort to identify factors with the ability to
up-regulate ADAM-TS12 expression, human KMST fetal fibroblasts were
incubated for 24 h in the presence of a series of cytokines and
growth factors, and total cellular RNAs were purified and analyzed by
Northern blot using a specific ADAM-TS12 radiolabeled probe. As shown
in Fig. 5B, TGF-
To examine the possibility that ADAM-TS12 was produced by human tumors,
we performed a preliminary survey of the expression of this gene in a
commercially available matched tumor/normal expression array. These
analyses indicated that ADAM-TS12 was widely expressed in
gastrointestinal carcinomas but not in the paired normal tissues (data
not shown). To further extend this observation, we performed reverse
transcription-PCR amplification with RNAs obtained from a panel of
paired primary gastric carcinomas and adjacent normal mucosa. As
illustrated in Fig. 5C, which shows some representative
cases, ADAM-TS12 was overexpressed in a significant number of gastric
carcinomas (9 of 12) when compared with the low or undetectable levels
observed in the paired adjacent normal tissues, thus confirming and
extending the previous results obtained after hybridization with the
tumor/normal expression array. Finally, preliminary analysis of
ADAM-TS12 expression in other tumor samples and cancer cell lines from
different sources revealed that some colorectal, renal, and pancreatic
carcinomas, as well as HeLa, A549 lung carcinoma, and Burkitt's
lymphoma (Daudi) cells, also have the ability to overexpress this gene
(data not shown).
In this work we describe the finding of ADAM-TS12, a novel member
of the ADAM-TS subfamily characterized by its complex pattern of
thrombospondin domains and its restricted expression in normal human
tissues. The strategy followed to identify ADAM-TS12 was first based on
an extensive scanning of the EST data bases, looking for sequences with
similarity to previously characterized ADAM family members. A sequence
presumably encoding the disintegrin region of a new ADAM was
identified, PCR amplified from human fetal lung cDNA, and used to
screen a cDNA library from the same source. After screening of this
library and further RACE-3' experiments, a full-length cDNA coding
for ADAM-TS12 was finally isolated and cloned. Structural analysis of
the identified sequence for ADAM-TS12 shows that it exhibits a series
of protein domains characteristic of ADAM-TS proteins, including a
prodomain, a catalytic domain, and metalloproteinase-like,
disintegrin-like, and cysteine-rich domains, as well as a series of TS
repeats. However, the number and organization of these repeats are
unique to ADAM-TS12. Thus, it contains a total of eight TS repeats
organized in three modules, whereas all remaining human ADAM-TS
proteins contain from one to four repeats, organized in one or two
modules (Fig. 2 and Refs.16-23). An additional distinctive feature of
the structure determined for ADAM-TS derives from the presence of an
additional domain called the spacer-2 region, with an absence of
overall sequence similarity to proteins present in the data bases. The
pattern of ADAM-TS expression in human tissues is also somewhat
unusual. Thus, in this work we have provided evidence that expression
of this gene is highly restricted in normal human tissues. The
observation that ADAM-TS12 is predominantly expressed in fetal lung
suggests that it could be involved in extracellular matrix remodeling
processes occurring within the fetal lung during development. The
restricted expression of ADAM-TS12 in normal tissues also suggests that
this gene is highly regulated. A survey of factors that could control ADAM-TS12 expression has shown that TGF- In contrast with its restricted expression in normal human tissues,
ADAM-TS12 is widely expressed in gastric carcinomas and in cancer cells
of diverse origin. These findings are suggestive of a role for this
enzyme in the progression of these tumors as proposed for ADAM-TS4 in
the case of human gliomas (20). Recent studies have shown that other
metalloproteases like MT1-matrix metalloproteinase are overexpressed in
gastric carcinomas and that their levels correlate with the grade of
vascular invasion (30). Further studies will be required to evaluate
the clinical significance of the expression of ADAM-TS12 in gastric
carcinomas as well as to extend the preliminary observations indicating
that this protease can be also overproduced by other malignant tumors such as colorectal, renal, and pancreatic carcinomas. Finally, we have
determined the chromosomal location of the ADAM-TS12 gene in an attempt
to explore further associations between this gene and tumor processes.
According to our mapping studies, the ADAM-TS12 gene is located at the
telomeric region of the long arm of chromosome 5. It is of interest
that this region (5q35) has been found to be a recurrent site of
translocations in hematological malignancies (31, 32). In addition, a
putative tumor suppressor gene involved in the development of
hepatocellular carcinomas without liver cirrhosis has been mapped to
5q35-qter (33). Furthermore, it is also remarkable in the context of
ADAM-TS12 overexpression in gastric carcinomas that minisatellite
probes corresponding to 5q35 have detected loss of heterozygosity or
new mutant alleles in gastrointestinal cancers (34). The localization
of ADAM-TS12 in this region will be useful for future studies aimed at
exploring the possibility that this gene can be directly involved in
any of these 5q35 alterations associated with tumor processes and especially with those of the gastrointestinal tract.
In summary, our results indicated that ADAM-TS12 is a novel member of
the disintegrin family of metalloproteases that contains a complex
array of thrombospondin domains. Most of these repeats are released
after a series of sequential proteolytic processing events that may be
of relevance for the in vivo function of this protein.
Further studies, now in progress, will be required to determine whether
this intracellular processing of ADAM-TS12 contributes to the
regulation of its activity in processes involving tissue remodeling and
cell adhesion and migration, as recently demonstrated for other members
of this growing family of proteases (21, 29, 35).
in KMST human fibroblasts. These
data suggest that ADAM-TS12 may play roles in pulmonary cells during
fetal development or in tumor processes through its proteolytic
activity or as a molecule potentially involved in regulation of cell adhesion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-converting enzyme), ADAM-12 (meltrin
), and ADAM-23, have been suggested to play important roles in the
development and progression of inflammatory and tumor processes
(8-15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11, a human matched tumor/normal expression array, and Northern
blots containing polyadenylated RNAs from different adult and fetal
human tissues were from CLONTECH (Palo Alto, CA).
Human tumors were obtained from patients who had undergone surgery for
diverse malignancies at the Hospital Clinico, Barcelona, Spain (Banco
de Tejidos y Tumores/Servicio Anatomía Patológica).
Restriction endonucleases and other reagents used for molecular cloning
were from Roche Molecular Biochemicals. All media and supplements for
cell culture were obtained from Sigma, except for fetal calf serum,
which was from Roche Molecular Biochemicals.
2-macroglobulin complex formation
assay. Lysates from cells transfected with ADAM-TS12 were solubilized
in 25 mM Tris-HCl, pH 7.4, 0.5% sodium deoxycolate, 0.1%
SDS, 100 mM NaCl, and 1% Triton X-100. The
2-macroglobulin substrate was added at a final concentration of 0.25 units/ml and incubated at 37 °C for 16 h in the presence or absence of 1 mM EDTA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phage DNA
prepared from a human fetal lung cDNA library. The 390-bp PCR
amplified product was cloned, and its identity was confirmed by
nucleotide sequence analysis. Then the cloned fragment was radiolabeled
and used as a probe to screen the same fetal lung cDNA library
employed for the previous PCR amplification experiment. Upon screening
of ~1 × 106 plaque forming units, a single positive
clone was identified and characterized. DNA was isolated from this
positive clone (called FL1), and its nucleotide sequence was determined
by automatic DNA sequencing. This analysis revealed that the length of
FL1 was 1.8 kb and contained the entire 469-bp sequence corresponding to the AI039653 EST. A comparative analysis of the identified sequence
with that corresponding to other ADAMs suggested that it was incomplete
at the 3'-end. To extend the partial cDNA sequence toward the
3'-end, we performed 3'-RACE experiments using a specific oligonucleotide deduced from the end of the FL1 clone and RNA from
human fetal lung as a template. Successive rounds of 3'-RACE experiments performed in similar conditions led us finally to obtain a
fragment containing an in frame stop codon. Nucleotide sequencing of
several independently isolated subclones of the RACE reaction did not
reveal any differences between them. Computer analysis of the obtained
sequence (Fig. 1) revealed an open
reading frame coding for a protein of 1,543 amino acids with a
predicted molecular mass of 177.5 kDa (EMBL accession number
AJ250725).
View larger version (76K):
[in a new window]
Fig. 1.
Nucleotide and amino acid sequences of
ADAM-TS12 from human fetal lung. The zinc-binding site
characteristic of metalloproteinases and the eight thrombospondin
domains present in the amino acid sequence are shaded. The
putative N-glycosylation sites are indicated with
asterisks.
View larger version (30K):
[in a new window]
Fig. 2.
Domain organization of ADAM-TS12. The
domain structures of the remaining members of the human ADAM-TS
subfamily of disintegrins are also shown. The structure of ADAM-TS10
has not been reported yet, whereas ADAM-TS11 is identical to
ADAM-TS5.
View larger version (33K):
[in a new window]
Fig. 3.
Chromosomal location of the human ADAM-TS12
gene. A, 100 ng of total DNA from the 24 monochromosomal somatic cell lines was PCR amplified with primers
described under "Experimental Procedures." DNA digested with
EcoRI and HindIII (Marker III, Roche Molecular
Biochemicals) was used as a size marker. The lower panel
shows a Southern blot of the material amplified above hybridized with
an ADAM-TS12 specific probe. B, cytogenetic ideogram of
human chromosome 5. The position of ADAM-TS12 gene at 5q35 is
indicated.
View larger version (46K):
[in a new window]
Fig. 4.
Intracellular processing and enzyme activity
of ADAM-TS12. A, Western blot analysis of COS-7 cells,
conditioned medium, and extracellular matrix derived from these cells
transfected with the indicated constructs. HA,
pcDNA3-ADAM-TS12-HA; FLAG, pcDNA3-ADAM-TS12-FLAG;
MUT, pcDNA3-ADAM-TS12-MUT; C-, pcDNA3
alone; WT, wild type. MUT indicates the mutations
carried out in the metalloprotease domain: H392Q and E393A.
B, SDS-polyacrylamide gel electrophoresis analysis of
ADAM-TS12 cDNA after in vitro transcription and
translation. C, Western blot analysis of furin-deficient
LoVo colorectal carcinoma cells transfected with the
pcDNA3-ADAM-TS12-FLAG construct. D, enzymatic activity
of ADAM-TS12 using 2-macroglobulin as substrate.
Extracts from COS-7 cells transfected with pcDNA3-ADAM-TS12-FLAG
were incubated with purified
2-macroglobulin and
analyzed by Western blot with anti-FLAG antiserum. The high molecular
weight species generated by covalent binding between ADAM-TS12 and
2-macroglobulin are indicated by arrows. The
binding was abolished in the presence of 1 mM EDTA. In all
cases, the sizes of the molecular weight markers are shown to the
left.
2-macroglobulin trapping assay previously employed for
demonstrating the proteolytic activity of other ADAMs (24, 25). To do
that, extracts from COS-7 cells transfected with ADAM-TS12 tagged with the FLAG epitope were incubated with purified
2-macroglobulin and then analyzed by SDS-polyacrylamide
gel electrophoresis. As shown in Fig. 4D, at least two high
molecular weight species immunoreactive against the FLAG antiserum were
detected in cells containing ADAM-TS12 but not in control cells nor in
cells incubated in presence of EDTA, an inhibitor of
metalloproteinases. Furthermore, these high molecular weight complexes
reacted with an anti-
2-macroglobulin antibody (data not
shown), confirming that they resulted from the covalent binding of the
ADAM-TS12 protein to
2-macroglobulin.
clearly induced the expression of this
gene, whereas other growth factors and cytokines, including TGF-
,
IL-1
, IL-1
, acidic fibroblast growth factor, and epidermal growth
factor, did not have any significant effect on ADAM-TS12 expression by
KMST human fetal fibroblasts.
View larger version (56K):
[in a new window]
Fig. 5.
Analysis of ADAM-TS12 expression and
regulation in human tissues. A, Northern blot analysis
of ADAM-TS12 in a panel of fetal and adult human tissues. 2 µg of
poly(A)+ RNA prepared from the indicated tissues was
analyzed by hybridization with a 1.8-kb probe corresponding to the
5'-end of the cDNA for human ADAM-TS12. The positions of RNA size
markers are shown. B, effect of TGF- 1 and other cytokines
and growth factors on ADAM-TS12 expression in KMST human fibroblasts.
Northern blot analysis was performed using 10 µg of total RNA from
KMST cells incubated for 24 h in the presence of TGF-
1 (5 ng/ml), TGF-
(50 ng/ml), IL-1
and IL-1
(5 ng/ml), epidermal
growth factor (EGF; 10 ng/ml), and acidic fibroblast growth
factor (aFGF; 10 ng/ml). Filters shown in A and
B were subsequently hybridized with a
-actin probe to
ascertain equal RNA loading for the different samples. C,
reverse transcription-PCR analysis of ADAM-TS12 expression in paired
normal gastric mucosa and gastric tumors. A 230-bp fragment
corresponding to a segment of ADAM-TS12 was amplified with primers
indicated under "Experimental Procedures" in a volume of 50 µl,
and 10 µl of the reaction were separated on a 1.5% agarose gel run
in Tris borate-EDTA buffer. Amplification of
-actin was used to
ascertain RNA integrity and equal loading. C
indicates
negative control. Marker V (MV; Roche Molecular
Biochemicals) was used as a size marker.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is able to up-regulate ADAM-TS12 in human fibroblast cells from fetal origin, whereas other
factors such as TGF-
, IL-1
, IL-1
, acidic fibroblast growth factor, and epidermal growth factor do not show any apparent
up-regulatory effect on the transcription of this gene. The finding
that TGF-
, widely assumed to be inhibitory for matrix
metalloproteinases expression, is able to induce ADAM-TS12 is not
unprecedented because recent studies have shown that this growth factor
up-regulates expression of potent metalloproteases associated with
tumor progression such as gelatinase A and collagenase-3 (26, 27).
Nevertheless, recent works have also shown that the activity of ADAM-TS
proteins can be regulated at other levels, including activation through the putative Cys switch or through furin cleavage during secretion (25,
28). Therefore, in this work we have examined the intracellular processing mechanisms of ADAM-TS12. Studies with cells transfected with
the full-length ADAM-TS12 cDNA have led us to conclude that this
protein is synthesized as a precursor molecule that is first activated
by cleavage of the prodomain in a furin-mediated process and
subsequently processed into two fragments of different size. One of
these fragments corresponds to the N-terminal region of the molecule
and contains the metalloproteinase and disintegrin domains of
ADAM-TS12. This fragment is proteolytically active as assessed by its
ability to interact with
2-macroglobulin. The second
fragment generated during the intracellular processing of ADAM-TS12
corresponds to the C-terminal region of the protein and contains most
of the TS-1 repeats. To date, the functional significance of this
processing mechanism in the context of putative normal or pathological
roles of ADAM-TS12 is unclear. In this regard, we are interested in the
recent observation that ADAM-TS1 also undergoes a complex processing
mechanism, leading to the formation of two distinct active forms (29).
Preliminary attempts to identify substrates for the proteolytically
active fragment of ADAM-TS12 have not provided positive results.
Nevertheless, the peculiarities of ADAM-TS12 in terms of structural
organization and tissue distribution may imply the possibility that
their substrates may be distinct from those hydrolyzed by other
proteases of this family. Similarly, the possibility that the released
C-terminal fragment containing a number of TS-1 repeats could regulate
angiogenic processes will require further exploration.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. P. Freije and E. Campo for helpful comments and support, Drs. A. Muñoz (Instituto Investigaciones Biomédicas-Madrid, Spain) and M. Namba (Okayama University, Okayama, Japan) for providing cell lines, and C. Garabaya and F. Rodríguez for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Comisión Interministerial de Ciencia y Tecnología-Spain Grant SAF97-0258 and Plan Fondos Europeos para el Desarrollo Regional Grant 1FD97-0214. The Instituto Universitario de Oncología is supported by Obra Social Cajastur-Asturias.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ250725.
§ Recipient of a research contract from Ministerio de Educación y Ciencia, Spain.
To whom correspondence should be addressed: Departamento de
Bioquímica y Biología Molecular, Facultad de Medicina,
Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: 34-985-104201;
Fax: 34-985-103564; E-mail: CLO@correo.uniovi.es.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M100534200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EST, expressed sequence tag; bp, base pair(s); PCR, polymerase chain reaction; TS, thrombospondin; TGF, transforming growth factor; RACE, rapid amplification of cDNA ends; kb, kilobase(s); HA, hemagglutinin; IL, interleukin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Werb, Z. (1997) Cell 91, 439-442[Medline] [Order article via Infotrieve] |
2. | Wolfsberg, T. G., Primakoff, P., Myles, D. G., and White, J. M. (1995) J. Cell Biol. 131, 275-278[Medline] [Order article via Infotrieve] |
3. | Blobel, C. P. (1997) Cell 91, 589-592[CrossRef] |
4. | Wolfsberg, T. G., and White, J. M. (1997) Dev. Biol. 180, 389-401[CrossRef] |
5. |
Gilpin, B. J.,
Loeche, F.,
Mattei, M. G.,
Engvall, E.,
Albrechtsen, R.,
and Wewer, U. M.
(1998)
J. Biol. Chem.
273,
157-166 |
6. |
Inoue, D.,
Reid, M.,
Lum, L.,
Krätzschmar, J.,
Weskamp, G.,
Myung, Y. M.,
Baron, R.,
and Blobel, C. P.
(1998)
J. Biol. Chem.
273,
4180-4187 |
7. | Mueller, C. G., Rissoan, M. C., Salinas, B., Ait-Yahia, S., Ravel, O., Bridon, J. M., Briere, F., Lebecque, S., and Liu, Y. J. (1997) J. Exp. Med. 189, 655-663[CrossRef] |
8. | Emi, M., Katagiri, T., Harada, Y., Saito, H., Inazawa, J., Ito, I., Kasumi, F., and Nakamura, Y. (1993) Nat. Genet. 5, 151-157[Medline] [Order article via Infotrieve] |
9. |
Krätzschmar, J.,
Lum, L.,
and Blobel, C. P.
(1995)
J. Biol. Chem.
271,
4593-4598 |
10. |
Yavari, R.,
Adida, C.,
Bray-Ward, P.,
Brines, M.,
and Xu, T.
(1998)
Hum. Mol. Genet.
7,
1161-1167 |
11. |
Cal, S.,
Pérez-Freije, J. M.,
López, J. M.,
Takada, Y.,
and López-Otín, C.
(2000)
Mol. Biol. Cell
11,
1457-1469 |
12. |
Iba, K,
Albrechtsen, R.,
Gilpin, B. J.,
Loechel, F.,
and Wewer, U. M.
(1999)
Am. J. Pathol.
154,
1489-1501 |
13. | Wu, E. N., Croucher, P. I., and McKie, N. (1997) Biochem. Biophys. Res. Commum. 235, 437-442[CrossRef][Medline] [Order article via Infotrieve] |
14. | Black, R. A., Rauch, C. T., Kozlosky, J. J. P., Slack, J. L., Wolfson, M. F., Castner, B. J., Stocking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Bolani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J., and Cerretti, D. P. (1997) Nature 385, 729-733[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Izumi, Y.,
Hirata, M.,
Hasuwa, H.,
Iwamoto, R.,
Umata, T.,
Miyado, K.,
Tamai, Y.,
Kurisaki, T.,
Sehara-Fujisawa, A.,
Ohno, S.,
and Mekada, E.
(1998)
EMBO J.
17,
7260-7272 |
16. |
Kuno, K.,
Kanada, N.,
Nakashima, E.,
Fujiki, F.,
Ichimura, F.,
and Matsushima, K.
(1997)
J. Biol. Chem.
272,
556-562 |
17. | Colige, A., Sieron, A. L., Li, S. W., Schwarze, U., Petty, E., Wertelecki, W., Wilcox, W., Krakow, D., Cohn, D. H., Reardon, W., Byers, P. H., Lapiere, C. M., Prockop, D. J., and Nusgens, B. V. (1999) Am. J. Hum. Genet. 65, 308-317[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Tortorella, M. D.,
Burn, T. C.,
Pratta, M. A.,
Abbaszade, I.,
Hollis, J. M.,
Liu, R.,
Rosenfeld, S. A.,
Copeland, R. A.,
Decicco, C. P.,
Wynn, R.,
Rockwell, A.,
Yang, F.,
Duke, J. L.,
Solomon, K.,
George, H.,
Bruckner, R.,
Nagase, H.,
Itoh, Y.,
Ellis, D. M.,
Ross, H.,
Wiswall, B. H.,
Murphy, K.,
Hillman, M. C., Jr.,
Hollis, G. F.,
Newton, R. C.,
Magolda, R. L.,
Trzaskos, R. M.,
and Arner, E. C.
(1999)
Science
284,
1664-1666 |
19. |
Abbaszade, I.,
Liu, R. Q.,
Yang, F.,
Rosenfeld, S. A.,
Ross, O. H.,
Link, J. R.,
Ellis, D. M.,
Tortorella, M. D.,
Pratta, M. A.,
Hollis, J. M.,
Wynn, R.,
Duke, J. L.,
George, H. J.,
Hillman, M. C., Jr.,
Murphy, K.,
Wiswall, B. H.,
Copeland, R. A.,
Decicco, C. P.,
Bruckner, R.,
Nagase, H.,
Itoh, Y.,
Newton, R. C.,
Magolda, R. L.,
Trzaskos, J. M.,
and Burn, T. C.
(1999)
J. Biol. Chem.
274,
23443-23450 |
20. |
Matthews, R. T.,
Gary, S. C.,
Zerillo, C.,
Pratta, M.,
Solomon, K.,
Arner, E. C.,
and Hockfield, S.
(2000)
J. Biol. Chem.
275,
22695-22703 |
21. |
Vazquez, F.,
Hastings, G.,
Ortega, M. A.,
Lane, T. F.,
Oikemus, S.,
Lombardo, M.,
and Iruela-Arispe, M. L.
(1999)
J. Biol. Chem.
274,
23349-23357 |
22. |
Hurskainen, T. L.,
Hirohata, S.,
Seldin, M. F.,
and Apte, S. S.
(1999)
J. Biol. Chem.
274,
25555-25563 |
23. | Melody, E. C., Gregory, S. K., Laurie, A. T., Antonia, B., Karen, C. A., and Richard, A. M. (2000) Genomics 67, 343-350[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Kuno, K.,
Terashima, Y.,
and Matsushima, K.
(1999)
J. Biol. Chem.
274,
18821-18826 |
25. |
Loechel, F.,
Overgaard, M. T.,
Oxvig, C.,
Albrechtsen, R.,
and Wewer, U. M.
(1999)
J. Biol. Chem.
274,
13427-13433 |
26. |
Overall, C. M.,
Wrana, J. L.,
and Sodek, J.
(1991)
J. Biol. Chem.
266,
14064-14071 |
27. |
Uría, J. A.,
Jiménez, M. G.,
Balbín, M.,
Freije, J. M. P.,
and López-Otín, C.
(1998)
J. Biol. Chem.
273,
9769-97677 |
28. |
Roghani, M.,
Becherer, J. D.,
Moss, M. L.,
Atherton, R. E.,
Erdjument- Bromage, H.,
Arribas, J.,
Blackburn, R. K.,
Weskamp, G.,
Tempst, P.,
and Blobel, C. P.
(1999)
J. Biol. Chem.
274,
3531-3540 |
29. |
Rodriguez-Manzaneque, J. C.,
Milchanowski, A. B.,
Dufour, E. K.,
Leduc, R.,
and Iruela-Arispe, M. L.
(2000)
J. Biol. Chem.
275,
33471-33479 |
30. | Nomura, H., Sato, H., Seiki, M., Mai, M., and Okada, Y. (1995) Cancer Res. 55, 3263-3266[Abstract] |
31. | Johansson, B., Brondum-Nielsen, K., Billstrom, R., Schiodt, I., and Mitelman, F. (1997) Cancer Genet. Cytogenet. 99, 97-101[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Jaju, R. J.,
Haas, O. A.,
Neat, M.,
Harbott, J.,
Saha, V.,
Boultwood, J.,
Brown, J. M.,
Pirc-Danoewinata, H.,
Krings, B. W.,
Muller, U.,
Morris, S. W.,
Wainscoat, J. S.,
and Kearney, L.
(1999)
Blood
94,
773-780 |
33. | Ding, S. F., Habib, N. A., Dooley, J., Wood, C., Bowles, L., and Delhanty, J. D. (1991) Br. J. Cancer 64, 1083-1087[Medline] [Order article via Infotrieve] |
34. | Nagel, S., Borisch, B., Thein, S. L., Oestreicher, M., Nothinger, F., Birrer, S., Tobler, A., and Fey, M. F. (1995) Cancer Res. 55, 2866-2870[Abstract] |
35. | Blelloch, R., and Kimble, J. (1999) Nature 399, 586-590[CrossRef][Medline] [Order article via Infotrieve] |