Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop IV

Abstract

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Title: Secretory clusterin (sCLU) expression as a sign of genomic instability, and a potential promoter of instability in bystander cells.

Authors: T. Criswell, M. Beman, S. Araki, K. Leskov, D. Klokov, and DA.
Boothman.

Institutions: Departments of Radiation Oncology, Pharmacology & Pathology, Lab of Molecular Stress Responses, Case Western Reserve University, Cleveland, OHIO 44106-4942; dab30@po.cwru.edu

Expression of the secretory form of clusterin (sCLU) is a sensitive marker of genetic instability created after extremely low doses of ionizing radiation (IR). In fact, up-regulation (>2-fold) of sCLU mRNA and protein in response to IR doses as low as 2 cGy have been reported (Yang et al., PNAS, 2000; Criswell et al., Cancer Biology and Therapy, 2003), with >200-fold after 1 Gy or more, 48-72 h after exposure. The CLU promoter is so responsive to low doses of IR that we are exploiting IR-inducible sCLU levels as a sensitive measure of damage, and have developed a ‘biodosimeter’, wherein real-time bioimaging of CLU promoter activity using human MCF-7 breast cancer cells with a stably integrated CLU promoterluciferase reporter construct in culture or as xenografts in nude mice can be monitored.

A transgenic animal with an incorporated CLU promoter-luciferase cassette that acts as a sentinel of low dose IR or other agent exposure, is also being generated. Unlike the pro-death nuclear form of the clusterin protein (nCLU) (Yang et al., NAR, 1999; PNAS, 2000; Leskov et al., 2001; JBC, 2003), the secreted form of clusterin (sCLU) is a pro-survival factor, as recent small interfering RNA (siRNA) gene repression studies from our laboratory have demonstrated (Fig. 1, Leskov et al., In Prep., 2004). Since large amounts of sCLU are secreted into the media and sera of cells and tissues, respectively, after IR (Klokov et al., In Press, 2003), the downstream bystander effects of sCLU could further contribute to a cascade of IR-induced effects, including in non-irradiated cells. Thus, improved understanding of the regulation and functions of sCLU is important for explaining the longterm genetic instability effects of IR in human cells after low doses of IR. This is further highlighted by the inverse relationship between functional p53 and sCLU levels, wherein loss of functional p53 results in dramatic elevations of basal and IR-inducible sCLU levels that we believe further contributes to the survival of IR-treated, genetically unstable cells (Criswell et al., Cancer Biology and Therapy, 2003).

Recent data from our laboratory indicate a complex regulatory pathway of sCLU gene expresson in response to low doses of IR. We are also investigating the downstream effects of sCLU secretion into the media or sera, and we hypothesize that this protein is a major factor in bystander effects reported by others in response to IR, or other types of cytotoxic stress. Our current data indicate the following regulatory processes are stimulated in human cancer and normal cells after low doses of IR that control sCLU gene expression.

sCLU signal transduction processes: Treatment of human MCF-7 or HCT116 p53-/- cancer cells or p53 mutant CT-5 mouse embryonic fibroblast (MEFs) cells with IR doses of 0.02-10 Gy results in the activation of the c-src and p38 MAPK signal transduction pathways, and expression of sCLU. Co-treatment of cells with
chemical inhibitors of these pathways prevented sCLU endogenous expression, as well as inhibited CLU promoter-luciferase activities. Forced transient expression of kinase dead c-src or P38 MAPK also suppressed sCLU expression 48-72 h post-IR treatment. Thus, IR stimulated c-src and p38 MAPK appear to regulate downstream sCLU expression. Experiments are underway to elucidate these signal transduction pathways, as well as the functiona l significance of abrogating these pathways in terms of cell survival and genetic instability (Criswell et al., In Prep., 2003).

The signal transduction process above can be suppressed by wild-type p53. A direct comparison between HCT116 parental cells expressing wild-type p53 and isogenic p53-/- HCT116 cells revealed that only cells lacking functional p53 induced sCLU. Expression of E6 (that binds and degrades p53) resulted in higher basal sCLU
levels, and a more pronounced IR-inducible level of this secreted protein. IR-inducible expression of sCLU and regulation of this protein by p53 was not a result of altered cell cycle checkpoint regulation, since IR-treated HCT116 parental and p21-/- HCT116 cells did not differ in their expression of sCLU (Criswell et al., CBT, 2003).

We are currently testing the theory that IR-inducible levels of sCLU can abrogate TGF-ß1 cell signaling. We recently showed that exposure of wild-type TGF-ß1 RII receptor-containing human colon or breast cancer cells with TGF-ß1 caused dramatic expression of sCLU, with time-course responses identical to those following IR, and growth suppression. In contrast, genetically matched cells lacking the RII receptor were non-responsive to TGF-ß1 treatments in terms of growth suppression and up-regulation of sCLU. Thus, TGF-ß1 can regulate sCLU expression in these cells. Interestingly, the presence or absence of the TGF-ß RII receptor did not affect sCLU protein level induction(Sang’s data?). This indicates that sCLU expression can be regulated by TGF-ß1 or IR, but the regulatory signal transduction processes are different. Since sCLU can bind the RI and RII receptors of TGF-ß1, we hypothesize (and are currently testing the theory) that sCLU represents a negative feedback loop acting to suppress normal TGF-ß1-mediated growth and gene regulation in irradiated or non-irradiated ‘bystander’ cells. This work was supported by DOE grant DE-FG-022179 to DAB.

References:
Criswell, T, Klokov, KS, and Boothman, DA.(2003) Transcriptional repression of clusterin by the p53 tumor suppressor protein. Cancer Biology and Therapy, 2(4): 25-31.

Criswell, T., Leskov, K., Miyamoto, S., Luo, G-B., and Boothman, DA. (2003) IR-inducible transcription factors in mammalian cells at clinically relevant doses. Oncogene, 22(37): 5813-5827

Klokov D, Criswell T, Sampath L, Leskov K, Frinkley K, Araki S, Beman M, Wilson D, and Boothman, DA. Clusterin: a protein with multiple functions as a potential ionizing radiation exposure marker. In: 1st Nagasaki Symposium of
International Consortium for Medical Care of Hibakusha and radiation Life sciences. (Shibata Y, Yamashita S, Watanabe M, Tomonaga M Eds.) Elsevier, Amsterdam, In Press, 2003.

Klokov, D, Kang, S-W, Criswell, T, and Boothman, DA.(2003) Regulation of secretory clusterin levels by TGF -ß1. Cancer Research, In Prep.,

Klokov D, Sampath L, Frinkley K, Wilson D, and Boothman, DA. (2003) Development of a sensitive biodosimeter for the detection of low doses of ionizing radiation. In Prep.
.
Leskov, K, Antonio, S, Criswell, T, Yang, C-R, Kinsella, TJ, Boothman, DA.(2001) Rad. Research 156: 441-442

Leskov, K., Criswell, T.A., Antonio, S. Li, J., Yang, C-R., Kinsella, T.J., and Boothman, D.A. (2001) When X-ray-inducible proteins meet DNA double strand break repair. Seminars in Radiation Oncology 11: 352-372

Leskov, KS, Klokov, DY, Li, J, Kinsella T J, and Boothman, DA. (2003) Synthesis and functional analyses of nuclear clusterin: a cell death protein. J. Biol. Chem. 278: 11590-11600


Sun, W, Sawada, M, Hayes, P, Leskov, K, Boothman, DA, and Matsuyama, S. (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nature Cell Biology 5: 320-329

Yang, C-R, Odegaard, E, Leskov, K, Hosley-Eberlein, K, Criswell, T, Kinsella, TJ, and Boothman, DA. (200) PNAS, USA, 97: 5907-5912