Characterization of p53-mediated Up-regulation of CD95 Gene Expression upon Genotoxic Treatment in Human Breast Tumor Cells
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m304397200
ISSN1083-351X
AutoresCarmen Ruiz‐Ruiz, Gema Robledo, Eva Cano, Juan Miguel Redondo, Abelardo López‐Rivas,
Tópico(s)Cancer therapeutics and mechanisms
ResumoDeath receptor CD95 gene expression is frequently low in human breast tumors and is up-regulated by genotoxic treatments in a p53-dependent manner. We have evaluated the relative contribution of promoter and intronic p53 consensus sites to the regulation of the human CD95 gene in breast tumor cells following doxorubicin treatment. Deletion constructs of the promoter region and site-directed mutagenesis of p53 consensus sites in a fragment spanning 1448 bp of the 5′-promoter demonstrate that these sites are not involved in the observed up-regulation of the CD95 gene upon doxorubicin treatment. In contrast, a p53 consensus site located within the first intron of CD95 gene is absolutely required for the inducible expression of CD95 upon genotoxic treatment in breast tumor cells. Analysis of the transcriptional activity of the two most common p53 mutants found in human breast tumors that are associated with resistance to doxorubicin reveals that these mutations completely eliminate the ability of p53 protein to transactivate CD95 gene expression. On the other hand, Bcl-2 overexpression albeit preventing doxorubicin-induced apoptosis, has no effect on p53-mediated CD95 up-regulation in breast tumor cells. Altogether, these results indicate the lack of involvement of p53 consensus sites of the CD95 promoter region and the pivotal role of intronic p53-responsive element in the regulation of human CD95 gene expression in breast tumor cells. Our results also suggest that in breast cancer patients with certain mutations in the p53 gene, expression of death receptor CD95 in response to genotoxic treatments could be severely compromised. Death receptor CD95 gene expression is frequently low in human breast tumors and is up-regulated by genotoxic treatments in a p53-dependent manner. We have evaluated the relative contribution of promoter and intronic p53 consensus sites to the regulation of the human CD95 gene in breast tumor cells following doxorubicin treatment. Deletion constructs of the promoter region and site-directed mutagenesis of p53 consensus sites in a fragment spanning 1448 bp of the 5′-promoter demonstrate that these sites are not involved in the observed up-regulation of the CD95 gene upon doxorubicin treatment. In contrast, a p53 consensus site located within the first intron of CD95 gene is absolutely required for the inducible expression of CD95 upon genotoxic treatment in breast tumor cells. Analysis of the transcriptional activity of the two most common p53 mutants found in human breast tumors that are associated with resistance to doxorubicin reveals that these mutations completely eliminate the ability of p53 protein to transactivate CD95 gene expression. On the other hand, Bcl-2 overexpression albeit preventing doxorubicin-induced apoptosis, has no effect on p53-mediated CD95 up-regulation in breast tumor cells. Altogether, these results indicate the lack of involvement of p53 consensus sites of the CD95 promoter region and the pivotal role of intronic p53-responsive element in the regulation of human CD95 gene expression in breast tumor cells. Our results also suggest that in breast cancer patients with certain mutations in the p53 gene, expression of death receptor CD95 in response to genotoxic treatments could be severely compromised. The CD95 (Fas/APO-1) receptor, a member of the TNF 1The abbreviations used are: TNF, tumor necrosis factor; PBS, phosphate-buffered saline; PS, phosphatidylserine; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay./NGF receptor superfamily, is a potent inducer of apoptosis in cells of the immune system upon interaction with its natural ligand, CD95L (1Suda T. Takahashi T. Golstein P. Nagata S. Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2449) Google Scholar). CD95 is also expressed in a broad panel of non-transformed cells outside the immune system (2Leithauser F. Dhein J. Mechtersheimer G. Koretz K. Bruderlein S. Henne C. Schmidt A. Debatin K.M. Krammer P.H. Moller P. Lab. Invest. 1993; 69: 415-429PubMed Google Scholar). Although the CD95/CD95L system could play a role in tumor regression (3Medema J.P. de Jong J. van Hall T. Melief C.J.M. Offringa R. J. Exp. 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The tumor suppressor protein p53 has been involved in the modulation of CD95 levels in response to chemo- and radiotherapy, not only in breast cancer cells but also in other tumor cell lines (14Ruiz-Ruiz M.C. López-Rivas A. Cell Death Diff. 1999; 6: 271-280Crossref PubMed Scopus (57) Google Scholar, 15Sheard M.A. Vojtesek B. Janakova L. Kovarik J. Zaloudik J. Int. J. Cancer. 1997; 73: 757-762Crossref PubMed Scopus (119) Google Scholar, 17Owen-Schaub L.B. Zhang W. Cusack J.C. Angelo L.S. Santee S.M. Fujiwara T. Roth J.A. Deisseroth A.B. Zhang W.-W. Kruzel E. Radinsky R. Mol. Cell. Biol. 1995; 15: 3032-3040Crossref PubMed Scopus (690) Google Scholar, 18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar). 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The p53 gene is one of the most often mutated in human tumors and the majority of these mutations are missense in one allele, producing full-length mutant inactive proteins with increased stability, while the other allele is often lost (20Levine A.J. Momand J. Finlay C.A. Nature. 1991; 351: 453-456Crossref PubMed Scopus (3662) Google Scholar, 22Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2571) Google Scholar, 23Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7475) Google Scholar). Major mutational "hot-spots" occur in four of five domains that are highly conserved in evolution and contain amino acids essential for p53 function. Therefore, these mutations usually create proteins that fail to bind to p53-specific DNA responsive sites and activate transcription (24Raycroft L. Schmidt J.R. Yoas K. Hao M.M. Lozano G. Mol. Cell. Biol. 1991; 11: 6067-6074Crossref PubMed Scopus (77) Google Scholar, 25Kern S.E. Pietenpol J.A. Thiagalingam S. Seymour A. Kinzler K.W. Vogelstein B. Science. 1992; 256: 827-829Crossref PubMed Scopus (891) Google Scholar). In breast cancer, p53 mutations have been found in about 20% of the cases (22Nigro J.M. Baker S.J. Preisinger A.C. Jessup J.M. Hostetter R. Cleary K. Bigner S.H. Davidson N. Baylin S. Devilee P. Glover T. Collins F.S. Weston A. Modali R. Harris C.C. Vogelstein B. Nature. 1989; 342: 705-708Crossref PubMed Scopus (2571) Google Scholar, 26Bartek J. Iggo R. Gannon J. Lane D.P. Oncogene. 1990; 5: 893-899PubMed Google Scholar) and they seem to be associated with poor prognosis and resistance to chemotherapeutic drugs (27Borresen A.-L. Andersen T.I. Eyfjörd J.E. Cornelis R.S. 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Oncogene. 1999; 18: 297-304Crossref PubMed Scopus (47) Google Scholar). However, despite the importance of p53 mutations and deregulation of Bcl-2 expression in breast tumor development, the impact of these changes on CD95 expression has not been evaluated. A consensus binding site for p53 (34el-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1751) Google Scholar) located in the first intron of the CD95 gene has been involved in the transcriptional regulation of this gene by p53 (18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar, 36Munsch D. Watanabe-Fukunaga R. Bourdon J.-C. Nagata S. May E. Yonish-Rouach E. Reisdorf P. J. Biol. Chem. 2000; 275: 3867-3872Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Furthermore, it has been suggested that other p53 consensus elements of the CD95 promoter region may collaborate with the intronic element in the regulation of CD95 expression (18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar). However, direct evidence supporting this proposition is still lacking. In this study, we analyzed the p53-mediated transcriptional regulation of the CD95 gene in human breast tumor cells. We have searched for p53 responsive elements in the upstream regulatory region (URR) of the CD95 gene that cooperate with the p53 intronic site in the transcriptional activation of this gene upon genotoxic stress. For this purpose we have generated constructs harboring different fragments of the URR of the CD95 gene fused to the p53 enhancer of the first intron. Despite the presence of several p53 consensus sequences, we have found no functional p53 responsive elements within a 1448 bp region of the URR of the CD95 gene. A minimal region of this promoter was sufficient to achieve proper transactivation of CD95 by the intronic element. EMSA and ChIP studies further demonstrated the importance of the intronic site. In addition, we analyzed the capacity of the two most common p53 missense mutants found in breast tumors to transactivate the CD95 gene, and investigated the effect of the anti-apoptotic Bcl-2 protein on p53-mediated up-regulation of CD95 in breast tumor cells. Cell Cultures and Treatments—The human breast tumor cell line MCF-7, expressing wild type p53, and the p53-null human lung carcinoma-derived cell line H1299 were kindly provided by Dr. M. Ruiz de Almodovar (University of Granada, Granada, Spain) and Dr. J. A. Pintor-Toro (Consejo Superior de Investigaciones Científicas, Sevilla, Spain), respectively. Cells were maintained in culture in RPMI 1640 medium containing 10% fetal bovine serum (Invitrogen), 1 mm l-glutamine, and gentamycin, at 37 °C in a humidified 5% CO2/95% air incubator. MCF-7 cells stably expressing the human papillomavirus type 16 E6 protein were generated as described previously (14Ruiz-Ruiz M.C. López-Rivas A. Cell Death Diff. 1999; 6: 271-280Crossref PubMed Scopus (57) Google Scholar). MCF-7 cells overexpressing human Bcl-2 protein were obtained as described previously (16Ruiz-Ruiz C. Muñoz-Pinedo C. López-Rivas A. Cancer Res. 2000; 60: 5673-5680PubMed Google Scholar). In some experiments, MCF-7 cells were treated with 500 ng/ml doxorubicin (Sigma Immunochemicals) for the indicated times. Plasmids—Several putative p53 responsive elements are found within the promoter and first intron of the human CD95 gene. The nucleotide sequences and position relative to the ATG start codon of these sites are: p53RE1 (AGACAAGCCT.... ctACAAGaCT, –1329/–1302), p53RE2 (GcGCAAGagT.AcACAgGTgT, –220/–200) and p53RE3 (AGGCcAGCCCtGGCTgcCCa, –110/–91) in the promoter region, and p53iRE (GGACAAGCCCtGACAAGCCa, +433/+452) in the first intron of the CD95 gene. Variant nucleotides compared with the perfect p53 consensus sequence are shown in lowercase letters. A 1448-bp fragment upstream from the ATG start codon of the CD95 gene (numbering according to Ref. 37Rudert F. Visser E. Forbes L. Lindridge E. Wang Y. Watson J. DNA Cell Biol. 1995; 14: 931-937Crossref PubMed Scopus (54) Google Scholar), was generated by PCR amplification with KlenTaq-LA polymerase Mix (Clontech laboratories, CA) and using the following primers: sense 5′-GGGGGATCCATACCATCCTCCTTAT-3′ and antisense 5′-GGGAGATCTGGTTGTTGAGCAATCCTCCGAA-3′. Genomic DNA obtained from Jurkat cells, was used as template. The fragment was purified and subcloned into the BglII site of the promoterless pXP2 luciferase reporter plasmid to generate the pCD95 1448Luc construct. Similarly, the pCD95 391Luc and pCD95 170Luc plasmids were generated by cloning into pXP2 the corresponding fragments amplified with the primers: sense 5′-GGGGGATCCGTGCAACGAACCCTGACTC-3′ and sense 5′-GGGGGATCCCGGTTTACGAGTGACTTGGCTGGA-3′, respectively, together with the antisense primer described above. Constructs pI-CD95 1448Luc, pI-CD95 391Luc and pI-CD95 170Luc were generated by subcloning a 500-bp region from the first intron of the CD95 gene into the BamHI site of the pXP2 polylinker, upstream from the promoter region of the previously described plasmids, pCD95 1448Luc, pCD95 391Luc and pCD95 170Luc, respectively. The 500-bp intronic fragment was also amplified by PCR from Jurkat genomic DNA using the primers: sense 5′-CGGGATCCGTGAGCCCTCTCCTGCCCGGGTG-3′, and antisense 5′-CGGGATCCCTGAAGGCTGCAGGCTCTCTCC-3′. The pC53-SN3 (wt p53) and pC53-SCX3 (143Ala mutant p53) plasmids were generously provided by Dr. M. Oren. The pRL-CMV plasmid was from Promega (Madison, WI). Mutant versions of the luciferase reporter plasmids and the p53 expression vector were prepared by QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using a Pfu Turbo DNA polymerase and two oligonucleotide primers, each complementary to opposite strands of the vector (template) and containing the desired mutation. To generate the mutants of pI-CD95 391Luc with substitutions within either the described intronic p53-binding element (18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar) (pmI-CD95 391Luc) or the putative promoter p53-responsive elements RE2 and RE3 (pI-CD95 m391Luc), the following primers were used, together with their respective complementary antisense oligonucleotides: 5′-AACTCCTGGAGGGGCCCTGACAAG-3′ and 5′-CCCTGACAATAAAAGCCAAAGGT-3′ for mutation of intronic p53-RE; 5′-CTGATCTCGCGGGGGAGTGACACACA-3′ and 5′-AGTGACACAAGGTTGTTCAAAGACG-3′ for mutation of p53-RE2; 5′-ACACCCTGAGGGGGGCCCTGGCTGCC-3′ for mutation of p53-RE3. The p53 mutant versions were prepared by using the following primers and their respective complementary oligonucleotides: 5′-CGGCATGAACTGGAGGCCCATCCTC-3′ for mp53–248Trp and 5′-GCTTTGAGGTGCATGTTTGTGCCTG-3′ for mp53–273His. Mutated nucleotides are underlined in the primers described above. The final products of all reactions were sequenced to confirm the introduction of the expected mutations. Cell Transfections and Luciferase Assays—Cells were plated at a density of either 2 × 105 (MCF-7 cells) or 105 (H1299 cells) per 35-mm tissue culture dish the day before transfection with FuGENE 6 reagent (Roche Applied Science, Mannheim, Germany) according to the manufacturer′s instructions. Cells were co-transfected with 0.5 μg of the luciferase reporter plasmids described above and 1 ng of the Renilla expression plasmid pRL-CMV as an internal control for transfection efficiency. Where indicated, the p53 expression plasmids or the pCMVneo vector were also co-transfected in a ratio of 1:1 (MCF-7 cells) or 1:5 (H1299 cells) versus the luciferase reporter plasmid. For luciferase assays, cells were detached with trypsin/EDTA, washed with PBS and lysed before determining luciferase activity according to the manufacturer (Dual-Luciferase Reporter Assay System, Promega) in a FB12 luminometer (Berthold Detection Systems, Germany). All transfection experiments were carried out in duplicate. Electrophoretic Mobility Shift Assay (EMSA)—For nuclear protein extractions, MCF-7 growing in 100-mm plates were treated with 500 ng/ml doxorubicin. Cells were lysed in 400 μl of hypotonic buffer (10 mm HEPES pH7.6, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm Na2MoO4, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 0,75 mm spermidine, and 0.15 mm spermine), containing 0.6% Nonidet P-40. Nuclei were then centrifuged and incubated with buffer C (20 mm HEPES pH 7.6, 400 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm Na2MoO4, and 2 μg/ml each pepstatin, leupeptin, and aprotinin) for 30 min in a rocking platform. Nuclei were centrifuged at 15,000 × g for 10 min, and the supernatants containing the nuclear extracts were immediately stored at –80 °C. The protein concentration was quantified by the Bradford procedure. EMSA was performed as described (38Lorenzo E. Ruiz-Ruiz C. Quesada A.J. Hernandez G. Rodriguez A. Lopez-Rivas A. Redondo J.M. J. Biol. Chem. 2002; 277: 10883-10892Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Nuclear proteins (3 μg) were incubated with 1.5 μg of poly(dI-dC) DNA carrier and 4 μl of 5× DNA binding buffer (DBD) (10% polyvinylethanol, 12,5% (v/v) glycerol, 50 mm Tris (pH 8), 2.5 mm EDTA, 2.5 mm dithiothreitol) in a final volume of 20 μl on ice for 10 min. Then, 2 μl (1 ng/μl) of 32P-labeled double-stranded oligonucleotide (40 × 106 cpm/μg) was added to the reaction mixture, and it was incubated at room temperature for 30 min. Where indicated, 3 μl of the anti-p53 antiserum pAb421, or 3 μl of conditioned medium from X63 myeloma cell line as control antibody, were added to the cellular extract before addition of the probe. The sequence of the oligonucleotide probe (5′-gatcCTCCTGGACAAGCCCTGACAAGCCAAGCCA-3′) has been described previously (38Lorenzo E. Ruiz-Ruiz C. Quesada A.J. Hernandez G. Rodriguez A. Lopez-Rivas A. Redondo J.M. J. Biol. Chem. 2002; 277: 10883-10892Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and corresponds to the p53 consensus sequence located within of the intronic enhancer of the human CD95 gen (39Müller 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 (734) Google Scholar). Chromatin Immunoprecipitation (ChIP)—ChIP was performed as previously described (40Weinmann A.S. Farnham P.J. Methods. 2002; 26: 37-47Crossref PubMed Scopus (302) Google Scholar) with some modifications. 2C. Gallego, personal communication. Briefly, MCF-7 cells (107) were incubated in 1% formaldehyde solution in PBS for 10 min at room temperature. Cross-linking was stopped by the addition of glycine to a final concentration of 0.125 m for 5 min, and monolayers were washed twice with PBS. Cells were scraped off following addition of 0.05% trypsin solution in PBS, washed once with PBS plus phenylmethylsulfonyl fluoride and incubated in lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 1% SDS) plus a protease inhibitor mixture, for 20 min on ice. Cell lysates were then sonicated to yield chromatin fragments of 500–1000 bp. After centrifugation for 30 min at 13,000 × g, supernatants were collected and diluted 1:10 in IP dilution buffer (16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl, 1.2 mm EDTA, 1.1% Triton X-100, 0.01% SDS) plus protease inhibitor mixture and precleared with protein A-Sepharose beads for 4 h with rocking at 4 °C. Immunoprecipitation was performed by rocking overnight at 4 °C with 5 μg of monoclonal antibody anti-p53 (Pab421, Oncogene Research Products). Antibody/protein/DNA complexes were collected with protein A-Sepharose beads, previously blocked with 1 μg/ml salmon sperm DNA and 1 μg/ml bovine serum albumin overnight at 4 °C, by rocking for 5 h at 4 °C and then washed once with 50 mm Tris-HCl, pH 8.0, 2 mm EDTA, twice with IP wash buffer (100 mm Tris-HCl pH 8.0, 500 mm LiCl, 1% Nonidet P-40, 1% sodium deoxycholate) and once with TE buffer. Precipitates were extracted twice with 50 μl of lysis buffer for 10 min at room temperature after which elutions were pooled, diluted 1:10 in IP dilution buffer, and immunoprecipitated as above. Precipitates were then extracted twice with 100 μl of elution buffer (50 mm NaHCO3, 1% SDS) by shaking on vortex for 20 min. Both elutions were combined, and 0.3 m NaCl and 10 μg of RNase A were added, and incubated overnight at 67 °C to reverse formaldehyde cross-linking. Samples were then treated with proteinase K for 1–2 h at 45 °C, extracted with phenol/chloroform/isoamyl alcohol, precipitated at –20 °C overnight with ethanol in the presence of glycogen and tRNA as carriers, and finally resuspended in 30 μl of water. For PCR amplification, 3-μl samples were used as template and 35 PCR cycles, each consisting of 1 min at 95 °C, 1 min at 65 °C, and 1 min at 72 °C, were performed by using the following primers: sense 5′-AACGCTGGAGGACTTGCTTT-3′and antisense 5′-CACCCGCGCCGGAGCGGACCTTTG-3′, which encompass the p53 responsive element within the first intron of the human CD95 gene. Northern Blot—Total RNA (20 μg) was run on 1% agarose/formaldehyde gel and transferred to nylon membranes (Hybond-N, Amersham Biosciences). Membranes were hybridized to a DNA probe for CD95 labeled with [α-32P]dCTP (Amersham Biosciences), using a random primer labeling kit (Roche Applied Science). The 0.7 kb XhoI-BamHI DNA fragment of the pBLF58-1 plasmid (41Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S.I. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Abstract Full Text PDF PubMed Scopus (2678) Google Scholar) was used as probe. Immunoblot Detection of Proteins—Following detachment with RPMI/EDTA, MCF-7 cells (5 × 105) were washed with PBS and lysed in 20 μl of Laemmli buffer. Cell samples were sonicated, and proteins were resolved on SDS-polyacrylamide minigels and detected as described previously (14Ruiz-Ruiz M.C. López-Rivas A. Cell Death Diff. 1999; 6: 271-280Crossref PubMed Scopus (57) Google Scholar). Blots were probed with mouse anti-human Bcl-2 mAb (DAKO, Denmark), anti-p53 sheep polyclonal IgG Ab (Oncogene Research Products, Cambridge, MA) and rabbit anti-CD95 polyclonal Ab (C-20, Santa Cruz Biotechnology). Determination of Apoptotic Cells—Phosphatydilserine (PS) exposure on the surface of apoptotic cells was detected by flow cytometry after staining with Anexin-V-FLUOS (Roche Applied Science). Flow cytometry was performed on a FACScan cytometer using the Cell Quest software (BD Biosciences, Mountain View, CA). Characterization of Genotoxic Stress-induced p53-mediated Up-regulation of CD95 Gene Expression in Human Breast Tumor Cells—Induction of a p53 response upon DNA damage leads to the up-regulation of several apoptotic proteins (29Miyashita T. Krajewski S. Krajewska M. Wang H.G. Lin H.K. Liebermann D.A. Hoffman B. Reed J.C. Oncogene. 1994; 9: 1799-1805PubMed Google Scholar, 42Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Muller H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (533) Google Scholar, 43Wu L. Levine A.J. Mol. Med. 1997; 3: 441-451Crossref PubMed Google Scholar). Among these, death receptor CD95 (Fas/APO-1) has been shown to be elevated in a number of tumor cells following genotoxic insults (15Sheard M.A. Vojtesek B. Janakova L. Kovarik J. Zaloudik J. Int. J. Cancer. 1997; 73: 757-762Crossref PubMed Scopus (119) Google Scholar, 18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar). In breast tumor cells, we recently reported that different DNA damaging treatments up-regulated the expression of CD95 protein in a p53-dependent manner (14Ruiz-Ruiz M.C. López-Rivas A. Cell Death Diff. 1999; 6: 271-280Crossref PubMed Scopus (57) Google Scholar). In Fig. 1A, we show that p53-dependent up-regulation of CD95 is also observed at the mRNA level in breast tumor MCF-7 cells. Treatment of these cells with the DNA-damaging drug doxorubicin up-regulated CD95 mRNA expression (Fig. 1A). However, doxorubicin-induced CD95 expression was completely abrogated in MCF-7 cells expressing the human papillomavirus E6 protein (14Ruiz-Ruiz M.C. López-Rivas A. Cell Death Diff. 1999; 6: 271-280Crossref PubMed Scopus (57) Google Scholar), that drives p53 to the proteasome-mediated degradation pathway. It has been suggested that p53 consensus sites in the region 5′ upstream of the ATG start codon may cooperate with a p53 intronic element in the regulation of CD95 expression in hepatoma cells (18Müller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (719) Google Scholar), although this hypothesis has not been demonstrated. In this report we have addressed this question by analyzing the relevance of putative p53 sites within the CD95 promoter
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