Kulsom Nuuri, Yoana Arroyo-Berdugo, and Maria Teresa Esposito
University of Roehampton Whiteland Campus Parkstead House Holybourne Avenue SW15 4JD London UK
University of Roehampton, Holybourne Avenue, SW15 4JD, London
ABSTRACT
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 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
1.1 Leukemia
Figure 1. A bar chart to show the most common cancers by gender in UK: 2015. Leukemia is the 12th common cancer and makes up 9900 new cases in the year of 2015, in UK (adapted from Cancer Research, 2018).
Leukemia is a malignant disease arising from the hematopoietic stem cells (HSC) that reside in the bone marrow and give rise to blood cells. Excessive production of immature HSCs in the bone marrow interrupts the normal production of the blood cells (Mwigiri et al., 2017). Acute leukemia is an aggressive form of cancer that can progress quickly and be fatal if left untreated. Leukemia is one of the twenty most common cancer in the UK (Figure 1) (Cancer Research, 2018) and it made up 437,033 new cases worldwide in the year 2018 (Worldwide cancer data, 2019).
1.2 Acute lymphoblastic leukemia
Acute lymphoblastic leukemia (ALL) affects the lymphocytes progenitors. B-cell Acute Lymphoblastic Leukemia (B -ALL) accounts for 80-85% of ALL and T-cell Acute Lymphoblastic Leukemia (T -ALL) accounts for 20 - 25% of ALL (Chiaretti et al., 2013). ALL is more prevalent in children aged 4 - 14 years and in adults aged over 50 (Mwirigi et al., 2017). Chromosomal and genetic alterations are hallmarks of ALL and affect differentiation and proliferation of lymphoid precursor cells derived from HSCs in the bone marrow. ALL is divided into subgroups depending on the chromosomal translocations (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 gene (Terwilliger and Abdul-Hay, 2017).
Figure 2. MLL partner genes. The N-terminal portion of MLL fuses with C-terminal portion of the partner gene. MLL-AF4 and MLL-ENL fusion genes belong to MLL-ALL (adapted from Zheng, 2013).
1.3 Mixed Lineage Leukemia
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 distinct 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 expressed during early development and hematopoiesis (Mohan et al., 2010). The C-terminal has transcriptional activation properties and is able to activate gene expression, for example, by methylating histone 3 on lysine 4 near gene 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 rearrangement composed of three or more fusion proteins (Sabiani et al., 2015). MLL-AF4 is the most prevalent MLL translocation in ALL and is present in SEM-1 human cell line, which is one of the experimental cell lines in our study (Mendelsohn 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 from B-ALL (Brown, 2005). HB-1119 is the second experimental cell line in our study.
Figure 3. MMR genes and their role in MMR pathway. The Mutsα recognizes single base mismatches and small IDLs (A and B). Mutsαβ recognizes larger IDLs (C) (adapted from Martin et al., 2010).
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; Boland et al., 2008). Mutations can occur in microsatellites due to the slippage of DNA polymerase causing small unnecessary 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 in the microsatellite, leading to a microsatellite instability (MSI) (Laghi et al., 2008).
Figure 4. MMR genes, heterodimer complexes and their role in MMR pathway. MSH2 and its partner (MSH6/MSH3) recognizes DNA mismatch (A). Recruitment of MLH1 and its partner PMS2 and other cofactors (PCNA) and enzymes (EXO1) conduct excision, resynthesis and ligation of the repaired DNA strand (B & C) (Wei et al., 2002).
MSH2 and MSH3 are important components of the MMR pathway (Burdova et al., 2015). Mismatches are recognized by MutS complexes. MutSα is composed by MSH2 and MSH6 and repairs small IDLs (Figure 3). MutSβ is composed by MSH2 and MSH3 and recognizes the IDLs that are1-13 nucleotides long. MSH2 interacts together with MSH3 (or with other proteins) (Figure 3) to give rise to the DNA mismatch-binding protein complex MutSβ. MutSβ can locate errors in the DNA produced during DNA replication (Burdova et al., 2015). MutS complexes (MSH2-MSH6 or MSH2-MSH3) allows to recruit MutL (consisting ofMLH1 and PMS2) (Figure 3) and the helicase MCM9. MCM9 helicase catalyze the removal of the mismatch- containing DNA strands. Replication factors such as proliferating cell nuclear antigen (PCNA), DNA polymerase δ (Polδ) and Replication protein A (RPA) repair the DNA strand (Figure 4) (Traver et al., 2015).
The loss of MMR proteins can cause accumulation of DNA replication errors and therefore contribute to MSI (Burdova et al., 2015; Surtees and Alani, 2006). MSI is mostly a result of under-activity of MutSβ (heterodimer of MSH2 and MSH3) rather than MutSα (heterodimer of MSH2 and MSH6), because defects in MSH6 protein do not lead to a high frequency of MSI due to the effect of the compensation provided by other secondary MMR molecules such as MSH3, MLH3 and PMS1 (Boland et al., 2008; Shia, 2008; Nakatani et al., 2015; Boland et al., 2008). MSH2 combined with MSH3 can compensate for the loss of MSH6 (Boland et al., 2008). In contrast, Kheirelseid et al. (2013) revealed that the MSH2-MSH3 heterodimer cannot compensate for MSH6 deficiency as MutSβ recognizes only IDLs. Nakatani et al., (2015) argues that the exact role of the MMR proteins remains controversial and it is different amongst the models being used in the studies. This may possibly explain why certain studies argue that MSH3 (combined with MSH2) can compensate for MSH6 loss while others do not.
1.5 Chemotherapy
Figure 5. Action mechanism of Analog Triphosphate (Ara-C). Ara-C, inhibits DNA strand synthesis by binding onto incorporating DNA chain- leading to cell death signalling (adapted from Ewald et al., 2008).
One of the common drugs used in childhood and infant ALL is Cytarabine also known as cytosine arabinoside (Ara-C), which is a nucleoside analogue (antimetabolite) (Matheson and Hall, 2003). Ara-C works by inhibiting DNA replication (Figure 5). Although Ara-C is widely used for leukemia treatment, 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). Genetic 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.
1.5.1 Chemotherapy problems associated with MMR
Defects in the MMR pathway has been shown to have a relationship with resistance of many cytotoxic drugs, including Ara-C (Matheson and Hall, 2003). A study conducted by 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 to the production of lethal double-strand DNA breaks (DSB) and apoptosis (Hewish et al., 2013).
Fordham et al. (2011) found that silencing of MSH2 and MSH6 in AML cells increased the sensitivity to Ara-C and Clorafabin. This study together with the study of Hewish et al. prompted us to investigate the expression of MSH2 and MSH3 in Ara-C resistant MLL-ALL cell lines (HB-1119 and SEM-1).
METHODS
In order to investigate the expression of MSH2 and MSH3 in the MLL experimental cell lines we began with culturing the cell lines in vitro. SEM-1, Kasumi-1 and K562 were purchased from DSMZ (DSMZ). The HB-1119 cell line was obtained through Professor. Eric So in 2015.The cell line was a gift of M. Cleary (Stanford university). The cell line is deposited in the depmap database (HB1119 DepMap Cell Line Summary, 2020). Cells were kept at 37OC and 5% CO2 in RPMI-1640 supplemented with 10% Fetal Bovine Serum and antibiotics. Actively growing cells were centrifuged at 500 g for 5 minutes, washed with PBS and then centrifuged again. Cell pellets were used for RNA isolation and subsequent generation of cDNA.
2.1 Making buffer and solutions
2.1.1 500ml of 50x stock of Tris-Acetate-EDTA(TAE)
Tris and EDTA were put in the beaker along with distilled water (Table 1). The TAE beaker was then placed on the magnetic field to mix the solution. Afterwards, 28.53ml of Acetic acid (Table 1) was added to the beaker. The pH of the TAE solution was brought to 8.5 by adding HCl.
2.1.2 Preparation of 1L 1x TAE from 50x stock for gel electrophoresis
The TAE prepared from 500 ml of 50x stock (step 2.1.1) was then diluted (Table 2).
2.2 RNA purification from cultured cells and RNA reading on spectrophotometer at 260nm
RNA was purified according to manufacturer’s methodology using the ‘RNA Mini Kit’ published by Bioline. The cells that were used are: Kasumi-1, HB-1119, K562 and SEM-1 (Table 3).
2.2.1 Cell homogenization and cell lysis
Next, 10 μL of DNA of reconstituted DNase I was added to 90 μL reaction buffer for DNase I; the tube was gently mixed. Next, 95 μL of DNase I mixture was placed onto the center of a silica membrane and incubated for 15 minutes at room temperature. The next step involved washing the silica membrane using three steps. In the first step, a 200 μL Wash Buffer RW1 was added to the ISOLATE II RNA Mini Column and centrifuged; the column was placed into the new collection tube. The second washing step was the same except that a 600 μL washing buffer RW2 was added; then at the end, the column was placed back into the collection tube. The third washing step involved adding 250 μL Washing buffer RW2 into the ISOLATE II RNA Mini column, centrifugation and placing column into the nuclease-free 1.5 ml collection 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 University of Roehampton.
2.3 Retro-transcription/ Reverse transcription
The purpose of Retro-transcription-Polymerase Chain Reaction (RT-PCR) was to use mRNA to generate cDNA (Leong et al., 2007).
RNA, Trans AMP buffer (Bioline), reverse transcriptase (Bioline) and distilled water as shown in Table 4, were added to the corresponding tubes. The tubes were then centrifuged; RT-PCR was run on the BioRad thermacycler to amplify cDNA. The cDNA was then diluted (Table 5).
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-Point PCR is an analysis when all cell cycles are completed. The results are collected at the plateau phase; therefore it does not detect gene expression qualitatively or quantitatively (Leong et al., 2007).
2.4.1 Preparing samples for End-Point-PCR
Three Eppendorf tubes were labelled as MSH2, MSH3 and GAPDH. These tubes were prepared for End-Point PCR. The ingredients (MyTaq™ Red Mix (Bioline), forward primer, reverse primer (Table 6) and distilled water) (Table 7) were added to the corresponding Eppendorf tubes. The forward and reverse primers for GAPDH, MSH2 and MSH3 were designed by our supervisor using a Basic Local Alignment Tool (BLAST) (Table 8A). All the Eppendorf tubes had same MyTaq™ Red Mix (Table 8A), but different forward and reverse primers (Table 8A). A 6 μL of master-mix that was prepared (for MSH2, MSH3 and GAPDH) were added to the corresponding PCR tubes. 4 μL of diluted cDNA was also added to the first four PCR tubes (Table 8A and 8B). The cDNA was also added to the corresponding PCR tubes. The fifth tube had distilled water (negative control) (Table 8A, Table 8B). The PCR tubes were then centrifuged, placed in a BioRad thermal cycler for PCR.
2.4.2 Gel preparation for End-Point PCR products
Gels were prepared to load End-Point PCR samples. First, 2 g of agarose powder was mixed with 100 ml 1x TAE and brought to boil then, 2 μL of 10.000x Syber Safe was added into the agarose solution. The agarose solution was left to turn into a gel; it then transferred to an electrophoresis tank. The samples and HyperLadder™ 100 base pairs (bp) (Bioline) was added to the wells of the gels; were left to run at 100 V to separate the PCR products into bands. The GelDoc, Image Lab machine was finally used to get the clear image of the bands- the genes that were expressed.
2.5 Semi-quantitative PCR
Figure 6. Summary of the whole method and the purpose behind each step.
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). The methodology of semi-quantitative PCR is exactly same as in End-Point PCR. The PCR results were quantified using ImageJ (Figure 6).
RESULTS
3.1 RNA concentration
The RNA from each cell line was purified and the absorbance at 260, 230 and 280 nm reported (Table 9). RNA absorbance is measured at 260 nm, protein at 280 nm and salt at 230 nm (Farrell, 2005). A260/A280 nm ratio of absorbance indicates the purity of RNA from proteins. If A260/A280 nm ratio of absorbance value is 2.000 or above, it indicates that RNA has been purified from protein (Farrell, 2005).This was the case in our experiment as all the ratios of absorbance at A260/A280 nm were 2.000 or above. The A260/A230 nm ratio of absorbance indicates purity of RNA from salts. If A260/A230 is below 1.8 ratio, it indicates that the samples were contaminated with salt (Farrell, 2005).This was the case in our experiment as all of the samples were contaminated with salt due to < 1.8 ratio of absorbance (Table 9).
3.2 End-Point PCR
The End-Point PCR results show whether the genes are expressed in the cells or not. The order of the samples in both Figures 7 and 8 is HyperLadder™ 100bp DNA molecular weight marker, Kasumi-1, HB-1119, K562, SEM-1 and water (the negative control). GAPDH was the housekeeping gene. Kasumi-1 and K562 were the positive control cell lines as GAPDH, MSH2 and MSH3 were expected to be expressed in those two cell lines. HB-1119 and SEM-1 were experimental cell lines. The two independent experiments indicate that MSH2 and MSH3 were expressed in all the four cell lines (Figures 7, 8).
3.3 Semi-quantitative PCR results
The semi-quantitative PCR for the genes GAPDH, MSH2 and MSH3 were run at 28 cycles. The three independent experiments show very consistent results (Figure 9, 10, 11).
Figure 11. Gel electrophoresis results. Based on the semi-quantitative PCR results of GAPDH, MSH2 and MSH3 at 28 cycles. Independent replica.
The GAPDH was expressed at comparable level in all four cell lines (Figure 9, 10, 11). The water samples did not show any bands, indicating no contamination. The slight short band in the water in the GAPDH PCR indicated an excess of primers (Figure 11). The size of the bands were comparedto the DNA molecular marker and it showed that size of the DNA fragments in all cell lines was around 100bp (Figure 7, 8, 9, 10, 11). MSH2 showed high expression in K562 and SEM-1 cell lines compared to HB-1119 cell lines. Kasumi-1 cell line showed a stronger MSH3 gene expression intensity than HB-1119. The intensity of MSH3 gene expression in K562 and SEM-1 was similar to Kasumi-1. However, these judgements were made visually. Semi-quantitative PCR does not allow a precise measurement of gene expression and this can be achieved by a real-time PCR (Marone et al., 2001). A quantification of the data presented in Figure 9, 10 and 11 was obtained by analyzing the images with ImageJ. The results indicate that there is a statistically significant increased expression of MSH2 in SEM1 compared to Kasumi-1 (p = 0.0066 unpaired T-test), whereas there is no difference between Kasumi-1 and HB-1119 (Figure 12). MSH3 expression is statistically significantly lower in HB1119 compared to Kasumi-1 (p = 0.0009 unpaired T-test) whereas there is no difference in MSH3 expression between Kasumi-1 and SEM1 (Figure 13).
DISCUSSION
The main focus of our study was to analyze the expression of MSH2 and MSH3 gene expression in the two MLL-ALL cell lines: HB-1119 and SEM-1, and to relate the gene expression to Ara-C resistance.
4.1 Analysis of MSH2 and MSH3 expression
The End-Point PCR showed that MSH2 and MSH3 were expressed in our experimental cell lines (Figures 7, 8). As in an End-Point PCR the results are analyzed at the plateau phase of the PCR reaction, this PCR could only provide an indication of whether the genes were expressed or not in the cell lines but not on the expression levels (Leong et al., 2007). We determined the expression levels of the genes by using the semi-quantitative PCR, where the results are analyzed at the exponential phase of the reaction, and therefore they are only dependent on the concentration of template. The results indicate that there is a statistically significant (p = 0.006) increased expression of MSH2 in SEM1 compared to Kasumi-1 (Figure 12) and statistically significantly (p = 0.0009.) decreased expression of MSH3 in HB-1119 compared to Kasumi-1 (Figure 13).
Previous studies have reported that cancerous cells defective in MSH2 expression had increased frequency of MSI (Ruszkiewicz et al., 2002; Shia et al., 2004).
The complete absence of MSH2 leads to complete loss of DNA MMR activity as MSH2 is the ‘primary gene’ in the MMR pathway (Boland et al., 2008). In our study, MSH2 was expressed at lower level in HB-1119 (MLL-ALL) and Kasumi-1 when compared to SEM-1 (MLL-ALL) and K562 (Figure 9, 10,11, 12). This could possibly mean that low expression of MSH2 can lead to MSI in some MLL-ALL. The HB-1119 did not completely lack MSH2 gene expression (Figure 9, 10, 11). The threshold of MSH2 gene expression needed for MSI, needs to be determined before making a conclusion.
Kasumi-1 is a cell line sensitive to Ara-C (Xie et al., 2010) and it showed expression of both MSH2 and MSH3 (Figure 9, 10, 11) (Xie et al., 2010). This may indicate that the lack of MMR genes is not necessarily required for Ara-C sensitivity.
Another study by Holt et al., (2009) used NALM-6 cell lines (B-ALL cell type) and found out that MSH2 was deficient in NAML-6 cell lines-indicating an increase chances 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 down-regulation 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 locations of replication errors generated during DNA synthesis or IDLs. However, the recognition of IDLs varies depending on 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 investigated 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 expression but was the cell line with the lowest amount of MSH2 expression (Figure 9, 10, 11, 12).
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 well as MLH1 and PMS2 should also be studied to appreciate the extent of MMR gene expression on Ara-C resistance.
ACKNOWLEDGMENTS
I would like to thank Dr Maria Teresa Esposito for allowing me to be part of this research project and for being the pil-lar of strength. The immeasurable support, guidance, teach-ing, and courage that she gave me throughout the years has made me a more independent, grateful, and productive young Biomedical scientist. I feel very lucky to have had such a caring and patient supervisor who gave me detailed feedback and responded promptly to my questions. I shall forever remain grateful for the knowledge and the laboratory skills that I gained through this project with the help of her. I would also like to extend my sincere gratitude to Dr Yoana Arroyo-Berdugo and my other fellow colleagues for helping me during laboratory practicals and my dear family for their immeasurable support throughout this project.
FUNDING
This work has been supported by the University of Roehampton.
AUTHORS’ CONTRIBUTION
K. N. performed the experiments, analyzed the data and wrote the manuscript. Y. A-B. performed the experiments and analyzed the data. M.T.E. designed the study, provided technical support, conceptual advice and edited the manuscript.
REFERENCES
Acute lymphoblastic leukaemia (ALL) statistics. (2018), available: https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/leukaemia-all#heading-Zero.
Armstrong, S., Staunton, J., Silverman, L., Pieters, R., den Boer, M., Minden, M., Sallan, S., Lander, E., Golub, T. and Korsmeyer, S. (2002). MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nature Genetics, 30(1), 41-47.
Blasco, M., Khan, M. and Pelengaris, S. (2006). The Molecular Biology of Cancer, Malden, MA: Blackwell Pub.
Boland, C., Koi, M., Chang, D. and Carethers, J. (2008). The biochemical basis of microsatellite instability and abnormal immunohistochemistry and clinical behavior in Lynch Syndrome: from bench to bedside. Familial Cancer, 7(1), 41-52.
Brown, P. (2005). FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression. Blood, 105(2), 812-820.
Burdova, K., Mihaljevic, B., Sturzenegger, A., Chappidi, N. and Janscak, P. (2015) The mismatch-binding factor MutSβ can mediate ATR activation in response to DNA double-strand breaks. Molecular Cell. 59(4) 603-614.
Cai, J., Damaraju, V., Groulx, N., Mowles, D., Peng, Y., Robins, M., Cass, C. and Gros, P. (2008). Two Distinct Molecular Mechanisms Underlying Cytarabine Resistance in Human Leukemic Cells. Cancer Research, 68(7), 2349-2357.
Cancer Research UK. (2018). Cancer incidence for common cancers, available: http://www.cancerresearchuk.org/health-professional/cancer-statistics/incidence/common-cancers-compared#heading-Zero [Accessed 14 Mar. 2018].
Català, A., Pastor-Anglada, M., Caviedes-Cárdenas, L., Malatesta, R., Rives, S., Vega-García, N., Camós, M., and Fernández-Calotti, P.(2016). FLT3 is implicated in cytarabine transport by human equilibrative nucleoside transporter 1 in pediatric acute leukemia. Oncotarget, 7(31), 49786-49799.
Chiaretti, S., Brugnoletti, F., Tavolaro, S., Bonina, S., Paoloni, F., Marinelli, M., Patten, N., Bonifacio, M., Kropp, M. G., Sica, S., Guarini, A., and Foà, R. (2013). TP53 mutations are frequent in adult acute lymphoblastic leukemia cases negative for recurrent fusion genes and correlate with poor response to induction therapy. Haematologica, 98(5), e59-e61.
Depmap.org. 2020. HB1119 Depmap Cell Line Summary. [online] Available at: https://depmap.org/portal/cell_line/ACH-001736?tab=fusion [Accessed 10 March 2020].
Dsmz.de. (no date). Details: ACC-10,available: https://www.dsmz.de/catalogues/details/culture/ACC-10.html [Accessed 26 Apr. 2018].
Dsmz.de. (no date). Details: ACC-546,available: https://www.dsmz.de/catalogues/details/culture/ACC-546.html [Accessed 26 Apr. 2018].
Dsmz.de. (no date.). Details: ACC-220,available: https://www.dsmz.de/catalogues/details/culture/ACC-220.html [Accessed 26 Apr. 2018].
Ewald, B., Sampath, D. and Plunkett, W. (2008). Nucleoside analogs: molecular mechanisms signaling cell death. Oncogene, 27(50), 6522-6537.
Farrell, R. (2005). RNA methodologies: a laboratory guide for isolation and characterization, 3rd ed. Amsterdam: Elsevier/Academic Press.
Fordham, S., Cole, M., Irving, J. and Allan, J. (2014). Cytarabine preferentially induces mutation at specific sequences in the genome which are identifiable in relapsed acute myeloid leukemia. Leukemia, 29(2), 491-494.
Fordham, S., Matheson, E., Scott, K., Irving, J. and Allan, J. (2010). Abstract 1962: DNA mismatch repair status modulates cellular response to cytarabine: Implications for treatment of acute myeloid leukemia. Cancer Research, 70(8 Supplement), 1962-1962.
Fordham, S., Matheson, E., Scott, K., Irving, J. and Allan, J. (2011). DNA mismatch repair status affects cellular response to Ara-C and other anti-leukemic nucleoside analogs. Leukemia, 25(6), 1046-1049.
Gu, T., Nardone, J., Wang, Y., Loriaux, M., Villén, J., Beausoleil, S., Tucker, M., Kornhauser, J., Ren, J., MacNeill, J., Gygi, S. P., Druker, B. J., Heinrich, M. C., Rush, J., and Polakiewicz, R. D. (2011). Survey of Activated FLT3 Signaling in Leukemia. PLoS ONE, 6(4), e19169.
Hewish, M., Martin, S., Elliott, R., Cunningham, D., Lord, C. and Ashworth, A. (2013). Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress. British Journal of Cancer, 108(4), 983-992.
Holt, S., Scemama, J., Panayiotidis, M. and Georgakilas, A. (2009). Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 674(1-2), 123-130.
Jaffe, E. S., Harris, N.L., Stein, H., Vardiman, J.W. (2001) World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues, 3rd ed., Lyon : IARC Press.
Kheirelseid, E. A. H., Miller, N., Chang, K. H., Curran, C., Hennessey, E., Sheehan, M., and Kerin, M. J. (2013). Mismatch repair protein expression in colorectal cancer. Journal of Gastrointestinal Oncology, 4(4), 397–408.
Kohlmann, A., Schoch, C., Dugas, M., Schnittger S., Hiddeman W., Kern W., and Haferlach T. (2005). New insights into MLL gene rearranged acute leukemias using gene gene expression profiling: shared pathways, lineage commitment and partner genes. Leukemia, 19(6), 953-964.
Krivtsov, A. and Armstrong, S. (2007). MLL translocations, histone modifications and leukemia stem-cell development. Nature Reviews Cancer, 7(11), 823-833.
Laghi, L., Bianchi, P., and Malesci, A. (2008). Differences and evolution of the methods for the assessment of microsatellite instability. Oncogene, 27(49), 6313-6321.
Larizza, L., Magnani, I., and Beghini, A. (2005). The Kasumi-1 cell line: a t(8;21)-kit mutant model for acute myeloid leukemia. Leukemia & Lymphoma, 46(2), 247-255.
Leong, D. T., Gupta, A., Bai, HF., Wang G., Too, HP., Chew, F.T. and Hutmacher, DW. (2007). Absolute quantification of gene expression in biomaterials research using real-time PCR. Biomaterials, 28(2), 203-210.
Lgcstandards-atcc.org. (no date). Kasumi-1 ATCC ® CRL-2724™ Homo sapiens peripheral blood acu. available: https://www.lgcstandards-atcc.org/Products/All/CRL-2724.aspx?geo_country=gb [Accessed 26 Apr. 2018].
Marone, M., Mozzetti, S., Ritis, D., Pierelli, L., and Scambia, G. (2001). Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biological Procedures Online, 3(1), 19-25.
Marschalek, R. (2011). Mechanisms of leukemogenesis by MLL fusion proteins. British Journal of Haematology, 152(2), 141-154.
Martin, S., Lord, C., and Ashworth, A. (2010). Therapeutic Targeting of the DNA Mismatch Repair Pathway. Clinical Cancer Research, 16(21), 5107-5113.
Matheson, E. C. and Hall, A. G. (2003). Carcinogenesis: Assessment of mismatch repair function in leukemic cell lines and blasts from children with acute lymphoblastic leukemia. 24(1), 31-38.
Mohan, M., Lin, C., Guest, E., and Shilatifard, A. (2010). Licensed to elongate: a molecular mechanism for MLL-based leukaemogenesis. Nature Reviews Cancer, 10(10), 721-728.
Mwirigi, A., Dillon, R., and Raj, K. (2017). Acute leukemia. Medicine, 45(5), 280-286.
Nakatani, R., Nakamori, M., Fujimura, H., Mochizuki, H., and Takahashi, M. (2015). Large expansion of CTG•CAG repeats is exacerbated by MutSβ in human cells. Scientific Reports, 5(1), 11020.
Naumann, S., Reutzel, D., Speicher, M., and Decker, H. (2001). Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization. Leukemia Research, 25(4), 313-322.
Negoro, E., Yamauchi, T., Urasaki, Y., Nishi, R., Hori, H., and Ueda, T. (2011). Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene gene expression and proteomic analyses. International Journal of Oncology, 38(4), 911-919.
Piloto, O., Wright, M., Brown, P., Kim, K., Levis, M., and Small, D. (2007). Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways. Blood, 109(4), 1643-1652.
Reese, N. and Schiller, G. (2013). High-Dose Cytarabine (HD araC) in the Treatment of Leukemia: a Review. Current Hematologic Malignancy Reports, 8(2), 141-148.
Ruszkiewicz, A., Bennett, G., Moore, J., Manavis, J., Rudzki, B., Shen, L., and Suthers, G. (2002). Correlation of mismatch repair genes immunohistochemistry and microsatellite instability status in HNPCC-associated tumours. Pathology, 34(6), 541-547.
Sabiani, S., Geppert, T., Engelbrecht, C., Kowarz, E., Schneider, G., and Marschalek, R. (2015). Unraveling the Activation Mechanism of Taspase1 which Controls the Oncogenic AF4–MLL Fusion Protein. EBioMedicine, 2(5), 386-395.
Mendelsohn, J., Gray, J., Howley, P., Israel, M., and Thompson, C. (2015). The molecular basis of cancer. 4th ed. Philadelphia: Elsevier Saunders.Shia J, Ellis NA. and Klimstra DS. (2004). The utility of immunohistochemical detection of DNA mismatch repair gene geneproteins. Virchows Arch, 445(5), 431–441.
Shia, J. (2008). Immunohistochemistry versus Microsatellite Instability Testing For Screening Colorectal Cancer Patients at Risk For Hereditary Nonpolyposis Colorectal Cancer Syndrome. The Journal of Molecular Diagnostics, 10(4), 293-300.
Siraj, AK., Kamat S., Gutiérrez I.M., Banavali S., Timpson, G., Sazawal, S., Bhargava, M., Advani, S., Adde, M., Magrath, I., and Bhatia, K. (2003) Leukemia. Frequencies of the Major Subgroups of Precursor B-Cell Acute Lymphoblastic Leukemia, 17, 1192–1193.
Stam, R., van den Heuvel-Eibrink, M., den Boer, M., Ebus, M., Janka-Schaub, G., Allen, J., and Pieters, R. (2003). Multidrug resistance genes in infant acute lymphoblastic leukemia: Ara-C is not a substrate for the breast cancer resistance protein. Leukemia, 18(1), 78-83.
Stumpel, D., Schneider, P., Pieters, R., and Stam, R. (2015). The potential of clofarabine in MLL -rearranged infant acute lymphoblastic leukaemia. European Journal of Cancer, 51(14), 2008-2021.
Surtees, J. A. and Alani, E. (2006) Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination. Journal of Molecular Biology, 360(3), 523-536.
Terwilliger, T. and Abdul-Hay, M. (2017). Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer Journal, 7(6), e577.
Traver, S., Coulombe, P., Peiffer, I., Hutchins, J., Kitzmann, M., Latreille, D., and Méchali, M. (2015). MCM9 Is Required for Mammalian DNA Mismatch Repair. Molecular Cell, 59(5), 831-839.
Verma, M. (2012). Personalized Medicine and Cancer. Journal of Personalized Medicine, 2(4), 1-14.
Wei, K., Kucherlapati, R., and Edelmann, W. (2002). Mouse models for human DNA mismatch-repair gene gene defectsdefects. Trends in Molecular Medicine, 8(7), 346-353.
Worldwide cancer data. (2019), availablehttps://www.wcrf.org/dietandcancer/cancer-trends/worldwide-cancer-data.
Xie, C., Edwards, H., Xu, X., Zhou, H., Buck, S., Stout, M., Yu, Q., Rubnitz, J., Matherly, L., Taub, J., and Ge, Y. (2010). Mechanisms of Synergistic Antileukemic Interactions between Valproic Acid and Cytarabine in Pediatric Acute Myeloid Leukemia. Clinical Cancer Research, 16(22), 5499-5510.
Yokoyama, A., Kitabayashi, I., Ayton, P., Cleary, M., and Ohki, M. (2002). Leukemia proto-oncoprotein MLL is proteolytically processed into 2 fragments with opposite transcriptional properties. Blood, 100(10), 3710-3718.
Zheng, J. (2013). Oncogenic chromosomal translocations and human cancer (Review). Oncology Reports, 30(5), 2011-2019.