(Received for publication, March 31, 1995; and in revised form, August 11, 1995)
From the
Interaction of certain cytokines with their corresponding
cell-surface receptors induces programed cell death. Interferon-
induces in HeLa cells a type of cell death with features characteristic
of programed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating
interferon-
-induced cell death. The rescue of this gene was
performed by a functional selection approach of gene cloning that is
based on transfection with an antisense cDNA expression library. The
antisense RNA-mediated inactivation of the DAP3 gene protected
the cells from interferon-
-induced cell death. This property
endowed the cells expressing it with a growth advantage in an
environment restrictive due to the continuous presence of
interferon-
and thus provided the basis of its selection. The gene
is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously
expressed in different tissues and codes for a 46-kDa protein carrying
a potential P-loop motif. Ectopic expression of DAP3 in HeLa
cells was not compatible with cell growth, resulting in a 16-fold
reduction in the number of drug-resistant stable clones. The data
presented suggest that DAP3 is a positive mediator of cell
death induced by interferon-
.
Programed cell death is a highly controlled process required for
the normal development, maintenance, and survival of the multicellular
organism. Among the physiological signals that induce inhibition of
cell proliferation and cell death are diffusible polypeptides such as
interferons, tumor necrosis factors, and transforming growth
factor- (1) . To date, relatively few of the positive
mediators of cell death in mammalian systems have been identified and
characterized. These include p53, interleukin-1
-converting enzyme, nedd2/ICH
, CPP32, nur77, BCL-Xs, BAX, and granzyme
B(2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
Recently, a functional approach of gene cloning developed in our
laboratory was used, as previously reported, to rescue genes that
mediate IFN-
(
)-induced cell death(12) . The
effect of IFN-
in epithelial cells is biphasic. The first phase
consists of cell proliferation arrest, which is subsequently followed
by a second phase of cell death, which has the characteristics of
programed cell death, including cell shrinkage, membrane blebbing, and
condensation and fragmentation of nuclear chromatin(13) . The
approach developed, termed technical knockout (TKO), is based on random
inactivation of genes by transfection of HeLa cells with an antisense
cDNA expression library, cloned into an Epstein-Barr virus-based
episomal vector (pTKO1). The HeLa cells were then exposed to the
selective pressure of IFN-
over a 4-week period. At the end of the
selection period, only cells that were protected from the effect of
IFN-
had survived.
The antisense cDNA clones rescued from these
cells were tested in secondary transfections for their ability to
reduce the susceptibility of HeLa cells to IFN-. Those that were
positively scored were sequenced, and novel fragments were then used to
probe a full-length cDNA library. Out of the genes rescued, by this
method, one turned out to be the gene coding for
thioredoxin(12) . Recently, two other novel genes have been
described. The first, DAP1 (death-associated protein-1), is a
basic, proline-rich 15-kDa protein. The second, DAPk (death-associated protein kinase), is a novel
Ca
/calmodulin-dependent serine/threonine kinase that
carries eight ankyrin repeats and a ``death''
domain(13, 14) . In this work, we describe the
isolation and characterization of a fourth gene, DAP3 (death-associated protein-3).
The sequence was used to search the data bases, applying FASTA (Genetics Computer Group package) at the nucleotide level and FASTS, BLASTP, and BLOCKS at the protein level(15, 16) . The MOTIF program (Genetics Computer Group software package) was used to analyze the sequence for potential motifs.
As described previously(13) , HeLa cells were
transfected with the antisense cDNA library, and selective pressure of
both IFN- (750 units/ml) and hygromycin B (200 µg/ml) was
applied over a 4-week period. The DNA plasmids extracted from the
surviving clones were classified into six nonoverlapping groups
according to cross-hybridization on Southern blots. The group of DAP3 was composed of only a single member carrying a novel DNA
fragment that was 252 base pairs long and was termed clone 259 ( Table 1in (13) ). The pTKO1-259 plasmid was then
transfected in duplicate into HeLa cells, and two stable polyclonal
cell populations were generated and maintained with hygromycin B
(designated 259-t1 and 259-t2).
The effect of pTKO1-259 in
protecting the cells from the IFN--induced antiproliferative and
cell death effects was then examined. This was accomplished by
comparing the residual cell viability, after IFN-
treatment,
between the antisense RNA-expressing cell populations and a nonrelevant
polyclonal population of HeLa cells transfected with a pTKO1-DHFR
control vector(12) . The assay was performed by using neutral
dye uptake into cells as a measure of viability (Fig. 1). During
the first 4 days after exposure to IFN-
, the cells stopped
proliferating, but remained viable as previously detailed(13) .
This is due to the initial cytostatic effect of IFN-
. During this
period, all cell populations behaved in a relatively similar manner, as
deduced from microscopic observations (data not shown) and from the
similar values of decline in neutral red dye uptake. However, there was
a clear difference in viability of the different cell populations from
day 4 after IFN-
treatment and later on. The
pTKO1-DHFR-transfected cells displayed rapid and massive cell death,
resulting in <5% cell viability on day 8 of IFN-
treatment. The
two polyclonal cell populations that expressed antisense RNA displayed,
however, reduced susceptibility to the death-inducing effect of
IFN-
(
40% cell viability on day 8 of IFN-
treatment).
These data indicate that expression of antisense RNA from DAP3 protects the HeLa cells exclusively from IFN-
-induced cell
death and not from its cytostatic action. The growth curves of 259-t1
and 259-t2 and of the pTKO1-DHFR-transfected cells, which were kept in
the absence of IFN-
, were indistinguishable (data not shown),
suggesting that antisense RNA expression has no effect on the normal
growth of the cells.
Figure 1:
DAP3 antisense RNA protects two HeLa populations from IFN--induced
cell death. The fraction of viable cells was determined by comparing
neutral dye uptake of IFN-
-treated cells and nontreated cultures
at the indicated time points. Control pTKO1-DHFR-transfected HeLa cells
(
) and two independently pTKO1-259-transfected HeLa cells
(
,
) were used. The decline in neutral dye uptake on day 4
is due to the cytostatic effect of IFN-
. Each point represents an
average of a quadruplicate (S.D. ranging from 0.5 to
8%).
The pattern of RNA expression was then
determined. The 252-base pair DNA fragment carried by pTKO1-259
hybridized on Northern blots to a single endogenous 1.7-kilobase mRNA
transcript in the control HeLa cell cultures (Fig. 2A).
In the 259-t1 cell culture, the antisense RNA was also detected, having
the predicted size of 1 kilobase (comprising the 252 bases of the cDNA
insert and 800 bases of sequence from the expression
cassette)(12) . The levels of antisense RNA exceeded by
5-fold that of the endogenous DAP3 sense mRNA. Addition
of IFN-
caused a further induction of antisense expression,
bringing it to exceed the DAP3 mRNA by 30-fold (Fig. 2A). This was due to the presence of an
interferon-stimulated response element in the pTKO1 vector. The
endogenous 1.7-kilobase mRNA did not appear to be influenced by
IFN-
; it appeared as a single transcript also when the full-length
cDNA was used as a probe.
Figure 2:
Expression of DAP3 RNA and DAP3
protein. A, total cellular RNA prepared from HeLa cells
transfected with pTKO1-DHFR (lane 1) and from
pTKO1-259-transfected cells that were treated with IFN- for 6 and
24 h (lanes 3 and 4, respectively) or left untreated (lane 2). Twenty micrograms of RNA were processed on Northern
blots. DNA fragment 259 was used as a probe. The results were
normalized according to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. The filter was exposed overnight to x-ray
film (Kodak) at -80 °C with an intensifying screen. B, in vitro translation of 1 µg of DAP3 RNA, transcribed from the full-length cDNA clone (lane
3). Lane 1, molecular mass markers; lane 2,
background obtained in the absence of RNA. The products were labeled
with [
S]methionine, separated by SDS-PAGE on a
10% gel, and exposed to x-ray film (Kodak) for 1 h at -80 °C
with an intensifying screen. C, immunoblot analysis of DAP3
protein levels in control and protected cell populations treated with
IFN-
. Lanes 1 and 2, HeLa pTKO1-DHFR with no
IFN-
or 48 h after addition of IFN-
to growth medium,
respectively; lanes 3 and 4, cells transfected with
pTKO1-259 with no IFN-
or 48 h after treatment, respectively. Two
bands are visible; the upper one (corresponding to a molecular
mass of 67 kDa) is a nonspecific cross-reactivity of the antibodies,
and the lower band (marked by an arrow) is that of DAP3
protein.
The antisense fragment was then used to
screen a K562 cDNA library constructed in the gt10 vector. A cDNA
clone that spanned 1560 base pairs was chosen for sequencing. The
sequence analysis of DAP3 cDNA confirmed the antisense
orientation of the HindIII-BamHI insert of pTKO1-259
and the fact that it lies within the coding region of the DAP3 mRNA (Fig. 3A). The sequence contains a single ORF
starting at position 74 and ending at position 1268 with a stop codon.
A polyadenylation signal (AATTA) is found at position 1517, and
polyadenylation itself starts at position 1555 (Fig. 3B). The ORF codes for a potential protein of 398
amino acids and a calculated molecular mass of
46 kDa.
Figure 3: Nucleotide sequence of DAP3 cDNA and deduced protein sequence. A, shown are a schematic representation and limited restriction map of DAP3 cDNA. B, BamHI; RI, EcoRI; H, HindIII; A, AvaI. The ORF and the position of antisense fragment 259 are indicated. B, the nucleotide sequence of DAP3 was determined for both strands. The deduced protein sequence is encoded below in one-letter code. The ATG initiation site is boxed, and the potential P-loop motif is underlined.
A search of the data bases for possible homologies has not resulted in any significant matches. However, analysis of the protein sequence with the MOTIF program indicated that the protein contains a potential P-loop motif. This potential domain was identified through the consensus sequence (G/A)XXXXGK(T/S) at positions 128-135 and implies that DAP3 is a potential ATP/GTP-binding protein. No indications of the presence of a signal peptide or a transmembrane domain have been found (SAPS prediction).
An in vitro translation assay in the reticulocyte lysate confirmed the ORF
prediction. The translation products of DAP3 cDNA were fractionated by
SDS-PAGE to two closely migrating bands (Fig. 2B). The
upper band corresponds to a protein of 46 kDa. The lower, less
abundant band is most probably due to alternative initiation of
translation from a ATG codon that is in frame 24 amino acids downstream
from the first ATG codon and hence represents a protein of
42 kDa.
This possibility is proposed since substitution of 2 base pairs
upstream to the first ATG codon changed the ratio between these two
bands in favor of the faster migrating form (data not shown).
The
expression of DAP3 was then measured in HeLa cells using polyclonal
antibodies made against the bacterially produced DAP3 protein. The
antibodies recognize in HeLa cells a prominent band of 46 kDa, a mass
consistent with the in vitro translated protein. In
pTKO1-DHFR-transfected cells, DAP3 protein levels were elevated by
2-fold at 48 h after addition of IFN-
. In contrast, the
259-t1 cell culture, which expresses DAP3 antisense RNA,
displayed a 2-3-fold reduction in DAP3 protein levels 48 h after
addition of IFN-
to the growth medium (Fig. 2C).
The high antisense RNA levels detected in the presence of IFN-
therefore reduced the steady-state levels of the corresponding DAP3
protein by
6-fold. As the DAP3 antisense RNA did not seem
to reduce endogenous DAP3 mRNA levels, it seems likely that
the antisense RNA exerts its effect by inhibiting, directly or
indirectly, the translation of the DAP3 mRNA transcript.
Since DAP3 is a mediator of IFN- cell death, it was plausible
that elevated levels of the protein, by ectopic expression, may cause
cell death on its own. To answer this question, HeLa cells were
transfected with DAP3 under the control of the cytomegalovirus
promoter and in parallel with a control vector, both carrying a gene
conferring resistance to G418. Following transfection, G418 was added
to the growth medium, and the number of colonies was subsequently
scored (Table 1). As evident from the data presented in Table 1, ectopic expression of DAP3 resulted in a
16-fold reduction in the number of G418-resistant HeLa colonies.
Examination of the DAP3-transfected cells early after
transfection (4-5 days, before mock transfectants are killed by
the drug) revealed that a large portion of the transfectants rounded up
and died. Moreover, at day 10, the colonies of the antibiotic-resistant
stable cytomegalovirus-DAP3 transfectants were much smaller
than the colonies of the control transfectants and grew thereafter at a
slow rate. These results were repeated in three independent
transfections, each carried out in triplicate and with different
preparations of plasmid DNA in each experiment.
In attempt to study the pattern of DAP3 expression, the tissue distribution of DAP3 mRNA was determined. A Northern blot containing mRNA from different human tissues was probed with the DAP3 antisense cDNA fragment (Fig. 4). The results indicate that DAP3 mRNA is ubiquitously expressed in all tissues analyzed. A 2-fold higher level of mRNA expression was found in lung tissue.
Figure 4:
Tissue distribution of DAP3 RNA
transcript. Five micrograms of poly(A) RNA (18) were processed on a Northern blot. DNA fragment 259 was
used as a probe. For quantitative analysis, glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used to normalize the DAP3 mRNA densities. The blot was exposed for 1 h to x-ray film (Fuji)
at -80 °C with an intensifying screen.
IFN- induces a type of cell death that has the
characteristics of programed cell death. These features include
chromatin condensation and segmentation, nuclear pyknosis, surface
blebbing, budding off of cytoplasmic projections, and disappearance of
surface microvilli(13) . This is consistent with previous
reports suggesting that IFN-
has a role in the negative selection
of T- and B-lymphocytes(22, 23) . Hence, study of the
genes mediating this effect should contribute to the elucidation of
basic mechanisms underlying programed cell death.
To date, only a
few positive mediators of cell death have been identified in mammalian
systems. Some, like p53 and c-myc, were well known
for their other functions, and only later was their involvement in
apoptotic cell death
established(2, 24, 25, 26) . Others,
like nur77, were isolated by the subtractive hybridization
approach of gene cloning and subsequently proved by functional assays
to be indispensable in certain apoptotic
systems(7, 8) . The powerful genetic tools available
in the nematode Caenorhabditis elegans, which led to the
rescue of positive mediators of cell death such as ced-3 and ced-4, were another starting point for the isolation of
mammalian homologs. This led to the identification of
interleukin-1-converting enzyme as a potential mediator of cell
death as well as to the recent isolation of other members of the
interleukin-1
-converting enzyme/ced-3 family(3, 5, 6, 27) .
Altogether, these findings demonstrate the importance of proteases in
the process of cell death(28) .
In this work, we demonstrate
that, consistent with our previous reports, applying the functional
approach of antisense knockout is a successful strategy for the
isolation of novel genes that function as positive mediators of cell
death. A growth advantage, conferred by expression of antisense RNA,
serves as a strong positive selection measure in an environment
restrictive due to the presence of IFN-. The isolation of DAP3 as a mediator of IFN-
-induced cell death is reported here.
Transfection of cells with the antisense cDNA protected them from cell
death in a clear manner, as judged by the nearly 10-fold enhanced
viability in the continuous presence of IFN-
. Yet, the antisense
RNA did not have an effect on the cytostatic effect of IFN-
,
implying that these two processes are distinct.
Ectopic expression of DAP3 alone caused massive cell death early after transfection and, as a consequence, a 16-fold reduction in the number of G418-resistant colonies. The ability of DAP3 to cause such a phenotype is consistent with its role as a mediator of cell death.
The single 1.7-kilobase mRNA of DAP3 has a single ORF
coding for a 46-kDa protein. This was confirmed by in vitro translation of the mRNA in the reticulocyte lysate. The mRNA is
ubiquitously expressed in all tissues examined at comparable levels.
This further suggests that the role of DAP3 as a positive
mediator of cell death is not restricted to certain tissues or inducers
or, alternatively, may hint at additional, yet undefined functions. As
the sequence of DAP3 shares no homology with known proteins, it is
difficult at this stage to assign a role for DAP3 in the
process of IFN--induced cell death. The potential P-loop motif,
which is the only motif found in DAP3 by use of computer searches, was
compared with the consensus P-loop domain in the seven classified
families of ATP- or GTP-binding proteins(29) . No similarity
was found, and further work is needed to verify that this element is
indeed a legitimate P-loop.
In our system, applying the approach of
functional inactivation of genes has so far resulted in the
identification of four different genes. One of these genes is the one
coding for thioredoxin, a protein that reduces the intramolecular thiol
groups that form disulfide bonds, suggesting that changes in the redox
state of certain proteins are crucial for mediating IFN--induced
cell death(12) . The other three genes identified so far code
for novel proteins: DAP1, DAPk, and DAP3.
This method of functional selection has proved fruitful in other
systems as well. For instance, expression selection by the use of
genetic suppressor elements was used to clone genes involved in
mediating the effect of the anticancer drug etoposide(30) . The
recent identification and isolation of Requiem, which is a
positive mediator of cell death induced by interleukin-3 deprivation,
was based on a similar strategy(31) . These data combine to
demonstrate the feasibility of isolating positive mediators of cell
death utilizing functional knockout approaches. Use of these approaches
will no doubt enhance the understanding of the pathways underlying the
mechanisms of cell death.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X83544[GenBank].