(Received for publication, August 30, 1996, and in revised form, February 3, 1997)
From the Laboratory of Molecular Pharmacology, Division of Basic Science, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 and the § Samyang Genex Research Institute, 63-2, Hwaam-Dong, Yusung-G, Taejeon 305-348, Korea
The mammalian cellular response to genotoxic stress is a complex process involving many known and probably many as yet unknown genes. Induction of the human DNA damage- and growth arrest-inducible gene, GADD34, by ionizing radiation was only seen in certain cell lines and correlated with apoptosis following ionizing radiation. In addition, the kinetics and dose response of GADD34 to ionizing radiation closely paralleled that of the apoptosis inhibitor, BAX. However, unlike BAX, the GADD34 response was independent of cellular p53 status. The carboxyl terminus of GADD34 has homology with the carboxyl termini of two viral proteins, one of which is known to prevent apoptosis of virus infected cells. The association of GADD34 expression with certain types of apoptosis and its homology with a known apoptosis regulator suggests that GADD34 may play a role in apoptosis as well.
The response to genotoxic stress in mammalian cells is complex and may include several processes, including enhanced DNA repair, transient growth arrest, and/or apoptosis (1-4). The p53 tumor supressor gene has been shown to be required for a G1 phase arrest following treatment with agents producing DNA strand breaks and other lesions in DNA (1, 5). It may play a role also in cell cycle arrest in the G2 and M phases but is not strictly required (6-8). p53 is required for apoptosis in certain cell types following certain genotoxic signals (9-11). However, other pathways for apoptosis are evident, since cells lacking normal p53 may also undergo apoptosis following various cellular stresses (9, 10). Models have been proposed whereby specific gene products are involved in various aspects of the DNA damage response. However, there are many missing pieces in all of these pathways that may include novel proteins.
The five gadd genes were originally isolated as UV-inducible transcripts in Chinese hamster ovary (CHO)1 cells. All five gadd genes were also found to be inducible by stressful growth arrest and by various types of DNA damage, hence the designation gadd. GADD33, GADD34, GADD45, and GADD153 are expressed in human cells and are inducible by a wide variety of genotoxic agents. The mouse homolog of GADD153, CHOP, has been shown to be a member of the CCAAT enhancer-binding protein family of transcription factors. The protein binds other CCAAT enhancer-binding protein family members and changes their DNA sequence binding specificity (12). A translocation that leads to a fusion between GADD153 and an RNA-binding protein is associated with myxoid liposarcomas, and this fusion protein shows altered biochemical properties relative to the normal GADD153 protein (13). The GADD45 protein is also a predominantly nuclear protein that binds proliferating cell nuclear protein and whose expression is regulated by the tumor supressor p53 as mentioned above (14, 15). GADD153, GADD45, and GADD34 all lead to growth inhibition as measured by colony formation when overexpressed in several human cells lines (16). Interestingly, combinations of these three genes, when overexpressed, lead to synergistic or cooperative effects, suggesting that they inhibit growth through different and perhaps complementary pathways. Apoptosis occurs in certain cell types after treatment with genotoxic agents. Since these treatments also induce the gadd genes, it has been suggested that the gadd genes may be involved in apoptosis as well as in growth arrest after DNA damage. The decrease in plating efficiency in response to overexpression of the gadd genes could be due to the stimulation of apoptosis, a permanent inhibition of cell growth, necrosis, or any combination of these.
GADD34 is the human/hamster homolog of the mouse MyD116 cDNA that was subsequently isolated as a primary response transcript expressed during myeloid differentiation of M1 cells (17). Treatment of M1 cells with MMS leads to apoptosis without differentiation, while treatment with interleukin-6 or lung conditioned medium leads to a myeloid terminal differentiation program, which culminates in apoptosis. In mouse M1 cells, both the gadd and the MyD gene groups respond to MMS with increased mRNA within several hours. Differentiation of M1 cells with interleukin-6 or lung-conditioned medium leads to a rapid increase in MyD gene mRNA, while the gadd response occurs much later at a time that corresponds to the onset of apoptosis. However, gadd34/MyD116 was unique in that it showed a biphasic response, which was suggestive of both the gadd and MyD kinetics (16). Therefore, in this system, increased expression of gadd34/MyD116 coincides with apoptosis and not with differentiation or DNA damage alone.
To further elucidate the regulation of GADD34 expression by DNA damage and possible functions of the GADD34 protein, a human GADD34 cDNA clone and genomic DNA clones for both the human and hamster genes were isolated and characterized. The human gene is inducible by various types of DNA damage and interestingly is induced by ionizing radiation (IR) in cells which undergo apoptosis following this treatment. The predicted human protein is 73.5 kDa and, like GADD45, GADD153, MDM2, and other damage-inducible proteins, is among the most highly negative charged of mammalian proteins (16).
The predicted protein product of the GADD34 gene has a
region of homology with two viral proteins. One of these, the herpes simplex virus-1 (HSV-1) protein 134.5, is required for
inhibition of apoptosis of HSV-1-infected neuroblastoma cells (18).
GADD34 has appreciable but less homology with the NL protein of the
African swine fever virus, whose function is as yet unknown (19). This area of homology is limited to an 84-amino acid stretch at the carboxyl
terminus of the two viral proteins and near the carboxyl terminus of
the 674-amino acid GADD34. Substitution of the carboxyl terminus of
134.5 in the HSV-1 genome with the corresponding domain
from MyD116, the mouse homolog of gadd34, prevented shutoff of protein synthesis and inhibited apoptosis similar to the wild type
HSV-1 virus (20). Based on this similarity, and the increased expression of GADD34 in cells treated with agents that elicit apoptosis, it is conceivable that GADD34 may be involved in apoptosis as well.
A full-length hamster
gadd34 probe, pXR34m, was used to screen a human ZAP XR
cDNA library (Stratagene) to isolate a cDNA corresponding to
the full-length human GADD34 transcript. This library was
made from mRNA from ML-1 cells treated with 20 Gy of ionizing
radiation harvested 3.5 h after treatment. A
CHO FIXII library
(Stratagene) was screened for hamster genomic clones. A human genomic
clone, p1-1812, was obtained by polymerase chain reaction screening of
a P1 library (Genome Systems) with GADD34-specific primers.
The 5
primer was CH040 (AAGAGGGAGTTGCTGAAGAGGAGGGAG), and the 3
primer was CH041 (TCCTCCGTGGCTTGATTCTCTTCCTCC). Several p1-1812
fragments were subcloned into pGEM3Zf(
).
Sequencing of the inserts of pHu34B and partially of pHG34B1 were done using the dideoxy chain termination method and by Lark Sequencing Technologies (Houston, TX). Sequence comparisons were done with the Genetics Computer Group Sequence Analysis Software Package (21) and the National Center for Biotechnology Information Blast program (22).
Plasmid ConstructspCMV-hu34S was made by inserting the 2.2-kb NotI/HindIII fragment from pHu34B into NotI/HindIII cut pCMV.3. For pPGK-Hygro, the hygromycin resistance gene was amplified from pREP4 (Invitrogen) by polymerase chain reaction and blunt end-ligated with the 4-kb PstI fragment of pKJ1 (23). Plasmid 3056, which contains the human BCL-2 cDNA driven by the pCMV promoter, was obtained from Stanley Korsmeyer (24). pCMV-ICP34.5 was made by inserting the 2-kb SphI fragment of pRB143 into pCMV.3. pZipBcl2-neo contains the human BCL-2 cDNA ligated into the BamHI site of pZipneoSV(X) and was obtained from J. Marie Hardwick (25).
Cells and Drug TreatmentsML-1 and WMN cells were grown in RPMI 1640 with 10% fetal bovine serum. A549, Ovcar-4, and RKO cells were grown in minimum essential medium with 10% fetal bovine serum. MMS (Aldrich) was diluted immediately before use to 10 mg/ml in water. Cells were irradiated with a Shepherd Mark I model 68 137-Cs irradiator at 5 Gy/min. Other cell lines were grown as described in Ref. 26.
RNA AnalysisFor RNA isolation, cells were rinsed briefly with phosphate-buffered saline and were then immediately lysed in situ with 5 ml of 4 M guanidine thiocyanate/150-cm tissue culture plate. Total cellular RNA was isolated by the acid phenol method (27). Poly(A) RNA was isolated by oligo(dT)-cellulose chromatography (28). Poly(A) RNA was size separated by electrophoresis in 1% agarose/formaldehyde gels and blotted to nylon membranes (Nytran, Schleicher & Schull) or dot-blotted directly onto nylon membranes (29). cDNA probes were labeled with [32P]dCTP using random nanomer primers and Klenow fragment and hybridized as described previously (29). The poly(A) content of all RNA samples was estimated by hybridization to a labeled polythymidylic acid probe (30).
For RNase protection assays, a T7 RNA polymerase template was made
using a 5 promoter primer (CH067 = GCTATAAAACGCTAGTGG) and
a 3
first exon primer containing a T7 promoter (CH068 = CCTAATACGACTCACTATAGGGAGACCACCCCGGGGGCAGG). T7 RNA polymerase
would generate a probe of 271 nucleotides, and this probe would protect
a GADD34 fragment of approximately 220 bases based on the
cDNA sequence. The glyceraldehyde phosphate dehydrogenase (GAPDH)
probe has been described previously (5). Probes were synthesized using
the Maxiscript RNA transcription kit and T7 RNA polymerase
(Ambion).
RNase protection assays were done using Ambion's Ribonuclease Protection Assay Kit with the following conditions: 0.2 µg of poly(A) RNA, 2 × 105 dpm of GADD34 probe (2 × 109 dpm/µg), and 1 × 104 dpm of GAPDH probe (2 × 107 dpm/µg) were hybridized overnight in 20 µl of 80% formamide hybridization buffer at 50 °C. RNA hybrids were digested for 30 min at 37 °C with 0.05 unit of RNase A and 10 units of RNase T1. Radioactivity for RNA dot blots, Northern blots, and RNase protection assays was counted on a Betascope 603 blot analyzer (Betagen).
Chromosomal LocalizationChromosomal localization was performed by Bios Inc. DNA from p1-1812 was labeled with biotin-dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized with normal metaphase chromosomes derived from PHA-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2 × SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated avidin. Following signal detection the slides were counterstained with propidium iodide and analyzed. A total of 120 metaphase cells were analyzed with 113 cells exhibiting specific labeling.
Based on the number of
clones obtained from the human cDNA library screen, the abundance
of GADD34 mRNA was estimated to be less than 0.01% of
polyadenylated messages in untreated ML-1 cells. Since the
GADD34 transcript size is 2.4 kb, several cDNA clones of
over 2.2 kb were considered to be nearly full length when the poly(A)
tail is taken into consideration. The 2330-bp insert of pHu34B was
predicted to comprise a nearly full-length GADD34 cDNA. This clone contains the entire open reading frame and 221 bp of 5
nontranslated sequence (Fig. 1). The 3
-untranslated
sequence is 87 bp. The human GADD34 cDNA has 67-68%
identity with the mouse (MyD116) and hamster homologs, while
the hamster and mouse have 77% identity between them. At the amino
acid level, the human protein has 52-55% identity and 67-70%
similarity with the rodent proteins using the GAP program of the
Genetics Computer Group Sequence Analysis Software Package (21). The
hamster and mouse proteins are 69% identical with 79% similarity. The
predicted human GADD34 protein is 674 amino acids with a predicted size of 73.5 kDa and a pI of 4.4. The protein would be expected to have a
net charge of
64 and a net charge per amino acid percent of
9.5.
This is similar to both hamster and mouse GADD34 proteins and also to
protein products from other gadd and related genes (16). The
hamster and mouse proteins are expected to be 64.5 and 71.8 kDa,
respectively. The human GADD34 protein, like the hamster and mouse
homologs, contains a series of repeated sequences in the center portion
of the predicted protein. These repeats are 34 amino acids long for the
human protein and 40 and 39 for the hamster and mouse proteins,
respectively. The hamster protein has 3.5 copies, and the mouse protein
4.5 copies of this sequence, while the human protein has 4 copies. The
repeats of the mouse and hamster proteins are arranged in tandem, while
the human repeats are separated by varying numbers of amino acids (data
not shown). The repeats of the hamster and mouse proteins show nearly
100% identity to each other. However, the human repeats show less
similarity to each other and to the rodent repeats (Fig.
2). There were two additional sequences in the human
cDNA that showed limited similarity with the other human repeats
(data not shown).
A search of the data bases for sequences homologous to the human
GADD34 yielded two viral proteins, one of which has nearly 50% identity to human GADD34 in an 90-amino acid region (Fig. 3). This protein, herpes simplex virus type-1-infected
cell protein 34.5 (134.5 or ICP34.5) was previously
known to have homology to the rodent GADD34 sequences (16, 31). The
other viral protein, the African swine fever virus NL protein, shows
less homology to GADD34, but this is in the same region as the homology
to
134.5. In GADD34, this region is the most
highly conserved between species with greater than 90% amino acid
homology. Several nucleolin proteins were somewhat homologous to
GADD34, but this similarity was limited mostly to aspartic acid and
glutamic acid residues present at high numbers in both GADD34 and
nucleolin (data not shown).
Gene Organization
GADD34 was found to be a single copy gene as only one band is visualized on genomic Southern blots when human DNA was cut with 6-bp recognition restriction enzymes, and the full-length cDNA was used as probe (data not shown). Genomic clone p1-1812 hybridized at high stringency with a probe made from the insert in pHu34B. Equivalent sized hybridizing bands were observed with p1-1812 and genomic DNA after DNA was cut with the 6-bp recognizing restriction enzymes HindIII, EcoRI, BamHI, XbaI, and KpnI (data not shown). Comparison of genomic and cDNA sequences revealed that the human GADD34 gene has two introns and that translation begins in the second exon (data not shown). To facilitate analysis of proximal genomic sequences, several genomic fragments were subcloned from p1-1812. pHG34B1 has a 2.1-kb insert containing approximately 600 bp of the promoter through the middle of the second exon. pHG34E contains a 9.1-kb fragment containing approximately 2.6 kb of GADD34 promoter and the entire coding region.
Comparison of the human and hamster gadd34 promoters
revealed only limited homology. Approximately 250 bp of the proximal promoter showed about 75% homology between species (Fig.
4). However, a TATA box was conserved at 48 and
51
in both human and hamster gadd34, respectively. At
77
(human) and
76 (hamster) a 10-bp binding site for the activating
transcription factor/cyclic AMP response element binding family of
transcription factors was also conserved. Just distal to this is a GC
box, which may bind the Sp1 transcription factor. There is one other GC
box in the promoter as well as one in the first intron, although these
are not conserved between species (data not shown). In the human
promoter, an Alu sequence occurred from
784 to
319.
To roughly map the transcription start site, an antisense genomic
riboprobe extending 58 bp more 5 than the start of the cDNA,
Hu34B, was hybridized to mRNA and digested with RNase. The size of
the protected fragment was very close to the expected size of 220 bases
by comparison with molecular size markers (data not shown).
The rodent
gadd34 transcript was previously found to be inducible by
treatment of cells with various types of DNA damaging agents (16, 32).
As expected, the human GADD34 transcript was increased also
by these agents in several cell types. In all cells tested, treatment
with the alkylating agent MMS led to increased GADD34
mRNA within several hours (Fig. 5). Previous data
had shown that maximum transcript levels are seen 4 h following
DNA damage in rodent cells (29). In human cells of lymphoid origin,
ionizing irradiation caused an increase in GADD34 mRNA
levels (Fig. 5, Table I). Similar to BAX,
induction of GADD34 by IR plateaued at a low dose (5 Gy),
while induction of the p53-regulated GADD45 continued to
increase with increasing dose (Fig. 6A).
Unlike previously published data for induction of GADD34 by
MMS, maximal levels of GADD34 following IR were seen at
8 h but decreased to near basal levels by 12 h (Fig.
6B). This is in contrast to the rodent gadd34
transcript, which was not increased by ionizing radiation in any rodent
cell type examined (data not shown). Induction of GADD34 by
IR was also not seen in multiple human monolayer cell lines of various
tissue origins (Fig. 5, Table I). However, induction by MMS was greater
in these adherent cells than in suspension cells (Fig. 5). As a control
transcript, levels of GAPDH mRNA varied by less than 2-fold.
|
To determine whether p53 can directly activate GADD34 expression, as is the case for GADD45, RKO cells were stably transfected with an expression vector encoding E6, a viral protein that targets p53 for turnover and effectively decreases the amount of wild type p53. These cells showed no difference in either the basal level of GADD34 or level after induction by MMS (data not shown). MMS induction of GADD45, for which p53 is required for IR but only contributes for MMS, was decreased with overexpression of E6 (33). GAPDH mRNA was not inducible by MMS or IR, and its basal levels remained unchanged.
Chromosomal LocalizationFluorescence in situ
hybridization of p1-1812 resulted in the specific labeling of the long
arm of a group F chromosome. To confirm the identity of the
specifically labeled chromosome two additional experiments were
performed: one with clone p1-1812 being co-hybridized with a chromosome
20 centromere-specific probe and the other with p1-1812 being
co-hybridized with E2A, a gene that has previously been localized to
19p13 (34). These experiments clearly demonstrated that p1-1812
hybridizes to the same chromosome as E2A. The fluorescent
hybridization signal was detected directly on chromosome 19 in most
banded metaphases (Fig. 7). Measurements of 10 specifically hybridized chromosomes 19 showed that GADD34 is
located 61% of the distance from the centromere to the telomere of
chromosome are 19q, an area that corresponds to band 19q13.2. It is
interesting to note that this area of chromosome 19 also contains a
cluster of known DNA repair genes including ERCC-1, ERCC-2, DNA ligase, and XRCC-1 (35). The
apoptosis-associated gene, BAX, is also localized near this
region (36).
GADD34 is a member of a group of genes whose transcript levels are increased following stressful growth arrest conditions and treatment with DNA-damaging agents, hence the designation gadd (32). In hamster cells, all five gadd genes appear to be coordinately regulated since they all show similar kinetics of induction and spectrums of inducing agents. In most human cells, however, only GADD34, GADD45, and GADD153 are expressed and increased following DNA damage (32). One major difference in expression of the gadd genes is that GADD45 induction following IR is strictly dependent on wild type p53. However, induction of GADD34 by IR occurs in certain human cell lines regardless of p53 status. For example, two cell lines with mutant or null p53 show increased GADD34 following IR treatment (Table I). Likewise, not all p53 wild type cells treated with IR exhibit an increase in GADD34 (Table I). For example, in Fig. 5, all cell lines have wild type p53 yet only two show induction of GADD34 by IR. One common property of cells, which showed increased GADD34, is that they all undergo apoptosis following this treatment, while cells that do not undergo apoptosis show no increase. Taxol, which can rapidly induce apoptosis regardless of p53 status, does not cause increased GADD34 in any of these cell lines (data not shown). Specifically, in RKO cells, very low concentrations of taxol (1 nM) induce apoptosis yet fail to induce GADD34 (data not shown). Therefore, not all signals for apoptosis cause increased expression of GADD34. Since the transcript is induced by various agents that damage DNA, it is possible that only these types of signals, when they lead to apoptosis, elicit the GADD34 response. On the other hand, treatments such as ultraviolet radiation and methyl methanesulfonate lead to increases in GADD34 in all cells examined yet fail to elicit detectable apoptosis in most cell lines (data not shown). This dual pathway of regulation is reminiscent of GADD45, which requires p53 for regulation of the IR response yet does not require p53 for other DNA damage responses.2 It is possible that increased Gadd34 may play a role in cellular responses to DNA damage other than apoptosis. Further work will ellucidate whether induction of GADD34 following IR is a consequence of apoptosis or if it somehow plays a role in apoptosis.
The regulation of GADD34 by DNA damage and its correlation with apoptosis resembles the regulation of GADD45 in some respects. Both are induced by MMS and UV in all cells but only by IR in some cells. For GADD45, it is known that the IR response is dependent on wild type p53. For GADD34, there may be some regulator which is present only in those cells which undergo apoptosis following IR, perhaps a lymphoid-specific factor. The apoptosis promoting gene, BAX, like GADD34, is also increased following IR induced apoptosis. However, the BAX response is seen only in cells having wild type p53. A reasonable explanation is that the BAX response requires both p53 and the IR/apoptosis proficiency factor required for the GADD34 response.
It is intriguing to note that GADD34 has nearly 50% identity over a
100-amino acid segment with a small herpes simplex virus protein,
134.5, that prevents apoptosis of virally infected cells
(18). This area of similarity occurs near the carboxy end of each
protein and comprises approximately 40% of
134.5 but
less than 15% of GADD34 (Fig. 3). This region of the mouse GADD34
protein (MyD116) can substitute for the equivalent region of
134.5 in suppression of HSV-1-induced apoptosis (20). However, overexpression of GADD34 suppressed cell growth in several human cells lines (16), suggesting that GADD34 has a growth inhibitory
effect rather than a survival effect.
It is intriguing that GADD34 was localized to a region of the human genome which harbors several genes which likely are involved in the response to DNA damage. These include four genes involved in the DNA repair process; XRCC-1, ERCC-1, ERCC-2, and Lig-1. The BAX gene, which is a positive effector of apoptosis, a common cellular response to treatments that damage DNA, is also localized just telomeric to this region. Deletions of chromosome bands on 19q have been reported to be associated with gliomas (38). GADD34, as well as the genes mentioned above, maps outside this deletion (37).3 Previous data have suggested that GADD34 may be involved in a signal transduction pathway controlling cell cycle arrest and/or apoptosis following DNA damage. Specifically, overexpression of GADD34 in human cells leads to both inhibition of cell growth and a change in colony morphology (16). Alterations in such a pathway might contribute to tumorigenesis.