p53 dependent apoptosis in glioma cell lines in response to hydrogen peroxide induced oxidative stress

Abstract

Generation of reactive oxygen species (ROS) is an important mode of action of many chemotherapeutic agents. Hydrogen peroxide (H₂O₂) is a model oxidant that has been used to study the response of cells to oxidative stress. The role of p53 in ROS induced cell death has not been consistent and has been shown to be cell type dependent. Study of cellular and molecular parameters and mechanisms involved in H₂O₂ induced cell death in glioma cells will contribute to the understanding of response of these cells to oxidative stress. We investigated induction of cell death by H₂O₂, and its relation to p53 in two human glial tumor derived cell lines U87MG (wild type p53) and U373MG (mutated p53). We observed that H₂O₂ was able to induce apoptosis (as shown by morphology, flow cytometry and DNA fragmentation studies) in U87MG in a dose dependent manner. Dimethyl sulfoxide (DMSO), a known ROS scavenger, was protective to the cells. H₂O₂ induced cell death was significantly reduced by antisense p53 oligonucleotide. Pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of the redox sensitive transcription factor NF-κB, abrogated the increased expression of p53 protein in response to H₂O₂, and enhanced cell survival. The U373MG cell line, having mutated p53, was comparatively resistant to H₂O₂ induced cell death. We conclude from the study that p53, activated by NF-κB, is essential for H₂O₂ induced apoptosis in glioma cells.

Introduction

Increased levels of intracellular ROS have been shown to be damaging to different biomolecules [1]. The hydroxyl radical, generated from H₂O₂ reacting with different transition metals, is particularly damaging to DNA, resulting in mutagenesis and leading to carcinogenesis [2], [3]. On the contrary, ROS have also been observed to take part in highly organized cellular functions like pathways of signal transduction and apoptosis [4].

H₂O₂ mediated cell death has been studied as a model for the ROS induced apoptosis in several experimental situations in order to elucidate the pathways involved in this process [5], [6], [7], [8], [9], [10]. H₂O₂ is stable and can easily penetrate lipid membrane [4]. Apart from its direct effect on intracellular redox status, H₂O₂ also acts by generating hydroxyl radicals leading to DNA damage [3]. Exogenous H₂O₂ is a potent inducer of single as well as double strand breaks in DNA [11]. ROS mediated DNA damage has been shown to induce apoptosis through accumulation of p53 in a number of situations [12], [13], [14], [15]. These also include the human fibroblast cell line WI38 [16], in human skin fibroblasts [10], in Jurkat and HeLa cells [6], and in cardiomyocytes [17]. Treatment of cells, having normal p53 or p53 null status, with a variety of ROS generating drugs and with ionizing radiation in vitro revealed a striking influence of p53 on the ability to trigger apoptosis [18]. Reports also suggest that p53 deficient cells are more resistant to exogenous H₂O₂ induced apoptosis [19].

However, the specific characteristic of the cell under study also determines the nature of the response. There are observations that the paradigm that H₂O₂ and other ROS induce p53 expression and thus lead to apoptosis is not always valid. Such reports include p53 independent induction of apoptosis in VSMC where cells were treated with H₂O₂+Fe(II) and in hepatoblastoma treated with H₂O₂ [8], [9]. p53 independent induction of apoptosis has also been observed due to oxidative stress induced by H₂O₂ in Jurkat cells [7] and by reduction of Cr(IV) in A549 cells [20], glucose deprivation induced altered redox state in rat fibroblasts [21], menadione induced oxidative stress in mouse splenocytes [22] and ROS generation by inhibition of the respiratory chain [23].

Clinically, malignant gliomas are among the least responsive of human tumors and for tumors of higher grades, complete remission and/or long term survival is rare [24], [25]. Accumulating evidence suggests that abnormalities in the control of cell cycle and apoptosis, a final common pathway through which cytotoxic agents exerts their lethal effect can lead to drug and/or radiation resistance [26]. p53 plays a crucial role in regulation of cell cycle and apoptosis. The role of p53 in modulating therapeutic response in glioma is debated. Transcription factor NF-κB has been shown to activate p53 in response to oxidative stress in different cell types [6]. However, this is not well studied in glioma.

Hypothesizing that the p53-mediated pathway is important for H₂O₂ induced cell death in gliomas, we have studied this phenomenon in glioma cell lines U87MG and U373MG differing in their p53 status. The modulating effect of DMSO, a known hydroxyl radical scavenger, was also looked at. The role of NF-κB in activating p53 was also determined using PDTC, an inhibitor of NF-κB. Experiments in U373MG (mutated p53) and with antisense p53 oligonucleotide in U87MG (wild type p53) provided further evidence for the role of this gene in glial cell lines.