From the Department of Clinical Biochemistry, Sackler
Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel, the
¶ Institute of Animal Science, Volcani Center, Bet-Dagan 50250, Israel, the
Department of Nephrology, Tel Aviv Medical Center,
Tel Aviv 64239, Israel, the ** Centre for Medical Genetics, Department
of Cytogenetics and Molecular Genetics, Women's & Children Hospital,
and the
Department of Genetics, University
of Adelaide, Adelaide 5006, South Australia, and the
§§ Section on Developmental and Molecular
Pharmacology, Laboratory of Developmental Neurobiology, NICHD, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, August 15, 2000, and in revised form, September 19, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have recently cloned the mouse
activity-dependent neuroprotective protein (ADNP). Here, we
disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA
library. Comparative sequence analysis of these two ADNP orthologs
indicated 90% identity at the mRNA level. Several single
nucleotide polymorphic sites were noticed. The deduced protein
structure contained nine zinc fingers, a proline-rich region, a nuclear
bipartite localization signal, and a homeobox domain profile,
suggesting a transcription factor function. Further comparative
analysis identified an ADNP paralog (33% identity and 46%
similarity), indicating that these genes belong to a novel protein
family with a nine-zinc finger motif followed by a homeobox domain. The
hADNP gene structure spans ~40 kilobases and includes five
exons and four introns with alternative splicing of an untranslated
second exon. The hADNP gene was mapped to chromosome 20q12-13.2, a
region associated with aggressive tumor growth, frequently amplified in
many neoplasias, including breast, bladder, ovarian, pancreatic, and
colon cancers. hADNP mRNA is abundantly expressed in distinct
normal tissues, and high expression levels were encountered in
malignant cells. Down-regulation of ADNP by antisense
oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced
the viability of intestinal cancer cells by 90%. Thus, ADNP is
implicated in maintaining cell survival, perhaps through modulation of p53.
Mouse activity-dependent neuroprotective protein
(mADNP),1 a novel
vasoactive intestinal peptide (VIP)-responsive gene, was recently
cloned (1). The relative enrichment of mADNP transcripts in the
cerebellum, cortex, hippocampus, medulla, and midbrain and the
increases found in the presence of VIP, an established neuroprotective
substance (2), implied a potential function in brain metabolism.
Specifically, mADNP mRNA increased 2-3-fold in astroglial cells
incubated for 3 h in the presence of nanomolar amounts of VIP (1).
Another tissue containing increased mADNP transcripts is the mouse
testis, a highly proliferative tissue, suggesting the involvement of
ADNP in cell division.
As deregulation of oncogenes has been associated with neurodegeneration
(3), pathways that regulate neuronal survival may impinge upon cancer
proliferation. VIP regulates both neuronal survival and cell division
(2). A system whereby labeled VIP is suggested as a tumor marker has
been proposed, localizing in vivo tumors of patients with
gastrointestinal neuroendocrine cancers as well as pancreatic and
colonic adenocarcinomas (4). Other studies have identified a very high
incidence of VIP receptor binding in breast, ovarian, endometrial,
prostate, bladder, lung, esophageal, colonic, and pancreatic tumors as
well as in neuroendocrine and brain tumors (5). However, the VIP effect
on cancer growth depends on the specific tumor and may be stimulatory
(6, 7) or inhibitory (8). In view of the high incidence of tumors containing VIP receptors, a potential intervention in tumor growth may
employ a gene downstream of VIP action that is directly associated with
stimulation of cell proliferation and survival.
This report mapped the human ADNP (hADNP) gene
(GenBankTM/EBI accession number AF250860) to a chromosomal
region amplified in cancer, and ADNP mRNA expression was found to
increase in proliferative tissues. Inhibition of ADNP protein
expression by antisense oligodeoxynucleotides resulted in marked
reduction in metabolic activity in the target cells coupled with
increases in the tumor suppressor p53 (3). Furthermore, a paralogous
protein was discovered, suggesting a novel protein family containing
zinc fingers and a homeobox domain.
RNA Preparation--
Neuroblastoma cells (6) were incubated in
the presence of 25 nM VIP in phosphate-buffered saline
(PBS) for 3 h. Total RNA was prepared using RNAzol B solution
(Tel-Test, Inc., Friendswood, TX). A similar extraction method
was used for tumor tissues, obtained fresh, post-surgery, and frozen
immediately on liquid nitrogen.
cDNA Isolation and Sequencing--
Oligodeoxynucleotide
primers were synthesized in accordance with the mADNP cDNA sequence
(GenBankTM/EBI accession numbers AF068198 and NM_009628)
(1). These primers (ACCTGCAGCAAAACAACTAT and GCTCGTTACAGATTGTAC, sense
and antisense, respectively, for the mADNP cDNA) were thereafter
used for reverse transcriptase-polymerase chain reaction with human neuroblastoma RNA, including murine mammary leukemia virus reverse transcriptase (Life Technologies, Inc.) and AmpliTaq DNA
polymerase (PerkinElmer Life Sciences). The resulting polymerase chain
reaction product was sequenced automatically (Applied Biosystems
sequencer) at the Weizmann Institute of Science Core Facilities
(Rehovot, Israel). A human neuroblastoma ADNP reverse transcriptase
polymerase chain reaction product utilizing primers
5'-ATCTGTAGGCCAGGGTTACA-3' and 5'-TTGAGGAAGTGTTACCTGGG-3' (sense
(positions 1350-1369) and antisense (positions 1653-1672),
respectively) (see Fig. 1) was labeled with [ Northern Blot Hybridization--
RNA (10-12 µg) was subjected
to electrophoresis followed by Northern blot hybridization on 0.45-µm
Nitran filters (Schleicher & Schüll, Dassel, Germany). For probe
labeling, the cDNA was subjected to polymerase chain reaction as
described above. rRNA stained with ethidium bromide and actin mRNA
amounts were used as internal standards (e.g. Ref. 1).
Chromosomal Mapping--
The chromosomal localization of hADNP
was performed using several methods, as follows: 1) radiation hybrid
mapping (Stanford Human Genome Center), 2) fluorescent in
situ hybridization (FISH) with a genomic human contig
(GenBankTM/EBI accession number dj914P20.02099), and 3)
FISH with hADNP. The H7 cDNA (see Fig. 1) was nick-translated with
biotin-14-dATP and hybridized in situ at a final
concentration of 20 ng/µl to metaphase cells from two normal
males. The FISH procedure was modified from that previously described
(9) in that no pre-reassociation was necessary, and chromosomes were
stained before analysis with both propidium iodide (as a counterstain)
and the fluorescent DNA stain 4,6-diamidino-2-phenylindole for
chromosome identification. Images of metaphase preparations were
captured by a cooled CCD camera using the ChromoScan image collection
and enhancement system (Applied Imaging International, Ltd.). FISH
signals and 4,6-diamidino-2-phenylindole banding were merged for figure preparation.
Western Analysis--
For ADNP analysis, cultures were washed
with PBS and subjected to lysis (15 min, 4 °C) in a buffer
containing 1 mM EDTA, 150 mM NaCl, 0.1 mM ZnCl2, 1 mM MgCl2,
50 mM Tris (pH 8.5), 0.1% SDS, and 0.1% Triton
X-100. Nuclear DNA was fragmented by sonication, and
supernatants (10,000 × g, 10 min) were collected and
frozen until further measurements. For p53 analysis, cells were washed with PBS, and cell lysis (10 min, 4 °C) was conducted in a buffer containing 5 mM EDTA, 150 mM NaCl, 10 mM Tris (pH 7.4), 1% Triton X-100, 0.23 units/ml
aprotinin, 10 mM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM benzamidine.
Protein supernatants were collected following sonication by
centrifugation (16,000 × g, 20 min, 4 °C). 5 µg
of the soluble proteins were separated by electrophoresis on a 10%
polyacrylamide gel and electrotransferred to nitrocellulose filters.
Membranes were treated with 10% milk + PBS/Tween (0.2%) for 1 h
and incubated overnight at 4 °C in 2% milk + PBS/Tween (0.2%) and
the appropriate antibody. After incubation with peroxidase-conjugated secondary antibodies (Roche Molecular Biochemicals), signals were revealed by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech).
Antibody Preparation--
The following commercial antibodies
were used: mouse monoclonal IgG anti-human p53 antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti- Cell Culture and Inhibition of Growth by Antisense
Oligodeoxynucleotides--
The human colon cancer cell line HT29 (10)
was cultured in Dulbecco's modified Eagle's medium supplemented with
10% heat-inactivated fetal calf serum, 2 mM
L-glutamine, and 1% Pen-Strep-Nystatin (Biological
Industries, Beit Haemek, Israel). The adherent cells were split when a
subconfluent monolayer was formed following treatment with 0.25 units
of trypsin and 0.02% EDTA and naturalization with serum-containing
medium. For growth inhibition experiments, subconfluent adherent cells
were washed with PBS, treated with trypsin as described above, and
resuspended in Dulbecco's modified Eagle's medium containing 5%
fetal calf serum to a final concentration of 50,000 cells/ml. 100-µl
aliquots were seeded into individual wells of 96-well microtiter plates
(Nunclon, Nunc Brand Products, Roskilde, Denmark). Each plate had a
blank column and the appropriate controls. Plates were incubated for
24 h in a humidified atmosphere containing 95% air and 5%
CO2 at 37 °C; the medium was then replaced to contain an
antisense oligodeoxynucleotide (10 µM) in Dulbecco's modified Eagle's medium without fetal calf serum. Following an additional 24-h incubation period, the medium was replaced again to
contain Dulbecco's modified Eagle's medium and 5% fetal calf serum,
and cells were subjected to a further 48-h incubation period. Viable
cell number was determined by a 3-h incubation period with the
MTS reagent (CellTiter 96 AQueous cell proliferation kit, Promega, Madison, WI). The MTS reagent is oxidized by active
mitochondria, resulting in increases in light absorbance at 490 nm
(evaluated by a Multiscan plate reader). For protein preparation, cells
were harvested (as described above) after a 30-h incubation period.
Statistical Analysis--
Analysis of variance with
Student-Neuman-Kuel's multiple comparison of means test was
used to assess the results.
hADNP Structure--
To isolate and characterize hADNP, the human
ortholog of mADNP (1), a cDNA library derived from human fetal
brain (19-23 weeks of gestation) was screened, and eight clones were
isolated. The complete sequences of two cDNA clones (clones H7 and
H3) indicated 90% identity to mADNP at the mRNA level. Fig.
1 shows the sequence of hADNP (clone H7)
with additional deduced upstream expressed sequence tags (AW453069,
AW452644, AW139427, and AW17331) (11), human genomic contig sequences
containing ADNP (dJ914P20 contig ID 02099 and genomic clone AL034553).
Table I shows the exon-intron junctions
of the five exons of the gene. The estimated gene size is 40,647 base
pairs. A CpG island that stretches over 1135 bases as predicted by
GRAIL was observed around exon 1 (69% GC). As particularly
CG-rich dinucleotides have been previously associated with
promoter regions, we tested this sequence using promoter prediction
programs TSSW and TSSG. Results gave low scoring promoter (TSSW
at base 106 with LDF 5.69 (LDF = statistical promoter score > 4.00 indicates a potential promoter); TSSG gave no promoter). Alternative splicing of the second exon has been observed in
expressed sequence tags (AI827420 and AW007743). Only the three
3'-exons are protein-coding. The proximal gene upstream of the ADNP
gene is DPM1
(dolichyl-phosphate
mannosyltransferase polypeptide 1 catalytic
subunit) separated by 3438 base pairs.
At the protein structure level (Fig. 1), nine potential zinc finger
motifs that are identical between hADNP and mADNP (1) were identified.
These zinc finger domains (12), a proline-rich region (12), a nuclear
bipartite localization signal (13), and a partial homeobox domain
profile (14) suggest nuclear localization (12-14). Furthermore, a
glutaredoxin active site (15) as well as a leucine-rich nuclear export
sequence (16) were found. One striking difference between mouse and
human was a polyglutamic acid stretch of nine residues in mouse (1)
shortened to one residue in human (position 931) (Fig. 1).
The second cDNA clone (H3) was identical to clone H7 except for
several polymorphic regions (Table
II) and utilization of a different
polyadenylation site (Fig. 1). Moreover, clone H3 contained a
frameshift mutation (an additional A nucleotide at position 3393) (Fig.
1), with a premature termination codon at position 3408 (Fig. 1).
Unexpectedly, the H3 cDNA contained an additional protein-coding
sequence downstream of a short poly(A) stretch, encoding the human
immunodeficiency virus Tat TBP1 protein (transactivator-binding
protein 1) (17, 18).
Comparative analysis utilizing BLAST identified part of rat ADNP
(GenBankTM/EBI accession number AAF40431) (90%
identity) (Fig. 2). Further analysis
revealed 33% identity and 46% similarity to the paralogous
brain protein KIAA0863 (GenBankTM/EBI accession number
AB020670) (19). This protein revealed similar nine-zinc finger domains
and a similar homeobox domain as found in ADNP, suggesting a new gene
family (Fig. 2).
hADNP Expression--
Northern blot hybridization utilizing mADNP
(1) and hADNP identified one major mRNA band (5.5 kilobases)
(Fig. 3A). This mRNA
showed increased expression in the heart, skeletal muscle, kidney, and
placenta. As ADNP was originally cloned from embryonic brain tissue
(see above and also Ref. 1), further analysis of different brain
regions was performed (Fig. 3B). The results indicated
increased expression in the cerebellum and cortex (Fig. 3B).
Serial analysis of gene expression was also performed. The results
obtained suggested increased expression in tumor tissues, adenocarcinoma (breast and ovaries), medulloblastoma (brain), and
glioblastoma (brain) and colon cancer. In normal tissues, ADNP
sequences were found in microvascular endothelial cells and in brain
(mostly white matter). Serial analysis of gene expression of the
related KIAA0863 (cDNA isolated from human brain;
GenBankTM/EBI accession number AB020670) revealed increased
expression in tumors (colon and prostate) and in brain white
matter as well as in the kidney and testis.
Chromosomal Localization--
20 metaphase cells from a
normal male were examined by FISH. All of these metaphase cells showed
signal on one or both chromatids of chromosome 20 in the region
20q12-13.2; 40% of this signal was at 20q12, 32% was at
20q13.1, and 28% was at 20q13.2 (Fig. 4). Similar results were obtained
utilizing public data bases, localizing the gene to chromosome 20q13.2
(with identity to the ordered markers G30243 and W45435 in linkage to
the Genome Data Base locus D20S831) and to 20q13.13-13.2 utilizing a
human contig sequence containing the hADNP gene. KIAA0863 was localized to human chromosome 18 using public data bases.
hADNP and Cancer--
Since serial analysis of gene expression
identified increased ADNP expression in cancer cell lines and since the
chromosomal region 20q12-13 is amplified in a wide variety of tumors
(19-23), we investigated the association of hADNP with cancer growth.
Three lines of experimental studies were conducted. 1) hADNP mRNA
was quantitated in human primary cancer tissue (breast and colon) in
comparison with adjacent normal tissue and was shown to be significantly increased in the cancer. A 2.5-3.5-fold increase was
observed in colon cancer (data not shown). The increased expression was
most evident in breast cancer and was 14.4 ± 4.6-fold (mean ± S.E.). When the ADNP mRNA content was compared with the actin mRNA content in the same breast cancer samples, the increase was 10.9 ± 5-fold (Fig. 5).
2) Six antisense oligodeoxynucleotides were synthesized (Fig. 1) and
further utilized to inhibit cell proliferation. The
oligodeoxynucleotides were chosen as complementary to the 5'-most
methionines (indicated in Fig. 1). The results showed that antisense
oligodeoxynucleotide 1 inhibited cell division (measured as metabolic
activity) in the human intestinal cancer cell line HT29
(p < 0.001) (Fig. 6). A
similar inhibition was observed with antisense oligodeoxynucleotide 8 (p < 0.001) (Fig. 6). Furthermore, antisense
oligodeoxynucleotide 9 inhibited by ~37.5 ± 3%, and antisense
oligodeoxynucleotide 68 also inhibited growth (by 45 ± 3%;
p < 0.001). In contrast to antisense
oligodeoxynucleotides 8 and 9, the sequence of antisense oligodeoxynucleotide 68 is shared by other cDNA sequences; hence, it may not be specific. Further specificity was determined with a control sense oligodeoxynucleotide complementary to antisense oligodeoxynucleotide 8 and with an antisense oligodeoxynucleotide 8 with all internucleotide bonds of the phosphorothioated type (Fig. 6). In addition, antisense oligodeoxynucleotides 7 and 67 did not inhibit growth.
3) To determine that the antisense oligodeoxynucleotides indeed
inhibited ADNP expression, Western blot analyses were performed with
actin and the tumor suppressor p53 as internal standards. The results
show that ADNP (114,000 Da) was decreased by ~3-fold in comparison
with actin (densitometric scan results: 1.11 ± 0.23 versus 0.31 ± 0.11, respectively; p < 0.023; n = 3), whereas p53 levels showed an apparent
increase (1.04 ± 0.04 versus 2.41 ± 0.41;
p < 0.029; n = 3) (Fig.
7).
This report characterizes the hADNP gene, encoding an mRNA
that is abundantly expressed in distinct normal tissues and that may be
alternatively spliced. The 5'-untranslated region of the mRNA is GC-rich, as has been recently shown for several other genes
(e.g. Refs. 24-26). hADNP was found to contain zinc fingers and a homeobox domain profile. Furthermore, a family including at least
two genes of significant homologies is described.
Based on cDNA and deduced protein sequence (12-14), hADNP and
KIAA0863 may represent nuclear DNA-binding proteins, putative transcription factors. The thiotransferase/glutaredoxin active site
(15) found in ADNP (Fig. 1) may modulate its own DNA binding activity
or that of other DNA-binding proteins in response to oxidative stress
and signal transduction pathways implicated in the redox state of the
cell (27). We have previously hypothesized that mADNP is a secreted
protein (1). To reconcile this discrepancy, one hypothesis may involve
alternate utilization of the seven putative initiator methionine
residues at the N terminus of hADNP (Fig. 1), resulting in processing
pathways that may yield secreted portions. An alternative hypothesis
was put forward by us in a recent report suggesting the existence of a
nuclear export signal within the ADNP mRNA (Fig. 1) (28). A
similar sequence was discovered in the engrailed transcription factor
(16) as well as in the ADNP-related protein KIAA0863.
The ADNP-containing locus, the 20q12-13.2 chromosomal region, is
amplified in many tumors (19-23). In breast tumors, comparative genomic hybridization revealed ~20 regions of recurrent increased DNA
sequence copy number (23, 29-31). These regions are predicted to
encode dominant genes that may play a role in tumor progression or
response to therapy. Three of these regions have been associated with
established oncogenes: ERBB2 at 17q12, MYC
at 8q24, and CCND1 and EMS1 at 11q13.
Amplification at 20q13 occurs in a variety of tumor types, but up to
date, does not involve a previously known oncogene (20).
Another aspect of ADNP/cancer/neuroprotection interaction is
the fact that ADNP and p53 expression may be interrelated, as shown
here, and both proteins may influence tumor growth as well as brain
function (1, 3).
The hADNP cDNA (clone H3) contained the TBP1
cDNA sequence downstream of the coding region of ADNP. Previously,
the TBP1 gene was localized to chromosome 11p12-13 (18),
and the TBP1 gene product was associated with the cell
cycle. The finding of TBP1 downstream of hADNP either may be
trivial, resulting from molecular cloning manipulations, or may
indicate translocation involved with cancer abnormalities.
The discovery of ADNP (1) as a VIP-responsive gene in astroglial cells
(a major component of brain white matter) is now extended to the serial
analysis of gene expression finding of ADNP-encoding sequences in brain
(mostly white matter) as well as in microvascular endothelial cells.
VIP-binding sites have been described in astrocytes (32) as well as in
endothelial cells (33). In both cases, developmental functions (33, 34) and proliferation (34-36)/survival (32, 37) functions have been
hypothesized. The homeobox-containing protein ADNP may thus mediate
some of the VIP developmental/survival-associated effects involving
normal growth and cancer proliferation. The abundance of ADNP mRNA
in heart, skeletal muscle, kidney, and placenta may represent, in part,
an astrocyte-like cell population (38) or enrichment in blood
microvessels (39). Indeed, the original characterization of VIP was as
a vasodilator (40); and since endothelial cells play a major role in
vasodilatation, endothelial ADNP points toward a new avenue for
research on potential VIP/ADNP interactions.
Our original findings related ADNP to VIP-mediated neuroprotection.
Thus, ADNP mRNA increased in glial cells incubated with VIP, and a
very short peptide fragment derived from ADNP (NAPVSIPQ, termed NAP)
provided potent neuroprotection (1). Given the abundant expression of
ADNP, future experiments are aimed at further assessing the question of
general normal cell protection and of secreted processed forms of ADNP
providing cellular protection against external toxicity. The
increased ADNP mRNA expression in the cerebellum (a structure
enriched in VIP-binding sites) (41) suggests a further avenue of
research dealing with tissue-specific expression and function.
From a clinical perspective, this report provides methods of using
hADNP nucleic acid probes to detect and identify pathologically proliferating cells, including cancer cells. Furthermore, our results
suggest that ADNP is important for cell survival, and the antisense
ADNP oligodeoxynucleotides may be developed as antitumor therapeutics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP
(3000 Ci/mmol; Amersham Pharmacia Biotech, Buckinghamshire, United
Kingdom). The labeled product was used to screen a cDNA library
derived from human whole fetal brain (male-female pooled, Caucasian,
19-23 weeks of gestation, cloned unidirectionally into the
Uni-ZAPTMXR vector (Stratagene, La Jolla, CA)).
-actin antibodies
(Sigma, Rehovot), peroxidase-conjugated goat anti-mouse IgG
(AffiniPure, Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA), and horseradish peroxidase-linked donkey anti-rabbit Ig
(Amersham Pharmacia Biotech). Anti-ADNP antibody was prepared against a
synthetic peptide
(989CEMKPGTWSDESSQSEDARSSKPAAKK1015) fused to
keyhole limpet hemocyanin through the N-terminal cysteine moiety. In a
parallel experiment, the carrier protein was bovine serum albumin.
Affinity chromatography was performed on the peptide attached to
Sepharose as described before (1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (81K):
[in a new window]
Fig. 1.
The hADNP cDNA and gene. Shown is
the hADNP sequence (based on clone H7). The left side of the sequence
shows nucleotides numbers, and the right side shows amino acid numbers.
The beginnings of the exons are labeled and indicated by
downward-pointing arrows. Alternative polyadenylation sites
are numbered and indicated by upward-pointing arrows: clone
H4 (1 ); clones H6 and H2 (2
); clone H10
(3
); and clones H3, H5, and H7 (4
). The
calculated molecular mass of the protein was 12,3562.8 Da, and the
theoretical pI was 6.97. Antisense oligonucleotides are
underlined and labeled (AS-1, AS-8,
AS-9, AS-7, AS-67, and
AS-68). Zinc finger domains are shown in boldface
with dotted underlining. The second,
sixth, and seventh dotted zinc finger domains are
designated as trusted by Pfam (protein families database of
alignments; alignments can be trusted
certain or potential). The
bipartite nuclear localization signal is shown in boldface
with dotted/dashed underlining. The homeobox domain is shown
in boldface with double underlining. The
proline-rich region is shown in boldface with dashed
underlining. The partial glutaredoxin (thiotransferase) active
site is shown in boldface with double-dotted and
dashed underlining. The leucine-rich nuclear export sequence
is as follows:
KLAASLWLWKSDIASHF
.
Exon-intron junctions of the hADNP gene
Polymorphic sites in hADNP (see Fig. 1)
View larger version (127K):
[in a new window]
Fig. 2.
The ADNP gene is conserved among
species. Comparative studies identified a new family member,
KIAA0863. Dashed lines are zinc finger domains; the
solid line is a presumptive homeobox domain region.
View larger version (65K):
[in a new window]
Fig. 3.
Patterns of expression of the hADNP
mRNA. A, master blot (human 12-lane multiple tissue
Northern blot 7780-1, CLONTECH, Palo Alto,
CA). Lane 1, brain; lane 2, heart;
lane 3, skeletal muscle; lane
4, colon; lane 5, thymus;
lane 6, spleen; lane 7,
kidney; lane 8, liver; lane
9, small intestine; lane 10, placenta;
lane 11, lung; lane 12,
peripheral blood leukocytes. B, hADNP mRNA in brain
tissues. The human brain RNA master blot (7755-1) was purchased from
CLONTECH. Hybridization was performed as described
under "Experimental Procedures." Lane 1, cerebellum;
lane 2, cerebral cortex; lane 3, medulla;
lane 4, spinal cord; lane 5, occipital lobe;
lane 6, frontal lobe; lane 7, temporal lobe;
lane 8, putamen. kb, kilobases.
View larger version (26K):
[in a new window]
Fig. 4.
Chromosomal localization of hADNP. Shown
are photographs and idiogram (insert) of the hybridization
sites of clone H7. A total of two nonspecific background dots were
observed in the 20 metaphases tested. A similar result was obtained
from hybridization of the probe to 10 metaphases from a second normal
male (not shown). Two representative pictures are shown.
View larger version (38K):
[in a new window]
Fig. 5.
ADNP mRNA content is increased in
tumors. RNA was extracted from human primary tumors
(breast) and from adjacent normal tissue and subjected to Northern blot
hybridization. C, control tissue; T, tumor. This
is a breast cancer sample from a 48-year-old female. Shown are
autoradiograms of ADNP and actin mRNAs and ethidium bromide-stained
RNA.
View larger version (11K):
[in a new window]
Fig. 6.
HT29 cell growth is inhibited in the presence
of antisense oligodeoxynucleotides specific for ADNP mRNA.
Five oligodeoxynucleotides were synthesized (see Fig. 1) and utilized
to inhibit cancer growth. A representative figure is shown. Bar
1, control; bar 2, antisense oligodeoxynucleotide 1;
bar 3, sense oligodeoxynucleotide 8; bar 4,
antisense oligodeoxynucleotide 8; bar 5, antisense 8 with
all internucleotide bonds of the phosphorothioated type.
View larger version (34K):
[in a new window]
Fig. 7.
Western blot analysis: reduction in ADNP in
HT29 cells in comparison with actin and p53. Experiments were
performed as described under "Experimental Procedures." , no
antisense oligodeoxynucleotide; +, cells incubated in the presence of
the antisense oligodeoxynucleotide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Prof. Samuel Berkovic for invaluable help with the chromosomal mapping. We are grateful to Joshua Steinerman and Sharon Furman for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the United States-Israel Binational Science Foundation (to I. G. and D. E. B.) and the Israel Science Foundation. Patents have been applied for hADNP and the antisense oligodeoxynucleotides.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) AF250860.
§ Performed this work in partial fulfillment of Ph.D. requirements at Tel Aviv University.
¶¶ Incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors. To whom correspondence should be addressed. Tel.: 972-3-6407240; Fax: 972-3-6408541; E-mail: igozes@post.tau.ac.il.
Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M007416200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: mADNP, mouse activity-dependent neuroprotective protein; VIP, vasoactive intestinal peptide; hADNP, human ADNP; PBS, phosphate-buffered saline; FISH, fluorescent in situ hybridization; contig, group of overlapping clones.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blat, C., Gibney, G., Glazner, G., Brenneman, D. E., and Gozes, I. (1999) J. Neurochem. 72, 1283-1293[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gozes, I., Fridkin, M., Hill, J. M., and Brenneman, D. E (1999) Curr. Med. Chem. 6, 1019-1034[Medline] [Order article via Infotrieve] |
3. |
Amson, R.,
Lassalle, J. M.,
Halley, H.,
Prieur, S.,
Lethrosne, F.,
Roperch, J. P.,
Israeli, D.,
Gendron, M. C.,
Duyckaerts, C.,
Checler, F.,
Dausset, J.,
Cohen, D.,
Oren, M.,
and Telerman, A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5346-5350 |
4. | Virgolini, I. (1997) Eur. J. Clin. Invest. 27, 793-800[Medline] [Order article via Infotrieve] |
5. | Reubi, J. C. (1996) Ann. N. Y. Acad. Sci. 805, 753-759[Medline] [Order article via Infotrieve] |
6. | Lilling, G., Wollman, Y., Goldstein, M. N., Rubinraut, S., Fridkin, M., Brenneman, D. E., and Gozes, I. (1995) J. Mol. Neurosci. 5, 231-239 |
7. | Zia, H., Hida, T., Jakowlew, S., Birrer, M., Gozes, Y., Reubi, J. C., Fridkin, M., Gozes, I., and Moody, T. W. (1996) Cancer Res. 56, 3486-3489[Abstract] |
8. |
Maruno, K.,
Absood, A.,
and Said, S. I.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14373-14378 |
9. | Callen, D. F., Baker, E., Eyre, H. J., Chernos, J. E., Bell, J. A., and Sutherland, G. R. (1990) Ann. Genet. 33, 219-221[Medline] [Order article via Infotrieve] |
10. | Tew, K. D., O'Brien, M., Laing, N. M., and Shen, H. (1998) Chem. Biol. Interact. 111-112, 199-211 |
11. | Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1998) DNA Res. 5, 277-286[Medline] [Order article via Infotrieve] |
12. | Taguchi, E., Muramatsu, H., Fan, Q. W., Kurosawa, N., Sobue, G., and Muramatsu, T. J. (1998) J. Biochem. (Tokyo) 124, 1220-1228[Abstract] |
13. | Williams, S. C., Angerer, N. D., and Johnson, P. F. (1997) Gene Expr. 6, 371-385[Medline] [Order article via Infotrieve] |
14. | Gehring, W. J., Affolter, M., and Burglin, T. (1994) Annu. Rev. Biochem. 63, 487-526[CrossRef][Medline] [Order article via Infotrieve] |
15. | Johnson, G. P., Goebel, S. J., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. (1991) Virology 181, 378-381[Medline] [Order article via Infotrieve] |
16. |
Maizel, A.,
Bensaude, O.,
Prochiantz, A.,
Joliot, A.,
and Nelbock, P.
(1999)
Development
126,
3183-3890 |
17. | Dillom, P. J., Perkin, A., and Rosen, C. A. (1990) Science 248, 1650-1653[Medline] [Order article via Infotrieve] |
18. | Hoyle, J., Tan, K. H., and Fisher, E. M. (1997) Hum. Genet. 99, 285-288[CrossRef][Medline] [Order article via Infotrieve] |
19. | Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1998) DNA Res. 5, 355-364[Medline] [Order article via Infotrieve] |
20. |
Collins, C.,
Rommens, J. M.,
Kowbel, D.,
Godfrey, T.,
Tanner, M.,
Hwang, S.,
Polikoff, D.,
Nonet, G.,
Cochran, J.,
Myambo, K.,
Jay, K. E.,
Froula, J.,
Cloutier, T.,
Kuo, W.-L.,
Yaswen, P.,
Dairkee, S.,
Giovanola, J.,
Hutchinson, G. B.,
Isola, J.,
Kallioniemi, O.-P.,
Palazzolo, M.,
Martin, C.,
Ericsson, C.,
Pinkel, D.,
Albertson, D.,
Li, W.-B.,
and Gray, J. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8703-8708 |
21. | Palmedo, G., Fischer, J., and Kovacs, G. (1997) Lab. Invest. 77, 633-668[Medline] [Order article via Infotrieve] |
22. | El-Rifai, W., Harper, J. C., Cummings, O. W., Hyytinen, E. R., Frierson, H. F., Jr., Knuutila, S., and Powell, S. M. (1998) Cancer Res. 58, 34-37[Abstract] |
23. | Sonoda, G., Palazzo, J., du Manoir, S., Godwin, A. K., Feder, M., Yakushiji, M., and Testa, J. R. (1997) Genes Chromosomes Cancer 20, 320-328[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Zabarovsky, E. R.,
Gizatullin, R.,
Podowski, R. M.,
Zabarovska, V. V.,
Xie, L.,
Muravenko, O. V.,
Kozyrev, S.,
Petrenko, L.,
Skobeleva, N.,
Li, J.,
Protopopov, A.,
Kashuba, V.,
Ernberg, I.,
Winberg, G.,
and Wahlestedt, C.
(2000)
Nucleic Acids Res.
28,
1635-1639 |
25. | Jeong, J., Choi, S., Gu, C., Lee, H., and Park, S. (2000) DNA Cell Biol. 19, 291-300[CrossRef][Medline] [Order article via Infotrieve] |
26. | Reichwald, K., Thiesen, J., Wiehe, T., Weitzel, J., Poustka, W. A., Rosenthal, A., Platzer, M., Stratling, W. H., and Kioschis, P. (2000) Mamm. Genome 11, 182-190[CrossRef][Medline] [Order article via Infotrieve] |
27. | Kallioniemi, A., Kallioniemi, O.-P., Piper, J., Tanner, M., Stokke, T., Chen, L., Smith, H. S., Pinkel, D., Gray, J. W., and Waldman, F. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2156-2160[Abstract] |
28. | Gozes, I., Zamostiano, R., Pinhasov, A., Bassan, M., Giladi, E., Steingart, R. A., and Brenneman, D. E. (2000) Ann N. Y. Acad. Sci. U. S. A., in press |
29. |
Bandyopadhyay, S.,
Starke, D. W.,
Mieyal, J. J.,
and Gronostajski, R. M.
(1998)
J. Biol. Chem.
273,
392-397 |
30. | Isola, J. J., Kallioniemi, O.-P., Chu, L. W., Fuqua, S. A., Hilsenbeck, S. G., Osborne, C. K., and Waldman, F. M. (1995) Am. J. Pathol. 147, 905-911[Abstract] |
31. | Albertson, D. G., Ylstra, B., Segraves, R., Collins, C., Dairkee, S. H., Kowbel, D., Kuo, W. L., Gray, J. W., and Pinkel, D. (2000) Nat. Genet. 25, 144-146[CrossRef][Medline] [Order article via Infotrieve] |
32. | Gozes, I., Mccune, S. K., Jacobson, L., Warren, D., Moody, T. W., Fridkin, M., and Brenneman, D. E. (1991) J. Pharmacol. Exp. Ther. 257, 959-966[Abstract] |
33. | Lange, D., Funa, K., Ishisaki, A., Bauer, R., and Wollina, U. (1999) Histol. Histopathol. 14, 821-825[Medline] [Order article via Infotrieve] |
34. | Gressens, P., Hill, J. M., Gozes, I., Fridkin, M., and Brenneman, D. E. (1993) Nature 362, 155-158[CrossRef][Medline] [Order article via Infotrieve] |
35. | Brenneman, D. E., Nicol, T., Warren, D., and Bowers, L. M. (1990) J. Neurosci. Res. 25, 386-394[Medline] [Order article via Infotrieve] |
36. | Zupan, V., Hill, J. M., Brenneman, D. E., Gozes, I., Fridkin, M., Robberecht, P., Evrard, P., and Gressens, P. (1998) J. Neurochem. 70, 2165-2173[Medline] [Order article via Infotrieve] |
37. | Ashur-Fabian, O., Giladi, E., Brenneman, D. E., and Gozes, I. (1997) J. Mol. Neurosci. 9, 211-222[Medline] [Order article via Infotrieve] |
38. | Buniatian, G., Gebhardt, R., Traub, P., Mecke, D., and Osswald, H. (1999) Biol. Cell 91, 675-684[CrossRef][Medline] [Order article via Infotrieve] |
39. | Said, S. I., and Mutt, V. (1972) Eur. J. Biochem. 28, 199-204[Medline] [Order article via Infotrieve] |
40. | Lee, T. J. (2000) J. Biomed. Sci. 7, 16-26[CrossRef][Medline] [Order article via Infotrieve] |
41. | Hill, J. M., Lee, S. J., Dibbern, D. A., Fridkin, M., Gozes, I., and Brenneman, D. E. (1999) Neuroscience 93, 783-791[CrossRef][Medline] [Order article via Infotrieve] |