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Molecular Biology and Genetics |
Department of Molecular Genome Analysis, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany [J. M., S. H., I. K., F. B., S. W., A. P.]; Immunology and Microbiology, Institute for Medical Biology, University of Southern Denmark, Odense University, 5000 Odense C, Denmark [U. H.]; Institute for Pathology, Neuropathology, Basel University, 4003 Basel, Switzerland [M. T.]; Molecular Neuro-Oncology, University Hospital, 4031 Basel, Switzerland [A. M., D. M.]; Department of Pathology, University of Southern Denmark, Odense University, 5000 Odense C, Denmark [H. D. S.]; and Department of Experimental Pathology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany [H-J. G.]
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ABSTRACT |
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INTRODUCTION |
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The majority of the SRCR proteins has been implicated in functions within the immune system. The Mac2-bp, for example, interacts with the lectin Mac-2 (galectin-3) and plays a role in the mucosal immune defense and in the activation of natural killer cells as well as in tumor suppression (5, 6, 7, 8) . The Mac2-bp further has been demonstrated to mediate cell-cell and cell-extracellular matrix adhesion (9 , 10) . Like the Mac2-bp, the SRCR protein gp-340, a binding protein for the lung collectins SP-D and SP-A, has been proposed to play a role in the mucosal immune defense (11 , 12) .
Ebnerin, CRP-ductin, and hensin are SRCR proteins in rat, mouse, and rabbit, respectively, that share homology with DMBT1, but have different expression patterns (13, 14, 15) . Whereas Ebnerin has been suggested to play a role in taste perception (13) , it has been reasoned that CRP-ductin might be involved in epithelial differentiation because of its differential expression in the intestinal epithelium (14) . Biochemical studies have established that hensin is able to switch the polarity and to induce processes of terminal differentiation in epithelial cells of the kidney collecting ducts (15 , 16) .
SRCR, ZP, and CUB domains are thought to mediate protein-protein interactions, and CUB domains have been recognized as a motif common to proteins involved in processes of embryogenesis and organogenesis (17, 18, 19) . DMBT1 codes for an extracellular protein with up to 14 SRCR domains, 2 CUB domains, and a ZP domain (1 , 20) . Alternative splicing takes place within the exons encoding the first 13 SRCR domains and the SIDs. These exons are part of locus-specific sequence repeats that potentially are prone to genomic instability (20) . The function of DMBT1, however, and the nature of the molecular events that might lead to an inactivation during tumorigenesis have remained unknown. In this study, we aimed at getting insight into the functions of DMBT1 as well as into its expression and localization in brain tumors. We further wanted to test the hypothesis that the repetitive structure of the gene gives rise to an increased susceptibility to genomic instability.
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MATERIALS AND METHODS |
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LOH and Deletion Analyses.
High molecular weight genomic DNA was prepared from the tumor
cell lines according to standard procedures. For the LOH analyses, the
homozygosity/heterozygosity of the DMBT1 flanking markers
D10S209 and D10S587 and of a bp polymorphism
(CCA/CCG) in codon 1707 of DMBT1 (referred to accession no.
AJ000342) was analyzed. We determined the heterozygosity of the bp
polymorphism to be 46% in the normal population (32 of 70
individuals). The combined heterozygosity of the three markers allowed
to predict LOH in the tumor cell lines with an error rate of 1/33. For
analyzing the bp polymorphism, PCR reactions were carried out with 50
ng of genomic DNA as the template, 1x Taq polymerase buffer and
2 units of Taq polymerase (both Perkin-Elmer), 1.5
mM MgCl2, 200
µM each dNTP, and 20 pmol each primer
(dmbt1ex54dsf2: 5'-CTTGGTGAGAGCTAAGGGG-3' and dmbt1ex54dsr3:
5'-CTCTGCACACCACCATTTTAC-3') in a final volume of 50 µl. After an
initial denaturation for 3 min at 94°C, 40 cycles consisting of
30 s at 94°C, 30 s at 60°C, and 1 min at 72°C a final
extension for 10 min at 72°C followed. One µl of the PCR was added
to 4 µl of dye marker (1x Tris-borate EDTA, 10% w/v Ficoll, and
0.01% w/v bromphenolblue) and 3 µl of H2O,
denatured for 3 min at 95°C, and stored on ice. The samples were
loaded on a 12% polyacrylamide gel (11 cm x 13 cm;
29:1 acrylamide:bisacrylamide; 1x Tris-borate EDTA; prerun for 5 min
at 100 V before loading), and electrophoresis was carried out for
24 h at 80 V at room temperature. Subsequently, the gels were
silver-stained to determine the heterozygosity/homozygosity of the
polymorphism. Tumor cell lines apparently homozygous for all three
markers were classified to have an LOH at the DMBT1 locus.
Southern blot analyses of the DMBT1 gene were carried out
with the probes DMBT1/sr1sid2 and DMBT1/59m21 as
described elsewhere (20)
.
Expression Analyses by RT-PCR.
Poly(A)+ RNAs from the different tissues of the immune system were
purchased from Clontech. Total RNA from the cell lines U937, Raji,
Jurkat, and HL-60 and from alveolar macrophages was prepared according
to standard procedures. Alveolar macrophages were obtained by cytospin
from human BAL. The reverse transcription was carried out with 1 µg
of total RNA or 20 ng of mRNA, respectively, as described earlier
(1)
. The subsequent PCR amplification was carried out in
two steps. The first PCR contained 5 µl of the 1:5 diluted
single-stranded cDNA from the reverse transcription, 1x PCR buffer
(Perkin-Elmer), 1.5 mM MgCl2, 200
µM each dNTP, 0.4 µl of a Taq:Pfu polymerase mix (9:1;
Taq from Perkin-Elmer, 5 units/µl; Pfu from Stratagene, 2.5
units/µl), and 20 pmol each primer (dmbt1ex50scf3:
5'-TGGGACATTGAGGTGCAAAAC-3' and dmbt1rtsr5: 5'-AGCTGACGTGAATACGGAGG-3')
in a final volume of 50 µl. Fifteen cycles with 30 s at 94°C,
30 s at 60°C, and 2 min at 72°C were carried out followed by a
final extension for 10 min at 72°C. Five µl of the first PCR were
used as the template in a second PCR that was set up as described above
except that 20 pmol each of the DMBT1-specific primers
dmbt1ex51scf2 (5'-GTTCCCCTCTCATTGCTCG-3') and dmbt1rtsr5 (PCR
product: 488 bp) were used in addition to 5 pmol each of the primers
TFR5 (5'-GTCAATGTCCCA-AACGTCACCAGA-3') and TFR3
(5'-ATTTCGGGAATGCTGAGAAAACAGACAGA-3'), which were specific for the
transferrin receptor. Thirty cycles with 45 s at 94°C, 45 s
at 60°C, and 2 min at 72°C were carried out in this second PCR,
followed by a final extension for 10 min at 72°C. Ten µl of the PCR
products were subsequently separated on 3% Nusieve agarose gels.
Generation of a DMBT1-specific Monoclonal Antibody.
A synthetic peptide, DMBT1/2640, corresponding to amino acids 26–40
(RTTDYASLIPSEVPLC) of DMBT1 was coupled to activated KLH (Pierce).
gp-340 was purified from BAL as described earlier (11)
.
Four BALB/c mice were immunized by four i.p. injections at 3-week
intervals (Eurogentec). As antigens, 50 µg of DMBT1/2640-KLH and 50
µg of purified gp-340, respectively, were administered with Freud’s
complete or incomplete adjuvant. Two mice were immunized with the KLH
peptide only, whereas the other two mice obtained one injection with
purified gp-340 and subsequently, three injections with the KLH
peptide. One of the mice immunized according to the latter protocol
produced the highest antibody titer against DMBT1, as determined by
direct ELISA on DMBT1/2640-coated microtiter plates and was selected
for the fusion of spleen cells. The fusion to the
Sp2/O-Ag-14 cell line and the selection of hybridomas were
carried out according to standard procedures at Eurogentec (Seraing,
Belgium). IgG-producing hybridomas were screened against the target
peptide antigen by direct ELISA. Hybridomas producing antibodies
against the peptide epitope were cloned by limit dilution and tested
for binding to a recombinantly expressed polypeptide containing the
epitope by Western blotting (see below), yielding the monoclonal
antibody anti-DMBT1h12. The subclass of the antibody was determined to
be IgG1.
Expression of Recombinant DMBT1 Polypeptides.
A 3.5-kb cDNA clone (DMBT1/c3.5) harboring the 5' part of
the 6-kb transcript was taken as the template for PCR. The primers
dmbt1F3–3 (5'-CAAGTTGAATTCACAGGTGGGTGGATCCCAAGGACT-3') and dmbt1F3–4
(5'-TCGGATTCTAGACCTGCTGTTGATGCATGTGAGGT-3') were used for the
amplification of the sequence that codes for a polypeptide comprising
the leader peptide up to domain SID2 and containing the peptide
sequence of DMBT1/2640. Primers dmbt1F3–5
(5'-GCATCGGAATTCGGATCCGAATCCAGTTTGG-CC-3') and dmbt1F3–6
(5'-TCGGATTCTAGACCTACTGTCAATGCCGGTAAGT-3') were used for the
amplification of the part encoding SRCR6 to SID7 of the 6-kb transcript
(accession no. AJ000342). PCRs were carried out with 25 ng of template,
1x Vent buffer, and 1 unit of Vent polymerase (both from NEB), 0.5
µl BSA (10 mg/ml), 200 µM each dNTP, and 20
pmol of each of the respective primers. After 21 cycles (1 min 95°C,
1 min 70°C, 1 min 70°C), the PCR product was digested with
XbaI and EcoRI and purified by preparative
agarose gel electrophoresis. The PCR products were then cloned into
XbaI/EcoRI-digested pPICZalphaA vector
(Invitrogen) that provides a combined myc and His(6)
tag COOH-terminal
to the DMBT1 polypeptides. The plasmids were transfected into
Escherichia coli strain XL-1 Blue. The orientation and the
integrity of the inserts were verified by sequencing. Pichia
pastoris strain GS115 was transfected with the plasmids according
to the suppliers instructions (Pichia Expression Kit,
Invitrogen). For each transformation, 50 colonies were transferred to
nitrocellulose membranes and analyzed for the expression of the myc
epitope with a horse-radish peroxidase-conjugated anti-myc antibody
(Invitrogen). One positive clone each, ppSRCR1/2 and ppSRCR6/7,
respectively, was selected for further analysis. The expression and
purification of the recombinant proteins were carried out as
recommended by the supplier (Invitrogen). The dialyzed supernatants
were applied on a column containing 1 ml of nickel chelate (Pharmacia)
in buffer A [25 mM Tris (pH 8.0), 1
M Urea, 500 mM NaCl, 1
mM mercaptoethanol]. The recombinant proteins
were eluted with a gradient of buffer A to buffer A with 1
M imidazole (60 min; flow rate, 1 ml/min), and
the fractions were tested for the presence of the polypeptides by
Western blotting and detection with an anti-myc antibody (Invitrogen).
The fusion proteins eluted at an imidazole concentration of 100–150
mM, and the positive fractions were pooled. The
predicted molecular weights of the two fusion proteins were
Mr 39,000 for ppSRCR1/2 and
Mr 31,000 for ppSRCR6/7. Both fusion
proteins displayed a band doublet at about
Mr 40,000 as well as several
additional bands up to Mr 85,000.
Western blot experiments with anti-DMBT1h12 revealed two closely spaced
bands at about Mr 80,000 for
ppSRCR1/2. PCR amplification with primers specific for the sequences
flanking the integration sites (5' AOX1 and 3' AOX1; Invitrogen)
delivered products of the predicted size, excluding that a
concatamerization of the plasmid inserts had taken place during
integration into the P. pastoris genome. The observed
increase in the sizes of the fusion proteins therefore most likely
results from posttranslational modification in P. pastoris.
Western Blotting.
Human BAL was obtained from a patient with alveolar proteinosis whose
lung was lavaged for therapeutic purposes, as described earlier
(11)
. For SDS-PAGE, the NuPAGE Gel system (Novex) was
used. Ten µl of the respective protein samples were mixed with 10
µl of loading buffer (NuPAGE Gel System, Novex) and heated for 10 min
at 70°C. The proteins were separated on 4–12% polyacrylamide
gradient gels with the 2-[N-morpholino]ethanesulfonic acid
buffer system for 1 h and 15 min at 200 V and subsequently
transferred to polyvinylidene difluoride membranes (Immobilon P,
Millipore). The membranes were preincubated with PBS-T (0.1% Tween 20
in PBS) containing 5% w/v skim milk powder for 1 h at room
temperature. As the primary antibody either anti-DMBT1h12 (8 µg/ml),
anti-DMBT1h12 (8 µg/ml) preadsorbed with the synthetic peptide
DMBT1/2640 (1 mg/ml), or monoclonal anti-gp-340 (50 ng/ml) in PBS-T
with 5% w/v skim milk powder was used. After an incubation for 1 h at room temperature, three washes with PBS-T, an incubation for
1 h with alkaline phosphatase-conjugated antimouse IgG (Santa
Cruz; diluted 1:1000 in PBS-T with 5% w/v skim milk powder), and three
further washes in PBS-T followed. The membranes were then developed
using nitro blue tetrazolium and potassium
5-bromo-4-chloro-3-indolylphosphate.
Immunohistochemistry.
Paraffin-embedded sections (thickness, 3–4 µm) of formalin-fixed
tissue samples were used for immunohistochemistry. Stainings with
anti-gp-340 as the primary antibody were carried out as described
earlier (11)
. For immunohistochemistry with anti-DMBT1h12,
sections were deparaffinized and rehydrated by xylene and a series of
graded ethanols. Endogenous peroxidase activity was blocked by
incubation for 20 min with 1%
H2O2 in PBS. For the
subsequent steps, the TSA-Indirect Kit was used following the
instructions of the supplier (NEN Life Science Products). Antibody
anti-DMBT1h12 was used at concentrations of 40 or 80 µg/ml in a final
volume of 200 µl/section and incubated overnight in a humidified
chamber at 4°C. Peroxidase activity was detected with
3-amino-9-ethylcarbazole as a substrate (Sigma Chemicals). Sections
were counterstained with Mayer’s Hematoxylin and mounted in crystal
mount (Biomeda) for microscopic examination. As a standard negative
control, anti-DMBT1h12 was substituted by equal amounts of normal mouse
IgG (Santa Cruz). All controls were negative. For image acquisition,
digital analySIS 3.0 software, a 3CCD color video camera (Sony), and an
Olympus BX-50 microscope (Olympus Optical) were used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The gp-340 cDNA has recently been isolated and shown to match the
DMBT1 sequence (22)
. Taken together, these data
demonstrate that gp-340 is encoded by one (or more) of the alternative
splice products of DMBT1 and thus suggest that DMBT1 plays a
role in the respiratory immune defense.
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. Both antibodies stained subsets of immune cells.
Anti-gp-340 reacted with perivascular macrophages in the cerebellum
(Fig. 3b)
, and anti-DMBT1h12 reacted with leukocytes within
an intact blood vessel in the vicinity of a tumor (Fig. 3c)
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. Two
of 10 GBMs analyzed with anti-DMBT1h12 were completely negative for
DMBT1 expression. The remaining GBMs were composed of negative cell
populations and cells that expressed the protein at high levels (Fig. 3e)
. DMBT1 localized to intracellular vesicles and/or to the
cell surface (Fig. 3, f and g)
. On some
occasions, the protein appeared to be confined to the sites of
cell-cell interactions (Fig. 3g)
.
DMBT1 Plays a Role in Epithelial Differentiation.
The three rodent proteins, Ebnerin, CRP-ductin, and hensin, share
homology with DMBT1 and have a similar domain organization. The
expression patterns of the four genes, however, substantially differ
from each other (13, 14, 15)
. When we aimed at isolating the
murine homologue of DMBT1 by using cDNA probes specific for
the 5'- and 3'-end of DMBT1, we retrieved a set of clones
from different mouse cDNA and genomic libraries that all turned out to
contain parts of the CRP-ductin coding sequence (not shown).
This suggested that CRP-ductin represents the closest
relative of DMBT1 in mice and in turn that also
Ebnerin and hensin might be DMBT1
homologues in the other rodents. Hensin has initially been
characterized as a protein localizing to the kidney collecting duct
epithelium (15)
, and both CRP-ductin and hensin localize
to the intestinal crypts and surface epithelium (14
, 16)
.
We therefore analyzed these tissues for the localization of DMBT1 by
immunohistochemistry. We found that anti-DMBT1h12 likewise stained
epithelial cells in the collecting ducts of the cortical kidney (Fig. 4a).
In the duodenum, the crypt bases showed strongest
staining, whereas in the colon, the crypt tops and the surface
epithelium were found to be positive (Fig. 4b,
Fig. 4c,
Fig. 4d).
DMBT1 therefore closely resembles the
distribution of CRP-ductin and hensin in these organs. Because both
rodent proteins have been implicated in epithelial differentiation
(14
, 16)
and a role for DMBT1 in the carcinogenesis of
epithelial tumors has been proposed (3
, 4)
, we compared
the DMBT1 expression and localization in fetal and adult epithelia.
Both the fetal intestinal epithelium (Fig. 4e
and
Fig. 4f)
and the fetal skin (Fig. 4g,
Fig. 4h,
Fig. 4i)
displayed higher protein amounts than the adult counterparts (Fig. 4b,
Fig. 4c,
Fig. 4d
and Fig. 4j,
respectively), and the
subcellular localization of DMBT1 was different in the fetal epithelia.
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and proposed that these may confer a susceptibility to
genomic instability (20)
. To test this hypothesis, we
analyzed a set of lung and brain tumor cell lines for LOH and genomic
alterations at the DMBT1 locus. LOH was observed in 3 of 12
lung and 6 of 9 brain tumors (included in Fig. 5b
).
Remarkably, 16 of 18 tumors had aberrations in DMBT1, as
revealed by Southern blot analyses using the restriction enzyme
RsaI (Fig. 5b)
. Five tumors (H69, H128, H4,
U118-MG, and U343-MG) showed internal homozygous deletions. For a
direct comparison, we analyzed the genomic DNA of 36 normal individuals
for the configuration of the gene using the same restriction enzyme. We
found that 72% (26 of 36) of the normal individuals had two
full-length copies of DMBT1, whereas 10 individuals (28%)
showed different hemizygous deletions (Fig. 5c)
. One of
these configurations (G3 in Fig. 5c
) is compatible with a
hemizygous loss of the region coding for SRCR4 to SRCR7, similar to the
deletion observed in cell line H4. This may indicate that one of the
mechanisms by which homozygous deletions arise is the presence of one
deleted copy in the constitutional DNA that is uncovered by an LOH that
eliminates the intact copy.
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DISCUSSION |
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Unambiguously, DMBT1 has at least two distinct functions, one in the immune system and a second one during epithelial differentiation, as suggested by several lines of evidence. The cloning of the gp-340 cDNA that has been shown to be identical to one of the 8-kb transcripts of DMBT1 (22) and the Western blot studies in the present report demonstrate that the collectin-binding protein gp-340 corresponds to one or more of the isoforms encoded by DMBT1. gp-340 and therefore DMBT1 is a putative receptor for bacteria, viruses, and allergens that are opsonized by the collectins SP-D and SP-A and has been demonstrated to have a stimulatory effect on alveolar macrophages. Hence, DMBT1 appears to play a role in the mucosal immune defense, which is in line with its predominant expression in tissues with large epithelial surfaces, such as the lung and the small intestine. However, DMBT1 must have further immune-related functions, as is indicated by its widespread expression in the immune system and by the finding that perivascular macrophages and peripheral blood leukocytes express the protein. Moreover, in 6 of 10 medulloblastomas, the tumor periphery was highly enriched with DMBT1 expressing macrophages. The collectin mannan-binding protein has been demonstrated to bind to glioma and colorectal carcinoma cells in vitro and to mediate a cellular antitumor response in vivo (23 , 24) . DMBT1/gp-340 secreted by tumor-associated macrophages could likewise participate in a cellular antitumor response, e.g., by interacting with the collectins SP-D and SP-A and/or other molecules possibly bound to altered carbohydrate structures on the surface of the tumor cells.
Tumor cells within medulloblastomas were negative for staining with anti-gp-340, whereas a fraction of astrocytes and neuronal cells within the cerebellum was found to be positive. It would be premature to conclude that a loss of DMBT1 expression is a general hallmark of medulloblastoma formation because the precursor cells of medulloblastomas are thought to be pluripotent stem cells that give rise to the different cell types of the cerebellum. Furthermore, there are some hints that the two monoclonal antibodies recognize epitopes that can be present in combination in some isoforms, as indicated by our Western blot experiments, but differentially used in other isoforms, as indicated by the finding that anti-DMBT1h12, in contrast to anti-gp-340, did not reveal staining of neuronal cells and only faint signals in astrocytes. The integrity of the signals detected by the two antibodies is confirmed by preliminary results obtained from the characterization of a third monoclonal antibody that does not appear to have a preference for specific variants and therefore recognizes the composite pattern of anti-DMBT1h12 and anti-gp-340.4 In summary, this raises the possibility that distinct DMBT1 isoforms might exist in the brain that are subjected to a differential regulation.
Our data demonstrate that the isoform(s) recognized by anti-DMBT1h12 are deregulated in GBM. Whereas a subset of the GBM is DMBT1-deficient, the major fraction contains cell populations that express these variants at levels higher than those observed in normal astrocytes. Furthermore, a surface-linked form appears to be ectopically expressed in a subset of the tumor cells. One possible explanation could be that the expression of aberrant forms or the ectopic expression of naturally occurring variants may even offer growth advantages to the tumor cells. However, DMBT1 apparently shares a set of features with the group A SRCR protein Mac2-bp that likewise is either secreted or surface-linked, has a function in the mucosal immune defense, interacts with a lectin, is expressed by both tumor and immune cells, and activates immune cells (5, 6, 7 , 9 , 25) . Mac2-bp is present at elevated levels in the sera of cancer patients (25 , 26) and has a local and systemic tumor suppressive effect in vivo probably due to the activation of natural killer cells (7 , 8) . It is therefore thought that Mac2-bp secreted by tumor cells acts as an alarm signal stimulating an antitumor response. Based on the parallels between DMBT1 and Mac2-bp, it is possible that DMBT1 secreted by tumor cells is likewise involved in mechanisms of tumor surveillance, e.g., by stimulating macrophages in the local environment. Therefore, a loss of expression or the overexpression of aberrant variants could alternatively interfere with such processes.
There are several lines of evidence that CRP-ductin and hensin represent the rodent orthologs of DMBT1. CRP-ductin and hensin (and also Ebnerin) share a high homology with DMBT1 and have an overall similar domain organization. Our initial efforts identified CRP-ductin as the genomic homologue of DMBT1. Likewise, Takito et al. (27) have shown in a recent report that DMBT1, CRP-ductin, hensin, and Ebnerin are homologues in men and rodents. Our analyses demonstrate that DMBT1 colocalizes with CRP-ductin and hensin in the gastrointestinal tract and the kidney, supporting that the three proteins share a common function. Detailed biochemical studies have revealed that hensin is able to switch the polarity of collecting duct epithelial cells and to induce processes of terminal differentiation (15 , 16) . Most likely, hensin exerts its effect by multimerization and interaction with the extracellular matrix (28) . Because differences in the protein levels and the spatial distribution of DMBT1 were found when comparing fetal and adult epithelia, we must anticipate that DMBT1 also participates in epithelial differentiation, probably by similar mechanisms.
However, whereas an involvement in processes of differentiation supports a potential role of DMBT1 as a tumor suppressor, it is not yet clear if and how the gene might be inactivated during tumorigenesis. As proposed by its repetitive genomic structure (20) , DMBT1 was found to be highly unstable in tumors. Most likely, the DMBT1 repeats give rise to an increased rate of recombination that eliminates part of the exons coding for the SRCR domains and SIDs or leads to other rearrangements within the gene. In most of the cell lines analyzed, the rearrangements are complex and a detailed determination of the genomic configuration of the gene is in progress, but is hampered by the repetitive genomic structure. Moreover, deletion polymorphisms that are observed in normal individuals further complicate these analyses and indicate that some of the homozygous deletions arise by the uncovering of a hemizygously deleted allele by an LOH at chromosome 10q. In fact, these results in part are challenging because they raise the question as to whether alterations in DMBT1 are a cause or a consequence of tumorigenesis. Alternatively, however, this could raise the possibility that a subset of the population is a carrier of alleles that increase the cancer risk or facilitate tumorigenic growth once the formation of a tumor has taken place.
SRCR, CUB, and ZP domains have been implicated in the mediation of protein-protein interactions (17, 18, 19) . DMBT1/gp-340 is secreted in the oligomeric form in the lung (11) , and hensin exerts its effects in dependency of its state of oligo- or multimerization (28) . The SRCR domains of DMBT1 therefore are likely involved in either the oligomerization of the protein or the mediation of interactions with ligands. Thus, major questions to be solved in the future are what the function of the SRCR domains and SIDs is and whether these repetitive domains have distinct or redundant functions.
In conclusion, our data indicate that DMBT1 is subjected to alterations in a high number of tumors and plays a role in epithelial differentiation, in the immune defense, and eventually, in the immune surveillance of tumors. In vitro studies are in progress to test these models and to determine whether the different DMBT1 variants differ in their functional properties. Moreover, knock-out mice that will help to further elucidate the functions of DMBT1 and its possible role in tumorigenesis are under construction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Deutsche Krebshilfe Grant
10-1260-Po2 (to A. P.), by the Wilhelm Sander-Stiftung Grant
99.018.1 (to A. P.), the Danish Medical Research Council (to
U. H.), the Benzon Foundation (U. H.), the Schweizerischer
Nationalfonds 31-053746.9811 (to A. M.), and the Theodore
Ott-Fonds (to A. M.). 
2 To whom requests for reprints should be
addressed, at Department of Molecular Genome Analysis, Deutsches
Krebsforschungszentrum, Kst. H0600, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany. Phone: 49-6221-424746; Fax: 49-6221-423454;
E-mail: j.mollenhauer{at}dkfz-heidelberg.de
3 The abbreviations used are: DMBT1, deleted in
malignant brain tumors 1; SRCR, scavenger receptor cysteine-rich;
Mac2-bp, Mac-2-binding protein; CUB, C1r/C1s Uegf Bmp-1; ZP, zona
pellucida; SID, SRCR interspersed domain; GBM, glioblastoma multiforme;
SP-A, surfactant protein A; SP-D, surfactant protein D; BAL,
bronchoalveolar lavage; RT-PCR, reverse transcription-PCR; LOH, loss of
heterozygosity; KLH, keyhole limpet hemocyanin.
4 U. Holmskov, unpublished data.
Received 8/27/99. Accepted 1/19/00.
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 G. Cannon, Y. Yi, H. Ni, E. Stoddard, D. A. Scales, D. I. Van Ryk, I. Chaiken, D. Malamud, and D. Weissman HIV Envelope Binding by Macrophage-Expressed gp340 Promotes HIV-1 Infection J. Immunol., August 1, 2008; 181(3): 2065 - 2070. [Abstract] [Full Text] [PDF]
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E. Stoddard, G. Cannon, H. Ni, K. Kariko, J. Capodici, D. Malamud, and D. Weissman gp340 Expressed on Human Genital Epithelia Binds HIV-1 Envelope Protein and Facilitates Viral Transmission J. Immunol., September 1, 2007; 179(5): 3126 - 3132. [Abstract] [Full Text] [PDF]
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P. Rosenstiel, C. Sina, C. End, M. Renner, S. Lyer, A. Till, S. Hellmig, S. Nikolaus, U. R. Folsch, B. Helmke, et al. Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion J. Immunol., June 15, 2007; 178(12): 8203 - 8211. [Abstract] [Full Text] [PDF]
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 A. C. Blackburn, L. Z. Hill, A. L. Roberts, J. Wang, D. Aud, J. Jung, T. Nikolcheva, J. Allard, G. Peltz, C. N. Otis, et al. Genetic Mapping in Mice Identifies DMBT1 as a Candidate Modifier of Mammary Tumors and Breast Cancer Risk Am. J. Pathol., June 1, 2007; 170(6): 2030 - 2041. [Abstract] [Full Text] [PDF]
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 N Gassler, D Newrzella, C Bohm, S Lyer, L Li, O Sorgenfrei, L van Laer, B Sido, J Mollenhauer, A Poustka, et al. Molecular characterisation of non-absorptive and absorptive enterocytes in human small intestine Gut, August 1, 2006; 55(8): 1084 - 1089. [Abstract] [Full Text] [PDF]
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 S. Zhang, N. M. Green, I. Sitkiewicz, R. B. LeFebvre, and J. M. Musser Identification and Characterization of an Antigen I/II Family Protein Produced by Group A Streptococcus Infect. Immun., July 1, 2006; 74(7): 4200 - 4213. [Abstract] [Full Text] [PDF]
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 W. Kang, O. Nielsen, C. Fenger, G. Leslie, U. Holmskov, and K. B.M. Reid Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer Carcinogenesis, June 1, 2005; 26(6): 1129 - 1137. [Abstract] [Full Text] [PDF]
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V. Loimaranta, N. S. Jakubovics, J. Hytonen, J. Finne, H. F. Jenkinson, and N. Stromberg Fluid- or Surface-Phase Human Salivary Scavenger Protein gp340 Exposes Different Bacterial Recognition Properties Infect. Immun., April 1, 2005; 73(4): 2245 - 2252. [Abstract] [Full Text] [PDF]
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T. E. Grys, M. B. Siegel, W. W. Lathem, and R. A. Welch The StcE Protease Contributes to Intimate Adherence of Enterohemorrhagic Escherichia coli O157:H7 to Host Cells Infect. Immun., March 1, 2005; 73(3): 1295 - 1303. [Abstract] [Full Text] [PDF]
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 S. Tynan, E. Pacia, D. Haynes-Johnson, D. Lawrence, M. R. D'Andrea, J.-Z. Guo, S. Lundeen, and G. Allan The Putative Tumor Suppressor Deleted in Malignant Brain Tumors 1 Is an Estrogen-Regulated Gene in Rodent and Primate Endometrial Epithelium Endocrinology, March 1, 2005; 146(3): 1066 - 1073. [Abstract] [Full Text] [PDF]
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C. van der Meer-van Kraaij, E. Kramer, D. Jonker-Termont, M.B. Katan, R. van der Meer, and J. Keijer Differential gene expression in rat colon by dietary heme and calcium Carcinogenesis, January 1, 2005; 26(1): 73 - 79. [Abstract] [Full Text] [PDF]
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 F.J. Bikker, J.E. van der Wal, A.J.M. Ligtenberg, J. Mollenhauer, J.M.A. de Blieck-Hogervorst, I. van der Waal, A. Poustka, and A.V. Nieuw Amerongen Salivary Agglutinin/DMBT1SAG Expression is Up-regulated in the Presence of Salivary Gland Tumors Journal of Dental Research, July 1, 2004; 83(7): 567 - 571. [Abstract] [Full Text] [PDF]
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 M. B. Genter, D. M. Burman, S. Vijayakumar, C. L. Ebert, and B. J. Aronow Genomic analysis of alachlor-induced oncogenesis in rat olfactory mucosa Physiol Genomics, December 26, 2002; 12(1): 35 - 45. [Abstract] [Full Text] [PDF]
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 K. Sasaki, K. Sato, Y. Akiyama, K. Yanagihara, M. Oka, and K. Yamaguchi Peptidomics-based Approach Reveals the Secretion of the 29-Residue COOH-Terminal Fragment of the Putative Tumor Suppressor Protein DMBT1 from Pancreatic Adenocarcinoma Cell Lines Cancer Res., September 1, 2002; 62(17): 4894 - 4898. [Abstract] [Full Text] [PDF]
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F. J. Bikker, A. J. M. Ligtenberg, K. Nazmi, E. C. I. Veerman, W. van't Hof, J. G. M. Bolscher, A. Poustka, A. V. N. Amerongen, and J. Mollenhauer Identification of the Bacteria-binding Peptide Domain on Salivary Agglutinin (gp-340/DMBT1), a Member of the Scavenger Receptor Cysteine-rich Superfamily J. Biol. Chem., August 23, 2002; 277(35): 32109 - 32115. [Abstract] [Full Text] [PDF]
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H. Sasaki, R. A. Betensky, J. G. Cairncross, and D. N. Louis DMBT1 Polymorphisms: Relationship to Malignant Glioma Tumorigenesis Cancer Res., March 1, 2002; 62(6): 1790 - 1796. [Abstract] [Full Text] [PDF]
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F.J. Bikker, A.J.M. Ligtenberg, J.E. van der Wal, P.A.M. van den Keijbus, U. Holmskov, E.C.I. Veerman, and A.V. Nieuw Amerongen Immunohistochemical Detection of Salivary Agglutinin/gp-340 in Human Parotid, Submandibular, and Labial Salivary Glands Journal of Dental Research, February 1, 2002; 81(2): 134 - 139. [Abstract] [Full Text] [PDF]
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J. Mollenhauer, S. Herbertz, B. Helmke, G. Kollender, I. Krebs, J. Madsen, U. Holmskov, K. Sorger, L. Schmitt, S. Wiemann, et al. Deleted in Malignant Brain Tumors 1 Is a Versatile Mucin-like Molecule Likely to Play a Differential Role in Digestive Tract Cancer Cancer Res., December 1, 2001; 61(24): 8880 - 8886. [Abstract] [Full Text] [PDF]
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 R. C. De Lisle, K. S. Isom, D. Ziemer, and C. U. Cotton Changes in the exocrine pancreas secondary to altered small intestinal function in the CF mouse Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G899 - G906. [Abstract] [Full Text] [PDF]
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H. Sasaki, M. C. Zlatescu, R. A. Betensky, Y. Ino, J. G. Cairncross, and D. N. Louis PTEN Is a Target of Chromosome 10q Loss in Anaplastic Oligodendrogliomas and PTEN Alterations Are Associated with Poor Prognosis Am. J. Pathol., July 1, 2001; 159(1): 359 - 367. [Abstract] [Full Text] [PDF]
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