Vol. 42, No. 5, August 1997
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THE TRANSCRIPTIONAL REPRESSION OF THE H U M A N Cu/Zn SUPEROXIDE DISMUTASE(sodl) GENE BY THE A N T I C A N C E R DRUG, MITOMYCIN C(MMC) Ginam Cho, Sangsun Kang, Seong Jin Seo, Younhee Kim and Guhung Jung* Department of Biology Education, Seoul National University, Seoul, 151-742, KOREA Received February 24, 1997 Received after revision April 1, 1997 Summary The Cu/Zn superoxide dismutase(sodl) is one of the key enzymes that protects cells against oxidative stress. In order to investigate the effects of mitomycin C(MMC) on the induction of apoptotic cell death and on the sod1 transcription level, the CATs activity of HepG2 cells transfected with sod1 promoter-CAT(chloramphenicol acetyl transferase) fusion reporter was measured after MMC treatment. The CAT assay showed that exposure of HepG2 cells to MMC decreased the transcription level of the sodl gene. The accumulation of p53 tumor suppressor protein by MMC treatment of HepG2 cells was noted, tn order to investigate the p53-negative response element in its promoter region, a p53 cotransfection experiment with serially deleted sodl promoter/CAT reporter constructs was performed. The results show a significant reduction of CAT activity in all deletion reporter constructs. The results show that MMC treatment inhibited sod1 gene transcription through p53-mediated transcriptional repression. Key Words : Cu/Zn superoxide dismutase(sodl), Mitomycin C(MMC), p53 protein. Introduction Reactive oxygen species are known to be implicated in the development and progression of cancer, inflammation, radiation injury and aging. As a defense system, cells have specific enzymes that remove reactive oxygen species. In mammals, there are three types of SOD(superoxide
dismutase)
including
Cu/Zn-SOD,
Mn-SOD
and
the
extracellular
SOD(1,2,3). Copper/Zinc superoxide dismutase(SODl) is one of the major defense enzymes of the cytoplasm that plays a role in the protection of cells
Abbreviations: SOD(superoxide dismutase), MMC(mitomycin C), CAT(Chloramphenicol acetyl transferase), FLAS(Familial Amyotrophic Lateral Sclerosis), * To whom correspondenceshould be addressed, Tel. 82-2-880-7773; FAX : 82-2-880-7773; E-mail : dlj
[email protected] 1039-9712/97/040949-08505.00/0 949
Copyright 9 1997 by Academic Press Australia. All rights of reproduction in any.form reserved.
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against the toxic effects of superoxide radical by catalytic dismutation of superoxide radical to 02 and H202, which is converted by catalase into water(3). It is known that the enzyme is associated with neuronal pathology
including Parkinson's
disease, Alzheimer's disease,
Down's syndrome and Familial Amyotrophic Lateral Sclerosis(FALS)(4). It is reported that Down's syndrome results from trisomy of chromosome 21 that contains the sod1 gene. In approx. 20% of FALS cases, mutations in the sodl gene are reported. FALS is a fatal degenerative disease characterized by the death of large motor neurons(5,6). Apoptosis is a physiological programmed cell death which is part of the control of normal development and contributes to the maintenance of homeostasis (7), Apoptosis may be induced by anticancer drugs including 5-fluorouracil, etoposide, adriamycin, mitomycin C(MMC) and others(8). In addition, several other methods including serum starvation, UV irradiation and growth factor starvation, induce apoptosis(9). However, the regulatory mechanism of apoptotic cell death is still unknown. It has been reported that p53 transcription is increased by MMC treatment(10). Gene p53 produces the tumor suppressor protein. Loss or mutation of the p53 gene is the most common single genetic change in cancer. P53 is also involved in a check point of the G1/S stage of the ceil cycle. Tetramers of p53 can DNA and activate the transcription of reporter genes. A crucial role of p53 is its involvement in apoptosis, in response to oncogenic stimuli. Because p53 has several roles in the control of cell, it is anecdotally described as a guardian of the cell. Two groups reported that the down-regulation of Cu/Zn-SOD caused apoptotic death in the PC 12 neuronal cell line and in spinal neurons(11,12). These findings are the indirect evidence that inhibition of Cu/Zn-SOD is associated with apoptosis. It is reported that copper, a cofactor of the Cu/Zn-SOD enzyme, is involved in the induction of apoptosis in vitro(13). The above information prompted an investigation of the effects of MMC treatment on the transcription of the sod] gene and of the relationship between p53 and the sodI gene. In previous reports(14,15), it was shown that the transcription of the sodl gene is basically regulated by Sp 1 and the C/EBP family transcription factor binding the region of nt -116 to 45 of the sod1 transcription start site. But the mechanism of transcriptional regulation and subsequent protective roles of the sodl gene have yet to be defined. As described above, it is speculated that the inhibition of the sodl transcription may be involved in apoptotic cell death.
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Therefore, it is proposed that sod1 transcription maybe suppressed by p53 increase in the cells after MMC treatment, and the resulting increase of free radical in cells resulting from the suppression ofsodl transcription by the p53 increase induces the apoptosis. In this report, the transcription of the sodl gene is repressed and the level of the p53 protein is elevated by MMC treatment Methods
Plasmid construction The sodl promoter-CAT(chloramphenicol acetyl transferase) fusion reporter constructs including - 1506CAT, - 117CAT, -82CAT, -43CAT was described in (14), the original name was changed into the name used above to facilitate easy recognition(Fig, l). The pCMVp53, the expression vector of wild type p53 under the control of CMV(cytomegalovirus early promoter) early promoter, was used for cotransfection(16). Cell culture, tranffection and CA T assay HepG2 cells were cultured in Mininmm Essential Medium supplemented with 10% Fetal Bovine Serum(Gibco BRL). Cells were transfected by a modified calcium phosphate coprecipitation method(l 7). After transfection , cells were cultured in serum-free medium. At 20hr after transfection, cells were treated with MMC (10microgram/ml). Cells were harvested and the same amount of proteins estimated by Bradford(18) was used for CAT activity assay. Each CAT activity was measured by phosphoimager(Fuji, BAS 1500). Transfection experiments were carried out several times with independent batches of DNA preparation. Although variations in the level of repression from batch to batch were noted, the pattern reported here was obvious. Western blot analysis Whole cell extracts were used. Cells were lysed by sonication in 100~1 of 0.25M Tris-HC1 (pH 7.8). The lysates were centrifuged and the protein concentration of the supernatant fluid was measured by Bradford assay(18). Twenty ~tg of extract was subjected to 8% SDS-PAGE, and transferred to a PVDF membrane(Immobilon-P, Millipore). It was incubated for lhr with monoclonal antibodies against p53(Santa Cruz Biotechnology, Inc., USA) diluted 1/5000 in phosphate buffered saline containing 0.1% Tween-20(PBST). Subsequently, the membrane was washed four times with PBST and incubated for lhr with HRP-anti rabbit antibody diluted 1/10000 in PBST. And then, membrane was washed four times with PBST, and incubated for lmin with the chemiluminescent ECL Detection System(Amersham). The membranes were exposed to X-ray film for 30sec. Results
The expression of the sodl gene is constitutively controlled. Although the expression of the sod1 gene is important for apoptotic cell death and oxidative strcss, its transcription regulation mechanism is less well studied. It has been shown that its transcription is regulated
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-1506
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Human Cu/Zn superoxidedismutase promoter region +221
Construct Designation CAT
-117 F -82 I
CAT
-4____~
CAT
CAT
I
-1506CAT
I
-117CAT
]
-82CAT
]
-43CAT
Fig. 1. Diagrams of the sodl-CAT constructs. These constructs were derived from Kim et,
aI(18) by Sp1 and the C/EBP family transcription factor binding the region o1' nt -117 to -43 of its promoter(IS). In order to explore the effect of MMC on sod] transcription, HepG2 ceils were transfected with 3 microgram of-1506CAT containing -1506 - +22 region of sod] promoter, and CAT activities were measured after exposure to MMC(10microgram/ml). in order to minimize the background effects of serum on the sodl promoter, HepG2 cells were treated with this agent under serum-free medium. As shown in Fig. 2, MMC treatment repressed CAT activity o f 1506CAT containing the sodi promoter-CAT reporter gene. Previous studies showed that the cellular p53 protein level is elevated upon exposure to various genotoxic agents including UV and anticancer drugs in fibroblast cell lines(8,9,I0). Also, the increased p53 concentration by p53 expression vector cotransfection not only activated the transcription of the promoter containing the p53 response element, but it also down-regulated a variety of promoters th.at lack p53 response element, including those for MDR-l,c-/os, b-actin, bcl-2, hsc70 and so on(19,20,21). The above report directed an examination of whether the p53 protein is involved in the MMC-mediated transcriptional repression of the sodl gene.
First, in order to investigate whether the MMC treatment
increased the p53 protein amount in the HepG2 cells, western blotting analysis using anti-p53 antibody was performed. As shown in Fig. 3, the level of p53 protein corresponding to 53kD on the SDS-PAGE was increased by MMC treatment of HepG2 cells containing the wild type p53 gene(22). These results were consistent with those of other groups, who showed that MMC treatment of a fibroblast cell line increased p53 protein(10). But, under serum fi'ee condition, p53 protein concentration was unchanged(Fig. 3). Secondly, in order to examine whether the p53 protein increased by MMC repressed transcription of the sod] gene, a p53-cotransfection and CAT assay were performed. As a
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Relative CAT Activity 100%
43%
46%
Repoder DNA
-1506CAT(3#g)
Time(hr)
0
8
16
MMC(10#g/ml) +
+
+
Fig. 2, Relative CAT activity of MMC treated HepG2 cells. Three microgram of-1506CAT was transfected into HepG2 cell. After transfection, The transfected HepG2 cells were treated with MMC for 0, 8 and 16hr. Relative CAT activity was normalized Io that of 0hr MMC treatment, which was arbitrarily set at 100%. The data represent the average of three independent experiments. MMC (10#g/ml) Time(hr)
0
+
+
+
3
8
16
16
200kD - 97kD-68kD ~:,.;~'~ ~
p53
43kD
Fig. 3. P53 levels in HepG2 cell treated with mitomycin C(MMC, 10 microgram/ml). At 3, 8 and 16111"after treatment, p53 levels were determined ~by western blot analysis. 40 microgram crude extracts were loaded on each lane. p53 protein was probed with p53-specific monoclonal antibody.
result of cotransfection,
it was observed that p53 protein repressed the transcription of
sodl(Fig. 4). In order to identify the p53- negative response element, the sodl/CAT deletion constructs including - 1506CAT, -117CAT, -82CAT and -43CAT, were prepared, and were cotransferred with pCMVp53. Comparison of the relative levels of CAT activity generated from these reporter gene constructs, when cotransfected with either p53 expression vector or empty vector(pBluescript II SK+), revealed that CAT activity of all deletion reporter gene were reproducibly decreased by the p53 protein. The results shown in Fig. 5 imply that the
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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Relative CAT Activity
100% 54%
Carrier DNA
21%
15gg 12gg 5~ag
Effector DNA (pCMVp53)
0p.g 3gg 10tag
Reporter DNA (-1506(~AT)
3gg
3tug 3gg
Fig. 4. Relative CAT activity of p53 cotransfection with -1506CAT. Three micrograms o f 1506CAT was cotransfected into HepG2 cells with 0, 3, 10 microgram of pCMVp53, respectively. Total DNA amount used in cotransfection is 18 micrograms. A pBluescript l] SK is used as carrier DNA. Relative CAT activity was normalized to that of 0 microgram pCMVp53-transfectant, which was arbitrarily set at 100%. The data represent the average of three independent experiments. Repression ratio 54%
43%
42%
52%
Reporter ONA -1506CAT -117CAT -82CAT -43CAT (3og) EffectorDNA 0/ag 5gg 0pg 5tag 0~g 51~g 0gg 5p.g (pCMVp53) Carrier DNA 5gg 0~g 5#g 0p.g 5#g 01.tg 5p_g 0#g.
:
:?:::
..
:
::
:
::
:
Fig. 5. p53 cotransfection experiment with several deleted sodl-CAT constructs. Each 1506CAT, -I17CAT, -82CAT and -43CAT were cotransferred with 5 microgram of pCMVp53 into HepG2 cells. Repression ratio formula is the percent of p53-cotransfection CAT activity/non-cotransfection CAT activity. The data represent the average of three independent experiments.
negative regulatory element is located in the minimal promoter region ofsodl(-43 nt. to +22). In summary, the results suggest that transcription repression of sod] gene by MMC is mediated through the p53 protein. Discussion
The results demonstrate that the transcription of sod] was repressed by the increase of p53 transcription induced by treatment with MMC. However, it was not possible to rule out that the possibility that the repression of the sod1 gene transcription by MMC treatment resulted 954
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from its cytotoxic effect. We tested cell viability with tryphane blue in order to investigate cytotoxicity. No differences in the cell viability between the normal cells and those cells treated with 10microgram/ml MMC for 16hr were noted. However, there is still no clear explanation for the biological meaning of the p53-mediated transcriptional repression and its repression mechanism. In recent work, it was reported that the p53 protein interacts with TBP(TATA-box Binding Protein)(23,24). Mack et al.(24) reported that the transcriptional repression is mediated by an interaction of p53 with basal transcription factor(s), ttowever, other groups suggested that the transcriptional repression by p53 is occurred by the interaction between p53 and the cis-acting element(20,21). It is proposed that the repression of sod1 gene expression by the p53 protein increase results from the interaction between p53 and TBP and that this process may be involved in apoptosis, ttowever, the biological significance of sod1 gene transcription repression by p53 protein induced by MMC is not yet clear.
Acknowledgments This work was supported by research grants from Seoul National University Daewoo Research Fund(96-06-2075) and Korea Science and Engineering Foundation(KOSEF) through the Research Center for Cell Differentiation(96K3-0401-04-03-1).
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