Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway

Abstract

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800 μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800 μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.

Introduction

Methyl methanesulfonate (MMS) is a typical methylating agent which can be used as an experimental research chemical and as a solvent catalyst in polymerization, alkylation, and esterification reactions (Wyatt and Pittman, 2006). MMS has also been tested as a cancer chemotherapeutic agent, and the monoester of methanesulfonic acid can be used as a human male contraceptive and an insect attractant and repellent (Kunz et al., 2010). MMS is a known genotoxic compound that can directly react with guanine and adenine bases of DNA to generate interstrand and intrastrand cross-links (Hosseinimehr et al., 2010). In dividing cells, a replication fork can be stalled at the sites of DNA cross-links, resulting in the formation of DNA double strand breaks (DSBs), which are regarded as one of the most detrimental forms of DNA damage (Yu et al., 2006). By disrupting the dynamic and structural properties of DNA, DSBs affect many aspects of DNA metabolism, including DNA replication, transcription, recombination and repair (Shanbhag et al., 2010). DSBs can also activate several signal transduction pathways that can eventually lead to cell tumorigenesis or, depending on the situation, to apoptosis (Suwaki et al., 2011). This DSB-induced apoptosis is a key event for many conventional chemotherapeutic agent applications (Waxman and Schwartz, 2003).

p53 plays a pivotal role in DSB-induced apoptosis, which is central to its function as a tumor suppressor (Chen et al., 2011). In response to DSB formation, p53 is rapidly phosphorylated by the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases, resulting in its activation (Shiloh, 2006). Upon activation, p53 can activate a series of pro-apoptotic proteins such as BCL2-associated X protein (BAX), p53 upregulated modulator of apoptosis (PUMA), p53-induced protein with a death domain (PIDD), and FAS receptor (Berube et al., 2005, Meulmeester and Jochemsen, 2008, Yee et al., 2009). However, the high frequency of p53 mutations in human cancer cells makes the exploration of p53-independent cell signaling pathways become increasingly urgent (Kashiwazaki et al., 1997). Previous studies have also demonstrated important roles for p53 in MMS-induced apoptosis (Tian et al., 2009, Ziv et al., 2006). Despite these findings, it is recognized that p53 may only be partially responsible for MMS-induced cytotoxicity. For instance, after treatment with MMS, the p53 mutant human lymphoblastoid cells (WTK1) exhibited a delayed apoptosis, as compared to the p53 proficient human lymphoblastoid cells (TK6) (Greenwood et al., 1998). In another case, both p53 knockout fibroblasts and the corresponding wild-type cells displayed a similar sensitivity to MMS-induced chromosomal aberrations and apoptosis (Lackinger et al., 2001). Thus, the apoptotic response to MMS-induced DNA damage involves both p53-dependent and -independent mechanisms. However, very little is known about the p53-independent mechanisms.

p73, as a p53 family member, is also responsive to the DNA damage that leads to apoptosis (Roos and Kaina, 2006). In contrast to p53, p73 is rarely mutated in human cancers, thus, p73 as a new candidate tumor suppressor is getting more and more interest (Ozaki and Nakagawara, 2005). The p73 gene encodes two isoforms that differ in their N-termini, and which can be functionally classified as trans-activation competent (TAp73) and dominant negative (DNp73) proteins (Moll and Slade, 2004). The TAp73 shares similarities with p53 with regards to DNA damage-induced apoptosis through its activation of p53 responsive genes (Nieto-Rementeria et al., 2009). The DNp73 has a very important regulatory role, as it exerts a dominant-negative effect on p53 and TAp73 by inhibiting their transactivation activity. Thus, DNp73 confers drug resistance to tumor cells harboring p53 and/or TAp73 (Stiewe et al., 2002). Its inhibitory function is exerted either at the oligomerization level, or by competing for binding to the p53/TAp73 DNA target sequence (Vossio et al., 2002). In addition, the DNp73 promoter contains an efficient p53/TAp73 responsive element that can be transactivated by p53 and/or TAp73, and therefore creates a dominant-negative feedback loop that regulates the function of p53 and/or TAp73 (Concin et al., 2004). Several lines of evidences indicate that the presence and participation of different spliced forms of p73 might be one reason for the different cell fates observed in response to DNA damage (Sayan et al., 2010). Moreover, previous studies have also demonstrated in response to different DNA-damaging stimuli, p73 isoforms can contribute to p53-dependent or -independent cell apoptotic signaling pathways (Costanzo et al., 2002, Murray-Zmijewski et al., 2006). However, it is still unknown whether TAp73 and/or DNp73 participate in MMS-induced apoptosis.

Caspase 2 is the only pro-caspase constitutively present in the nucleus, and it is unique among the caspases in that it has features of both upstream and downstream caspases (van Loo et al., 2002). In recent years, accumulating evidence indicates that in response to DNA damage, caspase 2 can act as an initiator regulatory enzyme upstream of the mitochondria-dependent apoptosis pathway (Vakifahmetoglu et al., 2008). For example, treatment of cells with 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a well-known oxidative agent that induces DNA damage, resulted in the release of cytochrome c from mitochondria, and this activity can be inhibited by the caspase 2 selective inhibitor (zVD-VAD-fmk) (Tamm et al., 2008). In addition, during cisplatin-induced apoptosis, using siRNA to inhibit caspase 2 expression caused decreased cytochrome c release, and reduced caspase 3 and caspase 9 activities (Vakifahmetoglu et al., 2008). Moreover, several observations suggested in some cell lines, caspase 2 is required for p53-mediated apoptosis induced by genotoxic agents (Ren et al., 2005). But the link between p73 isoforms and caspase 2 activation remains to be elusive. Despite the above evidence supporting roles for caspase 2 in stress-induced apoptosis, there is currently no model that links a given type of DNA damage with nuclear caspase 2 activation and mitochondria-dependent apoptosis. Furthermore, most reports have only investigated either the role of caspase 2 on the activation of down-stream genes, or the effect of genotoxic agents on the expression of caspase 2 in whole-cell lysates, rather than in specific sub-cellular compartments (Guo et al., 2002, Sidi et al., 2008).

In this current study, we sought to analyze the p53-independent mechanisms induced during MMS-induced apoptosis. Using the p53-deficient H1299 and Hep3B cells, we first established an MMS-induced apoptotic model. Then, we carried out a set of analyses assessing cell viability, percentages of apoptotic cells, extent of DNA damage, and mitochondrial membrane potentials (MMP). Finally, we analyzed the protein levels of TAp73, DNp73, pro-caspase 2, cleaved caspase 2, pro-caspase 9, cleaved caspase 9, pro-caspase 3, cleaved caspase 3, pro-PARP, cleaved PARP, and cytochrome c. Our results, as detailed below, suggest that there exists a caspase 2-associated, but p53- and p73-independent, apoptotic response to MMS-induced DNA damage, which is mediated through the mitochondrial pathway.