Signal transduction processes in response to low dose ionizing radiation doses expected during space flight

Tracy Criswell1, Eva Goetz2, Dmitri Klokov3, Yonglong Zou4, Xiuquan Luo4, and David A. Boothman4,*

4Departments of Radiation Oncology and Pharmacology, UT Southwestern Medical Center at Dallas, 6001 Forest Park Drive, ND2.210K, Dallas, Texas 75230-8807 David.Boothman@UTSouthwestern.edu

Current Author Affiliations: 1TC: Wake Forest Institute for Regenerative Medicine, Wake Forest, NC Richard H. Dean

Biomedical Building, 391 Technology Way Winston-Salem, NC 27101 2EG: Department of Medicine, Dana-Farber Cancer Institute, DA 1524, 44 Binney Street, Boston, MA

02115 3DK: 1Radiological Protection Research and Instrumentation Branch, Chalk River Laboratories,

Atomic Energy Canada Limited, Chalk River, ON, USA; *To Whom correspondence should be addressed Abstract A major issue affecting our plans to visit and one day inhabit other planets is that once outside the protective influence of our atmosphere, astronauts will experience dramatically increased exposure to ionizing radiation (IR). While shielding spacecraft can minimize effects, exposure to significant levels of both high and low linear energy transfer (LET) IR is inevitable. Understanding the effects of these doses at a mechanistic level is absolutely required to understand both dosimetry and long-term biological effects. While determining the physical dose is relatively straightforward using well worked out physic techniques, the biological effects of such exposures are far more complicated. Evidence now supports the notion that in addition to DNA repair sensors, other cellular processes (non-DNA targets) can be activated in response to low doses of IR (0.3 to 0.6 Gy), doses approximating levels that astronauts would experience in prolonged space travel, such as in a trip to Mars (~1.2 Gy total absorbed dose at 0.3-0.6 Gy over 2 years). Furthermore, IR exposure to one cell can stimulate bystander effects in neighboring cells. For example, we will use the induction of insulin-like growth factor 1-secretory clusterin expression (IGF-1-sCLU) to discuss low dose and low dose rate effects within tissue microenvironments, including within an emerging (and most likely unknown at the time of departure to Mars). It is clear that examining cellular responses to high doses of IR (e.g., clinically-relevant doses) cannot be used to ‘extrapolate down’ to low doses of IR (ranging from

axis as a mechanistic marker of the long-term consequences of low doses of both low and high LET IR.

Introduction Signal transduction processes have evolved to coordinate cellular responses following endogenous or exogenous insults to the cell. By definition (from Webster dictionary), ‘signal transduction’ means: (i) Signal: n, a sign for giving notice, especially at a distance; adj, memorable, remarkable; and (ii) Transduction: the transforming of one form of energy into another, as by the sensory mechanisms of the body. Thus, signal transduction processes, in the context of response to IR, means that the cell detects and amplifies sensory signals to stimulate a response, such as to stimulate growth, change cellular metabolism, stop cell division, induce expression of proteins required for repair (e.g., stimulated autophagy) or cell death (e.g., stress-induced premature senescence, SIPS); compared to most other exogenous insults, IR is a poor inducer of programmed cell death (i.e., apoptosis) in human epithelial cells. Importantly, signal transduction processes are context-dependent, and therefore, its processes cannot be investigated in their entirety in vitro using isolated cells in culture with 5-10% fetal bovine serum at 21% oxygen levels in culture. In addition, since various cell types have different sensitivities to IR, the cell type being investigated matters. The extent and type of IR used is also a major factor in the overall cell’s response. The cell cycle state is also a major determinant, and depending on what state the cell is in strongly affects cell responses to IR. Most cells in the human body, even with a growing tumor, are not dividing. Therefore, using logarithmically growing tumor cells to mimic effects of IR exposures is fraught with problems in interpreting physiological meaning. In conclusion, while initial investigations are commonly performed to determine mechanism using isolated normal or tumor cells in culture, the overall responses have to be performed in vivo using mouse models to simulate an appropriate microenvironment. A major determinant in understanding signal transduction processes is the dose used and damage created. An examination of the literature indicates that >1000 articles on IR-induced signal transduction processes have been published since 1995. The vast majority of publications (~99%), however, have used >2 Gy as doses (Figure 1). Approximately 1% of publications, or ~10 publications have used doses of

that may be experienced in space-related travel. Many studies have attempted to use high doses of IR, thinking that the responses noted can be simply extrapolated down to low doses. There are, in fact, few examples of successful extrapolation from high to low doses. In fact, there are very few examples of signal transduction processes activated by low doses of low LET IR (

DNA mismatch repair (MMR) system and DNA base excision repair (BER). These DNA repair systems detect extensive formation of base damage, including elevated levels of the mutagenic DNA lesion, 8-oxoguanine (8-OG). Once activated, these DNA sensing/repair systems cause cell cycle checkpoint alterations, altered gene expression, and changes in cellular metabolism (Figure 2). Depending on the cell type and extent of damage, cells can also respond by undergoing senescence or cell death, although IR is a poor inducer of apoptosis. DNA damage is not the only ‘signal’ created by low doses of IR that can stimulate responses in cells. Other lesions include lipid peroxidation, membrane alterations (pore composition and size changes), damage-induced conformational activation of receptors, and growth factors 3-15. These signals can ‘transduce’ (transfer) signals, including various post-translational modifications (i.e., phosphorylation, ubiquitinylation, sumoylation, S-nitrosylation), changes in protein conformation, alterations in second messengers (Ca2+, NO, and nucleotides (e.g., AMP) 3-11, 16-32. Such signal are, in general, secondary to the more dramatic responses mediated by DNA damage and sensory mechanisms of damage detection (Figure 2). Use of signal transduction processes for biodosimetry. Many researchers have been attempting to use signal transduction processes for biodosimetry 33-42, with the formation of gamma-H2AX (γH2AX) being a major endpoint of analyses. While such analyses are possible and can be a good measure of exposure, the short half-life and problems of basal level differences in humans are major issues. Furthermore, the biological significance of these early responses is not known in terms of long-term consequences. The short half-life of the responses also limits the use of these endpoints for biodosimetry. Signal transduction amplification: the IGF-1-sCLU secreted response Many of the issues discussed above are highlighted by our work with the IGF-1-sCLU expression response, which occurs in a complex, yet amplified manner to alter the tumor microenvironment. However, the mechanism of regulation of sCLU induction in response to IR (including high LET IR exposures), involving IGF-1 regulation was determined in vitro using a variety of normal and tumor cells in culture. sCLU/XIP8 induction. In the late 1980’s, using two-dimensional gel electrophoresis, a series of x-ray-inducible proteins (XIPs) were identified from growth-arrested human melanoma cells 43, 44, and their mRNAs isolated using subtractive hybridization 45. Steady state protein analyses demonstrated that XIP8 was secretory clusterin (sCLU, aka., apolipoprotein J), a secreted protein that mediated extracellular heat shock responses in which the protein cleared unfolded proteins created by injury, including reactive oxygen species (ROS)-induced damage 46-48. Importantly, sCLU levels increased dramatically after low doses of high or low LET IR (>0.001 Gy) 48. Second, induction was robust (>10,000-fold) and delayed, appearing in all cells after IR exposure at >48 h, with peak levels noted commonly at 96 h or later 48-56. This delayed response highlights the need, in general, to examine gene expression at both early (seconds to minutes), as well as later times (hours to days) after IR. Interestingly, peak induction times could be increased by higher doses of IR (or high LET), or lengthened by decreasing the doses of IR

Signal transduction processes in response to low dose ionizing radiation doses expected during space flight. Criswell T, Goetz E, Klokov D, Zou Y, Luo X, Boothman D. https://three.jsc.nasa.gov/articles/Boothman.pdf. Date posted: 05-02-2013.

used 56. The relative steady state levels of sCLU could be used, therefore, for biodosimetry reading, if induction were defined in vivo. IGF-1 controls sCLU induction. Defining the regulatory mechanisms by which a delayed inducible protein is controlled is not trivial. Working upstream and using various DNA damaging agents, we noted that while most DNA damaging agents caused induction of sCLU, those agents causing the greatest stimulation also were those that also dramatically induced ATM 50, 57. Further research demonstrated that sCLU was induced in wild-type, but not isogenetic cells in which ATM activity was deficient (i.e., AT cells corrected or not with ATM) 58. In contrast, loss of DNA-PK, ATR or c-abl did not influence sCLU. In addition, loss of functional p53 caused uncontrolled elevated basal levels of sCLU 53, 57, 58, suggesting that functional p53 (a tumor suppressor) negatively regulated expression of sCLU. We ultimately showed that sCLU was, in fact, controlled by IGF-1 expression and that all of the regulation of sCLU was controlled by regulating IGF-1 expression. Delineating IGF-1-sCLU expression mechanism in vitro. Use of genetic manipulation is key for investigating the mechanisms underlying the control of a gene expression axis, and without using select specific genetic alterations mechanisms are not likely to be accurately delineated. This is especially true when using inhibitors, which should only be combined with genetic alterations, to investigate given mechanisms. Finally, ‘circular genetics’ must be used in which genes are knocked down or deleted, and the cells are then isogenically ‘corrected’ using mutant versions of the cDNAs. An alternative approach is to knockdown a given gene using 3’- or 5’-untranslated regions (UTRs), where wild-type cDNAs can be re-inserted for ‘correction’ of isogenic cells. Using such techniques, as well as dominant-negative constructs or constitutively-active expression vectors, chromosome immunoprecipitation (ChIP) technology, DNA pull-down analyses, select point mutations of promoters and select use of specific inhibitors, we showed that IGF-1 was regulated by p53 in association with nuclear factor-Y (NF-Y) family

Figure 3. Mechanism for IGF-1-sCLU induction in response to low doses of IR.

Signal transduction processes in response to low dose ionizing radiation doses expected during space flight. Criswell T, Goetz E, Klokov D, Zou Y, Luo X, Boothman D. https://three.jsc.nasa.gov/articles/Boothman.pdf. Date posted: 05-02-2013.

members (Figure 3) 58. In its resting state, IGF-1 is suppressed by p53/NF-YA binding 50, 57, 58. Following the activation of ATM by IR, elevated p21 levels stimulate the inhibition of p53 phosphorylation through cdc2 (cyclin-dependent kinase) inhibition, which is required for the phosphorylation of NF-YA and association with p53. Dephosphorylated NF-YA binds less tightly to p53, decreasing p53/NF-YA binding to the IGF-1 promoter. Increased p53 stabilization results in minor induction of Mdm2, which further degrades p53 through its ubiquitin ligase activity 59. Increased levels of IGF-1, stimulate the IGF-1R/Src/MAPK/Egr-1 pathway, leading to Egr-1 protein binding to the clusterin promoter, resulting in CLU promoter transactivation and finally sCLU protein induction. IGF-1 stimulation of IGF-1R receptor also stimulated the AKT1 enzyme in the cell, which positively feeds back to stimulate phosphorylation of Mdm2, leading to further p53 degradation and further stimulation of sCLU expression levels. Expression of sCLU is a prosurvival factor, and suppression of its levels can greatly enhance lethality of clinically-relevant IR doses 51, 54, 55, 60. IGF-1-sCLU regulation in vivo. Having delineated the mechanism of sCLU induction in vitro, we delineated its induction in vivo. Surprisingly, the mechanism of induction in vivo occurred un-expectantly by the activation of transforming growth factor beta1 (TGFß1) 49, 56, and not by the mechanism delineated in vitro as shown in Figure 3. In vivo, especially at low doses, IR activates TGFß1, which ultimately controls IGF-1-sCLU expression. TGFß1 simultaneously induces the transcriptional activation and expression of Mdm2 and LEF1 (Zou et al., in preparation). Mdm2 induces p53 degradation, while LEF1 binds ß-catenin and these together cause the stimulation of the IGF-1-sCLU expression axis (Zou et al., in preparation). Induction was responsive in select tissue (colon, thymus, spleen, breast and bone marrow) in a robust and prolonged fashion so as to significantly alter the microenvironment of the irradiated tissue 56. IGF-1 can stimulate a host of tyrosine receptor kinases, and sCLU can bind numerous receptors, stimulating some and inhibiting others. Further research is ongoing to delineate the mechanism of induction in vivo and functions of IGF-1-sCLU in the microenvironment. Conclusions: Overall, there are significant alterations in the microenvironment in response to low or high doses of IR, which are under intense investigation in our laboratory as well as many others. These responses are likely to become even more important after low doses of high LET IR, where there will be sparse tracks and non-uniform damaged cells. Furthermore, responses in normal tissue (devoid of tumor cells) will likely be very different from microenvironments containing micro-nodules of benign or possibly more advanced tumors that are not detectable prior to a trip to Mars. Under these conditions low dose IR could induce IGF-1-sCLU responses that would promote the growth of these benign microenvironments. In terms of our discussion in this report, the major changes that occur are: (i) activation of TGFß1, enhanced levels of IGF-1 and dramatic and long-lasting secretion of sCLU. All of these affect the irradiated cells, but by definition, also affect the microenvironment as a whole since all three are secreted, extracellular proteins. Pre-irradiation of a given tissue can cause IGF-1-sCLU expression for months in the colons of irradiated mice after as little as

regimens during radiotherapies. Expression of the IGF-1-sCLU expression axis can arise from stroma and/or tumor tissue directly following low or high LET IR and at various doses56. Delineating these responses within a given microenvironment is essential for risk assessment, especially in individuals with given genetic variations.

Literature Cited: 1. Sahijdak, W.M., Yang, C.R., Zuckerman, J.S., Meyers, M. & Boothman, D.A. Alterations in

transcription factor binding in radioresistant human melanoma cells after ionizing radiation. Radiat Res 138, S47-51 (1994).

2. Criswell, T., Leskov, K., Miyamoto, S., Luo, G. & Boothman, D.A. Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation. Oncogene 22, 5813-5827 (2003).

3. Natarajan, M., Gibbons, C.F., Mohan, S., Moore, S. & Kadhim, M.A. Oxidative stress signalling: a potential mediator of tumour necrosis factor alpha-induced genomic instability in primary vascular endothelial cells. Br J Radiol 80 Spec No 1, S13-22 (2007).

4. Shao, C., Folkard, M. & Prise, K.M. Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells. Oncogene 27, 434-440 (2008).

5. Furweger, C., Hajek, M., Vana, N., Kodym, R. & Okayasu, R. Cellular signal transduction events as a function of linear energy transfer (LET). Radiat Prot Dosimetry 126, 418-422 (2007).

6. Wittlinger, M., Grabenbauer, G.G., Sprung, C.N., Sauer, R. & Distel, L.V. Time and dose-dependent activation of p53 serine 15 phosphorylation among cell lines with different radiation sensitivity. Int J Radiat Biol 83, 245-257 (2007).

7. Gil'iano, N., Konevega, L.V., Stepanov, S.I., Semenova, E.G. & Noskin, L.A. [The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles]. Radiats Biol Radioecol 47, 151-157 (2007).

8. Tichy, A. et al. Gamma-radiation-induced ATM-dependent signalling in human T-lymphocyte leukemic cells, MOLT-4. Acta Biochim Pol 54, 281-287 (2007).

9. Long, X.H. et al. Dose-dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells. Int J Mol Med 19, 607-615 (2007).

10. Portess, D.I., Bauer, G., Hill, M.A. & O'Neill, P. Low-dose irradiation of nontransformed cells stimulates the selective removal of precancerous cells via intercellular induction of apoptosis. Cancer Res 67, 1246-1253 (2007).

11. Colen, C.B. et al. Metabolic remodeling of malignant gliomas for enhanced sensitization during radiotherapy: an in vitro study. Neurosurgery 59, 1313-1323; discussion 1323-1314 (2006).

12. Mariotti, L.G., Bertolotti, A., Ranza, E., Babini, G. & Ottolenghi, A. Investigation of the mechanisms underpinning IL-6 cytokine release in bystander responses: the roles of radiation dose, radiation quality and specific ROS/RNS scavengers. Int J Radiat Biol 88, 751-762 (2012).

13. Joo, H.M. et al. The effects of low-dose ionizing radiation in the activated rat basophilic leukemia (RBL-2H3) mast cells. J Biol Chem 287, 27789-27795 (2012).

14. Hei, T.K., Zhao, Y., Zhou, H. & Ivanov, V. Mechanism of radiation carcinogenesis: role of the TGFBI gene and the inflammatory signaling cascade. Adv Exp Med Biol 720, 163-170 (2011).

15. Babicova, A. et al. Early changes in L-arginine-nitric oxide metabolic pathways in response to the whole-body gamma irradiation of rats. Int J Radiat Biol 87, 1067-1073 (2011).

16. Jeggo, P.A. Risks from low dose/dose rate radiation: what an understanding of DNA damage response mechanisms can tell us. Health Phys 97, 416-425 (2009).

17. Cogan, N. et al. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase. Mutat Res 683, 1-8 (2010).

Signal transduction processes in response to low dose ionizing radiation doses expected during space flight. Criswell T, Goetz E, Klokov D, Zou Y, Luo X, Boothman D. https://three.jsc.nasa.gov/articles/Boothman.pdf. Date posted: 05-02-2013.

18. Widel, M., Przybyszewski, W. & Rzeszowska-Wolny, J. [Radiation-induced bystander effect: the important part of ionizing radiation response. Potential clinical implications]. Postepy Hig Med Dosw (Online) 63, 377-388 (2009).

19. Mullenders, L., Atkinson, M., Paretzke, H., Sabatier, L. & Bouffler, S. Assessing cancer risks of low-dose radiation. Nat Rev Cancer 9, 596-604 (2009).

20. Postiglione, I., Chiaviello, A. & Palumbo, G. Twilight effects of low doses of ionizing radiation on cellular systems: a bird's eye view on current concepts and research. Med Oncol 27, 495-509 (2010).

21. Hei, T.K. et al. Mechanism of radiation-induced bystander effects: a unifying model. J Pharm Pharmacol 60, 943-950 (2008).

22. Haydont, V., Riser, B.L., Aigueperse, J. & Vozenin-Brotons, M.C. Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1. Am J Physiol Cell Physiol 294, C1332-1341 (2008).

23. Van Laere, K. et al. Whole-body biodistribution and radiation dosimetry of the human cannabinoid type-1 receptor ligand 18F-MK-9470 in healthy subjects. J Nucl Med 49, 439-445 (2008).

24. Kim, C.S. et al. Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt. Mol Cells 24, 424-430 (2007).

25. Gow, M.D., Seymour, C.B., Byun, S.H. & Mothersill, C.E. Effect of dose rate on the radiation-induced bystander response. Phys Med Biol 53, 119-132 (2008).

26. Suzuki, K., Kodama, S. & Watanabe, M. [Low-dose radiation effects and intracellular signaling pathways]. Yakugaku Zasshi 126, 859-867 (2006).

27. Mothersill, C. & Seymour, C. Radiation-induced bystander and other non-targeted effects: novel intervention points in cancer therapy? Curr Cancer Drug Targets 6, 447-454 (2006).

28. Trosko, J.E., Chang, C.C., Upham, B.L. & Tai, M.H. Low-dose ionizing radiation: induction of differential intracellular signalling possibly affecting intercellular communication. Radiat Environ Biophys 44, 3-9 (2005).

29. Shao, C., Folkard, M., Michael, B.D. & Prise, K.M. Targeted cytoplasmic irradiation induces bystander responses. Proc Natl Acad Sci U S A 101, 13495-13500 (2004).

30. Osuna, L., Coursol, S., Pierre, J.N. & Vidal, J. A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade. Biochem Biophys Res Commun 314, 428-433 (2004).

31. Liu, S. & Xie, F. Involvement of the Ca(2+)-protein kinase C and adenyilate cyclace signal pathways in the activation of thymocytes in response to whole-body irradiation with low dose X-rays. Chin Med Sci J 15, 1-7 (2000).

32. Kiyoshi, O. [Role of insulin-like growth factor-I receptor (IGF-IR) in survival kinetics and radioresistance of mouse embryo fibroblasts in a hypoxic environment]. Kokubyo Gakkai Zasshi 69, 107-118 (2002).

33. Zarybnicka, L. et al. Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats. Int J Radiat Biol 89, 110-117 (2013).

34. Ghardi, M., Moreels, M., Chatelain, B., Chatelain, C. & Baatout, S. Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells. Int J Mol Med 29, 769-780 (2012).

35. Ivashkevich, A., Redon, C.E., Nakamura, A.J., Martin, R.F. & Martin, O.A. Use of the gamma-H2AX assay to monitor DNA damage and repair in translational cancer research. Cancer Lett 327, 123-133 (2012).

36. Garty, G. et al. The RABiT: a rapid automated biodosimetry tool for radiological triage. II. Technological developments. Int J Radiat Biol 87, 776-790 (2011).

37. Sak, A. & Stuschke, M. Use of gammaH2AX and other biomarkers of double-strand breaks during radiotherapy. Semin Radiat Oncol 20, 223-231 (2010).

38. Roch-Lefevre, S. et al. Quantification of gamma-H2AX foci in human lymphocytes: a method for biological dosimetry after ionizing radiation exposure. Radiat Res 174, 185-194 (2010).

Signal transduction processes in response to low dose ionizing radiation doses expected during space flight. Criswell T, Goetz E, Klokov D, Zou Y, Luo X, Boothman D. https://three.jsc.nasa.gov/articles/Boothman.pdf. Date posted: 05-02-2013.

39. Garty, G. et al. The RABIT: a rapid automated biodosimetry tool for radiological triage. Health Phys 98, 209-217 (2010).

40. Bhogal, N. et al. Late residual gamma-H2AX foci in murine skin are dose responsive and predict radiosensitivity in vivo. Radiat Res 173, 1-9 (2010).

41. Andrievski, A. & Wilkins, R.C. The response of gamma-H2AX in human lymphocytes and lymphocytes subsets measured in whole blood cultures. Int J Radiat Biol 85, 369-376 (2009).

42. Marchetti, F., Coleman, M.A., Jones, I.M. & Wyrobek, A.J. Candidate protein biodosimeters of human exposure to ionizing radiation. Int J Radiat Biol 82, 605-639 (2006).

43. Boothman, D.A., Odegaard, E., Yang, C.R., Hosley, K. & Mendonca, M.S. Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy. Hum Exp Toxicol 17, 448-453 (1998).

44. Boothman, D.A., Bouvard, I. & Hughes, E.N. Identification and characterization of X-ray-induced proteins in human cells. Cancer Res 49, 2871-2878 (1989).

45. Boothman, D.A., Meyers, M., Fukunaga, N. & Lee, S.W. Isolation of x-ray-inducible transcripts from radioresistant human melanoma cells. Proc Natl Acad Sci U S A 90, 7200-7204 (1993).

46. Trougakos, I.P., Djeu, J.Y., Gonos, E.S. & Boothman, D.A. Advances and challenges in basic and translational research on clusterin. Cancer Res 69, 403-406 (2009).

47. Kalka, K., Ahmad, N., Criswell, T., Boothman, D. & Mukhtar, H. Up-regulation of clusterin during phthalocyanine 4 photodynamic therapy-mediated apoptosis of tumor cells and ablation of mouse skin tumors. Cancer Res 60, 5984-5987 (2000).

48.Yang, C.R. et al. Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death. Proc Natl Acad Sci U S A 97, 5907-5912 (2000).

49. Klokov, D. et al. IR-inducible clusterin gene expression: a protein with potential roles in ionizing radiation-induced adaptive responses, genomic instability, and bystander effects. Mutat Res 568, 97-110 (2004).

50. Criswell, T. et al. Delayed activation of insulin-like growth factor-1 receptor/Src/MAPK/Egr-1 signaling regulates clusterin expression, a pro-survival factor. J Biol Chem 280, 14212-14221 (2005).

51. Shannan, B. et al. Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer. Cell Death Differ 13, 12-19 (2006).

52. Boothman, D.A. & Reichrath, J. New basic science initiatives for improved understanding of radiation-induced multi-organ dysfunction syndrome (MODS). BJR Suppl 27, 157-160 (2005).

53. Araki, S. et al. Clusterin proteins: stress-inducible polypeptides with proposed functions in multiple organ dysfunction. BJR Suppl 27, 106-113 (2005).

54. Shannan, B. et al. Clusterin (CLU) and melanoma growth: CLU is expressed in malignant melanoma and 1,25-dihydroxyvitamin D3 modulates expression of CLU in melanoma cell lines in vitro. Anticancer Res 26, 2707-2716 (2006).

55. Shannan, B., Seifert, M., Boothman, D.A., Tilgen, W. & Reichrath, J. Clusterin and DNA repair: a new function in cancer for a key player in apoptosis and cell cycle control. J Mol Histol 37, 183-188 (2006).

56. Klokov, D. et al. Low dose IR-induced IGF-1-sCLU expression: a p53-repressed expression cascade that interferes with TGFbeta1 signaling to confer a pro-survival bystander effect. Oncogene 32, 479-490 (2013).

57. Criswell, T., Klokov, D., Beman, M., Lavik, J.P. & Boothman, D.A. Repression of IR-inducible clusterin expression by the p53 tumor suppressor protein. Cancer Biol Ther 2, 372-380 (2003).

58. Goetz, E.M. et al. ATM-dependent IGF-1 induction regulates secretory clusterin expression after DNA damage and in genetic instability. Oncogene 30, 3745-3754 (2011).

59. Araki, S. et al. TGF-beta1-induced expression of human Mdm2 correlates with late-stage metastatic breast cancer. J Clin Invest 120, 290-302 (2010).

60. Shannan, B., Seifert, M., Boothman, D.A., Tilgen, W. & Reichrath, J. Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro. J Steroid Biochem Mol Biol 103, 721-725 (2007).

Signal transduction processes in response to low dose ionizing radiation doses expected during space flight. Criswell T, Goetz E, Klokov D, Zou Y, Luo X, Boothman D. https://three.jsc.nasa.gov/articles/Boothman.pdf. Date posted: 05-02-2013.