DIO-1 is a gene involved in onset of apoptosis in vitro, whose misexpression disrupts limb development

ArticleinProceedings of the National Academy of Sciences 96(14):7992-7 · August 1999with19 Reads
Impact Factor: 9.67 · DOI: 10.1073/pnas.96.14.7992 · Source: PubMed

The DIO-1 (death inducer-obliterator-1) gene, identified by differential display PCR in pre-B WOL-1 cells undergoing apoptosis, encodes a putative transcription factor whose protein has two Zn finger motifs, nuclear localization signals, and transcriptional activation domains, expressed in the limb interdigitating webs during development. When overexpressed, DIO-1 translocates to the nucleus and activates apoptosis in vitro. Nuclear translocation as well as induction of apoptosis are lost after deletion of the nuclear localization sequences. DIO-1 apoptotic induction is prevented by caspase inhibitors and Bcl-2 overexpression. The in vivo role of DIO-1 was studied by misexpressing DIO-1 during chicken limb development. The most frequently observed phenotype was an arrest in limb outgrowth, an effect that correlates with the inhibition of mesodermal and ectodermal genes involved in this process. Our data demonstrate the ability of DIO-1 to trigger apoptotic processes in vitro and suggest a role for this gene in cell death during development.


Available from: Juan P Albar
Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 7992–7997, July 1999
Developmental Biology
DIO-1 is a gene involved in onset of apoptosis in vitro, whose
misexpression disrupts limb development
(transcription factors
interdigital webs)
*Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, Universidad Auto´noma, Campus de Cantoblanco, E-28049 Madrid,
Department of Immunology, Stockholm University, Stockholm, Sweden; and
The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037
Communicated by A. Garcia-Bellido, Autonomous University of Madrid, Madrid, Spain, April 23, 1999 (received for review March 3, 1999)
ABSTRACT The DIO-1 (death inducer-obliterator-1) gene,
identified by differential display PCR in pre-B WOL-1 cells
undergoing apoptosis, encodes a putative transcription factor
whose protein has two Zn finger motifs, nuclear localization
signals, and transcriptional activation domains, expressed in
the limb interdigitating webs during development. When
overexpressed, DIO-1 translocates to the nucleus and activates
apoptosis in vitro. Nuclear translocation as well as induction
of apoptosis are lost after deletion of the nuclear localization
sequences. DIO-1 apoptotic induction is prevented by caspase
inhibitors and Bcl-2 overexpression. The in vivo role of DIO-1
was studied by misexpressing DIO-1 during chicken limb
development. The most frequently observed phenotype was an
arrest in limb outgrowth, an effect that correlates with the
inhibition of mesodermal and ectodermal genes involved in
this process. Our data demonstrate the ability of DIO-1 to
trigger apoptotic processes in vitro and suggest a role for this
gene in cell death during development.
Apoptosis is a major form of cell death, characterized mor-
phologically by chromatin condensation, nuclear disruption,
and formation of cytosol containing apoptotic bodies. It is an
efficient mechanism for eliminating unwanted cells and is of
central importance for development and homeostasis in meta-
zoan animals (1). Many different signals within or from outside
the cell have been shown to influence the decision between life
and death (2). Most are controlled through triggering of
specific receptors, which leads to activation of specific medi-
ators; they may then act to suppress or promote activation of
the death program. It is hence not surprising that initiation of
apoptosis is precisely regulated.
A common meeting point for cell death signals is the
cytoplasm, where the caspases exert their function and are
blocked by their inhibitors (3–6). Very little is known as to how
these signals are transmitted to the nucleus. A caspase-
activated DNase (CAD) and its inhibitor (ICAD) have recently
been identified in the cytoplasmic fraction of a mouse lym-
phoma cell line. Caspase pathway activation by different
stimuli cleaves ICAD, allowing CAD to enter the nucleus and
degrade chromosomal DNA (7, 8). In addition to the caspases,
inducible gene products also appear to be required for apop-
totic death in some cell types (9). Evidence for this was derived
from experiments in which cell death could be suppressed by
the inhibition of RNA or protein synthesis in cells that should
otherwise die (10), suggesting that gene transcription and
RNA translation are required for death to occur in these cells.
Indeed, transcriptional activation of specific genes is absolutely
required for physiological apoptosis in both insect and verte-
brate embryos (10, 11). Several transcriptional regulators are
known to control apoptosis, among which p53 (12), Nur77
(13), the glucocorticoid receptor (14), STAT1 (15), c-myc (12,
16), c-jun (17), and NF-
B (18) have been identified to date.
Much of the natural cell death that occurs during insect and
vertebrate development appears to be mediated by the tran-
scriptional activation of killer genes. Although no such genes
have yet been identified in vertebrates, recent studies in the fly
Drosophila melanogaster have uncovered three components of
the genetic program controlling programmed cell death
(PCD), hid, grim, and reaper, whose transcriptional activation
precedes, induces, and is necessary for PCD by apoptosis
(19–21). The three genes map to a single genetic complex and
function as death switches that are regulated at the level of
transcription. Their ectopic activation triggers apoptosis in
otherwise viable cells, and their inactivation prevents apopto-
sis of cells that would normally undergo PCD. We and others
have recently demonstrated that the expression of one of these
genes, grim, activates apoptosis in mammalian cells, implying
conservation during metazoan evolution of both the gene and
the mechanisms required to trigger cell death (9).
The developing limb is perhaps one of the best-suited model
systems for the study of this process, because fine tuning is
required between cell proliferation and cell apoptosis to allow
proper limb modeling, a process subject to intervention with-
out endangering embryo viability. Whilst much has recently
been learned regarding factors involved in cell proliferation,
less is known about the mechanisms implicated in programmed
cell death during development. It has recently been shown that
inhibition of NF-
B translocation by viral overexpression of a
transdominant-negative I
B leads to perturbation of limb
outgrowth (22). We have now tested in vivo the effect of a, to
our knowledge, novel gene, DIO-1 (death inducer-obliterator-
1), identified by differential display PCR in pre-B cells under-
going apoptosis. Its mRNA and protein are present at very low
levels in the cytoplasm. Once an appropriate apoptotic signal
is detected, the protein translocates to the nucleus and up-
regulation is observed at both transcript and protein levels.
When overexpressed, it induces apoptosis in cell lines growing
in vitro, which is prevented by blocking caspase activity. The
protein encoded, DIO-1, is expressed in the limb interdigitat-
ing membranes during development. DIO-1 expression in
distal proliferating mesodermal cells of the developing chicken
limb bud prevents limb outgrowth, an effect that correlates
with inhibition of mesodermal and ectodermal genes involved
in limb outgrowth. These data demonstrate the ability of
DIO-1 to trigger apoptotic processes in vitro, as well as the
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PNAS is available online at www.pnas.org.
Abbreviations: AER, apical ectodermal ridge; E2, 17
-estradiol; NLS,
nuclear localization signal.
Data deposition: The sequence reported in this paper has been
deposited in the GenBank database (accession no. AJ238332).
To whom reprint requests should be addressed. e-mail cmartineza@
Page 1
utility of limb development as a model system to characterize
genes involved in apoptosis.
Cloning of DIO-1. Differential display experiments were
performed by using an RNAmap kit (GenHunter, Brookline,
MA) according to the manufacturer’s specifications. Briefly,
200 ng of total cytoplasmic RNA (after DNase treatment with
the MessageClean Kit; GenHunter) isolated from WOL-1 cells
at0,2,4,and8hafterIL-7 withdrawal were reverse tran-
scribed with oligo(dT) primers (T
MN) in the presence of
Moloney murine leukemia virus reverse transcriptase. They
were amplified with several combinations of 5 decamer
arbitrary primers and the T
MN used for reverse transcription
in the presence of [
S]dATP (1,200 Cimmol). Amplified
products were resolved in an 8-M urea6% polyacrylamide
DNA sequencing gel and analyzed by autoradiography. Bands
of interest were isolated, reamplified, cloned in the pCR-Script
SK() vector (Stratagene), and used for Northern analysis and
sequencing. DIO-1 cDNA was obtained from WOL-1 cDNA by
5 rapid amplification of cDNA ends (RACE) by using a
Marathon cDNA Amplification Kit (CLONTECH), with the
GAAAC-3). The resulting 2.6-kbp band was excised from the
gel and cloned in the TA-type vector pGEM-T (Promega).
Resulting clones were sequence analyzed for orientation, and
the oriented sense with respect to the T7 promoter was called
DIO-1pGEM-T. To confirm the ORF sequence obtained, a
cDNA library from mouse brain cloned in
ZAP II (Strat-
agene) was screened by probing with the RACE clone; the
same probe was used to screen a human fetal kidney cDNA
library (CLONTECH) from which the human DIO-1 homo-
logue was cloned.
Cells and Transfections. WOL-1 cells were derived from
adult BALBc mouse bone marrow. WOL-1 is an untrans-
formed IL-7-dependent stroma cell- independent pre-BI cell
line, capable of reconstituting irradiated severe combined
immunodeficient mice. Cells were cultured in Iscove’s modi-
fied Dulbecco’s medium supplemented with penicillin (100
unitsml)streptomycin (100
g/ml)1 mM sodium pyruvate
nonessential amino acids50
M 2-mercaptoethanol2mM
L-glutamine10% FCSIL-7 (3% supernatant from a murine
IL-7-producing cell line). The BaF3 and FL5.12 cell lines
were maintained in RPMI medium 1640 with 10% FCS and
5% supernatant of a murine IL-3-producing cell line, whereas
A20 and WEHI-231 grew in the same medium without IL-3.
The FL5.12hBcl-2 stable cell line was cultured in 1 mgml
G-418 (Calbiochem). MEF(10.1)Val5MycER cells were cul-
tured at 39°C in phenol red-free DMEM containing 10% FCS.
Where indicated, 1
-estradiol (E2) was added to
activate the MycER fusion protein after 24 h FCS starvation
(12). WOL-1, A20, BaF3, and FL5.12 cell lines were cultured
at 37°C, and all cell lines were maintained in a humidified
atmosphere with 5% CO
Transient DNA transfection was performed by electropo-
ration. For each transfection, 2 10
log phase cells were
collected by centrifugation and resuspended in 200
l of RPMI
medium 1640 without FCS. After addition of 10
g of plasmid
DNA (1 mgml), samples were gently shaken and electropo-
rated in a 0.4-cm electrode gap gene pulser cuvette at 960
and 320 V with a GenePulser (Bio-Rad). Samples were diluted
with 6 ml of the same medium supplemented with 10% FCS
and incubated at 37°C in a humidified atmosphere with 5%
. Cells were analyzed for cell-cycle staining by FACS at
48 h after electroporation.
Northern Blot Analysis. Total cytoplasmic RNA was pre-
pared as described (23). RNA (10
g) was Northern blotted by
using a
P-labeled DIO-1 riboprobe made by DIO-1pGEM-T
digestion with BglII and in vitro transcribed from SP6 by using
the Riboprobe In Vitro Transcription System (Promega). Hy-
bridization was performed in 50% formamide at 65°C; washes
were in 0.1 SSC 0.1% SDS at 80°C. Blots were exposed on
Kodak X-Omat AR film at 70°C with two intensifying
Antibody Production and Western Blot. We synthesized a
peptide corresponding to amino acids 58–72 of murine DIO-1
with an additional N-terminal cysteine (CSLRRSGRQP-
KRTERV); it was coupled to maleimide-activated keyhole
limpet hemocyanin and the purified conjugate injected into
New Zealand White rabbits. Polyclonal antibody was affinity
purified on a peptide-thiopropyl Sepharose column. For West-
ern blot, cells were collected at different times after IL-7
removal from culture medium; 5 10
cells were lysed with
RIPA buffer (0.15 mM NaCl0.05 mM Tris HCl, pH 7.21%
Triton X-1001% sodium deoxycholate0.1% SDS), and the
total extract separated in 8% SDSPAGE, transferred and
incubated with the affinity-purified polyclonal anti-DIO-1
antibody (1:100 dilution in TBS-1% nonfat dry milk). Protein-
loading equivalence was confirmed by Ponceau S staining.
In Situ Hybridization and Histology. Whole-mount in situ
hybridization was as described (24) with minor modifications
(25). The DIO-1 digoxigenin probe was made by BglII digestion
of the DIO-1pGEM-T and transcription from the SP6 pro-
moter. The probe used for Lhx-2 (700 bp) encompasses the
homeobox and the second LIM (lin-11, ISl-1, mec-3) domain.
The remaining probes have been described elsewhere and
include Msx-1 (26), Fgf-8 (27), and NF-
B (22). To visualize
cartilage, embryos were fixed in trichloroacetic acid after viral
infection, stained with 0.1% alcian green, and dehydrated
cleared in methyl salicylate.
Virus Production and Injection Protocols. Chicken embryos
(from MacIntyre Poultry, San Diego, CA, or SPAFAS, Pres-
ton, CT) were infected with a virus containing the DIO-1 ORF.
Virus preparation and injections were as previously described
(28). After injection, embryos were incubated at 37°C and fixed
at different time points for in situ hybridization or phenotypic
Isolation of DIO-1 cDNA: Protein Structure and Sequence
Relationships. To search for genes implicated in apoptosis, we
used the differential display PCR (DDRT-PCR) technique
(29) using mRNA obtained from the WOL-1 pre-B cell line as
a target. WOL-1 was derived from BALBc adult bone
marrow; it grows exponentially in the presence of IL-7 and
undergoes apoptosis on IL-7 withdrawal. The DDRT-PCR
technique gave rise to several positive bands, 10 of which were
initially identified as undergoing up- or down-regulation dur-
ing apoptotic death and were therefore considered candidates
for subsequent analysis. They were further amplified, se-
quenced, and compared with known gene sequences by using
the National Center for Biotechnology Information
program (30). Of these, one band (DIO-1) revealed that the
nucleotide sequence was a gene that showed no significant
identity to any known gene or translated products in the
databases. To confirm the sequence obtained by rapid ampli-
fication of cDNA ends (RACE), a murine cDNA library was
screened by using a labeled DIO-1 probe. Five positive clones
were identified and characterized by restriction mapping and
sequencing. Analysis of the cDNA revealed inserts identical in
sequence to the ORF cloned by RACE. The longest ORF
corresponds to a 614-aa protein (Fig. 1A) and shows a Kozak
consensus sequence before the ATG (considered as the 1
position) known to be crucial for initiation of translation (31).
It also comprises a putative nuclear localization signal (NLS)
and transcriptional activation domain in the N-terminal re-
gion, two central Zn finger motifs, and a lysine-rich carboxyl
terminus. Having analyzed the domains of the putative pro-
Developmental Biology: Garcı´a-Domingo et al. Proc. Natl. Acad. Sci. USA 96 (1999) 7993
Page 2
tein, we sought to determine their degree of conservation in
closely related species to assess their function. A human cDNA
library was screened by using the murine clone as probe;
positive clones were sequenced and showed strong similarity to
the murine gene in the ORF, with a high degree of structural
and compositional conservation (Fig. 1B).
DIO-1 Is Present in All Tissues and Its Levels Are Up-
Regulated During Apoptosis. To study DIO-1 gene regulation
during apoptosis, DIO-1 expression pattern was examined by
Northern blot analysis. Various tissues were analyzed to de-
termine DIO-1 transcript distribution, and two 9.5- and 5.4-Kb
mRNA species were detected in all tissues tested (Fig. 2A).
Southern blot analysis of genomic DNA showed that DIO-1 is
a single-copy gene in both mouse and human. RNA samples
were isolated from several cell lines in exponential growth or
undergoing apoptosis as a result of various experimental
treatments. In the exponential growth phase, WOL-1 cells
express low levels of DIO-1 mRNA, which increase after
induction of apoptosis (Fig. 2B). DIO-1 is up-regulated in
IL-7-deprived cells or those treated with IFN-
or dexameth-
asone, but not in cells treated with etoposide, UV irradiation,
or in those undergoing p53-induced cell death (Fig. 2B). It is
also up-regulated in anti-IgM-treated WEHI-231 cells. In
MEF(10.1)Val5MycER cells, up-regulation is observed in the
absence of serum after addition of E2, but not before or at 32°C
(even in the presence of E2 or serum). Up-regulation of DIO-1
mRNA levels in cells undergoing apoptosis was confirmed in
Western blot by using a polyclonal anti-DIO-1 antibody raised
against a synthetic peptide comprising amino acids 58–72. In
cell extracts derived from WOL-1 cells undergoing IL-7 dep-
rivation-induced apoptosis, a 67-kDa band was up-regulated 2
hr after induction (Fig. 2C), but not after etoposide-induced
cell death (data not shown). In all cases in which up-regulation
of the DIO-1 transcript or of the DIO-1 protein itself was
detected, there was a clear peak in the kinetic levels of DIO-1
up-regulation before any signs of cell death were detectable.
DIO-1-Induced Apoptosis Is Inhibited by Bcl-2 and Z-VAD
and Lost by Deletion of the NLS. The role of DIO-1 in the
apoptotic process was evaluated by transient transfection of
the gene into several cell lines and examination of cell death
kinetics. Transfection of a DIO-1 expression plasmid into
BaF3 cells results in a dramatic loss of cell viability at 48 hr
after transfection (Fig. 3). All cells displayed morphological
alterations characteristic of apoptosis, becoming rounded,
condensed, and finally dying. This effect was specific in that
transfection of BaF3 with an empty vector had no effect on
cell survival. To verify the generality of this observation, DIO-1
constructs were transfected into A20 or FL5.12 cells (Fig. 3).
In both cases, apoptosis was induced after kinetics similar to
those observed for BaF3. Transfection into
MEF(10.1)Val5MycER cells gave rise to apoptotic morphol-
ogy as assessed by 46-diamidino-2-phenylindole staining;
using the DIO-1-specific antibody, we found that endogenous
DIO-1 is located in the cytoplasm of MEF(10.1)Val5MycER
cells in exponential growth. When apoptosis is triggered in
these cells by addition of E2 at 39°C in the absence of serum,
DIO-1 is translocated to the nucleus (not shown). This trans-
location appears to be critical for activation of the apoptotic
pathway, as deletion of the NLS renders the DIO-1 protein
unable to translocate to the nucleus, thus impairing its ability
to trigger apoptosis (Fig. 3). When DIO-1 was transfected into
stable FL5.12 cells overexpressing human Bcl-2, cells were
resistant to apoptosis, showing that Bcl-2 coexpression inhibits
DIO-1 death-promoting activity, as has also been described for
other systems (32). We also incubated DIO-1-transfected
FL5.12 cells alone or in the presence of the caspase inhibitor
Z-VAD-fmk. After 48-h expression in the presence of IL-3, the
apoptosis induced by DIO-1 was completely blocked because
of caspase inhibition, an observation that again clearly suggests
that the death pathway induced by this gene requires caspase
activity. Finally, extensive efforts to derive stable DIO-1 trans-
fectants in these three cell lines were unsuccessful, suggesting
the lethality of DIO-1 expression in these cells. All together,
these results show that DIO-1 overexpression results in the
activation of a cell death program analogous to that operative
in other systems, and that activation of the death pathway
requires DIO-1 translocation to the nucleus, where it probably
performs a function compatible with its structure as a tran-
scription factor.
Alteration of Limb Development by DIO-1 Overexpression.
Using whole-mount in situ hybridization, we also studied
DIO-1 expression throughout mouse development, which is
expressed in the most distal limb cells on developmental day
10.5 (Fig. 4) and close to or within the limb interdigitating webs
on day 12.5 (Fig. 4 Inset). Based on the in vitro effects and in
vivo expression pattern described above, as well as on the
presence of DIO-1 transcripts in the limb cells undergoing
FIG. 1. Nucleotide and predicted amino acid sequences of murine
and human DIO-1.(A) The bipartite NLS sequence is boxed, and the
zinc finger motifs are underlined. (B) Schematic representation of the
predicted ORF of murine and human DIO-1. The start and end
positions of the amino acids defining the motifs are numbered above.
Percentages indicate degree of identity between human and mouse for
each domain. Overall similarity is 74%.
7994 Developmental Biology: Garcı´a-Domingo et al. Proc. Natl. Acad. Sci. USA 96 (1999)
Page 3
apoptotic cell death, we hypothesized that DIO-1 may influ-
ence the control of cell proliferation and death during verte-
brate limb development (see ref. 33 for a review on vertebrate
limb outgrowth).
Retroviral technology was used to misexpress DIO-1 in the
chicken limb. A replication-competent retroviral vector con-
taining the DIO-1 ORF was injected into limb primordia at
stages 10–23. The consequences of DIO-1 expression were
analyzed in embryo limb buds throughout development. At
60–72 h after injection, infected limb buds failed to develop a
normal apical ectodermal ridge (AER, the pseudo-stratified
epithelium located at the tip of the limb, required for normal
limb bud outgrowth) (Fig. 5 A and B). Maximal interference
with limb outgrowth was observed when embryos were in-
jected at stages 13–17. In 35% of the experiments performed
at this stage, truncation occurred in the most distal elements,
showing absence of digits, carpals, and metacarpals (Fig. 5 C
and D). In the majority of cases (65%), however, reduction in
size and malformation of the tibia and fibula are observed (Fig.
5 E and F). Misexpression before stage 13 caused alteration,
but not truncation, in 12% of the cases; misexpression of DIO-1
after stage 18 reduced malformation frequency to 40%, and no
truncations were observed. These data indicate that to affect
the phenotype of the developing limb bud, DIO-1 must be
expressed in a permissive environment and is not the conse-
quence of nonspecific toxic effects. Finally, we analyzed the
caspase activity level in the developing limbs. The maximal
effects of DIO-1 appear to correlate with maximum caspase
activity (not shown), reinforcing the view that execution of the
death program by DIO-1 is caspase dependent, although DIO-1
overexpression precedes caspase activity.
Because misexpression of DIO-1 can perturb AER forma-
tion, we would expect this process to be preceded by changes
in gene expression, in both the ectoderm and the underlying
limb bud mesoderm. In situ hybridization of embryos infected
with the RCAS-DIO-1 construct by using riboprobes for me-
FIG.3. DIO-1 induces apoptosis in vitro. The DIO-1 ORF was
cloned into the pcDNA3 mammalian expression vector (Invitrogen).
Both empty vector and the DIO-1 construct were transiently trans-
fected by electroporation into A20 and BaF3 cell lines. After 48-hr
expression, the cells were permeabilized and stained with propidium
iodide and cell cycle analyzed by FACS. FL5.12 wild-type and stably
transfected hBcl-2 cells were transiently transfected as before. DIO-
1NLSpcDNA3 encodes a mutant protein lacking amino acids 162–192,
which is therefore unable to translocate to the nucleus. Where
indicated, the general caspase inhibitor Z-VAD-fmk (50
M final
concentration; Bachem) was added immediately after transfection.
FIG. 4. Whole-mount in situ hybridization showing DIO-1 expres-
sion pattern during murine development. Staining is shown of a
10.5-day mouse embryo and a 12.5-day mouse embryo limb (Inset).
FIG.2. DIO-1 is differentially expressed under several apoptotic conditions and induces apoptosis when overexpressed. (A) DIO-1 expression
was analyzed in murine tissues by hybridization with the DIO-1 riboprobe of a mouse MTN blot (CLONTECH). Molecular size markers are indicated
on the left. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. (B) Northern blots containing 10
g per lane
of total cytoplasmic RNA from the indicated cell lines, treated with several apoptotic stimuli at different time points, were hybridized to the DIO-1
riboprobe. The blots were reprobed with an actin probe for normalization of the amounts loaded. (C) Western blot analysis of WOL-1 cells driven
to apoptosis by IL-7 starvation. The position of the DIO-1 gene product is indicated.
Developmental Biology: Garcı´a-Domingo et al. Proc. Natl. Acad. Sci. USA 96 (1999) 7995
Page 4
sodermal genes involved in limb outgrowth, such as Msx-1 (Fig.
6A), Lhx-2 (Fig. 6C), and NF-
B (Fig. 6D), showed down-
regulation in their transcript levels. Transcripts for ectodermal
genes involved in limb outgrowth, such as Fgf-8, are also absent
or down-regulated (Fig. 6B). It is not known whether DIO-1
misexpression is directly responsible for the down-regulation
of ectodermal gene markers (i.e., Fgf-8), or if this is a conse-
quence of the previously altered mesodermal gene expression.
The combination of these results indicates that DIO-1 may be
important during limb development, and its apoptotic function
may be a driving force in sculpting the final structure.
The apoptotic pathway is still elusive and, in many cases,
depends on the outcome of the balance between levels of
survival and apoptotic genes, at either the transcriptional or
translational level. Here we report the cloning and character-
ization of a gene involved in apoptosis, which is up-regulated
under certain apoptotic conditions that do not involve p53-
mediated cell death. This up-regulation is observed quite early
in cell death kinetics and always before any of the classical
characteristics of apoptosis, i.e., DNA laddering, haploid
cell-cycle peak, or alteration of cell membrane
polarity, can be detected. This indicates that DIO-1 acts very
early in the apoptotic cascade and suggests a key role in the
control of the initiating triggering mechanism. This gene is a
putative transcription factor, based on its sequence analysis,
which may have a role in regulating the cell death process at
the transcriptional level. Other genes with similar character-
istics have been reported, including p53 (34), c-myc (16), or
members of the glucocorticoid receptor family (35). Our
studies demonstrate that DIO-1 overexpression induces mas-
sive cell death, which can be blocked through overexpression
of hBcl-2, known to inhibit caspase activity (36–39). This
indicates that DIO-1 is upstream of the caspase cascade and
that the induction of apoptosis driven by this gene proceeds
through the main apoptotic route described so far. The Z-
VAD-fmk blockade of DIO-1-induced apoptosis supports
these conclusions.
The apoptotic mechanism activated by DIO-1 requires its
translocation to the nucleus; this finding, as well as its sequence
and differential localization in living and apoptotic cells, can be
used to draw inferences on its mode of function. DIO-1 may
thus be associated in the cytoplasm to a protein that prevents
its entry into the nucleus, to which it must presumably be
translocated to activate downstream mechanisms that initiate
a caspase-executed apoptotic pathway. Such a mechanism is
used by the nuclear transcription factor NF-
B, which is
maintained as a complex with I
B in the cytoplasm until a
given stimulus activates a caspase, leading to I
B phosphory-
lation, ubiquitination, and degradation, releasing the NF-
proteins to traverse to the nucleus and activate gene transcrip-
tion. A similar mechanism has been proposed for control of
caspase-activated DNase and its translocation to the nucleus
(7, 8).
To understand the in vivo role of this gene, we used the
developing limb as a model. This system, which has been
thoroughly characterized by developmental biologists, uses
interference with limb outgrowth through modification of the
gene expression pattern to analyze genes implicated in cell
death. Here we have demonstrated that DIO-1 affects chicken
limb formation by general disruption of growth.
Limb buds infected with DIO-1 constructs were reduced in
size and failed to develop a defined AER. Severely truncated
limbs with deformed andor absent zeugopodal elements
(most commonly the radius) and missing digits were also
observed. After budding, continued limb outgrowth depends
on correct AER formation. The reduced limb buds observed
after DIO-1 expression closely resemble the limb buds ob-
tained after AER removal. All together, our experiments
suggest that DIO-1 overexpression perturbs maintenance of
FIG.5. DIO-1 overexpression inhibits chicken limb outgrowth. A
retroviral vector containing the RCAS-DIO-1 construct was injected
into limb primordia of stage 8–12 chicken embryos. Embryos were
examined at different stages after infection. (A) Whole-mount prep-
aration showing the hind limb of a wild-type embryo. (B) Alcian green
staining of the same limb to visualize the normal cartilage pattern. (C)
An infected embryo 6 days after injection, showing extensive trunca-
tion of the distal elements of the leg. (D) The same embryo after
cartilage staining. Note the complete absence of elements distal to the
tibia–fibula joint. (E and F) Whole- mount and cartilage staining of an
embryo 8 days after infection with the RCAS-DIO-1 construct. The
infected limb is distorted and reduced in size, exhibiting an absence,
reduction or malformation of phalanges, tarsals, and metatarsals (dot).
In a few cases, the fibula was reduced in size (asterisk).
FIG.6. DIO-1 overexpression alters gene expression in the devel-
oping chicken limb bud. Misexpression of the RCAS-DIO-1 construct
leads to arrested limb outgrowth, preceded by changes in the expres-
sion of genes involved in outgrowth of the limb. Note the reduced size
of the infected limb buds (left limb buds in all cases). Transcripts for
Msx-1 (A) Fgf-8; (B) Lhx-2; (C) and NF-
B (D) are strongly down-
regulated (arrows) in the injected limb buds (compare with the normal
expression pattern in the uninjected limb bud, right limb bud in all
7996 Developmental Biology: Garcı´a-Domingo et al. Proc. Natl. Acad. Sci. USA 96 (1999)
Page 5
AER function and hence limb outgrowth. Interestingly, the
phenotypic changes caused by DIO-1 misexpression kinetics,
which result in altered limb development, appear to require the
presence of the caspase activity required for elimination of
interdigiting webs. Fgf-8, an AER-restricted gene involved in
initiation and maintenance of limb outgrowth, is down-
regulated or absent in infected limb buds, as are transcripts for
Msx-1 and Lhx-2 genes involved in limb outgrowth whose
expression is regulated by NF-
B. It thus appears that allowing
DIO-1 protein translocation to the nucleus, down-regulation is
observed for both mesodermal- and ectodermal-specific gene
expression. It is unclear, however, whether this down-
regulation occurs primarily in the mesoderm and indirectly in
the AER or, alternatively, whether down-regulation of gene
expression occurs initially in the AER and subsequently in the
mesoderm. Identification of the mechanisms of action of
DIO-1 may be instrumental in answering this question and may
provide another missing link in the identification of the
mechanism that controls cell proliferation and cell death. In
contrast to the consequences of DIO-1 overexpression, ectopic
B expression does not lead to significant morphological
perturbations (data not shown). In sum, it appears that some
proteins, such as NF-
B, control the cell proliferation and
outgrowth of the vertebrate limb, whereas others, such as
DIO-1, promote cell death. The integral process of limb
outgrowth would thus require a balanced interaction between
both forces.
We thank Drs. R. S. Geha, M. Izquierdo, D. Green, and J. Hurle´ for
reading the manuscript and C. Mark for editorial assistance. This work
was funded in part by a grant from the Direccio´n General de
Educacio´n Superior e Investigacio´n (DGESI) (Spain). D.G.-D. is the
recipient of a fellowship from the Spanish Ministerio de Educacio´n y
Ciencia. The Department of Immunology and Oncology, Centro
Nacional de Biotecnologı´a, Universidad Auto´noma, Madrid, was
founded and is supported by the Spanish Research Council (CSIC) and
Pharmacia & Upjohn.
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    • "In this study, we showed the low expression of anti-apoptotic BCL-2 and the pro-apoptotic BAX mRNA levels in ET and PMF. Therefore, we believe that the lower expression of DIDO 1 in ET and PV in comparison with CML may be associated with the resistance to cell death in MPN Ph negative once DIDO 1 presents the ability to trigger apoptosis [13]. The differences of DIDO expression between CML and MPN Ph negative may be associated with the presence of BCR-ABL1 gene in CML and absence in MPN Ph negative, what generates distinct phenotypes among these disorders and therefore different patterns of DIDO expression . "
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    • "B) Evaluation of the expression of protein markers by calculation of immunostaining index using the Metamorph software; graphs from left to right show the relative expression of KIAA1199, CASP3 and PCNA in control versus KIAA1199 knockdown tumor sections. the nucleus and activates apoptosis in cell culture [26]. KIAA1199 knockdown also led to up-regulation of Akinase anchor protein 8 (AKAP95) that is a potential carrier protein for active caspase 3, carrying it from the cytoplasm into the nuclei in apoptotic cells and is involved in the process of apoptotic nuclear morphological change [27]. "
    [Show abstract] [Hide abstract] ABSTRACT: KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer.
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    • "The fourth gene, DIDO1, is a target of hsa-miR-665, and encodes for death inducerobliterator 1 protein, which is identified as a gene upregulated early in apoptosis by several stimuli. The overexpression of DIDO-1 in cells induces massive apoptosis without any apoptotic stimuli (Garcia-Domingo et al., 1999). Among these four identified genes, three (CANK2D, AK1 and DIDO1) and nine others (ADK, PKLR, PKM2, STAT1, CAMK2B, CREB1, GRIN2B, GRIA1 and ACTN2) were grouped into a network of functional relevance. "
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