Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

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Abstract

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As3+) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53+/+ and p53−/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53−/− cells than in the p53+/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As3+. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53+/+ MEFs, As3+ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53−/− MEFs, As3+ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.

Introduction

Arsenic (As) is a well-known environmental toxicant that has been associated with numerous human health impacts including dermatosis, diabetes mellitus, cardiovascular disorders, and cancer (Tapio and Grosche, 2006). Within the last decade, arsenic has also been used as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL) (Chen et al., 1997, Miller et al., 2002) and has been shown to induce apoptosis in a relatively wide spectrum of tumors such as lung, ovarian, gastric, neuroblastoma, and breast cancer (Bode and Dong, 2002). Despite its well-known toxicity and carcinogenicity, arsenic trioxide is approved by the Food and Drug Administration as a chemotherapeutic agent for the treatment of APL (FDA, 2000). Currently, there are over 40 clinical trials underway to extend arsenic's therapeutic application (Litzow, 2008). The underlying mechanism of arsenic's paradoxical capability to act both as a carcinogen and as a chemotherapeutic agent, however, is unclear. Understanding this dichotomy has the potential to improve the ability to effectively use As3+ as a chemotherapeutic agent as well as protect against its toxicity.

Arsenic-induced cell cycle arrest and apoptosis is commonly associated with an increased generation of reactive oxygen species (ROS), depletion of the cellular antioxidant system, inhibition of DNA repair and DNA methylation, and decreased mitochondrial membrane potential accompanied by cytochrome c release and caspase activation (Miller et al., 2002). Research data imply that arsenic significantly affects specific signal transduction molecules that are involved in mediating cellular proliferation or apoptosis, including MAPKs, p53, AP-1, and NFκB. These changes in cellular signaling pathways have been associated with both arsenic's carcinogenicity and cancer-therapeutic effect (Kitchin, 2001, Qian et al., 2003).

The tumor suppressor gene p53, for example, has been linked to the DNA damage, cell cycle perturbations, and apoptosis that is seen with arsenic (Park et al., 2000, Yih and Lee, 2000). P53 is crucial in maintaining genome integrity through the induction of cell cycle arrest, allowing for the DNA repair or apoptosis for cells with irreparable damage (Vogelstein et al., 2000). In the presence of functional p53, exposure to 5 μM arsenite has been found to induce DNA strand breaks, increasing the p53 phosphorylation (Yih and Lee, 2000, Yih et al., 2002). Arsenic trioxide was also shown to inhibit proliferation of the human B lymphoma MBC-1 by up-regulating p53 expression and inducing apoptosis (Shen et al., 2000).

However, a number of alternate studies have demonstrated that As3+ also disrupts mitosis and induces apoptotic cell death in p53 deficient cells such as HeLa S3 and U937 cells (Huang and Lee, 1998, McCabe et al., 2000). In addition, P53 deficient myeloma cells were shown to be more sensitive to As3+ treatment exhibiting a higher level of apoptosis than p53+/+ cells (Liu et al., 2003). In an earlier study exploring the induction of p53 in cell lines with different genetic backgrounds including C-33A with mutations of the p53 gene, HeLa, Jurkat, and a transformed lymphoblast cell line, Salazar et al. found that C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat, and LCL-EBV cells showed similar cytotoxicity curves (Salazar et al., 1997). In addition, transfected Jurkat cells and human lymphocytes with mutated p53 genes showed increased sensitivity to arsenic cytotoxicity. Since treatment with arsenite or arsenate resulted in apoptosis in both wild-type p53 (p53+/+) and p53-deficient (p53−/−) cells, Bode and Dong suggested that p53 signaling pathway is not involved in arsenic-induced apoptosis (Huang et al., 1999, Bode and Dong, 2002). The use of cell lines to characterize the cellular response to arsenic has provided some clues but due to the complexity of the genetic background of these cell lines, there remains no firm consensus on the overall role of p53 in mediating As-induced toxicity. In this study, we applied mouse embryonal fibroblasts (MEFs) isolated from p53+/+and p53−/− mouse to examine the role of p53 in As3+-induced changes in signaling pathways. The ease of isolation of MEFs from mid-gestation mouse embryos and the ability to derive these cells from mice harboring various genetic alterations has been made the MEFs ideal model system for examining signaling pathways changes under different conditions (Vengellur et al., 2003). We have applied this culture system to investigate the molecular mechanism of metal-induced cell cycle arrest and apoptosis, as well as the role of p53 (Gribble et al., 2005).

High throughput gene expression profiling from mRNA expression microarray has become a powerful tool in the characterizations of biological processes, disease states, and response to drugs, as well as response to genetic perturbations (Lamb et al., 2006). In this study, we applied this approach to testify our hypothesis that although arsenic induces cell cycle arrest and apoptosis in both p53+/+ and p53−/− cells, the underlying molecular mechanisms or gene pathways are different. A systems-biology based examination of differential signaling pathways from the microarray study in the normal cells (p53 wildtype) and genetic abnormal MEFs (p53 knockout) may provide important information regarding the apparent dichotomy of arsenic's dual mechanisms potentially leading to further advancement of its utility as a chemotherapeutic agent.

Section snippets

Cell culture

Mouse embryonal fibroblasts (MEFs) were isolated following a modified protocol (Gribble et al., 2005). Briefly, embryos were separated from the uteri of pregnant females on day 14 of gestation. The torso and limbs were dissected to isolate fibroblasts. DNA obtained from the tail was used for PCR genotyping. Single cell suspensions were plated in 100 mm dishes in DMEM F-12 with 10% v/v Nu-Serum. The ease of isolation and the ability to derive these cells from mice harboring various genetic

As3+ induced cell death and cell cycle arrest in both p53-wildtype and knockout MEFs

In this study, arsenic-induced cytotoxicity and cell cycle arrest were examined in both p53+/+ and p53−/− MEFs. As shown in Fig. 1, As3+ treatment resulted in a consistent concentration-dependent compromise to morphological integrity. No significant morphological changes were observed with 5 μM in either genotype (Fig. 1A). Significant morphological changes were observed in p53−/− cell at 10 μM including irregular cell shape and condensation. Significant disruptions of morphology such as

Discussion

Our study demonstrates that As3+ induced dose-dependent cell cycle arrest and apoptosis in both p53+/+ and p53−/− cells, supporting findings of previous reports (Huang et al., 1999, Bode and Dong, 2002). Induced cell death in p53−/− cells appears to be unique to As3+, whereas this response is not seen in other metals such cadmium and methylmercury (Gribble et al., 2005), where less cytotoxicities were observed in p53−/− cells versus p53+/+ cells. Previous studies have demonstrated that

Acknowledgments

This work was supported by the National Institute of Environmental Health Sciences (2-P01-ES009601, P50 ES012762, P30 ES07033, R01-ES1063, U10 ES 11387 and T32 ES07032), the US Environmental Protection Agency (RD-83170901), University of Washington Royal Research Fund, and the Johns Hopkins University Center for Alternatives to Animal Testing (CAAT) Award. We thank Sean Quigley and Hannah Viernes in the Biomarker Lab for their excellent assistance with the microarray and qRT-PCR analyses.We

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