Determining the oncogenic activity of different Epstein-Barr virus proteins on the development of nasopharyngeal cancer by KONG EE LING A thesis presented in partial fulfilment of the requirement for the degree of Master of Science by Research at Swinburne University of Technology 2018
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Determining the oncogenic activity of
different Epstein-Barr virus proteins on
the development of nasopharyngeal
cancer
by
KONG EE LING
A thesis
presented in partial fulfilment of the requirement for the degree of
Master of Science by Research
at Swinburne University of Technology
2018
Abstract.
Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer arising from the
epithelium lining of the nasopharynx and has distinct geographical distribution and
multiple etiological cofactors. Epstein-Barr virus (EBV) infection is strongly associated
with nasopharyngeal carcinoma and EBV genes products are believed to play an
important role in the development of nasopharyngeal carcinoma. However, the definite
pathogenic roles of EBV genes products in premalignant nasopharyngeal epithelial cells
are still to be elucidated. In this study, the oncogenic activity of EBV genes products in
immortalized nasopharyngeal epithelial cells NP460hTert was examined. Four stable
EBV gene expressing NP460hTert was successfully generated. Here, it is shown that
EBV genes BARF1 and BHRF1 expression were able to promote cell proliferation, and
BARF1 expression can promotes cell migration in NP460hTert. In addition, the
autocrine/paracrine activity of secreted BARF1 was examined. Analysis of BARF1
expressing NP460hTert revealed that BARF1 was secreted by nasopharyngeal epithelial
cells into culture medium. Furthermore, it is shown that secreted BARF1 was capable to
stimulate cell proliferation and cell migration in parental NP460hTert. These findings
indicated that BHRF1 and BARF1 greatly affect cell proliferation and cell migration in
NP460hTert and may contribute to transformation of nasopharyngeal epithelial cells.
Acknowledgement
I would like to express my profound gratitude towards Swinburne University of
Technology for providing me this opportunity to conduct and complete my master’s
research project. I would also like to thank Swinburne University of Technology for
awarding me a scholarship and supporting the research with crucial funds and materials.
I would like to thank my supervisor Dr. Paul Neilsen for all his guidance and support
throughout this project. Without his wisdom and knowledge on cancer research, this
project could not be completed. I am also deeply grateful to my co-supervisor Dr. Yap
Lee Fah for expanding my horizons regarding method of research. She also contributed
valuable resources and materials to assist me in completing this project. I would also
like to thank University Malaya for allowing me to do my attachment at their
prestigious state of the art University Malaya Medical Centre. To Associate Professor
Peter Morin, I like to express my appreciation for providing essential materials at the
beginning of this project and moral support throughout the duration of the project.
Finally, I would like to express my sincere appriciation to Dr. Irine Henry for her
diligent proofreading of this thesis.
Last but not least, I am indebted to all my colleagues and staff from both Swinburne
University of Technology and University Malaya, my friends and family for their help
and support throughout the trying times.
Declaration
I, Kong Ee Ling, candidate of Master of Science by Research from Faculty of
Engineering, Computing and Science in Swinburne University of Technology Sarawak
Campus hereby declare that my master thesis entitled “Determining the oncogenic
activity of different Epstein-Barr virus proteins on the development of nasopharyngeal
cancer” is original writing outcome and contains no material or content which has been
accepted for the award to the stated candidate of any other degree or diploma studies,
except where due references are made in the text of the examinable outcomes; and
where the work is based on joint research or publications, the disclosed relative
contributions of the respective workers or authors.
Kong Ee Ling
As the principal coordinating supervisor, I hereby acknowledge and verify that the
above-mentioned statements are legitimate to the best of my knowledge.
Dr Paul Neilsen
Conference presentations/Publications
Kong, EL, Nissom, PM , Yap, LF, Neilsen PM 2016, “Determining the oncogenic
activity of different Epstein-Barr virus proteins on the development of nasopharyngeal
cancer”, 5th NPC Research Day 2016, Nasopharyngeal Carcinoma Society of Malaysia,
Institute for Medical Research, Kuala Lumpur, Malaysia, 3 March, 2016.
Table of contents
Chapter 1: Introduction 1
1.1 Nasopharyngeal carcinoma 1
1.1.1 Racial and geographical distribution 1
1.1.2 Histopathology of NPC 6
1.1.3 Clinical presentation and treatment for NPC 8
1.1.4 Etiological factors of NPC 9
1.1.4.1 Environmental factors 9
1.1.4.2 Genetic susceptibility 10
1.1.4.3 Epstein-Barr virus infection 11
1.2 Epstein-Barr virus 12
1.2.1 Route of EBV entry and infection 12
1.2.2 Genome structure and strain variation of EBV 13
1.2.3 EBV latent and lytic states 16
1.2.4 Contribution of EBV genes in NPC oncogenesis 17
1.2.4.1 Latent membrane proteins 18
1.2.4.1.1 LMP1 18
1.2.4.1.2 LMP2 19
1.2.4.2 Epstein-Barr nuclear antigens 20
1.2.4.2.1 EBNA1 20
1.2.4.2.2 EBNA2 and its co-activator EBNA-LP 21
1.2.4.2.3 EBNA3 family 21
1.2.4.3 BamHI-A rightward frame 1 (BARF1) 22
1.2.4.4 BamHI-H rightward frame 1 (BHRF1) 22
1.2.4.5 EBV immediate-early proteins 23
1.2.4.6 EBV-encoded small RNAs 24
1.2.4.7 EBV-encoded microRNAs 24
1.4 In vivo and in vitro model systems of NPC 25
1.5 Aims and objectives 26
Chapter 2: Methodology 27
2.1 Primers design 27
2.2 DNA sample preparation 30
2.2.1 Total RNA extraction for lytic stage NP460hTert-EBV 30
2.2.2 DNase I treatment and cDNA synthesis for PCR amplification 31
2.3 Polymerase chain reaction (PCR) 32
2.4 PCR free nucleotide removal 33
2.5 Agarose gel electrophoresis 33
2.6 Cloning 34
2.6.1 Restriction enzyme digestion 35
2.6.2 Gel purification 35
2.6.3 Ligation of PCR products into pcDNA 3.1 Hygro 36
2.7 Preparation of chemically competent cells 36
2.8 Transformation 37
2.9 Plasmid purification 37
2.10 Sub-cloning into pLVX-Puro 38
2.11 Maxi preparation of pLVX-EBV 39
2.11.1 Preparation of bacteria culture 39
2.11.2 Extraction of plasmid 39
2.11.3 Precipitation of plasmid 39
2.12 Cell lines and reagents 40
2.13 Cell lines maintenance 40
2.14 Cell lines cryopreservation and recovery from cryopreservation 41
2.15 Transfection of HEK293T and lentiviruses production 41
2.16 Establishment of NP460hTert puromycin selection concentration 42
2.17 Transduction of NP460hTert 42
2.18 RNA preparation for reverse-transcription PCR analysis 42
2.18.1 RNA extraction from transduced NP460hTert 42
Figure 3.28 Comparison of migration for NP460hTert expressing either BARF1 or vector control pLVX
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Figure 3.29 Analysis of micrographs by using Tscratch 104
Figure 3.30 The open wound area of NP460hTert-BARF1 and NP460hTert-pLVX at four different time points relative to zero hour
105
Figure 3.31 Optimization of conditioned medium experiments 107
Figure 3.32 Cell proliferation rates of NP460hTert in different conditioned medium
109
Figure 3.33 Comparison of migration for NP460hTert incubated in either conditioned medium form NP460hTert BARF1 (BARF1 CM) or vector control (pLVX CM).
111
Figure 3.34 Analysis of micrographs by using Tscratch 112
Figure 3.35 The open wound area of NP460hTert in conditioned medium form either NP460hTert-BARF1 or NP460hTert-pLVX at four different time points relative to zero hour.
113
List of Tables
Chapter 1: Introduction
Table 1.1 EBV gene expression in different type of latency programme 17
Chapter 2: Methodology
Table 2.1 List of cloning primers and gene expression primers 28
GSK3 Glycogen synthase kinase 3 GSTM1 Glutathione S-transferase Mu 1 GWAS Genome-wide association studies HDAC Histone deacetylases HIF-1 α Hypoxia-inducible factor 1-alpha HLA Human leukocyte antigen hOGG1 Human 8-oxoguanine DNA N-glycosylase 1 Hp1α Heterochromatin protein 1 alpha HRP Horseradish peroxidase ID1 Inhibitor of differentiation protein 1 IgA Immunoglobulin A IGF-1 Insulin-like growth factor 1 INF-γ Interferon gamma IRF3 Interferon regulatory factor 3 IRF7 Interferon regulatory factor 7 ITAM Immunoreceptor tyrosine activation motif JAK Janus kinase LCL Lymphoblasoid cell lines LMP Latent membrane protein MAPK Mitogen-activated protein kinase Mcl-1 Induced myeloid leukaemia cell differentiation protein MDM2 Mouse double minute 2 homolog MDS1-EVI1 Myelodyplasia 1 and ecotropic viral insertion site 1 fusion proteins miRs microRNAs MMPs Matrix metalloproteinase mTOR Mammalian target of rapamycin MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
Bromide, Thiazole Blue MVA Modified vaccinia Anakara NADPH Nicotinamide adenine dinucleotide phosphate NCBI National centre for biotechnology information NcoR Nuclear receptor co-repressor NF-κB Nuclear factor-kappa B NHEJ Non-homologous end joining NK/T Natural killer T cells NOX NADPH oxidase NPC Nasopharyngeal carcinoma ORF Open reading frame PCR Polymerase chain reaction PI3K Phosphoinositide 3-kinase PIGR Polymeric immunoglobulin receptor
PKR Protein kinase R PLUNC Palate, lung and nasal epithelium clone protein PML Promyelocytic leukaemia pRB Retinoblastoma protein PTEN Phosphatase and tensin homolog PUMA p53 upregulated modulator of apoptosis PVDF Polyvinylidene fluoride RAD51L1 DNA repair protein RAD51 homolog 2 RAGE Receptor for advanced glycation end products RASSF1A Ras association domain family member 1 A RBPJ Recombining binding protein suppressor of hairless RT-PCR Reverse transcription polymerase chain reaction SAHA Suberoylanilide hydroxamic acid SCID severely compromised immunedeficient SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sky Spleen tyrosine kinase STAT Signal transducer and activator of transcription TBS Tris buffered saline TCR T-cell receptor TGFβ Transforming growth factor beta TIMPs Tissue inhibitor of metalloproteinase TLRs Toll-like receptors TMED Tetramethylethylenediamine TNFRSF19 Tumour necrosis factor receptor superfamily, member 19 TNF-α Tumour necrosis factor alpha TP53 Tumour protein p53 TPA Tetradecanoyl phorbol USP7 Ubiquitin-specific protease UTR Untranslated region VCA Viral capsid antigen VEGF Vascular endothelial growth factor VEGF Vascular endothelial growth factor WHO World health organisation XRCC1 X-ray repair cross-complementing protein 1 ΔNp63 Tumour protein p63 isoform
1
Chapter 1
Introduction
1.1 Nasopharyngeal carcinoma
Nasopharyngeal carcinoma (NPC) is an epithelial cancer arising from epithelium lining
of the nasopharynx. NPC is distinguishable from other epithelial cancers arising from
the head and neck regions by its distinct clinical characteristics, histopathology, racial
and geographical distribution (Tsao et al. 2014).
1.1.1 Racial and geographical distribution
NPC is a rare malignancy throughout most regions of the world especially in Western
countries with an incidence rate of less than 1 per 100,000 per year (Chang & Adami
2006; Tsao et al. 2015; Tsao et al. 2014). NPC also only contributes to 0.6% of all
cancers and is more common in males than females with sex ratio of 2.3:1 (Ferlay et al.
2015). However, high incidences of NPC are observed in endemic regions such as
Southern China, South East Asia, Northern Africa and the Arctic. The incidences of
NPC in both sexes in the world are represented in Figure 1.1. Furthermore,
approximately 81% of global NPC cases are reported in Asia (Ferlay et al. 2013). The
highest incidence rates of NPC from a geographical prospective, demonstrated as age-
standardized rate (ASR) of 25 per 100,000 person-years for males and 9.0 per 100,000
person-years for females, come from Zhongshan city and Zhuhai, respectively (Bray et
al. 2017; Bray et al. 2015). Both endemic areas are located in Guangdong province of
southern China, where the majority of the population is Cantonese. The highest rates of
NPC were also reported among Cantonese in similar regions during 1998-2002 (Feng
2013). Comparable ASRs were observed in nearby cities including Jiangmen,
Guangzhou and Hong Kong, whereas the incidences in Northern china is significantly
lower than that in the South (Bray et al. 2017).
An intermediate incidence rate of NPC has been observed among Southeast Asian
countries. However, the risk of NPC in Southeast Asia varies due to diverse ethnic
backgrounds and social admixture with southern Chinese. Notably, elevated incidences
of NPC were reported among the population of Chinese descent and the populations
2
which have a history of intermarriage with Chinese ancestors (Chang & Adami 2006).
The comparison of NPC incidence for top 10 Southeast Asian countries, demonstrated
as age-standardized rate, is illustrated in Figure 1.2.
3
Figure 1.1: Estimated age-standardized rate of NPC incident in both sexes in the world. Data were taken from the Cancer Incidence in Five
Continents, Volume XI (Bray et al. 2017).
4
Figure 1.2: Top 10 Age standardized incidence rates of NPC in Southeast Asian
countries. Malaysia, Sarawak data are taken from Malaysian National Cancer Registry
Report (Azizah et al. 2016). Unless otherwise stated, data were taken from the Cancer
Incidence in Five Countries, Volume XI (Bray et al. 2017).
Malaysia is one of the endemic regions of NPC with incidence rates in males and
female of 6.4 and 2.2 per 100,000 person-years, respectively (Figure 1.2). Furthermore,
NPC is the fifth most common cancer in Malaysia and third most common in males
(Azizah et al. 2016). In Malaysia, the ethnic group with the highest risk of NPC is
Malaysian Chinese with ASR of 11.0 and 3.5 for male and female, respectively. The
possible factors that contributed to this higher incidence in Malaysian Chinese might be
5
due to their ancestors who originated from endemic regions in Southern China.
Malaysian Malays and Indians come in distant second in NPC incidences with ASR of
3.3 and 1.3 for Malay males and females, respectively; 1.1 and 0.6 for Indian males and
females, respectively. Notably, The Bidayuh people, one of the Sarawak native ethnic
groups, exhibit the highest ASR (ASR 31.5 f or male and 11.8 for female) recorded by
any population-based registry in the world during 1998-2002 (Devi et al. 2004). Other
indigenous groups, the Iban people, also show a high risk of NPC with ASR of 13.1 for
males and 5.6 for females. The unique distribution of NPC incidence among the
Malaysia ethnic groups might have an influence on variation in NPC incidence across
different states. High population of Southern Chinese descendent was known to have
significant correlation with the regional NPC incidence in Southeast Asia (Armstrong,
Kutty & Dharmalingam 1974; Chang & Adami 2006). Indeed, in the Malaysian state of
Selangor during 1968-1972, it had the highest rate of NPC while the population of
Selangor Chinese was also higher than the corresponding proportion for Peninsular
Malaysia as a whole (Armstrong, Kutty & Dharmalingam 1974). Similar racial pattern
for NPC was also observed during 2007-2011. The reported NPC incidence during
2007-2011 was demonstrated in Figure 1.3.
Figure 1.3: Age standardized incidence rates of NPC across Malaysia states during
2007-2011. The data was taken from Malaysia National Cancer Registry Report (Azizah
et al. 2016).
6
NPC incidences are high among the states such as Penang, Johor, and Perak which has
high population of Chinese of 45.6%, 33.6% and 30.4%, respectively (Department of
Statistics, Malaysia). Correspondingly, NPC incidencess in Pahang, Kedah, Perlis,
Terengganu and Kelantan (16.2%, 13.6%, 8.0%, 3.4% and 2.6% population of Chinese,
respectively) are roughly half of those in Penang, Johor and Perak, where a higher
proportion of population is Chinese (Department of Statistics, Malaysia). Likewise, the
NPC incidences in Selangor during 2007-2011 are lower than same state during 1968-
1972 due to the decline in regional Chinese population from 46% to 28% (Armstrong,
Kutty & Dharmalingam 1974). Noteworthy, while Sarawak had the highest NPC
incidences in Malaysia, the state only has Chinese population of 24.5%. The high risk of
NPC in Sarawak was strongly associated with its native population. Sarawak native
population especially Bidayuh and Iban were once reported to have the highest
incidence rate recorded by any population-based registry (Devi et al. 2004). Similarly,
the incidence rates of NPC are high in other Malaysia states on the island of Borneo
(Sabah and WP Labuan) where the major population are indigenous people (Devi et al.
2004).
1.1.2 Histopathology of NPC
Studies in the early 20th century has revealed that NPC does not only consists of a single
type of epithelium cell but is composed of multiple morphologically different
carcinomas (Nicholls & Niedobitek 2013). Thus, identification of nasopharyngeal
biopsies from NPC endemic populations provides a significant pathological diagnosis
for the clinician to enable both appropriate treatment and clinical follow up. Therefore,
it is important to have a universal histopathological classification for terminology,
definition, and classification of different types of NPC (Shanmugaratnam et al. 1979).
In 1978, the World Health Organization proposed an international classification which
classified NPC into three subtypes: keratinizing squamous cell carcinoma, non-
keratinizing carcinoma, and undifferentiated carcinoma (Shanmugaratnam et al. 1979;
(Figure 3.10b). Single-digestion of pLVX-Puro with restriction enzyme HindIII
produced seven DNA fragments with the sizes of 3285 base pairs, 1947 base pairs, 639
base pairs, 588 base pairs, 577 base pairs, 556 base pairs and 510 base pairs (Figure
3.10a). Notably, the multiple cloning site of pLVX-Puro was located within the 577
base pairs fragments. Thus, the size of this fragment will be shifted depending on the
size of insert.
After the sub-cloning primers were synthesised by IDT technology, eight EBV genes
were PCR amplified from pcDNA 3.1 Hygro, doubled digested with restriction enzymes
EcoRI and XbaI and then ligated into pLVX-Puro with T4 DNA ligase. Only 6 out of 8
EBV genes (LMP1, LMP2A, LMP2B, BARF1 and BRLF1) were successfully PCR
amplified from pcDNA 3.1 Hygro and subsequently cloned into pLVX-Puro (Figure
3.1). The recombinant pLVX-Puro were analysed with single-digestion with restriction
enzyme HindIII. LMP1, LMP2A and LMP2B were shown to be in the expected size of
1767 base pairs, 2145 base pairs and 1788 base pairs respectively (Figure 3.11a).
BARF1, BHRF1 and BRLF1 were also shown to be in the expected size of 1317 base
pairs, 1277 base pairs and 2469 base pairs respectively (Figure 3.11b).
EBNA1 and EBNA2 were PCR amplified from respective pcDNA 3.1 Hygro, but the
sizes of their genes fragments were not in expected range (Figure 3.12). EBNA1 and
EBNA2 were expected to have the size of 1868 base pairs and 1535 base pairs
respectively. However, the bands amplified from pcDNA 3.1 Hygro were shown be in
the ranges between the 1000 and 1500 base pairs markers in the Promega DNA ladder.
68
Figure 3.10: The restriction enzyme digestion of pLVX-Puro. A: Gel image of pLVX-
Puro digested with restriction enzyme HindIII. B: The vector map of circular pLVX-
Puro with seven recognition sites for HindIII.
69
Figure 3.11: Gel images of restriction enzymes digestion for recombinant pLVX-Puro.
A: The single-digestion of recombinant pLVX-Puro containing LMP1, LMP2A and
LMP2B with the restriction enzyme HindIII. B: The single-digestion of recombinant
pLVX-Puro containing BARF1, BHRF1 and BRLF1 with the restriction enzyme
HindIII.
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Figure 3.12: Gel image of PCR amplification for EBNA1 and EBNA2 from
recombinant pcDNA 3.1 Hygro.
3.5.1 Discussion
Six EBV genes, LMP1, LMP2A, LMP2B, BARF1, BHRF1 and BRLF1 were
successfully shuttled from pcDNA 3.1 Hygro into pLVX-Puro. The sizes of the inserts
were checked by restriction enzyme digestion with HindIII and matched the theoretical
sizes. The other two EBV genes, EBNA1 and EBNA2 were not cloned into pLVX-Puro
due to unforeseen complication in preserving the recombinant pcDNA 3.1 Hygro.
PCR amplification of EBNA1 and EBNA2 from pcDNA 3.1 Hygro produced two
fragments that have shorter sizes, which is in between 1000 and 1500 base pairs,
compared to the expected sizes (Figure 3.12). This result suggests that the PCR
amplification with new primers was successful since it specifically targets the c-Myc
sequence of EBNA1 and EBNA2. Without EBNA1 and EBNA2 inserts in pcDNA 3.1
Hygro, there will be no PCR amplicons when amplified with sub-cloning primers.
Furthermore, the sizes of EBNA1 and EBNA2 in pcDNA 3.1 Hygro were identified in
previous sections. The shortening of EBNA1 and EBNA2 sizes could be explained by
few bases deletion in the middle region of the genes segments. The reason for this
deletion could be due to tandem repeat rearrangement by homologous recombination in
E.coli (Bzymek & Lovett 2001). Repetitive sequences are susceptible to tandem repeat
rearrangement and this sequence rearrangement could result in deletion of sequence
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repeats. EBNA1 has an IR3 domain that contains approximately 570 base pairs
repetitive sequence which encode for only glycine and alanine repeat (Falk et al. 1995).
ENBA2 has two regions of repetitive sequences of total 160 base pairs that encode for
a polyproline domain and arginine-glycine repeat motif (Wang et al. 2012a). The length
of both repetitive sequences in EBNA1 and EBNA2 matches the length of the missing
fragments. Therefore, the deletion in these repeats might randomly occur during
propagation of Top10F’ and resulted in shorter EBNA1 and EBNA2 PCR amplicons as
observed (Figure 3.12).
3.6 Sequence analysis of 6 EBV genes in pLVX-Puro
All six recombinant pLVX generated from previous section (pLVX-LMP1, pLVX-
LMP2A, pLVX-LMP2B, pLVX-BARF1, pLVX-BHRF1 and pLVX-BRLF1) were
sequenced with ABI 3730XL capillary DNA Sequencer (Figure 3.1). Raw sequences
were quality filtered using Chromas (Technelysium) and aligned with ClustalW
multiple alignment algorithm (GenomeNet). The aligned sequences were then converted
into protein sequence with ExPASy translate tools (Swiss Institute of Bioinformatics).
The sequences of amino acids in 5’ to 3’ direction at Frame 1 or the longest open
reading frame were selected. Final protein sequences were then aligned against the
protein sequences from either Akata or B95.8 EBV genome using ClustalW2 (EMBL-
EBI) and checked using BLAST against the NCBI protein database.
As shown in Figure 3.1, only 4 out of 6 EBV genes (LMP1, LMP2A, BARF1 and
BHRF1) passed the sequence validation. LMP2A and BHRF1 sequences have 100%
homology with the respective reference sequences (Figure 3.13 and Figure 3.14).
Although pass the sequence validation, LMP1 have a single DNA point mutation and
BARF1 has 2 DNA point mutations in their DNA sequences. These sequence mutations
lead to the third amino acid mutation in LMP1 translated protein and the 39 and 41
amino acids mutation in BARF1 translated protein (Figure 3.15 and Figure 3.16). The
other two EBV genes, LMP2B and BRLF1 were shown to have multiple sequence
mutations that lead to premature termination in translated proteins (Figure 3.17 and
Figure 3.18).
72
Figure 3.13: Sequence analysis of LMP2A in pLVX-Puro. The alignment of pLVX
LMP2A translated protein sequence (LMP2ASEQ) against B95.8 EBV LMP2A protein
sequence (LMP2AWT) using ClustalW2.
Figure 3.14: Sequence analysis of BHRF1 in pLVX-Puro. The alignment of pLVX
BHRF1 translated protein sequence (BHRF1SEQ) against Akata EBV BHRF1 protein
sequence (BHRF1WT) using ClustalW2.
73
Figure 3.15: Sequence analysis of LMP1 in pLVX-Puro. The alignment of pLVX LMP1
translated protein sequence (LMP1SEQ) against B95.8 LMP1 protein sequence
(LMP1WT) using ClustalW2.
Figure 3.16: Sequence analysis of BARF1 in pLVX-Puro. The alignment between
pLVX BARF1 translated protein sequence (BARF1SEQ) and Akata EBV counterpart
(BARF1WT).
74
Figure 3.17: Sequence analysis of LMP2B in pLVX-Puro. The alignment between
pLVX LMP2B translated protein sequence (LMP2BSEQ) and B95.8 EBV LMP2B
protein sequence (LMP2BWT).
75
Figure 3.18: Sequence analysis of BRLF1 in pLVX-Puro. The alignment between
pLVX BRLF1 translated protein sequence (BRLF1SEQ) and Akata EBV BRLF1
protein sequence (BRLF1WT).
76
3.6.1 Discussion
Four out of six EBV genes (LMP1, LMP2A, BARF1 and BHRF1) in pLVX passed the
sequence analysis when compared to their respective reference sequences. LMP2A and
BHRF1 have 100% homology to the reference sequences and no mutation was observed
in their translated protein sequences.
Acceptable point mutations were found in LMP1 and BARF1. From the sequencing
data (Figure 3.15), LMP1 translated protein has almost identical sequence compared to
B95.8 LMP1. The only difference between two protein sequences is LMP1 has an
arginine as third amino acid while B95.8 LMP1 has histidine at the same position. This
modification in amino acid was unintentionally introduced when designing the cloning
primers. The forward cloning primer was designed based on Akata EBV LMP1
sequence instead of B95.8 EBV sequences. Akata LMP1 sequence has a guanine at 8
bases while B95.8 LMP1 has an adenine at same position. Thus, this error in designing
cloning primers inevitably creates a point mutation in LMP1. The point mutation in
LMP1 was considered as a conservative point mutation according to GONNET PAM
(point accepted mutation) 250 matrix (Gonnet, Cohen & Benner 1994). Since both
arginine and histidine are basic amino acids, this substitution is acceptable in term of
GONNET PAM matrix. Besides, the arginine residue is present in native LMP1
sequences of Akata EBV. Therefore, this mutation might not affect the final structure of
LMP1 generated form these sequences.
The sequencing results of BARF1 from pLVX shown that it has two amino acid
residues difference compared to Akata EBV BARF1 protein (Figure 3.16). The
differences were asparagine (serine in Akata EBV variant) and serine (glycine in Akata
EBV variant) at amino acids 39 and 41 respectively. These point mutations were
considered as semi conservative substitution according to GONNET PMA 250 matrix.
These substitutions of amino acids might not be introduced during PCR amplification
but were due to variation in BARF1 sequences. Sequence variations of BARF1 have
been reported in NPC (Wang et al. 2012b) and NK/T cell lymphoma (Sun et al. 2015)
biopsies from northern China. Several amino acid mutations were detected in multiple
samples. Among these mutations, V29A where valine at 29 amino acids substituted
with alanine was the most frequent amino acid mutation observed. V29A mutation in
BARF1 does not change its final protein structure and might not have any effect on its
77
biological functions. Besides V29A, other minor amino acid mutations such as V46A,
D79G and V113I were sporadic and only found in a few samples. Interestingly, the
amino acid mutations observed in BARF1 has not been described in any of the previous
studies. These mutations might be cell lines specific since the BARF1 cDNA obtained
in this study was from Akata infected nasopharyngeal epithelial cells. Furthermore, the
mutations could be novel and have a non-destructive amino acid modification to
BARF1 protein. Therefore, BARF1 was selected for further downstream experiments
even though it has certain level of amino acid modifications.
Two of the EBV genes, LMP2B and BRLF1 were excluded from the downstream
experiments due to nonsense mutation in protein sequences. LMP2B protein sequence
has a nonsense mutation that results in premature stop codon at 226 amino acid residues
(Figure 3.17). This nonsense mutation is due to nucleotide substitution at 678 bases of
LMP2B DNA sequence where thymine in triplet codon for cysteine (TGT) changes to
adenine and create a stop codon (TGA). The stop codon will produce an incomplete and
non-functional protein when introduced into a cell system. Therefore, LMP2B was
excluded from downstream experiments. A total of eight DNA mutations were
identified within BRLF1 and have led to the mutations in its protein sequences (Figure
3.18). Four-point mutations were conservative and semi conservative amino acid
substitution. Three amino acid substitutions were neither conservative nor semi
conservative. Unfortunately, the amino acid substitution at 489 residues led to nonsense
mutation in BRFL1. This mutation was due to the substitution of adenine at 1452 bases
in BRLF1 with thymine which in turn created a stop codon (TAG) instead of lysine
(AAG). Since BRLF1 will not be able to generate complete functional protein, it was
excluded from the downstream experiments.
The protein generated from nonsense mutation will be incomplete, non-functional and is
not valuable for our expression study. Multiple DNA sequence mutations were observed
during the sequencing analysis. Based on this observation, it is concluded that the PCR
does not produce satisfactory cloning results because of the amount of mutations in
DNA sequences. One probable source of the mutation is the quality of DNA polymerase
used. DNA polymerase used in this study was Taq polymerase. Taq polymerase is a
relatively low fidelity enzyme that has an error rate of (2 x 10-5) and lack of 3’ to 5’
proofreading exsonuclease (Flaman et al. 1994; McInerney, Adams & Hadi 2014).
78
Without proofreading exsonuclease, Taq polymerase could not remove mismatches
during PCR nucleotides extension and thus produced an error prone PCR amplicon
Therefore, high fidelity enzymes such as Pfu with 3’ to 5’ proofreading exsonuclease
should be used to amplify target genes during PCR cloning. High fidelity enzymes
could be used to minimize PCR generated errors and produce credible replication of
gene of interest. Besides DNA polymerase, our polymerase reactions might not have the
optimized conditions for cloning. Several variables in PCR could be refined to minimize
the mutation frequency during PCR cloning. First, low concentration of MgCl2 and
dNTP could be incorporated to reduce the error frequency. Next, since the PCR error
rates is directly proportional to cycle number, number of PCR cycle could also be
reduced to 25 or 30 cycles instead of 35 cycles. Lastly, an increase in number of PCR
template might improve the fidelity of polymerase extension.
3.7 The generation of 4 EBV genes expressing NP460hTert
The EBV genes expressing NP460hTert cell lines were generated by lentiviral
transduction as described in methodology (Section 2.17). Stably transduced clones were
selected and maintained as polyclonal populations under puromycin selection. The EBV
expressing cell lines were NP460hTert-LMP1, NP460hTert-LMP2A, NP460hTert-
BARF1 and NP460hTert-BHRF1. Vector control was also generated by transducing
pLVX-Puro into NP460hTert.
Once established, the EBV gene expression in the stable transduced cell lines was
analysed by reverse-transcription PCR (Figure 3.1). The cDNA synthesised from total
RNA of NP460hTert-LMP1, NP460hTert-LMP2A, NP460hTert-BARF1 and
NP460hTert-BHRF1 were PCR amplified with cloning primers and gene expression
primers. The GAPDH gene was also PCR amplified as a loading control for reverse-
transcription PCR.
For NP460hTert-BARF1 and NP460hTert-BHRF1, cDNA from four passages
NP460hTert-BARF1 (passage 1, passage 2, passage 3 and passage 4) and two passages
NP460hTert-BHRF1 (passage 3 and passage 4) were selected for reverse-transcription
PCR analysis (Figure 3.19). cDNA from IgG activated Akata cell and vector control
were included as references. BARF1 was expected to have a size of 740 base pairs and
183 base pairs when amplified with cloning primer and gene expression primer
79
respectively. BHRF1 was expected to have a size of 650 base pairs and 190 base pairs
when PCR amplified with cloning primer and gene expression primer respectively.
From Figure 3.19, all cells lines were found to express BARF1 and BHRF1 and theirs
size were in the expected ranges in Promega DNA ladder. The levels of GAPDH were
consistent among all the cell lines
For NP460hTert-LMP2A, two passages of NP460hTert-LMP2A (passage 3 and passage
6) were selected for reverse-transcripttion PCR analysis (Figure 3.20). For comparison,
pLXSN vector and cDNA from LMP2A expressing CNE2 and HONE1 were included
as reference (kindly provided by Dr. Yap). LMP2A was expected to have a size of 1568
base pairs when PCR amplified with cloning primer and to have a 152 base pairs short
amplicon when amplified with gene expression primer. As shown in Figure 3.20, full
length of LMP2A was expressed in all the cells except HONE1 cells whereas short
amplicons of LMP2A were found in all the cells. The sizes of LMP2A expressed were
shown to be in the expected ranges in Promega DNA ladder. The levels of GAPDH
were consistent among all the cell lines.
For NP460hTert-LMP1, cDNA from two passages of transduced cells (passage 1 and
passage 2) were extracted and PCR amplified with cloning primers and gene expression
primers (Figure 3.21). For comparison, cDNA from LMP1 expressing HONE1 cells
(kindly provided by Dr. Yap) and pLXSN vector were included as reference. LMP1 was
expected to have a size of 1190 base pairs when PCR amplified with cloning primer and
to produce a short amplicon of 160 base pairs when PCR amplified with gene
expression primer. From Figure 3.21, undetectable levels of LMP1 were displayed in all
NP460hTert cells and HONE1 cell. Low level of LMP1 short amplicon was observed in
passage 1 but not in passage 2 of NP460hTert-LMP1. The levels of GAPDH were
consistent among all the cell lines.
80
Figure 3.19: Reverser-transcription PCR analysis of NP460hTert-BARF1 and
NP460hTert-BHRF1. A: Gel images of full length (FL) and short amplicons (Short) for
BARF1 and BHRF1 from PCR amplification with cloning primers and gene expression
primers respectively. B: Gel images of GAPDH amplification for all the cell lines.
81
Figure 3.20: Reverser-transcription PCR analysis of LMP2A. Gel images of full length
(FL) and short amplicons (Short) for LMP2A from PCR amplification with cloning
primers and gene expression primers respectively.
Figure 3.21: Reverser-transcription PCR analysis of LMP1. Gel images of full length
(FL) and short amplicons (Short) for LMP1 from PCR amplification with cloning
primers and gene expression primers respectively.
82
3.7.1 Discussion
From the reverse-transcription analysis of EBV genes expressing NP460hTert, it is
found that BARF1 and BHRF1 were expressed in all the respective transduced
NP460hTert. LMP2A was also expressed in NP460hTert. Interestingly, the reference
for LMP2A, which is the cDNA from HONE1, did not produce full length of LMP2A
but the expression of LMP2A can be detected by PCR amplification with gene
expression primer.
The complete LMP1 was not detected in any of the transduced NP460hTert. However,
the expression of LMP1 was detected in early passage of transduced NP460hTert as a
short amplicon. The expression of LMP1 became undetectable after additional passages.
During the puromycin selection of transduced NP460hTert, only a few NP460hTert-
LMP1 cells were able to survive the selection and further propagated. From these
observations, it is postulated that LMP1 could be cytotoxic to the NP460hTert and
therefore most of the cells which expressed high level of LMP1 died and those with low
levels of LMP1 survived. Ectopic expression of LMP1 at high level was toxic to cells
and induced cell death in the form of apoptosis (Hammerschmidt, Sugden & Baichwal
1989). Several studies have reported that LMP1 could inhibit cell growth in NPC cells
(Liu et al. 2002) and induce cell death in epithelial cells (Lu et al. 1996), monocytes
cells and lymphoblastoid cell line (Brocqueville et al. 2013). Previous study shows two
transformation effectors in C terminal of LMP1 were involved in LMP1 induced
apoptosis (Brocqueville et al. 2013). Low level expression of LMP1 in MDCK cells
allowed them to survive the cytotoxic event and then acquire LMP1 oncogenic
properties such as epithelia-mesenchymal transition phenotype and anti-apoptotic effect.
Contrary to their results, the surviving cells with low level of LMP1 expression in the
present study did not display any LMP1 oncogenic properties such as acquisition of
epithelial mesenchymal transition phenotype or enhanced survival and the LMP1
expression is further silenced at later NP460hTert passage. The cells might suppress the
LMP1 expression through gene regulating mechanism such as DNA methylation in
order to survive. The surviving cells could be those with minimum expression of LMP1
but sufficient expression of the puromycin resistant gene.
83
Thus, NP460hTert-LMP2A, NP460hTert-BARF1 and NP460hTert-BHRF1 were
chosen to further protein analysis with western blot whereas LMP1 was excluded due to
the complication in transduced cells (Figure 3.1).
3.8 Western blot analysis of 3 EBV genes in transduced NP460hTert
Having examination of LMP2A, BARF1 and BHRF1 expression in transduced
NP460hTert, western blot analysis was performed to confirm the findings made from
reverse-transcription PCR analysis (Figure 3.1).
To perform western blot analysis, protein lysates were extracted from three different
passage of NP460hTert-LMP2A (passage 3, passage 4 and passage 6), two different
passage of NP460hTert-BHRF1 (passage 3 and passage 4) and four different passages
of NP460hTert-BARF1 (passage 1, passage 2, passage 3 and passage 4). The proteins
lysates were blotted against anti c-Myc antibody and anti beta-actin antibody. Protein
lysate from vector control was included as reference.
Western blot analysis reveals that BHRF1 protein was detected in between the range of
15 kDa and 20 kDa (Figure 3.22). No LMP2A and BARF1 protein were detected in all
the cell lines (Figure 3.22 and Figure 3.23). The beta-actin references were consistent
among all the cell lines. Multiple unknown protein bands were observed in all the blot
images.
84
Figure 3.22: Western blot of LMP2A and BHRF1 in transduced NP460hTert. A: Western blot detection of LMP2A and BHRF1 with anti c-Myc monoclonal antibody. B: Immunoblot detection of the loading control beta actin by reprobing with anti-beta actin antibody.
85
Figure 3.23: Western blot analysis of BARF1 in transduced NP460hTert. A: Western blot detection of BARF1 with anti c-Myc monoclonal antibody. B: Immunoblot detection of loading control beta actin by reprobing with anti-beta actin antibody.
86
3.8.1 Discussion
Multiple strong bands observed in all the samples including the vector control. The
reason for these nonspecific bindings of anti c-Myc monoclonal antibody (9E10) was
not clear. Several factors in the analysis of protein expression might play a role in this
anomaly. First, it could be due to the amount of protein loaded into the SDS-PAGE.
High concentration of protein samples could induce formation of nonspecific
antibodies-antigen complexes and thus results in multiple bands on immunoblot.
However, testing on different concentrations of protein lysates for SDS-PAGE and
western blot produced the same results. Multiple bands were still observed in
immunoblot even with the lower concentration of protein lysates.
Secondly, the blocking agents might not be efficient in preventing the nonspecific
binding of both primary and secondary antibodies. PVDF membranes have a high
binding affinity for protein and this membrane property will allow the transfer of
proteins from the gel to PVDF membranes during blotting (Gultekin & Heermann 1988).
On the other hand, antibodies will bind to the PVDF membrane as well. Therefore, it is
important to block the unbound surface of the membrane to prevent nonspecific binding
of antibodies. The blocking reagent used in this study was 5% commercial skim milk. In
order to troubleshoot this problem, alternative blocking agents such as different brands
of commercial skim milk and 1% BSA were used. Unfortunately, the attempt of using
different blocking agents to eliminate the multiple bands was to no avail.
In addition to previously mentioned troubleshooting, it is also speculated that anti c-
Myc antibody could be the main reason for multiple bands. The antibody used might not
be specific for only c-Myc epitope but also have cross reaction on immunoblot. Thus,
two different anti c-Myc antibodies were used in an attempt to minimize the background
issues. They were anti c-Myc antibodies 9E10 from SantaCruz and 9B11 from Cell
Signalling Technologies. Regrettably, multiple bands were still observed in the
immunoblot with different anti c-Myc antibodies. Anti c-Myc antibody from SantaCruz
was shown to develop an immunoblot with inferior resolution while the antibody from
Cell Signalling Technologies provided the similar blot images as Figure 3.22 and Figure
3.23.
87
Since the multiple bands were nonspecific binding and they were also found in mock
transduced NP460hTert at different optimization, it was decided that the current
immunoblot was used for the current study of transduced construct.
From the immuonoblot analysis, there were only two distinct bands that can be detected
in all four images and both bands were observed at BHRF1 transduced NP460hTert.
Detectable strong distinct bands were absent in all BARF1 and LMP2A transduced
NP460hTert.
3.8.1.1 BHRF1
BHRF1 was detected in between the range of 15 kDa and 20 kDa (Figure 3.22). This is
smaller than the predicted size of BHRF1 protein. BHRF1 protein is predicted to have a
size of 22 kDa which consists of 192 amino acids. The reduction in BHRF1 protein size
was not due to posttranslational modification but because of the anomalous migration of
BHRF1 protein in SDS-PAGE (Austin et al. 1988; Pearson et al. 1987). BHRF1 protein
is a transmembrane protein which contains a hydrophobic transmembrane domain
located at 166 to 186 amino acids (Khanim et al. 1997). This helical transmembrane
domain enables BHRF1 protein to localize into the mitochondria membrane. The
hydrophobic transmembrane domain will also interact with detergents such as SDS in
SDS-PAGE gel. SDS loading in transmembrane domain was shown to have a positive
correlation with protein PAGE mobility (Rath et al. 2009). Increase in bound SDS
within protein structure will reduce the protein gel migration ability, thus have a slower
shift and higher molecular weight than theoretical protein size (Rath et al. 2009).
Interaction between SDS and BHRF1 protein transmembrane domain could have altered
its hydropathy states which changes the SDS partitioning within the transmembrane
domain and leads to loss of SDS aggregation thus resulting in faster than predicted
protein gel shift.
3.8.1.2 LMP2A
LMP2A was not detectable in three different passages of transduced NP460hTert
(Figure 3.22). The LMP2A transcript was however confirmed in the transduced
NP460hTert with RT-PCR. There were several possibilities for this observation such as
the LMP2A protein losses its c-Myc tag during maturation, c-Myc tag was masked after
88
posttranslational modification or the mature LMP2A was degraded by unknown cell
mechanism. After extensive literature review, it is concluded that the LMP2A was in
fact ubiquitinated and was either secreted in exosomes or digested in endolysosome
expressing cells. The mitogenic activity of BARF1 was consistent with previous studies,
which stated that the expression of BARF1 induced cell proliferation in human
epithelial cells, gastric carcinoma and NPC (Sakka et al. 2013; Sall et al. 2004; Seto et
al. 2005). This study further expanded the understanding regarding BARF1 by
demonstrating that ectopic expression of BARF1 could upregulate the cell proliferation
of nasopharyngeal epithelial cells.
Although studies have addressed the anti-apoptotic effects of BHRF1 in different cell
lines, mitogenic roles of BHRF1 in epithelial cells were in contradiction. BHRF1
expression was shown to promote cell proliferation in human squamous cell carcinoma
but reduced the NPC cell proliferative rate. In this regard, the data from this study
provided additional insight on the proliferative activity of BHRF1 that might be
dependent on the nature of cells. For instant, the data from this study showed that the
proliferative activity of BHRF1 in non-tumorigenic nasopharyngeal epithelial cells was
similar to non-tumorigenic human squamous cell carcinoma but totally opposite to
malignant NPC cells. In addition, possible HeLa contamination might have unforeseen
effects on BHRF1 proliferation in NPC cells.
In contrast with the effect of the increase of cell proliferation, BHRF1 expression did
not increase cell migration in NP460hTert. Similar to mitogenic role of BHRF1,
BHRF1 migration capability was not well reported in previous studies. That BHRF1
promote cell proliferation rather than cell migration suggests that it may only affect
117
proliferation signalling pathway but not migration signalling pathway. The interaction
between BHRF1 and cell proliferation regulator is clearly an area for future study. On
the other hand, it was observed that BARF1 expressing nasopharyngeal epithelial cells
closed its wound faster than the vector control. In the past, most studies only described
BARF1 as a strong mitogenic agent and an earlier report showed that BARF1 did not
improve the cell migration of gastric carcinoma cells (Chang et al. 2013). Our
observation suggested that BARF1 could induce both cell migration and proliferation in
nasopharyngeal epithelial cells. Interestingly, this discrepancy might indicate that
BARF1 expression may have different biological effects depending on cell types.
Furthermore, the results in this study suggested that the cell migration induced by
BARF1 was independent from cell proliferation.
The increased cell proliferation and migration observed in BARF1 expressing
nasopharyngeal epithelial cells might be contributed by secreted BARF1. In order to
verify the autocrine or paracrine activity of secreted BARF1 on cell proliferation and
migration, the parental nasopharyngeal epithelial cells were cultivated in conditioned
medium generated from either BARF1 expressing cells or the vector control.
Interestingly, parental nasopharyngeal epithelial cells with BARF1 conditioned medium
appeared to have greater proliferation rate and faster migration than cells in conditioned
medium from the vector control. These findings indicated that secreted form of BARF1
could be produced from BARF1 expressing nasopharyngeal epithelial cells and could
promote both proliferation and migration in parental cells. The findings also
demonstrated that secreted BARF1 have mitogenic activity that in comparison to
growth supplement found in complete medium for nasopharyngeal epithelial cells. The
precise signalling pathways involved in BARF1 related cell proliferation and migration
remains to be elucidated.
4.2 Technical conclusions
Recombinant DNA technology has revolutionized the molecular science by providing
tools that allows us to understand the structures, expressions and functions of genes
(Pasternak 2005). It also has a profound impact in medical science through the
understanding of the molecular basis of genetic disorder and development of new
therapeutic strategies (Khan et al. 2016). Generation of recombinant DNA typically
involved extraction of DNA from source organism, then digesting the DNA with
118
restriction enzyme and finally ligation of the DNA to specific designed vector. In the
present study, the isolation of targeted EBV genes from EBV infected nasopharyngeal
epithelial cells, the generation of four transduced NP460hTert cell lines including vector
control and the determination of the molecular effects of three EBV genes were made
possible with the help of recombinant DNA technology. The novelty of this project lies
in its use of mammalian cell culture and to perform cloning of DNA constructs. Several
adaptations, optimizations and changes were made in order to overcome the challenges
faced to accomplish the objectives of this study.
In this study, it was demonstrated that specific primers could be designed to target and
amplify genes of interest based on the cDNA sequences found in GenBank database.
The functional cDNA must first be synthesised from source mRNA to exclude all the
introns from the gene. Furthermore, several primer modifications such as a tag,
restriction sites and the Kozak sequence could be added into the primers depending on
the objectives of current study. These modifications wrere added to the cDNA during
PCR amplification. Kozak sequence, added to the 5’ end of cDNA, was incorporated to
initiate the translation and to improve the translation efficiency. Since innate cDNA
lacked restriction sites, two restriction sites were added to flank both ends of the cDNA.
The restriction sites added could facilitate the cloning of cDNA into vector or shuttle
the cDNA across different vectors.
The c-Myc epitope tag sequence was placed in between Kozak sequence and forward
primers sequence. The choice of the tag placement has an important impact on
experimental outcome. Initially, the objective was to integrate a similar tag in every
EBV genes and use only one antibody for the entire detection assay. This design has a
few advantages such as a streamlined detection process with only one antibody and
reducing the expenses from purchasing multiple costly antibodies. However, there are
some underlying issues that were beyond our considerations during the design of the tag
system. First, protein stability is regulated by the microenvironment of its amino
terminal. Addition of a tag in the amino terminal of protein might block the
ubiquitination of protein and prevent the subsequent degradation or induce a rapid
unbuqitination of the tagged protein. In this study, the addition of c-Myc tag at the
amino terminal end of LMP2A had dramatically increased the LMP2A ubiquitination
and resulted in establishment of inadequate LMP2A expression in the NP460hTert cell
119
line. Secondly, the tag could be subjected to proteolytic cleavage during post-
translational modification of secreted protein. The cleavage of amino terminal
methionine and specific signal peptide is important for maturation and secretion of
protein (Rogers & Overall 2013). As a secreted protein, the removal of first twenty
amino acids is essential for maturation and secretion of BARF1. Because of this
cleavage event, the amino terminal c-Myc tag was removed during the secretion of
BARF1 and the detection of secreted BARF1 using antibody was hindered in the
conditioned medium from NP460hTert-BARF1. Therefore, comprehensive and
thorough understanding of protein biosynthesis is recommended for designing the
cloning primers.
The cDNA generated were incorporated into the cloning vectors through restriction
digestion and ligation. The cDNA was first cloned into mammalian expression vector
pcDNA 3.1 Hygro and then shuttled into lentiviral vector pLVX-Puro. The host
independent self-replicating attribute of cloning vectors allowed us to perpetuate the
insert by transforming the recombinant DNA into bacterial host cells. In addition,
cloning vectors have few selectable markers that enable the isolation of host cells with
proper DNA construct. Escherichia coli has been developed as the universal bacterial
host for cloning of recombinant DNA (Hanahan 1983). However, the process when
recombinant DNA was introduced into E. coli, transformation, is extremely inefficient.
A competent state of E. coli must be first induced before the uptake of exogenous DNA
through transformation. In the present study, E. coli strain TOP10F’ was induced to a
highly competent state for transformation through chemical induction. The utilization of
manganese, magnesium and potassium in addition to calcium was able to produce
competent TOP10F’ with higher transformation efficiency. Furthermore, this project
was able to generate highly competent TOP10F’ consistently among different batches of
competent cells by using similar chemical treatments.
Multiple DNA sequence mutations were observed in the recombinant DNA produced in
this study. A few of the mutations were likely introduced during the PCR amplification
while one of the mutations was incorporated unintentionally. High fidelity DNA
synthesis was an important technique for cloning of the targeted gene with superb
accuracy. Precise DNA sequences are required for in vitro synthesis of functional
protein from gene of interest. However, PCR cloning is susceptible to PCR generated
120
errors especially during the cloning of large pool DNA templates with low fidelity
polymerase. Therefore, utilising high fidelity polymerase that contains proofreading
exonuclease is crucial to synthesise the DNA free from mismatched nucleotides. This
would allow to overcome the challenges arising from PCR larger templates and to
generate error free EBV cDNA.
The nasopharyngeal epithelial cells NP460hTert was the only cell line used in the
present study. NP460hTert was established from primary non-malignant
nasopharyngeal biopsies and immortalized with overexpression of human telomerase
(Li et al. 2006b). NP460hTert harbours several genetic alterations such as inactivation
of p16 and RASSF1A that has been previously identified in premalignant
nasopharyngeal epithelial cells (Lo, To & Huang 2004). Furthermore, due to the lack of
representative models, previous studies of EBV infection in epithelial cells were mainly
conducted in established cancer cell lines (Tsang et al. 2010). Thus, NP460hTert was
considered as an excellenct model for the investigation of EBV involvement in the
pathogenesis of NPC.
Lentiviral vectors have become one of the most widely used vectors for transgene
expression due to their relative broad range of tropism coupled with the ability to infect
both diving and non-dividing cells (Merten, Hebben & Bovolenta 2016). By using
second generation lentiviral system, this study successfully transduced nasopharyngeal
epithelial cells NP460hTert and generated five different clones including the vector
control. Thus, this study demonstrated that lentiviral system was albe to transduce
nasopharyngeal epithelial cells efficiently, producing cells with stable expressed gene of
interest, and without any complication.
121
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