Androgen deprivation modulates the inflammatory response induced by irradiation
© Wu et al; licensee BioMed Central Ltd. 2009
Received: 31 July 2008
Accepted: 25 March 2009
Published: 25 March 2009
The aim of this study was to determine whether radiation (RT)-induced inflammatory responses and organ damage might be modulated by androgen deprivation therapies.
The mRNA and tissue sections obtained from the lungs, intestines and livers of irradiated mice with or without androgen deprivation were analyzed by real-time PCR and histological analysis. Activation of NF-kappa B was examined by measuring nuclear protein levels in the intestine and lung 24 h after irradiation. We also examined the levels of cyclooxygenase-2 (COX-2), TGF-β1 and p-AKT to elucidate the related pathway responsible to irradiation (RT) -induced fibrosis.
We found androgen deprivation by castration significantly augmented RT-induced inflammation, associated with the increase NF-κB activation and COX-2 expression. However, administration of flutamide had no obvious effect on the radiation-induced inflammation response in the lung and intestine. These different responses were probably due to the increase of RT-induced NF-κB activation and COX-2 expression by castration or lupron treatment. In addition, our data suggest that TGF-β1 and the induced epithelial-mesenchymal transition (EMT) via the PI3K/Akt signaling pathway may contribute to RT-induced fibrosis.
When irradiation was given to patients with total androgen deprivation, the augmenting effects on the RT-induced inflammation and fibrosis should take into consideration for complications associated with radiotherapy.
Although radiotherapy is an important cancer treatment modality, the cell killing induced by radiation is not tumor- or cell-type specific. Treatment of cancer patients with radiation can be significantly compromised by the development of severe acute and late damage to normal tissues. Normal tissue complications induced by irradiation differ depending on the target organ and cell types. Sequelae of pelvic RT include small bowel obstruction, enteritis, proctitis and radiation cystitis [1, 2], and the lung is one of the most critical dose- limiting organs after thoracic RT. Recent studies have led towards a better understanding of the molecular mechanisms underlying radiation injury [3–5]. The response to radiation is dynamic and involves several mediators of inflammation and fibrosis that are produced by macrophages, epithelial cells and fibroblasts. Not surprisingly, pro-inflammatory cytokines are highly prominent among the panoply of molecules expressed in tissue after irradiation, and has been demonstrated to contribute to the significant complications associated with radiotherapy [5–7]. Since persistent accumulation and activation of immune cells is a hallmark of chronic inflammation, early manipulation of inflammatory responses could be useful for modification of the subsequent late effects [8, 9].
In many respects, the tissue responses to irradiation mimic the cytokine storms generated by many other tissue damaging insults, such as hemorrhagic shock and sepsis. Hemorrhagic shock results in excessive production of pro-inflammatory mediators, which play a critical role in the development of multiple organ dysfunctions under such conditions. After hemorrhagic shock and sepsis, gonadal steroids have a significant effect on the maladaptive changes in immune cell function [10–12]. Gender-dimorphic immune and organ responsiveness have been reported [13, 14]. Androgens might mediate the immunosuppression following trauma-hemorrhagic shock in males, whereas female sex steroids have immunoprotective properties after hemorrhagic shock and sepsis. Testosterone reportedly has key roles in fibrosis and wound healing via cell-specific and differential regulation [15, 16]. Furthermore, a combination of hormone treatment and curative radiation treatment was often given to patients with prostate cancer and breast cancer [17, 18]. Based on these reports, it triggers an unsolved issue; if the inflammation response and fibrosis induced by irradiation (RT) might be regulated by hormone manipulation through surgical or medical method. Therefore, the aim of the present study was to test the hypothesis that the inflammatory response and organ damage induced by irradiation can be modulated by androgen deprivation therapies including administration with flutamide (anti- androgen) and castration.
Mice, radiation and androgen deprivation (flutamide administration or castration)
RNA isolation and real-time RT-PCR
At the indicated times after irradiation, Four mice from each group were sacrificed by cervical dislocation and the lungs, intestine and liver were dissected and stored at -80°C pending analysis. Specific Assay-on-Demand Gene Expression Assay mixes (including primer and Taqman MGB probes) for IL-1α/β, IL-6, TNF-α and TGF-β were used for real-time PCR analysis (Applied Biosystems, Foster, CA, USA). The first strand cDNA was amplified through 40 cycles (95°C for 15 s and 60°C for 1 min) with the TaqMan Universal PCR Master mix and the specific Assay-on-Demand Gene Expression Assay mix for each gene according to the manufacturer's instructions. To compare loading differences, a GAPDH primer was used as the internal control. Optimized PCR was performed on an iCycler iQ multicolor real-time PCR detection system. Significant PCR fluorescent signals were normalized to a PCR fluorescent signal obtained from the mean value of the sham-irradiated control mice. Two- sided log- rank analysis was used to assess statistical significance, and the association between discrete variables was tested using t-test.
Assessment of myeloperoxidase (MPO) activity
MPO activities were determined in whole lung, liver and intestinal homogenates as described by Toth et al. . Tissue samples were collected, frozen in liquid nitrogen and stored at -80°C pending analysis. The samples were sonicated on ice, then centrifuged at 12,000 g for 15 min at 4°C. Aliquots were divided into 180 μl phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The reactions were monitored by spectrophotometry at 460 nm (model 550, Bio-RAD, Hercules, CA) for 10 min.
Histological analysis and immunochemical staining
Cellular aspects of inflammation were measured in lung and intestinal tissue samples using hematoxylin and eosin (H&E) and immunochemical staining. Treated and control mice were sacrificed by cervical dislocation 24 h after exposure to 20 Gy irradiation. Three mice for each group were used, and the lungs and intestines were fixed pre-autopsy with 10% formalin. Moreover, the lung and intestinal tissue samples from mice with Lupron treatment (with Lupron Depot (0.4 mg/Kg) at two weeks before irradiation) were also analyzed. For histological analysis, the tissues were fixed in 10% buffered formalin, paraffin-embedded and sectioned at an average thickness of 5 μm. The mounted sections were subjected to H&E and immunochemical staining. Briefly, samples were incubated overnight with goat anti-mouse TGF-β1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:100 in 0.01 M RPMI at room temperature. After washing three times with PBS, the sections were incubated with biotinylated anti-goat IgG (1:100) for 10 min followed by peroxidase-avidin staining. Samples were washed with PBS, followed by addition of 3-amino-9 ethylcarbazole.
For western blot analysis of whole cell extracts, cells were lysed in a lysis buffer and the nuclear and cytoplasmic proteins were separated using an NE-PER kit (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were incubated with antibodies specific for TGF-β1 and COX-2 (Santa Cruz Biotechnology, Inc), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using enhanced chemiluminescence. To normalize the protein loading, the membrane was re-probed with mouse anti-r-tubulin antibody (1:1000).
Electrophoretic mobility gel shift assays (EMSA)
Nuclear proteins were collected from BALB/c mice lung, intestinal and liver tissues 24 h after exposure to 20 Gy. Four murine lung tissues from each group were checked. The protein content was measured using the Bradford method. The DNA oligonucleotide used for NF-κB binding was 5'-AGTTGAGGGACTTTCCCAGGC-3', a sequence specific to NF-κB binding for mouse. The NF-κB binding activity was evaluated using a LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL, USA). Anneal oligo by mixing together equal amounts of the labeled complementary oligos and incubating the mixture for 1 h at room temperature. Nuclear extracts were incubated with the biotin-labeled DNA probe for 20 min at room temperature. The DNA-protein complex was separated from free oligonucleotides on a 5% polyacrylamide gel, transferred to a nylon membrane and cross-linked by UV. The membrane was incubated with streptavidin-horseradish peroxidase conjugate and detected by Enhanced chemiluminescence (Pierce, Rockford, IL, USA).
Effects of androgen deprivation including castration and flutamide administration on the radiation-induced expression of pro-inflammatory cytokines
Effect of androgen deprivation on the radiation-induced inflammatory response by Histologic examination
Androgen deprivation by castration augmentation radiation-induced NF-κB activation in the lung and intestine
We hypothesized that the EMT induced by TGF-β1 might contribute to the RT-induced fibrosis. To test this hypothesis, we used western blot analysis to compare p-Akt and β-catenin levels in irradiated mrine intestine with different treatments. Increased p-Akt and decreased β-catenin levels were observed after stimulation by TGF-β1 or irradiation compared to controls. Moreover, inhibition of Phosphoinositide-3 kinase by wortmannin was associated with attenuation the increased p-Akt and vimentin and decreased β-catenin stimulated by irradiation and TGF-β1 (Figure 5C).
Radiation pneumonitis and radiation enteritis are two of the most significant complications associated with chest and abdomen-pelvic irradiation, respectively. RT-induced production of pro-inflammatory cytokines including IL-1β, TNF-α, TGF-β1 and IL-6 have been shown to contribute significantly to the complications associated with radiotherapy [4, 20, 22, 23]. Early overproduction of both pro-inflammatory IL-1 and TNF-α and pro-fibrogenic TGF-β1 during radiotherapy in animal studies suggests a role in the development of acute and late radiation toxicities . In humans, some clinical series have shown changes in the plasma concentrations of TGF-β1 and IL-6 during radiotherapy suggesting that these variations could identify patients at risk of radiation pneumonitis [24–26]. Such data indicate that the RT-induced response in vivo is associated with increased expression and activity of inflammatory cytokines.
In the present study, we demonstrated that the RT-induced inflammatory response involved increased expression of the pro-inflammatory cytokines IL-1α/β, TNF-α, TGF-β1 and IL-6 and increased MPO activity, consistent with previous studies [22, 23, 27]. We found that androgen deprivation by castration significantly augmented the RT-induced inflammatory response in the lung and intestine, but not in the liver. The discrepancy between the modulating effects on the lung, intestine and liver might be due to organ specificity.
TGF-β1 is autoinductive and chemotactic to monocytes and macrophages and may lead to increased growth factor expression at the site of injury [28, 29]. In addition, TGF-β1 is a potent chemoattractant for fibroblasts and triggers the expression of extracellular matrix components in fibrosis. Rube et al.  have suggested that the localization of TGF-β1 indicates areas with inflammatory cell infiltrates that are involved in the pathogenesis of RT-induced fibrosis. In the present study, we show that androgen deprivation by castration (surgical and chemical with Lupron Depot) increased TGF-β1 immunoreactivity. To examine further whether the pro-inflammatory effect of castration was related to androgen deprivation by androgen receptor blockade, we examined inflammation and TGF-β1 immunoreactivity in irradiated mice with or without flutamide administration. However, androgen deprivation by flutamide, a blocker of androgen receptor, did not augment RT-induced inflammation. Therefore, we propose there should be other mechanisms are responsible for the augmented pro-inflammatory effects in castrated mice, rather than flutamide treatment.
NF-κB is activated by many different stimuli such as radiation and oxidative stress, which induce the phosphorylation of IκB . NF-κB activation is widely recognized as a key regulator of immune and inflammatory responses. Several studies have shown that NF-κB is a key transcription factor in the activation of genes encoding inflammatory cytokines including IL-1β, TNF-α, TGF-β1, IL-6 and IL-8 and induces their expression [30, 31]. Thus, NF-κB is thought to have a pivotal role in the induction of cytokine expression in the inflammatory response after irradiation. We found that DNA binding activity of NF-κB after irradiation was more augmented by castration than flutamide administration. Similarly, Shimizu et al.  reported that the attenuation of pro-inflammatory cytokines production by flutamide is associated with inhibiting NF-κB- DNA complex. Furthermore, several studies [33, 34] have reported that COX-2 is the important gene regulated by NF-κB activation and mediating the subsequent inflammation. According to Cai's report , combined androgen blockade induced the increased COX-2. In the present study, the increased NF-κB binding in castrated mice was associated with the more induction of COX-2 expression, in contrast to flutamide treatment. Although the mechanism responsible for this response still requires further elucidation, the increased NF-κB binding may at least partially explain why androgen deprivation by castration modulated RT-induced inflammation.
Radiation-induced fibrosis is an untoward effect of high dose therapeutic and inadvertent exposure to ionizing radiation. TGF-β is the master switch cytokine, which once activated after radiation promotes a train of cellular events that result in radiation-induced fibrosis [20, 36]. Moreover, TGF-β1 was reported to play an important role in the induction of epithelial-mesenchymal transition (EMT) [24–28]. Several studies have reported that TGF-β1 induces EMT via the PI3K/Akt signaling pathways [37, 38], which are associated with loss of β-catenin, a hallmark of EMT. In this study, decreased β-catenin with concurrent increases in vimentin and p-Akt were observed in murine intestine with TGF-β1 treatment, similar to that induced by irradiation. Moreover, pretreatment with wortmannin, which is a general inhibitor of PI3K family proteins, led to attenuate the increased p-Akt and vimentin and decreased β-catenin stimulated by irradiation and TGF-β1. Based on these findings, we propose that induction of EMT by TGF-β1 via the PI3K/Akt signaling pathway may contribute at least partially to RT-induced fibrosis.
In summary, we show that androgen deprivation by castration, rather than flutamide administration, augmented the RT-induced inflammatory response. In contrast to flutamide, the increased NF-κB activity and subsequent elevated COX-2 by castration might be the underlying mechanism responsible to the increase in RT-induced inflammatory response. Our data also indicate that RT-induced fibrosis is related to TGF-β1-induced EMT and is probably mediated via the PI3K/Akt signaling pathway. These results suggest that sex differences play an important role in the inflammatory response. When androgen deprivation is concurrently used with irradiation treatment, the modulating effects on the RT-induced inflammation and fibrosis should take into consideration for complications associated radiotherapy. In future, we will further investigate the modulating effect of androgen on the RT- induced chronic toxicity with longer follow- up.
The work was supported by CMRPG 660171 from Chang Gung Memorial Hospital
- Bandy LC, Clarke-Pearson DL, Soper JT, Mutch DG, MacMillan J, Creasman WT: Long-term effects on bladder function following radical hysterectomy with and without postoperative radiation. Gynecol Oncol. 1987, 26: 160-168. 10.1016/0090-8258(87)90269-1.View ArticlePubMed
- Barter JF, Soong SJ, Shingleton HM, Hatch KD, Orr JW: Complications of combined radical hysterectomy-postoperative radiation therapy in women with early stage cervical cancer. Gynecol Oncol. 1989, 32: 292-296. 10.1016/0090-8258(89)90627-6.View ArticlePubMed
- Rubin P, Johnston CJ, Williams JP: A perpetual cascade of cytokines post irradiation leads to pulmonary fibrosis. Int J Radiat Oncol Biol Phys. 1995, 33: 99-109.View ArticlePubMed
- Rodemann HP, Blaese MA: Responses of normal cells to ionizing radiation. Semin Radiat Oncol. 2007, 17: 81-88. 10.1016/j.semradonc.2006.11.005.View ArticlePubMed
- Brush J, Lipnick SL, Phillips T, Sitko J, McDonald JT, McBride WH: Molecular mechanisms of late normal tissue injury. Semin Radiat Oncol. 2007, 17: 121-30. 10.1016/j.semradonc.2006.11.008.View ArticlePubMed
- Fu XL, Huang H, Bentel G, Clough R, Jirtle RL, Kong FM, Marks LB, Anscher MS: Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V(30) and transforming growth factor beta. Int J Radiat Oncol Biol Phys. 2001, 50: 899-908. 10.1016/S0360-3016(01)01524-3.View ArticlePubMed
- Arpin D, Perol D, Blay JY, Falchero L, Claude L, Vuillermoz-Blas S, Martel-Lafay I, Ginestet C, Alberti L, Nosov D, Etienne-Mastroianni B, Cottin V, Perol M, Guerin JC, Cordier JF, Carrie C: Early variation of circulating interleukin-6 and interleukin-10 levels during thoracic radiotherapy are predictive radiation pneumonitis. J Clin Oncol. 2005, 23: 8748-8756. 10.1200/JCO.2005.01.7145.View ArticlePubMed
- McBride WH: Cytokine cascades in late normal tissue radiation responses. Int J Radiat Oncol Biol Phys. 1995, 33: 233-234.View ArticlePubMed
- Anscher MS, Vujaskovic Z: Mechanisms and potential targets for prevention and treatment of normal tissue injury after radiation therapy. Seminar in Oncol. 2005, 32 (Suppl 3P): S86-91. 10.1053/j.seminoncol.2005.03.015.View Article
- Angele MK, Schwacha MG, Ayala A, Chaudry IH: Effect of gender and sex hormones on immune responses following shock. Shock. 2000, 14: 81-90. 10.1097/00024382-200014020-00001.View ArticlePubMed
- Knöferl MW, Angele MK, Diodato MD, Schwacha MG, Ayala A, Cioffi WG, Bland KI, Chaudry IH: Female sex hormones regulate macrophage function after trauma-hemorrhage and prevent increased death rate from subsequent sepsis. Ann Surg. 2002, 235: 105-112. 10.1097/00000658-200201000-00014.PubMed CentralView ArticlePubMed
- Homma H, Hoy E, Xu DZ, Lu Q, Feinman R, Deitch EA: The female intestine is more resistant than the male intestine to gut injury and inflammation when subjected to conditions associated with shock states. Am J Physiol Gastrointest Liver Physiol. 2005, 288: G466-472. 10.1152/ajpgi.00036.2004.View ArticlePubMed
- Yokoyama Y, Kuebler JF, Matsutani T, Schwacha MG, Bland KI, Chaudry IH: Mechanism of the salutary effects of 17beta-estradiol following trauma-hemorrhage: direct downregulation of Kupffer cell proinflammatory cytokine production. Cytokine. 2003, 21: 91-97. 10.1016/S1043-4666(03)00014-0.View ArticlePubMed
- Hildebrand F, Hubbard WJ, Choudhry MA, Thobe BM, Pape HC, Chaudry IH: Effects of 17beta-estradiol and flutamide on inflammatory response and distant organ damage following trauma-hemorrhage in metestrus females. J Leukoc Biol. 2006, 80: 759-765. 10.1189/jlb.0406254.View ArticlePubMed
- Ashcroft GS, Mills SJ: Androgen receptor-mediated inhibition of cutaneous wound healing. J Clin Invest. 2002, 110: 615-624.PubMed CentralView ArticlePubMed
- Gilliver SC, Ashworth JJ, Mills SJ, Hardman MJ, Ashcroft GS: Androgens modulate the inflammatory response during acute wound healing. J Cell Sci. 2006, 119: 722-732. 10.1242/jcs.02786.View ArticlePubMed
- Roach M: Current status of androgen suppression and radiotherapy for patients with prostate cancer. J Steroid Biochem Mol Biol. 1999, 69: 239-45. 10.1016/S0960-0760(99)00040-0.View ArticlePubMed
- Lee AK: Radiation therapy combined with hormone therapy for prostate cancer. Semin Radiat Oncol. 2006, 16: 20-8. 10.1016/j.semradonc.2005.08.003.View ArticlePubMed
- Toth B, Alexander M, Daniel T, Chaudry IH, Hubbard WJ, Schwacha MG: The role of gammadelta T cells in the regulation of neutrophil-mediated tissue damage after thermal injury. J Leukoc Biol. 2004, 76: 545-552. 10.1189/jlb.0404219.View ArticlePubMed
- Fleckenstein K, Gauter-Fleckenstein B, Jackson IL, Rabbani Z, Anscher M, Vujaskovic Z: Using biological markers to predict risk of radiation injury. Semin Radiat Oncol. 2007, 17: 89-98. 10.1016/j.semradonc.2006.11.004.View ArticlePubMed
- Baeuerle PA, Henkel T: Function and activation of NF-kappa B in the immune system. Annu Rev Immunol. 1994, 12: 141-179.View ArticlePubMed
- Linard C, Marquette C, Mathieu J, Pennequin A, Clarencon D, Denis M: Acute induction of inflammatory cytokine expression after gamma-irradiation in the rat: effect of an NF-κB inhibitor. Int J Radiat Oncol Biol Phys. 2004, 58: 427-434.View ArticlePubMed
- Chen MF, Keng PC, Lin PY, Liao SK, Wu CT, Chen WC: Differential effects of caffeic acid phenethyl ester for normal lung and lung cancer treated with irradiation: in vitro and in vivo study. BMC Cancer. 2005, 5: 158-10.1186/1471-2407-5-158.PubMed CentralView ArticlePubMed
- Anscher MS, Kong FM, Andrews K, Clough R, Marks LB, Bentel G, Jirtle RL: Plasma transforming growth factor beta 1 as a predictor of radiation pneumonitis. Int J Radiat Oncol Biol Phys. 1998, 41: 1029-1035.View ArticlePubMed
- Chen Y, Williams J, Ding I, Hernady E, Liu W, Smudzin T, Finkelstein JN, Rubin P, Okunieff P: Radiation pneumonitis and early circulatory cytokines markers. Semin Radiat Oncol. 2002, 12: 26-33. 10.1053/srao.2002.31360.View ArticlePubMed
- Chen Y, Rubin P, Williams J, Hernady E, Smudzin T, Okunieff P: Circulating IL-6 as a predictor of radiation pneumonitis. Int J Radiat Oncol Biol Phys. 2001, 49: 641-646.View ArticlePubMed
- Brink N, Szamel M, Young AR, Wittern KP, Bergemann J: Comparative quantification of IL-1β, IL-10, IL-10r, TNF and IL-7 mRNA levels in UV-irradiated human skin in vivo. Inflamm Res. 2000, 49: 290-296. 10.1007/PL00000209.View ArticlePubMed
- Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J: Adenovirus- mediated gene transfer of active transforming factor-β induces prolonged severe fibrosis in rat lung. J Clin Invest. 1997, 100: 768-766. 10.1172/JCI119590.PubMed CentralView ArticlePubMed
- Rube CE, Uthe D, Schmid KW, Richter KD, Wessel J, Schuck A, Willich N, Rube C: Dose-dependent induction of transforming growth factor β (TGF-β) in the lung tissue of fibrosis-prone mice after thoracic irradiation. Int J Radiat Oncol Biol Physics. 2000, 47: 1033-1042.View Article
- Neurath MF, Becker C, Barbulescu K: NF-κB in immune and inflammatory responses in the gut. Gut. 1998, 43: 856-860.PubMed CentralView ArticlePubMed
- Phal HL: Activators and target genes of Rel/NF-κB transcription factors. Oncogene. 1999, 18: 6853-6866. 10.1038/sj.onc.1203239.View Article
- Shimizu T, Yu HP, Hsieh YC, Choudhry MA, Suzuki T, Bland KI, Chaudry IH: Flutamide attenuates pro-inflammatory cytokine production and hepatic injury following trauma-hemorrhage via estrogen receptor-related pathway. Ann Surg. 2007, 245 (2): 297-304. 10.1097/01.sla.0000232523.88621.17.PubMed CentralView ArticlePubMed
- Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS: Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res. 2001, 480–481: 243-268.View ArticlePubMed
- Chiu FL, Lin JK: Tomatidine inhibits iNOS and COX-2 through suppression of NF-kappaB and JNK pathways in LPS-stimulated mouse macrophages. FEBS Lett. 2008, 582: 2407-12. 10.1016/j.febslet.2008.05.049.View ArticlePubMed
- Cai Y, Lee YF, Li G, Liu S, Bao BY, Huang J, Hsu CL, Chang C: A new prostate cancer therapeutic approach: combination of androgen ablation with COX-2 inhibitor. Int J Cancer. 2008, 123: 195-201. 10.1002/ijc.23481.View ArticlePubMed
- Kovacs EJ: Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol Today. 1991, 12: 17-23. 10.1016/0167-5699(91)90107-5.View ArticlePubMed
- Cho HJ, Baek KE, Saika S, Jeong MJ, Yoo J: Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway. Biochem Biophys Res Commun. 2007, 353: 337-343. 10.1016/j.bbrc.2006.12.035.View ArticlePubMed
- Gal A, Sjoblom T, Fedorova L, Imreh S, Beug H, Moustakas A: Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis. Oncogene. 2008, 27: 1218-1230. 10.1038/sj.onc.1210741.View ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/92/prepub
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