Department of Microbiology, College of Natural Sciences, Pusan National University, Pusan 609-735, Republic of Korea1
Author for correspondence: Kyung Lib Jang. Fax +82 51 514 1778. e-mail kljang{at}hyowon.cc.pusan.ac.kr
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Abstract |
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Introduction |
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Cell cycle progression is driven by the sequential activation of cyclin-dependent kinases (CDKs), which are subject to regulation by positive (cyclins) and negative (CDK-inhibitory proteins) effectors (Morgan, 1995 ). One such negative effector is the universal CDK inhibitor p21 (Xiong et al., 1993
). While p21 can be transcriptionally regulated by the p53 tumour repressor protein (El-Deiry et al., 1993
), and is thus believed to participate in the execution of p53 effects, its induction can also be activated via p53-independent pathways (Michiell et al., 1994
; Liu et al., 1996
; Zeng & El-Deiry, 1996
). Through binding to cyclin-CDK complexes, p21 prevents CDK-dependent phosphorylation, and subsequent inactivation, of the retinoblastoma protein (RB), which negatively regulates cell cycle progression. p21 also interacts with proliferating cell nuclear antigen (PCNA) to inhibit PCNA-dependent DNA replication (Li et al., 1994
). Thus, p21 exerts antiproliferation activity through inhibition of cyclin-CDK complexes and/or PCNA functions (Luo et al., 1996
; Cayrol et al., 1998
).
Recently, Ishido & Hotta (1998) reported that the HCV NS3 could form a complex with wild-type p53, both in the absence and the presence of NS4a viral cofactor. Therefore, the initial aim of this study was to determine whether the complex formation between p53 and NS3 affects the trans-regulatory activity of p53 on the transcription of p21. Next, we tried to determine the region of the NS3 protein responsible for the transcriptional repression of the p21 promoter. For this purpose, we generated several mutants in which amino acid residues of NS3 were either deleted or changed into other residues and tested their activity on repression of p21 transcription. Finally, we investigated whether repression of p21 transcription by NS3 is connected to cell growth stimulation.
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Methods |
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Transfection and luciferase assay.
NIH3T3 and other cell lines used in this study were grown in Dulbeccos modified Eagles medium supplemented with 10% foetal calf serum. Cells were seeded at 2x105 cells per 60 mm diameter plate and transfected the next day with a calcium phosphateDNA precipitate containing target and effector plasmid DNAs as previously described (Gorman, 1986 ). To control for variation in transfection efficiency, 2 µg of plasmid pCH110 (Pharmacia), containing the E. coli lacZ gene under control of the SV40 promoter, was cotransfected. After 48 h, the level of expression from the target was analysed by either CAT assay (Gorman, 1986
) or luciferase assay (Otsuka et al., 2000
), depending on the reporter construct used, and values obtained were normalized to the
-galactosidase activity measured in the corresponding cell extracts. A quantitative estimate of chloramphenicol acetylation was obtained by excision of the substrate and products from thin-layer chromatography plates, and subsequent measurement by liquid scintillation counting in an LKB 216 scintillation counter. Each experiment was repeated at least twice, and although there were variations in the absolute reporter activity from one experiment to the next the profile of the response was always consistent.
Generation of stable cell lines.
NIH3T3 cells (2x105 cells per 60 mm diameter plate) were transfected with 10 µg of plasmid DNA as described above. After 48 h, the transfected cultures were split in a ratio of 1:5, and were selected with 400 µg/ml of G418 sulfate (Gibco BRL) in Dulbeccos modified Eagles medium (DMEM) supplemented with 5% foetal calf serum (FCS). After incubating for 23 weeks, colonies were selected and amplified to obtain cell lines. The expression level of transfected genes was checked by Western blotting analysis.
Semi-quantitative RT-PCR and Western blotting analysis.
For RT-PCR, total cellular RNA was extracted from NIH 3T3 cell lines using Trizol (Gibco BRL). 3 µg of RNA was reverse transcribed with the corresponding antisense primer. One-quarter of the reverse transcribed RNA was amplified with Taq polymerase (95 °C, 5 min; 30 cycles of 95 °C, 1 min 56 °C, 1 min 72 °C, 30 s; 72 °C, 5 min) using 5' ATG GGA TCC CCG GAC GAT ATT GAA 3' as sense primer and 5' TCG CTT AGT GGA TCC TGG GGG CAG 3' as antisense primer to compare the endogenous p53 RNA level. To determine the endogenous level of p21 RNA 5' GGA TGG CTT CGA CAC CAT TC 3' was used as sense primer and 5' GAC AGC TTC TCT TCT GAC AGA 3' as antisense primer. All PCR products were separated by 1·2% agarose gel electrophoresis, stained with 0·5 mg/ml ethidium bromide and photographed on a UV transilluminator. For Western blotting analysis, total cell lysates were prepared from NIH 3T3 cells and 10 µg of protein was separated by SDS-PAGE. The protein concentration of the extracts was measured using the BSA protein assay kit (Bio-Rad). The samples were loaded on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond PVDF; Amersham) by semi-dry electroblotting for 1 h at 15 V. The membranes were incubated in PBS with 0·1% (v/v) Tween 20 and 5% skimmed milk for 1 h at room temperature. p53, p21 and actin were detected using p53 monoclonal antibody (Santa Cruz Biotechnology), p21 rabbit polyclonal IgG (Santa Cruz) and actin mouse monoclonal IgG (Santa Cruz), respectively. The primary antibodies were detected by using the appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham). Immunoblots were developed by addition of ECL-plus reagent (Amersham) according to the instructions given by the manufacturer and exposed to Hyperfilm ECL (Amersham).
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Results and Discussion |
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p53-dependent repression of p21 by NS3 protein
Next, we tried to determine the regions of the p21 promoter responsible for the transcriptional repression by HCV NS3. The p53 binding sites present on the p21 promoter might be the most plausible candidate because binding of p53 onto one of these sites is very important for the induction of p21 transcription (Macleod et al., 1995 ). To examine this possibility, a series of progressive 5' promoter deletion mutants of the p21 promoter were tested (El-Deiry et al., 1995
; Fig. 2a
) These constructs were transfected into NIH 3T3 cells in the presence or absence of the NS3-expressing construct and CAT activity was measured. CAT activity from p21A CAT, which contains a full-length p21 promoter with two p53-binding sites, was repressed approximately 9-fold by NS3. Although removal of a p53 binding site reduced the effect of NS3, the repression from p21C CAT was still high, up to 3·7-fold. However, the repressive activity of NS3 was almost completely lost on p21D CAT, which does not contain any p53 binding site. Therefore, the regions of the p21 promoter responsible for repression by NS3 were mapped to two segments ranged from -2326 to -1892 and from -1481 to -1184, respectively.
p53 binding sites present on the p21 promoter might play a critical role in the effect because successive removal of p53 binding site-containing segments resulted in a stepwise reduction in the NS3 responsiveness. Therefore, we could suggest that repression of the p21 promoter by NS3 is mediated by modification of p53 activity. To demonstrate that the p53 binding sites present on the p21 promoter are involved in the action of NS3, p53 binding sites were used in an attempt to confer NS3 responsiveness to an heterologous promoter. A luciferase construct (pT81-luc) containing a basic promoter element (TATA box) was not responsive to NS3 (Fig. 2c). However, as we expected, 13 copies of the p53 binding site in pG13 (Kern et al., 1992
) were sufficient to confer a strong NS3 responsiveness. Furthermore, when the p21A CAT construct was tested on Hep 3B cells, in which the p53 gene has been reported to be truncated and p53 protein absent, CAT activity from p21A CAT was not affected by co-expression of NS3 (data not shown). Therefore, we concluded that NS3 represses the transcription of p21 by modulating the activity of p53.
NS3 domains responsible for repression of p21
To determine the region of NS3 protein responsible for the transcriptional repression of p21 promoter, several effector plasmids containing a deleted segment of the NS3 gene were generated (Fig. 3a). NS310271260 and NS310271206 encode a serine protease domain and a protease minimal domain of NS3 protein, respectively (Yamada et al., 1998
). They effectively repressed p21 promoter activity (Fig. 3b
), indicating that the NS3 protein domain required for the majority of p21 repression is located on the protease domain and the helicase/NTPase domain located at the C-terminal half is dispensable for the effect. The repression activities of both NS310271260 and NS310271206 were actually much higher than that of full-length NS3 although no significant differences in their expression levels were observed (data not shown). It was a novel finding that NS310271206 has transcription repressor activity because no other functions except proteinase activity have been described so far for this region.
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Connection between p21 repression and stimulation of cell growth
Because the tumour repressor p21 protein is a universal inhibitor of cyclin-CDK complexes and DNA replication that induces cell cycle arrest at the G1-S checkpoint, the repression of p21 by HCV NS3 protein may result in stimulation of cell growth. To test this possibility, we prepared several NIH 3T3 cell lines which stably express NS3 and measured their growth rates. Five different cell lines with each plasmid were selected and tested to show that differences in their growth rates are not just due to the chance selection of cell clones that grow at different rates.
As expected, the growth rate of NS3-expressing cell lines was at least twice as fast as that of the parent NIH 3T3 cells (Fig. 4a). Of the NS3-expressing cell lines, those expressing NS310271206 R1135G showed the highest growth rate. Considering that NS310271206 R1135G repressed the p21 promoter most strongly in transient transfection experiments (Fig. 3b
), it seems likely that the repression of p21 is accurately reflected by the stimulation of cell growth. Furthermore, we investigated whether NS3 affects expression of the endogenous p53 gene. According to a semi-quantitative RT-PCR analysis of the cell lines (Fig. 4b
), the cellular p53 RNA level was not affected by expression of NS3. A similar result was obtained when the level of p53 protein was measured in the same cells (Fig. 4b
). These results, in addition to a previous report (Ishido & Hotta, 1998
), strongly suggest that NS3 represses transcription of p21 by inhibiting the regulatory activity of p53 via proteinprotein interaction(s) but not by affecting the protein level of p53 through either transcriptional repression or protein degradation.
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Received 27 March 2001;
accepted 29 May 2001.