From the La Jolla Institute for Allergy and
Immunology, San Diego, California 92121 and § Human Genome
Sciences, Inc., Rockville, Maryland 20850
Received for publication, December 4, 2000, and in revised form, March 29, 2001
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ABSTRACT |
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Galectins are a family of Galectins are members of a large family of animal lectins defined
by their shared consensus sequences and Diverse biological functions have been demonstrated for various
galectins in vitro, including extracellular functions that are consistent with the lectin properties. For example, galectin-1 and
-3 have been shown to activate various cell types, through cross-linkage of appropriate cell surface glycoproteins, and to modulate cell adhesion, probably through interactions with cell surface
glycoproteins, including cell adhesion molecules (reviewed in Refs. 3
and 5). Galectin-3 has been shown to promote neurite growth (8) and
induce differentiation and angiogenesis of endothelial cells (9).
Galectin-3 is also a chemoattractant for monocytes (10) and endothelial
cells (9), while galectin-9 is a chemoattractant for eosinophils (11).
Galectin-1 and -9 have been demonstrated to induce apoptosis of T-cells
and thymocytes (12-14), via recognition of cell surface glycoproteins.
Intracellular functions of galectins are also noteworthy. Consistent
with the absence of a classical signal sequence, many galectins are
found to primarily reside intracellularly. Moreover, the presence of some, such as galectin-1 and -3, in the nuclei of proliferating cells
has been reported (15, 16). Both galectin-1 and -3 have been shown to
be active in vitro in inducing pre-mRNA splicing (17,
18). Galectin-3 has been found to suppress apoptosis, possibly through
interaction with Bcl-2, a well characterized antiapoptotic protein with
which it shares sequence similarity (19-21). Galectin-7, on the other
hand, has been demonstrated to promote apoptosis in transfected cells
(22) and may be responsible for the proapoptotic function of p53 (23).
Relevant to the intracellular functions is the fact that galectins have
been shown to recognize intracellular proteins, including Bcl-2 (19),
nuclear lectins (24), and cytokeratins (25).
Another notable feature of galectins is their association with
neoplastic transformation. Galectin-3 is overexpressed in some types of
cancers, in which the normal parental cells do not express the protein,
including specific types of lymphomas (26, 27), thyroid carcinoma (28,
29), and hepatocellular carcinoma (30). However, it is down-regulated
in other kinds of neoplasms, including colon carcinoma (31, 32), breast
carcinoma (33), ovarian carcinoma (34), and uterine carcinoma (35).
Galectin-7, which is highly inducible by p53 (23) and normally
expressed in stratified epithelia, is down-regulated in squamous cell
carcinomas (36). Galectin-8 is overexpressed in prostate cancer (37),
and galectin-9 is overexpressed in Hodgkin's lymphomas (38, 39).
Whether the altered expression of galectins contributes to the
neoplastic transformation or is a consequence of the transformation
awaits elucidation. Studies of cells transfected with galectin-3
cDNA or treated with specific antisense oligonucleotides, however, have provided evidence for the involvement of galectin-3 in tumor development and metastasis (40, 41). Galectins may play a major role in
the biological behaviors of cancer cells in which the lectins are
either up-regulated or down-regulated because of the various functions
of galectins mentioned above.
Cell cycle control is a major theme of normal development and
differentiation. Most cancers arise due to faulty cell cycle regulation
(42). Under genotoxic assaults, cell cycle arrest prevents transmission
of damaged genomic DNA that could otherwise cause widespread mutations
of vital genes controlling cell growth in daughter cells, a major cause
of cancer (43). In conditions not permissive for productive
proliferation, e.g. growth factor deficiency, cells invoke
G1 arrest to stay alive until conditions improve. Premature
entry into the cell cycle leads to apoptosis (44, 45). Cell
differentiation is also usually associated with irreversible cell cycle
exit (46).
Identification of new cell cycle regulation proteins is
therefore important for the understanding of normal cellular
homeostasis and pathogenesis of neoplasms as well as the development of
new methods for diagnosis and treatment of cancers. Here we report the
isolation of a cDNA for such a protein from human T-cells in
G1 phase, identify that this protein is a member of the
galectin family, and demonstrate that its ectopic expression arrests
the cell cycle at the G1 phase.
Cells and Cell Lines--
All cell lines used were from ATCC
(Manassas, VA). The breast cancer cell line MCF-7 was grown in
Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal
bovine serum (FBS). Human T leukemic lines and U937 were cultured in
RPMI 1640 medium containing 10% FBS and 2 mM glutamine.
HeLa cells were grown in McCoy's 5A medium with 10% FBS and 2 mM glutamine. Other cell lines were grown in Dulbecco's
modified Eagle's medium containing 10% FBS and 2 mM
glutamine. The human tumor cell lines used to evaluate galectin-12
expression were cultured under logarithmic growth conditions and are as
follows: B leukemic lines Daudi, Raji, and Wil-2; T leukemic lines CEM,
HuT-78, MOLT-3, and MT-2; promonocytic line U937; basophilic line
KU-812; myelomonocytic line HL-60; ovarian line SKOV3; pancreatic line
CAPAN-2; prostate line PC-3; hepatic line HepG2; colon line HT-29;
breast cell lines 435, T47D, HBL-100, and MCF-7. Human blood was
obtained from three unrelated healthy volunteers following informed
consent and pooled. Mononuclear cells were prepared by isopycnic
centrifugation on Histopaque 1077 (Sigma). Polymorphonuclear cells were
prepared by dextran sedimentation and isolated from the high density
cell pellet that also contained erythrocytes, followed by removal of
erythrocytes by osmotic shock.
Cloning of cDNAs for Full-length Galectin-12--
Randomly
selected EST clones from a G1 phase Jurkat T-cell V
cDNA library (47) were sequenced, and the sequences were compared with those in the public data base. A partial clone HJACE54
(GenBankTM accession number AA311108) was found to have
sequence similarity to galectins. Based on the sequence of this clone,
cloning of the full-length cDNA was attempted by 5'-rapid
amplification of cDNA ends (RACE) from a human retina Marathon
cDNA library using the Advantage cDNA PCR kit
(CLONTECH, Palo Alto, CA) with a touchdown PCR
protocol recommended by the manufacturer. An oligonucleotide GSP
(CCTGGAACCATCCTCAGGAGTGGACACAGTAGAGCTGG), which corresponds to a
sequence in the HJACE54 cDNA encompassing the translation stop
codon (antisense to nucleotides 942-979 in Fig. 1), was used as the
downstream primer. The purified RACE product was subjected to another
round of PCR using the same primers in the presence of Pfu
polymerase (Stratagene, La Jolla, CA) to generate blunt-ended DNA. The
DNA products from the second PCR were digested with NotI and
cloned into NotI/SmaI-digested pBlueScript SK
vector. Transformants were screened by PCR using T3 and
GGCTCATCAGCAGGAAAGG as primers, the latter corresponding to nucleotides
41-59 in the HJACE54 cDNA (antisense to nucleotides 553-571 in
Fig. 1). Positive clones were sequenced with the ABI PRISM Rhodamine
Terminator Cycle Sequencing Ready kit (PE-Applied Biosystems, Foster
City, CA) on the ABI PRISM 310 Genetic Analyzer per the manufacturer's
protocols. Three positive clones were found to contain the same insert,
whose 3' regions were identical to the HJACE54 cDNA.
In the EST data base dbEST, a putative mouse homolog of the above
described cDNA was found. Sequencing the entire EST clone, however,
revealed that it contained additional 5' sequence not present in the
human clones. In GenBankTM, a cosmid clone (cSR187d6;
GenBankTM Accession Number U73641) from human chromosome 11 was found to contain the genomic DNA for the cloned cDNA. In this
cosmid DNA, a segment homologous to the leading sequence found in the mouse homolog was identified. The full-length cDNA was then
obtained by PCR of human retina Marathon cDNA with an Advantage 2 PCR kit (CLONTECH), using the upstream primer
(AGTTGGAGTTGCCCCAC), specified from the upstream cosmid sequence, and
the downstream primer GSP. The PCR product was cloned into pCR-Script
with the pCR-Script Amp Cloning kit (Stratagene) to generate pCR-Gal12
and sequenced as described above.
Analysis of Galectin-12 mRNA Expression--
PCR on Multiple
Tissue cDNA panels (CLONTECH) was carried out
per the manufacturer's protocol using the following primers: for
galectin-12, CCAGCTCTACTGTGTCCACTCCTGAGGATGGTTCCAGG (nucleotides 942-979 in Fig. 1) and TAGTCTACAACACTTGCCTCTGTGAGTGCAGTCCAGGC (antisense to nucleotides 1211-1248 in Fig. 1); for galectin-3, CTTATAACCTGCCTTTGCC and CAGATTATATCATGG; and for
glyceraldehyde-3-phosphate dehydrogenase, TGAAGGTCGGAGTCAACGGATTTGGT
and CATGTGGGCCATGAGGTCCACCAC. PCR was performed using the Advantaq Plus
PCR kit from CLONTECH. Cycling conditions were as
follows: for galectin-12, 94 °C for 30 s and then 38 cycles at
94 °C for 30 s and 68 °C for 1 min; for galectin-3, 94 °C
for 30 s and then 30 cycles at 94 °C for 10 s, 55 °C
for 5 s, and 72 °C for 30 s; for
glyceraldehyde-3-phosphate dehydrogenase, 94 °C for 30 s and
then 22 cycles at 94 °C for 30 s and 68 °C for 2 min. The
PCR products were visualized on 1.5% agarose gels. For RT-PCR of
mRNA from cell lines, total RNA was isolated from cell lines by a
one-step method using Tri Reagent (Molecular Research Center,
Cincinnati, OH), and mRNA was reverse transcribed with SuperScript
IITM (Life Technologies, Inc.) according to the manufacturer's
instructions. One µl of the RT reaction was amplified by PCR using
Taq polymerase (Promega, Madison, WI) together with upstream
(AGGTGCCCTGCTCACATGCTC; nucleotides 581-601 in Fig. 1) and downstream
(CCATCCTTCAGGAGTGGACACAG; antisense to nucleotides 951-972 in Fig. 1)
primers for galectin-12 and upstream (TCCTGTGGCATCCACGAAACT) and down
stream (GAAGCATTTGCGGTGGACGAT) primers for Cell Cycle Synchronization--
Synchronization of Jurkat cells
was performed by treatment of the cells (in logarithmic growth)
overnight with 0.1 mM thymidine or 1 mM
hydroxyurea to obtain cells at the G1/S boundary (48), with
0.1 µg/ml nocodazole to block cells at mitosis, or with 0.2 mM theophylline plus 0.5 mM dibutyryl-cAMP to
obtain cells at G1 (49). RNA was extracted, and reverse
transcription was done as described above. PCR on the reverse
transcribed products was performed using the same primers for
galectin-12 and glyceraldehyde-3-phosphate dehydrogenase as described above.
Preparation of Anti-galectin-12 Antibody and Immunoblot
Analysis--
A peptide corresponding to residues 162-175 in the
linker region of galectin-12 (Fig. 1) was synthesized, and antibody was produced in rabbits by standard procedures (50). The specific antibody
was affinity-purified with an adsorbent made by conjugating the peptide
through the sulfhydryl group to sulfolink gel using the kit provided by
the manufacturer (Pierce). The immunoblot analysis of galectin-12 was
performed by using this antibody at 0.2 µg/ml. The immunoblot
analysis for detecting HA-tagged proteins was performed by using the
mouse monoclonal antibody C12A5 (Roche Molecular Biochemicals) at 0.2 µg/ml. All blots were developed with the SuperSignal West Femto
Maximum Sensitivity Substrate Kit from Pierce.
Lactose-binding Assay--
This was performed as described
(51).
Constructs for Mammalian Expression--
The insert from
pCR-Gal12 (see "Cloning of cDNAs for Full-length Galectin-12")
was directly cloned into pEF (51) to generate pEF1-Gal12. pCMV was
generated by deleting the internal ribosome entry site and
neo gene from pIRES1neo (CLONTECH).
cDNA for HA-tagged galectin-9 was generated by PCR using
ACCATGTACCCATACGACGTCCCAGACTACGCTATGGCCTTCAGCGG and
AGCCGCCTATGTCTGCACATGGG as primers and the plasmid DNA from clone
HTPBR22 as template. cDNA for HA-tagged galectin-12 was generated
by PCR using ACCATGTACCCATACGACGTCCCAGACTACGCTATGTCACCTGGAG and
GSP as primers and pCR-Gal12 as template. PCR products were cloned into
pCR-Script and sequenced to generate pCR-HA9 and pCR-HA12, inserts from
which were finally cloned into pCMV to generate pCMV-HA9 and pCMV-HA12,
respectively. pCMV-puro was made by replacing the BamHI-XhoI fragment in pIRES1neo with the
BamHI-SalI fragment from pWE3 (ATCC).
Transfection and Cell Cycle Analysis--
Cells from
subconfluent cultures in the log growth phase were transfected by
electroporation following published protocols with some modifications
(52, 53). HeLa cells at 70-80% confluency were split 1:3 the day
before transfection. On the day of transfection, cells were trypsinized
and resuspended in culture medium (McCoy's 5A medium with 10% FBS and
glutamine). Aliquots of 0.4 ml of cell suspension containing
0.75-1 × 106 cells were transferred to 0.4-cm
electroporation cuvettes. Plasmid DNA (a total of 30 µg) was added
and electroporation was carried out on a Gene Pulser-II electroporator
(Bio-Rad) at 250 V, 1070 microfarads. Cells were then resuspended in 5 ml of complete tissue culture media and cultured for 24 h.
Cells were subsequently cultured in the presence or absence of 0.1 µg/ml nocodazole for an additional 24 h before harvest. For cell
cycle analysis, trypsinized cells were fixed in 70% ethanol at 4 °C
overnight. Nuclear DNA was then stained for 20 min at 37 °C with 50 µg/ml propidium iodide (Calbiochem) in phosphate-buffered saline
containing 50 µg/ml RNase A. Samples were analyzed by flow cytometry
for propidium iodide width versus area fluorescence, which
allowed the exclusion of doublets and weakly fluorescent cell debris.
The above gated cells were acquired into a red fluorescence area
histogram on a FACS Calibur (Becton-Dickinson, Franklin Lakes, NJ) and
analyzed with the CellQuest software (Becton-Dickinson).
Cell Growth Assay--
HeLa cells (0.75 × 106)
were transfected with a mixture of 6 µg of pCMV-puro and 24 µg of
test constructs by electroporation and cultured in 100-mm tissue
culture dishes. Puromycin was added 24 h afterwards to a final
concentration of 5 µg/ml, and the cells were cultured for an
additional 5 days. For quantitative determination of viable cells, the
transfected cells were trypsinized and resuspended in 2 ml of medium.
The MTS assay was performed using the Cell Titer 96 aqueous
nonradioactive cell proliferation assay kit (Promega), following the
manufacturer's instructions. Absorbance at 490 nm was read on a
SpectraMAX 250 ELISA reader (Molecular Devices, Inc., Sunnyvale, CA)
with SOFTmaxPRO software (Molecular Devices).
Cloning of Galectin-12 cDNA--
One of the EST clones from a
human T-cell line, Jurkat, at the G1 phase was found to
have sequence homology with galectins. This clone, HJACE54, has an
insert of 865 bp containing an open reading frame of 402 bp (134 amino
acids). A search of an EST data base with this sequence revealed that
the gene is also expressed in human retina. We then attempted to clone
the full-length cDNA from a human retina cDNA library by
5'-RACE, and a clone with an open reading frame coding for 275 amino
acids was obtained. A BLAST search showed that the deduced protein is
homologous to the galectin family members with two CRDs separated by a
linker sequence. The N-terminal domain of this protein, however,
appeared to be incomplete, because it lacks the sequence to form the S1
Sequence comparisons between our cDNA and homologous genomic DNA in
a cosmid clone and with a mouse cDNA homolog, both identified in
the nonredundant GenBankTM data base, revealed the complete
5' sequence of the human cDNA. This complete sequence, however, did
not contain the 5' sequence of the RACE product obtained from the
retina cDNA library. The full-length cDNA was then cloned by
PCR using a primer designed on the basis of this extra sequence
information. The complete nucleotide sequence and deduced protein
sequence of the novel gene product, designated as galectin-12, are
presented in Fig. 1. Like all other known
galectins, the sequence of galectin-12 does not contain a transmembrane
domain or a classical signal sequence.
RT-PCR with mRNA from cell lines, using a primer corresponding to a
sequence at the 5'-end of the cDNA generated by 5'-RACE, failed to
yield any detectable product. In the same experiments, RT-PCR using
primers specific for full-length galectin-12 resulted in predicted
products (data not shown), and this is the method used for full-length
cloning. The cDNA from 5'-RACE thus probably represents an
alternatively spliced gene product with a retained intron, and the
expression of the corresponding mRNA in cell lines may be
insignificant compared with the authentic full-length mRNA.
The first ATG in the open reading frame of galectin-12 cDNA is not
in a favorable context for translation initiation, according to the
Kozak rule (54). Significantly, the 3'-untranslated region contains
five AT-rich motifs (ATTTA) (Fig. 1), which are initially identified in
inflammatory cytokine cDNAs (55) and are also detectable in many
other cDNAs coding for proteins of growth regulatory functions,
such as oncoproteins and growth factors (56). These motifs are known to
confer instability to mRNA (56, 57).
Organization of Galectin-12 Genomic DNA--
Alignment of the
full-length galectin-12 cDNA sequence with the genomic DNA sequence
revealed that the galectin-12 gene consists of nine exons (Fig.
2A). Comparison of the coding
exon sequences of the galectin-12 gene with those of other known
galectin genes revealed conservation in exon organization (Fig.
2B). This comparison allowed us to classify exons for the
galectin CRD into two groups. The lengths of the three exons coding for
the N-terminal CRD of galectin-12 correspond well to those coding for
the N-terminal CRD of mouse galectin-6 and the single CRD of
galectin-10. The lengths of the three exons coding for the C-terminal
CRD of galectin-12 correspond well to those coding for the C-terminal
CRD of galectin-6 and the CRD of galectin-1, -2, and -3. Therefore,
galectin-12 and other known galectins appear to be derived from an
ancestral gene.
Exon by Exon Protein Sequence Comparison between Galectin-12 and
Other Galectins--
Crystal structures of galectin-1 (58), galectin-2
(59), galectin-3 (60), galectin-7 (61), and galectin-10 (62) determined by x-ray diffraction showed that each carbohydrate recognition domain
is composed of a five-stranded (F1-F5) and a six-stranded (S1-S6)
Comparison of each CRD in galectin-12 with CRDs of other members of the
galectin family demonstrates that the N-terminal domain has significant
homologies with other galectins (Fig. 3C). The C-terminal
domain (galectin-12C) displays greater divergence from other members,
as evident from lower percentage identities (in comparison, among all
other galectin domains listed in Fig. 3C, the median
sequence identity is 38%, with the upper decile of 45% and the lower
decile of 32%). These homologies are reflected in the evolutionary
analysis of the galectin family as shown in the phylogenetic tree in
Fig. 3D.
Galectin-12 Exhibits Lactose Binding Activity--
The sequence
analysis described above suggests that galectin-12 is likely to have
lactose binding activity because of the contribution of the N-terminal
domain. To clarify this point, lysate from Jurkat cells transfected
with the galectin-12 cDNA was mixed with lactosyl-Sepharose 4B, and
the bound protein was eluted with the SDS sample buffer. The eluted
material was analyzed by immunoblotting using antibodies specific for
an internal peptide of galectin-12. We found that galectin-12 bound to
lactosyl-Sepharose 4B (Fig. 4). The
binding is specific, because galectin-12 did not bind to
sucrosyl-Sepharose 4B, and its binding to lactosyl-Sepharose 4B was
inhibited by lactose but not by sucrose (Fig. 4).
Tissue Distribution of Galectin-12 mRNA--
Northern blot
analysis showed that galectin-12 mRNA was nearly undetectable in
many tissues tested, in contrast to galectin-3 mRNA, which was
detected in almost all tissues, when the same membranes were reprobed
(data not shown). However, using a more sensitive procedure, RT-PCR,
galectin-12 mRNA was detectable in the heart, pancreas, spleen,
thymus, and peripheral blood leukocytes. It was also present at lower
levels in the lung, skeletal muscle, kidney, prostate, testis,
ovary, and colon but virtually undetectable in the brain,
placenta, and liver (Fig. 5A).
Galectin-12 mRNA was not detected in many cell lines tested, but
its expression was confirmed by RT-PCR in the peripheral blood
mononuclear and polynuclear cells as well as myeloid cell lines, U937,
HL-60, and KU-812; the B-cell line Wil-2; and the breast cancer cell line HBL-100 (Fig. 5B). Galectin-12 was weakly expressed in
the human T cell line HuT-78, the hepatic cell line HepG2, the human ovarian cancer line SKOV3, and the human breast cancer line T47D. Immunoblot analysis with the anti-peptide antibodies, however, failed
to detect galectin-12 protein in these cell lines (data not shown),
although this procedure detected the protein in the lysates from
galectin-12 transfectants (Fig. 4).
Up-regulation of Galectin-12 Expression by Cell Cycle
Synchronization at G1 or G1/S
Boundary--
Since a partial galectin-12 cDNA clone was isolated
from a Jurkat cell library arrested at G1, we wished to
determine whether the expression of the message is up-regulated under
conditions that induce cell stasis. Jurkat cells were treated with
hydroxyurea or thymidine to synchronize cells at the G1/S
boundary (48), with theophylline plus dibutyryl-cAMP to synchronize
cells at G1 (49), or with nocodazole to block cells at
mitosis. As shown in Fig. 6,
synchronization at G1 or G1/S boundary, but not
mitosis, induced galectin-12 expression.
Cell Cycle Arrest by Ectopic Expression of
Galectin-12--
Changes in galectin-12 expression in response to cell
cycle synchronization prompted us to test the possible function of this protein in cell cycle transition. We developed a highly efficient transfection method that can reproducibly transfect the human cervical
cancer cell line HeLa with efficiencies between 80 and 90%, regardless
of the DNA constructs used in the transfection (Fig.
7A), using a protocol similar
to that used for cell cycle studies of the breast cancer cell line
MCF-7 (53). This allowed us to perform transient transfection
experiments without cotransfection of a selection marker. This is well
suited for cell cycle analysis, because the effects of the transfected
gene on the cell cycle need only 1-2 days to manifest.
The effect of galectin-12 on the cell cycle distribution was compared
with another two-CRD galectin, galectin-9. Thus, the cells were
transfected with cDNA constructs designed in such a way that the
expressed proteins would be tagged with an epitope of the influenza
hemagglutinin (HA). The expression levels of the two proteins after
transfection could thus be compared by immunoblotting using the same
antibody, mouse anti-HA clone C12A5. As shown in Fig. 7B,
cells transfected with HA-galectin-9 and HA-galectin-12 expressed
comparable levels of the two galectins. However, there were significant
differences in cell cycle distribution in the resultant transfectants
(Fig. 7C). Transfection with both 15 and 30 µg of the
HA-galectin-9 construct barely altered the percentages of cells in G1,
S, and G2/M. In significant contrast, transfection with 15 µg of the HA-galectin-12 construct resulted in a considerably higher
percentage of cells in G1, with compensatory decreases in
cells in S and G2/M. A more pronounced effect was seen when
the cells were transfected with 30 µg of the HA-galectin-12 construct.
In order to demonstrate that the accumulation of cells at
G1 was a result of G1 arrest rather than accelerated M to
G1 transition, we employed a protocol commonly used in
studies of G1 cell cycle arrest (e.g. see Ref.
53), in which nocodazole is added to the cells after transfection. This
drug disrupts the formation of spindle fibers and results in a block at
mitosis, thus preventing cells from recycling back to G1.
Thus, 1 day after transfection with different doses of the constructs,
HeLa cells were cultured in the presence of nocodazole. Under these
conditions, an even more dramatic effect of galectin-12 expression on
the cell cycle distribution was seen, as demonstrated by the
substantial increases in the percentages of cells in G1
(Fig. 7C). There was also a minor effect at the higher dose
of HA-galectin-9, but the effects of HA-galectin-12 were substantially greater.
Growth Suppression by Ectopic Expression of Galectin-12--
Cell
cycle arrest invariably results in cell growth suppression; therefore,
the above results suggested that transfection with galectin-12 would
result in retarded cell growth. To test this, we compared the growth
rates of cells transfected with galectin-9 and galectin-12,
respectively. In transient transfection, cells gradually lose
expression of the transfected gene; thus, the procedure is not suited
for long term analysis. Therefore, we cotransfected HeLa cells with a
control vector, the HA-galectin-9 construct, or the HA-galectin-12
construct, together with a puromycin resistance gene, so that cells
expressing the transfected genes could be selected by culturing in the
presence of puromycin. The advantage of this system is that it requires
only 3-5 days to eliminate all the cells that do not express the
puromycin resistance gene, in contrast to another more commonly
employed system, using G418 as the selection marker, which usually
takes 1-2 weeks to achieve the selection. As shown in Fig.
8, while cells transfected with the
vector or HA-galectin-9 continued to grow during 5 days of culturing,
those transfected with HA-galectin-12 failed to proliferate. After 5 days of selection, the number of HA-galectin-12 transfectants was about
one-sixth of that of HA-galectin-9 transfectants or control vector
transfectants.
A number of structural features of galectin-12 support the
possibility that this protein is evolutionarily related to galectins. First, it contains a basic two-CRD type of structure characteristic of
a subfamily of galectins, with two homologous domains separated by a
linker. Second, the exon organization and the size of each exon
correspond well to those of other galectins. Third, many residues of
the consensus sequences found in other galectins are present in this
protein, particularly in the N-terminal domain. These structural
similarities, plus the observation that the full-length protein binds
lactose, make galectin-12 a bona fide galectin. However, significant divergence from galectins is also evident. While
the N-terminal domain of galectin-12 contains all of the elements to
form a CRD, the C-terminal domain strays significantly from the
consensus sequences.
Several features of galectin-12 mRNA suggest that the expression of
the protein is tightly regulated. The start codon of full-length galectin-12 is predicted to be a weak initiator for translation under
normal conditions. Significantly, galectin-12 mRNA carries AU-rich
motifs in the 3'-untranslated region, which confer instability to
mRNA (56, 57). Northern blot analysis indeed showed that galectin-12 mRNA is barely detectable in many tissues tested. The
results were not due to the quality of the galectin-12 cDNA probe,
since strong signals were observed when mRNA from cells transfected
with galectin-12 cDNA were analyzed using the same probe (data not
shown). Also, we have not been able to detect galectin-12 protein in a
number of cell lines by immunoblotting with an anti-peptide antibody.
However, we cannot determine quantitatively the levels of the protein
in these cells, because we do not know the detection limits of this
antibody in immunoblot analysis.
AU-rich motifs are found in the 3'-untranslated regions of many
messenger RNAs of proto-oncogenes and genes coding for nuclear transcription factors and cytokines. They represent the most common determinant of RNA instability in mammalian cells (56, 57). Messenger
RNA degradation directed by these motifs is influenced by many
exogenous factors, including phorbol esters, calcium ionophores, cytokines, and transcription inhibitors. The protein products of many
messenger RNAs carrying these motifs have critical roles in cell
growth and differentiation (56, 57). Consistent with this, several
lines of evidence strongly point to the role of galectin-12 in cell
cycle regulation. First, its cDNA is cloned from cells arrested at
the G1 phase. Second, its expression is increased in cells
undergoing cell cycle arrest. Most importantly, its ectopic expression
induces cell cycle arrest as clearly demonstrated by transfection
experiments (Fig. 7). The ability of galectin-12 to induce cell cycle
arrest is also supported by the finding that cell lines ectopically
expressing galectin-12 fail to proliferate (Fig. 8).
Determination of how galectin-12 affects cell cycle progression awaits
further investigation, but it may involve interactions with other
intracellular proteins important for cell growth control. Preliminary
data showed down-regulation of cyclin A levels and retinoblastoma
protein (Rb) phosphorylation by galectin-12 by immunoblotting from
lysates of galectin-12 transfectants using antibodies specific for
cyclin A, Rb, and phosphorylated Rb (data not shown). Previously, we
found that galectin-3 shares sequence similarity with Bcl-2 and binds
Bcl-2 (19). Galectin-3-Bcl-2 interaction is inhibitable by lactose,
although Bcl-2 is not glycosylated. The results suggest that galectin-3
may employ its carbohydrate-binding site to interact with intracellular
proteins, by recognizing peptide sequences that are structurally
similar to the carbohydrate ligands of the lectin. In the case of
galectin-12, however, whether the carbohydrate-binding site is involved
in its cell growth-regulatory function remains to be determined.
Galectin-12 may play an important role in host response to adverse
situations through its up-regulated expression, leading to cell cycle
arrest. The possible role of this protein in tumorigenesis is
particularly attractive. The galectin-12 gene is found in several human
chromosome 11q13 clones by BLAST search. This is a region frequently
altered in many human cancers (see the Cancer Genome Anatomy Project
site on the World Wide Web). Within this region lies the
CCND1 gene encoding cyclin D1, ectopic expression of which
is implicated in several types of leukemia. Tumor suppressor gene(s) in
this region, however, have yet to be identified. The ability of
galectin-12 to arrest cell cycle and inhibit cell proliferation of
several cancer cell lines makes it a candidate tumor suppressor in
chromosome 11q13. Moreover, the findings suggest that gene therapy
using galectin-12 cDNA may prove to be a valuable approach for the
treatment of cancers.
Recently, other investigators reported that galectin-3 regulates cell
cycling (63). They found that while parental BT549 cells undergo
apoptosis (anoikis) when they are grown as suspension cultures,
galectin-3-overexpressing transfectants undergo G1 arrest, without detectable cell death, in response to the loss of adhesion. The
effect of galectin-3 appears to be mediated by down-regulation of
G1-S cyclin levels and up-regulation of their inhibitory
protein levels. Galectin-1 has also been shown to induce cell cycle
arrest when added to human mammary cancer cells (64) and alter the cell
cycle when added to human T lymphocytes (65). In these cases, the
lectin appears to act through extracellular mechanisms by binding to
cell surface proteins, which then transmit signals to the intracellular
machinery, culminating in cell cycle arrest. Therefore, regulation of
the cell cycle may be a common and important function of members of the
galectin family. Although galectin-9 was found not to have significant
cell cycle regulation activity in the cell line tested (Figs. 7 and 8),
we cannot exclude the possibility that it regulates cell cycle in other
cell types.
Our findings shed light on the understanding of the functions of
galectins. An emerging picture is that they may play important roles in
regulation of cellular homeostasis. As mentioned in the Introduction,
four galectins, galectin-1, -3, -7, and -9, have been shown to either
induce or inhibit apoptosis, although by different mechanisms. Also,
the intracellular sites may be the primary location in which the
galectins normally function. These proteins have been shown to localize
intracellularly, consistent with the absence of a classical signal
sequence, and some of them (galectin-1, -3, and -7) have been ascribed
with important intracellular functions, such as pre-mRNA splicing
and regulation of apoptosis. Interestingly, these functions may not be
dependent on the carbohydrate recognition properties. It is conceivable
that the ancient galectin CRD is utilized as a template in evolution to
generate many galectin-related proteins in which the carbohydrate
recognition motif is adapted for protein-protein interactions.
-galactoside-binding
animal lectins with conserved carbohydrate recognition domains (CRDs).
Here we report the identification and characterization of a new
galectin, galectin-12, which contains two domains that are homologous
to the galectin CRD. The N-terminal domain contains all of the sequence elements predicted to form the two
-sheets found in other galectins, as well as conserved carbohydrate-interacting residues. The C-terminal domain shows considerable divergence from the consensus sequence, and
many of these conserved residues are not present. Nevertheless, the
protein has lactose binding activity, most likely due to the contribution of the N-terminal domain. The mRNA for galectin-12 contains features coding for proteins with growth-regulatory functions. These include start codons in a context that are suboptimal for translation initiation and AU-rich motifs in the 3'-untranslated region, which are known to confer instability to mRNA. Galectin-12 mRNA is sparingly expressed or undetectable in many tissues and cell lines tested, but it is up-regulated in cells synchronized at the
G1 phase or the G1/S boundary of the cell
cycle. Ectopic expression of galectin-12 in cancer cells causes cell
cycle arrest at the G1 phase and cell growth suppression.
We conclude that galectin-12 is a novel regulator of cellular homeostasis.
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-galactoside binding activities (reviewed in Refs. 1-5). Members can be classified into
three groups: those containing one carbohydrate recognition domain
(CRD),1 those containing two
homologous CRDs in tandem separated by a short linker, and the chimeric
type, which has a proline/glycine-rich repetitive sequence connected to
a CRD (1). Members of this evolutionarily highly conserved family are
found in all vertebrates as well as lower organisms, including
nematodes, sponges, and mushrooms (2). In mammals, 11 members have been
designated as galectins (galectin-1 through -11); a large number of
additional members are likely to be discovered, and many identifiable
homologues are present in the published data base (6). Proteins
containing sequence similarities with galectins but without
demonstrable lectin activity also exist. For example, a novel protein
structurally related to galectins, but lacking
-galactoside
binding activity, has been identified in the lens and designated as
GRIFIN (galectin-related interfiber protein) (7).
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-actin. Products were
visualized on 5% polyacrylamide gels.
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-strand that is present in all other known galectins.
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Fig. 1.
Human galectin-12 cDNA and the deduced
protein sequence. The two domains homologous to the galectin CRD
are separated by a linker of 27 amino acids (underlined).
AT-rich motifs in the 3'-untranslated region are indicated by a
wavy underline.
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Fig. 2.
Exon organization of galectin-12 gene in
comparison with other cloned galectin genes. A, nine
exons (bars) separated by introns. B, exon
comparisons. The exon structures of galectin-12 are compared with other
known galectin structures (66-70). The numbers
below each closed bar indicate the
lengths of exons, portions of which shown in shaded regions
constitute the galectin domains.
-sheet. These studies also identified residues in the proteins that
are in contact with the carbohydrate ligands. The N-terminal domain of
galectin-12 contains all of the sequence elements predicted to form the
two
-sheets as well as the conserved carbohydrate-contact residues
(Fig. 3A). The sequence of the
C-terminal domain, however, strays significantly from the consensus
sequence, and many of these conserved residues are not present (Fig.
3B).
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Fig. 3.
Protein sequence comparison between
galectin-12 and other galectins of known genomic structures.
A, alignment of N-terminal CRD amino acid sequences of
galectin-12 with corresponding exons in galectin-6 (68) and -10 (70).
B, alignment of C-terminal CRD sequences of galectin-12 with
corresponding exons in galectin-1 and -2 (67), -3 (66), and -6 (68).
Suffixes in Roman numerals indicate the exon number for each member.
The predicted strands are overlined, and residues
corresponding to those shown to interact with lactose in galectin-1
(58) and -2 (59) are marked by an asterisk. C,
percentages of protein identities among CRDs of human galectin family
members were calculated with Gap in GCG version 10. Interspecies
comparisons are shown for rat galectin-5 (71), murine galectin-6 (68),
ovine galectin-11 (72), and rat GRIFIN (7). PP13, placental protein 13 (73). D, a phylogenetic tree of galectin family members,
constructed with Phylip (74) from alignments performed with ClustalW
(75). hu, human; mu, murine; ov,
ovine; -N, N-terminal CRD; -C, C-terminal
CRD.
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Fig. 4.
Lactose binding activity of galectin-12.
Lysate from Jurkat E6-1 cells transfected with either control
vector (pEF1) or galectin-12-expressing construct (pEF1-Gal12) were
incubated with either sucrosyl- or lactosyl-Sepharose 4B, in the
absence or presence of 25 mM sucrose or lactose. After
extensive washing, the bound protein was eluted with SDS-sample buffer
and immunoblotted with anti-galectin-12 antibody.
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Fig. 5.
Analysis of galectin-12 mRNA expression
in various tissues. mRNA expression of galectin-12 in human
tissues (A) and cell lines (B) was analyzed by
RT-PCR. Mononuclear and polynuclear peripheral blood cells were pooled
from three normal donors. RNA was isolated, and RT-PCR was performed
for galectin-12, -actin, or glyceraldehyde-3-phosphate dehydrogenase
(G3PDH), and the products were analyzed on agarose
(A) or polyacrylamide gels (B).
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Fig. 6.
Up-regulation of galectin-12 mRNA
expression by cell cycle synchronization. Jurkat cells were
cultured overnight with 1 mM hydroxyurea, 0.1 mM thymidine, 0.2 mM theophylline plus 0.5 mM dibutyryl-cAMP, or 0.1 µg/ml nocodazole. Following
each treatment, RNA was isolated, and RT-PCR was performed. Similar
results were obtained in four experiments.
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Fig. 7.
Effect of galectin-12 expression on cell
cycle distribution. A, HeLa cells were transfected with
30 µg of pCMV, 15 µg of pEGFP-C3 (a plasmid coding for green
fluorescent protein) together with 15 µg of either pCMV,
pCMV-HA-galectin-9 (HA-9), or pCMV-HA-galectin-12
(HA-12). The percentage of green fluorescent
protein-positive cells in each transfection as shown on each dot plot
was determined 1 day later by flow cytometry. B, expression
of protein after transfection. HeLa cells were transfected with the
indicated doses of the test constructs and variable amounts of pCMV for
a total of 30 µg of DNA for each transfection. 1 day after
transfection, proteins were extracted with SDS-sample buffer, and
samples equivalent to 104 cells were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting with 0.2 µg/ml mouse anti-HA antibody. C, one day after
transfection, as described for B, cells were cultured for
another day in the presence or absence of 0.2 µg/ml nocodazole, and
the cell cycle distribution was analyzed by flow cytometry. The two
peaks correspond to cells at G1 and G2/M. Cells
that lie between the two peaks are at the S phase. The
number below each histogram indicates
the percentage of cells at each phase of the cell cycle. The results
are representative of three separate experiments.
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Fig. 8.
Inhibition of cell growth by
galectin-12. HeLa cells were cotransfected with 24 µg of the
indicated constructs together with 6 µg of pCMV-puro and then
cultured in a puromycin-containing medium for 5 days. Viable cells
surviving the selection were quantitated by the MTS assay. Each data
point represents the mean ± S.D. of triplicates. Similar results
were obtained in three independent experiments.
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FOOTNOTES |
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* This work was supported by NIAID, National Institutes of Health, Public Health Service Grants RO1 AI20958 and AI39620. The General Clinical Research Center at the Scripps Clinic is supported by Public Health Service Grant MO1RR0833.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.
¶ To whom correspondence should be addressed. La Jolla Institute for Allergy and Immunology, San Diego, CA 92121. Tel.: 858-678-4640; Fax: 858-678-4581; E-mail: ftliu@liai.org.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF310686.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M010914200
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ABBREVIATIONS |
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The abbreviations used are: CRD, carbohydrate recognition domain; EST, expressed sequence tag; FBS, fetal bovine serum; HA, influenza hemagglutinin; PCR, polymerase chain reaction; RT, reverse transcription; RACE, rapid amplification of cDNA ends.
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1. |
Barondes, S. H.,
Cooper, D. N. W.,
Gitt, M. A.,
and Leffler, H.
(1994)
J. Biol. Chem.
269,
20807-20810 |
2. | Kasai, K., and Hirabayashi, J. (1996) J. Biochem. (Tokyo) 119, 1-8[Abstract] |
3. | Perillo, N. L., Marcus, M. E., and Baum, L. G. (1998) J. Mol. Med. 76, 402-412[CrossRef][Medline] [Order article via Infotrieve] |
4. | Rabinovich, G. A. (1999) Cell Death Differ. 6, 711-721[CrossRef][Medline] [Order article via Infotrieve] |
5. | Liu, F.-T. (2000) Clin. Immunol. 97, 79-88[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Cooper, D. N. W.,
and Barondes, S. H.
(1999)
Glycobiology
9,
979-984 |
7. |
Ogden, A. T.,
Nunes, I.,
Ko, K.,
Wu, S. J.,
Hines, C. S.,
Wang, A. F.,
Hedge, R. S.,
and Lang, R. A.
(1998)
J. Biol. Chem.
273,
28889-28896 |
8. | Pesheva, P., Kuklinski, S., Schmitz, B., and Probstmeier, R. (1998) J. Neurosci. Res. 54, 639-654[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Nangia-Makker, P.,
Honjo, Y.,
Sarvis, R.,
Akahani, S.,
Hogan, V.,
Pienta, K. J.,
and Raz, A.
(2000)
Am. J. Pathol.
156,
899-909 |
10. |
Sano, H.,
Hsu, D. K., Yu, L.,
Apgar, J. R.,
Kuwabara, I.,
Yamanaka, T.,
Hirashima, M.,
and Liu, F. T.
(2000)
J. Immunol.
165,
2156-2164 |
11. |
Matsumoto, R.,
Matsumoto, H.,
Seki, M.,
Hata, M.,
Asano, Y.,
Kanegasaki, S.,
Stevens, R. L.,
and Hirashima, M.
(1998)
J. Biol. Chem.
273,
16976-16984 |
12. | Perillo, N. L., Pace, K. E., Seilhamer, J. J., and Baum, L. G. (1995) Nature 378, 736-739[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Perillo, N. L.,
Uittenbogaart, C. H.,
Nguyen, J. T.,
and Baum, L. G.
(1997)
J. Exp. Med.
185,
1851-1858 |
14. |
Wada, J.,
Ota, K.,
Kumar, A.,
Wallner, E. I.,
and Kanwar, Y. S.
(1997)
J. Clin. Invest.
99,
2452-2461 |
15. | Vyakarnam, A., Lenneman, A. J., Lakkides, K. M., Patterson, R. J., and Wang, J. L. (1998) Exp. Cell Res. 242, 419-428[CrossRef][Medline] [Order article via Infotrieve] |
16. | Moutsatsos, I. K., Wade, M., Schindler, M., and Wang, J. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6452-6456[Abstract] |
17. | Vyakarnam, A., Dagher, S. F., Wang, J. L., and Patterson, R. J. (1997) Mol. Cell. Biol. 17, 4730-4737[Abstract] |
18. | Dagher, S. F., Wang, J. L., and Patterson, R. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1213-1217[Abstract] |
19. |
Yang, R.-Y.,
Hsu, D. K.,
and Liu, F.-T.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6737-6742 |
20. | Akahani, S., Nangia-Makker, P., Inohara, H., Kim, H. R. C., and Raz, A. (1997) Cancer Res. 57, 5272-5276[Abstract] |
21. |
Hsu, D. K.,
Yang, R.-Y., Yu, L.,
Pan, Z.,
Salomon, D. R.,
Fung-Leung, W.-P.,
and Liu, F.-T.
(2000)
Am. J. Pathol.
156,
1073-1083 |
22. |
Bernerd, F.,
Sarasin, A.,
and Magnaldo, T.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11329-11334 |
23. | Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W., and Vogelstein, B. (1997) Nature 389, 300-305[CrossRef][Medline] [Order article via Infotrieve] |
24. | Seve, A.-P., Felin, M., Doyennette-Moyne, M.-A., Sahraoui, T., Aubery, M., and Hubert, J. (1993) Glycobiology 3, 23-30[Abstract] |
25. |
Goletz, S.,
Hanisch, F.-G.,
and Karsten, U.
(1997)
J. Cell Sci.
110,
1585-1596 |
26. | Hsu, D. K., Hammes, S. R., Kuwabara, I., Greene, W. C., and Liu, F.-T. (1996) Am. J. Pathol. 148, 1661-1670[Abstract] |
27. | Konstantinov, K. N., Robbins, B. A., and Liu, F.-T. (1996) Am. J. Pathol. 148, 25-30[Abstract] |
28. | Fernádez, P. L., Merino, M. J., Gómez, M., Campo, E., Medina, T., Castronovo, V., Cardesa, A., Liu, F.-T., and Sobel, M. E. (1997) J. Pathol. 181, 80-86[CrossRef][Medline] [Order article via Infotrieve] |
29. | Xu, X. C., El-Naggar, A. K., and Lotan, R. (1995) Am. J. Pathol. 147, 815-822[Abstract] |
30. | Hsu, D. K., Dowling, C. A., Jeng, K. C. G., Chen, J. T., Yang, R. Y., and Liu, F. T. (1999) Int. J. Cancer 81, 519-526[CrossRef][Medline] [Order article via Infotrieve] |
31. | Lotz, M. M., Andrews, C. W., Jr., Korzelius, C. A., Lee, E. C., Steele, G. D., Jr., Clarke, A., and Mercurio, A. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3466-3472[Abstract] |
32. | Castronovo, V., Campo, E., van den Brûle, F. A., Claysmith, A. P., Cioce, V., Liu, F.-T., Fernandez, P. L., and Sobel, M. E. (1992) J. Natl. Cancer Inst. 84, 1161-1167[Abstract] |
33. | Castronovo, V., Van den Brûle, F. A., Jackers, P., Clausse, N., Liu, F. T., Gillet, C., and Sobel, M. E. (1996) J. Pathol. 179, 43-48[CrossRef][Medline] [Order article via Infotrieve] |
34. | van den Brûle, F. A., Berchuck, A., Bast, R. C., Liu, F.-T., Pieters, C., Sobel, M. E., and Castronovo, V. (1994) Eur. J. Cancer 30A, 1096-1099 |
35. | van den Brûle, F. A., Buicu, C., Berchuck, A., Bast, R. C., Deprez, M., Liu, F. T., Cooper, D. N. W., Pieters, C., Sobel, M. E., and Castronovo, V. (1996) Hum. Pathol. 27, 1185-1191[CrossRef][Medline] [Order article via Infotrieve] |
36. | Magnaldo, T., Fowlis, D., and Darmon, M. (1998) Differentiation 63, 159-168[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Su, Z.-Z.,
Lin, J.,
Shen, R.,
Fisher, P. E.,
Goldstein, N. I.,
and Fisher, P. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7252-7257 |
38. | Sahin, U., Tureci, O., Schmitt, H., Cochlovius, B., Johannes, T., Schmits, R., Stenner, F., Luo, G., Schobert, I., and Pfreundschuh, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11810-11813[Abstract] |
39. |
Tureci, O.,
Schmitt, H.,
Fadle, N.,
Pfreundschuh, M.,
and Sahin, U.
(1997)
J. Biol. Chem.
272,
6416-6422 |
40. | Raz, A., Zhu, D., Hogan, V., Shah, N., Raz, T., Karkash, R., Pazerini, G., and Carmi, P. (1990) Int. J. Cancer 46, 871-877[Medline] [Order article via Infotrieve] |
41. | Bresalier, R. S., Mazurek, N., Sternberg, L. R., Byrd, J. C., Yunker, C. K., Nangia-Makker, P., and Raz, A. (1998) Gastroenterology 115, 287-296[Medline] [Order article via Infotrieve] |
42. |
Sherr, C. J.
(1996)
Science
274,
1672-1677 |
43. | Levine, A. J. (1997) Cell 88, 323-331[Medline] [Order article via Infotrieve] |
44. | Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., and Hancock, D. C. (1992) Cell 69, 119-128[Medline] [Order article via Infotrieve] |
45. | Gibson, A. W., Cheng, T., and Johnston, R. N. (1995) Exp. Cell Res. 218, 351-358[CrossRef][Medline] [Order article via Infotrieve] |
46. | Lipinski, M. M., and Jacks, T. (1999) Oncogene 18, 7873-7882[CrossRef][Medline] [Order article via Infotrieve] |
47. | Adams, M., et al.. (1995) Nature 377 Suppl. 6547, 3-174[Medline] [Order article via Infotrieve] |
48. | Tobey, R. A., and Crissman, H. A. (1972) Exp. Cell Res. 75, 460-464[Medline] [Order article via Infotrieve] |
49. | Coffino, P., Gray, J. W., and Tomkins, G. M. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 878-882[Abstract] |
50. | Cooper, H. M., and Paterson, Y. (1995) in Current Protocols in Immunology (Coligan, J. E. , Kruisbeek, A. M. , Margulies, D. H. , Shevach, E. M. , and Strober, W., eds) , pp. 2.4.1-2.4.9, John Wiley and Sons, Inc., New York |
51. | Yang, R. Y., Hill, P. N., Hsu, D. K., and Liu, F. T. (1998) Biochemistry 37, 4086-4092[CrossRef][Medline] [Order article via Infotrieve] |
52. | Baum, C., Forster, P., Hegewisch-Becker, S., and Harbers, K. (1994) BioTechniques 17, 1058-1062[Medline] [Order article via Infotrieve] |
53. | Agami, R., and Bernards, R. (2000) Cell 102, 55-66[Medline] [Order article via Infotrieve] |
54. | Kozak, M. (1989) J. Cell Biol. 108, 229-241[Abstract] |
55. | Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S., and Cerami, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1670-1674[Abstract] |
56. | Chen, C.-Y. A., and Shyu, A.-B. (1995) Trends Biochem. Sci 20, 465-470[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Laroia, G.,
Cuesta, R.,
Brewer, G.,
and Schneider, R. J.
(1999)
Science
284,
499-502 |
58. | Liao, D.-I., Kapadia, G., Ahmed, H., Vasta, G. R., and Herzberg, O. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1428-1432[Abstract] |
59. |
Lobsanov, Y. D.,
Gitt, M. A.,
Leffler, H.,
Barondes, S. H.,
and Rini, J. M.
(1993)
J. Biol. Chem.
268,
27034-27038 |
60. |
Seetharaman, J.,
Kanigsberg, A.,
Slaaby, R.,
Leffler, H.,
Barondes, S. H.,
and Rini, J. M.
(1998)
J. Biol. Chem.
273,
13047-13052 |
61. | Leonidas, D. D., Vatzaki, E. H., Vorum, H., Celis, J. E., Madsen, P., and Acharya, K. R. (1998) Biochemistry 37, 13930-13940[CrossRef][Medline] [Order article via Infotrieve] |
62. | Leonidas, D. D., Elbert, B. L., Zhou, Z., Leffler, H., Ackerman, S. J., and Acharya, K. R. (1995) Structure 3, 1379-1393[Medline] [Order article via Infotrieve] |
63. |
Kim, H. R. C.,
Lin, H. M.,
Biliran, H.,
and Raz, A.
(1999)
Cancer Res.
59,
4148-4154 |
64. | Wells, V., Davies, D., and Mallucci, L. (1999) Eur. J. Cancer 35, 978-983[CrossRef][Medline] [Order article via Infotrieve] |
65. |
Allione, A.,
Wells, V.,
Forni, G.,
Mallucci, L.,
and Novelli, F.
(1998)
J. Immunol.
161,
2114-2119 |
66. | Kadrofske, M. M., Openo, K. P., and Wang, J. L. (1998) Arch. Biochem. Biophys. 349, 7-20[CrossRef][Medline] [Order article via Infotrieve] |
67. | Gitt, M. A., and Barondes, S. H. (1991) Biochemistry 30, 82-89[Medline] [Order article via Infotrieve] |
68. |
Gitt, M. A.,
Xia, Y. R.,
Atchison, R. E.,
Lusis, A. J.,
Barondes, S. H.,
and Leffler, H.
(1998)
J. Biol. Chem.
273,
2961-2970 |
69. |
Gitt, M. A.,
Massa, S. M.,
Leffler, H.,
and Barondes, S. H.
(1992)
J. Biol. Chem.
267,
10601-10606 |
70. | Dyer, K. D., Handen, J. S., and Rosenberg, H. F. (1997) Genomics 40, 217-221[CrossRef][Medline] [Order article via Infotrieve] |
71. |
Gitt, M. A.,
Wiser, M. F.,
Leffler, H.,
Herrmann, J.,
Xia, Y.-R.,
Massa, S. M.,
Cooper, D. N. W.,
Lusis, A. J.,
and Barondes, S. H.
(1995)
J. Biol. Chem.
270,
5032-5038 |
72. |
Dunphy, J. L.,
Balic, A.,
Barcham, G. J.,
Horvath, A. J.,
Nash, A. D.,
and Meeusen, E. N.
(2000)
J. Biol. Chem.
275,
32106-32113 |
73. | Than, N. G., Sumegi, B., Than, G. N., Berente, Z., and Bohn, H. (1999) Placenta 20, 703-710[CrossRef][Medline] [Order article via Infotrieve] |
74. | Felsenstein, J. (1989) Cladistics 5, 164-166 |
75. | Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-4680[Abstract] |