Abstract
Background/Aim: Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme which has an essential function in promoting antibody diversification. Its overexpression is strongly associated with B-cell derived malignancies including Burkitt lymphoma, where AID is required for the characteristic c-MYC/IGH translocation. This study aimed at defining AID’s oncopathogenic role which is still poorly understood. Materials and Methods: We created over-expressing and knock-down cell culture models of AID, and used cellular assays to provide insight into its contribution to lymphomagenesis. Results: We showed that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers and that ectopic overexpression of AID leads to rapid cell death. Using a knock-down model, we revealed that AID expression significantly impacts genomic stability, proliferation, migration and drug resistance. Conclusion: AID is an important driver of lymphoma, impacting multiple cellular events, and is potentially a strong candidate for targeted therapy in lymphoma.
Almost a decade ago a 24-kDa protein was described by Muramatsu et al. (1), and has since been demonstrated to be fundamental in the process of antibody specificity and diversification in B-cells (2-4). Activation-induced cytidine deaminase (AID) is an enzyme and nucleic acid modifier belonging to the evolutionary conserved APOBEC family of cytidine deaminases, with it being the only member having DNA-modifying activities. While V(D)J recombination ensures that the naïve B cell repertoire represents a tremendous diversity of antibody molecules, two additional DNA alteration events are needed to enhance the specificity and functionality of antibodies once B-cells reach secondary lymphoid organs. These processes are somatic hypermutation (SHM) and class-switch recombination (CSR) which ensure the production of antibodies with high-affinity to specific antigens, and AID plays an essential role in both (5). By deaminating cytidines AID inserts mismatches in single-strand DNA which, through various mechanisms of DNA replication and repair, gets modified, creating new mutations and recombination events in specific loci.
Because of its powerful mutagenic effect on DNA, the expression of AICDA/AID requires fine regulation to prevent aberrant expression. At the transcriptional level four conserved regulatory regions have been described for the human AICDA loci which contain binding elements for various transcription factors including Sp1, STAT6, NFkB and c-MYB (6). Tight regulation is achieved through the balance between B-cell-specific and stimulation-responsive elements as well as ubiquitous silencers. Post-translationally, AID activity is controlled by phosphorylation at serine, threonine and tyrosine residues while cellular localization in the form of nuclear cytoplasmic shuttling provides yet another level of AID regulation (7, 8). Several protein partners of AID have been described; these include replication protein A (RPA), DNA repair proteins UNG and MSH2/6, as well as the spliceosome protein CTNNBL1 which influence the enzyme’s activity and stability (9).
Due to its mutagenic nature, aberrant expression of AID is highly detrimental to the genome and its deregulation is associated with the development of several cancer types, but it is in cancers of the B cell lineage that it has been shown to play a notable role (2, 10-12). Burkitt Lymphoma (BL), a highly aggressive Non-Hodgkin lymphoma (NHL) of B cell origin, is highly over-represented in the HIV-positive population and is especially pertinent to the sub-Sahara African region (13). It is also a common pediatric cancer, accounting for ~30% of pediatric lymphomas. A hallmark of BL is the occurrence of translocations, particularly between c-MYC and the IGH loci. This leads to overexpression of the MYC oncogene, which is considered to have a major role in driving the cancer. Studies in IL6 transgenic mice showed that AID is necessary for this process (14). This role has been assigned to its ability to promote DNA double strand breaks, a compulsory step in the translocation event. In recent years AID’s role in epigenetic regulation has been highlighted, attributable to its ability to alter cytosines at CpG sites (15). The deamination of a 5-mC by AID yields thymidine which would then be removed through the DNA mismatch repair pathway and replaced by an unmethylated cytidine. Consequently, this gives AID the potential to alter gene expression. The role of AID as an oncoprotein is, therefore, multifaceted and hence its exact role in the oncogenic process, and the cellular consequences of its aberrant expression remains largely undefined. This study seeks to clarify the involvement of AID in the oncogenic process. We firstly showed that the overexpression of AID is highly specific to B cell-derived cancers, and that AID is expressed at very low levels, if at all, in non-lymphoid cells. Using a cell culture model, we demonstrated that constitutive ectopic expression of this protein is detrimental to cells, causing rapid cell death. We further show, using a knock down approach, that alterations in AID expression have consequences on genomic DNA integrity, cellular proliferation, migration, and susceptibility to chemotherapeutic drug. Our results provide insights into the pathobiology of B cell lymphomas where AID is deregulated and show a pleiotropic involvement of this factor in the development and progression of cancer.
Materials and Methods
Cell lines, reagents and plasmid constructs. All cells lines were maintained in Roswell Park Memorial Institute media supplemented with 10% Fetal Bovine Serum (FBS), with the exception of L1439A, Du145, and Caco-2, which were maintained in Dulbecco’s Modified Eagle Medium. The L1439A was produced at the University of Cape Town and require 20% FBS. The Ramos cell line was obtained from ATCC (Manassas, VA, USA). BL2, P3HR1 and Seraphina were donated by Professor Bernheim (French Institute of Health and Medical Research), PNT1A, PNT2 and PC-3 were donated by Professor Sharon Prince (University of Cape Town), LnCaP and Du145 were donated by Dr Luiz Zerbini (University of Cape Town), Caco-2, DLD-1, Colo205, Colo206F, HT-29 and LoVo, KYSE-30, KYSE-520, OE19 and OE33, UMSCC22b were donated by Professor Stoecklein (Heinrich-Heine University Düsseldorf). Doxorubicin and G418 was purchased from (Sigma-Aldrich, St. Louis, MI, USA). pEGFP-N3-AID and pEGFP-N3-Empty were obtained from Professor Bernhard Lüscher from RWTH Aachen University, Germany. The pcDNA-3.1-AID and pcDNA-3.1-Empty plasmids were obtained from Professor Reuben Harris from the University of Minnesota in the United States.
Quantitative real-time PCR. Total RNA was isolated using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) with 200 ng RNA used for reverse transcription using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) to produce cDNA. Standard curves of AICDA and GAPDH were produced by conventional PCR using MyTaq™ DNA Polymerase (Bioline, London, UK). KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, SA) was used for the qRT-PCR and analysed on the LightCycler® 480 (Roche). Primers were: AICDA-F 5’-CCAAACCATCTCTCCAAAGC-3’; AICDA-R 5’-CATCCCCACCCATAACAATC-3’; GAPDH-F 5’-GAAGGCTGG GGCTCATTT-3’; GAPDH-R 5’-CATCCCCACCCATAACAATC-3’.
Western blotting. Radio-Immunoprecipitation Assay buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 10 mM Tris pH 7.5 and 1% Deoxycholate powder) combined with 7X complete™, Mini, EDTA-free, Protease Inhibitor (Roche, Basel, Switzerland) was used for total protein extraction. Protein concentrations were determined using the Pierce™ BCA Assay kit (Thermo Scientific™, Waltham MA, USA) and separated using 12% SDS-PAGE gels, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) using the Mini-PROTEAN 3 casting apparatus (Bio-Rad, California, USA). Primary antibodies used were anti-c-MYC (SC-764; Santa Cruz Biotechnology, Texas, USA; 1:1,000); anti-AID (392500; Thermofisher Scientific, Massachusetts, USA; 1:1,000); anti- γ-H2AX (CST2577S Phospho-Histone H2AX (Ser139); Cell Signaling Technology, Beverly, MA, USA; 1:1,000); anti-p38 (M0800; Sigma-Aldrich, St. Louis, USA; 1:5,000). Densitometric analysis of signal intensity of bands was done using the Li-Cor Image Studio Lite software (Version 4.0, Germany)
Nucleofection, transfection and cell sorting using flow cytometry. The Amaxa® Cell Line Nucleofector® Kit V was used on the Nucleofector™ II system (Lonza, Basel, Switzerland). Nucleofection conditions were as per Thapa et al., using programme O-006 (16). The X-tremeGENE HP (Roche) transfection reagent was used for siRNA knockdown using the FlexiTube AID siRNA kit and AllStars Negative Control siRNA (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For cell sorting, the FACS Aria (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer was used to isolate either GFP expressing cells, or live cells via 7-AAD dye exclusion.
Annexin V incorporation assay. The Annexin V Apoptosis Kit I (Becton Dickinson) was used which contained the Annexin V protein conjugated to the Phycoerythrin (PE) fluorochrome according to manufacturer’s instructions. Cells were analysed on the FACSCaliber flow cytometry instrument (Becton Dickinson). Controls for the experiment were unstained cells to allow for compensation and size determination, cells only stained with PE Annexin V (no 7-AAD) and cells only stained with 7-AAD (no PE Annexin V).
WST-1 proliferation assay. The WST-1 proliferation (Roche, Basel, Switzerland) was used to measure cell viability (for IC50 determination) and proliferation, and used according to the manufacturer’s instructions. The WST-1 reagent contains tetrazolium salts which are cleaved to formazan dye in the presence of cellular enzymes produced by metabolically active cells, thus the more salt cleaved, the greater the colour change which indirectly indicates cell viability and proliferation. Absorbance was measured using the GloMax®-Multi microplate reader (Promega, Madison, WI, USA).
BrdU incorporation assay. Bromodeoxyuridine (BrdU) is a thymine analog which is incorporated into replicating DNA in place of thymidine, and can then be detected using a secondary antibody conjugated to a fluorochrome. Coverslips were coated with poly-L-Lysine (Sigma-Aldrich) to enhance the ability of the cells to adhere to the surface. The cells were incubated with 10 μM BrdU for 8 h followed by fixation and then denaturation. The slides were blocked (PBS/0.05 % Tween-20+5% human serum), and BrdU incorporation was detected using an anti-BrdU mouse monoclonal antibody (6 μg/ml) (Roche) diluted in 0.1 % BSA at 37°C for 30 min. and the donkey anti-mouse secondary antibody (AEC-Amersham) conjugated to Cy3 (1:1,000 diluted in 0.1% BSA). The cells were counter stained using Hoechst 33342 stain (#H1399; Invitrogen, Carlsbad, CA, USA) for 10 min. For visualization, cells were mounted on slides in Movial fluid containing anti-fade and visualised under a fluorescent microscope (Zeiss Axiovert 200M, Carl Zeiss Microimaging).
Transwell migration assay. The Corning® Transwell® assay kit was used (Sigma-Aldrich) to determine migratory abilities of cells. A total of 15 Transwell inserts were used, with five inserts for the AID-siRNA3 knockdown cells, five for the siNeg RNA control cells and five for the untreated Ramos cells. Approximately 2.2×105 cells per ml were plated into each Transwell insert and migration was allowed to proceed for 48 h. Two of each condition was used as a total control, whereby all the cells were stained to determine total number of cells in each well. Following incubation, each insert was fixed in 100% methanol for five min, washed in water, and then stained using 0.1% crystal violet for 30 min. Remaining cells in the upper chambers were removed and the inserts were then placed overnight in 50% acetic acid. The absorbance of 100 μl of each sample was determined at 600 μm using the Glo-Max®-Multi+ multiplate reader (Promega).
Statistical analyses. Standard error and significance were determined using the two sample t-test (Microsoft Excel for Office 365 or GraphPad Prism version 8).
Results
Expression of AID is highly specific to B-cell derived cancers. The normal physiological function of AID is clearly defined in B cells, and its overexpression in these cells is linked to B cell-derived cancers particularly Burkitt lymphoma, directly linking it to the c-MYC/IGH signature translocation event (14). Although a few reports implicate AID in certain non-lymphoid cancers, including gastric and colon cancer, it has as yet no defined normal or oncogenic role in these tissues (2, 3, 17, 18). The extent of its expression in these cancer types, compared to B cell-derived cancers, could be an indication of its significance in contributing to a cancer phenotype in these cells. We, thus, sought to assess the abundance of AID expression in panels of cell lines representing various epithelial-derived cancers where AID has been implicated, relative to B-cell derived lines, as an indication of its importance in normal cellular processes as well as in cancer. Both mRNA and protein expressions were measured in a panel of 16 cell lines representing cancers of the prostate, colon, oesophageal and Head and Neck, and compared to AID expression in a panel of B cell lines. We found that, except for two oesophageal cancer cell lines (OE19 and KYSE-30), AICDA expression was undetectable in all cell lines of epithelial origin, while high expressions of the transcript were found in the four BL cell lines (BL2, P3HR1, Ramos and Seraphina) and the Epstein-Barr virus (EBV)-immortalised lymphoblastoid cell line (L1439A) (Figure 1a). Among the B cell panel, Seraphina had the lowest number of AICDA copies (132) while P3HR1 had the most (2,415). Using a specific antibody to the human AID protein, no AID could be detected in any of the non-B cell lines (Figure 1b) while the protein was easily detectable in all of lymphocyte cell lines (Figure 1c). This indicates that the role of AID in these epithelial-derived cancers may not be as consequential as its role in cancers of B-cell origin.
Expression of AID in B cells compared to cell lines of epithelial origin. (a) Bar graphs showing AICDA mRNA expression determined by qPCR in B cell derived cell lines, as well as prostate, colon, oesophageal, and Head and Neck (H&N) cell lines. In all cases, AICDA expression is normalised to the housekeeping gene GAPDH. (b) Western blot analysis displaying the AID protein expression levels in the indicated epithelial cell lines with either the B cell extract from Seraphina or HT1080 cells transfected with pcDNA-3.1-AID as a positive control to show detection of AID. The p38 protein was used as a loading control and detected using the rabbit anti-p38 antibody and the HRP bound goat anti-rabbit secondary antibody. (c) Western blot displaying AID protein expression levels in B cell derived cell lines. The p38 protein was used as a loading control. Bars represent densitometric analysis of signal intensity of bands analysed using the Li-Cor Image Studio Lite software normalised to the loading control p38.
Ectopic expression of AID in B lymphocytes is toxic, leading to rapid cell death. The oncogenic function of AID has not been comprehensively explored in Burkitt lymphoma and we, therefore, sought to do this using an AID-overexpression cellular model. The use of overexpression cell lines as models are useful in studying the role of specific factors in the mechanisms of disease and oncogenic development (19). Overexpression of AID in the EBV-immortalized lymphoblastoid cell line L1439A, which was the lowest expressor of the endogenous protein (Figure 1c) since these cells are derived from a healthy donor, led to rapid cell death following nucleofection using an expression vector constitutively expressing GFP-tagged AID protein. We then ectopically expressed GFP-tagged AID in the BL cell line Ramos. While a pure population of GFP-only expressing Ramos cells could be obtained following cell expansion and FACS sorting two weeks postnucleofection, no GFP-positive cells could be detected in the population where the AID expressing plasmid was introduced. Once again, this indicated that the overexpression of the mutagenic enzyme in these cells was highly toxic and led to cell death (Figure 2a). When the cell population was assessed 3 days post-nucleofection we found that more than half of the AID expressing cells stained positive for Annexin V (Figure 2b). Nucleofection of the Ramos cells using an AID-expression plasmid without GFP, and selection through neomycin resistance yielded similar results, excluding the possibility that the effect could be due to AID being linked to GFP (data not shown). These results, therefore, indicate that AID is lethal when expressed constitutively and at high doses which correlates with its function as a powerful nucleic acid mutator and gene expression modifier. We next proceeded to produce a knock-down model of AID to continue to study its oncogenic function.
Ectopic expression of AID leads to cell death. (a) Cell sorting analysis of Ramos cell, either untransfected (control) or transfected with pEGFP-N3-Empty or pEGFP-N3-AID plasmids. LHS panels represent flow cytometric analysis showing forward and side scatter; in middle panels P2 represents the live GFP expressing cells; RHS panels show histograms of GFP-expressing cells where cells within the P3 quadrants are positive for GFP expression. A-C are the control cells which underwent nucleofection without plasmid, D-F are the cells nucleofected with pEGFP-N3-Empty, G-I are the sorted cells from D-F and a check purity after sorting, and J-L are cells nucleofected with pEGFP-N3-AID. (b) Flow cytometry analysis of Annexin V expression of Ramos cells 3 days post nucleofection; A shows cells nucleofected with empty vector and B shows cells expressing AID. The values in each block represent the percentage of cells counted.
Reduced AID expression leads to decreased DNA lesions and slows down proliferation. RNA interference was used to knock-down AID expression in the Ramos cell line. Of the four commercially available siRNAs designed specifically for the human AID, only one, AID-siRNA3, achieved a significant knock down of the protein (Figure 3a). The consequence of this knock down on specific cellular features was studied. Through its deaminase function one of the primary outcomes of AID is the incorporation of base pair mismatches, leading to double strand breaks (DSB) through the DNA repair pathway (20). DSB is the gravest form of DNA lesion in eukaryotic cells and if not repaired timeously and appropriately will lead to genomic instability which is a major contributor to tumorigenesis (21). The phosphorylated histone H2AX (p-γH2AX) is widely used as a biomarker for the presence of and cellular response to DSBs (22). A significant reduction in p-γH2AX was observed in Ramos cells when AID was knocked down compared to cells transfected with a non-specific siRNA (Figure 3b) which indicates that the elevated level of AID in lymphoma cells keeps the genome under significant stress.
AID down-regulation leads to reduced DNA damage. (a) Upper panel: Western blot analysis displaying AID protein expression in Ramos cells harbouring four commercially produced AID-siRNA (3, 5, 6 and 7) compared to parental line. The p38 protein was used as a loading control. Lower panel: Densitometric analysis of signal intensity of bands analysed using the Li-Cor Image Studio Lite software, normalised to p38, and relative to parental (Ramos). (b) Left Panels: Western blot analysis showing expression of phosphorylated γ-H2AX in Ramos cells harbouring siRNA Neg or AID-siRNA3. Right Panels: Western blot analysis showing expression of AID protein in the same cells (siRNA Neg or AID-siRNA3) used for detecting phosphorylated γ-H2AX. The p38 protein was used as a loading control in both instances.
One of the major hallmarks of cancer cells is the ability to proliferate faster than normal (23). In Ramos cells where AID was knocked down, the proliferation rate reduced significantly, by approximately 30%, compared to control cells (Figure 4a). This was confirmed using BrdU incorporation, where we show that the cells with reduced AID expression displayed very low BrdU staining compared to both parental Ramos cells and cells harbouring a non-specific siRNA (siRNA Neg) (Figure 4b). The overexpression of c-MYC is a key feature of BL (13) and serves as a “master regulator” of cellular metabolism and proliferation (24). We, therefore, expected a concomitant decrease in c-MYC expression upon AID knock down. Indeed, we found that c-MYC protein was markedly reduced in Ramos cells where AID is knocked down, with a more pronounced reduction 48 h post nucleofection, where c-MYC protein expression in AID-siRNA3 cells was about half that of control cells (Figure 4c).
AID knock-down in Ramos cells leads to a significant reduction in proliferation and c-MYC expression. (a) WST-1 cell proliferation assay to measure proliferation rate of Ramos cells harbouring siRNA Neg or AID-siRNA3 relative to parental cells. **p≤0.01 for AID-siRNA3 value, relative to siRNA Neg. Error bars represent the standard deviation. (b) BrdU staining (LHS panel) using Cy3-tagged antibody showing proliferating cells and Hoechst staining (middle panel) showing nuclei of cells of Ramos cells and cells harbouring siRNA Neg or AID-siRNA3. RHS panel show merged images. Cells were visualised under the fluorescent microscope at 40x magnification. (c) Western blot analysis displaying AID and c-MYC protein expression levels in Ramos cells harbouring siRNA Neg or AID-siRNA3 at 24 h and 48 h post nucleofection. The p38 protein was used as a loading control. Bars represent densitometric analysis of signal intensity of bands analysed using the Li-Cor Image Studio Lite software, normalised to p38, and relative siRNA Neg cells.
Lymphoma cells are sensitized to doxorubicin and have reduced migratory ability when AID is knocked down. The use of anthracyclines such as doxorubicin (Dox) in the therapeutic regimen of later stage disease (III and IV) and aggressive lymphomas such as Burkitt lymphoma has proven beneficial for patients (25). We sought to establish whether high expression of AID in these cancers influenced the response to therapy using the susceptibility of cells to Dox as a proxy. Using a Dox concentration of 0.5 ug/ml, which inhibited the viability of Ramos cells by ~50% over a 48 hour period (data not shown), cells with reduced AID expression were found to be significantly more susceptible to the drug, compared to controls (Figure 5a). A similar effect was observed when the amount of apoptotic (Annexin V) and necrotic (7AAD) cells were measured within the various cell populations. In the cells exposed to doxorubicin, there was an increase in Annexin V positive cells from 49.75% in the siRNA Neg cells compared to 56.08% in the AID-siRNA3 cells, thus showing an increase in apoptosis when AID expression is reduced (Figure 5b). A similar trend is observed for necrotic cells.
Reduced levels of AID sensitize lymphoma cells to doxorubicin and limits migratory ability. (a). Measurement of proliferation using the WST-1 assay of Ramos cells harbouring siRNA Neg or AID-siRNA3 relative to parental cells in the presence of doxorubicin (0.5 μg/ml over 48 h). Error bars represent the standard deviation. *p≤0.05 for AID-siRNA3 value. (b) Flow cytometric analysis using Annexin V and 7-AAD markers of Ramos cells harbouring siRNA Neg or AID-siRNA3 treated with or without doxorubicin. Panels A-D display results of untreated cells, while panels E-H display results of cells exposed to doxorubicin. A, C, E and G are profiles for Annexin V expression; B, C, F and H are profiles for 7-AAD expression. (c) Migration using the Transwell migration assay of Ramos cells harbouring siRNA Neg or AID-siRNA3 relative to parental cells. The result was normalised against the total number of cells plated, as determined by counting cells in both the upper and lower chambers. Error bars represent the standard error of the mean and *p≤0.05 for AID-siRNA3 value.
The ability for cells to migrate and invade new sites is another pertinent feature of advanced-stage aggressive lymphomas characterized by extranodal involvement and bone marrow infiltration. We assessed the migratory potential of cells using an in vitro transwell assay and found that cells with reduced expression of AID migrated slower through the matrix (Figure 5c). Our results, therefore, show that AID is an important driver of the lymphoma phenotype, enhancing features of advanced-stage disease and impacting multiple cellular phenotypes including proliferation, chemotherapeutic drug resistance and metastasis.
Discussion
The enzyme activation-induced cytidine deaminase is known to deaminate cytidine residues in DNA by converting them to uridine, leading to DNA cleavage. This is fundamental in the process of antibody specificity and diversification (2-4). Overexpression of AID has the potential to have catastrophic consequences in cells, being the candidate gene described to drive the c-MYC/IGH translocation in BL (14). Although the reported involvement of AID in carcinogenesis has not only been restricted to lymphomagenesis and B cells, its only described physiological role is in the production of a diverse antibody repertoire by B lymphocytes.
Our study supports the notion that the expression of AID is mainly confined to B lymphocytes as no AID could be detected, neither at the transcript nor protein level, in cell lines representing cancer of the prostate, colon, oesophageal and Head and Neck. In contrast, high expressions of AID were easily detectable in our B cell derived cell lines, with higher expressions found in the Burkitt lymphoma cells compared to the lymphoblastoid cell line. More recently, AID has been proposed as an oncogenic agent in another NHL, namely diffuse large B cell lymphoma (DLBCL), where its expression level was found to have prognostic impact (26). Furthermore, another recent study in DLBCL found AID to be responsible for recruiting the demethylation enzyme TET2 to the promoter of the FANCA proto-oncogene, leading to its overexpression (27). These reports, as well as the current study, support accumulating evidence of AID as having an important and composite role in lymphoma.
The BL cells Ramos, despite harbouring deficient p53 (28), start to undergo apoptosis very soon after ectopic AID overexpression. On the other hand, when AID was knocked down, phosphorylated γH2AX expression could barely be detected, indicating that an excess of AID, beyond physiological levels, leads to a very unstable genome. In this study, we define some of the downstream phenotypic consequences associated with this altered genomic state. Our results show that AID promotes proliferation and migration, which are two major hallmark features of cancer cells (23) and is supported by the recent report which shows that silencing AID in bladder cancer negatively impacted cell proliferation, invasion and migration (29). Importantly, we demonstrated a direct correlation between the expression of AID and c-MYC, not only supporting the association between AID and the translocation of c-MYC but also suggesting that AID may be indirectly driving proliferation, invasion and migration via up-regulation of c-MYC which is a driver of these cellular features (20).
This study is the first to demonstrate, in vitro, that lowered levels of AID in Burkitt lymphoma cells display an increased sensitivity to the chemotherapeutic drug doxorubicin, indicating that overexpression of AID contributes to drug resistance. Doxorubicin is one of the chemotherapeutic agents used in the management of BL patients as well as being one of the drugs used in first-line therapy for the treatment of DLBCL (31). This acquisition of drug resistance can be attributed to AID’s ability to promote genetic instability, which contributes to hypermutation of tumour suppressor genes leading to impairment of cell death pathways (32). A previous study found dramatic alteration of the gene expression profile of plasmacytoma cells when AID was silenced, which enhanced their susceptibility to immunotherapy by cytotoxic T lymphocytes (32). Another example supporting our finding is shown in a study done in chronic myeloid leukemia, where drug resistance of B lymphoid blast crisis cells was attributed to enhanced expression of AID in these cells, leading to the acquisition of BCR-ABL1 imatinib-resistant mutations (33). In a more recent study, AID expression in the lymph nodes of lymphoma patients was found to be a better predictive marker of DLBCL prognosis, compared to stage classification reports. Our findings therefore demonstrate that AID is an important driver of lymphoma and supports this gene as a potential target for preventing cancer progression and drug resistance.
Acknowledgements
The Authors wish to acknowledge the Flow Cytometry Core Facility located at Institute of Infectious Disease and Molecular Medicine, University of Cape Town. They also wish to acknowledge the University of Cape Town Confocal and Light Microscope Imaging Facility.
Footnotes
Authors’ Contributions
Author Shaheen Mowla conceptualized the research, developed the methodology, performed experiments and data analysis, sourced the funding, curates the data, supervised the research, and wrote and edited the manuscript. Author Grant Godsmark performed experiments and data analysis, developed methodology, and wrote an early draft of the manuscript. Author Leonardo Alves de Souza Rios performed experiments and data analysis, and assisted with manuscript editing.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received November 24, 2020.
- Revision received December 15, 2020.
- Accepted December 16, 2020.
- Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.