Human lymphocytes

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Oxaliplatin is known to form DNA crosslinks in vitro (wonynarowski,2000).Thus,our aim was to determine the oxaliplatin-induced DNA crosslinks within human lymphocytes by using an modified alkaline Comet assay .Such modification allowed to detect the formation of DNA crosslinks caused by Oxaliplatin via the reduction of Mean Olive Tail Moment ( OTM ) induced by H2O2 .

Lymphocytes were treated with 50µM of H2O2for 30min after a conventional incubation for 1 HR with oxaliplatin at 37°C.Tow control setups were used, one without any added chemical, the second control only treated with 50µM H2O2 without oxaliplatin .

Human lymphocytes were incubated with H2O2 (control) or increasing concentrations of oxaliplatin (0.02 -200 µM) for 30 min at 37&deg;C. As shown in Figure 1, our data indicate that oxaliplatin incubations result in decreased OTM in a dose dependent manner where the OTM decreases as the dose of oxaliplatin increases. Oxaliplatin concentrations of 0.2, 2.0, 20.0 and 200µM resulted in approximately 18%, 25%, 30% and 44% decreased OTM, respectively, compared to the H2O2 control. The lowest dose of oxaliplatin that induces a significant increase in OTM is 2.0 µM with p<0.01 relative to the H2O2 control. The 0.02 µM and 0.2uM doses did not result in a significant decrease in OTM relative to the H2O2 control. As indicated, the greatest decrease in MOTM was observed in human lymphocytes incubated with the highest dose of 200µM oxaliplatin. This concentration is statistical significant in decreasing the oxaliplatin OTM relative to control treatment with H2O2 with p<0.001.

Oxaliplatin is frequently used in the therapy of cancer. In DNA, oxaliplatin induces, like cisplatin, the formation of crosslinks, which are commonly accepted as being responsible for the cytotoxicity of platinum agents. The detection of oxaliplatin-induced DNA crosslink formation and repair could be a good measure of assessing how a patient is responding to the agent. In the study, Gabriela et al., 2005, used a validated modification of the alkaline comet assay for detecting the presence of these crosslinks in vitro and in cancer patients. The H460 tumour cell line was treated in vitro with a range of oxaliplatin and cisplatin doses, and the subsequent crosslink formation and repair compared between the two agents. In addition, lymphocytes from cancer patients undergoing oxaliplatin-based chemotherapy were assayed for the formation and repair of oxaliplatin induced crosslinks. A dose-response was observed in the in vitro samples, with cisplatin producing more cross links than oxaliplatin at equimolar concentrations and lesions induced by both agents showing different repair efficiencies. Furthermore, evidence of crosslink formation and repair was observed in the peripheral blood lymphocytes of all cancer patients studied, along with the detection of inter individual variability in crosslink formation and repair efficiencies. This is the first time that oxaliplatin DNA cross links have been detected either in vitro or in patient samples using the alkaline comet assay.

In H460 cells, maximum crosslink formation for both agents was determined to occur at 6 h post the 1-h incubation with the agents. The study also shown that cisplatin induces a higher amount of crosslinks than oxaliplatin at equimolar concentrations, However, in the in vitro system, the percentage of crosslinks repaired at 72 h is higher for oxaliplatin than for cisplatin. This observation might be a direct consequence of the presence of fewer oxaliplatin-induced crosslinks, in that less damage might be easier to repair than more damage. DNA crosslink formation and repair in response to oxaliplatin and cisplatin was investigated in a NSCLC cell line, H460, by use of the modified alkaline comet assay that allows for the detection of these lesions. The typical comet images obtained when treating cells with oxaliplatin are shown in Fig. 2. The unirradiated non-oxaliplatin treated control (Fig. 2A) and the unirradiated drug treated sample (Fig. 2D) show a small OTM that reflects the presence of only intact, unbroken DNA. However, when the samples are irradiated (10 Gy) prior to analysis, strand breaks are formed and the released DNA migrates upon electrophoresis leading to an increase in OTM as seen in the irradiated non-drug treated control (Fig. 2B). When cells are treated with oxaliplatin the subsequent formation of crosslinks retards the electrophoretic mobility of DNA after irradiation and, therefore, the OTM is somewhat smaller in this sample (Fig. 2E) than in the corresponding irradiated non-drug treated control (Fig. 2B). When cells are allowed to repair the crosslinks (e.g. 72 h post-treatment) there is an increase in OTM when compared with the maximal crosslink formation at previous incubation times (Fig. 2F). Repair will be close to completion when the OTM for the drug treated irradiated sample is similar to the one obtained for the irradiated control sample (Fig. 2C). Oxaliplatin- and cisplatin-induced crosslink formation and repair were investigated in the H460 cell line by treating cells with an equimolar dose (250_M) of the agents for 1 h followed by up to 72 h incubation in drug-free media. Background damage was subtracted from the irradiated samples at each time point, and results are expressed as the percentage decrease in MOTM±S.E.M. (of two to four independent cultures) of the drug treated irradiated sample when compared with the non-drug treated irradiated control for each time point. Cell viability was assessed at each incubation period and determined to be >95%. No additional damage was detected in the non-irradiated oxaliplatin or cisplatin treated samples when compared with the non-irradiated controls (data not shown). At the radiation dose used, 10 Gy, the percentage of DNA in the tail was always below 50% (i.e. well below the saturation limit of the comet assay) and a good linear correlation was observed between radiation dose and both MOTM or percentage DNA in the tail (data not shown); therefore, the percentage decrease in MOTM is directly related to the amount of repair. Results show that the maximum crosslink formation occurs at 6 h after treatment, in response to both oxaliplatin and cisplatin (Fig. 3). Thereafter, the number of crosslinks decreased, with apparently more rapid removal of damage after cisplatin than oxaliplatin (Fig. 3). However, repair was more complete in the case of oxaliplatin (72% by 72 h) compared to cisplatin (56% by 72 h). A noticeable difference in the amount of crosslinks being formed in response to both drugs is observed, with cisplatin inducing a higher number of these lesions when compared to oxaliplatin (Fig. 3). This is also evident in Fig. 4, where the effect of increasing concentrations of oxaliplatin and cisplatin was investigated at the maximum time of crosslink formation for both agents. A dose-response effect was observed for both agents, where an increase in dose leads to a higher percentage decrease in MOTM, which is indicative of an increased formation of DNA crosslinks. The assay can, therefore, be used to detect crosslinks induced by a range of both oxaliplatin and cisplatin concentrations. At equimolar concentrations, cisplatin formed approximately twice as many crosslinks as oxaliplatin (Fig. 4).

The oxaliplatin induced DNA damage was investigated with sister chromatid exchange (SCE)assay. Human lymphocytes were incubated with 0.2 µM oxaliplatin or without any treatment (control) for 48 hr. SCE induction was used to measure oxaliplatin-induced DNA damage. SCE data for control and oxaliplatin incubated lymphocytes are summarized in the Figure 4. Incubation of lymphocytes in the presence of 0.2 µM oxaliplatin significantly increased the mean number of sister chromatid exchanges to approximately 17 SCEs compared to 2 SCEs measured in the control cells incubated in the absence of oxaliplatin. The data indicated that oxaliplatin induced an increase in SCE by exchanged between the sister chromatid by 8- fold. Further, the data demonstrate the induction of DNA damage by oxaliplatin, as measured by SCE, is statistically significant with p<0.001 compared to the control.

The Sister Chromatid Exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome. SCEs represent the interchange of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. Detection of SCEs requires some means of differentially labelling sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles.(Kligerman, A.D., et al.,1993). The in vivo sister chromatid exchange (SCE) assay detects the ability of a chemical to enhance the exchange of DNA between two sister chromatids of a duplicating chromosome. Sister chromatid exchange means a reciprocal interchange of the two chromatid arms within a single chromosome. This exchange is visualized during the metaphase portion of the cell cycle and presumably requires the enzymatic incision, translocation and ligation of at least two DNA helices.(Latt SA et al.,1981)

In the present study, the Oxaliplatin treated lymphocytes are showing more occurrence of SCE with respect to the control group that is without the Oxaliplatin. This result demonstrates that the oxaliplatin has introduced more number of DNA breaking and cross linking with respect to the control group. As the number of SCE is directly proportional to the amount of DNA damage and cross linking, the increased SCE in the study is proving the action of Oxaliplatin as it induces the morebDNA strand breaking as compared to the control sample.

The in vitro micronucleus assay is a mutagenic test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e.micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The purpose of the micronucleus assay is to detect those agents which modify chromosome structure and segregation is such a way as to lead to induction of micronuclei in interphase cells.

Analysis of micronucleus (MN) frequencies has since many years been applied for in vitro genotoxicity testing of new chemicals and for biomonitoring of human populations exposed to different environmental, occupational or lifestyle factors. MN are found in interphase cells as small, extranuclear bodies resulting from chromosome breaks (leading to acentric fragments) and/or whole chromosomes that did not reach the spindle poles during cell division. At telophase, when the nuclear envelope is reconstituted around the two daughter cells, these lagging chromosomes or fragments are not incorporated into the main nucleus but encapsulated into a separate, smaller nucleus, a MN. MN represents therefore a measure of both chromosome breakage and chromosome loss and can be used to classify chemicals into clastogens or aneugens. Since the formation of a MN requires a nuclear division, it is necessary to be able to distinguish dividing cells from resting cells. The cytokinesis-block micronucleus (CBMN) methodology developed by Fenech and Morley uses cytochalasin B to identify cells that have divided in culture. Cytochalasin B is an inhibitor of actin polymerization which is required for the formation of the microfilament ring that constricts the cytoplasm between the daughter nuclei during cytokinesis. The CBMN methodology allows distinction between a mononucleated cell, that did not divide, and a binucleated cell that has divided once. micronuclei present in mononucleated cells (MNMONO) may provide an indication of the genome instability accumulated in vivo, while micronuclei in binucleated cells (MNBN) indicate the chromosome/genome mutations accumulated before cultivation plus lesions expressed during in vitro culture.

The formation of micronuclei generated by oxaliplatin was studied using the cytokinesis-block micronucleus (CBMN) assay in the present study.

Cells were treated with 0.2 µM oxaliplatin or without oxaliplatin as negative control .However,0.2 µM Mitomycin C was used as positive control. The number of micronuclei in 1000 binucleated cells was determined. The results are summarized in the bar graph represented in Figure 2. In the control group there were approximately 21% mononucleated cells, 75% binucleated cells, and 1% multinucleated cells present (Figure 2). Oxaliplatin treatment with 0.2 µM resulted in a significant decrease in mononucleated cells compared with untreated cells. Likewise, oxaliplatin decreased the number of binucleated cells by approximately 50% compared to the control group levels. Conversly, oxaliplatin significantly increased the number of mulitnucleated cells by approximately 50-fold compared to the control group with p<0.001. The predominate cell type after oxaliplatin treatment was multinucleated cells while binucleated cells were the predominant cell type in control untreated cells. These results indicate that oxaliplatin treatment specificically increases multinucleated cells while decreasing the number of mononucleated and binucleated cells.

The effect of oxaliplatin on the micronuclei formation was measured in 1000 human binucleated lymphocytes.Human lymphocytes were treated with 0.2µM oxaliplatin and the formation of micronuceli was measured in the presence and absence of oxaliplatin. The results are summarised in the bar graph represented in Figure 3. In untreated cells, approximately 5% of the cells measured showed micronuclei formation with the remaining 95% having no micronuclei formation (Figure 3). In contrast, oxaliplatin treatment resulted in approximately 25% micronuclei formation in the 1000 binucleated cells with the remaining 75% having no micronuclei formation. Oxaliplatin treatment of human lymphocytes results in a significant increase in the the number of micronuclei formation with with p<0.001 .In accordance with an increase in the percent of micronuclei positive cells, there was an increase in the nuclear division index (NDI) from 1.79 to 2.38 with oxaliplatin (approximately a 75% increase).The increase in NDI supported the findings of oxaliplatin increasing micronuclei formation. Likewise, there was a decrease in the number of micronuclei negative lymphocytes with oxaliplatin treatment.These data demonstrate that oxaliplatin treatment increases micronuclei formation in human lymphocytes.

Polyploidy cells are usually tetraploid. These mostly arise due to the failure of cytokinesis, defects in the cytokinetic regulating proteins, errors in the chromosomal disjunction/ segregation( Ganem et al.,2007). Cells can also become tetraploid if there is arrest of mitosis is there at spindle assembly check point. Tetraploidy promotes genetic instability. Tetraploid cells can arise through cytokinesis failure, cell fusion or mitotic slippage. Although cytokinesis failure and cell fusion give rise to binucleate cells, mitotic slippage occurs without karyokinesis and results in tetraploid cells with a single large nucleus. Regardless of the mechanism of tetraploid formation, a common consequence is supernumerary centrosomes. These supernumerary centrosomes can lead to chaotic multipolar mitoses in which chromosomes are haphazardly segregated into two or more daughter cells, a defect that directly causes whole-chromosome aneuploidy. Alternatively, it has been observed that some tetraploid cells can cluster supernumerary centrosomes into two poles, thereby promoting a bipolar mitosis even in the presence of extra centrosomes( Ganem et al.,2007). Tetraploid cells can also be generated by cell fusion, like endoreplication, cell fusion is a normal, physiologically important process that occurs during development in certain tissues of the body and results in the formation of terminally differentiated polyploid cells. Ogle et al.,2005). that tetraploid cells often undergo a p53-dependent cell cycle arrest in the G1 phase following cytokinesis failure . Andreassen et al., 2001,after demonstrating that inhibition of cytokinesis with DCB in primary rat fibroblasts results in a p53-dependent G1 cell cycle arrest, proposed that a 'tetraploidy checkpoint' monitors the number of chromosomes or centrosomes.

Centrosome abnormalities are known to trigger G1 arrest and a p53 response. A recent study demonstrates that p53 activation by abnormal centrosomes is mediated, at least in part, by p38 kinase, which phosphorylates and stabilizes p53 in response to a variety of cellular stresses. Additionally, after mitotic slippage abnormal microtubules induce the translocation of the tumor suppressor Lats2 from the centrosome to the nucleus, where it promotes p53 stabilization. In addition to cell cycle arrest, the abnormalities associated with tetraploidy can also activate cell death pathways. Inhibition of apoptosis in a variety of cell types results in an increased prevalence of spontaneously tetraploid cells. One recent study by castededo et al.,2006,demonstrated that newly generated tetraploid cells are prone to activate Bax-dependent mitochondrial membrane permeabilization and apoptosis.

In the absence of functional mitotic spindle and consequent failure of chromatid migration to the poles lead to the inactivation of maturation promoting factor in the cells leading mitotic arrest being known as Mitotic slippage. This process leads to the process of poly ploidy. Cells do not arrest in mitosis indefinitely; rather, by a phenomenon known as mitotic slippage, cells slowly recover from the spindle checkpoint arrest and re-enter G1 as tetraploids. An interesting recent study provides insight into the mechanism of mitotic slippage (Brito et al., 2006). After spindle checkpoint activation, cyclin B is slowly but continuously degraded by proteasome-mediated proteolysis until its abundance is too low to maintain mitosis, resulting in exit from mitosis without anaphase or cytokinesis. Checkpoint is a regulatory mechanism to ensure genomic integrity and prevent the propagation of transformed cells (Hartwell and Kastan, 1994). Loss of checkpoint induces genomic instability and, consequently, alters ploidy of cells (Hartwell and Kastan,1994; Paulovich et al., 1997). The p53 tumor suppressor protein has been shown to play crucial roles in DNA damage-induced checkpoint functions. Recent studies demonstrated that p53 is a key molecule, not only for G1, G2 and S checkpoints, but also the spindle assembly checkpoint (Cross et al., 1995). Loss of p53 function permits murine fibroblasts exposed to spindle inhibitor to undergo multiple rounds of DNA synthesis, becoming hyperploid (Cross et al., 1995; Di Leonardo et al., 1997). Recent alternative explanations imply that exposing cells to microtubule inhibitor leaves transient mitotic arrest at similar rates regardless of the p53 status, and that p53 prevents transition of the abnormal cells from G1 to S phase (Minn et al., 1996; Lanni and Jacks, 1998; Sablina et al., 1999). Formation of polyploid or aneuploid cells is a pathological hallmark of malignant tumors.