Oxaliplatin Genotoxicity on Human Lymphocytes - Research Paper Example

Oxaliplatin Genotoxicity On Human Lymphocytes Abstract Aim To address the effects of Oxaliplatin genotoxicity on human lymphocytes by using various cytogenetic techniques, such as, Comet Assay, Sister Chromatid Exchange, Micronuclei Assay (MN Assay) and micronuclei –FISH Assay. Method The predominant cytotoxicity demonstrated by oxaliplatin is genotoxicity. In vitro studies indicated formation of DNA adducts by oxaliplatin. Modified alkaline Comet assay detected strand breaks and DNA migration in a dose-dependent manner. SCE assay indicated defective DNA synthesis. It also demonstrated defective DNA mismatch repair following treatment with oxaliplatin. Oxaliplatin causes bulky DNA adducts. The absence of mismatch repair also indicated loss of p53 dependent apoptosis. CBMN could detect micronuclei which are indicative of DNA damage. Detection of micronuclei through CBMN indicated chromosome loss, breakage, rearrangement, nondisjunction, and apoptosis. All of these could mean specific genotoxic effects. These all are chromosomal aberrations, which may be caused by oxaliplatin. Mitotic catastrophe was a plausible mechanism of DNA toxicity caused by oxaliplatin through endocycling indicated in this experiment. This has been described as a process of mitotic failure induced by DNA damage leading to cell death. The other plausible mechanism that has been suggested is through decreased expression of the antiapoptotic protein, survivin, leading to cell death. Conclusion Apart from formation of chromosomal aberrations, oxaliplatin adducts also prevent DNA mismatch repair. The final common pathway of such DNA damage is enhanced apoptosis. However, other mechanisms also have been recognised in these experiments, which include increased micronuclei formation, mitotic catastrophe, inhibition of survivin leading to increased rates of apoptosis, although some of them may be putative calling for further studies to be suggestive of a plausible mechanism. Discussion Compounds of the platinum-based class of anticancer drugs such as cisplatin and carboplatin work through formation of DNA crosslinks, which in turn result in specific cellular responses, namely, apoptosis and inhibition of transcription leading to arrest of cell replication. This is essentially the basis of antitumour activity exhibited by these agents (Almeida et al, 2005; Todd and Lippard, 2009). Oxaliplatin, chemically described as 1R, 2R-diaminocyclohexane oxalatoplatinum (II), is a third generation platinum-based drug used for chemotherapy in many cancers. It is unique among platinum-based agents because of its tumour cell growth inhibition which requires fewer DNA disruptive lesions than cisplatin ( Malina et al, 2007) and thus results in significantly less toxicity (Hah et al, 2007). This makes oxaliplatin a more effective and favoured chemotherapeutic agent as opposed to cisplatin. Presently, oxaliplatin has been approved by FDA for use in the treatment of advanced colorectal cancer (Ibrahim, 2004). Although, predictably the manifested cytotoxicity of oxaliplatin will be minimal, the predominant toxicity cited is genotoxicity. The aim of the present study was to determine the genotoxicity of oxaliplatin in human lymphocytes by various cytogenetic endpoints. In vitro Crosslinking Action induced by Oxaliplatin It has been amply indicated that the primary mechanism of cell death induced by platinum compounds is through the formation of DNA adducts (Almeida et a.l, 2005; Hah et al., 2007; Boysen et al., 2009). These DNA adducts are comprise of intrastrand and interstrand crosslinks that result from covalent bonding between the platinum atom and DNA bases (Hah et al., 2007). The ability of a modified alkaline Comet assay was investigated to measure the crosslinks that are induced by oxaliplatin. For this, 50 µM H2O2 was used with the consideration that it would strongly induce strand and double strand breaks, and the reduced detection of these breaks would serve to identifythe action of a cross-linking agent. It was demonstrated that DNA breaks were reduced in presence of cross-links.REF Hence, lymphocytes were pre-treated in vitro with different concentrations of oxaliplatin to induce cross-links in order to decrease the H2O2-induced DNA migration with the Comet assay. Our data showed that an inverse relation exits between the increased concentration of oxaliplatin-induced DNA crosslinks and H2O2-induced DNA migration as measured by decreased OTM (Figure 1). However, direct relations between oxaliplatin-induced DNA crosslinks and oxaliplatin concentrations have been also detected. This can be explained by the fact that oxaliplatin forms intrastrand crosslinks between two adjacent guanine residues and between guanine-adenosine. Oxaliplatin also forms interstrand crosslinks, which represent the smaller percentage(10%) of the total number of adducts (Scheeff et al., 1999). It has been observed that DNA adduct formation occurs most frequently at the reactive N7-position of guanine residue (Spingler et al., 2001). X-ray crystallography studies determined differences in conformation between the cisplatin-GG adduct and the oxaliplatin-GG adducts (Scheeff et al, 1999; Malina et al., 2007). Specifically, oxaliplatin was shown to form 1,2-GG intrastrand DNA crosslinks (Malina et al., 2007). X-ray crystallography studies have additinaly shown that bulky 1, 2-diaminocyclohexane (DACH) group of oxaliplatin occur when DNA adducts were formed occupying much of the major groove of the bound DNA. This conformation restricts DNA bending toward the major groove in the area of the DNA adduct and results in a less polar major groove (Scheeff et al., 1999; Malina et al., 2007). Spingler and colleagues (2001) showed that oxaliplatin, but not cisplatin, formed a hydrogen bond directly to the DNA in the area of the lesion. DNA bending towards the major groove is required for 1, 2-intrastrand crosslink formation, and restriction of the bending results in the reduced binding of the high mobility group (HMG) domain protein (Vaisman et al., 1999). Further, the 1,2-GG intrastrand crosslink of oxaliplatin results in more DNA polymerase dysfunction (Malina et al., 2007) . In agreement with our data, Almeida et al. (2006) demonstrated that oxaliplatin-induced crosslinks were detected by alkaline Comet assay in the H460 tumour cell line and patient lymphocytes, which suggests that the Comet assay could be a means for predicting patient response to chemotherapy with platinum compounds. A correlation between mitomycin C (MMC) induced crosslinks and cellular response of DNA repair and apoptosis in H460 non-small cell carcinoma and RT4 bladder cancer cells was demonstrated by the same alkaline Comet assay used in our study (Volpato et al., 2005). Similar studies indicated that the alkaline Comet assay is widely applicable for detecting DNA crosslinking action induced by a variety of DNA crosslinking agents (Cemeli et al., 2009) Sister Chromatid Exchange Assay Sister chromatid exchange (SCE) to occur requires the passage of the cell through DNA synthesis where this phenomenon occurs at the replication fork of the DNA. If there is a DNA lesion at the moment of replication, the likelihood of sister chromatid exchange increases (Kadyk and Hartweel, 1992). It has been suggested that (SCEs) most frequently occur during a defective DNA synthesis, where replication forks occur due to the closer of homologous double strands, which increase the chances of a homologous recombination event. The SCE assay evaluates the harlequin staining pattern of the sister chromatids for detection of persistence or repair sister chromatid exchange (Painter, 1980). During chromosome duplication, DNA exchanges can occur between sister chromatids where DNA breaks from one chromatid and attaches to the reciprocal sister chromatid. The (SCE) assay detects these DNA exchanges through differential stain of the sister chromatids due to bromodeoxyuridine (BrdU) incorporation in the metaphase of the second mitosis. The process of formation of SCEs can be correlated with cytotoxicity, gene amplification, mutations at the points of induction, and recombinational repair (Perry and Wolff, 1974). We used this method to determine whether oxaliplatin can induce such exchanges. In the present study, we reported for the first time that treatment of lymphocytes with the subpharmacological dose of 0.2 µM oxaliplatin (Hah et al., 2007) was enough to induce a significant 8 fold increase in the number of SCEs (Figure 5). Oxaliplatin leads to bulky lesions with cellular DNA which are generally repaired through nucleotide-excision repair (Sancar, 1994). However, lesions that escape repair by nucleotide-excision repair (NER) can stall the process of replication leading to fork collapse, which may culminate into genetic instability (Flores-Rozas and Kolodner, 2000). Chaney et al, (2005) have stated that DNA replication does not stop due to the formation of platinum adducts, rather replication of DNA continues through leading to DNA translesion synthesis and to miscoding errors in the newly synthesised DNA. Translesional DNA synthesis is a pathway of post-replication repair that allows replication of damaged DNA, which had been induced by the platinum drug. This may occur without removal of the adduct (Visman and Chaney, 2000). Platinum-GG intrastrand adducts may be successfully bypassed by translesional DNA polymerases (Visman et al., 2001). Several translesional DNA polymerases have been shown to bypass platinum-GG intrastrand di-adducts (Visman et al., 1999; Bassett et al., 2002; Havener et al., 2003). The ability to bypass these adducts may serve as a double-edged sword for the cell in that it allows replication and at the same time promotes mutations as some DNA polymerases can insert nonsense bases while bypassing a platinum adduct (Bassett et al., 2002). The efficiency of bypassing these platinum adducts in human cells has been measured by the primer extension assay for pol η (eta) > pol μ (mu) > pol β (beta) >> pol γ (gamma) (Visman et al, 2001). DNA polymerase β is able to elongate the arrested replication products of polymerases α and δ, which can complete the stalled DNA replication. However, DNA polymerase β completes this stalled replication in an error-prone manner (Bassett et al., 2002). DNA polymerase β also synthesises DNA with oxaliplatin adducts with greater efficiency than that with cisplatin adducts (Visman et al, 2001). The efficiency and fidelity of such a bypass by these translesional polymerases is likely to affect the mutagenicity of these induced adducts (Havener et al, 2003). RNA polymerases which have been stalled at platinum adducts, on the other hand, would produce various cellular responses, such as, nucleotide excision repair, polymerase degradation, and apoptosis (Jung et al., 2003). These stalled RNA polymerases can dissociate from the DNA by subsequent polymerases initiating from the point of origin on the same template (Todd and Lippard, 2009). A polymerase which had been stalled at a platinum DNA adduct may resume transcription following the removal of the platinum adduct from the template (Jung et al., 2003). However, although this concept appears very reasonable, there appears to be no studies that examined the bypass phenomenon of RNA polymerases involving platinum adducts as the DNA polymerases are evidenced to do. In agreement with our result, it can be stated that we demonstrated adduct lesions induced by oxaliplatin producing fork collapse may bypass replication process successfully. Such bypass is mediated by translesional DNA polymerases through the involvement of homologous recombination repair (HR) of the lesion which had stalled the replication fork, and this way accomplished easily. Subsequently, HR involving RAD51-dependent strand invasion may then lead to the formation of SCE. Mismatch repair (MMR) is the repair system that is responsible for recognising and correcting the newly synthesised DNA strand. Mismatch repair is a process which can determine the fidelity of translesional synthesis after the formation of platinum-DNA adducts (Wu et al, 2004). It has been demonstrated that the (MMR) complex has ability to recognise preferentially the mismatch opposite of these adducts (Wu et al, 2005). However, it is also to be noted that (MMR) proteins cannot recognise bulky DNA adducts caused by oxaliplatin (Nebel, 1997). In addition, the absence of (MMR) in human ovarian tumour models correlates with loss of p53-dependent apoptosis (Anthoney, 1996) and reduced G2 arrest (Brown, 1997). Loss of (MMR) correlates with loss of G2 arrest induced by certain drugs (Hawn, 1995), even though there is no evidence that the loss of G2 arrest is linked to the loss of drug-induced apoptosis. The cytokinesis-block assay (CBMN) Chromosomal aberrations formed during mitosis can generate small distinct extranuclear bodies called micronuclei (MN) (Fenech et al, 2000; Chung et al 2002) .which become visible in binucleated cells after blocking cytokinesis. Fragments lacking centromeres (acentric chromosomes) or whole chromosomes that fail to interact with metaphase spindles resulting from chromosomal damage are not included within the nucleus when the nuclear membrane’s newly formed during telophase. Instead the form separate smaller micronuclei (MN) ( KirschVolders et al,1997; Fenech, 2000). The presence of MN has been used to detect DNA damage for more than 20 years (Fenech, 2007). Recently, there has been a great deal of focus on using high throughput of CBMN method for determining the genotoxic effects of chemicals and for biomonitoring of DNA changes in individuals exposed to various chemical compounds (Fenech, 2006 ; Decordier et al, 2009). The induction of MN are significant since they can be very significant from the experimental point of view since they can identify chromosome loss, breakage, rearrangement, non-disjunction and apoptosis (Fenech, 2007), all of which may be very relevant in detecting specific genotoxic effects of chemotherapeutic agents. The cytokinesis-block assay (CBMN), developed by Fenech and Morley uses cytochalasin B (Cyt-B) an inhibitor of actin polymerisation to block cytokinesis of dividing cells. This agent leaves the cells to assume a divided a binucleated (BN) appearance, hence this provides a distinguishing characteristic from undivided, mononucleated cells . The CBMN assay allows for the detection of chemically induced chromosomal aberrations through the presence of MN in binucleated cells or even detection of in vivo mutations as defined by MN presence in mononucleated cells (Chung et al 2002; Fenech, 2002). The goal of our study was to investigate the possible mechanism of oxaliplatin genotoxicity on the human lymphocytes. We used the CBMN assay as one method of measuring DNA damage induced by oxaliplatin. We observed an increase in MN formation following lymphocyte incubation with 0.2 µM oxaliplatin and a corresponding decrease in MN negative cells (Figure 3). Interestingly, our data indicated that the most preminant cell types were multinucleated cells after oxaliplatin treatment and binucleated cells within untreated control. Further, there was a 75% increase in the NDI driven by the high number of multinucleated cells. Chromosomal aberrations play a critical role in the progression of cancer. The inability of the cells to separate at the end of mitosis, results in tetraploidy, an event occurring in human cells with no reported checkpoints (Uetake and Studer, 2004). It is important to note that tetraploidy can precedes aneuploidy, and aneuploidy has been identified to be one of the cellular initiating events in cancer. This can be correlated to DNA damage, which can predict the risk of an individual developing cancer (Uetake and Studer, 2004; Gamen et al, 2007). In addition to aneuploidy, DNA damage biomarkers can include chromosome aberrations, telomere shortening, DNA oxidation and methylation, and DNA strand breaks (Fenech, 2002). Oxaliplatin may induce human lymphocyte cell death through mitotic catastrophe(MC). Mitotic catastrophe has been defined as a process of mitotic failure induced by DNA damage leading to cell death (Hunag et al,2005;Vakifahmetogul et al, 2008). It has been recently shown that treating with oxaliplatin can induce G2-M cell cycle arrest (Fujie, 2005). However, prolonged G2 arrest can cause mitotic cell death which is also called mitotic catastrophe that can be described as an aberrant form of mitosis associated with the formation of multinucleate giant cells that are temporarily viable but reproductively dead (Kondo, 1995). This thus provides a reasonable explanation for the multinucleated predominant cells found after treating with oxaliplatin demonstrated in the present study. Still, other publications state that mitotic catastrophe is pre-determined in G2 and characterised by an abortive short cut into metaphase arrest (Vakifahmetoglu et al, 2008) that occurs when cells resistant to genotoxic damage, such as in the case of p53 mutant tumours, bypass G2 and S phase arrests and accumulate chromosomal aberrations. When these aberrations are undetected by G2/M checkpoints, cells inappropriately enter mitosis (Erenpreisa and Cragg, 2001) and divide. This process is known as endocycling, and it is known to result in endopolyploid cells. These cells are nonviable, and hence eventually undergo cell death often at the spindle checkpoint after completing a number of cell divisions. Defects in mitosis or cell fusion can result in multinucleated giant cells (Erenpreisa and Cragg, 2001) and have been linked to human cancers (Hunag et al, 2005). Ngan et al. and colleagues (2008) demonstrated with oxaliplatin-induced oesophageal carcinoma cell lines TE7 and TE3 that they divide by mitotic catastrophe. Our data suggest that oxaliplatin may induces mitotic catastrophe (NDI=2.31) prior to cell death. In fact, MC is proposed to be the main route of cell death by DNA damaging therapeutic agents(Hunag et al., 2005). Another mechanism by which oxaliplatin may induce cell death in human lymphocytes is through decreased expression of the anti-apoptotic protein, survivin. Survivin is a multifunctional protein and member of the inhibitor of apoptosis protein (IAP) family (Mita et al, 2008) that prevent mitotic death by stabilising microtubules during cell division (Cheung et al, 2009). Survivin accumulates at the central spindle during anaphase. Survivin interacts with other proteins such as aurora B (Lens et al, 2006). The survivin/aurora B complex functions as a spindle checkpoint playing an important role in regulating microtubule tension during chromosome segregation and therefore in maintaining genomic stability (Li et al, 2009). Genomic stability studies demonstrated that lack of survivin results in cell division defect resulting in polyploidy. (Li et al, 1999 ; Yang et al, 2004). Interestingly, oxaliplatin was shown to inhibit survivin in the gastric cancer cell line MKN45 (Ngan et al, 2008). Survivin expression is increased in most human cancers including cancers of the lung, breast, pancreas, colon, brain, skin, and blood through various mechanisms, such as, gene amplification, enhanced promoter activity, DNA demethylation, and through activation of the phoshpatidylinositol 3-kinases and/or mitogen activated protein kinases. Increased survivin expression and subcellular localisation thus may predict a poor prognostic outcome for cancer recurrence, metastasis, and death (Mita et al, 2008). In addition to playing a role in spindle checkpoint, survivin additionally promotes angiogenesis and has been reported to be responsible for demonstrable chemoresistance to various anticancer therapeutic agents, including cisplatin and paclitaxel in human oral carcinoma cells (Mita et al, 2008; Cheung et al, 2009). Furthermore, Cheung et al. (2009) reported that overexpression of survivin could counteract microtubules destabilising drugs, while decreased survivin expression in human oral carcinoma cells increased the efficacy of such drugs. Li (2009) and colleagues reported downregulation of survivin sensitised BX-PC3 human pancreatic cancer cells to cisplatin-induced apoptosis. However, scope of therapy with such agents is limited since pharmacological survivin inhibitors have low antitumour activity alone. However, they are being tested in combination with conventional chemotherapeutic agents including oxaliplatin (Mita et al, 2008) for synergistic action with a higher antitumour potential. Combination of the cytokinesis-block assay and fluorescent in situ hybridisation(FISH) To fully understand the genotoxic and DNA damaging effects of oxaliplatin, we must understand the origin of chromosomal material of MN. It is crucial to identify whether whole chromosomes from malsegregation of chromosomes during mitosis or accentric chromosomal fragments. This may be determined by using antibodies to kinetochore proteins in order to determine the centromeric chromosomal regions. Unfortunately, this method cannot distinguish unique chromosomes or chromosome loss due to missing or dysfunctional centromeres. (Fenech 2007). In our study the CBMN assay was combined with FISH to identify the alpha-satellite repeats of centromeres within MN, using pan-centromeric DNA probes. Thus MN containing whole chromosomes can be identified and distinguished from those without any centromeric signals (weier et al., 1991). We report that oxaliplatin incubation of human lymphocytes results in MN formation, consistent with our CBMN assay study (Figure 3 and 4). By combining FISH with the CBMN assay, we observed a 2-fold higher MN formation which was statically significant (p Read More