Dna Damage Repair Responses Acute Myeloid Leukaemia Cells Biology Essay

Acute myeloid Leukaemia ( AML ) is a malignant upset of the myeloid line of blood cells, characterized by the rapid unnatural proliferation of the leukocytes ( WBC ) that accumulate in the bone marrow interrupting the production of normal blood cell. It ‘s one of the most common ague leukemia impacting the grownups and disease incidence additions with age. AML is frequently associated with familial instability which is characterized by a diverseness of chromosomal and molecular alterations ( 1 ) . It has been reviewed that FLT3 cistron mutants are one of the most common abnormalcies which are found to be associated with AML. Approximately 30 % of AML patients have mutants of the FLT3 cistron in their blast cells, composed of FLT3-ITD mutants, 24 % ( 2 ) or FLT3 a point mutant within the activation cringle, 7 % ( 2 ) .

FLT3 is a individual transmembrane receptor with five-immunoglobulin like creases. The extracellular sphere binds to its growing factor, known as FLT3 ligand or FL. A individual sphere traverses the membrane, and so a kinase sphere is split by the kinase insert. The cistron location of FLT3 is 13q12 ; the FLT3 kinase sphere belongs to the type III receptor tyrosine kinase household, which includes KIT, FMS and 2 cistrons for the thrombocyte derived growing factor receptors ( PDGFR ) . Its ligand stimulates the proliferation of hematopoietic root primogenitor and dendritic cells. Surveies have shown that FLT3 is constitutively over expressed in most acute leukemia ( 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 mutant occurs in the juxtamembrane ( JM ) sphere of the FLT3 cistron which interrupts the suppression and constitutively activates the cistron. Another mutant which accounts for 8-12 % of AML patients is a mutant in the activation cringle of the cistron, most often affecting aspartic acid 835 or immediate next isoleucine 836 ( 6, 7, and 8 ) .

FLT3 transduces the signal from the membrane via activation of multiple downstream tracts. Normally, FLT3 is a monomeric protein on the cells surface. Upon ligand binding, dimerization occurs and kinase activity is initiated by autophosphorylation and later phoshorylating the other substrate molecules in the downstream signalling procedures. In the instance of the mutated signifier of FLT3 cistron the kinase is constitutively activated, which in bend activates the PI3 kinase/AKT tract, the RAS/MAP kinase tract, and the STAT5 phosphorylation tracts. All these tracts are involved in the procedure of programmed cell death, distinction and proliferation. ( Figure 1 ) . STAT5 tract is merely activated in mutant FLT3 tyrosine kinase, instead than the wild type ( 42 ) .

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Figure 1: Mutated FLT3 signals via activation of multiple downstream tracts

Dimerization of FLT3 protein occurs as FTL3 ligand binds to it, originating the procedure of

autophosphorylation and kinase activity. FLT3 mutant constitutively activates the kinase sphere and activates legion tracts, including the PI3 kinase/AKT tract, the ras/MAP kinase tract and the STAT 5 phosphorylation tract. All of these tracts interrupt the procedures of programmed cell death, distinction, and proliferation. All except STAT5 tract 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 undertaking is concerned with difference in DNA harm and response tract between FLT3 mutation and wild type cells. Deoxyribonucleic acid amendss can happen due to several beginnings. These amendss can either happen via endogenous or exogenic beginnings. DNA fix systems act otherwise for individual strand DNA amendss and dual strand DNA amendss. In instance of individual strand harm, the other strand is used as a templet to steer the right fix procedure. There are chiefly three types of individual base DNA harm fix mechanisms. They are, basal deletion fix, nucleotide deletion fix and mismatch fix. Double strand harm fix involves different theoretical accounts which include homologous recombination, the vacation theoretical account and not homologous terminal connection ( NHEJ ) tract etc.

Figure 2: DNA-repair tracts. Several DNA-repair tracts exist and cover with assorted types of Deoxyribonucleic acid abuses. These tracts include 1 ) the direct reversal tract, 2 ) the MMR tract, 3 ) the NER tract, 4 ) the BER tract, 5 ) the HR tract, and 6 ) the NHEJ tract. This diagram was copied from the publication by Kanaar et Al, 2008 ( Ref: 20 )

The induction of dual isolated interruption checkpoints is dependent on the enlisting of MRE11 / RAD50 /NBS1 ( MRN ) composite at the harm site. And that is followed by the enlisting or activation of ataxia-telangiectasia mutated ( ATM ) , a member of the household of phosphoinositide-3-kinase-related kinases ( PIKKs ) ( 17 ) . And most significantly, two other PIKKs, DNA-dependent protein kinase ( DNA-PK ) and ATR ( ATM and Rad3 related ) , are besides activated and involved in the response to DSBs. The primary map of ATR is to originate the DNA harm response at stalled reproduction forks ( RFs ) ( 18 ) . ATM, ATR and DNA-PK are the first proteins that act when DNA harm occurs and that is via phoshorylating different marks of the downstream processes.

NHEJ or Non Homologous End Joining tract is an mistake prone DSB fix tract that is active in G1 stage and is the prevailing tract for DSB in mammalian cells ( 20 ) . The nucleus protein constituents of the mammalian NHEJ include the Ku fractional monetary units ( Ku70 and Ku80 ) , DNA-PKcs, XRCC4, DNA ligase IV ( LigIV ) , Artemis, and the late identified Cernunnos-XLF ( besides known as NHEJ1 ) ( 21 ) . NHEJ joins together double isolated DNA ends after the bases are corrected, and individual stranded tempering ( SSA ) occurs through deletion of damaged bases in a individual DNA strand and filling in the losing bases ( 22 ) . Homologous recombination or HR tract is a multistep tract which requires several proteins and it acts on S stage or G2 stage. HR lone histories for 10 % of DSBs in mammalian cells but defect in HR tract cistrons can hold terrible effects that include deadliness ( 42 ) . There are several studies that implicate DSB and their fix by NHEJ in the coevals of some cardinal chromosomal translocations in both childhood and grownup ague myeloid and lymphoid leukemia ( 23 ) .

Surveies showed that FLT3 mutated AML cells respond to, and fix, chemotherapy-induced harm more expeditiously than AML cells with wild type FLT3 ( 1, 9 ) . As a consequence FLT3 mutated AML cells may non acquire killed by the chemotherapeutic drug and could later impact the clinical result of the patients. It has been found in old surveies that backsliding affects over 50 % of patients with AML and patients with a FLT3 mutant have higher backsliding rates than patients without a mutant ( 10 ) . Studies done utilizing the S-phase drug clofarabine showed that FLT3 mutant AML cells do non collar the cell cycleand repair the fix the DNA harm expeditiously when incubated with clofarabine for a short clip period. It was found that cell rhythm apprehension in response to DNA harm by clofarabine in S stage is affected via loss of the transcriptional regulator called cdc25A. FLT3-ITD mutant cells have the ability to cut down or counterbalance this loss when treated with clofarabine. Small interfering RNA against the FLT3-ITD provides sufficient grounds that the message is about reduced by 87.5 % ( 1 ) . An extra survey on DNA fix in FLT3-ITD cells shows that AML cells holding the FLT-ITD mutant to hold increased reactive O species ( ROS ) production. As a effect increased DNA dual strand interruptions ( DSBs ) and choice of mistake prone fix pathway leads to aggressive AML in FLT3-ITD patients. AML cell lines with FLT3-ITD mutants produce ROS via STAT5 signalling pathway and by the activation of RAC1, which is an indispensable constituent of ROS-producing NADPH oxidases. A possible mechanism for the coevals 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 ensuing in decreased DSB and increased fix and fidelity ( 11 ) . Another survey found that look of RAD51, a cistron which is involved in the homologous recombination DNA fix system, was found to be associated with FLT3-ITD mutant AML cell lines. Inhibition of FLT3 by utilizing PKC412 significantly down regulated the RAD51 transcript and this was purely seen in FLT-ITD mutant AML cell lines, non the wild type AML cell lines. Use of FLT3-short interfering RNA ( siRNA ) besides came with the same consequence ( 9 ) .

In this undertaking, the response of different AML cell lines to DNA harm of different AML cell lines utilizing chemotherapeutic drug and specific inhibitor were analyzed. The harm response was measured in two different classs of AML cell lines, one with a FLT3-ITD and the other with a wild type FLT3 position. Several curative DNA fix inhibitors are being developed ; one of these inhibitors was used to detect the fix abnormalcies in AML cells with common FLT3 mutant.

Several molecular biological science techniques which included DNA harm responses, DNA harm degree measuring and mensurating cell viability were used in this undertaking. Basal expression degree of several DNA harm antiphonal cistrons were measured and analyzed for their engagement in different DNA fix mechanisms or tracts.

This undertaking was aimed to see the DNA harm responses, step DNA harm degrees and cell viability by utilizing several molecular biological science techniques. In add-on basal look degree of several DNA harm antiphonal cistrons were besides measured and analyzed for their engagement in different DNA fix mechanisms or tract which was compared with the old determination in this field.

Materials & A ; Methods:

Cell lines

MOLM 13 ( DMSZ ) , M07e ( DMSZ ) , OCI-AML-3 ( DMSZ ) , MV4-11 ( ATCC ) and U937 ( ECACC ) AML cell lines were used for experimentation intents. Of these five cell lines MOLM13 and MV4-11 have got FLT3-ITD mutants, the remainder are all FLT-3-WT genotype. On the other manus U937 cell line is p53 mutation and the remainder are wild type for p53 position. All the cell lines were maintained in Rosewell Park Momorial Institute ( RPMI ) -1640 medium with 10 % fetal calf serum ( FCS ) , 1 % penicillin and 1 % streptomycin. In the leukemia lab all the cell lines used are in suspension, so the attachment factor is omitted here.

Viability Assays

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 bluish check was followed as suggested by the makers. The cell lines used were MOLM13 and M07e. The cytotoxic drug etoposide was used for this check. 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 number the unrecorded cells manually. Two cell lines, MOLM13 and M07e were used in this check where drug concentrations for MOLM13 500ng/ml and for M07e 1000ng/ml ( IC20 determined from the alamar bluish check ) were used along with the phosphor-ATM kinase inhibitor, 10mM/ml DMSO ( Calbiochem ) and controls. The concentration of the inhibitor used was 1AµM.The initial cell concentration for this experiment was 5×105 /ml. After one twenty-four hours the drug and the drug plus inhibitor treated cell samples were counted and washed twice with RPMI-1640 and so divided into two more samples incorporating the drug and the drug plus inhibitor. Cells were grown in complete medium incorporating RPMI 1640 with 10 % FCS and 1 % Pen/Strep and Glutamine on cell civilization flasks and counted after a fit clip period to find the viability. Cell count was done until there were two close cell counts and the norm was taken.

Alkaline Comet Assay

MOLM 13, M07e and U937 cell lines were used for comet check analysis. From old surveies the concentration for Clofarabine was determined to utilize on these cell lines ( 1 ) . Another drug, Etoposide was used as it produces DNA harm which can be detected by the comet check and the concentration ( IC20 ) of the drug for these two cell lines were determined from the alamar bluish viability check. The cells were fed a twenty-four hours earlier before handling with drugs ( Etoposide and Clofarabine ) . Clofarabine was used at a concentration of 0.3AµM/ml for MOLM13 and 1AµM/ml for M07e and cell were incubated for an hr. On the other manus 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 hr. Cells were taken at 5×105/ml concentration. Control cell lines were thawed in a 37A°C H2O bath and were assorted with agarose merely before lading them to the comet slides. Control cell lines are cell samples that have got specific degree of DNA harm done by specific dose of drug intervention. Trevigen ( AMS Biotechnology Ltd, Abingdon, UK ) comet assay slides and lysis buffer were used for the comet check and harmonizing to the maker ‘s instructions the protocol was followed. For alkaline and impersonal comet assay the cataphoresis parametric quantities were 400mA for 20 proceedingss and 20V for 15 proceedingss severally. 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 utilizing Comet Assay III image analysis package ( Perspective Instruments, Suffolk, UK ) . Fifty comet images were recorded form each of the two gel musca volitanss and each experiment status ; hence, 100 images were in entire for each intervention. Mean tail minute of the comets was used in all analysis and it was referred that the higher the average comet tail minute the greater the harm.

Quantitative RT-PCR

Ribonucleic acid from six different cell lines ( MOLM13, M07e, OCI-3, KG-1a, HL-60 and MV4-11 ) was prepared utilizing QIAmp RNA kits with DNAase intervention harmonizing to the maker ‘s instructions ( Qiagen ) . Up to 2000ng RNA was used in a rearward RNA polymerase reaction with MMLV contrary RNA polymerase ( Invitrogen ) and random hexamers ( GE Healthcare ) . Quantitative PCR was done on an ABI Prism 7700 ( Applied Biosystems ) utilizing Excite Real-time Mastermix with SYBR Green ( Biogene ) ..Thermal cycler conditions included incubation at 95A°C 10 proceedingss followed by 40 rhythms of 95A°C 15 s and 60A°C 1 minute. Following the 40 rhythms, the merchandises were heated from 60A°C to 95A°C over 20 proceedingss to let runing curve analysis to be done. This measure allowed the specificity of the merchandises to be determined as a individual thaw extremum and besides confirmed the absence of primer-dimmers.

The housework cistron I?2 microglobulin ( I? 2M ) was used to standardise the samples and the comparative look degree of PARP1, DNA ligIII, DNA ligIV, ATM, WRN, OGG1, XRCC6 and Artemis, ( The primers for these 9 cistrons were bought from Qiagen ) . Therefore it was calculated as the ratio between the degrees of these trials cistrons and the control cistron ( I?2M ) . All Negative controls ( no templet ) were included in each experiment and all reactions were run in triplicates.

Flow cytometry

Phoshpho ATM & A ; Phospho p53 checks

Cells ( MOLM13 & A ; M07e ) were incubated at 5×105/ml for 24 hours with and without the particular doses ( for MOLM13: 500ng/ml & A ; M07e: 1000ng/ml ) of etoposide followed by two rinses in cold RPMI-1640. A part of the cells was so fixed instantly to measure the baseline harm and the remainder was resuspended in fresh civilization medium and was left in the brooder to let clip ( 2hr, 4hr, 24hr ) for fix to happen. After these recovery clip intervals phosphorylation of serine 1981 at ATM and phosphorylation of serine 15 at p53 were measured. Counterstaining to mensurate the intracellular DNA harm content analysis was done with 25Aµg/ml 7-AAD in PBS. For quantitative analysis, fluorescence values obtained from the untreated control cells were compared to the drug treated trial. For both of these checks leucoperm ( AbD Serotec ) kit arrested development method was used and the protocol was followed as the maker ‘s direction. Phospho ATM antibody mouse monoclonal MAB3806 from Millipore at 1mg/ml was used at 1:400 dilution and for Phospho p53 check, 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 ) ) , 100Aµg/ml was isotype matched for both of the experiments. 5Aµl 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 pulverization. Solution was made as 1mg/ml utilizing ice cold PBS.

Consequences

Comet Assay

Comet Assay or Single Cell Gel Electrophoresis measures DNA harm from single cells based on the migration of denaturized DNA through an cataphoretic field ( 12, 13 ) . Single cells or karyons are embedded in agarose gel, lysed to expose their Deoxyribonucleic acid content, alkali treated to denature or loosen up the Deoxyribonucleic acid and electrophoresed to divide the Deoxyribonucleic acid. The damaged DNA incorporating strand interruptions migrates further in the gel than integral DNA and that creates an image resembling a heavenly comet ( 14, 15 ) . Fluorescent dye, SYBR Green, makes the damaged Deoxyribonucleic acid to be visualized as comets on fluorescent microscope. Alkali comet check can observe dual stranded and individual stranded DNA amendss, on the other manus impersonal comet check detects individual DNA harm and cross links ( 17, 18 ) .

The intent of the comet check in this undertaking was to detect the DNA harm degree of the drug clofarabine on a FLT3-ITD AML cell line and compare it with a FLT3-WT cell line. Etoposide was besides used because it is known to bring forth DNA harm which is noticeable by the comet check. The control cells ( CC0, CC1, CC2, and CC3 ) used in this experiment had exponential DNA harm degree as they came in treated with different drug concentrations prior use.

Figure 3: Alkaline Comet check 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 harm ( n=1 )

In the cell lines MOLM13 & A ; M07e no harm 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 harm sensing 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 harm was observed even though the control cells produced expected consequences, that is an addition in comet minute with increased concentrations of drug. Unfortunately clip restraints did n’t let optimizing the comet assay farther.

Phospho-ATM and Phospho p53 Assaies

Phospho-ATM and phospho-p53 sensing checks were performed utilizing a flow cytometry technique to mensurate the DNA harm response on MOLM13 and M07e cell lines. Different recovery periods were set up to buttockss fix of the damaged Deoxyribonucleic acid and analyze fluctuations in DNA harm responses.

Figure 4: Phosphorylated Ser-1981 degree of ATM in MOLM13 ( A ) and M07e ( B ) cell lines with etoposide intervention 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 surveies, n=3 and for 0hr survey n=1. In this graph the mean of 2hr, 4hr and 24hr surveies were plotted with standard deviatioin as mistake bars

As it can be seen from the graph ( figure 4 ) that MOLM13 has got less DNA harm response than M07e and besides it can be observed that the DNA harm response gets unusually lower by 4 hours recovery period compared to the M07e cell line where phosphorylated ATM degree is still, about, more than the half of MOLM13 cell line ‘s highest phosphorylated ATM degree.

Figure 5: Phosphorylated Ser-15 degree of p53 in MOLM13 ( A ) and M07e ( B ) cell lines with etoposide intervention 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 check, n=1 ) Standard divergence was used as mistake bars for 24 hr check.

Similar consequences came from the phospho-p53 check with a difference in both cell lines, as during 4 hr 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 province of M07e is about two times higher than MOLM13 cell line.

Viability Assay

Viability assay utilizing the manual cell count technique gave the thought of the growing and decease in cell samples incorporating 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 consequence on MOLM13 cell lines than M07e. This is surprising as the flow cytometry checks 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 information here, it can be seen that MOLM13 had more cell kill than M07e. It was besides found that entirely Inhibitor has got more consequence on MOLM13 as the cell count went below the basal degree ( 5×105 cells/ml ) , on the other manus M07e cells still grew over the radical degree. Whilst these experiment demand repeatation, these consequences steadfastly suggest that MOLM13 cell line might be more dependent on the ATM tract than the M07e cell line.

Figure 6: Vaibility assay utilizing drug ( Etoposide ) & A ; Inhibitor ( ATM kinase ) MOLM13 & A ; M07e Cell lines ( Day-1 ) . Here C, I, ( C-D ) & A ; ( D+I ) represents sample which is control, inhibitor entirely, 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 consequence of the inhibitor in drug treated cell samples. Here drug concentrations for MOLM13 and MO7e were 500ng/ml and 1000ng/ml severally, the concentration of the ATM kinase inhibitor was 1AµM and the initial cell concentration ( 5×105 /ml ) was taken as the base line.

After twenty-four hours one the samples were washed and divided into farther two sets with drug merely and drug plus inhibitor samples. From the graph ( Figure 7 ) it can be seen that the figure of cells increasingly gets reduced in cells treated with drug entirely and drug plus inhibitor. It was besides found that MOLM13 had more dead cells than MO7e, back uping the old implicative analysis.

Figure 7: Vaibility assay utilizing drug ( Etoposide ) & A ; Inhibitor ( ATM kinase ) MOLM13 & A ; M07e Cell lines ( Day 2 ) . the first twenty-four hours sample count is represented by the first two bars of each cell lines. Following, each of the samples of first twenty-four hours were washed and divided into two farther 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 severally and the concentration of the ATM kinase inhibitor was 1AµM.

These consequences are besides displayed in Table 1 and 2 where the growing column represents the growing of cells in different samples through clip. As the get downing concentration of cell sample was taken as one hundred per centum so below 100 per centum implies cell decease. From Table 2, sample Drug & A ; Inhibitor ( D+I ) , which had drug and inhibitor for the whole clip, was found to be incorporating the least growing or upper limit cell decease among all other samples. MOLM13 and M07e had 3 % and 18 % growing severally which implies 97 % and 82 % cell decease on twenty-four hours 2.

Table 1: Viability assay MOLM13 & A ; M07e cell lines ( Control & A ; Inhibitors treated samples )

Sample

Day 1

Average

Growth

( day1/0 )

Day 2

Average

Growth

( Day2/1 )

MOLM13 Control

8.7×105

6.4 x105

7.55×105

151 %

1.54 x106

1.68 x106

1.61 x106

213 %

MOLM13- Inhibitor

4.2 x105

3.8×105

4 x105

80 %

7.8 x105

7.4 x105

7.6 x105

190 %

M07e Control

1.1×106

0.9 x106

1×106

200 %

1.14 x106

9.8 x105

1.06 x106

106 %

M07e -Inhibitor

A­A­A­A­6.8 x105

7.2 x105

7 x105

127 %

7.3 x105

7.2 x105

7.25 x105

103 %

Table2: Viability assay MOLM13 & A ; M07e cell lines ( Drug and Inhibitor treated samples )

Sample

Day 1

Average

Growth

Day1/0

Day 2-Drug was washed away and new drug was added with or without inihibitor.

Day 2

Average

Growth Day2/1

MOLM13 Drug

3.1×105

3.9 x105

3.5×105

70 %

MOLM13 Drug ( D )

1.3 x105

1.5 x105

A­A­A­A­1.4 x105

40 %

MOLM13 Drug ( D+I )

6 x104

7 x104

6.5 x104

18.5 %

MOLM13 Drug+

Inhibitor

2.6 x105

2.8 x105

2.7 x105

54 %

MOLM13 Drug Inhibitor ( D )

4 x104

6 x104

A­A­A­5 x104

18.5 %

MOLM13 Drug & A ; Inhibitor ( D+I )

1 x104

1 x104

1 x104

3 %

M07e Drug

A­A­4.7 x105

5.9 x105

5.3 x105

106 %

M07eDrug ( D )

1.8 x105

1.7 x105

1.75 x105

33 %

M07e Drug ( D+I )

1.5 x105

1.6 x105

1.55 x105

29 %

M07e Drug+ Inhibitor

2 x105

1.9 x105

1.95 x105

39 %

M07e Drug & A ; Inhibitor ( D )

6 x104

7 x104

6.5 x104

33 %

M07e Drug & A ; Inhibitor ( D+I )

4 x104

3 x104

3.5 x104

18 %

As these two cell lines might hold different growing rate that is why cell count and growing per twenty-four hours was measured to find the per centum of cell viability.

Quantitative RT-PCR

Chiefly radical looks of eight DNA fix cistrons were analyzed in this undertaking in context to four different cell lines. The cistrons 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 Deoxyribonucleic acid dual strand interruption fix protein that is a member of RecQ helicase household and contains 3 ‘ — & gt ; 5 ‘ helicase and 3 ‘ — & gt ; 5 ‘ exonuclease activities and binds with PARP1 foremost so interacts with Ku70 and Ku86 proteins fractional monetary units of NHEJ fix system ( 26-28 ) . DNA ligase III or Lig3 is a member of Nucleotide Excision Repair ( NER ) tract which can straight adhere to the N-terminal part of PARP1 ( 29 ) . It has besides been found that in NER pathway interaction of Lig3 with XRCC1 plays an of import function by making stableness ( 30, 31 ) . Deoxyribonucleic acid ligase IV or Lig4 cistron is involved in DSB fix tract which acts via NHEJ mechanism ( 32 ) . It is of import to advert that it was found to be associated with DNA-PKs and Ku proteins ( 33 ) . Another cistron involved in the DSB fix tract is ATM ; this cistron merchandise has been found to be associated with BRCA1, p53, Chk2, DNA-PKcs and Artemis phosphorylation ( 34, 34, 35 ) . PARP1 is member of SSB fix tract cistrons which interacts with WRN and Ku70/86 composite of NHEJ tract ( 28 ) . Another survey found that in order to adhere to PARP1, p53 must be phosphorylated bespeaking their interaction with each other ( 37 ) . OGG1 is involved in Base Excision Repair ( BER ) tract and recent surveies found that it can be used as a predictive marker in AML as its look is associated with higher reactive O species ( ROS ) and extra mutation p21 and p53, which leads to bad forecast ( 38 ) . Artemis is another cistron of NHEJ tract which has been found to interact with DNA-PKc and mutant in Artemis cistron consequences in hypersensitivity to DSBs and immunodeficiency ( 39 ) . Artemis was besides found to interact with Ku70 and DNA ligase IV and ATM cistrons merchandises ( 40 ) . Another DSB fix cistron 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 look survey of DNA fix cistrons were done to detect the different degree of DSB fix cistrons and SSB fix cistrons among the six different AML cell lines. Of these six cell lines two were FLT3 mutation ( MOLM13 & A ; MV4-11 ) and two other were FLT3-WT ( M07e and OCI-AML-3 ) . These four cell lines have wild type p53 position. Of the eight cistron looks observed, five of them are involved in DSB harm response and/or fix tract and the other three are involved in SSB response and/or fix tract.

A

Bacillus

Figure 8 ( A & A ; B ) : SSB harm antiphonal cistron look degree in FLT3 mutation cell lines ( MOLM13 & A ; MV4.11 ) and FLT3 wild type cell lines ( OCI-AML-3 and M07e ) . n=1

Bacillus

A

Figure 9 ( A & A ; B ) : DSB harm antiphonal cistron look degree in FLT3 mutation cell lines ( MOLM13 & A ; 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 cistron look degree. These cistrons are involved in NHEJ dual strand interruption fix pathway. MV4-11 is homozygous for FLT3 mutant position on the other manus MOLM13 is heterozygous for FLT3 position. MOLM13 manus significantly lower degrees of look of the cistrons mentioned above but it had relatively higher ATM look than the other FLT3 mutation cell line, MV4-11. On the other manus OCI-AML-3 cell line had high WRN look and relatively high PARP1 look. And the same was observed for M07e cell line which even had high look of ATM. WRN protein binds to PARP1 and upon adhering it activates it and following interacts with XRCC6 ( Ku70 ) of NHEJ tract ( 26-28 ) . This might be a transverse talk among the DSB and SSB fix cistrons which is interesting.

Discussion:

Mutant in AML is a really of import factor for predictive and intervention intents. Several mutants are associated with AML. As of import as FLT3, NPM1 is another of import mutant that has been found at least in half of the normal karyotype AML patients, taging the most common molecular marker in AML to day of the month ( 16 ) . FLT3-ITD incorporating patients have a significantly greater hazard of backsliding and ensuing in hapless forecast ( 10 ) . Analyzing FLT3 mutant is a really of import research field in analyzing acute myloid leukemia. The chief aim of this undertaking was to detect the differences between DNA harm response degree in FLT3 mutation and wild type cell lines and compare them.

The undertaking started by looking at the DNA dual strand interruption harm by making comet check and as the drug clofarabine was non working another drug ; etoposide was used to optimise the check. Comet assay done in this undertaking was non consistent were jobs with a faulty cataphoresis power battalion, hence although efforts were made to optimise the impersonal and alkalic comet check, this was non achieved due to clip restraints. Consequences from the viability count check provided the thought that MOLM13 cell line might be more dependent on ATM tract for DSB fix response than M07e cell line and as from the cell count it was found that by the terminal of two twenty-four hours experimentation, MOLM13 had got more killed cells than MO7e cell lines. Supporting this information DNA harm response measured by phospho-ATM and phosphor p53 assay found that MO7e had more DNA harm response than MOLM13. It was found in the old surveies that FTL3-ITD mutant incorporating AML cells respond more to chemotherapy induced harm and fix harm more expeditiously than wild type FLT3 cells ( 1, 9 ) . Data found from the harm response check and viability assay might back up these old surveies because if FLT3-ITD incorporating cells have other active fix mechanisms, they may originate their harm fix procedure but as the procedure is error prone an AML patient with a FLT3 mutant ends up with bad forecast. Non-homologous terminal fall ining fix mechanism is one of the mistake prone DSB fix processes that are prevailing in mammalian system ( 20 ) . Further surveies with different sets of mutation and wild type cell lines can be done to prove this hypothesis.

Real clip PCR information showed the heterogenous look of DNA fix cistrons in different AML cell lines. High look degree of NHEJ tract cistrons were observed in FTL3 mutation cell line ( MV4-11 ) and it was besides found that compared to other cell lines MOLM13 had higher look of ATM. Although MOLM13 and MV4-11 have got mutated FLT3 position but similar look degree was non observed. Cell lines wild type for FLT3 and p53 position had been observed with higher WRN and PARP1 look. Studies antecedently found that WRN binds straight to PARP1 and so interacts with Ku70 ( XRCC6 ) of the NHEJ tract ( 26-28 ) . The basal fix cistron look survey can be taken farther where drugs and specific inhibitor or monoclonal antibody might be used to find the predominant or active tract and cistrons. Different sets of cell lines or transgenic cell lines can be used as to reproduce the determination dependability and variableness. As these consequences did non bring forth important statistical grounds farther experimentations are suggested to make.

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