Cancer Therapy: Preclinical

Radiation and Transforming -B Cooperate inTranscriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene Jurre Hageman,1Bart J. Eggen,3 Tom Rozema,1, 2 Kevin Damman,1 Harm H. Kampinga,1and Robert P. Coppes1, 2

Abstract Radiation-induced is an important side effect in the treatment of cancer. Profibrotic pro- teins, such as plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-h (TGF-h), and tissue type inhibitor of metalloproteinases-1 (Timp-1), are thought to play major roles in the development of fibrosis via the modulation of integrity.We did a detailed anal- ysis of transcriptional activation of these profibrotic genes by radiation and TGF-h. Irradiation of HepG2 cells led to a high increase in PAI-1mRNA levels and a mild increase in Timp-1mRNA lev- els. In contrast,TGF-h1and Smad7 were not increased. Radiation and TGF-h showed strong co- operative effects in transcription of the PAI-1 gene. The TGF-b1 gene showed a mild cooperative activation, whereas Timp-1and Smad7 were not cooperatively activated by radiation and TGF-h. Analysis using the proximal 800 bp of the human PAI-1promoter revealed a dose-dependent increase of PAI-1levels between 2 and 32 Gy g-rays that was independent of latent TGF-h acti- vation. Subsequent site-directed mutagenesis of the PAI-1promoter revealed that mutation of a -binding element abolished radiation-induced PAI-1 transcription. In line with this, PAI-1 was not activated in p53-null Hep3B cells, indicating that p53 underlies the radiation-induced PAI-1activation and the cooperativity with theTGF-h/Smad pathway. Together, these data show that radiation and TGF-h activate PAI-1 via partially nonoverlapping signaling cascades that in concert synergize on PAI-1 transcription. This may play a role in patient-to-patient variations in susceptibility toward fibrosis after radiotherapy.

Radiation induced fibrosis, an important side effect of cancer complex and tight regulation of ECM degradation. Dysregula- treatment, is characterized by a progressive and excessive tion of factors implicated in these systems may result in an accumulation of extracellular matrix (ECM; ref. 1), which alteration of degrading capacity (5, 6). To date, many different may eventually severely compromise tissue function. Factors cytokines have been identified that regulate the transcription that play a role in remodeling of the ECM include structural of genes implicated in the regulation of the ECM integrity. Of ECM , inhibitors of ECM breakdown, and cytokines these, transforming growth factor-h1 (TGF-h1) has been shown influencing transcription of profibrotic genes (2). Two main to be a potent transcriptional activator of ECM regulators, such degrading extracellular protease systems play a crucial role: the as PAI-1 and tissue type inhibitor of metalloproteinases-1 plasminogen-activating system with plasminogen activator (Timp-1; refs. 7–9). TGF-h is ubiquitously expressed and inhibitor-1 (PAI-1) as negative regulator and the matrix secreted as a latent complex in the connective tissues (10). metalloproteinase system with tissue type inhibitors of metal- Upon activation, the TGF-h1 homodimer binds to a cell surface loproteinases (3, 4). Together, these two systems form a highly complex that activates cytosolic Smads that translocate to the nucleus, bind DNA, and facilitate gene-specific transcription (11). Optimal binding is achieved with the four-nucleotide sequence AGAC or its reverse complement 1 Authors’ Affiliations: Departments of Radiation and Stress Cell Biology and GTCT (12, 13). Smads have a relatively weak affinity for DNA 2Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 3Department of Developmental Genetics, (13) and use other transcription factors to form a robust Biological Center, Haren, the Netherlands complex with high affinity and specificity to cognate DNA Received 2/25/05; revised 5/19/05; accepted 5/20/05. sequences in regulatory regions of genes. These include many Grant support: Interuniversitair Instituut voor Radiopathologie en Stralen- profibrotic genes, such as Col1a2, PAI-1, and the TGF-b1 gene bescherming grant IRS 9.0.18and University of Groningen. itself (14, 15). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance Several studies show that radiation can induce transcription with18U.S.C. Section1734 solely to indicate this fact. of some profibrotic genes consistent with its induction of Requests for reprints: Robert P. Coppes, Department of Radiation and Stress fibrosis. However, the transcriptional activation of most of Cell Biology, University Medical Center Groningen, University of Groningen, 5th these genes by radiation is often only weak and conflicting Floor, Building 3215, Ant. Deusinglaan 1, 9713 AV Groningen, the Netherlands. Phone: 31-50-3632709; Fax: 31-50-3632913; E-mail: r.p.coppes @med.umcg.nl. reports on activation of some of these genes exist in the F 2005 American Association for Cancer Research. literature (16–18). Moreover, the molecular mechanism doi:10.1158/1078-0432.CCR-05-0427 underlying activation of profibrotic genes by radiation remains

Clin Cancer Res 2005;11(16) August 15, 2005 5956 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Radiation and TGF-b Cooperate in PAI-1 Transcription to be elucidated. Interestingly, studies done by Barcellos-Hoff previously described (27). Site-directed mutagenesis was done using et al. (19) showed a rapid activation of latent TGF-h by the QuickChange Site-Directed mutagenesis kit (Stratagene, La Jolla, radiation-induced reactive oxygen species in mouse mammary CA) according to the protocol of the manufacturer by using the PAI- glands. This implies that radiation-induced profibrotic gene Luc plasmid as a template unless otherwise specified. The following oligonucleotides (Isogen, Maarssen, The Netherlands) were used: AP1- transcription might be mediated via the TGF-h/Smad signaling 1-for: CAGACAAGGTTGTTGCAGCAAGAGAGCCCTCAG, AP1-1-rev: pathway. It seems, however, likely that other transcription CTGAGGGCTCTCTTGCTGCAACAACCTTGTCTG; AP1-2-for: CTGGAC- factors need to be activated by radiation concurrently to explain ACGTGGGGACACAGCCGTGTATCATC, AP1-2-rev: GATGATACACGG- the strong profibrotic effects. Indeed, radiation can induce CTGTGTCCCCACGTGTCCAG; AP1-3-for: GAGTCAGCCGTGCAGCAT- the activation of a subset of transcription factors, such as p53, CGGAGGCGGCC, AP1-3-rev: GGCCGCCTCCGATGCTGCACGGCTG- AP-1, NF-nB, SP1, EGR-1, and Oct-1 (20). Interestingly, bind- ACTC; AP1-3-for2: GGACACAGCCGTGCAGCATCGGAGGCGG (tem- ing sites of some of these transcription-factors are reported in plate AP1-2 mutated), AP1-3-rev2: CCGCCTCCGATGCTGCACGGCTG- a number of profibrotic genes, including Timp-1, TGF-b1, and TGTCC (template AP1-2 mutated); AP1-4-for: GGGTGGGGCTGGAA- PAI-1 (7, 9, 17, 21). CATGCAAACATCTATTTCCTGCCCACATC, AP1-4-rev: GATGTGGGCA- The PAI-1 gene is rapidly activated by radiation (22, 23). GGAAATAGATGTTTGCATGTTCCAGCCCCACCC; p53-for: CACACAT- However, (s) conferring the radiation GCCTCAGAAATTCCCAGAGAGGGAGGT, p53-rev: ACCTCCCTCTCT- GGGAATTTCTGAGGCATGTGTG. All mutations were sequence verified response have not been identified. Several putative binding (Base-Clear, Leiden, the Netherlands). AP1-1,2 mutated was constructed sites of radiation-responsive transcription factors, such as SP1, using AP1-1 mutated as a template. AP1-1,2,3 mutated was constructed n AP1, NF- B, and p53 (7, 21, 24, 25), are present in this using AP1-1,2 as a template with primers AP1-3-for2 and AP1-3-rev2 promoter as well as Smad-binding elements (SBE). The that harbored the necessary mutated AP1-2 sites due to close proximity radiation responsiveness of this gene may thus be mediated of these two AP1-sites. AP1-1,2,3,4 mutated was constructed by via activation of latent TGF-h followed by Smad-mediated transferring a SphI fragment from AP1-1,2,3 mutated to AP1-4 mutated. transcriptional activation and/or by activation of other radia- Sequence analysis was done to verify the mutations and the orientation. tion-responsive transcription factors. A p53 overexpression plasmid was constructed as follows; the p53 Here, we investigated the radiation responsiveness of a coding sequence was amplified from HepG2 cDNA using primers number of profibrotic genes. Specifically, we show that PAI-1 p53-cds-for: ACCAACAAGCTTACCATGGAGGAGCCGCAGTCAGA responded to either radiation or TGF-h alone, but impor- and p53-cds-rev: ACCAACGAATTCTCAGTCTGAGTCAGGCCCTT and cloned in the EcoRI and HindIII sites of the pCDNA3.1(+) vector tantly that radiation and TGF-h synergized in PAI-1 gene (Invitrogen). activation. The radiation-mediated induction of PAI-1 was Luciferase reporter assay. Stable clones were seeded (50,000 cells) h independent of latent TGF- activation/Smad signaling and in coated tubes in tetra-replicates in 500 AL medium. After 24 hours, the cis-element conferring the radiation response was mapped they were either sham-irradiated or irradiated using a 137Cs irradiation at the proximal 800 bp promoter part. Site-directed muta- device. Directly after irradiation, cells were left untreated or were genesis revealed the requirement of a conserved DNA element stimulated with recombinant derived active TGF-h (1 ng/mL; R&D with a p53-binding motif. In agreement with this, a related Systems, Minneapolis, MN). For a fractionated radiation regime, cells p53-null cell line showed a severely reduced radiation were irradiated for 4 subsequent days with a dose of 2 Gy. The medium response and the cooperative effect with TGF-h was com- was replaced daily before irradiation. Directly after irradiation, the h h pletely abolished, indicating that Smad/p53 cooperativity medium with or without TGF- (1 ng/mL) was added. TGF- – neutralizing antibodies (1 Ag/mL) were added 1 hour before irradiation underlies the molecular mechanism of TGF-h/radiation where indicated. Latent TGF-h was activated by adding HCl (80 mmol/L) synergy. to the medium and incubated for 1 hour at 4jC. Thereafter, the solution was neutralized by the addition of NaOH (80 mmol/L) to the Materials and Methods medium. Serum deprivation experiments were done by seeding the cells on day 1. The same day, cells were washed twice with DMEM without Cell culture and transfections. Mv1Lu (CCL-64), HepG2 (HB-8065), serum and incubated for 18 hours. Thereafter, cells were treated with and Hep3B (HB-8064) cells were obtained from the American Type irradiation and/or TGF-h as described above. In all cases, after Culture Collection (Manassas, VA) and were maintained in DMEM administration of different stimuli, the cells were incubated for another (complete medium) supplemented with 10% fetal bovine serum. Cells 24 hours and lysed. Cell lysis and luciferase activity measurements were were stably transfected using the calcium phosphate transfection done as previously described (28). Luciferase activity was corrected for method. Briefly, 10 Ag plasmid was cotransfected with 1 Ag pSV2neo cell number and plotted as relative increases compared with the to 5 Â 105 cells seeded on 9 cm plates. Twenty-four hours after (untreated) control. transfection, the medium was replaced with a complete medium Quantitative PCR. HepG2 or Hep3B cells were seeded and after containing geneticin (400 Ag/mL). Approximately 2 weeks after 24 hours they were either sham-irradiated or irradiated using a 137Cs transfection, colonies were picked, expanded, and screened for irradiation device. Three, 6, or 9 hours after treatment, total RNA was luciferase activity. Luciferase-positive clones were expanded and assayed isolated using the Absolutely RNA isolation kit (Stratagene). Subse- as described further. Results shown are representative effects of a quently, the RNA was validated using primers corresponding to minimum of at least three independent, luciferase-positive clones genomic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tested to minimize the effect of clonal variation. In all cases, a sequences for the presence of genomic DNA contamination by qualitatively similar induction pattern after radiation, TGF-h, or both regular PCR. Thereafter, 1 Ag of total RNA was transcribed in first- stimuli was found for all clones tested. Transient transfection was strand cDNA using M-MLV reverse transcriptase (Invitrogen). The done using LipofectAMINE (Invitrogen, Carlsbad, CA) according to the cDNA synthesis was primed by oligo(dT)12-18 (Invitrogen). Relative protocol of the manufacturer. changes in transcript level were determined on the Icycler (Bio-Rad, Plasmid construction and manipulations. The PAI-Luc plasmid Hercules, CA) using SYBR green supermix (Bio-Rad). Calculations were containing 800 bp of the human PAI-1 promoter was a generous gift done using the comparative CT method according to User Bulletin 2 of Prof. Loskutoff (The Scripps Research Institute, La Jolla, CA) and (Applied Biosystems, Foster City, CA). For each set of primers, the PCR was described previously (26). The SBE-Luc plasmid was also efficiency was determined. Primer sequences used in this study were as

www.aacrjournals.org 5957 Clin Cancer Res 2005;11(16) August 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical follows: GAPDH-for: TGCACCACCAACTGCTTAGC, GAPDH rev: GG- CATGGACTGTGGTCATGAG; hypoxanthine phosphoribosyltransferase (HPRT)-for: TGGCGTCGTGATTAGTGATG, HPRT-rev: GATGTAATCC- AGCAGGTCAG; PAI-1-for: GGAATGACCGACATGTTCAG, PAI-1-rev: ACTCTCGTTCACCTCGATCT; TGF-h1-for: CCGCTTCACCAGCTCCAT- GT, TGF-h1-rev: TGCTACCGCTGCTGTGGCTA; Timp-1-for: GATGC- CGCTGACATCCGGTT, Timp-1-rev: AACTCCTCGCTGCGGTTGTG; Smad7-for: CCAGGACGCTGTTGGTACAC, Smad7-rev: CGTCCACG- GCTGCTGCATAA. The PCR efficiencies for all primers used were between 89% and 96%. A derivative of the comparative CT method was used to validate reference gene alteration by irradiation or TGF-h as described elsewhere (29). Neither GAPDH nor HPRT was significantly affected (<1.3-fold induction for HPRT and <1.1-fold induction for GAPDH) by either radiation (15 Gy), TGF-h (1 ng/mL), or both stimuli. Data are expressed as fold induction corrected for GAPDH. Western blot analysis. Standard Western blot procedures were used as previously described (28). p53 was detected with the monoclonal DO-1 p53 antibody purchased from Santa Cruz Biotechnology (Santa Cruz, CA). GAPDH was detected using a monoclonal antibody purchased from RDI Research Diagnostics (Concord, CA).

Results Fig. 1. Radiation and/or TGF-h causes activation of genes implicated in ECM remodeling. Quantitative PCR analysis of PAI-1 (A), TGF-h1(B), Timp-1 (C), and Radiation and TGF-b show cooperative effects on PAI-1 and Smad7 (D) transcript levels. HepG2 cells were treated with15Gy g-radiation and/or active TGF-h (1ng/mL). Cells were harvested 3, 6, and 9 hours poststimulation. TGF-b1 promoter activation. Both radiation and TGF-h are Neither GAPDH nor HPRT responded significantly to these stimuli. mRNA levels known to activate a number of profibrotic genes. The are corrected for GAPDH. Columns, mean; bars, SE (n =2). mechanisms of radiation-induced activation, however, still remains to be resolved. We initiated the present study to siveness of PAI-1 in more detail, we transfected a luciferase investigate in detail the transcriptional activation of a number gene-reporter construct containing the major regulatory part of profibrotic genes by radiation and TGF-h.Weused of the human PAI-1 promoter (Fig. 2A) stable in Mv1Lu cells. quantitative PCR to investigate the induction of the PAI-1, Several stable clones were selected and irradiated with a dose TGF-b1, and Timp-1 genes by radiation (15 Gy) and/or TGF-h ranging from 2 to 32 Gy. Twenty-four hours after irradiation, a (1 ng/mL) in HepG2 cells. In addition, the induction of the dose-dependent increase of the PAI-1 promoter was found as putative antifibrotic gene Smad7 was examined. Total RNA was shown by a representative stable clone (Fig. 3A). Furthermore, a harvested at 3, 6, and 9 hours after stimulation and reverse radiation dose–dependent increase of the cooperative effect transcribed in first-strand cDNA. Validation of the normaliza- was found after cells were treated with both radiation (2-32 Gy) tion genes revealed that neither GAPDH nor HPRT transcript and TGF-h (1 ng/mL; Fig. 3A). Thus, similar to the quantitative levels were significantly affected by radiation, TGF-h, or both PCR data obtained in HepG2 cells, stimulation of Mv1Lu stimuli (data not shown). Subsequently, relative PAI-1, TGF-h1, PAI-Luc cells with both irradiation and TGF-h resulted in Timp-1, or Smad7 transcript levels were determined and cooperative effects on PAI-1 promoter activation, resulting in a normalized to GAPDH transcript levels. We found that both highly increased PAI-1 promoter activity. These results indicate radiation and TGF-h activated the human PAI-1 gene (Fig. 1A). that the regulatory region mediating the radiation response Interestingly, radiation and TGF-h stimulation were found to and the cooperation with TGF-h is located within the 800 bp have a cooperative effect on PAI-1 transcription. This cooper- proximal promoter part. One could argue, however, that the ative effect increased in time and resulted in a very strong relatively high irradiation conditions that were used do not increase in PAI-1 transcription after 9 hours. The TGF-b1 gene reflect the effects that may occur under clinically relevant showed very low, if any, responsiveness toward irradiation irradiation regimes (20). In these regimes, the total dose is (Fig. 1B) but a relatively mild synergistic activation in response usually delivered in a fractionated daily dose of 2 Gy. Although to radiation and TGF-h was observed (Fig. 1B). The Timp-1 gene a clear dose response was observed for the entire dose range did not respond to TGF-h and showed a weak induction by applied, a single dose of 2 Gy did not show a significant radiation (Fig. 1C). The Smad7 gene showed a TGF-h response PAI-1 promoter activation on its own (Fig. 3A). Therefore, but not a radiation response (Fig. 1D). These data show that we investigated whether fractionated doses of 2 Gy have the radiation and TGF-h cause a very strong cooperative effect on potency to activate the PAI-1 promoter and to cooperate with PAI-1 transcription. This effect was also observed for the TGF-h. Hereto, Mv1Lu PAI-Luc cells were stimulated with a TGF-b1 gene although the inductions were mild compared with daily dose of 2 Gy for 4 subsequent days. Medium with or the PAI-1 gene. Because the effects were strongest on PAI-1 without TGF-h was refreshed daily before irradiation. After 4 transcription and because this gene is clearly implemented days, we found that the PAI-1 promoter was activated and that in tissue fibrosis as shown by previous studies (30, 31), we TGF-h and irradiation cooperated under these conditions decided to concentrate on the radiation-induced transcriptional (Fig. 3B). The fractionated radiation scheme used (4Â 2 Gy) activation of this gene. in fact resulted in a stronger level of gene transcription than Radiation dose dependence and kinetics of radiation and TGF- after the corresponding 8 Gy single-dose irradiation (compare b–induced PAI-1 transcription. To study the radiation respon- Fig. 3A and B). This is likely due to the longer time allowed for

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did not block TGF-h activated in the close vicinity of target cells. To exclude this possibility, we used Mv1Lu cells stably transfected with an artificial reporter construct containing eight SBE repeats in front of a minimal promoter (Fig. 2B). The advantage of using such a reporter system is that any TGF-h activation, including activation in close vicinity of target cells, would be detected. No radiation-induced activation of the SBE promoter was found (Fig. 4B). Addition of 1 ng/mL of active TGF-h showed that the SBE promoter was induced >50-fold over basal activity, demonstrating that this construct was very responsive to TGF-h. Also, no cooperative effect was found after a combined treatment of radiation and TGF-h, indicating that the radiation effect on PAI-1 promoter acti- vation is TGF-h/Smad independent. To investigate whether serum components played a role in Fig. 2. Schematic representation of the PAI-Luc (A)andtheSBE-Luc(B) reporter radiation-induced PAI-1 activation, cells were cultured without constructs. Putative transcription factor binding sites are indicated.The PAI-Luc serum for 18 hours and subsequently treated with radiation, construct contains 800 bp of the human PAI-1promoter and the natural transcription TGF-h, or both stimuli. Radiation effects were still observed, initiation site.The SBE-Luc contains several SBEs in front of an artificial minimal promoter that initiates transcription. Arrow, luciferase coding sequence. which indicates that serum components do not contribute to the radiation effects (Fig. 4C). We were, however, still mRNA and subsequent formation in the fractionated interested whether latent TGF-h activation could be observed experiment compared with single-dose experiments (96 versus in this cell system. First, we used acid treatment to induce 24 hours) allowing accumulation of luciferase. Furthermore, dissociation of active TGF-h from the latency-associated the classic concept of split dose recovery between fractions may peptide complex of serum-containing medium (32). Medium not apply to the transcriptional activation in our experimental with serum was acidactivated, pH-neutralized, and added to set-up. In any case, our data show that the cooperative effects of radiation and TGF-h are found under clinically relevant irradiation schemes at least as efficient as after (higher) single doses of radiation. For practical reasons, however, all further experiments were done with a single dose of 15 Gy. To determine the rate of radiation and TGF-h–induced PAI-1 promoter activation, a time course luciferase reporter experi- ment was done using the PAI-Luc cell line. Cell extracts were prepared 3, 6, 12, 24, and 48 hours after treatment followed by determination of luciferase activity. Cooperative effects of radiation and TGF-h were found to be maximal between 12 and 48 hours poststimulation, where both radiation and TGF-h also individually activated the PAI-1 promoter. The cooperative effects declined at 48 hours poststimulation (Fig. 3C). All subsequent experiments were therefore done at 24 hours poststimulation. In vitro activation of the human PAI-1 promoter by radiation is independent of latent TGF-b activation. The possibility that the observed radiation-induced activation of the PAI-1 promoter is dependent on extracellular TGF-h activation and, therefore, purely Smad dependent was tested using TGF-h–neutralizing antibodies. These antibodies were added to the medium of PAI-Luc–transfected cells before irradiation or TGF-h stimula- tion to inactivate any potentially radiation-activated TGF-h. As shown in Fig. 4A, the addition of TGF-h–neutralizing antibodies completely blocked TGF-h–induced PAI-1 reporter activation but did not block the radiation-induced PAI-1 Fig. 3. A, radiation and TGF-h cooperate on PAI-Luc activation and these promoter activation. Moreover, the addition of TGF-h– effects are radiation dose dependent. Doses from 2 to 32 Gy were applied on neutralizing antibodies to cells stimulated with a combined PAI-Luc ^ containing Mv1Lu cells with or without administration of TGF-h (1ng/mL). Cell extracts were prepared 24 hours posttreatment and luciferase treatment of radiation and TGF-h resulted in a block of the activity was determined. Activity is expressed as fold above control. B, fractionated TGF-h response, as expected, leaving an activation of PAI-1 radiation doses of 2 Gy activates the PAI-1promoter and synergizes with TGF-h. PAI-Luc ^ containing Mv1Lu cells were treated with a daily dose of 2 Gy g-radiation promoter activity as found with radiation alone (Fig. 4A). Thus, for 4 subsequent days.Tothose cells that were treated with TGF-h,1ng/mLTGF-h whereas the TGF-h antibodies completely neutralized TGF-h was added daily after radiation. C, cooperation of PAI-1activation by radiation and signaling, the radiation response was not affected, indicating TGF-h is temporal. PAI-Luc ^ containing Mv1Lu cells were treated with 15 Gy h g-radiation and/or1ng/mL TGF-h. Cell extracts were prepared 3, 6, 12, 24, and that the radiation response was independent of latent TGF- 48 hours poststimulation. Luciferase activity is expressed as fold above control. activation. However, it could be possible that the antibodies Columns, mean; bars, SE (n =4).

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Fig. 4. No latent TGF-h activation upon irradiation was detected. A, administration of TGF-h neutralizing antibodies did not abrogate the radiation effect. Two micrograms of TGF-h neutralizing antibodies were added to the medium 1hour before irradiation (15 Gy) and/or TGF-h stimulation (1ng/mL). After 24 hours, the cells were lysed and assayed for luciferase activity. B, SBE-Luc ^ harboring Mv1Lu cells were treated with radiation (15 Gy) and/or TGF-h (1ng/mL); 24 hours later, cells were lysed and luciferase activity was measured. C, serum depletion did not result in an abrogation of the radiation response. PAI-Luc ^ harboring Mv1Lu cells were serum depleted for18 hours. Thereafter, they were treated with radiation (15 Gy) and/or TGF-h (1ng/mL). D and E, the serum contains large amounts of latent TGF-h. Medium was acid activated, neutralized, and placed on PAI-Luc ^ harboring Mv1Lu(D)or SBE-Luc ^ harboring Mv1lu (E)cells.After 24 hours, the cells were lysed and luciferase activity was measured. Two micrograms per milliliter of TGF-h ^ neutralizing antibodies were added where indicated. F and G, irradiation of the serum-containing medium with a dose up to 50 Gy did not result in detectable latent TGF-h activation. Medium was irradiated, placed on PAI-Luc ^ harboring Mv1Lu(F)or SBE-Luc ^ harboring Mv1Lucells (G)and luciferase activity was assayed 24 hours postirradiation. Columns, mean; bars, SE (n = 4). Note that irradiation was conducted directly on cells in medium in (A), (B), and (C), whereas in (F)and(G) serum-containing medium without cells was irradiated and subsequently placed on PAI-Luc ^ or SBE-Luc ^ harboring cells.

PAI-Luc Mv1Lu cells. After 24 hours, we found that the acid- another signaling route must be involved in this activation activated serum induced PAI-1 promoter activity (Fig. 4D). This process. Therefore, we searched the literature for known binding effect could be blocked by TGF-h–neutralizing antibodies, sites of radiation-activated/induced transcription factors in the which confirmed that the acid-activated compound that proximal 800 bp of the human PAI-1 promoter. We found that activated the PAI-1 promoter was indeed TGF-h (Fig. 4D). AP-1, SP1, and p53 are all involved in PAI-1 activation. Similar data were obtained when we added acid-activated Furthermore, they can be activated, induced, or stabilized by medium to SBE-Luc–transfected cells (Fig. 4E). No activation radiation and have experimentally verified binding sites in the effects were observed after treating medium without serum proximal part of the human PAI-1 promoter (17, 20, 21, 25). with acid (data not shown). From this, we concluded that the Mithramycin, an inhibitor of SP1 binding, did not block the serum contains high amounts of latent TGF-h. In the next radiation response that makes it unlikely that this transcription experiments, we investigated whether this latent TGF-h from factor is involved (data not shown). The proximal part of the serum can be activated by the applied irradiation doses. Serum- PAI-1 promoter contains three AP1-like sites located at positions containing medium without cells was irradiated and added À716, À670, and À659 bp from transcriptional start albeit none directly to the PAI-Luc–harboring Mv1Lu cells. Despite the of them matches the exact consensus site (7). Furthermore, a presence of latent TGF-h in the serum, irradiation of the CRE-like element is positioned at À58 (Fig. 5A; ref. 33). CRE medium without cells with doses up to 50 Gy did not lead to elements are 8 bp in length in contrast to the 7 bp AP-1–like any detectable PAI-1 promoter activation (Fig. 4F) or SBE-Luc sites and are preferentially bound by heterodimers composed of activation (Fig. 4G). Jun and activating transcription factor members (34). We mut- Altogether, these data show that PAI-1 promoter activation ated each AP-1 site according to previous literature (Fig. 5B). by radiation in our in vitro –cultured cells takes place Mv1Lu cells were transfected stably with the respective mutated independently of extracellular latent TGF-h activation. Despite plasmids whereafter a minimum of three independent clones the presence of latent TGF-h in the medium, irradiation up to were tested. The results of representative clones are shown in Fig. 50 Gy did not result in any detectable activation. This implies 5C. Although the magnitude of the responses was variable, that the radiation-mediated PAI-1 activation is dependent on probably due to clonal variations, the patterns were similar and another route than the TGF-h/Smad showed that mutation of individual AP1/CRE-like sites or the signaling pathway and other factors than the Smad family of combined mutation of all four sites did not abolish the transcriptional activators. radiation effect or its cooperative effect with TGF-h (Fig. 5C). A p53-binding element is involved in the radiation-induced A p53-binding element was shown to be involved in PAI-1 activation of PAI-1. The finding that radiation activates the transcription induced by the DNA-damaging agent N-methyl- PAI-1 promoter in a Smad-independent manner indicates that NV-nitro-N-nitrosoguanidine (25). Binding of p53 to this

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Fig. 5. p53 is required for radiation induced PAI-1activation.A,schematic representation of binding positions of different transcription factors (not to scale). B, site-directed mutagenesis of the PAI-1 promoter. Introduced mutations are underlined. Positions are indicated relative to the transcriptional start site. C, luciferase analysis of Mv1Lucells, stably transfected with the mutated constructs according to (B). Induction patterns displayed are of a representative clone of at least three independent investigated clones. Luciferase activities were measured 24 hours poststimulation. Columns, mean; bars, SE (n =4).

element has been shown in two studies (25, 35) using gel-shift Hep3B cells. Furthermore, no synergistic effect on PAI-1 induc- assays. Therefore, we disrupted this p53-like element according tion by a treatment of both irradiation and TGF-h was observed to these reports (two-nucleotide substitution; Fig. 5B; refs. (Fig. 6B). We next complemented the Hep3B cells with wild- 25, 35). Mutation of the p53 element completely abolished the type p53. This experiment was technically hampered by the fact irradiation response as well as the synergy observed with that p53 expression is toxic for Hep3B cells leading to massive simultaneous TGF-h treatment (MUT E; Fig. 5C). To further apoptosis at 48 hours (37). Therefore, stable expression cell eliminate the risk of clonal effects, we analyzed pooled lines could not be generated and fluorescence-activated cell transfected clones. No radiation effect was detected for the sorting isolation of green fluorescent protein cotransfected PAI-Luc construct with a mutated p53-binding element in this cells did not result in a viable fraction. Nevertheless, transient pooled population, not even after using a dose as high as 30 Gy expression of wild-type p53 in Hep3B cells partially restored (data not shown). PAI-1 mRNA induction by either radiation or both TGF-h and It is interesting to see that the induction by TGF-h was reduced radiation (Fig. 6C). Fluorescence-activated cell sorting analysis but not abolished (MUT E; Fig. 5C). The latter suggests that of cotransfected green fluorescent protein showed that 5% of p53 is needed as coactivator of Smads in TGF-h–induced PAI-1 the cells were transfected (data not shown). According to transcription, which is in agreement with a previous report (36). Western blot analysis, this fraction contained a high amount of p53 is required for radiation-induced PAI-1 activation. Be- p53 (Fig. 6A). Summarizing these data, we show that p53-null cause the p53-binding site was essential for radiation-induced cells show an impaired response to PAI-1 transcription by PAI-1 transactivation, we tested whether p53 was required for either radiation, TGF-h, or both stimuli and introducing p53 the radiation-induced PAI-1 induction. First, we tried knock- in a fraction of these cells partially restored these responses. down of p53 in HepG2 cells by small interfering RNA. In agreement with the mutagenesis data, these results suggest Although we found a f70% knockdown of p53 protein levels, that p53 is involved in both the radiation-induced transcrip- it did not result in any detectable change on PAI-1 transcript tion of the human PAI-1 gene as well as the cooperation with induction by radiation, TGF-h, or both stimuli (data not the TGF-h/Smad signaling pathway. shown). We reasoned that 70% knockdown might not be enough and that activation of the remaining 30% by radiation Discussion might be sufficient to confer a full radiation response. Therefore, we analyzed PAI-1 induction in Hep3B cells, a Radiation is known to activate gene transcription of several human hepatoma p53-deficient cell line closely related to profibrotic genes. To date, molecular mechanisms underlying HepG2 (Fig. 6A; ref. 37). As shown in Fig. 6B, quantitative PCR this phenomenon remain largely unknown. In this study, we analysis indicated that the induction of PAI-1 by radiation was investigated the induction of transcription of a small set of completely abrogated and by TGF-h was severely reduced in genes that have been implicated in fibrotic disease. Of these,

www.aacrjournals.org 5961 Clin Cancer Res 2005;11(16) August 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical we found that the PAI-1 gene shows a rapid and dose- Radiation-induced activation of PAI-1 is unrelated to activa- dependent activation by radiation. Importantly, we found tion of latent TGF-b. A possible mechanism for radiation- that costimulation with TGF-b resulted in a strong cooper- induced activation of PAI-1 involves the activation of latent ative effect on PAI-1 transcription. The same results, although TGF-h by radiation followed by receptor-mediated activation less dramatic, were found for the TGF-b1 gene. These data of Smad signaling. Using immunohistochemical analysis with may have important implications for understanding the an antibody against active TGF-h, Barcellos-Hoff et al. (19) development of radiation-induced tissue fibrosis because showed that radiation (5 Gy) could activate latent TGF-h in situ. several studies provided direct evidence that PAI-1 plays a However, they also found that activation of recombinant pivotal role in the pathogenesis of tissue fibrosis (5). The derived latent TGF-h in vitro was inefficient and required observation that PAI-1 knockout mice are significantly strikingly high doses of 50 to 200 Gy (39). Here, we found that protected from the development of bleomycin-induced lung latent TGF-h indeed is present in the serum, but this could not fibrosis stresses the clinical importance of this gene in the be activated with radiation doses as high as 50 Gy. Therefore, development of fibrotic disease (30, 31). In addition, PAI-1 radiation-induced PAI-1 activation does not require latent TGF- knockout mice showed a rapid removal of a fibrin-rich h activation. However, our data do not exclude the possibility matrix compared with wild-type mice, resulting in reduced that activation of latent TGF-h may differ in vitro from that fibrotic tissue accumulation (38). This observation implies in tissues. that PAI-1 is particularly important at the onset of fibrotic Radiation-induced activation of PAI-1 and synergy with TGF- disease, and indicates that it may be an important target for b/Smad signaling is p53 mediated. Despite the lack of latent future therapeutic approaches. In line with this, we found TGF-h activation in serum, we found that radiation up-regulates that the PAI-1 gene is rapidly and temporarily activated by PAI-1 transcription. The lack of an effect on a reporter construct either radiation or TGF-h. Therefore, the rapid and dose- containing SBE elements, the absence of an effect of anti–TGF- dependent PAI-1 induction might be of therapeutic value in h as well as the lack of a radiation effect on the TGF-h– the prevention of radiation side effects and stresses the need responsive Smad7 gene clearly showed that another signal to elucidate the molecular mechanism underlying the trans- transduction route must be involved in mediating this event. criptional synergy by radiation and TGF-h. Mutation of a p53-binding element completely abolished the

Fig. 6. p53 is involved in radiation-induced PAI-1transcription.A,Westernblot analysis of p53 in HepG2-, Hep3B-, and p53-transfected Hep3B cells. B, quantitative PCR analysis of PAI-1levels in HepG2 (p53-wild type) or Hep3B (p53-null). Cells were treated with irradiation (15 Gy) and/or activeTGF-h (1ng/mL) as indicated. C, transfection of transient p53 expression partially restored the radiation effect in Hep3B cells. PAI-1 transcript levels were analyzed using quantitative PCR. Columns, mean; bars, SE (n =2).

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radiation effect on PAI-1 transcription. Cordenonsi et al. (36) which circulating levels of latent TGF-h (produced by tumors) were the first to show that Smads and p53 cooperate on PAI-1 were linked to a higher risk of radiation-induced fibrosis. Our transcription. They found that knockdown of p53 abrogated data imply that, if indeed radiation would activate this latent the induction of PAI-1 by the TGF-h family member activin. TGF-h in situ (of which the molecular mechanism remains This is consistent with our observation that induction of PAI-1 unclear) and simultaneously activate a synergizing other by TGF-h was severely reduced by mutating the p53-binding pathway, patients bearing such a tumor may be predisposed element of the PAI-1 promoter and in p53-null cells. Moreover, for developing radiation fibrosis. It needs to be realized, we observed that radiation effects were severely reduced in the however, that whereas some clinical studies looking at plasma Hep3B cell line and that the cooperative effects were completely TGF-h levels indeed have suggested such a predisposition abrogated. Complementation of these cells with p53 partially (46, 47), others have not confirmed this (48, 49). However, restored the radiation effects with respect to PAI-1 transcription. measuring overall plasma TGF-h levels may not be accurate These results all strongly suggest that p53 is involved in enough for this purpose and local concentrations in the tissue conferring the radiation-induced activation of the human surrounding the tumor and within the radiation field may PAI-1 promoter. be more relevant. Furthermore, promoter polymorphisms Implications for radiation-induced fibrosis. Transcriptional (e.g., in the PAI-1 promoter) have been described in a number synergy is a relatively common phenomenon in eukaryotes of profibrotic genes (50) and it would be interesting to (40). It enables the organism to adapt quickly to changes in the investigate if polymorphisms exist that alters the response to environment. Interestingly, transcriptional cooperation of either radiation or TGF-h on a patient-to-patient basis (50). Smads and other transcription factors have been reported in Therefore, it is necessary to know the individual variations in previous studies (15, 21, 36, 41–44) and the cooperation with responsiveness of profibrotic genes to TGF-h and/or radiation p53, in particular, has been recently reviewed (45). To our next to the local concentrations of (tumor-produced) TGF-h to knowledge, we are the first to show that radiation and TGF-h, be able to develop reliable predictive tests for the chance of both known to activate a number of profibrotic genes patients to develop radiation-induced fibrosis. individually, cooperate in activation of PAI-1 transcription in vitro. Yet, future in vivo experiments are warranted to confirm Acknowledgments our findings. If extrapolated to the in vivo situation, our data would We thank Prof. Loskutoff (The Scripps Research Institute, La Jolla, CA) for support correlative evidence from some clinical studies in providing us with the PAI-Luc construct.

References 1. Morgan GW, Breit SN. Radiation and the lung: a independent pathways in TGF-h family signalling. forming growth factor-h-induced physical and func- reevaluation of the mechanisms mediating pulmonary Nature 2003;425:577 ^ 84. tional interactions between smads and Sp1. J Biol injury. Int JRadiat Oncol Biol Phys 1995;31:361 ^ 9. 12. ZawelL,DaiJL,BuckhaultsP,etal.HumanSmad3 Chem 2000;275:40014^9. 2. Rodemann HP, Bamberg M. Cellular basis of radi- and Smad4 are sequence-specific transcription acti- 22. Zhao W, O’Malley Y, Wei S, Robbins ME. Irradiation ation-induced fibrosis. Radiother Oncol 1995;35: vators. Mol Cell 1998;1:611^ 7. of rat tubule epithelial cells alters the expression 83^90. 13. Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, of gene products associated with the synthesis and 3. Dobrovolsky AB,Titaeva EV. The fibrinolysis system: Pavletich NP. Crystal structure of a Smad MH1domain degradation of extracellular matrix. Int J Radiat Biol regulation of activity and physiologic functions of its bound to DNA: insights on DNA binding in TGF-h 2000;76:391^402. main components. Biochemistry (Mosc) 2002;67: signaling. Cell1998;94:585^94. 23. Zhao W, Spitz DR, Oberley LW, Robbins ME. Redox 99^108. 14. Zhang W, Ou J, Inagaki Y, Greenwel P, Ramirez F. modulation of the pro-fibrogenic mediator plasmino- 4. Lijnen HR. Matrix metalloproteinases and cellular Synergistic cooperation between Sp1 and Smad3/ gen activator inhibitor-1 following ionizing radiation. fibrinolytic activity. Biochemistry (Mosc) 2002;67: Smad4 mediates transforming growth factor h1stim- Cancer Res 2001;61:5537 ^ 43. 92 ^ 8. ulation of a 2(I)-collagen (COL1A2) transcription. 24. HouB,ErenM,PainterCA,etal.TNFa activates the 5. Loskutoff DJ, Quigley JP. PAI-1, fibrosis, and the J Biol Chem 2000;275:39237 ^ 45. human plasminogen activator inhibitor-1 through elusive provisional fibrin matrix. J Clin Invest 2000; 15. Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 a distal NFnB site. JBiol Chem 2004;279:18127^36. 10 6:14 41 ^ 3. cooperate with c-Jun/c-Fos to mediate TGF-h- 25. Parra M, Jardi M, Koziczak M, NagamineY, Munoz- 6. Martin M, Lefaix J, Delanian S. TGF-h1andradia- induced transcription. Nature 1998;394:909 ^ 13. Canoves P. p53 phosphorylation at serine 15 is re- tion fibrosis: a master switch and a specific thera- 16. Gault N,Vozenin-Brotons MC, Calenda A, Lefaix JL, quired for transcriptional induction of the plasminogen peutic target? Int J Radiat Oncol Biol Phys 2000; Martin MT. Promoter sequences involved in trans- activator inhibitor-1 (PAI-1) gene by the alkylating 47:277 ^ 90. forming growth factor b1 gene induction in HaCaTker- agent N-methyl-N-nitro-V N-nitrosoguanidine. J Biol 7. Keeton MR, Curriden SA, van Zonneveld AJ, atinocytes after g irradiation. Radiat Res 2002;157: Chem 2001;276:36303 ^ 10. Loskutoff DJ. Identification of regulatory sequences 249 ^ 55. 26. van Zonneveld AJ, Curriden SA, Loskutoff DJ. in the type 1 plasminogen activator inhibitor gene 17. Martin M,Vozenin MC, Gault N, Crechet F, Pfarr CM, Type 1 plasminogen activator inhibitor gene: responsive to transforming growth factor h.JBiol Lefaix JL. Coactivation of AP-1 activity andTGF-b1 functional analysis and glucocorticoid regulation of Chem1991;266:23048 ^ 52. gene expression in the stress response of normal skin its promoter. Proc Natl Acad Sci U S A 1988;85: 8. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, cells to ionizing radiation. Oncogene 1997;15:981 ^ 9. 5525 ^ 9. Gauthier JM. Direct binding of Smad3 and Smad4 to 18. Boerma M, Bart CI, Wondergem J. Effects of ioniz- 27. Jonk LJ, Itoh S, Heldin CH, ten Dijke P, Kruijer W. critical TGF h-inducible elements in the promoter of ing radiation on gene expression in cultured rat heart Identification and functional characterization of a human plasminogen activator inhibitor-type 1 gene. cells. Int J Radiat Biol 2002;78:219^ 25. Smad binding element (SBE) in the JunB promoter EMBO J 1998;17:3091 ^ 100. 19. Barcellos-Hoff MH, Derynck R, Tsang ML, Weather- that acts as a transforming growth factor-h, activin, 9. Hall MC, Young DA, Waters JG et al. The compara- beeJA.Transforming growth factor-h activation in irra- and bone morphogenetic protein-inducible enhancer. tive role of activator protein 1and Smad factors in the diated murine mammary gland. J Clin Invest 1994; J Biol Chem 1998;273:21145 ^ 52. regulation of Timp-1 and MMP-1 gene expression by 93:892 ^ 9. 28. Michels AA, Nguyen VT, Konings AW, Kampinga transforming growth factor-h1. J Biol Chem 2003; 20. CriswellT, Leskov K, Miyamoto S, Luo G, Boothman HH, Bensaude O.Thermostability of a nuclear-targeted 278:10304^13. DA. Transcription factors activated in mammalian cells luciferase expressed in mammalian cells. Destabilizing 10. Gleizes PE, MungerJS, Nunes I, et al.TGF-h latency: after clinically relevant doses of ionizing radiation. On- influence of the intranuclear microenvironment. Eur J biological significance and mechanisms of activation. cogene 2003;22:5813^ 27. Biochem 1995;234:382^ 9. Stem Cells 1997;15:190 ^ 7. 21. Datta PK, Blake MC, Moses HL. Regulation of 29. Livak KJ, Schmittgen TD. Analysis of relative 11. Derynck R, Zhang YE. Smad-dependent and Smad- plasminogen activator inhibitor-1expression by trans- gene expression data using real-time quantitative

www.aacrjournals.org 5963 Clin Cancer Res 2005;11(16) August 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. Cancer Therapy: Preclinical

PCR and the 2(ÀDDC(T)) method. Methods 2001; sors: p53 is required for TGF-b gene responses by 44. Lopez-Rovira T, Chalaux E, Rosa JL, Bartrons R, 25:402^ 8. cooperating with Smads. Cell 2003;113:301 ^14. Ventura F. Interaction and functional cooperation of 30.Eitzman DT, McCoy RD, Zheng X, et al. Bleomycin- 37. Stahler F, Roemer K. Mutant p53 can provoke NF-nB with Smads. Transcriptional regulation of the induced pulmonary fibrosis in transgenic mice apoptosis in p53-deficient Hep3B cells with delayed junB promoter. J Biol Chem 2000;275:28937 ^ 46. that either lack or overexpress the murine plas- kinetics relative to wild-type p53. Oncogene 1998; 45. Dupont S, Zacchigna L, Adorno M, et al. Conver- minogen activator inhibitor-1 gene. J Clin Invest 1996; 17:3507^12. gence of p53 and TGF-h signaling networks. Cancer 97:232^ 7. 38. Chuang-Tsai S, Sisson TH, Hattori N, et al. Reduc- Lett 2004;213:129^ 38. 31. Hattori N, Degen JL, SissonTH, et al. Bleomycin- tion in fibrotic tissue formation in mice genetically defi- 46. Anscher MS, Kong FM, Andrews K, et al. Plasma induced pulmonary fibrosis in fibrinogen-null mice. cient in plasminogen activator inhibitor-1. Am J Pathol transforming growth factor h1as a predictor of radia- J Clin Invest 2000;106:1341 ^ 50. 2003;163:445^52. tion pneumonitis. Int J Radiat Oncol Biol Phys 1998; 32. van Waarde MA, van Assen AJ, Kampinga HH, 39. Barcellos-Hoff MH, Dix TA. Redox-mediated acti- 41:1029^ 35. Konings AW, Vujaskovic Z. Quantification of trans- vation of latent transforming growth factor-h1. Mol 47. Anscher MS, Marks LB, ShafmanTD, et al. Risk of forming growth factor-h in biological material using Endocrinol 1996;10:1077 ^ 83. long-term complications after TFG-h1-guided very- cells transfected with a plasminogen activator 40.Wagner A. Genes regulated cooperatively by one or high-dose thoracic radiotherapy. Int J Radiat Oncol inhibitor-1 promoter-luciferase construct. Anal Bio- more transcription factors and their identification in Biol Phys 2003;56:988 ^ 95. chem 1997;247:45 ^ 51. whole eukaryotic genomes. Bioinformatics 1999;15: 48. Novakova-Jiresova A, Van Gameren MM, Coppes 33. Olman MA, Hagood JS, Simmons WL, Fuller GM, 776 ^ 84. RP, Kampinga HH, Groen HJ. Transforming growth Vinson C,White KE. Fibrin fragment induction of plas- 41. Verrecchia F,Vindevoghel L, Lechleider RJ, Uitto J, factor-h plasma dynamics and post-irradiation lung in- minogen activator inhibitor transcription is mediated Roberts AB, Mauviel A. Smad3/AP-1 interactions jury in lung cancer patients. Radiother Oncol 2004; by activator protein-1 through a highly conserved control transcriptional responses to TGF-h in a 71:183 ^ 9. element. Blood1999;94:2029^ 38. promoter-specific manner. Oncogene 2001;20: 49. De Jaeger K, Seppenwoolde Y, Kampinga HH, 34. Reddy SP, Mossman BT. Role and regulation of 3332^40. Boersma LJ, Belderbos JS, Lebesque JV.Significance activator protein-1 in toxicant-induced responses of 42. Hua X, Miller ZA, Benchabane H, Wrana JL, Lodish of plasma transforming growth factor-h levels in radio- the lung. Am J Physiol Lung Cell Mol Physiol 2002; HF. Synergism between transcription factorsTFE3 and therapy for non-small-cell lung cancer. Int J Radiat 283:L1161 ^ 78. Smad3 in transforming growth factor-h-induced tran- OncolBiolPhys2004;58:1378^87. 35. Kunz C, Pebler S, OtteJ, derAhe D. Differential reg- scription of the Smad7 gene. J Biol Chem 2000;275: 50. Dawson SJ, Wiman B, Hamsten A, Green F, ulation of plasminogen activator and inhibitor gene 33205 ^8. Humphries S, Henney AM. The two allele sequences transcription by the tumor suppressor p53. Nucleic 43. Hua X, Liu X, Ansari DO, Lodish HF. Synergistic of a common polymorphism in the promoter of the Acids Res 1995;23:3710^ 7. cooperation of TFE3 and smad proteins in TGF-h- plasminogen activator inhibitor-1 (PAI-1) gene re- 36. Cordenonsi M, Dupont S, Maretto S, Insinga A, induced transcription of the plasminogen activator spond differently to interleukin-1in HepG2 cells. J Biol Imbriano C, Piccolo S. Links between tumor suppres- inhibitor-1 gene. Genes Dev 1998;12:3084 ^ 95. Chem 1993;268:10739^45.

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