DNA damage response pathway in radioadaptive response

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Abstract

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.

Introduction

The adaptive response is a phenomenon whereby the detrimental effects of DNA-damaging agents can be mitigated if the cells are exposed to a prior stress. Among other triggering stressors, low-dose ionizing radiation was first identified to have an effect [1] and has been most extensively studied (reviewed in [2]). This adaptive response, called radioadaptive response, is the acquirement of cellular resistance to the genotoxic effects of radiation by prior exposure to low-dose radiation. Its ubiquitous nature across a variety of eukaryotic cells has attracted attention in the context of a programed genome defense system, and moreover, its long-lasting nature [3] presents an impact to the genetic risk assessment of low dose and low-dose rate exposure to radiation. In mammalian cells, the radioadaptive response has an optimum dose range below ∼0.1 Gy [4], [5], occurs in metabolically active cells but not in dormant G0 cells [6], [7], represents an immediate early response being expressed maximally at 4–6 h after irradiation [5], [6] and continues for more than 20 h [5], [6], or more than 40 days in some instances [3]. Higher doses are not only incapable of inducing adaptation, but also immediately erase the adapted condition [5], indicating an involvement of some feedback regulatory mechanisms. In spite of a large body of data characterizing the response in a variety of organisms and cell types, its molecular mechanism still remains largely unknown. For the adaptive response to occur, the incident stimuli should be recognized by cellular sensing systems and transduced to a long-lasting response where some effector molecules should mitigate the potentially harmful damage inflicted by the subsequent radiation insult. Recently, evidence for the activation of a wide variety of cellular radiation response networks has been accumulated, including activation of cytoplasmic protein kinases and phosphatases, transcriptional activation of early response genes, activation of plasma membrane receptors and cytokines (reviewed in [8], [9]). However, most studies have been based on the experiments with lethal or supralethal doses and do not provide an immediate information on their significance in the adaptive response to low doses while they may suggest the activation of death signals.

We previously demonstrated in cultured mouse cells that the adaptive response was mediated by a rapid and robust circuitous feedback signal transduction pathway involving activation of PKCα and p38MAPK with a possible feedback via p38MAPK-associated PLCδ1, where a breakage of the pathway was elicited by a down-regulation of a labile PKCα at high dose [10]. Further, to elucidate the downstream effector pathway involved in the radioadaptive response, we studied the cytogenetic adaptive response to low-dose X-rays in cultured mouse and human cells with different genetic background which might be relevant to the repair of DSBs. With these analyses, we show that p53 tumor suppressor protein plays a key role in the radioadaptive response and may channel the DSB repair into legitimate DNA end-joining pathway, which otherwise links to an illegitimate rejoining leading to chromosome aberration formation and signal to an apoptotic cell death.

Section snippets

Cells and cell culture

The m5S cell line is an immortalized, but non-tumorigenic near diploid cell line established from mouse embryonic skin cells [11] and retains the authentic mouse Trp53 gene (mouse homolog of human TP53 gene), which is highly expressed and activated upon X-ray irradiation [12]. Primary cultures of Trp53 knockout mouse embryonic fibroblasts with allelic constitutions of Trp53−/−, Trp53+/− and Trp53+/+ were established as described [13]. Severe combined immunodeficiency (SCID) primary cultures

Results

Fig. 1 shows the adaptive response as assayed by the induction of chromosome aberrations (dicentrics) in cultured mouse cells with different Trp53 and scid genotypes. In this experiment, the cells pretreated with priming dose of 0.02 Gy X-rays were subsequently irradiated with challenging dose of 3 Gy with various time interval between two doses. The priming irradiation with low doses clearly rendered the cells less susceptible to the challenging dose in Trp53+/−, +/scid and wild type (+/+) cells

Discussion

The results of present experiments highlight a requirement of p53 in the induction of adaptive response. So far studied, the adaptive response was absent only in the p53 deficient cells, such as Trp53 knockout mouse cells and the pSV immortalized AT cells which were suspected to have a functional abrogation of p53 by association with large T antigen. In accordance with these observations, in a separate experiment we observed that the adaptive response was not elicited in the malignant

Acknowledgements

We thank Dr. Shinichi Aizawa, Kumamoto University for providing Trp53 knockout mice. We also thank Dr. Sohei Kondo and Dr. Shunichi Takeda for critical reading of the manuscript and valuable comments. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture and the research grant from the Ministry of Health and Welfare, Japan.

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