- Email: [email protected]
Synergistic induction of profibrotic PAI-1 by TGF-β and radiation depends on p53
Radiotherapy and Oncology 97 (2010) 33–35
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Molecular genetics of RT side-effects
Synergistic induction of profibrotic PAI-1 by TGF-b and radiation depends on p53 Maarten Niemantsverdriet a,b, Edwin de Jong a, Johannes A. Langendijk b, Harm H. Kampinga a, Robert P. Coppes a,b,* a
Department of Cell Biology, Section Radiation and Stress Cell Biology; and b Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 30 March 2010 Accepted 5 April 2010
a b s t r a c t Radiation-induced fibrosis is a severe side effect of radiotherapy. TGF-b and radiation synergistically induce expression of the profibrotic PAI-1 gene and this cooperation potentially involves p53. Here, we demonstrate that p53 is both indispensable and sufficient for the radiation effect inducing synergistic activation of PAI-1 by radiation and TGF-b. Ó 2010 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 97 (2010) 33–35
Keywords: P53 Radiation TGF-b PAI-1 Fibrosis
Cancer therapy has improved considerably in the past decades with increasing patient survival. However, this also allows the development of late therapy-induced side effects such as radiation fibrosis. This late sequel of radiation therapy may affect irradiated lung, breast, kidney, skin and other tissues [1,2]. Extracellular matrix integrity is maintained in normal, healthy tissues as a tight balance between production and degradation of collagen and other extracellular matrix (ECM) proteins [3]. The progressive and excessive accumulation of extracellular matrix which lies at the basis of radiationinduced fibrosis severely compromises the functioning of these tissues, leading to morbidity and sometimes even death [1,2]. To be able to find strategies effectively preventing or curing radiation-induced fibrosis, a good insight into the molecular mechanisms involved in fibrosis induction is required. It is known that one of the major players in induction of fibrosis is TGF-b [2], a cytokine regulating transactivation of multiple genes, including PAI-1 [3–5]. PAI-1 is the main inhibitor of the fibrinolytic system and is required for proper degradation of ECM components [5]. Alteration of PAI-1 expression therefore disturbs the ECM balance. Accordingly, PAI-1 overexpressing mice are very sensitive to bleomycin-induced pulmonary fibrosis, whereas PAI-1 deficient mice are exceptionally resistant [4]. Interestingly, PAI-1 is synergistically induced by radiation and TGF-b. It was suggested that p53, the most investigated tu-
* Corresponding author at: Department of Cell Biology, Section Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, A.Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail address: [email protected] (R.P. Coppes). 0167-8140/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2010.04.002
mor suppressor that is known to be stabilized upon radiation to regulate gene expression of many down-stream targets [6,7], might play a role in the radiation effect inducing this synergy [8,9]. However, TGF-b responses depend on cellular context and are usually highly cell-type specific [10], and radiation also induces many factors other than p53 [11]. The aim of this study was to investigate the mechanism of cooperative activation of PAI-1 by TGF-b and radiation and to establish the importance of p53. Methods and materials Cell culture Human embryonic kidney cell line Hek293, human lung carcinoma cell line A549, human breast cancer cell line MCF7 and human colorectal cancer cell line HCT 116, human cervix carcinoma cell line HeLa (ATCC) were cultured in DMEM supplemented with 10% fetal calf serum and primary human fibroblasts (VH25) were cultured in Ham’s F10 supplemented with 15% fetal calf serum as described [12]. Tetracycline regulated p53 inducible p53-19 cells were cultured and p53 induced as described [13]. Cells were treated with 10 Gy of c-radiation with a Cs137 source and/or with 1 ng/ml recombinant human TGF-b (R&D systems) as indicated. RT-PCR and QPCR Conventional RT-PCR (p53 and GAPDH) was performed as described in [13] and (relative) PAI-1 QPCR as described in [9].
34
Profibrotic PAI-1 induction by TGF-b and p53
Results and discussion Radiation-induced ser15 p53 phosphorylation is detectable in different cell lines but not in HeLa cells Fig. 1. P53 protein levels and activation in different cell lines. MCF7, A549, Hek293, Primary human fibroblasts, HCT 116 and HeLa cells were left untreated or irradiated (10 Gy) and lysed 24 h later. Lysates were immunoblotted, detecting serine 15 phosphorylated p53 protein, total p53 protein and c-tubulin (loading control).
Immunoblotting For protein extracts, cell lines were irradiated 24 h after seeding and 24 h after irradiation cells were lysed and immunoblotted as described in [13], using a-p53 DO-1 (Santa Cruz Biotechnology, Inc.), a-c-tubulin (Sigma–Aldrich) and a-p53 pser 15 Ab-3 (Calbiochem) primary antibodies.
P53 activity and stability is regulated by phosphorylation at different sites and initial phosphorylation at p53 serine 15 is essential for its full activation in response to ionizing radiation [14]. To investigate as to what extent synergistic induction of the profibrotic PAI-1 gene by radiation and TGF-b depends on p53, we investigated p53 protein levels and the phosphorylation state of serine 15 after radiation in cultured cell lines derived from diverge tissues (Fig. 1). MCF7 (breast cancer cell line) and A549 (lung cancer cell line) both express wild-type p53 [15]. P53 levels in these cells are low, but detectable (Fig. 1). After radiation, p53 serine 15 phosphorylation and total p53 levels increase in these cells (Fig. 1). The human embryonic kidney cell line Hek293 also expresses wildtype p53 [16] and increased levels of serine 15 phosphorylated
Fig. 2. Synergistic PAI-1 induction by radiation and TGF-b1 depends on p53. (a) Human breast cancer cells (MCF7), (b) human lung epithelial cells (A549), (c) human embryonic kidney cells (Hek293), (d) primary human fibroblasts, (e) human colorectal cancer cells (HCT 116) and (f) human cervix carcinoma cells (HeLa) were left untreated (–), irradiated (R), treated with TGF-b1 (b) or irradiated and treated with TGF-b (R + b). Relative PAI-1 mRNA levels were quantified by QPCR. (g,h) P53 inducible p53-19 cells were left uninduced or p53 induced by washing away tetracycline from the growth-medium. Cells were also treated with TGF-b1 where indicated and/or irradiated (samples on the right). (g) p53 RTPCR using a specific primer set detecting tetracycline induced p53. GAPDH RT-PCR was used as control. (h) Relative PAI-1 expression levels in p53-19 cells quantified by QPCR.
M. Niemantsverdriet et al. / Radiotherapy and Oncology 97 (2010) 33–35
p53 were also found after irradiation in these cells (Fig. 1). We also used primary human fibroblasts (Fig. 1). These cells also contain wild-type p53 [12], and show similar serine 15 phosphorylation of p53 in response to radiation as MCF7, A549 and Hek293 cell lines (Fig. 1). The p53 wild-type [15] human colon cancer cell line HCT 116 also shows an increase in total p53 levels and serine 15 phosphorylation after radiation (Fig. 1). This cell line was used because it is deficient for external TGF-b signaling [17]. In contrast to all other cells used, HeLa human cervix carcinoma cells in which p53 is constitutively downregulated [18] showed no p53 or serine 15 phosphorylation upon radiation (Fig. 1). Complete p53 activation depends on multiple modifications and detection of serine 15 phosphorylation alone is therefore insufficient to investigate to what extent p53 is exactly activated [6]. However, phosphorylation of p53 at serine 15 in all cell lines except HeLa does show that p53 is present and responds to radiation in these cells.
35
quired and sufficient to induce PAI-1 transactivation in response to radiation and for synergy with TGF-b1, without the requirement for other factors induced by radiation. Since multiple mechanisms involving TGF-b signaling have been implicated in fibrosis [2] and cooperation between p53 and TGF-b signaling has been shown for other targets in addition to PAI-1 [8], it can be speculated that other profibrotic factors are also regulated by a similar or the same mechanism. Very recent findings by others using cell-type specific knock-out mouse models also suggest a specific role for p53 in the development of late radiation injury [21]. The molecular mechanism described here may be a potential candidate for therapeutic targeting and precise elucidation of molecular mechanisms contributing to radiation-induced fibrosis like the one we describe here may contribute to the development of such therapies. Conflict of interest notification
Synergistic PAI-1 induction by TGF-b and radiation depends on p53 Next, we treated the various cells with a combination of radiation and/or TGF-b1 and relative PAI-1 mRNA levels were measured (Fig. 2a–f). Breast cancer cells (MCF7), lung carcinoma cells (A549) and human embryonic kidney cells (Hek293) all showed induction of PAI-1 after irradiation and TGF-b1 and always synergy after both (Fig. 2a–c). Because the behaviour of fibroblasts after irradiation is especially important for the induction of fibrosis [3,19,20], we also investigated the PAI-1 induction in response to radiation and TGF-b1 in primary human fibroblasts (Fig. 2d). Here, radiation and TGF-b1 both induced PAI-1 expression and a combination induced additive expression, similar to the synergistic expression in MCF7, A549 and Hek293 cell lines (Fig. 2a–c). In contrast, HCT 116 cells showed a clear increase in PAI-1 expression in response to radiation, but not to TGF-b1 (Fig. 2e). HCT 116 cells contain a homozygous mutation in the gene encoding the TGF-b Receptor II which is required for downstream signaling of external TGF-b responses [17]. This lack of external TGF-b signaling thus explains why PAI-1 was not induced by TGF-b1 and why no cooperativity was found between radiation and TGF-b1 in these cells (Fig. 2e). The results in HeLa cells are contrary to those seen in HCT 116 cells: in these cells that lack p53 (Fig. 1), PAI-1 was not induced by radiation and although TGFb1 alone did activate the PAI-1 promoter, no synergy for PAI-1 activation was found when radiation and TGF-b1 were combined (Fig. 2f). These results are consistent with previous work suggesting that p53 may have a role in the induction of the PAI-1 gene in response to radiation and its synergy with TGF-b signaling [8,9] and the results from these two studies combined with those presented here indicate the generality of this synergistic response. To conclusively demonstrate that p53 is crucial for the above described effects of radiation on PAI-1 expression, we used SCC15, a squamous cell carcinoma cell line that does not express endogenous p53 at all, also not after irradiation [13]. In these cells, a tetracyclinerepressed p53 gene was stably integrated (p53-19 cells) and p53 levels are undetectable in the presence of tetracycline but are induced after removal of tetracycline from the medium (Fig. 2g). The protein expression and activity of induced p53 in the p53-19 cell line was tested very thoroughly in another study [13]. In this cell line, p53 actions can be monitored in the absence of other factors induced by radiation [13]. Induction of p53 alone without any radiation was already sufficient to induce PAI-1 expression (Fig. 2h). In the absence of p53, TGF-b1 also induced PAI-1 expression and, as expected, PAI-1 induction was greatly enhanced upon expression of p53 (Fig. 2h, left). The induction levels were comparable to those seen after radiation alone or after combined radiation and TGF-b1 treatment (Fig. 2a–d). Interestingly, radiation did not further increase the PAI-1 expression (with or without co-stimulation with TGF-b1) when p53 was already induced (Fig. 2h). This suggests that p53 is re-
None. Acknowledgement The authors thank Dr. J. Hageman for advice. References [1] Delanian S, Lefaix JL. Current management for late normal tissue injury: radiation-induced fibrosis and necrosis. Semin Radiat Oncol 2007;17:99–107. [2] Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target? Int J Radiat Oncol Biol Phys 2000;47:277–90. [3] Rodemann HP, Bamberg M. Cellular basis of radiation-induced fibrosis. Radiother Oncol 1995;35:83–90. [4] Eitzman DT, McCoy RD, Zheng X, et al. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest 1996;97:232–7. [5] Loskutoff DJ, Quigley JP. PAI-1, fibrosis, and the elusive provisional fibrin matrix. J Clin Invest 2000;106:1441–3. [6] Meek DW. Multisite phosphorylation and the integration of stress signals at p53. Cell Signal 1998;10:159–66. [7] Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell 2009;137:413–31. [8] Cordenonsi M, Dupont S, Maretto S, et al. Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. Cell 2003;113:301–14. [9] Hageman J, Eggen BJ, Rozema T, et al. Radiation and transforming growth factor-beta cooperate in transcriptional activation of the profibrotic plasminogen activator inhibitor-1 gene. Clin Cancer Res 2005;11:5956–64. [10] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [11] Snyder AR, Morgan WF. Gene expression profiling after irradiation: clues to understanding acute and persistent responses? Cancer Metastasis Rev 2004;23:259–68. [12] van Waarde-Verhagen MA, Kampinga HH, Linskens MH. Continuous growth of telomerase-immortalised fibroblasts: how long do cells remain normal? Mech Ageing Dev 2006;127:85–7. [13] Niemantsverdriet M, Jongmans W, Backendorf C. Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes. Exp Cell Res 2005;310:237–47. [14] Wittlinger M, Grabenbauer GG, Sprung CN, et al. Time- and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity. Int J Radiat Biol 2007;83:245–57. [15] O’Connor PM, Jackman J, Bae I, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res 1997;57:4285–300. [16] Hamid T, Kakar SS. PTTG/securin activates expression of p53 and modulates its function. Mol Cancer 2004;3:18. [17] Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336–8. [18] Nomine Y, Masson M, Charbonnier S, et al. Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirusmediated pathogenesis. Mol Cell 2006;21:665–78. [19] Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer 2006;6:702–13. [20] Delanian S, Lefaix JL. The radiation-induced fibroatrophic process: therapeutic perspective via the antioxidant pathway. Radiother Oncol 2004;73:119–31. [21] Kirsch DG, Santiago PM, di Tomaso E, et al. p53 controls radiation-induced gastrointestinal syndrome in mice independent of apoptosis. Science 2010;327:593–6.
Contents lists available at ScienceDirect
Radiotherapy and Oncology journal homepage: www.thegreenjournal.com
Molecular genetics of RT side-effects
Synergistic induction of profibrotic PAI-1 by TGF-b and radiation depends on p53 Maarten Niemantsverdriet a,b, Edwin de Jong a, Johannes A. Langendijk b, Harm H. Kampinga a, Robert P. Coppes a,b,* a
Department of Cell Biology, Section Radiation and Stress Cell Biology; and b Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 14 December 2009 Received in revised form 30 March 2010 Accepted 5 April 2010
a b s t r a c t Radiation-induced fibrosis is a severe side effect of radiotherapy. TGF-b and radiation synergistically induce expression of the profibrotic PAI-1 gene and this cooperation potentially involves p53. Here, we demonstrate that p53 is both indispensable and sufficient for the radiation effect inducing synergistic activation of PAI-1 by radiation and TGF-b. Ó 2010 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 97 (2010) 33–35
Keywords: P53 Radiation TGF-b PAI-1 Fibrosis
Cancer therapy has improved considerably in the past decades with increasing patient survival. However, this also allows the development of late therapy-induced side effects such as radiation fibrosis. This late sequel of radiation therapy may affect irradiated lung, breast, kidney, skin and other tissues [1,2]. Extracellular matrix integrity is maintained in normal, healthy tissues as a tight balance between production and degradation of collagen and other extracellular matrix (ECM) proteins [3]. The progressive and excessive accumulation of extracellular matrix which lies at the basis of radiationinduced fibrosis severely compromises the functioning of these tissues, leading to morbidity and sometimes even death [1,2]. To be able to find strategies effectively preventing or curing radiation-induced fibrosis, a good insight into the molecular mechanisms involved in fibrosis induction is required. It is known that one of the major players in induction of fibrosis is TGF-b [2], a cytokine regulating transactivation of multiple genes, including PAI-1 [3–5]. PAI-1 is the main inhibitor of the fibrinolytic system and is required for proper degradation of ECM components [5]. Alteration of PAI-1 expression therefore disturbs the ECM balance. Accordingly, PAI-1 overexpressing mice are very sensitive to bleomycin-induced pulmonary fibrosis, whereas PAI-1 deficient mice are exceptionally resistant [4]. Interestingly, PAI-1 is synergistically induced by radiation and TGF-b. It was suggested that p53, the most investigated tu-
* Corresponding author at: Department of Cell Biology, Section Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, A.Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail address: [email protected] (R.P. Coppes). 0167-8140/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2010.04.002
mor suppressor that is known to be stabilized upon radiation to regulate gene expression of many down-stream targets [6,7], might play a role in the radiation effect inducing this synergy [8,9]. However, TGF-b responses depend on cellular context and are usually highly cell-type specific [10], and radiation also induces many factors other than p53 [11]. The aim of this study was to investigate the mechanism of cooperative activation of PAI-1 by TGF-b and radiation and to establish the importance of p53. Methods and materials Cell culture Human embryonic kidney cell line Hek293, human lung carcinoma cell line A549, human breast cancer cell line MCF7 and human colorectal cancer cell line HCT 116, human cervix carcinoma cell line HeLa (ATCC) were cultured in DMEM supplemented with 10% fetal calf serum and primary human fibroblasts (VH25) were cultured in Ham’s F10 supplemented with 15% fetal calf serum as described [12]. Tetracycline regulated p53 inducible p53-19 cells were cultured and p53 induced as described [13]. Cells were treated with 10 Gy of c-radiation with a Cs137 source and/or with 1 ng/ml recombinant human TGF-b (R&D systems) as indicated. RT-PCR and QPCR Conventional RT-PCR (p53 and GAPDH) was performed as described in [13] and (relative) PAI-1 QPCR as described in [9].
34
Profibrotic PAI-1 induction by TGF-b and p53
Results and discussion Radiation-induced ser15 p53 phosphorylation is detectable in different cell lines but not in HeLa cells Fig. 1. P53 protein levels and activation in different cell lines. MCF7, A549, Hek293, Primary human fibroblasts, HCT 116 and HeLa cells were left untreated or irradiated (10 Gy) and lysed 24 h later. Lysates were immunoblotted, detecting serine 15 phosphorylated p53 protein, total p53 protein and c-tubulin (loading control).
Immunoblotting For protein extracts, cell lines were irradiated 24 h after seeding and 24 h after irradiation cells were lysed and immunoblotted as described in [13], using a-p53 DO-1 (Santa Cruz Biotechnology, Inc.), a-c-tubulin (Sigma–Aldrich) and a-p53 pser 15 Ab-3 (Calbiochem) primary antibodies.
P53 activity and stability is regulated by phosphorylation at different sites and initial phosphorylation at p53 serine 15 is essential for its full activation in response to ionizing radiation [14]. To investigate as to what extent synergistic induction of the profibrotic PAI-1 gene by radiation and TGF-b depends on p53, we investigated p53 protein levels and the phosphorylation state of serine 15 after radiation in cultured cell lines derived from diverge tissues (Fig. 1). MCF7 (breast cancer cell line) and A549 (lung cancer cell line) both express wild-type p53 [15]. P53 levels in these cells are low, but detectable (Fig. 1). After radiation, p53 serine 15 phosphorylation and total p53 levels increase in these cells (Fig. 1). The human embryonic kidney cell line Hek293 also expresses wildtype p53 [16] and increased levels of serine 15 phosphorylated
Fig. 2. Synergistic PAI-1 induction by radiation and TGF-b1 depends on p53. (a) Human breast cancer cells (MCF7), (b) human lung epithelial cells (A549), (c) human embryonic kidney cells (Hek293), (d) primary human fibroblasts, (e) human colorectal cancer cells (HCT 116) and (f) human cervix carcinoma cells (HeLa) were left untreated (–), irradiated (R), treated with TGF-b1 (b) or irradiated and treated with TGF-b (R + b). Relative PAI-1 mRNA levels were quantified by QPCR. (g,h) P53 inducible p53-19 cells were left uninduced or p53 induced by washing away tetracycline from the growth-medium. Cells were also treated with TGF-b1 where indicated and/or irradiated (samples on the right). (g) p53 RTPCR using a specific primer set detecting tetracycline induced p53. GAPDH RT-PCR was used as control. (h) Relative PAI-1 expression levels in p53-19 cells quantified by QPCR.
M. Niemantsverdriet et al. / Radiotherapy and Oncology 97 (2010) 33–35
p53 were also found after irradiation in these cells (Fig. 1). We also used primary human fibroblasts (Fig. 1). These cells also contain wild-type p53 [12], and show similar serine 15 phosphorylation of p53 in response to radiation as MCF7, A549 and Hek293 cell lines (Fig. 1). The p53 wild-type [15] human colon cancer cell line HCT 116 also shows an increase in total p53 levels and serine 15 phosphorylation after radiation (Fig. 1). This cell line was used because it is deficient for external TGF-b signaling [17]. In contrast to all other cells used, HeLa human cervix carcinoma cells in which p53 is constitutively downregulated [18] showed no p53 or serine 15 phosphorylation upon radiation (Fig. 1). Complete p53 activation depends on multiple modifications and detection of serine 15 phosphorylation alone is therefore insufficient to investigate to what extent p53 is exactly activated [6]. However, phosphorylation of p53 at serine 15 in all cell lines except HeLa does show that p53 is present and responds to radiation in these cells.
35
quired and sufficient to induce PAI-1 transactivation in response to radiation and for synergy with TGF-b1, without the requirement for other factors induced by radiation. Since multiple mechanisms involving TGF-b signaling have been implicated in fibrosis [2] and cooperation between p53 and TGF-b signaling has been shown for other targets in addition to PAI-1 [8], it can be speculated that other profibrotic factors are also regulated by a similar or the same mechanism. Very recent findings by others using cell-type specific knock-out mouse models also suggest a specific role for p53 in the development of late radiation injury [21]. The molecular mechanism described here may be a potential candidate for therapeutic targeting and precise elucidation of molecular mechanisms contributing to radiation-induced fibrosis like the one we describe here may contribute to the development of such therapies. Conflict of interest notification
Synergistic PAI-1 induction by TGF-b and radiation depends on p53 Next, we treated the various cells with a combination of radiation and/or TGF-b1 and relative PAI-1 mRNA levels were measured (Fig. 2a–f). Breast cancer cells (MCF7), lung carcinoma cells (A549) and human embryonic kidney cells (Hek293) all showed induction of PAI-1 after irradiation and TGF-b1 and always synergy after both (Fig. 2a–c). Because the behaviour of fibroblasts after irradiation is especially important for the induction of fibrosis [3,19,20], we also investigated the PAI-1 induction in response to radiation and TGF-b1 in primary human fibroblasts (Fig. 2d). Here, radiation and TGF-b1 both induced PAI-1 expression and a combination induced additive expression, similar to the synergistic expression in MCF7, A549 and Hek293 cell lines (Fig. 2a–c). In contrast, HCT 116 cells showed a clear increase in PAI-1 expression in response to radiation, but not to TGF-b1 (Fig. 2e). HCT 116 cells contain a homozygous mutation in the gene encoding the TGF-b Receptor II which is required for downstream signaling of external TGF-b responses [17]. This lack of external TGF-b signaling thus explains why PAI-1 was not induced by TGF-b1 and why no cooperativity was found between radiation and TGF-b1 in these cells (Fig. 2e). The results in HeLa cells are contrary to those seen in HCT 116 cells: in these cells that lack p53 (Fig. 1), PAI-1 was not induced by radiation and although TGFb1 alone did activate the PAI-1 promoter, no synergy for PAI-1 activation was found when radiation and TGF-b1 were combined (Fig. 2f). These results are consistent with previous work suggesting that p53 may have a role in the induction of the PAI-1 gene in response to radiation and its synergy with TGF-b signaling [8,9] and the results from these two studies combined with those presented here indicate the generality of this synergistic response. To conclusively demonstrate that p53 is crucial for the above described effects of radiation on PAI-1 expression, we used SCC15, a squamous cell carcinoma cell line that does not express endogenous p53 at all, also not after irradiation [13]. In these cells, a tetracyclinerepressed p53 gene was stably integrated (p53-19 cells) and p53 levels are undetectable in the presence of tetracycline but are induced after removal of tetracycline from the medium (Fig. 2g). The protein expression and activity of induced p53 in the p53-19 cell line was tested very thoroughly in another study [13]. In this cell line, p53 actions can be monitored in the absence of other factors induced by radiation [13]. Induction of p53 alone without any radiation was already sufficient to induce PAI-1 expression (Fig. 2h). In the absence of p53, TGF-b1 also induced PAI-1 expression and, as expected, PAI-1 induction was greatly enhanced upon expression of p53 (Fig. 2h, left). The induction levels were comparable to those seen after radiation alone or after combined radiation and TGF-b1 treatment (Fig. 2a–d). Interestingly, radiation did not further increase the PAI-1 expression (with or without co-stimulation with TGF-b1) when p53 was already induced (Fig. 2h). This suggests that p53 is re-
None. Acknowledgement The authors thank Dr. J. Hageman for advice. References [1] Delanian S, Lefaix JL. Current management for late normal tissue injury: radiation-induced fibrosis and necrosis. Semin Radiat Oncol 2007;17:99–107. [2] Martin M, Lefaix J, Delanian S. TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target? Int J Radiat Oncol Biol Phys 2000;47:277–90. [3] Rodemann HP, Bamberg M. Cellular basis of radiation-induced fibrosis. Radiother Oncol 1995;35:83–90. [4] Eitzman DT, McCoy RD, Zheng X, et al. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest 1996;97:232–7. [5] Loskutoff DJ, Quigley JP. PAI-1, fibrosis, and the elusive provisional fibrin matrix. J Clin Invest 2000;106:1441–3. [6] Meek DW. Multisite phosphorylation and the integration of stress signals at p53. Cell Signal 1998;10:159–66. [7] Vousden KH, Prives C. Blinded by the light: the growing complexity of p53. Cell 2009;137:413–31. [8] Cordenonsi M, Dupont S, Maretto S, et al. Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. Cell 2003;113:301–14. [9] Hageman J, Eggen BJ, Rozema T, et al. Radiation and transforming growth factor-beta cooperate in transcriptional activation of the profibrotic plasminogen activator inhibitor-1 gene. Clin Cancer Res 2005;11:5956–64. [10] Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol 2000;1:169–78. [11] Snyder AR, Morgan WF. Gene expression profiling after irradiation: clues to understanding acute and persistent responses? Cancer Metastasis Rev 2004;23:259–68. [12] van Waarde-Verhagen MA, Kampinga HH, Linskens MH. Continuous growth of telomerase-immortalised fibroblasts: how long do cells remain normal? Mech Ageing Dev 2006;127:85–7. [13] Niemantsverdriet M, Jongmans W, Backendorf C. Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes. Exp Cell Res 2005;310:237–47. [14] Wittlinger M, Grabenbauer GG, Sprung CN, et al. Time- and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity. Int J Radiat Biol 2007;83:245–57. [15] O’Connor PM, Jackman J, Bae I, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res 1997;57:4285–300. [16] Hamid T, Kakar SS. PTTG/securin activates expression of p53 and modulates its function. Mol Cancer 2004;3:18. [17] Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336–8. [18] Nomine Y, Masson M, Charbonnier S, et al. Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirusmediated pathogenesis. Mol Cell 2006;21:665–78. [19] Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer 2006;6:702–13. [20] Delanian S, Lefaix JL. The radiation-induced fibroatrophic process: therapeutic perspective via the antioxidant pathway. Radiother Oncol 2004;73:119–31. [21] Kirsch DG, Santiago PM, di Tomaso E, et al. p53 controls radiation-induced gastrointestinal syndrome in mice independent of apoptosis. Science 2010;327:593–6.