(MLL)-Acute Lymphoblastic Leukemia (ALL) Human Cell Lines

Journal of Young Investigators Research

Analysis of expression of MSH2 and MSH3 genes in Mixed Lineage Leukemia (MLL)-Acute Lymphoblastic Leukemia (ALL) human cell lines

Kulsom Nuuri1, Yoana Arroyo-Berdugo1, and Maria Teresa Esposito1*

Around 803 new cases of Acute Lymphoblastic Leukemia (ALL) are diagnosed in the United Kingdom every year (Cancer Research UK, 2018). Cytarabine (Ara-C) is a chemotherapeutic drug used to treat leukemic patients, however, chemotherapy resistance occurs frequently. Mechanisms of chemotherapy resistance to Ara-C are poorly understood. DNA mismatch repair (MMR) pathway is one of the pathways responsible to repair errors caused during DNA replication. MMR can also repair damage due to Ara-C and therefore a proficient MMR can confer resistance to Ara-C. Our project aims to investigate the expression of MMR genes MSH2 and MSH3 in ALL cells carrying Mixed Lineage Leukemia (MLL), also known as KMT2A, chromosomal translocations. The purpose of this study was to examine a potential correlation between the expression of these genes in MLL-ALL and Ara-C resistance. RNA was isolated from cultured cell lines: Kasumi-1 and K562 (positive non- MLL controls) and HB-1119 and SEM-1 (experimental MLL-cell lines), retro-transcribed into cDNA. End-Point and Semi- quantitative PCR were employed to amplify the cDNA and analyze the expression of MSH2 and MSH3 in the experimental cell lines. The results indicate that MSH2 and MSH3 are expressed in both Ara-C resistant MLL-ALL cell lines HB-1119 and SEM-1 and that the expression levels in HB-1119 were lower than SEM1. Further research is needed to understand the contribution of MMR to Ara-C response in ALL. This research should be focused on testing the functionality of MMR in Ara-C resistant and sensitive leukemic cell lines. Furthermore, the contribution of each MMR genes to Ara-C resistance should be addressed by knock-down and over-expression studies.

INTRODUCTION 1.2 Acute Lymphoblastic Leukemia 1.1 Leukemia Acute lymphoblastic leukemia (ALL) affects the lymphocytes Leukemia is a malignant disease arising from the hemato- progenitors. B-cell Acute Lymphoblastic Leukemia (B -ALL) poietic stem cells (HSC) that reside in the bone marrow and accounts for 80 - 85% of ALL and T-cell Acute Lymphoblastic give rise to blood cells. Excessive production of immature Leukemia (T -ALL) accounts for 20 - 25% of ALL (Chiaretti HSCs in the bone marrow interrupts the normal production et al., 2013). ALL is more prevalent in children aged 4 - 14 of the blood cells (Mwigiri et al., 2017). Acute leukemia is years and in adults aged over 50 (Mwirigi et al., 2017). Chro- an aggressive form of cancer that can progress quickly and mosomal and genetic alterations are hallmarks of ALL and be fatal if left untreated. Leukemia is one of the twenty most affect differentiation and proliferation of lymphoid precursor common cancer in the UK (Figure 1) (Cancer Research, cells derived from HSCs in the bone marrow. ALL is divided 2018) and it made up 437,033 new cases worldwide in the into subgroups depending on the chromosomal transloca- year 2018 (Worldwide cancer data, 2019). tions (Siraj et al., 2003); these translocations include t(12;21) [ETV6-RUNX1], t(1;19) [TCF3-PBX1], t(9;22) [BCR-ABL1] and translocations affecting 11q23 which rearrange the MLL Address correspondance to: gene (Terwilliger and Abdul-Hay, 2017). 1University of Roehampton Whiteland Campus Parkstead House Holybourne Avenue SW15 4JD 1.3 Mixed Lineage Leukemia London UK * [email protected] MLL is a leukemia that can phenotypically resemble both ALL and Acute Myeloid Leukemia (AML) (Jaffe et al., 2001). However, from a genetic perspective, MLL is unique and dis- doi:10.22186/jyi.37.4.38-48 tinct from ALL and AML (Jaffe et al., 2001; Armstrong et al., 2002). The MLL gene (also known as KMT2A, ALL-1, HRX, and TRX1) is located at chromosome 11q23 and encodes MLL, an epigenetic regulator of gene expression mainly ex- Except where otherwise Acceptance date: October 2019 noted, this work is licensed pressed during early development and hematopoiesis (Mo- under https://creativecom- han et al., 2010). The C-terminal has transcriptional activa- Publication date: April 2020 mons.org/licenses/by/4.0 tion properties and is able to activate gene expression, for example, by methylating histone 3 on lysine 4 near gene

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promoters and bodies, whereas the N-terminal can recruit transcriptional repression proteins (Yokoyama et al., 2002; Ballabio and Milne, 2012). Chromosomal translocations affecting MLL generate oncofusion proteins. In these proteins, MLL loses its C-terminal, whereas the N-terminal is fused to partner proteins (Mohan et al., 2010) (Figure 2). The oncofusion protein is a potent regulator of gene expression and recruits several epigenetic regulators including Dot1L (Krivtsov and Armstrong, 2007; Sabiani et al., 2015) (Figure 2). Mixed-Lineage leukemia- Acute lymphoblastic leukemia fused gene from chromosome 4 (MLL-AF4) fusion protein is generated by t(4;11), an abnormal rearrangement between MLL and AF4 (also known as AFF1) (Sabiani et al., 2015). MLL–AF4 and the reciprocal AF4–MLL fusion alleles are found in 80% of t(4;11) leukemia patients. The other 20% fusion alleles in t(4);11 leukemia patients show more complex Figure 1. A bar chart to show the most common cancers by rearrangement composed of three or more fusion proteins gender in UK: 2015. Leukemia is the 12th common cancer and (Sabiani et al., 2015). MLL-AF4 is the most prevalent MLL makes up 9900 new cases in the year of 2015, in UK (adapted from translocation in ALL and is present in SEM-1 human cell line, Cancer Research, 2018). which is one of the experimental cell lines in our study (Men- delsohn et al., 2015; Stumpel et al., 2015). MLL can also fuse with other partner genes such as ENL to generate the oncofusion protein MLL-ENL (Figure 2). This is the result of chromosomal translocation t(11;19) (q23;p13). This translocation is found in HB-1119 human cell line that is derived Figure 2. MLL partner genes. The N-terminal portion of MLL fus- from B-ALL (Brown, 2005). HB-1119 is the second experi- es with C-terminal portion of the partner gene. MLL-AF4 and MLL- mental cell line in our study. ENL fusion genes belong to MLL-ALL (adapted from Zheng, 2013).

1.4 Mismatch repair pathway- MSH2 and MSH3 MMR allows the repair of mismatched bases and insertion- deletion loops (IDLs), produced during DNA replication. Mutation rates are higher if the MMR pathway is missing in tumor cells (Laghi et al., 2008). Deficiency in MMR can be detected via microsatellites which are short repetitive DNA sequences generated by faulty MMR-(Laghi et al., 2008; Bo- land et al., 2008). Mutations can occur in microsatellites due to the slippage of DNA polymerase causing small unneces- sary deletions or expansions within the sequence (Blasco et al., 2006). Normally, the replication errors in the microsatellites are repaired by the MMR. However, deficiencies in the MMR pathway cause DNA residues to increase or decrease Figure 3. MMR genes and their role in MMR pathway. The in the microsatellite, leading to a microsatellite instability Mutsα recognizes single base mismatches and small IDLs (A and (MSI) (Laghi et al., 2008). B). Mutsαβ recognizes larger IDLs (C) (adapted from Martin et al., 2010). MSH2 and MSH3 are important components of the MMR pathway (Burdova et al., 2015). Mismatches are rec- MSH2-MSH3) allows to recruit MutL (consisting of MLH1 ognized by MutS complexes. MutSα is composed by MSH2 and PMS2) (Figure 3) and the helicase MCM9. MCM9 heli- and MSH6 and repairs small IDLs (Figure 3). MutSβ is com- case catalyze the removal of the mismatch- containing DNA posed by MSH2 and MSH3 and recognizes the IDLs that strands. Replication factors such as proliferating cell nuclear are1-13 nucleotides long. MSH2 interacts together with antigen (PCNA), DNA polymerase δ (Polδ) and Replication MSH3 (or with other proteins) (Figure 3) to give rise to the protein A (RPA) repair the DNA strand (Figure 4) (Traver et DNA mismatch-binding protein complex MutSβ. MutSβ can al., 2015). locate errors in the DNA produced during DNA replication The loss of MMR proteins can cause accumulation of (Burdova et al., 2015). MutS complexes (MSH2-MSH6 or DNA replication errors and therefore contribute to MSI (Bur-

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ment, 33 - 50% of patients relapse. This means that Ara-C is not very effective (Verma, 2012). Drug resistance is more common in children who suffers from ALL than in adults; though the reasons are not clear (Stam et al., 2003). Ge- netic background, diseases severity, efficacy of absorption, metabolism and elimination of the drug can account for the effectiveness of Ara-C (Cai et al., 2008). Ara-C is adsorbed into the intracellular space through membrane transporters of the human nucleoside transporter (hENT) family (Català et al., 2016). High hENT1 expression has been linked to Ara-C sensitivity in infant and adult MLL-ALL (Català et al., 2016). Català et al. (2016) demonstrated that hENT expression and activity can be modulated by inhibitors of Tyrosine kinase receptor FLT3. This can affect Ara-C cytotoxicity. Figure 4. MMR genes, heterodimer complexes and their role in MMR pathway. MSH2 and its partner (MSH6/MSH3) recog- 1.5.1 Chemotherapy problems associated with MMR nizes DNA mismatch (A). Recruitment of MLH1 and its partner Defects in the MMR pathway has been shown to have a PMS2 and other cofactors (PCNA) and enzymes (EXO1) conduct relationship with resistance of many cytotoxic drugs, includ- excision, resynthesis and ligation of the repaired DNA strand (B & ing Ara-C (Matheson and Hall, 2003). A study conducted by C) (Wei et al., 2002). Hewish et al. (2013) revealed that cytosine-based nucleoside analogues are harmful for MSH2-deficient tumor cells and other MMR deficient cells by changing mitochondrial membrane potential (ΔΨm) and generating reactive oxygen species (ROS). Absence of MSH2 leads to uncontrolled ROS levels and inadequate antioxidant response leads to apoptosis. Incapability of repairing oxidatively damaged DNA leads Figure 5. Action mechanism of Analog Triphosphate (Ara-C). to the production of lethal double-strand DNA breaks (DSB) Ara-C, inhibits DNA strand synthesis by binding onto incorporating and apoptosis (Hewish et al., 2013). DNA chain- leading to cell death signalling (adapted from Ewald Fordham et al. (2011) found that silencing of MSH2 and et al., 2008). MSH6 in AML cells increased the sensitivity to Ara-C and dova et al., 2015; Surtees and Alani, 2006). MSI is mostly Clorafabin. This study together with the study of Hewish et a result of under-activity of MutSβ (heterodimer of MSH2 al. prompted us to investigate the expression of MSH2 and and MSH3) rather than MutSα (heterodimer of MSH2 and MSH3 in Ara-C resistant MLL-ALL cell lines (HB-1119 and SEM-1). MSH6), because defects in MSH6 protein do not lead to a high frequency of MSI due to the effect of the compensa- METHODS tion provided by other secondary MMR molecules such as In order to investigate the expression of MSH2 and MSH3 MSH3, MLH3 and PMS1 (Boland et al., 2008; Shia, 2008; in the MLL experimental cell lines we began with cultur- Nakatani et al., 2015; Boland et al., 2008). MSH2 combined ing the cell lines in vitro. SEM-1, Kasumi-1 and K562 were with MSH3 can compensate for the loss of MSH6 (Boland purchased from DSMZ (DSMZ). The HB-1119 cell line was et al., 2008). In contrast, Kheirelseid et al. (2013) revealed obtained through Professor. Eric So in 2015.The cell line that the MSH2-MSH3 heterodimer cannot compensate for was a gift of M. Cleary (Stanford university). The cell line is MSH6 deficiency as MutSβ recognizes only IDLs. Nakatani deposited in the depmap database (HB1119 DepMap Cell et al., (2015) argues that the exact role of the MMR proteins Line Summary, 2020). Cells were kept at 37OC and 5% CO2 remains controversial and it is different amongst the models in RPMI-1640 supplemented with 10% Fetal Bovine Serum being used in the studies. This may possibly explain why and antibiotics. Actively growing cells were centrifuged at certain studies argue that MSH3 (combined with MSH2) can 500 g for 5 minutes, washed with PBS and then centrifuged compensate for MSH6 loss while others do not. again. Cell pellets were used for RNA isolation and subse- 1.5 Chemotherapy quent generation of cDNA. One of the common drugs used in childhood and infant ALL 2.1 Making buffer and solutions is Cytarabine also known as cytosine arabinoside (Ara-C), which is a nucleoside analogue (antimetabolite) (Matheson 2.1.1 500 ml of 50x stock of Tris-Acetate-EDTA (TAE) and Hall, 2003). Ara-C works by inhibiting DNA replication Tris and EDTA were put in the beaker along with distilled wa- (Figure 5). Although Ara-C is widely used for leukemia treat- ter (Table 1). The TAE beaker was then placed on the mag-

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Table 1. Summary of calculations needed to prepare 500ml of Table 3. Summary of the cell lines: cell type, fusion genes and 50x stock of TAE. translocations. Moles Formula Volume Equation Final Cell Experi- Dis- Translo- Fusion Sensitive/ Refer- (M) weight (L) Quantity line mental ease cation gene resistant ences (FW) (g/ sample/ to Ara-C mol) positive control TRIS 2 121.14 0.5 M*FW*Volume 121g SEM-1 Ex- MLL- T(4;11) KMT2A- Resistant (Stumpel perimetal ALL AFF1 (MLL- et al., sample AFF1;MLL- 2015) AF4) Acetate 0.872 59.04 0.5 1) M*FW*Volume 28.53 ml 2) density=weight/ HB-1119 Ex- MLL- t(11;19) MLL-ENL Resistant (Brown, volume perimetal ALL (q23;p13) 2005; 3) volume=weight/ sample Piloto et al., 2007) density Kasumi-1 Positive AML t(8;21) AMLI-ETO Sensitive (Lar- EDTA 0.05 292.24 0.5 M*FW*Volume 7.3g control (q22;q22) (also known izza et as AML1- al., 2005; MTG or Xie et al., Table 2. Summary of the calculation needed to prepare 1L 1x RUNX1- 2010) TAE from 50x stock of TAE. CBF2TI) K562 Positive CML t(9;22) BCR-ABL1 Resistant (Naumann Initial con- Final Initial Formula Final control (q34;q11) e14-a2 et al., centration concen- volume volume (b3-a2) 2001; tration (L) (ml) Negoro et al., 2011) TAE 50x 1x 1 Final 20 volume/ Table 4. Summary of calculations of retro-transcription for dilution cDNA preparation. factor Cell Initial Final Final volume line amount amount (μL) netic field to mix the solution. Afterwards, 28.53ml of Acetic Kasumi-1 RNA 131.2 ng/μL 2 ug 15.2 acid (Table 1) was added to the beaker. The pH of the TAE Trans AMP buffer 5x 1x 4 solution was brought to 8.5 by adding HCl. Reverse transcriptase 1 Dnase/RNAse free 0.8 2.1.2 Preparation of 1L 1x TAE from 50x stock for gel water electrophoresis Final total volume 20 The TAE prepared from 500 ml of 50x stock (step 2.1.1) was HB119 RNA 99.2 ng/μL 2 ug 10.0 then diluted (Table 2). Trans AMP buffer 5x 1x 4

2.2 RNA purification from cultured cells and RNA read- Reverse transcriptase 1 ing on spectrophotometer at 260nm Dnase/RNAse free 5 RNA was purified according to manufacturer’s methodology water using the ‘RNA Mini Kit’ published by Bioline. The cells that Final total volume 20 were used are: Kasumi-1, HB-1119, K562 and SEM-1 (Table 3). K562 RNA 255.6 ng/μL 2 ug 7.8 Trans AMP buffer 5x 1x 4 2.2.1 Cell homogenization and cell lysis All cells were centrifuged and lysed by adding 350 μL Lysis Reverse transcriptase 1 Dnase/RNAse free 7.2 Buffer RLY and 3.5 μl of β-ME. water 2.2.2. Filtering lysate and adjusting RNA binding condi- Final total volume 20 tions SEM-1 RNA 111.6 ng/μL 2 ug 9 An ISOLATE II Filter was placed into a collection tube and Trans AMP buffer 5x 1x 4 lysate was loaded and centrifuged. The ISOLATE II Filter Reverse transcriptase 1 was then removed and 350 μL ethanol (70%) was added to Dnase/RNAse free 6 the homogenized lysate and was then mixed. water Final total volume 20 2.2.3 RNA binding and desalinizing silica membrane One ISOLATE II RNA Mini Column was placed into the col- 2.2.4 DNA digestion, washing, drying silica membrane; lection tube. Lysate was mixed, loaded into the column and eluting RNA centrifuged. A 350 μL membrane desalting buffer was added Next, 10 μL of DNA of reconstituted DNase I was added to and centrifuged. 90 μL reaction buffer for DNase I; the tube was gently mixed.

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Table 5. Dilution of cDNA for the four cell lines. a 600 μL washing buffer RW2 was added; then at the end, Initial Final Final total Dilution Final the column was placed back into the collection tube. The concentra- amount volume of factor volume of third washing step involved adding 250 μL Washing buffer tion of of RNA RNA samples cDNA for RW2 into the ISOLATE II RNA Mini column, centrifugation RNA (ng/ (ug) (μL) dilution μL) (μL) and placing column into the nuclease-free 1.5 ml collection HB1119 99.2 2 20 1:5 80 tube. The RNA was then eluted with 60 μL distilled water and centrifuged. RNA concentration readings were done on a NanoVue spectrophotometer at 260 nm, supplied by the SEM-1 111.6 2 20 1:5 80 University of Roehampton. 2.3 Retro-transcription/ Reverse transcription The purpose of Retro-transcription-Polymerase Chain Re- Kasumi-1 131.2 2 20 1:10 180 action (RT-PCR) was to use mRNA to generate cDNA (Le- K562 255.6 2 20 1:10 180 ong et al., 2007). RNA, Trans AMP buffer (Bioline), reverse transcriptase Table 6. Forward and reverse primer sequences for GAPDH, MSH2 and MSH3. (Bioline) and distilled water as shown in Table 4, were added to the corresponding tubes. The tubes were then cen- Gene Forward primer sequence Reverse primer sequence trifuged; RT-PCR was run on the BioRad thermacycler to amplify cDNA. The cDNA was then diluted (Table 5). Human CCTGTTCGACAGT- CGACCAAATCCGTT- GAPDH CAGCCG GACTCC 2.4 End-Point PCR/ Traditional PCR The purpose of End-Point PCR was to determine if the genes (MSH2, MSH3 and GAPDH) were expressed or not. End- Human CACTGTCTGCGGTAAT- CTCTGACTGCTG- Point PCR is an analysis when all cell cycles are completed. MSH2 CAAGT CAATATCCAAT The results are collected at the plateau phase; therefore it does not detect gene expression qualitatively or quantita- Human GTGGCAAAAGGATATA- AAAGGGCAG- tively (Leong et al., 2007). MSH3 AGGTGGG CAATTTCCGGG 2.4.1 Preparing samples for End-Point-PCR Table 7. Summary of the master mix for GAPDH, MSH2 and Three Eppendorf tubes were labelled as MSH2, MSH3 and MSH3. GAPDH. These tubes were prepared for End-Point PCR. Initial Final Volume neces- Volume The ingredients (MyTaq™ Red Mix (Bioline), forward primer, concen- concen- sary for one of master reverse primer (Table 6) and distilled water) (Table 7) were tration tration reaction (1 mix (μL) (6 added to the corresponding Eppendorf tubes. The forward sample) (μL) samples) and reverse primers for GAPDH, MSH2 and MSH3 were de- MyTaqtm 99.2 2 20 1:5 signed by our supervisor using a Basic Local Alignment Tool Red Mix (BLAST) (Table 8A). All the Eppendorf tubes had same My- Forward 111.6 2 20 1:5 Taq™ Red Mix (Table 8A), but different forward and reverse primer 10 primers (Table 8A). A 6 μL of master-mix that was prepared uM (for MSH2, MSH3 and GAPDH) were added to the corre- Reverse 131.2 2 20 1:10 sponding PCR tubes. 4 μL of diluted cDNA was also added primer 10 to the first four PCR tubes (Table 8A and 8B). The cDNA was uM also added to the corresponding PCR tubes. The fifth tube DNAse/ 255.6 2 20 1:10 had distilled water (negative control) (Table 8A,Table 8B). Rnase free The PCR tubes were then centrifuged, placed in a BioRad waer thermal cycler for PCR. 2.4.2 Gel preparation for End-Point PCR products Next, 95 μL of DNase I mixture was placed onto the center Gels were prepared to load End-Point PCR samples. First, of a silica membrane and incubated for 15 minutes at room 2 g of agarose powder was mixed with 100 ml 1x TAE and temperature. The next step involved washing the silica mem- brought to boil then, 2 μL of 10.000x Syber Safe was added brane using three steps. In the first step, a 200 μL Wash Buf- into the agarose solution. The agarose solution was left to fer RW1 was added to the ISOLATE II RNA Mini Column and turn into a gel; it then transferred to an electrophoresis tank. centrifuged; the column was placed into the new collection The samples and HyperLadder™ 100 base pairs (bp) (Bio- tube. The second washing step was the same except that line) was added to the wells of the gels; were left to run at

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Table 8A. Volume of PCR reaction needed for Kasumi-1, SEM- Table 9. Results of the RNA concentration for the four cell 1, HB-1119, K562 and Water sample (negative control). lines at 280nm and ratios A260/A280 and A260/A230.

Initial concen- Final concen- Final volume Final volume (μL) of MSH2, MSH3, and GAPDH actin tration tration (ul) for an end-point PCR products cDNA 10 ng/μL 40 ng/10 μL 4 Cell line RNA concentration A260/A280 A260/A230 per reaction (ng/μL) Master mix 2x 1x 5 Kasumi 131.2 2.050 1.147 Forward 10 μM 0.4 μM 0.4 primer 10 uM HB-1119 99.2 2.000 0.379 Reverse 10 μM 0.4 μM 0.4 K562 255.6 2.081 0.864 primer 10 uM DNAse/Rnase 0.2 SEM-1 111.6 2.051 0.790 free waer Total final 10 volume

Table 8B. Summary of the final volume (μl) of master mix, cDNA and distilled water. Altogether, 15 PCR tubes were used, and the 5th tube in each sample was the negative control. Final volume (μL) of MSH2, MSH3, and GAPDH actin for an end-point PCR products Master mix cDNA Water Tube 1-Kasumi 6 4 0

Tube 2-HB1119 6 4 0 Tube 3-K562 6 4 0

Tube 4-SEM-1 6 4 0 Tube 5-negative control 6 0 4

100 V to separate the PCR products into bands. The Gel- Doc, Image Lab machine was finally used to get the clear image of the bands- the genes that were expressed.

2.5 Semi-quantitative PCR The purpose of semi-quantitative PCR is to interrupt the PCR reaction at an intermediate level, so the results were analyzed at 28 cycles rather than 35 (as in End-Point PCR). Figure 6. Summary of the whole method and the purpose be- The methodology of semi-quantitative PCR is exactly same hind each step. as in End-Point PCR. The PCR results were quantified using ImageJ (Figure 6). case in our experiment as all the ratios of absorbance at A260/A280 nm were 2.000 or above. The A260/A230 nm ra- RESULTS tio of absorbance indicates purity of RNA from salts. If A260/ 3.1 RNA concentration A230 is below 1.8 ratio, it indicates that the samples were The RNA from each cell line was purified and the absorbance contaminated with salt (Farrell, 2005).This was the case in at 260, 230 and 280 nm reported (Table 9). RNA absorbance our experiment as all of the samples were contaminated with is measured at 260 nm, protein at 280 nm and salt at 230 nm salt due to < 1.8 ratio of absorbance (Table 9). (Farrell, 2005). A260/A280 nm ratio of absorbance indicates 3.2 End-Point PCR the purity of RNA from proteins. If A260/A280 nm ratio of The End-Point PCR results show whether the genes are ex- absorbance value is 2.000 or above, it indicates that RNA pressed in the cells or not. The order of the samples in both has been purified from protein (Farrell, 2005).This was the Figures 7 and 8 is HyperLadder™ 100 bp DNA molecular

Figure 7. Results of the 2% agarose gel electrophoresis. Based Figure 8. Results of the 2% agarose gel electrophoresis. Based on the End-Point PCR results of GAPDH, MSH2 and MSH3 at 35 on the End-Point PCR results of GAPDH, MSH2 and MSH3 at 35 cycles. The order of each sample is: DNA hyperladder, Kasumi-1, cycles. Independent replica. HB-1119, K562, SEM-1 and water.2010). of the DNA fragments in all cell lines was around 100bp (Fig- weight marker, Kasumi-1, HB-1119, K562, SEM-1 and water ure 7, 8, 9, 10, 11). MSH2 showed high expression in K562 (the negative control). GAPDH was the housekeeping gene. and SEM-1 cell lines compared to HB-1119 cell lines. Ka- Kasumi-1 and K562 were the positive control cell lines as sumi-1 cell line showed a stronger MSH3 gene expression GAPDH, MSH2 and MSH3 were expected to be expressed intensity than HB-1119. The intensity of MSH3 gene expres- in those two cell lines. HB-1119 and SEM-1 were experimen- sion in K562 and SEM-1 was similar to Kasumi-1. However, tal cell lines. The two independent experiments indicate that these judgements were made visually. Semi-quantitative MSH2 and MSH3 were expressed in all the four cell lines PCR does not allow a precise measurement of gene expres- (Figures 7, 8). sion and this can be achieved by a real-time PCR (Marone 3.3 Semi-quantitative PCR results et al., 2001). A quantification of the data presented in Fig- The semi-quantitative PCR for the genes GAPDH, MSH2 ure 9, 10 and 11 was obtained by analyzing the images with and MSH3 were run at 28 cycles. The three independent ImageJ. The results indicate that there is a statistically sig- experiments show very consistent results (Figure 9, 10, 11). nificant increased expression of MSH2 in SEM1 compared The GAPDH was expressed at comparable level in all to Kasumi-1 (p = 0.0066 unpaired T-test), whereas there four cell lines (Figure 9, 10, 11). The water samples did not is no difference between Kasumi-1 and HB-1119 (Figure show any bands, indicating no contamination. The slight 12). MSH3 expression is statistically significantly lower in short band in the water in the GAPDH PCR indicated an ex- HB1119 compared to Kasumi-1 (p = 0.0009 unpaired T-test) cess of primers (Figure 11). The size of the bands were com- whereas there is no difference in MSH3 expression between paredto the DNA molecular marker and it showed that size Kasumi-1 and SEM1 (Figure 13).

Figure 9. Results of the 2% agarose gel electrophoresis. Based Figure 10. Gel electrophoresis results. Based on the semi-quan- on another trial End-Point PCR results of GAPDH, MSH2 and titative PCR results of GAPDH, MSH2 and MSH3 at 28 cycles. In- MSH3 at 28 cycles. dependent replica. DISCUSSION Previous studies have reported that cancerous cells de- The main focus of our study was to analyze the expression fective in MSH2 expression had increased frequency of MSI of MSH2 and MSH3 gene expression in the two MLL-ALL (Ruszkiewicz et al., 2002; Shia et al., 2004). cell lines: HB-1119 and SEM-1, and to relate the gene ex- The complete absence of MSH2 leads to complete loss pression to Ara-C resistance. of DNA MMR activity as MSH2 is the ‘primary gene’ in the MMR pathway (Boland et al., 2008). In our study, MSH2 4.1 Analysis of MSH2 and MSH3 expression was expressed at lower level in HB-1119 (MLL-ALL) and The End-Point PCR showed that MSH2 and MSH3 were ex- Kasumi-1 when compared to SEM-1 (MLL-ALL) and K562 pressed in our experimental cell lines (Figures 7, 8). As in (Figure 9, 10,11, 12). This could possibly mean that low ex- an End-Point PCR the results are analyzed at the plateau pression of MSH2 can lead to MSI in some MLL-ALL. The phase of the PCR reaction, this PCR could only provide an HB-1119 did not completely lack MSH2 gene expression indication of whether the genes were expressed or not in (Figure 9, 10, 11). The threshold of MSH2 gene expression the cell lines but not on the expression levels (Leong et al., needed for MSI, needs to be determined before making a 2007). We determined the expression levels of the genes by conclusion. using the semi-quantitative PCR, where the results are ana- Kasumi-1 is a cell line sensitive to Ara-C (Xie et al., 2010) lyzed at the exponential phase of the reaction, and therefore and it showed expression of both MSH2 and MSH3 (Figure they are only dependent on the concentration of template. 9, 10, 11) (Xie et al., 2010). This may indicate that the lack of The results indicate that there is a statistically significant (p MMR genes is not necessarily required for Ara-C sensitivity. = 0.006) increased expression of MSH2 in SEM1 compared Another study by Holt et al., (2009) used NALM-6 cell to Kasumi-1 (Figure 12) and statistically significantly p ( = lines (B-ALL cell type) and found out that MSH2 was defi- 0.0009.) decreased expression of MSH3 in HB-1119 com- cient in NAML-6 cell lines-indicating an increase chances pared to Kasumi-1 (Figure 13). of endogenous oxidative stress and accumulation of highly

mutagenic DNA lesions (Holt et al., 2009). Comparing this study with our MLL-ALL cell types, HB-1119 might be more vulnerable to development of mutagenic DNA lesions (due to low MSH2 expression) than SEM-1 that has a higher MSH2 expression compared to HB-1119 (Figure 9, 10, 11, 12). Overall, HB-1119 showed both MSH2 and MSH3 downregulation compared to the other MLL-ALL cell type-SEM-1 (Figure 9, 10, 11, 12 and 13). As mentioned before, together MSH2 and MSH3 makes up MutSβ and the role of MutSβ is to find the location of errors produced during DNA synthesis and MutSβ recognizes IDLs (Burdova et al., 2015). This indicates that HB-1119 might be more vulnerable to MMR deficiency than SEM-1 by not adequately recognizing loca- Figure 11. Gel electrophoresis results. Based on the semi-quan- tions of replication errors generated during DNA synthesis or titative PCR results of GAPDH, MSH2 and MSH3 at 28 cycles. IDLs. However, the recognition of IDLs varies depending on Independent replica. their nucleotide length as MLH1-PMS2/1 and MSH2-MSH6 can recognize small IDLs and MLH1-PMS2/1 complex recognizes larger IDLs (Martin et al., 2010) (Figure 3 and 4). Proteins responsible for compensating for MSH2/MSH3 downregulation in HB-1119 and/or in SEM-1 should be in- vestigated to determine if the MMR pathway is completely deficient in MLL-ALL cells.

4.2 Cytarabine relationship with MSH2 and MSH3 gene expression (based on semi-quantitative PCR results) Fordham et al. (2011) demonstrated that, one of the reasons for Ara-C resistance could be the presence of MMR genes. Ara-C induces DNA lesions, and these DNA lesions could be repaired by MMR proteins (Fordham et al., 2011). The authors showed that MSH2 and MSH6 deficient lymphoma cell line models showed increased sensitivity to Ara-C (Fordham et al.,2011). In our study, HB-1119 was not deficient in MSH2 Figure 12. Relative expression of MSH2. The graph represents expression but was the cell line with the lowest amount of the relative expression of MSH2 in the cell lines. Data represent the MSH2 expression (Figure 9, 10, 11, 12). average of three independent experiments +/- Std Dev, p <0.01. Another study by Hewish et al. (2013) demonstrated that MSH2-deficient cancerous cells are more prone to Ara- C toxicity due to alteration of mitochondrial membrane potential and generation of ROS. Comparing the Hewish et al. (2013) study with our findings, HB-1119 could be more prone to Ara-C toxicity than SEM-1, due to its MSH2 downregulation (Figure 9, 10, 11, 12). In contrast, Fordham et al. (2010) revealed that MSH3 knockdown granted resistance to cytotoxic effects induced by Ara-C (Fordham et al., 2010). In our study, MSH3 was highly expressed in SEM-1 (Figure 13), but MSH3 downregulation was observed in HB-1119 (Figure 9, 10, 11). The results highlight that the analysis of expression of a single gene might not be sufficient to explain the molecular resistance to Ara-C and that functional assays should be used to relate MMR activity to resistance to chemotherapeutic drugs. In addition, real-time PCR, should be used to obtain a quantitative measurement of gene expression in the cell lines. The compensatory genes MSH6, as Figure 13. Relative expression of MSH3. The graph represents well as MLH1 and PMS2 should also be studied to appreci- the relative expression of MSH3 in the cell lines. Data represent the ate the extent of MMR gene expression on Ara-C resistance. average of three independent experiments +/- Std Dev, p < 0.001

ACKNOWLEDGMENTS Chiaretti, S., Brugnoletti, F., Tavolaro, S., Bonina, S., Paoloni, F., Marinelli, M., Patten, N., Bonifacio, M., Kropp, M. G., Sica, S., Guarini, A., and I would like to thank Dr Maria Teresa Esposito for allowing Foà, R. (2013). TP53 mutations are frequent in adult acute lympho- me to be part of this research project and for being the pil- blastic leukemia cases negative for recurrent fusion genes and cor- lar of strength. The immeasurable support, guidance, teach- relate with poor response to induction therapy. Haematologica, 98(5), ing, and courage that she gave me throughout the years e59-e61. has made me a more independent, grateful, and productive Depmap.org. 2020. HB1119 Depmap Cell Line Summary. [online] Available at: https://depmap.org/portal/cell_line/ACH-001736?tab=fusion [Ac- young Biomedical scientist. I feel very lucky to have had cessed 10 March 2020]. such a caring and patient supervisor who gave me detailed Dsmz.de. (no date). Details: ACC-10,available: https://www.dsmz.de/cata- feedback and responded promptly to my questions. I shall logues/details/culture/ACC-10.html [Accessed 26 Apr. 2018]. forever remain grateful for the knowledge and the laboratory Dsmz.de. (no date). Details: ACC-546,available: https://www.dsmz.de/cata- skills that I gained through this project with the help of her. I logues/details/culture/ACC-546.html [Accessed 26 Apr. 2018]. Dsmz.de. (no date.). Details: ACC-220,available: https://www.dsmz.de/cat- would also like to extend my sincere gratitude to Dr Yoana alogues/details/culture/ACC-220.html [Accessed 26 Apr. 2018]. Arroyo-Berdugo and my other fellow colleagues for helping Ewald, B., Sampath, D. and Plunkett, W. (2008). Nucleoside analogs: mo- me during laboratory practicals and my dear family for their lecular mechanisms signaling cell death. Oncogene, 27(50), 6522- immeasurable support throughout this project. 6537. Farrell, R. (2005). RNA methodologies: a laboratory guide for isolation and FUNDING characterization, 3rd ed. Amsterdam: Elsevier/Academic Press. Fordham, S., Cole, M., Irving, J. and Allan, J. (2014). Cytarabine prefer- This work has been supported by the University of Roe- entially induces mutation at specific sequences in the genome which hampton. are identifiable in relapsed acute myeloid leukemia. Leukemia, 29(2), 491-494. AUTHORS’ CONTRIBUTION Fordham, S., Matheson, E., Scott, K., Irving, J. and Allan, J. (2010). Ab- stract 1962: DNA mismatch repair status modulates cellular response K. N. performed the experiments, analyzed the data and to cytarabine: Implications for treatment of acute myeloid leukemia. wrote the manuscript. Y. A-B. performed the experiments Cancer Research, 70(8 Supplement), 1962-1962. and analyzed the data. M.T.E. designed the study, provided Fordham, S., Matheson, E., Scott, K., Irving, J. and Allan, J. (2011). 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