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FLT3 (fms like tyrosine kinase 3) is constitutively activated via mutations in about 30% patients with acute myloiod leukaemia. Association of FLT3 with AML leads to bad prognosis. FLT3-ITD (internal tandem duplication) is one of the most common FLT3 mutations that are found in about 24% AML patients and are associated with bad prognosis. Constitutive activated FLT3 interacts with several downstream signalling molecules of different biological pathways which are involved in differentiation, apoptosis and proliferation. Activated STAT5 phosphorylation pathway is involved in reactive oxygen species (ROS) production which can lead to DNA damage and genetic instability. In this project the DNA damage response of a FLT3-ITD cell line was compared with a FTL3- wild type control and the different level of response were measured. Etoposide and an ATM kinase inhibitor were used for DNA damage induction and blocking of the ATM pathway to observe the involvement of different pathways in the DNA damage response. Basal expression level of these DNA damage response genes were also analyzed in association with mutation FLT3 status.
Acute myeloid Leukaemia (AML) is a malignant disorder of the myeloid line of blood cells, characterized by the rapid abnormal proliferation of the leucocytes (WBC) that accumulate in the bone marrow disrupting the production of normal blood cell. It's one of the most common acute leukaemia affecting the adults and disease incidence increases with age. AML is often associated with genetic instability which is characterized by a diversity of chromosomal and molecular changes (1). It has been reviewed that FLT3 gene mutations are one of the most common abnormalities which are found to be associated with AML. Approximately 30% of AML patients have mutations of the FLT3 gene in their blast cells, composed of FLT3-ITD mutations, 24% (2) or FLT3 a point mutation within the activation loop, 7% (2).
FLT3 is a single transmembrane receptor with five-immunoglobulin like folds. The extracellular domain binds to its growth factor, known as FLT3 ligand or FL. A single domain traverses the membrane, and then a kinase domain is split by the kinase insert. The gene location of FLT3 is 13q12; the FLT3 kinase domain belongs to the type III receptor tyrosine kinase family, which includes KIT, FMS and 2 genes for the platelet derived growth factor receptors(PDGFR). Its ligand stimulates the proliferation of haematopoietic stem progenitor and dendritic cells. Studies have shown that FLT3 is constitutively over expressed in most acute leukaemias (3, 4, 5) and overall it was found to be over expressed in 98% of pre-B ALL and about 90% of AML patients (3). The FLT3-ITD mutation occurs in the juxtamembrane (JM) domain of the FLT3 gene which interrupts the inhibition and constitutively activates the gene. Another mutation which accounts for 8-12% of AML patients is a mutation in the activation loop of the gene, most frequently involving aspartic acid 835 or immediate adjacent isoleucine 836 (6, 7, and 8).
FLT3 transduces the signal from the membrane via activation of multiple downstream pathways. Normally, FLT3 is a monomeric protein on the cells surface. Upon ligand binding, dimerization occurs and kinase activity is initiated by autophosphorylation and subsequently phoshorylating the other substrate molecules in the downstream signalling processes. In the case of the mutated form of FLT3 gene the kinase is constitutively activated, which in turn activates the PI3 kinase/AKT pathway, the RAS/MAP kinase pathway, and the STAT5 phosphorylation pathways. All these pathways are involved in the process of apoptosis, differentiation and proliferation. (Figure 1). STAT5 pathway is only activated in mutant FLT3 tyrosine kinase, rather than the wild type (42).
Figure 1: Mutated FLT3 signals via activation of multiple downstream pathways
Dimerization of FLT3 protein occurs as FTL3 ligand binds to it, initiating the process of
autophosphorylation and kinase activity. FLT3 mutation constitutively activates the kinase domain and activates numerous pathways, including the PI3 kinase/AKT pathway, the ras/MAP kinase pathway and the STAT 5 phosphorylation pathway. All of these pathways interrupt the processes of apoptosis, differentiation, and proliferation. All except STAT5 pathway can be activated by FLT3 ligand and receptor dimerization in wild type cells. The diagram was copied from the publication by Small D, 2008 (Ref: 3).
This project is concerned with difference in DNA damage and response pathway between FLT3 mutant and wild type cells. DNA damages can occur due to several sources. These damages can either occur via endogenous or exogenous sources. DNA repair systems act differently for single strand DNA damages and double strand DNA damages. In case of single strand damage, the other strand is used as a template to guide the correct repair process. There are mainly three types of single stand DNA damage repair mechanisms. They are, base excision repair, nucleotide excision repair and mismatch repair. Double strand damage repair involves different models which include homologous recombination, the holiday model and non homologous end joining (NHEJ) pathway etc.
Figure 2: DNA-repair pathways. Several DNA-repair pathways exist and deal with various types of DNA insults. These pathways include 1) the direct reversal pathway, 2) the MMR pathway, 3) the NER pathway, 4) the BER pathway, 5) the HR pathway, and 6) the NHEJ pathway. This diagram was copied from the publication by Kanaar et al, 2008 (Ref: 20)
The initiation of double stranded break checkpoints is dependent on the recruitment of MRE11 / RAD50 /NBS1 (MRN) complex at the damage site. And that is followed by the recruitment or activation of ataxia-telangiectasia mutated (ATM), a member of the family of phosphoinositide-3-kinase-related kinases (PIKKs) (17). And most importantly, two other PIKKs, DNA-dependent protein kinase (DNA-PK) and ATR (ATM and Rad3 related), are also activated and involved in the response to DSBs. The primary function of ATR is to initiate the DNA damage response at stalled replication forks (RFs)(18). ATM, ATR and DNA-PK are the first proteins that act when DNA damage occurs and that is via phoshorylating different targets of the downstream processes.
NHEJ or Non Homologous End Joining pathway is an error prone DSB repair pathway that is active in G1 phase and is the predominant pathway for DSB in mammalian cells (20). The core protein components of the mammalian NHEJ include the Ku subunits (Ku70 and Ku80), DNA-PKcs, XRCC4, DNA ligase IV (LigIV), Artemis, and the recently identified Cernunnos-XLF (also known as NHEJ1)(21). NHEJ joins together double stranded DNA ends after the bases are corrected, and single stranded annealing (SSA) occurs through excision of damaged bases in a single DNA strand and filling in the missing nucleotides (22). Homologous recombination or HR pathway is a multistep pathway which requires several proteins and it acts on S phase or G2 phase. HR only accounts for 10% of DSBs in mammalian cells but defect in HR pathway genes can have severe consequences that include lethality (42). There are several reports that implicate DSB and their repair by NHEJ in the generation of some key chromosomal translocations in both childhood and adult acute myeloid and lymphoid leukaemias (23).
Studies showed that FLT3 mutated AML cells respond to, and repair, chemotherapy-induced damage more efficiently than AML cells with wild type FLT3 (1, 9). As a result FLT3 mutated AML cells may not get killed by the chemotherapeutic drug and could subsequently affect the clinical outcome of the patients. It has been found in previous studies that relapse affects over 50% of patients with AML and patients with a FLT3 mutation have higher relapse rates than patients without a mutation (10). Studies done using the S-phase drug clofarabine showed that FLT3 mutant AML cells do not arrest the cell cycleand repair the repair the DNA damage efficiently when incubated with clofarabine for a short time period. It was found that cell cycle arrest in response to DNA damage by clofarabine in S phase is affected via loss of the transcriptional regulator called cdc25A. FLT3-ITD mutant cells have the ability to reduce or compensate this loss when treated with clofarabine. Small interfering RNA against the FLT3-ITD provides sufficient evidence that the message is almost reduced by 87.5 % (1). An additional study on DNA repair in FLT3-ITD cells shows that AML cells having the FLT-ITD mutation to have increased reactive oxygen species (ROS) production. As a consequence increased DNA double strand breaks (DSBs) and selection of error prone repair pathway leads to aggressive AML in FLT3-ITD patients. AML cell lines with FLT3-ITD mutations produce ROS via STAT5 signalling pathway and by the activation of RAC1, which is an essential component of ROS-producing NADPH oxidases. A possible mechanism for the generation of ROS was found by the direct association of RAC-GTP which binds to phosphorylated STAT5 (pSTAT5). A FLT3 inhibitor blocked increased ROS production in FLT3-ITD cells resulting in decreased DSB and increased repair and fidelity (11). Another study found that expression of RAD51, a gene which is involved in the homologous recombination DNA repair system, was found to be associated with FLT3-ITD mutant AML cell lines. Inhibition of FLT3 by using PKC412 significantly down regulated the RAD51 transcript and this was strictly seen in FLT-ITD mutant AML cell lines, not the wild type AML cell lines. Use of FLT3-short interfering RNA (siRNA) also came with the same result (9).
In this project, the response of different AML cell lines to DNA damage of different AML cell lines using chemotherapeutic drug and specific inhibitor were analyzed. The damage response was measured in two different categories of AML cell lines, one with a FLT3-ITD and the other with a wild type FLT3 status. Several therapeutic DNA repair inhibitors are being developed; one of these inhibitors was used to observe the repair abnormalities in AML cells with common FLT3 mutation.
Several molecular biology techniques which included DNA damage responses, DNA damage level measurement and measuring cell viability were used in this project. Basal expression level of several DNA damage responsive genes were measured and analyzed for their involvement in different DNA repair mechanisms or pathways.
This project was aimed to see the DNA damage responses, measure DNA damage levels and cell viability by using several molecular biology techniques. In addition basal expression level of several DNA damage responsive genes were also measured and analyzed for their involvement in different DNA repair mechanisms or pathway which was compared with the previous finding in this field.
Materials & Methods:
MOLM 13 (DMSZ), M07e (DMSZ), OCI-AML-3 (DMSZ), MV4-11 (ATCC) and U937 (ECACC) AML cell lines were used for experimentation purposes. Of these five cell lines MOLM13 and MV4-11 have got FLT3-ITD mutations, the rest are all FLT-3-WT genotype. On the other hand U937 cell line is p53 mutant and the rest are wild type for p53 status. All the cell lines were maintained in Rosewell Park Momorial Institute (RPMI)-1640 medium with 10% foetal calf serum (FCS), 1% penicillin and 1% streptomycin. In the leukaemia lab all the cell lines used are in suspension, so the adherence factor is omitted here.
Alamar Blue Assay
In this viability assay Alamar Blue dye( AbD Serotec), complete medium RPMI 1640 with 10% FCS and 1% Pen/Strep and Glutamine and plate reader with 560Ex/590Em were used. Standard protocol for alamar blue assay was followed as suggested by the manufacturers. The cell lines used were MOLM13 and M07e. The cytotoxic drug etoposide was used for this assay. Drug concentrations for MOLM13 cell line were 50, 100,250, 500 and 1000ng/ml and for M07e cell line were 100, 250, 500, 1000 and 2000ng/ml. From this assay the IC20 for both the cell lines were determined.
Viability Assay Using Trypan Blue Dye by Manual Counting Method
Trypan Blue solution, 0.4% (Sigma Aldrich) was used for cell viability assay as light microscope was used to count the live cells manually. Two cell lines, MOLM13 and M07e were used in this assay where drug concentrations for MOLM13 500ng/ml and for M07e 1000ng/ml (IC20 determined from the alamar blue assay) were used along with the phosphor-ATM kinase inhibitor, 10mM/ml DMSO (Calbiochem) and controls. The concentration of the inhibitor used was 1ÂµM.The initial cell concentration for this experiment was 5x105 /ml. After one day the drug and the drug plus inhibitor treated cell samples were counted and washed twice with RPMI-1640 and then divided into two more samples containing the drug and the drug plus inhibitor. Cells were grown in complete medium containing RPMI 1640 with 10%FCS and 1% Pen/Strep and Glutamine on cell culture flasks and counted after a set time period to determine the viability. Cell count was done until there were two close cell counts and the average was taken.
Alkaline Comet Assay
MOLM 13, M07e and U937 cell lines were used for comet assay analysis. From previous studies the concentration for Clofarabine was determined to use on these cell lines (1). Another drug, Etoposide was used as it produces DNA damage which can be detected by the comet assay and the concentration (IC20) of the drug for these two cell lines were determined from the alamar blue viability assay. The cells were fed a day earlier before treating with drugs (Etoposide and Clofarabine). Clofarabine was used at a concentration of 0.3ÂµM/ml for MOLM13 and 1ÂµM/ml for M07e and cell were incubated for an hour. On the other hand the drug etoposide was administered for 24 hours and the concentration for MOLM13 was 500ng/ml and for M07e was 1000ng/ml.
The U937 cell line was treated with 50ng/ml and 300ng/ml of etoposide for an hour. Cells were taken at 5x105/ml concentration. Control cell lines were thawed in a 37Â°C water bath and were mixed with agarose just before loading them to the comet slides. Control cell lines are cell samples that have got specific level of DNA damage done by specific dosage of drug treatment. Trevigen (AMS Biotechnology Ltd, Abingdon, UK) comet assay slides and lysis buffer were used for the comet assay and according to the manufacturer's instructions the protocol was followed. For alkaline and neutral comet assay the electrophoresis parameters were 400mA for 20 minutes and 20V for 15 minutes respectively. Slides were stained with SYBR Green 1, fluorescent dye (Sigma) at a concentration of 1in 5000 dilutions in PBS. Images were visualized under a fluorescent microscope and the comets were analyzed using Comet Assay III image analysis software (Perspective Instruments, Suffolk, UK) . Fifty comet images were recorded form each of the two gel spots and each experiment condition; therefore, 100 images were in total for each treatment. Mean tail moment of the comets was used in all analysis and it was referred that the higher the mean comet tail moment the greater the damage.
RNA from six different cell lines (MOLM13, M07e, OCI-3, KG-1a, HL-60 and MV4-11) was prepared using QIAmp RNA kits with DNAase treatment according to the manufacturer's instructions (Qiagen). Up to 2000ng RNA was used in a reverse transcriptase reaction with MMLV reverse transcriptase (Invitrogen) and random hexamers (GE Healthcare). Quantitative PCR was done on an ABI Prism 7700 (Applied Biosystems) using Excite Real-time Mastermix with SYBR Green (Biogene)..Thermal cycler conditions included incubation at 95Â°C 10 minutes followed by 40 cycles of 95Â°C 15 s and 60Â°C 1 minute. Following the 40 cycles, the products were heated from 60Â°C to 95Â°C over 20 minutes to allow melting curve analysis to be done. This step allowed the specificity of the products to be determined as a single melting peak and also confirmed the absence of primer-dimmers.
The housekeeping gene Î²2 microglobulin (Î² 2M ) was used to standardize the samples and the relative expression level of PARP1, DNA ligIII, DNA ligIV, ATM, WRN, OGG1, XRCC6 and Artemis, (The primers for these 9 genes were bought from Qiagen). Therefore it was calculated as the ratio between the levels of these tests genes and the control gene (Î²2M). All Negative controls (no template) were included in each experiment and all reactions were run in triplicates.
Phoshpho ATM & Phospho p53 assays
Cells(MOLM13 & M07e) were incubated at 5x105/ml for 24 hours with and without the specific doses(for MOLM13: 500ng/ml & M07e: 1000ng/ml) of etoposide followed by two rinses in cold RPMI-1640. A portion of the cells was then fixed immediately to assess the baseline damage and the rest was resuspended in fresh culture medium and was left in the incubator to allow time (2hr, 4hr, 24hr) for repair to occur. After these recovery time intervals phosphorylation of serine 1981 at ATM and phosphorylation of serine 15 at p53 were measured. Counterstaining to measure the intracellular DNA damage content analysis was done with 25Âµg/ml 7-AAD in PBS. For quantitative analysis, fluorescence values obtained from the untreated control cells were compared to the drug treated test. For both of these assays leucoperm (AbD Serotec) kit fixation method was used and the protocol was followed as the manufacturer's instruction. Phospho ATM antibody mouse monoclonal MAB3806 from Millipore at 1mg/ml was used at 1:400 dilution and for Phospho p53 assay, Phospho-p53(Ser 15)(16G8) mouse mAb from Cell Signalling Technology was used at 1:100 dilution. For non-specific isotype control, IgG1 control abX0931(Dako)), 100Âµg/ml was isotype matched for both of the experiments. 5Âµl of Rabibit anti Mouse IgG-FITC Rabbit F(ab`)2, 0.55g/L was used The 7-AAD, A9400-5MG, 079K4046 , was bought from the Sigma Life Science as powder. Solution was made as 1mg/ml using ice cold PBS.
Comet Assay or Single Cell Gel Electrophoresis measures DNA damage from individual cells based on the migration of denatured DNA through an electrophoretic field (12, 13). Single cells or nuclei are embedded in agarose gel, lysed to expose their DNA content, alkali treated to denature or relax the DNA and electrophoresed to separate the DNA. The damaged DNA containing strand breaks migrates farther in the gel than intact DNA and that creates an image resembling a celestial comet (14, 15). Fluorescent dye, SYBR Green, makes the damaged DNA to be visualized as comets on fluorescent microscope. Alkali comet assay can detect double stranded and single stranded DNA damages, on the other hand neutral comet assay detects single DNA damage and cross links (17, 18).
The purpose of the comet assay in this project was to observe the DNA damage level of the drug clofarabine on a FLT3-ITD AML cell line and compare it with a FLT3-WT cell line. Etoposide was also used because it is known to produce DNA damage which is detectable by the comet assay. The control cells (CC0, CC1, CC2, and CC3) used in this experiment had exponential DNA damage level as they came in treated with different drug concentrations prior use.
Figure 3: Alkaline Comet assay of drug treated U937 cell line and the control cell lines. Etoposide was used on this cell line with a concentration of 50ng/ml and 300ng/ml. Here CCO-CC3 are control cell lines with exponential DNA damage (n=1)
In the cell lines MOLM13 & M07e no damage could be detected by the comet assay when it was treated with clofarabine. So, etoposide was used on U937 cell line as it is already known that DNA damage detection by the comet assay can be detected in this cell line when treated with etoposide. The IC30 for etoposide in this cell line was already known in this lab. No damage was observed even though the control cells produced expected results, that is an increase in comet moment with increased concentrations of drug. Unfortunately time constraints didn't allow optimising the comet assay further.
Phospho-ATM and Phospho p53 Assays
Phospho-ATM and phospho-p53 detection assays were performed using a flow cytometry technique to measure the DNA damage response on MOLM13 and M07e cell lines. Different recovery periods were set up to asses repair of the damaged DNA and analyze variations in DNA damage responses.
Figure 4: Phosphorylated Ser-1981 level of ATM in MOLM13(A) and M07e (B) cell lines with etoposide treatment and different recovery period. Here the for both of the cell lines the recovery periods were 0hr, 2hr, 4hr and 24hr. For 2hr, 4hr and 24hr studies, n=3 and for 0hr study n=1. In this graph the mean of 2hr, 4hr and 24hr studies were plotted with standard deviatioin as error bars
As it can be seen from the graph (figure 4) that MOLM13 has got less DNA damage response than M07e and also it can be observed that the DNA damage response gets remarkably lower by 4 hours recovery period compared to the M07e cell line where phosphorylated ATM level is still, almost, more than the half of MOLM13 cell line's highest phosphorylated ATM level.
Figure 5: Phosphorylated Ser-15 level of p53 in MOLM13(A) and M07e (B) cell lines with etoposide treatment and different recovery period. Here the for both of the cell lines the recovery periods were 0hr, 2hr, 4hr and 24hr. (For 24hr assay n=2 and 0hr, 2hr and 4hr assay, n=1) Standard deviation was used as error bars for 24 hour assay.
Similar results came from the phospho-p53 assay with a difference in both cell lines, as during 4 hour recovery period the most p53 phosphorylation was observed and as for Phospho-ATM it was within the first two hours for MOLM13 and upto 4 hours for M07e. And after 24 hours of recovery period still the phosphorylated p53 state of M07e is almost two times higher than MOLM13 cell line.
Viability assay using the manual cell count technique gave the idea of the growth and death in cell samples containing drug, Inhibitor, drug plus inhibitor and the control samples.
From the graph (Figure 6) it can be seen that the drug, inhibitor and both of them combined together had more effect on MOLM13 cell lines than M07e. This is surprising as the flow cytometry assays suggested that MOLM13 cell lines had less DNA phosphorylated ATM damge response than M07e and the same goes for activated phosphorylated p53. But from the data here, it can be seen that MOLM13 had more cell kill than M07e. It was also found that alone Inhibitor has got more effect on MOLM13 as the cell count went below the basal level (5x105 cells/ml), on the other hand M07e cells still grew over the basal level. Whilst these experiment need repeatation, these results firmly suggest that MOLM13 cell line might be more dependent on the ATM pathway than the M07e cell line.
Figure 6: Vaibility assay using drug (Etoposide) & Inhibitor (ATM kinase) MOLM13 & M07e Cell lines (Day-1). Here C, I, (C-D) & (D+I) represents sample which is control, inhibitor alone , the difference in cell count between control and drug treated samples and the sample that was treated with both drug and inhibitor. The last two bars of each cell line represents the effect of the inhibitor in drug treated cell samples. Here drug concentrations for MOLM13 and MO7e were 500ng/ml and 1000ng/ml respectively, the concentration of the ATM kinase inhibitor was 1ÂµM and the initial cell concentration (5x105 /ml) was taken as the base line.
After day one the samples were washed and divided into further two sets with drug only and drug plus inhibitor samples. From the graph (Figure 7) it can be seen that the number of cells progressively gets reduced in cells treated with drug alone and drug plus inhibitor. It was also found that MOLM13 had more dead cells than MO7e, supporting the previous suggestive analysis.
Figure 7: Vaibility assay using drug (Etoposide) & Inhibitor (ATM kinase) MOLM13 & M07e Cell lines (Day 2). the first day sample count is represented by the first two bars of each cell lines. Next, each of the samples of first day were washed and divided into two further samples, one had drug(D) and the other had drug plus inhibitor (D+I). Here drug concentrations for MOLM13 and MO7e were 500ng/ml and 1000ng/ml respectively and the concentration of the ATM kinase inhibitor was 1ÂµM.
These results are also displayed in Table 1 and 2 where the growth column represents the growth of cells in different samples through time. As the starting concentration of cell sample was taken as one hundred percent so below hundred percent implies cell death. From Table 2, sample Drug & Inhibitor (D+I), which had drug and inhibitor for the whole time, was found to be containing the least growth or maximum cell death among all other samples. MOLM13 and M07e had 3% and 18% growth respectively which implies 97% and 82% cell death on day 2.
Table 1: Viability assay MOLM13 & M07e cell lines (Control & Inhibitors treated samples)
Table2 : Viability assay MOLM13 & M07e cell lines (Drug and Inhibitor treated samples)
Day 2-Drug was washed off and new drug was added with or without inihibitor.
MOLM13 Drug Inhibitor (D)
MOLM13 Drug & Inhibitor (D+I)
M07e Drug+ Inhibitor
M07e Drug & Inhibitor (D)
M07e Drug & Inhibitor (D+I)
As these two cell lines might have different growth rate that is why cell count and growth per day was measured to determine the percentage of cell viability.
Mainly basal expressions of eight DNA repair genes were analyzed in this project in context to four different cell lines. The genes were WRN, DNA ligase III, DNA ligase IV, ATM, PARP1, OGG1, Artemis and XRCC6. The cell lines were MOLM13, M07e, OCI-AML-3 and MV4-11. The protein, WRNp is a DNA double strand break repair protein that is a member of RecQ helicase family and contains 3'-->5' helicase and 3'-->5' exonuclease activities and binds with PARP1 first then interacts with Ku70 and Ku86 proteins subunits of NHEJ repair system (26-28). DNA ligase III or Lig3 is a member of Nucleotide Excision Repair (NER) pathway which can directly bind to the N-terminal region of PARP1 (29). It has also been found that in NER pathway interaction of Lig3 with XRCC1 plays an important role by creating stability (30, 31). DNA ligase IV or Lig4 gene is involved in DSB repair pathway which acts via NHEJ mechanism (32). It is important to mention that it was found to be associated with DNA-PKs and Ku proteins (33). Another gene involved in the DSB repair pathway is ATM; this gene product has been found to be associated with BRCA1, p53, Chk2, DNA-PKcs and Artemis phosphorylation (34, 34, 35). PARP1 is member of SSB repair pathway genes which interacts with WRN and Ku70/86 complex of NHEJ pathway (28). Another study found that in order to bind to PARP1, p53 must be phosphorylated indicating their interaction with each other (37). OGG1 is involved in Base Excision Repair (BER) pathway and recent studies found that it can be used as a prognostic marker in AML as its expression is associated with higher reactive oxygen species (ROS) and additional mutant p21 and p53, which leads to bad prognosis (38). Artemis is another gene of NHEJ pathway which has been found to interact with DNA-PKc and mutation in Artemis gene results in hypersensitivity to DSBs and immunodeficiency (39). Artemis was also found to interact with Ku70 and DNA ligase IV and ATM genes products (40). Another DSB repair gene which acts via the NHEJ mechanism is XRCC6 or Ku70, it has been found to be associated with the DNA-PKc proteins, Ku86, PARP1 and WRN, DNA ligase IV proteins (26, 28, 33, 40).
The basal expression study of DNA repair genes were done to observe the different level of DSB repair genes and SSB repair genes among the six different AML cell lines. Of these six cell lines two were FLT3 mutant (MOLM13 & MV4-11) and two other were FLT3-WT (M07e and OCI-AML-3). These four cell lines have wild type p53 status. Of the eight gene expressions observed, five of them are involved in DSB damage response and/or repair pathway and the other three are involved in SSB response and/or repair pathway.
Figure 8 (A & B): SSB damage responsive gene expression level in FLT3 mutant cell lines (MOLM13 & MV4.11) and FLT3 wild type cell lines (OCI-AML-3 and M07e). n=1
Figure 9 (A & B): DSB damage responsive gene expression level in FLT3 mutant cell lines (MOLM13 & MV4.11) and FLT3 wild type cell lines (OCI-AML-3 and M07e). n=1
MV4-11 cell line had high XRCC6 (Ku70), WRN and Artemis gene expression level. These genes are involved in NHEJ double strand break repair pathway. MV4-11 is homozygous for FLT3 mutant status on the other hand MOLM13 is heterozygous for FLT3 status. MOLM13 hand significantly lower levels of expression of the genes mentioned above but it had comparatively higher ATM expression than the other FLT3 mutant cell line, MV4-11. On the other hand OCI-AML-3 cell line had high WRN expression and comparatively high PARP1 expression. And the same was observed for M07e cell line which even had high expression of ATM. WRN protein binds to PARP1 and upon binding it activates it and next interacts with XRCC6 (Ku70) of NHEJ pathway (26-28). This might be a cross talk among the DSB and SSB repair genes which is interesting.
Mutation in AML is a very important factor for prognostic and treatment purposes. Several mutations are associated with AML. As important as FLT3, NPM1 is another important mutation that has been found at least in half of the normal karyotype AML patients, marking the most common molecular marker in AML to date (16). FLT3-ITD containing patients have a significantly greater risk of relapse and resulting in poor prognosis (10). Studying FLT3 mutation is a very important research field in studying acute myloid leukaemia. The main objective of this project was to observe the differences between DNA damage response level in FLT3 mutant and wild type cell lines and compare them.
The project started by looking at the DNA double strand break damage by doing comet assay and as the drug clofarabine was not working another drug; etoposide was used to optimize the assay. Comet assay done in this project was not reproducible were problems with a faulty electrophoresis power pack, therefore although attempts were made to optimize the neutral and alkaline comet assay, this was not achieved due to time constraints. Results from the viability count assay provided the idea that MOLM13 cell line might be more dependent on ATM pathway for DSB repair response than M07e cell line and as from the cell count it was found that by the end of two day experimentation, MOLM13 had got more killed cells than MO7e cell lines. Supporting this data DNA damage response measured by phospho-ATM and phosphor p53 assay found that MO7e had more DNA damage response than MOLM13. It was found in the previous studies that FTL3-ITD mutation containing AML cells respond more to chemotherapy induced damage and repair damage more efficiently than wild type FLT3 cells (1, 9). Data found from the damage response assay and viability assay might support these previous studies because if FLT3-ITD containing cells have other active repair mechanisms, they may initiate their damage repair process but as the process is error prone an AML patient with a FLT3 mutation ends up with bad prognosis. Non-homologous end joining repair mechanism is one of the error prone DSB repair processes that are predominant in mammalian system (20). Further studies with different sets of mutant and wild type cell lines can be done to test this hypothesis.
Real time PCR data showed the heterogeneous expression of DNA repair genes in different AML cell lines. High expression level of NHEJ pathway genes were observed in FTL3 mutant cell line (MV4-11) and it was also found that compared to other cell lines MOLM13 had higher expression of ATM. Although MOLM13 and MV4-11 have got mutated FLT3 status but similar expression level was not observed. Cell lines wild type for FLT3 and p53 status had been observed with higher WRN and PARP1 expression. Studies previously found that WRN binds directly to PARP1 and then interacts with Ku70(XRCC6) of the NHEJ pathway (26-28). The basal repair gene expression study can be taken further where drugs and specific inhibitor or monoclonal antibody might be used to determine the predominant or active pathway and genes. Different sets of cell lines or transgenic cell lines can be used as to reproduce the finding reliability and variability. As these results did not produce significant statistical evidence further experimentations are suggested to do.