The Human Caspase-8 Promoter Sustains Basal Activity through SP1 and ETS-like Transcription Factors and Can Be Up-regulated by a p53-dependent Mechanism

Caspase-8, also known as MACH/FLICE/Mch5, is the most upstream-located cysteine-aspartyl-protease (caspase) in a caspase cascade involved in apoptosis triggered by members of the tumor necrosis factor receptor superfamily or other stimuli such as chemotherapeutic agents. Regulation of caspase-8 expression on a post-translational level has been studied in detail, whereas only little information is available on its control by gene transcription. We identified and cloned the human caspase-8 promoter, determined the transcriptional start site of the caspase-8 gene, and examined the regulatory mechanisms of the promoter with respect to its basal activity as well as to its inducibility upon apoptotic stimuli in human hepatoma cells. We identified two minimal sequences essential for basal transcription of caspase-8 and demonstrate that a single SP1 and an ETS-like binding motif mediate this effect. We further show that the caspase-8 promoter is inducible and demonstrate that adenoviral infection increases caspase-8 mRNA levels. However, the increase in caspase-8 gene transcription after adenoviral infection absolutely depends on the p53 status of the hepatoma cell line, implying that caspase-8 is a target gene of p53. We show that delivery of exogenous p53 alone is sufficient to induce the caspase-8 promoter even in p53-deficient Hep3B hepatoma cells. Subsequent promoter deletion analysis in combination with luciferase reporter assays identified a p53-responsive element downstream of the transcriptional start site. We demonstrate that this p53-responsive sequence overlaps with the ETS-like binding site and suggest that an additional p53-inducible, yet unknown factor interacts with this region of the caspase-8 promoter. In summary, our study contributes to the understanding of the transcriptional regulation of the caspase-8 gene by basal (SP1- and ETS-dependent) and inducible (p53-dependent) mechanisms. Caspase-8, also known as MACH/FLICE/Mch5, is the most upstream-located cysteine-aspartyl-protease (caspase) in a caspase cascade involved in apoptosis triggered by members of the tumor necrosis factor receptor superfamily or other stimuli such as chemotherapeutic agents. Regulation of caspase-8 expression on a post-translational level has been studied in detail, whereas only little information is available on its control by gene transcription. We identified and cloned the human caspase-8 promoter, determined the transcriptional start site of the caspase-8 gene, and examined the regulatory mechanisms of the promoter with respect to its basal activity as well as to its inducibility upon apoptotic stimuli in human hepatoma cells. We identified two minimal sequences essential for basal transcription of caspase-8 and demonstrate that a single SP1 and an ETS-like binding motif mediate this effect. We further show that the caspase-8 promoter is inducible and demonstrate that adenoviral infection increases caspase-8 mRNA levels. However, the increase in caspase-8 gene transcription after adenoviral infection absolutely depends on the p53 status of the hepatoma cell line, implying that caspase-8 is a target gene of p53. We show that delivery of exogenous p53 alone is sufficient to induce the caspase-8 promoter even in p53-deficient Hep3B hepatoma cells. Subsequent promoter deletion analysis in combination with luciferase reporter assays identified a p53-responsive element downstream of the transcriptional start site. We demonstrate that this p53-responsive sequence overlaps with the ETS-like binding site and suggest that an additional p53-inducible, yet unknown factor interacts with this region of the caspase-8 promoter. In summary, our study contributes to the understanding of the transcriptional regulation of the caspase-8 gene by basal (SP1- and ETS-dependent) and inducible (p53-dependent) mechanisms. Caspase-8 is the most upstream-located cysteine-aspartyl-protease (caspase) involved in apoptosis mediated by Fas, TNF, 1The abbreviations used are: TNF, tumor necrosis factor; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; wt, wild type; STAT, signal transducers and activators of transcription. and related death receptors of the TNF superfamily (1Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2111) Google Scholar, 2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar, 3Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2741) Google Scholar). Caspase-8 is highly regulated on a posttranslational level. Upon apoptotic stimulation and subsequent death-inducing signaling complex (DISC) formation, an unprocessed procaspase-8 is coupled to the adapter molecule Fas-associated death domain protein (FADD) via two death effector domains, which is a prerequisite for processing of procaspase-8 to the active caspase by proteolytic cleavage leading to the active subunits p18 and p10 (2Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1041) Google Scholar). These subunits are released to the cytosol and mediate caspase activity via a C-terminal protease domain. Caspase-8-dependent apoptosis can be modulated by expression of c-FLIP, a molecule sharing homology to caspase-8 but lacking a functional protease domain (4Scaffidi C. Schmitz I. Krammer P.H. Peter M.E. J. Biol. Chem. 1999; 274: 1541-1548Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). For downstream signaling of caspase-8 leading to apoptosis two different pathways have been described (5Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.-M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-3596Crossref PubMed Scopus (2631) Google Scholar), one via activation of Bid involving mitochondria and cytochrome c release and the other via direct activation of effector caspases as caspase-3. Details of apoptosis signaling and caspase-8 activation are reviewed in detail elsewhere (6Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (774) Google Scholar, 7Strasser A. O'Connor L. Dixit V.M. Annu. Rev. Biochem. 2000; 69: 217-245Crossref PubMed Scopus (1383) Google Scholar). At present, the transcriptional regulation of the caspase-8 gene has not been examined in detail. However, it has been demonstrated in vitro that ectopic overexpression of caspase-8 is sufficient to induce apoptosis (3Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2741) Google Scholar). This implies that a self-processing mechanism might be activated if a critical threshold of pro-caspase-8 is expressed. In addition it has been shown that in certain childhood neuroblastomas and neuroectodermal brain tumors as well as in some types of lung cancers, caspase-8 expression is rather low or even abolished, which was explained by both somatic gene mutations and deletions as well as silencing due to hypermethylation of genomic caspase-8 sequences (8Takita J. Yang H.W. Bessho F. Hanada R. Yamamoto K. Kidd V. Teitz T. Wei T. Hayashi Y. Med. Pediatr. Oncol. 2000; 35: 541-543Crossref PubMed Scopus (37) Google Scholar, 9Teitz T. Wei T. Valentine M.B. Vanin E.F. Grenet J. Valentine V.A. Behm F.G. Look A.T. Lahti J.M. Kidd V.J. Nat. Med. 2000; 6: 529-535Crossref PubMed Scopus (699) Google Scholar, 10Zuzak T.J. Steinhoff D.F. Sutton L.N. Phillips P.C. Eggert A. Grotzer M.A. Eur. J. Cancer. 2002; 38: 83-91Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 11Shivapurkar N. Toyooka S. Eby M.T. Huang C.X. Sathyanarayana U.G. Cunningham H.T. Reddy J.L. Brambilla E. Takahashi T. Minna J.D. Chaudhary P.M. Gazdar A.F. Cancer Biol. Ther. 2002; 1: 65-69Crossref PubMed Scopus (8) Google Scholar). The aim of this study was to examine the regulation of the caspase-8 promoter. Because acute liver failure and viral hepatitis are often associated with Fas/TNF-dependent apoptotic mechanisms (12Kanzler S. Galle P.R. Semin. Cancer Biol. 2000; 10: 173-184Crossref PubMed Scopus (135) Google Scholar), we focused our analysis on the transcriptional mechanisms that are involved in controlling caspase-8 expression in hepatoma cells. Plasmids and Recombinant DNA Techniques—Standard recombinant DNA techniques were carried out as described elsewhere (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY1989Google Scholar). For intermediate cloning steps the vectors pBluescript SK+ (Stratagene) and pCR2.1-TOPO (Invitrogen) were used. For construction of luciferase-reporter plasmids we amplified appropriate genomic fragments of the caspase-8 promoter using a PCR approach and oligonucleotides that are flanked by KpnI and HindIII restriction sites. The resulting fragments were then cloned into the KpnI and HindIII sites of the pGL2-Basic vector (Promega). As the template for the amplification of the caspase-8 promoter we used male human genomic DNA (Promega). Promoter sequences amplified by PCR were confirmed by sequencing using an ABI Prism 310 (Applied Biosystems). Oligonucleotides—Oligonucleotides used in this study were purchased from MWG Biotech (Ebersberg, Germany). For amplification of genomic promoter fragments and for primer extension analysis we designed oligonucleotides of 20–30 bp in length and a GC content of ∼50%. Primer sequences for amplifying promoter fragments are as follows (only the distal base position is given; restriction sites are indicated in bold): +76 (antisense), 5′-AAGCTTGTACAGGCAGGCAGGAAGCTC-3′; +76mutETS (antisense), 5′-AAGCTTGTACAGGCAGGCAGAAAGCT-3′; +66 (antisense), 5′-AAGCTTGCAGGAAGCTCAGTCTGAAC-3′; +58 (antisense). 5′-AAGCTTCTCAGTCTGAACAACCAGCC-3′; +48 (antisense), 5′-AAGCTTACAACCAGCCTTCAGACCAG-3′; –972 (sense), 5′-GGTACCGTCACCAAAGAGAACAGAGTG-3′; –470 (sense), 5′-GGTACCGTCACTATGCTTCCTGTACAGC-3′; –370 (sense), 5′-GGTACCGATGAGCTGATACAGTTTCC-3′; –307 (sense), 5′-GGTACCCAGATTGCTTAGTAATTAAAACGC-3′; –171 (sense), 5′-GGTACCAAACGACAACTCACAGTGCC-3′; –121 (sense), 5′-GGTACCGTGGAGAATGGGAGCACTCTCCGC-3′; –111 (sense), 5′-GGTACCGGAGCACTCTCCGCAGTGGG-3′; –101 (sense), 5′-GGTACCCCGCAGTGGGCGGGAGGAGACG-3′; –101SP1mut (sense) 5′-GGTACCCCGCAGTGTTCGGGAGGAGACGAG-3′; –91 (sense), 5′-GGTACCCGGGAGGAGACGAGGAGGGCG-3′; –81 (sense): 5′-GGTACCCGAGGAGGGCGTTCCCTGGGGAG-3′; –71 (sense), 5′-GGTACCGTTCCCTGGGGAGTGGCAGTGGTTGG-3′; +34 (antisense), 5′-AAGCTTCAGACCAGCTCAGACACAAGGTGAAAC-3′; –24 (antisense), 5′-AAGCTTTTACCTCCTCCAAACCTTTGC-3′; –99 (antisense), 5′-AAGCTTGGAGAGTGCTCCCATTCTCC-3′. PCR and Duplex RT-PCR of Human Caspase-8/GAPDH—RNA was isolated using a RNeasy kit (Qiagen) according to the manufacturer's instructions. PCR was performed according to standard procedures using the Expand High Fidelity PCR system (Roche Applied Science) with 5′-3′ proofreading activity for exact amplification of the promoter fragments. PCR was carried out for 30 cycles with denaturing at 92 °C for 15 s, an amplification step of 1 min/kilobase at 68 °C, and a final extension step at 68 °C for 7 min. For RT-PCR we performed first-strand synthesis using Oligo(dT)15 primer (Promega) and the Omniscript RT kit (Qiagen) in combination with 1 μg of total RNA per reaction. Amplification of caspase-8 and GAPDH cDNA were carried out in one reaction using the following primers: caspase-8 (sense), 5′-CTTGGATGCAGGGGCTTTGACC-3′; caspase-8 (antisense), 5′-GTTCACTTCAGTCAGGATGG-3′; GAPDH (sense), 5′-TGATGACATCAAGAAGGTGGTGAAG-3′; GAPDH (antisense), 5′-TCCTTGGAGGCCATGTAGGCCAT-3′. PCR was performed for 10 cycles with first-strand DNA and caspase-8 primers at a T anneal of 53 °C. Then the reaction was interrupted, primers for GAPDH were added, and the reaction was continued for another 25 cycles. PCR products were documented and quantitated using a Gel Doc 1000 apparatus (Bio-Rad) and Molecular Analyst software (Bio-Rad). For direct comparison, the GAPDH/caspase-8 ratio was determined. Cell Culture, Transfection Experiments, and Luciferase Assays— HepG2, HuH7, and Hep3B hepatoma cell lines were obtained from the American Type Culture Collection (ATCC) and differ with respect to their p53 status as described in Muller et al. (14Muller M. Wilder S. Bannasch D. Israeli D. Lehlbach K. Li-Weber M. Friedman S.L. Galle P.R. Stremmel W. Oren M. Krammer P.H. J. Exp. Med. 1998; 188: 2033-2045Crossref PubMed Scopus (733) Google Scholar). The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. DNA transfection was performed using a modified calcium phosphate precipitation method as described previously (15Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar). Cells were grown on 60-mm dishes to ∼50% confluence when used for transfection experiments. In general, 2.5 μg of reporter plasmid were used for transfection. All transfections contained 0.5 μg of the β-galactosidase reporter pCMV-Gal as an internal standard. By adding pBSK+ DNA (Stratagene) to a total of 5 μg of DNA to the transfection mix, the total amount of DNA was kept constant in each transfection experiment. To measure luciferase activity, cells were washed twice with phosphate-buffered saline and lysed by adding 350 μl of extraction buffer (25 mm Tris-H3PO4, pH 7.8, 2 mm EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, and 2 mm dithiothreitol) for 10 min. The lysates were cleared by centrifugation. 50 μl of the supernatant were assayed by adding 300 μl of measuring buffer (25 mm glycylglycine, 15 mm MgSO4, and 5 mm ATP). The light emission was measured in duplicate for 10 s in a Lumat LB 9501 (Berthold, Bad Wildbad, Germany) by injecting 100 μl of 250 μm luciferin. Each experiment was performed in duplicate and repeated at least three times. The data were compared with appropriate controls, calculated as relative luciferase activity, and represent the average of three independent experiments. Adenoviral Vectors Used in This Study and Infection of Hepatoma Cells—Recombinant adenoviral vectors carrying GFP (adv-GFP) and p53 (adv-p53) were kindly provided by Dr. B. Mundt and Dr. L. Zender (Hannover, Germany) and have been described in Mundt et al. (16Mundt B. Kuhnel F. Zender L. Paul Y. Tillmann H. Trautwein C. Manns M.P. Kubicka S. FASEB J. 2003; 17: 94-96Crossref PubMed Scopus (141) Google Scholar) and Zender et al. (17Zender L. Kock R. Eckhard M. Frericks B. Gosling T. Gebhardt T. Drobek S. Galanski M. Kuhnel F. Manns M. Kubicka S. Gastroenterology. 2002; 123: 608-618Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For adenoviral infection, hepatoma cells were seeded to a density of 2 × 105 cells/ml in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Before infecting the cells at a multiplicity of infection of 50, the medium was changed to Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum. Nuclear Extracts and Gel Retardation Assays—HepG2 and HuH7 nuclear extracts were prepared using the modified Dignam C method (18Trautwein C. Rakemann T. Malek N.P. Plumpe J. Tiegs G. Manns M.P. J. Clin. Invest. 1998; 101: 1960-1969Crossref PubMed Scopus (69) Google Scholar). For gel retardation assays, nuclear extracts were used as indicated. Binding buffer for SP1 EMSA consisted of 25 mm HEPES, pH 7.6, 5 mm MgCl2, 34 mm KCl, 2 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 1 μg/μl poly(dI-dC), and 2 μg/μl bovine serum albumin. As probes, 32P-end-labeled oligonucleotides were used at an activity of 30000 cpm/μl. The oligonucleotides used for SP1 and ETS EMSA are shown under Results. The binding reaction was performed for 30 min at 30 °C. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel. Supershift experiments were performed with a specific SP1 antibody or an antibody directed against a broad range of ETS family members, respectively (Santa Cruz Biotechnology, Santa Cruz, CA). For p53- and ETS-specific EMSA experiments, respectively, modified protocols were used (19Kubicka S. Kuhnel F. Zender L. Rudolph K.L. Plumpe J. Manns M. Trautwein C. J. Biol. Chem. 1999; 274: 32137-32144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Primer Extension Analysis—For mapping the transcriptional start site of the caspase-8 promoter we used the commercial available Primer Extension System (Promega) and performed primer extension analysis according to the manufacturer's protocol. Briefly, 10 pmol of the oligo 5′-TGGCGGGGTACAGGCAGGCAGGAAGCTC-3′ (compare Fig. 1A) as well as a DNA marker mix were end-labeled using 10 units of T4 polynucleotide kinase and 3000 Ci/mmol [γ-32P]ATP. The primer extension reaction was performed using 10 μg of total RNA from HepG2 or HuH7 cells and 0.2 pmol of labeled oligo. The reaction was analyzed on a denaturing polyacrylamide gel containing 8% acrylamide, 7 m urea in 1× Tris-buffered EDTA. After autoradiography overnight, the size of the primer extension product was determined by direct comparison to the provided DNA size marker. Computational Analysis—The human caspase-8 5′-untranslated region was identified by using the BLAST server (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70758) Google Scholar) at the NCBI and the human caspase-8 cDNA sequence (accession number NM 001228) as a query. For a prediction of putative binding sites for transcription factors we used the MatInspector professional software (Genomatix) as described in Quandt et al. (22Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2424) Google Scholar). Cloning and Analysis of the 5′-Upstream Region of the Human Caspase-8 Gene—To isolate the human caspase-8 promoter we performed a BLAST search with the human genome using the longest isoform of the caspase-8 cDNA (variant A, accession number NM 001228). The 5′ sequence of the caspase-8 cDNA matched perfectly to a genomic sequence located on chromosome 2 (accession number NT 005403). This is in agreement with recent reports showing that the caspase-8 gene maps at chromosome 2 (band 2q33-q34) (23Grenet J. Teitz T. Wei T. Valentine V. Kidd V.J. Gene (Amst.). 1999; 226: 225-232Crossref PubMed Scopus (82) Google Scholar, 24Kischkel F.C. Kioschis P. Weitz S. Poustka A. Lichter P. Krammer P.H. Cytogenet. Cell Genet. 1998; 82: 95-96Crossref PubMed Google Scholar). Therefore we concluded that the 5′ upstream region of the match may contain the caspase-8 promoter. The sequence containing the putative caspase-8 promoter as well as the noncoding exons 1 and 2 was deposited to the GenBank™ data bank under accession number AY 291598 and is displayed in Fig. 1A. Performing an initial computer analysis of the promoter sequence using MatInspector software (22Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2424) Google Scholar), binding sites for transcription factors including NFκB, STAT1, SP1, and ETS were predicted and are shown in Fig. 1A. Next we were interested to map the transcriptional start site of the caspase-8 gene. For this experiment we used an antisense primer (see Fig. 1A, arrow) located within the noncoding exon 1. Primer extension analysis with total RNA isolated from both HepG2 and HuH7 cells revealed a 78-bp primer extension product (Fig. 1B, see lanes 2 and 3, respectively). Accordingly, we identified the sequence TTTCTG as the transcriptional start site of the caspase-8 gene. This site is located 29 bp upstream of the 5′ end of the published cDNA. The position of the transcriptional start site was arbitrarily set to +1. A Sequence Located between –121 and –71 Contributes to the Basal Activity of the Caspase-8 Promoter—For further experiments, we amplified an ∼1000-bp fragment containing the putative caspase-8 promoter region and a small part of the first, noncoding exon 1 using primers –972 and +76. The resulting fragment was cloned into the HindIII and KpnI sites of the pGL2 luciferase vector (Promega) and was termed –972/+76 with respect to the 5′ and 3′ positions related to the transcriptional start site. To map the core region of the caspase-8 promoter we first introduced 5′ deletions (Fig. 2A) in the –972/+76 construct using a PCR approach (respective primers are listed under "Experimental Procedures"). All constructs were transfected in HuH7 cells including the empty pGL2 vector as a control. Luciferase activity of the –972/+76 construct was nearly 300-fold increased compared with the pGL2 control, whereas the strongest activity was found with the –121/+76 construct (Fig. 2B). A further 5′ deletion of 50 bp (–71/+76 construct) resulted in a 8-fold reduction in luciferase activity, indicating that this region is involved in controlling basal caspase-8 promoter activity. Because our data suggested that the region located between bp –121 and –71 is essential for basal caspase-8 promoter activity, we further concentrated on the role of this sequence. We performed a detailed analysis of this area by introducing increasing 10-bp 5′ deletions in the –121/+76 construct (Fig. 2C). Transfection experiments in HuH7 cells using these constructs revealed that the region located between position –101 and –91 plays a major role for basal caspase-8 promoter activity (Fig. 2D). A Unique SP1 Binding Motif Is Essential for Basal Activity of the Caspase-8 Promoter—MatInspector software analysis identified a potential SP1 binding site between position –101 and –91 (Fig. 2C, underlined sequence). To confirm SP1 DNA binding to this motif, we next performed gel retardation analysis with 32P-labeled oligonucleotides representing the putative SP1 site found in the caspase-8 promoter and nuclear extracts isolated from HuH7 cells. A SP1 consensus oligonucleotide (Fig. 3A) served as a control. Complex formation with the SP1 consensus oligonucleotide revealed three SP1-specific complexes, where only the upper slow-migrating complex could be supershifted by anti-SP1 (Fig. 3B, lanes 3 and 4). Gel shift experiments using the putative SP1 binding site of the caspase-8 promoter (Fig. 3A) revealed complex formation comparable with the pattern found with the SP1 consensus oligonucleotide. The slower migrating complex also could be supershifted with an SP1 antibody. In contrast, introducing two G → T mutations within the core sequence of the putative SP1 binding motif (Fig. 3A) in the caspase-8 promoter abolished SP1-dependent complex formation (Fig 3B, lane 10). To further support our data, we performed competition experiments using the SP1 consensus motif as a 32P-labeled probe and competed for complex formation by using either the wt or the mutant putative binding site as derived from the caspase-8 promoter (Fig. 3A). A 5, 25, and 100 m excess of the respective cold oligonucleotide was used for competition analysis. When the wt sequence was used as competitor, a 5 m excess was already able to compete for binding of the three putative SP1-specific complexes (Fig. 3C, lane 3–5), whereas the mutant oligonucleotide was not able to inhibit formation of these specific complexes in the same molar range (lane 6–8). These data together with the transfection experiments indicated that SP1 is involved in controlling the basal activity of the caspase-8 promoter. To further support our findings, we introduced the mutated SP1 motif in the caspase-8 promoter of reporter-construct –101/+76, resulting in –101SP1mut/+76. Transfection experiments using the –101/+76, the –101/+76mut, and the –91/+76 caspase-8 construct and subsequent luciferase assays revealed a significant reduction (–60%) in promoter activity of the mutant compared with the respective wt construct (Fig. 3D). However, the activity of the –91/+76 caspase-8 construct was still lower compared with –101/+76mut. An ETS-like Element Is Located in the 3′ End of the Caspase-8 Promoter and Contributes to Basal Activity—To identify further elements involved in controlling basal caspase-8 promoter activity, we introduced several 3′ deletions in the –470/+76 construct and compared the respective luciferase activities after transfection into HuH7 cells. Luciferase activity completely dropped when the deletion contained the start site of transcription (Fig. 4, A and B, plasmid –470/–24 and –470/–99, respectively). Interestingly, a 3′ deletion of 28 bp in plasmid –470/+48 reduced the basal activity to ∼50% compared with the –470/+76 construct. Because this sequence contains a predicted ETS-like binding motif, we analyzed this sequence in more detail. By introducing subsequent 8 –10 bp 3′ deletions in this region we could demonstrate that luciferase activity was diminished when the predicted ETS-like motif and the surrounding sequences were missing, indicating that this site might be involved in controlling basal promoter activity (Fig. 4, C and D). To further test this hypothesis we next performed EMSA experiments using modified binding conditions for ETS (20Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) with oligonucleotides representing either the wt promoter sequence (ETSput, Fig. 5A, upper sequence) or a modified sequence carrying a mutation in the predicted ETS site (ETSmut, Fig. 5A, lower sequence). The 32P-labeled oligonucleotides were incubated with HuH7 nuclear extracts. Complex formation could be detected with the oligonucleotide representing the wt promoter sequence, which was not found with the mutant sequence (Fig. 5B, compare lanes 1 and 3). Complex formation with the wt oligonucleotide could be blocked by an antibody directed against a broad range of ETS-like factors (anti-ETS), indicating that an ETS-like factor binds to this sequence in the caspase-8 promoter. In contrast, the anti-ETS antibody had no effect on complex formation when the mutant oligonucleotide was used (Fig. 5B). Additionally, supershift analysis was performed with an ETS-1-specific antibody. However, no effect on complex formation was found (data not shown), indicating that this element is recognized by another ETS family member. Because our experiments suggested that an ETS-like factor binds to this region in the caspase-8 promoter, we introduced the mutant sequence in the –470/+76 construct (–470/+76mutETS). Transfection experiments of the mutant compared with the wt and –470/+48 construct demonstrated that luciferase activity of 470/+76mutETS was reduced to a range as found with the –470/+48 construct (Fig. 5C). These results indicate that besides the SP1, the ETS site in the promoter also contributes to the basal activity of caspase-8-dependent gene transcription. The Caspase-8 Promoter Is Inducible upon Adenoviral Infection in p53 wt Hepatoma Cells—Adenoviral gene transfer into the liver and hepatoma cells is an established approach for effective gene delivery (19Kubicka S. Kuhnel F. Zender L. Rudolph K.L. Plumpe J. Manns M. Trautwein C. J. Biol. Chem. 1999; 274: 32137-32144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70758) Google Scholar, 22Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2424) Google Scholar). However, several disadvantages have been described including strong cytotoxic effects mainly due to Fas-mediated apoptosis and induction of p53 (19Kubicka S. Kuhnel F. Zender L. Rudolph K.L. Plumpe J. Manns M. Trautwein C. J. Biol. Chem. 1999; 274: 32137-32144Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 25Kuhnel F. Zender L. Paul Y. Tietze M.K. Trautwein C. Manns M. Kubicka S. J. Biol. Chem. 2000; 275: 6421-6427Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). To further evaluate the molecular mechanisms responsible for adenoviral-induced apoptosis in the liver, we wanted to examine if caspase-8-dependent gene transcription is increased after adenoviral challenge. Three hepatoma cell lines (HepG2, HuH7, Hep3B) with a different p53 status (14Muller M. Wilder S. Bannasch D. Israeli D. Lehlbach K. Li-Weber M. Friedman S.L. Galle P.R. Stremmel W. Oren M. Krammer P.H. J. Exp. Med. 1998; 188: 2033-2045Crossref PubMed Scopus (733) Google Scholar) were transfected with the caspase-8 –470/+76 promoter construct. 24 h after transfection we infected the cells with of a GFP expressing adenovirus at a multiplicity of infection of 50. Efficiency of infection was controlled by GFP fluorescence (data not shown). Cells were harvested before and 8 and 24 h after adenoviral infection, and luciferase activity was measured (Fig. 6A). In HepG2 cells expressing wt p53 protein, an up to 10-fold induction of the caspase-8 promoter was observed 24 h after infection. In contrast, in hepatoma cells carrying a mutant p53 allele (HuH7 cells) or a p53 deletion (Hep3B cells) no induction was found after adenoviral infection. To test if the report