[CANCERRESEARCH57.5137-5142, November15, 19971 The Transactivation and p53-interacting Functions of Virus X Protein Are Mutually Interfering but Distinct1

Yong Lin, Takahiro Nomura, Tatsuya Yamashita, Dorjbal Dorjsuren, Hong Tang, and Seishi Murakami2 Department of Molecular Biology, Cancer Research Institute, Kanazawa University, Takara-machi /3-1, Kanazawa 920, Japan

ABSTRACT teins that interact with p53. These include the SV4O large T antigen, adenovirus E1B, EBV EBNA-5, human cytomegalovirus 1E84, and Transactivation of viral and host genes expression by the human papillomavirus E6 proteins (28—30).Disruption of the p53 x protein(HBx)isbelievedtobeinvolvedinhepatocarcinogenesis.The functions, such as transcriptional activation and p53-dependent apop interaction of HBx with the tumor suppressor p53 and Its inhibitory effect on p53 functIons have been reported recently. However, the question of tosis by viral oncoproteins, is not only beneficial to virus infection but whetherp53 is directlyinvolvedin HBxtransactivationhasnot yet been also important in virally induced carcinogenesis. addressed. l.a this study, we delineated the interaction sites of HBx and The direct interaction of HBx and p53 both in vitro and in vivo has p53 using far-Western blotting and glutathione S-transferase-resm pull been reported recently (31—35).HBx was shown to inhibit p53 tans down assays The results indicate that the HBx-binding sites are located activation and p53-dependent (32, 33, 36). However, there within the oligomerization and specific DNA-binding domains of p53 and have been no reports showing that HBx tansactivation is affected by that the p53-binding site was confined to a small region in the HBx p53. The relationship between transactivation and the p53-binding transactivatlon domain. Mutual interference of the transactivations by functions of HBx has not yet been addressed. In this report, we HBx and p53 was detected by CAT assays in a transient transfection mapped the interaction sites of both p53 and HBx and investigated system. Strikingly, transactivation by HBx was observed in the p53- negativecells,Saos-2and Hep3B,indicatingthatthe transactivationand whether p53 binding is involved in HBx transactivation. The results the p53-inhibitingfunctionsof HBxare mutuallyinterferingbutdistinct. show that the p53-binding site is within the transactivation domain of HBx, and p53 has two independent HBx-binding sites located in the specific DNA-binding domain and oligomerization domain, respec INTRODUCTION tively. p53 and HBx interfere with each other's transactivation func There is a great deal of epidemiological evidence to suggest that tion. However, because HBx tansactivation was clearly detected in chronic HBV3 infection is a major risk factor associated with hepa p53-negative cells (Saos-2 and Hep3B), HBx tansactivation is ‘us tinct from its p53-binding function. tocarcinogenesis (1). However, the mechanisms by which HBV con tributes to liver cell transformation remain elusive. No HBV gene acts as a dominant or acutely transforms liver cells. The wood MATERIALS AND METHODS chuck and ground squirrel hepatitis viruses, the genomes of which harbor the X gene, have been associated with the development of Plasmid Constructions. The full-length and truncatedHBx-5Dl, -5D2, HCC, whereas the oncogenic potential of avian hepadnaviruses, -3D5, -3D39, -Dl6, and -D26 expression plasmids have been described pre which are devoid of the X gene, has not been established (2, 3). viously (37—39). Construction of the truncated HBx mutant HBx-Dl9 (5 1—99 Importantly, the HBV X gene has been shown to have the ability to an) was carried out by using PCR cloning. p53 and TBP cDNA derived from induce transformation in rodent cells (4—6)and HCC in some tans pSGHS3 and HlO (40), which were kindly provided by Drs. M. Fujii (Tokyo Medical and Dental University, Tokyo, Japan) and M. Hirokoshi (Tokyo genic mice lines or to potentiate oncogenesis by oncogene or chemical University, Tokyo, Japan), respectively, were inserted into the the EcoRI and carcinogens (7—10).The X gene is frequently integrated into the host BamHI sites of pSG5UTPL and pGENKI (37). The resultant plasmid, genome and expressed in HCC (1 1—13).HBx, a 154-na protein pSG5UTPL-p53, was used to construct p53-dl (1—100 as), pS3-d5 (101—314 encoded by the HBV X gene, has been shown to deregulate an), and p53-d3 (315—393 an) using PCR cloning. The p53 mutant R273H checkpoint controls (14). On the other hand, HBx is a viral transac coding sequence was constructed by PCR-directed mutagenesis. The histidine tivator that can activate the expression of many viral and cellular tagged protein expression plasmid pLHis was derived from pETlld by replac genes through a wide variety of cis-elements (15—19).Although the ing the NdeI—BamHI fragment with the annealed complementary oligonucleo mechanism has not been definitely elucidated, HBx transactivation tides, TATGAATI'CCATGAAGCTTGGATC, so as to create the EcoRI, seems to be important for its oncogenic role, because HBx tansacti HindIII, and BamHI digestion sites. Plasmids pLHis-HBx and pLHis-p53 were yates not only HBV genes but also various host genes that engage in constructed by inserting the HBx and p53 coding sequence into the EcoRI and cell proliferation and acute inflammatory responses (15, 20—23). BamHI sites of pLHis. All of the constructs were sequenced using Taq sequencing kits and a DNA sequencer (370A; Applied Biosystems). The tumor suppressor p53 is involved in maintenance of the Preparation of Recombinant Proteins. GST-fused proteins were cx genomic integrity of cells (24). p53 has multiple functions, including pressed in Escherichia coli by induction with 0.4 mM isopropyl @3-D-thioga transcriptional activation and repression, modulation of factors en lactopyranoside for 3 h at 30°C. Cells were harvested and sonicated in PBST gaged in DNA repair, cell cycle control, and apoptosis regulation buffer (PBS containing 1% Triton X-lOO). After centrifugation, the extract (25—27).Various DNA tumor viruses encode transforming oncopro (supematant) was collected and stored at —80°C.Forpurification, the extracts were incubated with glutathione-Sepharose 4B resin (Pharmacia) for 1 h at Received 6/12/97; accepted 9/19/97. room temperature. The beads were collected and washed four times with PBST Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage buffer and then eluted with 10mMreduced glutathione in 50 nmiTris-HCI (pH charges. This article must therefore be hereby marked advertisement in accordance with 8.0). The eluted proteins were divided into aliquots and stored at —80°C. 18U.S.C.Section1734solelyto indicatethisfact. Histidine-tagged HBx and p53 were expressed in the BL2I (DE3 pLys) I This study was partly supported by a Science Grant from the Ministry of Education andCultureof Japan. transformants of pLHis-HBx and pLHis-p53 by 0.4 mM isopropyl @-D-thioga 2 To whom requests for reprints should be addressed. Phone: 8 1-76-265-273 1 ; Fax: lactopyranoside induction at 30°C.Cells were harvested 3 h postinduction and 81-76-234-4501; E-mail: [email protected]. sonicated in denaturing binding buffer (6 M guanidine chloride, 20 m@i sodium 3 The abbreviations used are: HBV, hepatitis B virus; HBx, HBV X protein; HCC, phosphate, and 500 nmiNaC1,pH 7.8). Histidine-tagged proteins were purified hepatocellular carcinoma; as, (s); NF-.cB, nuclear factor KB; GST. glutathione S-transferase; TBP, TATA box binding protein; RPB5, RNA polymerase II subunit 5; by incubating the sonication supernatant with nickel resin and following TFIIB, factor IIB. extensive washing with denature binding buffer and then denaturing washing S 137

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buffer (6 Mguanidine chloride, 20 mMsodium phosphate, and 500 mr@iNaCl, have been described previously. (42). Total cell lysates were prepared from pH 6.0) and were eluted with denaturing elution buffer (20 msi sodium cells that were harvested 48 h after transfection. The CAT assay reactions phosphate and 500 mM NaCl, pH 4). The eluted proteins were renatured by proceeded for 60 mm at 37°Cusing20 @gofprotein from the transfected cell dialysis against a buffer with a sequentially reducing concentration of urea (20 lysates (42). The fractionated TLC plates were exposed to imaging plates, and mM sodium phosphate and 500 mr@iNaCl, pH 7.8). After the final dialysis in the CAT activities were measured as the percentage of the conversion to PBS, the proteins were divided into aliquots and stored at —80°C. acetylated forms of [‘4C]chlorumphethcol (% acetylation; Amersham) using a Far-WesternBlotting.Thefar-Westernblottingwasperformedasprevi Bioimage analyzer (BAS1000; Fuji). Transfection and CAT assays were ously described (39). The target proteins (200 ng each) were fractionated on performed at least three times with each combination of transactivator and SDS-PAGE gels and then electrotransferred onto nitrocellulose membranes, CAT reporter constructs. Representative data are shown. which were denatured, renatured, and blocked with5% skim milk in modified GBT buffer (10% glycerol, 50 nmiHepes-NaOH, pH 7.5, 170mi@KC1,7.5 mai RESULTS MgCl2,0.1 IflMEDTA, 0.1 mMDU, and 1% Triton X-lOO)before subjected to far-Western blot (38). The binding reaction proceeded in modified GBT HBx and p53 Directly Interact with Each Other in Vitro. We at buffer containing 32P-labeledprobe (40—100ng/mlof protein with 2 X 106 first investigated the direct interaction between HBx and p53. By cpm/,.@g of protein), 1% BSA, 2 nmi of unlabeled AlP, and the sonicated far-Western blotting, the HBx probe bound to p53, RPB5, and itself supernatant ofE. coli JM1O9transformed by pGENK1, which contained a final but not to TBP or GST (Fig. 1A). The specific binding of p53 to HBx GST protein concentration of I mg/mI. The membranes were washed five was also detected by far-Western blotting using the p53 probe, which times with modified GBT buffer and exposed to X-ray films (XAR Omat, Kodak) or imaging plates (Fuji). bound to HBx, TBP, and itself but not to RPBS or GST (Fig. lB). GST Resin Pull-downAssay. Equalamounts(approximately1 @g)of Similar results were obtained with the GST resin pull-down assay, GST and GST-fused proteins immobilized on 10 p1 of glutathione resin were which showed that histidine-tagged p53 was efficiently retained by incubated with 100 ng of target proteins (p53-His6 in Figs. 1C and 3C, GST-HBx but not by GST-RPB5 and GST (Fig. 1D). These results HBx-His6 in Fig. 2C) in modified GBT buffer containing 1% BSA for 2 h at suggested a direct interaction between HBx and p53 in vitro and are 4°C.Afterfour washes with modified GBT buffer, the bound proteins were consistent with the reports of other groups, who published data on eluted with elution buffer (10 mMTris-HCI, pH 7.4, 2% SDS, 0.3 MNaCl, and physical and functional interactions between HBx and p53 while this I ma@EDTA), separated by SDS-PAGE, and then transferred onto nitrocellu study was being conducted (3 1—35). lose membranes for Western blotting to detect HBx or p53. The proteins were HBx Binding Sites Reside in the Specific DNA-binding and visualized by ECL (Amersham) according to the manufacturer's instructions. Oligomerization Domains of p53. The regions of interaction be Preparation of anti-HBx was described previously (39). The anti-p53 mono clonal antibody (Ab-2) was purchased from Oncogene Science. tween HBx and p53 were mapped by far-Western blotting. p53 has at Transfection and CAT Assay. The p53-responsive CAT reporter least three functional domains, namely, the transactivation domain in pG13CAT was kindly provided by Dr. B. Vogelstein (The Johns Hopkins the NH2-terminal part, the specific DNA-binding domain in the mid University, Baltimore, MD) (41). The HBx-responsive CAT reporters, dle part, and the oligomerization domain in the COOH-terminal part pHECx2CAT and NF-KBx3CAT, and the transient transfection and CAT assay (43, 44). Three truncated mutants of p53 covering each of the three

A B C

!cH'i@kDs #@c@I kD. U —116 ic@I —116 -66

@@@ -42 -. —42 @ -42 Fig. 1. HBx and p53 bind to each other in vitro. A and B, far-Westem blotting. Equal amounts of ; .. GST(Lane5) andGST-fusedTBP,p53,HBx,and I —17 RPB5 (Lanes 1—4)wereblotted with each of the —17 probesindicatedasdescribedinthe“Materialsand —17 12345 123456 @ Methods.―A, binding by HBx probe; B, binding by 12345 GSTp53 p53 probe; C, Coomassie staining showing that GST-HBx prob. comparable amounts of the proteins were applied for blotting in A and B; D, pull-down of histidine Far-Wsstsrn blotting Cooni@sIo staining tagged p53 (p53-His6) by GST, GST-HBx, GST RPB5, and GST-TBP (Lanes 2—5).Lane1, 10% of the input p53-His6 protein. One @sgof GST or GST-fused proteins immobilized on glutathione Sepharose resin was incubated with 100 ng of p53- Hisóin GBT buffer for 2 h at 4°C.After extensive D washing, the bound proteins were eluted followed by Westem blotting to detect p53. The molecular mass markers are indicated in kDa to the right of ii iii kO. each panel. @@@ “1*p53-Hl.6-66

-42

12345 GST-rs&n puil-down sony

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Fig. 2. Delineation of the HBx-interacting sites — 116 onp53byfar-Westernblotting.A,HBxbindstothe ,@ p53 oligomerization and specific DNA-binding do mains. p53 full (Lane 1), its truncated mutants p53-di, -d3, and -d5 (Lanes 2—4),and substituted B/:::c:? mutant R273H (Lane 5) were blotted with the GST -@ HBx probe as described in the legend to Fig. 1. GST was used as a negative control (Lane 6). B, I 23456 Coomassie staining showing that comparable 1234@6 amounts of the proteins were applied for blotting in Far-Wsstsrn blat: hiBa probs Casm@ Wus A. C, pull-down of histidine-tagged HBx (HBx His6) by GST-fused HBx mutants. Lane 1. 10% of the input HBx-His6 protein. Lanes 2—7,pull-down by GST.GST-p53-full,andp53-dl, -d3,-d5, and -R273H, respectively. Pull-down experiment was as D described in the legend to Fig. JD. D, schematic c/I4,$@ @,P4*@o,42 IIBa binding I 40100 315@ mapof the p53 constructs.0, transactivationdo — k@XX@X@@ + main; @,specificDNA binding domain; W, oh gomerizationdomain.HBx-bindingabilitiesare dl@°°r - shown to the right. The molecular mass markers are 101 314 @@ indicated in kDa to the right of A, B, and C. — on @-HBz4*eS dO s.―,.'s.@:',x'sX1 + — 17 d3 3@3 + I 234567 GST-msln puII.down assay

domains were constructed in GST fused forms and expressed in E. dispensable for HBx-binding. The deletion of either of the binding coli. The HBx probe bound to p53-d3 (3 15—393aa) and p53-d5 sites and the single point mutant p53-R273H reduced the HBx (101—314 aa) but not to p53-di (1—100aa; Fig. 2A). This result was binding ability (Fig. 2, A and C, compare -d3, -d5, and -R273H to confirmed by the alternative approach of the GST-resin pull-down p53 full), suggesting that both binding sites contribute to the HBx assay. Histidine-tagged HBx was expressed in and purified from E. binding of the full-length p53. coli. GST-fused forms of wild-type and mutated p53 proteins p53-binding Site Resides in the HBx Transactivation Domain. immobilized on glutathione resin were incubated with histidine We previously reported that HBx consists of a functional transacti tagged HBx. After extensive washing, the bound proteins were vation domain (51—148an) and a regulatory domain (1—50an; Ref. eluted and Western blotted. In agreement with the results of 37). The p53 probe bound to HBx-5Dl(51—l54 an) but not to HBx far-Western blotting, wild-type p53, p53-d3, and p53-d5 but not 3D5 (1—50an), indicating that the p53-binding site is located within p53-dl bound to HBx (Fig. 20. The tumor-derived p53 mutant, the transactivation domain of HBx but not the regulatory domain (Fig. p53-R273H, also bound HBx (Fig 2, A, Lane 5, and C, Lane 7). 3A). Further delineation indicated that the p53-binding region is These results indicate that p53 has two independent HBx-binding located in a small region of the COOH-terminal portion of HBx sites located in either the specific DNA-binding domain or the (102—136an) because the p53 probe bound to the truncated HBx oligomerization domain and that the transactivation domain is proteins harbor 102—136an (HBx full, -SD1, -5D2, -5D4, -5D12,

A @ B

/i#@kDs

Fig. 3. p53 binds to a small region of the HBx —42 “@4. .@ —42 @ transactivation domain. A, HBx full (Lane 1) and its •1.@ truncated mutants HBx-5Dl, -5D2, -5D4,. -3D5, -30 -3D39, -Dl9, -Dl6, and -D26 (Lanes 2—9)were blotted with GST-p53 probe as described in the I 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 legendto Fig. 1. B, Coomassiestainingshowing Far.Wsstsm blot: p53 probs that comparable amounts of the proteins were ap Coom.ss@ bin. staining pliedfor blottingin A. C, pull-downofhistidine tagged p53 (p53-His6) by GST-fused HBx mutants. Lane 1, 10% of the input p53-His6 protein. Lanes D 2—7,pull-down by Iffix full, GST, and HBx-5Dl, C @ -3D5, -Dl9, and -D26, respectively. Pull-down cx 305 @° p53blndlng periment was as described in the legend to Fig. JD. 154p@ D, schematic map of the p53-binding regions in NSa fuN + HBx. HBxfull: 0. transactivation domain; , reg 501 @ ulationdomain.p53-bindingabilitiesareshownto *--- @-p63@s6 + 502 + theright.Themolecularmassmarkersareindicated @72 in kDa to the right of A, B, and C. . -42 504 + Dig 1M‘ifI@1-@@n2

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C 0 U) a- Fig. 4. p53 represses HBx transactivation in C HepG2 cells. The transfected plasmid DNA was 5 0 iLg of PHECx2CAT together with 5 @sgof 0 pSG5UTPL (Lane 1); 1, 2, and 3 @gof pSG5UTPL-p53 (Lanes 2—4,respectively);0, 1, 2, @ and 3 of pSG5UTPL-p53 plus 1 @igof pSG5UTPL-HBx (Lanes 5—8.respectively); 0, 1, 2. and 3 g.tg of pSG5UTPL-p53 plus 1 @gof U pSG5UTPL-HBx-5D1 (Lanes 9—12,respectively); U) and 2 @.tgof pSG5UTPL-p53 plus 1, 2, and 3 @gof pSG5UTPL-HBx (Lanes 13—15,respectively). The total amount of DNA added per transfection was C.) adjusted to 10 @gwith the control vector, pSG5UTPL. The CAT activity was measured 48 h after transfection and measured as percentage of conversion. I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 @ r@-i;fl HBx I HBx-501 I HBx 1p531 ri@:1

-3D39, -dl6, and -D26) but not to -D19 (51—99an;Fig. 3A). A similar background of the p53-responsive reporter was high in HepG2 cells, requirement for this region of HBx for p53 binding was also detected perhaps due to the endogenous p53, it was difficult to observe the using the GST-resin pull-down assay (Fig. 3C). effect of HBx. Therefore, we choose Saos-2, a p53-negative osteo PS3 Interferes with the Transactivation Activity of HBx. Next, sarcoma cell line. The introduction of p53 transactivated the p53- we examined whether p53 has an effect on HBx transactivation by a responsive reporter pG13CAT efficiently in this cell line, whereas CAT assay using the reporter pHECx2CAT, which harbors the HBx HBx had no effect on the reporter (Fig. 5, compare Lanes 5—7to2—4). responsive element derived from the HBV enhancer I core (42). HBx However, the transacting activity of p53 was repressed by HBx when and p53 were transiently coexpressed in HepG2 cells, and the tans HBx and p53 were coexpressed (Fig. 5, Lanes 9—11).HBx-5D1, acting activity of HBx was examined by CAT assay. p53 had no which harbors the p53-binding site, exhibited a repression similar to detectable effect on the reporter (Fig. 4, Lanes 2—4).However, the the full-size HBx (Fig. 5, Lanes 12—14).HBx-3D5 and -D19, which HBx transactivation activity was severely repressed by p53 in a could not bind to p53, showed no effect on p53 transactivation (Fig. dose-dependent manner (Fig. 4, Lanes 5—8).A similar effect was 5, compare Lanes 15—17to Lanes 18—20). The transactivation-defec observed when p53 and HBx-Dl, which constitutes the transactivation tive mutant HBx-5D4, which harbors the p53-binding site, exhibited domain of HBx, were coexpressed (Fig. 4, Lanes 9—12). The repres similar repression (Fig. 5, Lanes 21—23).These results indicate that sion by p53 could be alleviated by an excess amounts of HBx (Fig. 4, HBx interferes with p53 functionally and that the p53-interacting site Lanes 13—15).These results indicate that p53 and HBx interact in the tansactivation domain of HBx is responsible for the inhibitory functionally in vivo. effect of HBx on p53 transactivation. HBx Represses p53 Transactivation Activity. The effect of HBx HBx Exerts Transactivating Activity in p53-negative Cells. Be on p53 tansactivation was investigated by CAT assay. Because the cause the p53-binding site was mapped in the HBx transactivation

Fig. 5. HBx represses p53 transactivation in Saos-2 cells. The transfected plasmid DNA was 5 ,.&g of pG13CAT together with 5 @gofpSG5UTPL (Lane I); I, 2, and 3 ,.@gof pSG5UTPL-HBx (Lanes 2—4,respec lively); I, 2, and 3 pg of pSG5UTPL-p53 (Lanes 5—7. respectively); 0, 1, 2, and 3 @gofpSG5UTPL-HBx plus 1 @gofpSG5UTPL-p53 (Lanes 8—11,respectively); 1, 2, and 3 @tgof pSG5UTPL-HBx-5Dl plus 1 @gof pSG5UTPL-p53 (Lanes 12—14.respectively);I, 2, and 3 ILg of pSG5UTPL-HBx-3D5 plus 1 @gof pSG5UTPL p53 (Lanes 15—17,respectively); 1, 2, and 3 @sgof pSG5UTPL-HBx-D19 plus I @gof pSG5UTPL-p53 (Lanes 18—20,respectively); and I, 2, and 3 @agof pSG5UTPL-HBx-5D4 plus I pg of pSG5UTPL-p53 (Lanes 21—23,respectively). The total amount of DNA added per transfection was adjusted to 10 @agwith the control vector, pSG5UTPL. The CAT activity was meas ured 48 h after transfection and measured as percentage of conversion.

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A B

C I

I

HBx: (rig) r-@1ri;;1

Fig. 6. HBx exhibits transactivation activity in p53-negative cells. A, p53-negative hepatoma cells (Hep3B) were transfected with pHECx2CAT or NF-KBx3CAT together with pSG5UTPL-HBx. The amount of pSG5UTPL-HBx is shown in @.tgatthe bottom of the figure. The total amount of DNA added per transfection was adjusted to 10 @gwiththe control vector,pSGSUTPL.B,introductionofp53to Saos-2cells(p53negative)suppressesHBxtransactivation.ThetransfectedplasmidDNAwas5 @sgofpHECx2CATtogetherwith5@sg ofpSG5UTPL(Lane 1); 1, 2, and 3 g.&gofpSG5UTPL-p53 (Lanes 2—4,respectively); 1, 2, and 3 @.tgofpSG5UTPL-HBx (Lanes 5—7,respectively); 1, 2, and 3 @gofpSG5UTPL-p53 plus I @gof pSG5UTPL-HBx (Lanes 8—10.respectively); 1, 2, and 3 @gof pSG5UTPL-p53-d3 plus I @agof pSG5UTPL-HBx (Lanes 11—13.respectively); I, 2, and 3 @.&gof pSG5UTPL-p53-d5 plus 1 @.&gofpSG5UTPL-HBx (Lanes 14—16,respectively);and I. 2, and 3 @gofpSG5UTPL-p53-R273H plus I @gofpSG5UTPL-HBx (Lanes I 7—19. respectively). The total amount of DNA added per transfection was adjusted to 10 @gwiththe control vector, pSG5UTPL. The CAT activity was measured 48 h after transfection and measured as percentage of conversion. domain, we addressed whether p53 interaction is involved in the HBx p53 and HBx may prevent p53 from accessing the regulation element transactivation. In Hep3B, a p53-negative hepatoma cell line, the reporter of the p53-responsive genes (32, 33), and thus communicating with having HBV enhancer I core or NF-i

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1997 American Association for Cancer Research. HBx-p53 INTERACTION p53 and HBx by coexpression of these factors in p53-negative cells. The through acting on nuclear factor KB and CCAAT/enhancer-binding protein-like cis-elements. J. Biol. Chem., 266: 13759—13763, 1991. expressed p53 level may be different from that of wild-type p53-positive 21. Hu, K. Q., Vierhing, J. M., and Siddiqui, A. Trans-activation of HLA-DR gene by cells under physiological conditions. However, the amount of p53 pro hepatitis B virus X gene product. Proc. Nath. Acad. Sci. USA, 87: 7140—7147, 1990. tein, which is regulated at several levels, varies when cells are exposed to 22. Twu, J. S Lai, M. Y., Chen, D. S., and Robinson, W. S. Activation of proto-oncogene c-jun by the X protein of hepatitis B virus. Virology, 192: 346—350, 1993. different stimuli (48—51).Similarly, expression of the X gene is under 23. Yen, B. T. S. Hepadnaviral X protein: review of recent progress. J. Biomed. Sci., 3: autoregulation of HBx and is affected by various extracellular stimuli, 20—30,1996. such as tumor promoters and inflammation (42). Thus, the results of this 24. Lane, D. P. Cancer: p53, guardian of the genome. Nature (Land.), 358: 15—16,1992. 25. Wu, X., and Levine, A., J. p53 and E2F-l cooperate to mediate apoptosis. Proc. NatI. study reveal the significant functional interactions of p53 and HEx in Acad. Sci. USA, 91: 23602—23606, 1994. cells. The significance of the two functions of HBx, namely p53 binding 26. Wang, X. W., Vermeulen, W., Coursen, J. D., Gibson, M., Lupold, S. E., Forrester, K., Xu, G., Elmore, L., Yeh, H., Hoeijmakers, J. H., and Harris, C. C. The XPB and and HBx transactivation, will be better understood by isolating HBx XPD DNA hehicasesarecomponentsof the p53-mediatedapoptosispathway.Genes substitution mutations that abolish either of these two functions. Dcv.,10:1219—1232,1996. 27. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1997 American Association for Cancer Research. The Transactivation and p53-interacting Functions of Hepatitis B Virus X Protein Are Mutually Interfering but Distinct

Yong Lin, Takahiro Nomura, Tatsuya Yamashita, et al.

Cancer Res 1997;57:5137-5142.

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