Modulation of NF-κB, p53 and Bcl-2 in apoptosis induced by cisplatin in HeLa cells

Abstract

Cisplatin exposure induces apoptosis in HeLa cells. Since the interaction of this drug with DNA produces reactive oxygen species, we performed an analysis of the oxidative stress-responsive factors AP-1 and NF-κB. Although AP-1 levels were not modified during cisplatin exposure, electrophoretic mobility shift assays demonstrated an increase in NF-κB DNA binding activity that correlated with a decrease of the inhibitory protein IκBα and a specific relocalization of c-Rel, as assessed by immunoblotting and immunofluorescence. No changes in the levels or localization of p65 were found. Interestingly, IκBα relocalized to the nucleus, probably in order to regulate the binding of specific complexes. This process was accompanied by a decrease of the antiapoptotic protein Bcl-2, and a relocalization of p53 protein to the nucleus. Since HeLa cells lost most of their p53 protein due to a specific E6-dependent degradation, cisplatin could be inhibiting this degradation, since the p53 total levels were not increased during the exposure to the drug.

Introduction

Apoptosis is an active cell death process. Morphologically, it is characterized by condensation of nuclear chromatin and cytoplasm, and formation of membrane-enclosed apoptotic bodies, containing well-preserved organelles. The biochemical hallmark of apoptosis is the formation of distinct DNA fragments of oligonucleosomal size (180–200 bp) 1, 2. The process is active and requires specific gene regulation. A critical role for p53 in the execution of some forms of apoptosis has been suggested 3, 4, 5, 6, 7. This protein is a sequence-specific DNA-binding protein, active as a transcription factor. It has been proposed that p53 may be involved in the cellular response to DNA damage, producing arrest in the G1 phase of the cell cycle to allow efficient repair of the DNA before entry to S phase, or cell death if the damage is too large to be repaired [3]. Cells containing negligible amounts of wild-type p53 protein as a result of specific degradation induced by infection with human papillomavirus, or cells expressing mutant p53, are not able to arrest the cell cycle progression and continue to proliferate, leading to genetic instability and eventually malignancy [4]. Another gene implicated in apoptosis is Bcl-2 [8]. The Bcl-2 gene product functions as an anti-apoptotic signal, suppressing apoptosis induced by a wide variety of stimuli, including chemotherapeutic drugs and γ radiation 9, 10. The exact mechanism of Bcl-2 in preventing apoptosis is still not clear. However, Bcl-2 has been implicated in cellular control of their redox state11, 12.

Apoptosis plays a role in a number of disease processes, including cancer. It has been suggested that antineoplastic drugs may exert their effects by inducing apoptotic cell death. In this regard cisplatin (CDDP) has been shown to induce apoptosis in diverse cell lines 13, 14, 15. The drug is an effective crosslinking agent that generates: intra- and inter-strand DNA crosslinks, DNA–protein crosslinks, cisplatin–DNA–glutathione crosslinks and DNA-monoadducts [16]. The mechanism of CDDP cell toxicity has not been completely elucidated although inhibition of DNA synthesis, synthesis of specific mRNA and generation of active oxygen species have been considered critical for its toxicity 17, 18, 19, 20. A central role for reactive oxygen species (ROI) in cell death has been proposed 21, 22; therefore, the transcriptional factors NF-κB and AP-1, both implicated in the oxidative stress response, could play a role in the apoptotic process.

Recently, we have demonstrated that exposure of HeLa cells to CDDP induces apoptosis. After 18 h of treatment with the drug, these cells undergo specific DNA fragmentation [14]. We investigated the DNA binding activity of the transcriptional factors NF-κB and AP-1 upon CDDP treatment, since they might be implicated in the oxidative stress response [22]and the apoptotic process elicited by this drug 19, 20. We also analyzed the distribution of p53, which is activated by NF-κB [23]and is involved in the apoptotic process in several cell lines. Finally, since Bcl-2 protein has been widely related to cell death and is down-regulated by p53 we also analyzed its expression [24].