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Supplement: Free Radicals: The Pros and Cons of Antioxidants

Redox-Sensitive Signaling Factors and Antioxidants: How Tumor Cells Respond to Ionizing Radiation¹

Molecular Radiation Oncology Section, National Cancer Institute, Bethesda, MD 20892

²To whom correspondence should be addressed. E-mail: giusd{at}mail.nih.gov.

KEY WORDS: • ionizing radiation • stress response • redox • thioredoxin reductase • thioredoxin • AP-1

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Addressing the molecular aspects of redox signaling and the role of antioxidants in radiation exposure, there is a paradigm that in response to ionizing agents, prosurvival genes are expressed and activation of these genes can alter phenotypes in cells. A model utilizing the AP-1 DNA-binding transcriptional complex, containing one protein from the fos family and one protein from the jun family, illustrates this paradigm (Fig. 1). This complex is activated by various outside factors, such as ionizing radiation (IR),³ that produce oxidative stress. Hydroxyl radicals produced from water by ionizing radiation probably act as a signal to the cell that has been initiated by oxidative stress.

View larger version (32K): [in this window] [in a new window]   FIGURE 1 A model utilizing the AP-1 DNA-binding transcriptional complex, containing 1 protein from the fos family and 1 protein from the jun family.

Antioxidants and oxidative stress activate proteins such as TR/TRX and Ref-1 through modification of sulfur atoms on cysteines, primary targets for redox reactions. The critical redox-sensitive signaling proteins and their cysteines transport a signal from the cytoplasm to the nucleus to turn on the transcription factor. For example, thioredoxin interacts in the nucleus with a second signaling protein, Ref-1 (i.e., an endonuclease), a protein that has a 5' critical cysteine that is necessary for its signaling activity. Investigations have confirmed this observation. There is a physical interaction between Ref-1 and the fos and jun proteins of the transcriptional complex of AP-1, and the DNA-binding activity of the complex is increased, as is transcription.

Hydrogen peroxide (H₂O₂) stimulates many cytoplasmic signaling factors (e.g., erk families, p38, ras and raf). Hence, it seems logical to determine whether H₂O₂ and IR would use these pathways to respond to the damaging effects of oxidative stress. To address this issue, cell lines that overexpress wild type or cysteine mutant forms of TR were used. The mutant form of TR lacked critical N-terminal cysteine residues that presumably are involved with the passage of electrons from NADPH to TRX, effectively inhibiting the ability of TR to reduce TRX. The results of the experiments with cells overexpressing wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity and reporter gene expression (relative to vector controls), with little further induction following exposure to IR. In contrast, cell lines overexpressing mutant TR showed no increase in constitutive AP-1 DNA-binding activity and reporter gene expression (relative to vector controls) and no induction following IR. In addition, similar results were observed with the permanently transfected cell lines expressing the wild type and mutant TRX genes. Interestingly, the observed increase in AP-1 DNA-binding activity is independent of increased total TRX or c-Fos and c-Jun protein levels. Finally, preliminary results suggest that TR may regulate AP-1 activity by a mechanism involving the regulation of TRX subcellular localization. The results of these experiments, combined with earlier results, strongly support the hypothesis that, following exposure to IR, TR mediates an alteration in the redox state of TRX that participates in the activation of AP-1 DNA-binding activity and gene expression. In addition, it appears that the critical cysteines in TR and TRX are targets for this signaling process, further suggesting a mechanism involving alterations in the redox status of these proteins.

Based on these results, it is appealing to hypothesize that TR is a signaling factor in a cascade that begins with IR-induced ROI in the cytoplasm, then activates transcription factors in the nucleus that, in turn, regulate downstream genes that protect the cell from the oxidative stress induced by free radicals. This raises several interesting questions regarding the mechanisms involved in cytoplasmic signaling cascades activated by H₂O₂ produced from IR as well as the specific factors that pass the signal from the cytoplasm to the nucleus. The results presented in this report identify the cysteine residues located in the N-terminal regions of TR and TRX as critical for IR-induced activation of AP-1 activity. Thus, it would appear that these critical cysteine residues are targets for the passage of redox-sensitive cellular signals to transcription factors in response to stress. In this model, subtle changes in cellular redox potential induced by a stressing agent could alter the flow of electrons through the cysteine residues of TR and TRX, resulting in profound changes in protein activity. The critical cysteine(s) would appear to act as redox-sensitive "sulfhydryl switches" that reversibly modulate protein activity and allow signal transduction cascades to redirect metabolism in response to radiation-induced stress using redox-sensitive transcription factors.

To summarize the model, H₂O₂ and IR produce free radicals; the NADP level is altered in cytoplasm and mitochondria (not proven); TRX is activated and passes the signal on to TR, which is transported into the nucleus; TR forms a physical interaction with REF1; REF1 passes the signal to the AP-1 transcriptional complex, which is composed of fos and jun, each of which have critical cysteine in the DNA-binding domain; and DNA-binding activity is increased.

	FOOTNOTES

 
¹ Presented as part of the conference "Free Radicals: The Pros and Cons of Antioxidants," held June 26–27 in Bethesda, MD. This conference was sponsored by the Division of Cancer Prevention (DCP) and the Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Department of Health and Human Services (DHHS); the National Center for Complementary and Alternative Medicine (NCCAM), NIH, DHHS; the Office of Dietary Supplements (ODS), NIH, DHHS; the American Society for Nutritional Science; and the American Institute for Cancer Research and supported by the DCP, NCCAM, and ODS. Guest editors for the supplement publication were Harold E. Seifried, National Cancer Institute, NIH; Barbara Sorkin, NCCAM, NIH; and Rebecca Costello, ODS, NIH.

³ Abbreviations used: IR, ionizing radiation; ROI, reactive oxygen intermediate; TR, thioredoxin; TRX, thioredoxin reductase.

	LITERATURE CITED

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1. Abate, C., Pate, L., Rauscher, F. J., III & Curran, T. (1990) Redox regulation of fos and jun DNA-binding activity in vitro. Science 249:1157-1161.[Abstract/Free Full Text]

2. Xanthoudakis, S. & Curran, T. (1992) Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J 11:653-665.[Medline]

3. Kirkpatrick, D. L, Ehrmantraut, G., Stettner, S., Kunkel, M. & Powis, G. (1997) Redox active disulfides: the thioredoxin system as a drug target. Oncol. Res. 9:351-356.[Medline]

4. Hirota, K., Matsui, M., Murata, M., Takashima, Y., Cheng, F. S., Itoh, T., Fukuda, K. & Yodoi, J. (2000) Nucleoredoxin, glutaredoxin, and thioredoxin differentially regulate NF-kappaB, AP-1, and CREB activation in HEK293 cells. Biochem. Biophys. Res. Commun. 274:177-182.[Medline]

5. Wei, S. J., Botero, A., Hirota, K., Bradbury, C. M., Markovina, S., Laszlo, A., Spitz, D. R., Goswami, P. C., Yodoi, J. & Gius, D. (2000) Thioredoxin nuclear translocation and interaction with redox factor-1 activates the activator protein-1 transcription factor in response to ionizing radiation. Cancer Res 60:6688-6695.[Abstract/Free Full Text]

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