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Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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UV irradiation is one of the well-known agents that damage DNA by formation of pyrimidine dimers, leading to somatic mutation and cancer (1) . Exposure of mammalian cells to UV irradiation elicits the induction of a number of genes that exert a protective effect known as the UV response (2) . The five gadd4 genes are a group of genes that were originally isolated from Chinese hamster ovary cells on the basis of rapid induction by UV irradiation (3) . Subsequently, they have been identified in human and rodent tissues and found to be induced by many other types of stresses that elicit growth arrest, including DNA-damaging agents such as MMS (4) , and hydrogen peroxide or starvation (4, 5, 6) . Among them, only gadd45 is inducible by IR, and this response is regulated transcriptionally by the tumor suppressor p53 protein, probably via a p53 consensus binding site in the third intron of the gadd45 gene (7) . Zhan et al. (8) have found recently that abrogation of p53 function by using dominant-negative vectors reduced the induction of gadd45 mRNA and protein by MMS or UV irradiation in human cell lines. More recently, they also have demonstrated that p53 interacts with WT1, a transcription factor, to form a complex that binds directly to the proximal promoter of gadd45 (9) . Thus, it has been clarified that gadd45 can be induced through a p53-dependent pathway.
On the other hand, the existence of a p53-independent pathway in the UV response has been already implied by the fact that the first identified gadd genes, including gadd45, were isolated as UV-inducible genes in a hamster cell line that lacks functional p53 (3) . Several reports have indicated that appreciable induction of gadd45 occurs after UV irradiation or MMS treatment in all cell lines examined, including those lacking functional p53 (10, 11, 12) . In addition, other reports revealed that agents such as MMS, UV irradiation, or glucose starvation consistently activate the human and hamster gadd45 promoters in p53 null cell lines (5 , 13) . Presumably, the region of the proximal promoter might be sufficient for the response induced by MMS or UV irradiation in the p53-independent pathway. However, specific sequences that confer this response have not yet been identified.
In the present study, we tried to identify the UV-responsive element that confers p53-independent induction to the human gadd45 promoter by using a luciferase assay involving various proximal and internal deletion constructs and EMSAs. The results suggested that the Oct-1 regulatory element at -99 bp relative to the transcription start site might be at least one of the essential elements for the activation of the gadd45 promoter by UV irradiation in a p53-independent manner.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For cell growth studies, 2 x 105 HeLa cells were seeded per 35-mm-diameter tissue culture dish. Twenty-four h after plating, the cells were irradiated by UV at 254 nm using a Stratalinker UV cross-linker (2400 model; Stratagene, La Jolla, CA). The number of viable cells was counted by a trypan blue dye exclusion test. For colony formation assays, 1 x 105 HeLa cells were seeded in 10-mm-diameter tissue culture dishes. Twenty-four h after plating, the cells were irradiated with UV as per the cell growth studies. After 1 week of culture, the number of colonies was counted.
RNA Isolation and Northern Blot Analysis.
Total RNA from HeLa cells was isolated using the Trizol RNA isolation
kit (Life Technologies, Inc., Rockville, MD), and 10 µg of each RNA
were separated by electrophoresis on a 1.5% agarose gel, transferred
to a membrane (GeneScreen Plus; DuPont New England Nuclear, Boston,
MA), and hybridized with a 32P-labeled
XbaI/HindIII fragment from pCMV-45 (a kind gift
from Dr. A. J. Fornace, Jr.; Ref. 14
). The same
filter was rehybridized with 32P-labeled
glyceraldehyde 3-phosphate dehydrogenase cDNA probe (Clontech
Laboratories, Inc., Palo Alto, CA). Hybridization was carried out at
65°C in 6x SSC, and the filter was washed at 65°C in 2x SSC.
Construction of Plasmid DNA.
The construction of the human gadd45 promoter-luciferase
fusion plasmid, pG45-luc, was described previously (15)
.
pG45-1326, pG45-817, pG45-234, pG45-81, and pG45-10 were generated by
digesting pG45-luc with the restriction enzymes KpnI,
BamHI, BssHII, BalI, and
ApaI, respectively, and religation. To construct pG45-172,
two pairs of complementary oligonucleotides corresponding to the
sequence between -172 and -125 bp, -124 and -82 bp were
synthesized, annealed, and cloned into the MluI and
BalI sites of pG45-luc. To generate pG45-234/-81, the 2.2-kb
fragment of pG45-luc was digested with SacI and
BssHII, end-filled with Klenow fragment, and cloned into the
SacI and BalI sites of pG45-172. To construct
pG45-172/-81, the 2.2-kb fragment of pG45-luc was digested with
SacI and BssHII, and complementary
oligonucleotides corresponding to the sequence between -234 and -172
bp were annealed and inserted into the SacI and
BalI sites of pG45-luc. pG45-128/-81 was constructed by
annealing complementary oligonucleotides corresponding to the sequence
between -172 and -128 bp and ligation into the BalI site
of pG45-172/-81. To generate several tandem repeat mutants,
complementary pairs of synthesized oligonucleotides were annealed,
phosphorylated, ligated, blunted, and inserted into the SmaI
site of IL8-mini Luc, a kind gift from Dr. H. Kojima
(16)
. The sequences of the sense strand of the
oligonucleotides were the same as those shown in Fig. 7A
.
The sequences of the antisense strand are as follows: for
IL8-OCx6, 5'-ATCACCATTGGGCTATGCAA-3'; for
IL8-OmCx6,
5'-ATCACCACAGGGCTATGCAA-3'; and for
IL8-mOCx6,
5'-GTCACCATTGGGCTCATGTA-3'. Plasmids containing six tandem
repeats were selected by sequencing.
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EMSA.
After HeLa cells (2 x 106 cells)
had been irradiated by 50 J/m2 of UV radiation
and cultured for 24 h, nuclear extracts were prepared using the
method of Andrews and Faller (17)
. The nuclear extracts (2
µg) were preincubated with 1 µg of
poly(deoxyinosinic-deoxycytidylic acid) for 5 min at room temperature
in 10 mM HEPES-KOH (pH 7.8) containing 50 mM
KCl, 5 mM MgCl2, 5 mM
DTT, 1 mM EDTA, 0.7 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml
leupeptin, and 10% glycerol, and 
2 ng of a
32P-labeled probe (20,000 cpm) were added to the
mixture and incubated at room temperature for an additional 30 min. The
reaction mixtures were loaded on native 4.5% polyacrylamide gels at
4°C and run at 150 V for 2 h in 0.5x TBE. The gels were dried
and then analyzed using a BAS 2000 bioimaging analyzer (Fujix, Tokyo,
Japan).
Complementary oligonucleotides were synthesized, annealed, and
radiolabeled at the 5' ends with [
-32P]ATP
using T4 polynucleotide kinase (Toyobo, Tokyo,
Japan). Competition and antibody supershift experiments were performed
as described previously (18)
. Supershift assays used 1
µl of antibody against C/EBP-
(sc-61X; Santa Cruz Biotechnology,
Santa Cruz, CA) and Oct-1 (YL15; Upstate Biotechnology, Lake Placid,
NY).
The sequences of the various complementary oligonucleotides used for
EMSAs are listed in Fig. 7A
. The Oct.cons primers contain an
Oct-1-binding consensus sequence, which was commercially available
(Santa Cruz Biotechnology).
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RESULTS |
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A, cell growth was inhibited in an UV dose-dependent manner.
The treatment of cells with 25 J/m2 or more of UV
irradiation resulted in significant inhibition of cell growth, and 75
or 100 J/m2 of UV irradiation were lethal, with
cell death occurring within 24 h of irradiation. At 50
J/m2 irradiation, the cell number decreased to
14% of the control on day 4, but most of the cells remained viable.
In colony formation assays (Fig. 1B)
, the treatment of cells
with 25 J/m2 or more of UV irradiation resulted
in significant inhibition of colony formation. At 50
J/m2 irradiation, the number of colonies
decreased to 13% of the control.
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A, 24 h after exposure to UV irradiation at about 10–25
J/m2 or more, the level of the gadd45
mRNA increased and reached a maximum after exposure to about 50–75
J/m2. A time course study showed that the
gadd45 mRNA was induced markedly 12 h after 50
J/m2 of UV irradiation (3-fold compared with
the 0-h value), and the induction was maintained at 24 h (Fig. 2B)
. In untreated cells, the level of gadd45 mRNA
remained constant at all time points (data not shown). These results
indicate that UV irradiation apparently enhances the level of
gadd45 mRNA through a p53-independent pathway in HeLa cells.
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10-fold) was observed after UV exposure of
50 J/m2 (Fig. 3A)
. A time course analysis indicated that treatment with 50
J/m2 of UV irradiation for 6 h caused a
slight increase in the activity of the gadd45 promoter,
whereas treatment for 12–24 h led to a significant increase in
gadd45 promoter activity (Fig. 3B)
. These results
are consistent with gadd45 mRNA being induced by UV
irradiation (Fig. 2)
.
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. We also examined whether UV irradiation up-regulates
gadd45 mRNA and promoter activity in WiDr human colon
adenocarcinoma cells, which harbors a point mutation at codon 273 of
p53 (22)
. Fifty J/m2 or more of UV
irradiation significantly stimulated the level of gadd45
mRNA level and promoter activity in WiDr cells as well as in HeLa cells
(Fig. 4, B and C)
. These results confirmed that
UV irradiation activates the gadd45 promoter and induces the
transcription in a p53-independent manner.
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A, a construct containing the region -234/+289 bp relative
to the transcription start site (13)
retained 5-fold
activation after UV irradiation, a level comparable with that of the
full-length promoter (pG45-luc), whereas a construct containing the
region -81/+289 bp completely lost its responsiveness. To narrow down
the region responding to UV irradiation, we performed further deletion
analysis of the promoter region between -234 and -81 bp. A construct
containing the region -172/+289 bp maintained the significant
induction (5.8-fold) after UV irradiation (Fig. 5B)
,
suggesting that a 91-bp region from -172 to -81 bp includes the major
UV-responsive element.
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, constructs that contain deletions in the region between -128 and -81
bp were defective in the induction of luciferase activity by UV
irradiation. This was consistent with the results of the sequential
deletion of the 5'-flanking region as shown in Fig. 5
and suggests that
the major UV-responsive element exists in the 47-bp region between
-128 and -81 bp relative to the transcription start site.
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. The result revealed that this region was sufficient for
the functional activity of the UV response (IL8-OCx6). To define the
crucial element, we performed point mutation analysis in either element
to examine the effect on the UV-response. The activity of a construct,
which contained a mutated CCAAT sequence (IL8-OmCx6), was still
stimulated by UV irradiation, but mutation of the Oct-1 consensus
sequence (IL8-mOCx6) significantly reduced the UV-induced
stimulation of the activity. These results indicate that the Oct-1
consensus sequence is at least one of the essential elements for the
stimulation by UV irradiation.
Identification of a Protein Interacting with the UV-responsive
Element.
Because an Oct-1 consensus sequence is involved in the UV-induced
activation of the gadd45 promoter, we examined whether Oct-1
or other proteins can interact with the element using EMSAs. EMSAs of
nuclear extracts isolated from both control and UV-stimulated HeLa
cells were performed using a double-stranded oligonucleotide probe,
named OC, corresponding to the sequence between -101 and -82 bp from
the transcription start site of the gadd45 gene (Fig. 8A)
. Specific protein-DNA complexes could be detected, but UV
irradiation did not change either the intensity or the mobility pattern
of the complex (Fig. 8
A, Lanes 2 and 7). A molar
excess of unlabeled oligonucleotides encoding a consensus sequence for
Oct-1 (Oct.cons) displaced the major complex in both control and
UV-stimulated extracts (Fig. 8
A, Lanes 5, 6, 10, and
11). Conversely, the complex was also detected when Oct.cons
was used as a probe, and OC efficiently competed with the binding (data
not shown). Moreover, the major specific protein-DNA complex was
displaced by a molar excess of unlabeled OmC oligonucleotides, which
contained a mutated CCAAT sequence and an intact Oct-1 sequence,
whereas unlabeled oligonucleotides of mOC and mOmC, both of which
contained a mutated Oct-1 sequence, could not displace the complex
(Fig. 8, B and C)
. Taken together with the result
that the mobilities of the complexes using the OC and Oct.cons probes
were the same (data not shown), it is suggested that the major binding
protein for OC is identical to that for Oct.cons, and the protein
recognizes the Oct-1 sequence.
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D, the complex was supershifted in the
presence of an anti-Oct-1 antibody but not an anti-C/EBP- antibody
as a control (Lanes 5, 6, 10, and 11). This
result clearly shows that Oct-1 binds to the Oct-1 consensus sequence
of the gadd45 promoter. |
DISCUSSION |
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In our examination, gadd45 was activated markedly at 12 h after UV irradiation in HeLa and WiDr cells, as demonstrated at the
level of promoter activity and mRNA expression (Figs. 2
3
4)
. A previous
report by Zhan et al. (11)
showed that the
induction of gadd45 after UV irradiation peaked at 4–8 h in
several cell lines. This difference might be attributable to the cell
lines, i.e., they used wild-type p53 cell lines and we used
inactivated p53 cell lines such as HeLa and WiDr cells. Actually, when
Zhan et al. (11)
used mutant p53 cell lines
RKO, ML-1, MG536, and MCF-7, they could not observe enough induction at
4 h after UV treatment. These results might suggest two distinct
pathways, depending on p53 status, and the induction mediated by p53
might occur earlier.
We have shown that UV irradiation inhibits cell growth and specifically
induces the gadd45 mRNA and its promoter activity in HeLa
cells. It is well known that in HeLa cells the human papillomavirus E6
oncoprotein inactivates p53 by inducing its degradation
(19, 20, 21)
. According to the recent report of Butz et
al. (22)
, gadd45 gene expression could not
be stimulated by -irradiation in HeLa cells, suggesting that p53 is
completely inactivated in these cells. However, several reports
revealed that p53 is still detectable in HeLa cells (23)
,
and UV irradiation may produce too much p53 to be completely
inactivated by the E6 oncoprotein. Therefore, we performed p53
overexpression experiments in HeLa cells. The luciferase activity of a
reporter plasmid containing six tandem Oct-1 sites (data not shown) as
well as a full-length promoter of the gadd45 was not
stimulated but was inhibited by ectopic expression of p53 (Fig. 4A)
. These results strongly suggest that p53 is irrelevant
to the activation of the gadd45 promoter by UV irradiation,
although p53 activity remains in HeLa cells. Moreover, we also
confirmed the induction of the gadd45 mRNA and the marked
activation of the gadd45 promoter by UV irradiation in WiDr
cells, in which the p53 gene has a point mutation and p53 is
functionally inactivated (Fig. 4, B and C
; Ref.
24
). In MG63 human osteosarcoma cells, in which the
p53 gene is rearranged and inactivated (25)
, UV
irradiation also activated the gadd45 promoter, remarkably
in the same conditions (data not shown). From these results, a clear
argument can be made that the gadd45 promoter is activated
by UV irradiation irrespective of the function of p53.
To identify the element in mediating the response to UV irradiation, we
analyzed a series of mutations of the gadd45 promoter and
found that the major UV-responsive element is the Oct-1 site between
-99 and -92 bp relative to the transcription start site. In addition,
we revealed that UV irradiation could stimulate the luciferase activity
of a reporter plasmid containing six tandem Oct-1 sites. Furthermore,
competition and antibody supershift analysis in EMSAs revealed that
Oct-1 could interact specifically with this UV-responsive element in
the gadd45 gene. Thus, it is supposed that Oct-1 might be
involved in the transcriptional activation of the gadd45
promoter in response to UV irradiation. However, we cannot rule out the
possibility that other responsive element exists within the
gadd45 promoter, because subtle reduction of the activation
by UV irradiation was observed in the region between -817 and -234 bp
(Fig. 5A)
. In EMSA (Fig. 8A)
and Western blotting
using the anti-Oct-1 antibody (data not shown), both the intensity and
mobility pattern of the related bands were not changed by UV
irradiation. This means that activation of the gadd45
promoter by UV irradiation does not appear to be attributable to an
increase in the binding or the modification of Oct-1. Hence, there is a
possibility that Oct-1-related or other unknown factors might be
subject to modulation, such as phosphorylation, resulting in the
activation of the gadd45 promoter in response to UV
irradiation. Further studies will be required to elucidate the
mechanism of how UV irradiation modulates the proteins that regulate
through the Oct-1 site.
Oct-1 belongs to the POU (pit-oct-unc)-homeodomain family of transcription factors that bind to 8-bp octamer sequences (26) . In particular, the POU-domain of Oct-1 has high homology with that of Oct-2 and Oct-3/4. The antibody we used does not cross-react with Oct-2 or Oct-3/4. Furthermore, Oct-1 is expressed ubiquitously, whereas Oct-2 is only expressed in lymphoid cells and Oct-3/4 is expressed specifically in undifferentiated cells. We therefore conclude that the factor binding to the Oct-1 consensus sequence of the gadd45 promoter is actually Oct-1.
Hollander et al. (13) cloned hamster, human, and mouse gadd45 genes and found that the promoter was induced by MMS, but they could not define the element responsible. However, they compared the sequences of the three species and pointed out that a high percentage of homology is observed in 100-bp of the proximal promoter region, and two Oct-1 consensus sequences are completely conserved among these species. This indicates that Oct-1 sites are probably significant in regulation of the promoter of the gadd45 gene in multiple species. More recently, the chromatin structure and binding of transcription factors in vivo to the gadd45 gene have been monitored (27) . This revealed that the gadd45 gene is highly organized with nucleosomes positioned around but not within the promoter and intron 3 regions, and DNA-protein interactions were identified at the octamer binding site in the promoter and the p53 binding site within intron 3. This suggests that the Oct-1 binding sites in the promoter, as well as the p53-binding site within intron 3, may play an important role in the transcriptional regulation of gadd45.
It is known that Oct-1 regulates the transcription of several genes
such as histone H2B genes (28
, 29)
, the
small nuclear RNA gene (30)
,
immunoglobulin gene (31)
, the von
Willebrand factor gene (32)
, and vascular cell
adhesion molecule gene (33
, 34)
. We examined the
effect of UV irradiation on the expression of the genes containing
octamer-binding sites. The IL-8 gene, which contains an
Oct-1 sequence in the promoter (35)
, was not expressed in
HeLa cells in the basal state, and 50 J/m2 of UV
irradiation gave no activation of IL-8
gene.5
On the other hand, histone H2B genes were expressed
constitutively in HeLa cells, but their expression was suppressed at
3 h after UV irradiation to 19% of the basal level, and the
suppression continued until at least 24 h after
treatment.5
Because the suppressive response of
the H2B genes was more rapid than the activating response of
gadd45, it is presumable that another mechanism is involved
in the suppression of H2B genes. The regulation through an
Oct-1 element in response to UV irradiation may be an event specific to
the activation of gadd45.
The function of the gadd45 protein has been examined because it is a downstream gene of p53. The gadd45 protein binds to the proliferating cell nuclear antigen and the p21waf1 protein (10 , 36, 37, 38) , which are important for DNA replication and cell cycle regulation (39 , 40) , and gadd45-/- cell lines or cells expressing antisense gadd45 RNA failed to arrest at G2-M checkpoints after exposure to UV radiation (41 , 42) . In addition, it has been proposed that gadd45 is directly responsible for the DNA excision repair process, although this hypothesis is rather controversial (36 , 43, 44, 45, 46) . Recently, it has been strongly suggested that gadd45 is involved in apoptosis and mediates the activation of the JNK/p38 pathway in response to environmental stresses (47 , 48) . From these results, we can hypothesize that when cells are exposed to DNA damage by environmental stresses such as UV, alkylating agents, or glucose starvation, gadd45 elicits cell cycle arrest to allow DNA repair or leads cells into apoptosis so as not to propagate a genetic defect. Thus, gadd45 appears to play an important role in the protective response of the cells to environmental stresses. Our results demonstrate that the gadd45 gene is activated specifically in response to UV irradiation in a p53-independent pathway, and this pathway would be of great value in protecting cells or inducing apoptosis, because the p53 gene itself could be damaged by stresses and p53 is frequently mutated in common human tumors (49 , 50) . This pathway could be exploited in tumors with abnormal p53 function in future therapeutic or chemopreventive strategies, which we have termed "gene-regulating chemotherapy or chemoprevention" (51) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants from
the Ministry of Education, Science, Sports and Culture of Japan; the
Smoking Research Foundation Grant for Biomedical Research; the Nissan
Science Foundation; the Sasakawa Scientific Research Grant from The
Japan Science Society; and the Hayashi Memorial Foundation for Female
Natural Scientists. 
2 The first two authors contributed equally to
this work.
3 To whom requests for reprints should be
addressed, at Department of Preventive Medicine, Kyoto Prefectural
University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan. Phone:
81-75-251-5339; Fax: 81-75-241-0792; E-mail: tsakai{at}basic.kpu-m.ac.jp
4 The abbreviations used are: gadd, growth arrest
and DNA damage; MMS, methylmethane sulfonate; IR, ionizing radiation;
EMSA, electrophoretic mobility shift assay; IL, interleukin; C/EBP,
CCAAT/enhancer binding protein.
Received 11/16/99. Accepted 12/ 8/00.
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), 25 (
), 50 (•), 75 (
), or
100 (
) J/m2, and the cell growth was compared with
control culture (
). B, colony formation assay. After
exposing cells to the indicated doses of UV irradiation, single cells
were seeded and incubated for 1 week, and the number of colonies was
counted. Data represent means of triplicate experiments;
bars, SD.
