If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* This work was supported by the Ministry of Science and Technology of Korea and the Korea Science and Engineering Foundation through the Research Center for Proteineous Materials. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This study investigated the role of oncogenic H-Ras in DNA repair capacity in NIH3T3 cells. Expression of dominant-positive H-Ras (V12-H-Ras) enhanced the host cell reactivation of luciferase activity from UV-irradiated and cisplatin-treated plasmids and also increased the unscheduled DNA synthesis following cisplatin or UV treatment of cells. This observed enhancement of DNA repair capacity was inhibited by transient transfection with dominant-negative H-Ras (N17-H-Ras) or Rac1 (N17-Rac1) plasmids. Moreover, stable transfection of dominant-positive Rac1 (V12-Rac1) further enhanced DNA repair capacity. Because reactive oxygen species (ROS) are known to be a downstream effector of oncogenic Ras, we examined the role of ROS in DNA repair capacity. We found that ROS production by V12-H-Ras expression was mediated by the Ras/phosphatidylinositol 3-kinase (PI3K)/Rac1/NADPH oxidase-dependent pathway and that pretreatment of V12-H-Ras-transformed cells with an antioxidant (N-acetylcysteine) and an NADPH oxidase inhibitor (diphenyleneiodonium) decreased DNA repair capacity. Similarly, treatment with PI3K inhibitors (wortmannin and LY294002) inhibited the ability of oncogenic H-Ras to enhance DNA repair capacity. Furthermore, inhibition of the Ras/PI3K/Rac1/NADPH oxidase pathway resulted in increased sensitivity to cisplatin and UV in V12-H-Ras-expressing NIH3T3 cells. Taken together, these results provide evidence that oncogenic H-Ras activates DNA repair capacity through the Ras/PI3K/Rac1/NADPH oxidase-dependent pathway and that increased ROS production via this signaling pathway is required for enhancement of the DNA repair capacity induced by oncogenic H-Ras.
The cellular Ras protein, which is normally activated by growth factor receptors, is a mediator of those intracellular signaling pathways that are responsible for regulating cell proliferation (
). Point mutation in the ras gene occurs at high frequency in mammalian cells, resulting in transformation and malignant progression to cancer, with oncogenic Ras mutations occurring in ∼30% of all human tumors (
). Although a number of studies concerning the effect of Ras on DNA repair activity have been performed, the precise role of Ras in the regulation of DNA repair activity has not been fully elucidated. Several prior studies have provided evidence indicating that the Ras signaling pathway is involved in the down-regulation of DNA repair capacity (
). In this study, we therefore directly addressed the question of whether oncogenic Ras contributes to the regulation of DNA repair capacity. Because oncogenic Ras is known to participate in the development of carcinogenesis in many human cancers, understanding the molecular basis of oncogenic Ras-regulated DNA repair capacity could lead to strategies that improve anticancer therapeutic benefits.
), have therefore been generally considered toxic to cells. However, recent studies have demonstrated that ROS play a role as second messengers in regulating mitogenic signal transduction in various cell types (
In this study, we sought to determine whether oncogenic Ras is involved in the regulation of DNA repair capacity in NIH3T3 cells. The results show that expression of dominant-positive V12-H-Ras both protects NIH3T3 cells from UV- and cisplatin-induced cytotoxicity and enhances DNA repair capacity through the Ras/PI3K/Rac1/NADPH oxidase pathway, and increased ROS production via this signaling pathway is required for enhancement of the DNA repair capacity induced by oncogenic H-Ras.
The major findings of this study are that DNA repair is enhanced by oncogenic H-Ras expression in NIH3T3 cells; that this enhancement of DNA repair occurs via a Ras/PI3K/Rac1/NADPH oxidase-dependent pathway; and, most importantly, that stimulation of ROS generation via this signaling pathway is required for enhancement of DNA repair activity. To our knowledge, the findings of this study represent the first evidence demonstrating that ROS serve as a Ras effector to enhance DNA repair activity.
Several prior studies have reported that oncogenic Ras is associated with altered cellular response to DNA damage and DNA repair. However, the results of these studies are contradictory. Overexpression of activated Ras in NIH3T3 cells resulted in an increase in resistance to DNA-damaging agents such as UV light and cisplatin (
). The mechanisms of the observed increase in Ras-mediated resistance to DNA-damaging agents are unclear, but may involve the influence of oncogenic Ras on enhancement of the DNA repair activity because the higher survival of the Ras-transformed cells appears to be associated with a lower amount of cisplatin- or UV-induced DNA lesions and a higher efficiency of DNA repair capacity (
), which are believed to be involved in the DNA repair system. However, others have reported either no change or increased sensitivity to DNA-damaging agents in response to overexpression of active ras genes in NIH3T3 cells (
) have shown that the modulation of ErbB-2 (receptor tyrosine kinase family) activity significantly enhances the cytotoxicity of cisplatin by mechanisms involving down-regulation of DNA repair, and this down-regulation of DNA repair is mediated by the Ras signaling pathway. More recently, two groups have reported that oncogenic Ras has no effect on the DNA repair activity (
). In this study, we tried to determine the role of activated V12-H-Ras in the regulation of DNA repair activity in NIH3T3 cells. To better study the potential role of oncogenic H-Ras in the DNA repair capacity, NIH3T3 cells were stably transfected with the pIND-V12-H-Ras plasmid under the control of ponasterone A. In this study, we have demonstrated that expression of oncogenic H-Ras enhances host cell reactivation of luciferase activity from UV-irradiated and cisplatin-treated pGL3-Luc reporter plasmids (Figs. 1 and 2) and that dominant-negative N17-H-Ras blocks V12-H-Ras-mediated enhancement of host cell reactivation (data not shown). We also have shown that oncogenic H-Ras expression leads to an increase in the unscheduled DNA synthesis in UV-irradiated or cisplatin-treated NIH3T3 cells (Fig. 2). These results have provided evidence that expression of oncogenic V12-H-Ras is involved in enhancement of DNA repair activity in NIH3T3 cells.
Recent research has suggested a linkage between MAPK and DNA repair systems. For example, ERCC-1, which is required for the excision step necessary to remove damaged DNA, is induced by the activation of the Ras/ERK-dependent pathway (
). Thus, we have asked whether enhancement of the DNA repair activity induced by oncogenic H-Ras is primarily mediated through activation of a single branch, being either the Raf/MEK/ERK pathway or the PI3K pathway. In this study, we have demonstrated that the PI3K inhibitors wortmannin and LY294002 effectively inhibit V12-H-Ras-induced host cell reactivation of luciferase activity from UV-irradiated or cisplatin-treated pGL3-Luc reporter plasmids, whereas the ERK/MAPK inhibitors PD98059 and U0126 do not (Fig. 3). These results suggest that PI3K activity is involved, at least in part, in the oncogenic H-Ras-mediated DNA repair capacity. We also observed that transient transfection of dominant-negative N17-Rac1 reduced DNA repair capacity in V12-H-Ras-expressing NIH3T3 cells (Fig. 4) and that expression of dominant-positive V12-Rac1 increased DNA repair capacity (Fig. 5). Taken together, these results demonstrate that enhancement of DNA repair capacity by oncogenic H-Ras is mediated through the Ras/PI3K/Rac1-dependent pathway.
ROS have been demonstrated to serve as a downstream effector of Ras (
). This ROS production is thought to be mediated by Rac-dependent activation of NADPH oxidase, a multicompartment enzyme, and localized to the cell membrane in non-phagocytic cells. We confirmed that expression of oncogenic H-Ras significantly enhanced ROS production and that this enhancement of ROS production was blocked by transient transfection of dominant-negative N17-Rac1 as well as by treatment with DPI, an NADPH oxidase inhibitor (Fig. 6). Moreover, we found that expression of dominant-positive V12-Rac1 led to stimulation of ROS production (Fig. 6A). Thus, the results of our study, together with those of previous studies, suggest that ROS production by oncogenic H-Ras is mediated by the Ras/Rac1/NADPH oxidase pathway. NADPH oxidase-mediated bursts in neutrophil cells are mediated by p38 MAPK activity (
) have established that protein-tyrosine kinase activity is required for superoxide production by activated H-Ras expression in human lung WI-38VA-13 cells. In this study, however, we found that treatment of V12-H-Ras-expressing NIH3T3 cells with a PI3K inhibitor (wortmannin or LY294002), but not with an ERK inhibitor (PD98059 or U0126), decreased V12-H-Ras-mediated ROS production (Fig. 7). These results suggest that enhancement of ROS production by oncogenic H-Ras is mediated through the Ras/PI3K/Rac1/NADPH oxidase pathway.
The correlation of enhanced DNA repair capacity with induced oncogenic H-Ras expression as well as with elevated levels of ROS led us to hypothesize that raising the level of oncogenic H-Ras may enhance DNA repair capacity via increased intracellular ROS. A role for ROS in enhancement of DNA repair capacity by activation of the Ras/PI3K/Rac1 signaling pathway was suggested by the finding that transient transfection of dominant-negative N17-Rac1 and inhibition of PI3K activation with a PI3K inhibitor (wortmannin or LY294002) led to the attenuation of ROS production (Figs. 3 and 4) as well as a decrease in DNA repair activity (Figs. 6A and 7). Furthermore, overexpression of V12-Rac1 caused an increase in ROS generation (Fig. 6A) as well as an enhancement of DNA repair activity (Fig. 5). Direct evidence for the ability of ROS to enhance DNA repair was obtained in V12-H-Ras-expressing NIH3T3 cells using the antioxidant N-acetylcysteine and the NADPH oxidase inhibitor DPI. Because the ras oncogene produces ROS via the NADPH oxidase-dependent pathway, inhibition of NADPH oxidase can inhibit the major source of ROS generation in oncogenic H-Ras-expressing NIH3T3 cells. In this experiment, removed intracellular ROS were associated with reduced DNA repair capacity (Fig. 8). These data allowed us to delineate the following relationship: oncogenic H-Ras → PI3K → Rac1 → NADPH oxidase → stimulates DNA repair capacity in NIH3T3 cells (Fig. 10).
Enhanced DNA repair capacity can contribute to drug resistance, and inhibition of DNA repair can enhance cytotoxicity and induce apoptosis (
). In our system, expression of oncogenic H-Ras significantly increased resistance to cisplatin and UV (Fig. 9). Upon blocking activated H-Ras-mediated ROS production, the observed cell resistance to cisplatin and UV treatment suggested that ROS act as a cisplatin and UV resistance signal to promote cell survival. However,N-acetylcysteine treatment prevented cytotoxicity in a large number of cisplatin- and UV-treated cells. The effects of ROS on cellular activity appear to depend on the dose and cell type. A high ROS concentration (100 μm to mm) results in a parallel up-regulation of poly(ADP-ribose) polymerase, which is a marker of apoptosis (
). The concentration of ROS acting as signaling agents in the regulation of cell proliferation is in the nanomolar to micromolar range and is therefore significantly lower than the concentration necessary to induce apoptosis (
). Thus, ROS may exert different biologic effects, which are dependent on their intracellular concentration. UV-C and cisplatin are known to stimulate the generation of intracellular ROS. Thus, the high level of UV and cisplatin treatment results in the generation of a large amount of ROS. Under these conditions, the physiological role of ROS may be related to the induction of apoptosis. The use of such high levels of UV-C and cisplatin raises concerns regarding the biologic relevance of the responses, as such concentrations can be markedly toxic for cells and thus lead to apoptosis.
ROS play a regulatory role in the cellular signaling pathway. Although a large number of signaling pathways are regulated by ROS, the signaling molecules targeted by ROS are far from clear. There is growing evidence, however, that transiently increased ROS production is functionally associated with the regulation of gene expression and the activation of transcription factors (
). For example, ROS have been implicated in the activation of transcription factors such as nuclear factor-κB, AP-1 (activator protein-1), Sp1, Nrf2, and p53. Interestingly, the promoters for many DNA repair genes contain redox-sensitive transcription factor-binding sites. The promoter for the nucleotide excision repair gene (xeroderma pigmentosum A, B, C, and D and Cockayne's syndromes A and B) contains Sp1, Ets1 (AP-1-like family), and p53 sites; that for human OGG1(oxoguanine-DNA glycosylase) contains Ets1 and Nrf2 sites; that for NTH1 (human endonuclease III homolog) contains Ets1 and SP1 sites; and that for MSH2 (mismatch repair-related gene) contains p53, Ets1, and AP-1 sites. According to such promoter analysis experiments, these redox-sensitive transcription factor-binding sites are essential for their gene expression (
). Thus, it is likely that increased ROS production may be involved in the regulation of DNA repair activity through the activation of redox-sensitive transcription factors. In conclusion, this work has demonstrated that oncogenic H-Ras enhances DNA repair capacity in NIH3T3 cells and that this enhanced DNA repair capacity is required, at least in part, for increased ROS production, which is mediated by the Ras/PI3K/Rac1/NADPH oxidase pathway.