Identification and characterization of IFI30 as a glioblastoma specific promoter for glioma gene therapy

IDENTIFICATION AND CHARACTERIZATION OF IFI30 AS A GLIOBLASTOMA SPECIFIC PROMOTER FOR USE IN GLIOMA GENE THERAPY MADHUMITHA RENGASAMY [MSc.. Promoter activity was studied through lucife

IDENTIFICATION AND CHARACTERIZATION OF

IFI30 AS A GLIOBLASTOMA SPECIFIC PROMOTER FOR USE

IN GLIOMA GENE THERAPY

MADHUMITHA RENGASAMY [MSc Microbiology]

A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE

DEPT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2009

I would like to thank my co-supervisor Dr Jerome Boulaire for his support and encouragement I would also like to thank Dr Poonam Balani for being a constant source of motivation I would like to thank Farah for help with the initial experiments Special thanks to all the members in my lab for their willingness to discuss my work and teaching me various experimental techniques

I would like to thank my friends Aparna, Chrishan, Mohammad, Sriram, Yukti and Ghayathri for being there for me

Table of contents

Acknowledgements I

Table of Contents II

Summary V

List of Tables VII

List of Figures VIII

Abbreviations X

1 Introduction 2

1.1 Glioma 2

1.1.1 Characteristics of Glioma 2

1.1.2 Existing therapies for Glioblastoma 3

1.2 Gene therapy for GBM 4

1.3 Transcriptional Targeting 5

1.3.1 Tissue-specific promoters 7

1.3.2 Tumour-specific promoters 9

1.4 Vectors for Gene Therapy 14

1.4.1 Baculovirus as a vector for Gene Therapy 17

1.5 Aim of the Study 19

2 Materials and Methods 21

2.1 Cell Culture and Tissue samples 21

2.2 Microarray and Real -Time PCR analyses 22

2.3 Cloning of the IFI30 promoter 23

2.3.1 PCR amplification of IFI30 promoter from Genomic DNA 23

2.3.2 Cloning into TOPO and pGL4.11 vectors 24

2.3.3 Cloning of the deletion variants of IFI30 26

2.3.4 Cloning of the 0.68 kb IFI30 promoter into pFastBac Dual vector 28

2.4 Preparation and Amplification of Recombinant Baculovirus 32

2.5 Luciferase Assays 33

2.6 Cell Viability Assays 34

2.7 Western Blot Analysis 35

3 Results 37

3.1 Data Mining 37

3.2 Microarray and Real Time PCR results 39

3.3 Cloning of the IFI30 promoter 42

3.4 Luciferase assays …… 44

3.5 Cell viability assays………… 47

3.6 Western Blot Analysis 52

4 Discussion 54

4.1 Interferon Gamma-Inducible protein 30 54

4.2 IFI30 and Cancer 56

4.3 Characterization of the IFI30 promoter 58

4.3.1 Analysis of the promoter sequence 58

4.3.2 Promoter activity in different cell lines 60

4.3.3 Transcriptional Targeting 63

4.3.4 Western Blot Analysis 66

5 Conclusion 70

6 References 73

Summary

Transcriptional targeting involves the use of tissue-specific or specific promoters to drive gene expression selectively in cancer cells Though substantial work has been done in transcriptional targeting of hepatoma and breast cancer, little work has been done in the characterization of a glioblastoma specific promoter

tumour-This study deals with the identification, isolation and characterization of a glioblastoma specific promoter for suicide gene therapy The gene coding for IFI30, interferon gamma‐inducible protein 30 (also known as GILT) was found to be up regulated in glioblastoma with respect to normal brain tissue through Microarray and Real Time PCR analyses

The 2 kb region upstream of the transcriptional start site of IFI30 was then taken to check for promoter activity Promoter activity was studied through luciferase assays in glioma cell lines, normal human astrocytes and other cell lines including breast and cervical cancer cell lines Deletion analysis was carried out to find the shortest fragment of the promoter that showed high activity in glioma cell lines and minimal activity in normal human astrocytes

The 0.68 kb fragment of the IFI30 promoter was chosen to drive

expression of the suicide gene-Herpes Simplex Virus Thymidine kinase (HSV-Tk) This IFI30-HSVTk construct was used in the generation of recombinant baculoviruses (BV-IFI30-HSVTk) U87 (glioma cell line) and NHA (normal human astrocytes) cells were transduced with BV-IFI30-HSVTk and subjected to ganciclovir (GCV) treatment Thirty-six hours after the addition of 50μM GCV, BV-IFI30-HSVTk was able to kill 67% of the U87 cells with minimal toxicity to NHA To confirm that cell death was due to expression of the HSV-Tk protein, a western blot assay was performed using whole cell protein from transduced NHA and U87 cells

As expected, there was high expression of the HSV-Tk protein in U87 cells and minimal expression in NHA

This is the first study that characterizes the use of the IFI30 promoter for transcriptional targeting in glioma and paves way for further research in the use of this promoter for in-vivo therapy

Table 2.4- PCR conditions for cloning the IFI30 promoter

(0.68 kb) in pFastBac Dual vector 29

Table 3.1- Results of Microarray showing up regulation of

IFI30 in different glioma cell lines 40

List of figures

Figure 2.1- Schematic representation of the pGL4.11

plasmid constructs 30

Figure 2.2- Schematic representation of the pFastBac Dual plasmid constructs 31

Figure 3.1- Gene Expression data set GDS2728………37

Figure 3.2- Gene expression data sets GDS1813, GDS1815, GDS1962 ………… ……… 38

Figure 3.3- Microarray analysis showing genes over expressed in glioma with respect to NHA 40

Figure 3.4- Real Time PCR quantification of IFI30 mRNA levels 42

Figure 3.5- Agarose gel photographs of IFI30 clones 43

Figure 3.6- IFI30 promoter activity in glioma cell lines 45

Figure 3.7- IFI30 promoter activity in other cell lines 46

Figure 3.8- Cell survival plots and images of NHA (24h after GCV treatment) 48

Figure 3.9- Cell survival plots and images of U87 (24h after GCV treatment) 49

List of figures(Contd.)

Figure 3.10- Cell survival plots of NHA and

U87 (36h after GCV treatment) 50

Figure 3.11- Western Blot Analysis 52  

Figure 4.1- Sequence of the Interferon-gamma

inducible protein 30……… ……… …54  

Abbreviations

AcMNPV Autographa californica nuclear polyhedrosis virus CMV Cytomegalovirus

DMEM Dulbecco’s modified Eagle’s Medium

FBS Fetal bovine serum

EGFP Emerald Green fluorescent protein

GCV Ganciclovir

HMGB2 High mobility group protein

HSV Herpes simplex virus

IFI30 Interferon gamma inducible protein

NHA Normal Human Astrocytes

PBS Phosphate buffered saline

NCBI National Center for Biotechnology Information

CHAPTER I INTRODUCTION

et al., 2006)

patients with glioma is often very poor (only ~2% of patients aged 65 years or older and only 30% of those under the age of 45 years at GM diagnosis survive for 2 years or more) Despite surgery, radiotherapy and/or chemotherapy, death results in 80% of patients from tumour recurrence within 6–12 months In this report the high grade gliomas are collectively referred  to  as  “glioblastoma”  or 

“GBM”

1.1.2 Existing therapies for Glioblastoma

The characteristic resistance to treatment shown by GBM resides in the fact that the cells undergo constant genotypic and phenotypic changes and are highly

infiltrative in nature (Sonabend et al., 2007) The effect of surgery is

compromised due to the lack of a defined tumour edge and its proximity to vital anatomical structures Glial tumour cells intersperse among the surrounding normal brain parenchyma and cause the recurrence of tumour within the surgical

site (Giese et al., 1996)

Normal tissues in the brain can tolerate up to 60 Gy of radiation However this may be not be enough to kill all the GBM cells and hence the risk of residual tumour cells is high Stereotactic radio surgery or radiotherapy, interstitial radiotherapy and boron neutron capture therapy that attempt to enhance the effect of radiotherapy have not provided significant improvements GBM tends to

be chemoresistant partly due to the blood-brain barrier which may act as a physical barrier or operate different efflux pumps hindering the transport of chemotherapeutic agents into the CNS The main aim of chemotherapy is to control tumour growth and maintain satisfactory performance of patients for as long as possible The drugs procarbazine, lomustine, and vincristine and temozolomide have demonstrated significant prolongation of survival Generally there is no curative therapy for GBM and long-term control is rarely achieved with current therapies (Pulkkanen and Yla-Herttuala, 2005)

1.2 Gene Therapy for GBM

Although gliomas show extensive infiltration within the brain, they are regarded

as a local lesion within the Central Nervous System (CNS) as metastatic spread

to other organs is rarely seen These unique features of GBM such as the presence of mitotically active tumour cells in an essentially post mitotic background and absence of metastases outside the central nervous system make it an ideal target for selective gene therapy As gene therapy for GBM is localised there is minimum risk of systemic toxicity (Pulkkanen and Yla-Herttuala, 2005)

Gene therapy is the transfer of exogenous genes, called transgenes, into somatic cells of a patient to obtain a therapeutic effect Initially gene therapy was considered as an approach for treating hereditary diseases, but it's potential in the treatment of acquired diseases like cancer is now widely recognised (Robson

et al., 2003)

Ignoring the innate resistance of GBM to chemotherapy and radiotherapy, there

is a narrow therapeutic index (TI) of the existing treatments Therapeutic index is the chance of a beneficial outcome compared to the risk of a serious adverse event For surgical resection and radical radiotherapy, the TI is dictated by the need to preserve vital adjacent normal structures, whereas for chemotherapy, it

is governed by systemic treatment related toxicity Therefore the development of gene therapy is seen as a more efficient replacement as it is based on the

understanding of the molecular biology of the disease It is expected that toxicity would be reduced and TI will be increased as a result of the precise targeting of

the therapy (Harrington et al., 2000)

1.3 Transcriptional targeting

With a few exceptions, every cell of the human body contains a complete copy of the genome It is through the regulation of gene expression that the genotypically homogenous cells show phenotypic heterogeneity Cancer cells transcriptionally activate many genes that are important for uncontrolled proliferation The transcriptional machinery in tumour cells usually consists of increased expression/activity of transcription factors Transcriptional targeting of cancer cells aims at using tissue-specific or tumour-specific promoters that allows the selective expression of the transgene in the tumour cells while the normal cells remain relatively unaffected

This in particular is relevant for cancer suicide gene therapy in which the therapeutic gene is toxic or encodes an enzyme which converts an inactive prodrug into a toxic metabolite Ideally, cancer specific killing can be achieved by delivering a therapeutic gene under the control of the DNA elements that can be activated by transcription factors that are over expressed and/or constitutively

activated in cancer cells (Lo et al, 2005)

The most widely used suicide gene transfer system in preclinical investigations,

as well as human clinical trials, is the one based on herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV-1-Tk/GCV) HSV-1-Tk encodes a viral enzyme that is foreign for human cells and is able to convert the inactive and relatively less toxic prodrug ganciclovir (GCV) into its mono phosphate form Herpes simplex thymidine kinase (Tk)  has  significant  affinity  for  GCV  while  normal cellular thymidine kinase does not, and thus the drug is preferentially

phosphorylated in cells expressing the viral gene (Ishii-Morita et al., 1997)

Intracellular host kinases metabolize this GCV monophosphate into di- and triphosphates The GCV triphosphate can be incorporated into dividing cells and inhibits DNA polymerase, which results in chain termination, termination of DNA

synthesis and cell death (Fillat et al., 2003) The phosphorylated GCV can move

to neighbouring cells via gap junctions and cause cell death of nearby cells This property of enzyme/prodrug systems is referred to as "bystander effect" which refers to the death of unmodified dividing tumour cells adjacent to genetically

modified cells (Dilber et al, 1997)

To achieve tumour specific expression, cancer specific vectors are generally composed  of  promoters,  enhancers,  and/or  5′-UTR that are responsive to

tumour-specific transcription factors (Lo et al., 2005) Promoter elements that

have been used in cancer gene therapy can be broadly classified as specific and tumour specific promoters

tissue-1.3.1 Tissue-specific promoters

Tissue specific promoters (TSP) should have some desired characteristics for their use in cancer therapy Firstly, the TSP must have high activity restricted to the target tissue Secondly, as low level of activity is expected in normal tissue, the tissue should be expendable, replaceable or located far away from the site of gene delivery/expression For example, killing of melanocytes or prostrate cells can be tolerated but killing of liver or neuronal cells would lead to disastrous

consequences (Harrington et al., 2000)

The glial fibrillary acidic protein (GFAP) promoter is a tissue-specific promoter which has been used for transcriptional targeting in GBM The GFAP gene is highly expressed, almost exclusively, in astrocytes and the protein is found in

high levels in many glioma cell lines and brain tumours (Yung et al., 1985)

Studies in cell culture and transgenic mice have demonstrated that a 2.2 kb

5'-flanking region of the GFAP gene, gfa2, is sufficient to mediate astrocyte specificity (Brenner et al., 1994) Transfection of gfa2-HSV-Tk construct into

GFAP positive glioma cell line and GFAP negative ovarian cancer cell line showed selective toxicity in glioma cell line on treatment with GCV This restricted expression of the HSV-Tk gene has the advantage that the HSV-Tk gene is expressed in GFAP-positive infiltrating tumour cells, with reduced toxicity

to the surrounding normal cells (Vandier et al., 1998) Another advantage of

restricted expression of the suicide gene is the use of general delivery systems

Retroviruses are used for tumour-specific delivery of therapeutic genes as they transduce only dividing cells but they have the disadvantage of low titres and

poor in-vivo transduction efficiency (Harsh et al., 2000) Especially in GBM which

has a heterogeneous tumour population and the number of mitotically active cells varies, the transduction efficiency is low as retroviruses transduce only dividing

cells (Rainov et al., 2000) In contrast other viral vectors like adenoviruses and

baculoviruses can be produced with high titres and they transduce both dividing

and non-dividing cells (Kost et al., 2005, Pulkkanen and Yla-Herttuala, 2005)

Recombinant adenoviruses engineered to express the HSV-Tk gene under the

control of gfa2 promoter (Adgfa2Tk), was found to cause inhibition of glioma cells in-vitro and in-vivo (Vandier et al., 2000)

Tissue-specific promoters have the disadvantage that a low level of transgene expression would occur in the normal tissue as well Although normal astrocytes would also express gfa2-HSVTk, their low rate of cell division would render them relatively immune to GCV treatment Adult mice carrying gfa2-Tk transgene have been shown to be insensitive to high doses of GCV as astrocyte mitosis is rare in

adult cerebellum (Delaney et al., 1996)

However a subsequent in-vivo study showed that subcutaneous GCV delivery to uninjured adult transgenic mice genetically engineered to express HSV-Tk under the control of the GFAP promoter, proved fatal within 19 days in the absence of CNS pathology Subsequent examination of vital peripheral organs by RT-PCR

screening for expression of transgene derived HSV-Tk revealed expression in gut, heart, lung, liver, kidney, adrenal gland and spleen It was also shown that HSV-Tk caused depletion of the enteric glia and this resulted in severe

inflammation and hemorrhagic necrosis of the jejunum and ileum (Bush et al.,

1998)

1.3.2 Tumour-specific promoters

There is no clear distinction between tissue-specific and tumour-specific promoters Tumour-specific promoters can be broadly defined as promoters that are highly active in tumour cells but show little or no activity in normal tissues Tumour-specific promoters can be further classified into different types Promoters which are specific for the malignant process, but which do not have any tissue specificity are referred to as "cancer-specific promoters" Promoters of genes which encode oncofetal antigens and have well defined pattern of tissue specificity are referred to as "tumour-type specific promoters" while there other promoters that are up regulated due to the pathophysiological conditions associated with regions of tumour formation (e.g hypoxia responsive promoters) Lastly, there are promoters specific to the tumour vascular endothelium

Cancer specific genes are up regulated among different tumours in a specific manner A typical example of a cancer specific gene would be that of telomerase Telomerase is not expressed by normal tissues (except germ cells

non-and stem cells) but is reactivated in all major types of cancer Telomerase enables tumour cells to maintain telomere length hence circumventing the process of senescence The use of the promoter of human telomerase reverse transcriptase gene (hTERT) for cancer therapy is attractive as it can be used to target therapeutic genes to a wide variety of malignant tissues Telomerase-specific replicative adenovirus has been developed as oncolytic virus (Telomelysin, OBP-301) that replicates selectively in cancer cells and causes cell death via viral toxicity However, systemic toxicity is a concern in this treatment modality as telomerase activity has been reported to exist in some normal cells, such as hematopoietic crypt and endometrial cells which have high regenerative

potentials (Kyo et al., 2008)

Another gene up regulated in a cancer -specific manner is midkine Midkine is a heparin-binding growth factor that is induced by retinoic acid in embryonal carcinoma cells It has diverse biological roles including in neural development

(Li et al., 1990) and pathogenesis of neurodegenerative diseases Midkine is

involved in the development of cancer because of its mitogenic effect, promotion

of angiogenesis, anti-apoptotic activity (Qi et al., 2000) and transforming activity (Kadomatsu et al., 1997) Midkine expression is increased in a number of

malignant tumours including oesophageal, stomach, colon, hepatocellular, breast and pancreatic carcinoma when compared with the level in non-cancerous

tissues (Tsutsui et al., 1993, Toyoda et al., 2008) In contrast, the expression of

midkine in normal human tissues is limited to moderate expression in the kidneys

and low expression in the lungs, colon and thyroid gland (Aridome et al., 1995, Muramatsu et al., 1993) Midkine promoter based conditionally replicative

adenovirus therapy has been tested for its efficacy in the treatment of pancreatic

cancer, bladder cancer and malignant glioma (Toyoda et al., 2008, Kohno et al.,

2004)

With respect to transcriptional targeting in GBM, apart from GFAP the other widely studied promoter is that of the survivin gene Survivin is a member of the Inhibitor of Apoptosis (IAP) protein family and plays a vital role in the survival of cancer cells and the progression of malignancy Survivin is normally expressed during embryogenesis, is undetectable in fully differentiated adult tissues and is over expressed among a wide range of cancers including cancers of the oesophagus, pancreas, bladder, central nervous system, lung, prostrate gland,

breast, uterus, melanoma and soft tissue sarcomas (Fukuda et al., 2006)

Survivin is considered an attractive target because of it's differential expression in cancer cell versus normal cells and its potential requirement for maintaining cancer cell viability Patients whose tumour expressed survivin had a decreased overall survival, an increased rate of recurrence, resistance to therapy and a reduced apoptotic index in-vivo (Altieri 2003) Survivin-mediated suppression of apoptosis and growth-factor independent cell survival is implicated in the

resistance of the tumour to standard therapy (Van Houdt et al., 2006) In this

regard, 80% of GBM cells demonstrate abundant survivin expression In glioma

tumours, survivin expression is also correlated with resistance to chemo and

radiotherapy and with poor prognosis (Kajiwara et al.,, 2003) Survivin

promoter-based conditionally replicative adenoviruses (CRAds) have been found to

selectively replicate in and kill glioma tumour cells in-vitro and in-vivo (Van Houdt

et al., 2006) A comparison between midkine and survivin promoters for GBM

specificity revealed that the survivin promoter was superior in it's enhanced

activity in glioma cells and low expression in normal brain (Ulasov et al., 2007)

However recent studies show that survivin levels in normal tissues can be up regulated by cytokines indicating that survivin may have a physiological role in regulating proliferation and survival Survivin expression has been reported in liver, endometrium, breast, gastrointestinal mucosa, lung, neutrophils and brain among other adult tissues In the brain, survivin expression has been observed in neuronal precursor cells, neurons, astrocytes, oligodendrocytes, ependymal cells

and in the choroid plexus (Fukuda et al., 2006) The role of survivin in the neural

precursors in the adult brain and its impact in the regulation of adult

neurogenesis is under investigation (Pennartz et al., 2004) Hence transcriptional

targeting of GBM using survivin may prove harmful to the normal cells in the CNS

Recently, members of our lab published the results of characterization of a glioblastoma specific promoter, a 0.5 kb fragment of the human high mobility

group box2 (HMGB2) promoter (Balani et al., 2009) This promoter was found to

be effective in driving transgene expression in glioblastoma cells while showing negligible activity in human neurons and normal human astrocytes Recombinant baculoviral constructs expressing HSV-Tk under the control of HMGB2 promoter were found to be effective in killing glioma cells in-vitro and in-vivo

1.4 Vectors for Gene Therapy

Vectors which are used to carry the transgene into the tumour cells can be viral

or non-viral in nature Viral vectors used in gene therapy can be replicative or non-replicative

The most commonly used non-replicative viruses for GBM gene therapy are replication deficient retroviruses and adenoviruses Retrovirus vectors are derived primarily from Moloney murine leukemia virus (MoMLV) These are enveloped RNA viruses which can transfer genes to a wide spectrum of dividing cell types Retrovirus-mediated herpes simplex virus type 1 thymidine kinase gene therapy (RV-HSV-Tk) delivered by intra-tumoural injections of viral producing cells (VPC), followed by systemic administration of ganciclovir (GCV) was used for gene therapy of GBM Although initial studies seemed promising

(Ram et al., 1997, Klatzmann et al., 1998), the VPC approach failed because of

poor transduction of tumour cells VPCs themselves do not have any tumour

homing abilities and probably get killed by the GCV (Rainov et al., 2000) As

GBM has a heterogeneous tumour population and the number of mitotically active cells varies, the transduction is low as retroviruses replicate only in dividing cells

Adenoviruses are non-enveloped DNA viruses and there are more than 50 human adenovirus serotypes However, most of the recombinant adenoviral vectors used in gene therapy application are based on serotypes 2 and 5

Studies using replication-deficient adenoviral mediated HSVTk (Ad.HSVTk) gene

therapy (Sandmair et al 2000, Immonen et al 2004) showed that it caused a

significant increase in the mean survival time of patients However in an earlier study involving glioblastoma model in Lewis rats it was shown that although Ad.HSVTk mediated gene therapy was very efficient, the brains of long-term survivors showed the presence of chronically active brain inflammation, strong and widespread HSV-1-Tk immunoreactivity and loss of myelinated fibres and

axons (Dewey et al., 1999) In a study involving Phase I trial of Ad.HSVTk mediated gene therapy (Trask et al., 2000), pronounced CNS toxicity was

observed in patients who received the highest dose of virus

Oncolytic viral therapy is based on the concept of using live viruses that selectively infect and replicate in cancer cells with minimal destruction of non-cancerous cells Although safety of intra-tumoural injections of oncolytic viruses has been demonstrated, significant therapeutic efficiency remains to be

established (Forsyth et al., 2008)

Non-viral gene delivery systems include naked DNA, cationic lipids poly cationic polymers and cellular vectors The most commonly used is cellular vectors Neural and mesenchymal stem/progenitor cells, isolated from various tissue sources, possess a remarkable inherent tumour tropism which would prove to be

an advantage while targeting infiltrating tumour cells A study in nude mice however showed that NSCs localized to the ventricular compartments in the

brain in the absence of observable tumour burden (Tyler et al., 2009) In a recent

study a patient who received neural stem cell therapy developed tumours in his

brain and spinal cord (Amariglio et al., 2009) This underscores the need to study

and properly describe the molecular mechanisms by which NSCs migrate through the brain parenchyma in response to tumour pathophysiology

1.4.1 Baculovirus as a vector for gene therapy

There is an increasing use of the insect baculovirus Autographa californica

multiple nucleopolyhedrovirus (AcMNPV)-based vectors as gene delivery vectors

for mammalian cells (Kost et al., 2005) The baculovirus/insect cell system is very

popular for the production for recombinant proteins The first successful transduction of mammalian cells with recombinant baculoviruses was reported in

hepatocytes (Hofmann et al., 1995) and it was shown that the endocytotic

pathway was essential in the uptake of baculovirus by the cells Subsequent studies showed that baculovirus was capable of transducing a wide variety of

human cell types including keratinocytes (Condreay et al 1999), primary neural cells (Sarkis et al., 2000), embryonic stem cells (Zeng et al., 2007) and cancer

cells like HeLa, HepG2 (Boyce and Bucher, 1996) It has been shown to have high transduction efficiency in glioma cells transducing up to 98% of the cells

(Wang et al., 2006)

Baculovirus (BV) mediated gene delivery approach has the advantage that the virus does not replicate within mammalian cells and baculoviral infection does not result in the expression of any viral genes Although certain sequences in BV genome could function as promoters or enhancers, they remain silent in mammalian cells due to the absence of supporting transcription factors Thus it is less likely that the cell type specificity of a mammalian promoter would be

affected (Liu et al., 2006) Having a large 130 kb genome, baculovirus AcMNPV

has a large cloning capacity and can be used to transfer a large functional gene

or multiple genes Other advantages of baculovirus include the ease of construction of recombinant viral vector, simple procedure for purification of large volumes of high titre viruses and the virtual absence of cytotoxicity even at a high

multiplicity of infection( MOI) (Zheng et al., 2007)

1.5 Aim of the study

The IFI30 (Interferon gamma-inducible protein) gene has been shown to be over

expressed in glioblastoma (Rhodes et al., 2004) In this study, over expression of

the IFI30 gene was shown by microarray analysis and verified by Real Time PCR The 5'-flanking region of the IFI30 gene was taken for characterization for use as a glioblastoma specific promoter

Deletion analysis of the promoter was done to check for the smallest length of the promoter fragment that showed high activity and retained specificity for glioma cell lines This shortened promoter fragment was then cloned upstream of the HSV-Tk gene and used in the generation of recombinant baculoviruses This recombinant baculovirus (BV-IFI30-HSVTk) was then used to transduce glioma cells and normal human astrocytes to check for selective toxicity A western blot assay was performed to check the expression levels of the HSV-Tk protein in normal human astrocytes and glioma cells

CHAPTER II MATERIALS AND METHODS

2 Materials and Methods

2.1 Cell culture and Tissue samples

The cells used in this study include the human glioblastoma cell lines: U87 cell line from American Type Culture Collection (ATCC, VA) and U251 cell line from the Chinese Academy of Science (Shanghai, China) The other cancer cell lines HeLa (human cervical cancer cell line) and MCF-7 (breast cancer cell line) were obtained from ATCC Normal Human Astrocytes (NHA) was obtained from Clonetics Primary Cell Systems (Lonza, Switzerland) Human Embryonic Stem cells HES-3 (NIH code:ES03) was obtained from ES Cell International (Singapore) The neural stem cell lines NSC-1 and NSC-3 were derived from HES-1 (NIH code: ES01) and HES-3 by other members in the lab

The tumour cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), 1% penicillin- streptomycin and 1% L-glutamine at 37°C, 5% CO2 Normal Human Astrocytes were cultured in Astrocyte Basal Medium supplemented with AGM SingleQuots (Lonza, Switzerland) at 37°C, 5% CO2 HES-1 was maintained on mitotically inactivated mouse embryonic fibroblasts from mouse strain CF-1 and NSC-1 was maintained in DMEM/F12 (1:1) supplemented with bFGF, hEGF, penicillin- streptomycin and L-glutamine

For the Real Time PCR analysis, the primary glioblastoma tissue samples 65482A1 (OCT embedded, malignant glioma), 77696A2 (fresh-frozen glioblastoma), 113246A1 (fresh-frozen astrocytoma) and 77672A1 (fresh-frozen astrocytoma) were obtained from Asterand (Detroit, MI) The formalin fixed paraffin embedded tissue samples Glioblastoma FFPE-133727 and Glioblastoma FFPE-126038-41 were obtained from Capital Biosciences (Gaithersburg, MD)

The microarray data was gathered by Dr Jerome Boulaire (IBN) The Affymetrix Gene Chip Array System (Affymetrix, CA) was used to obtain the expression profiles of the human sourced glioblastoma cell lines- H4, SW1088, SW1783, U251, U87MG,U118, U138, T98G, A172 and CCF-ST and NHA In order to achieve a manageable number of target genes from a list of 5600 entries, filters were used to sieve out genes whose expression was at least 2-fold higher in glioma cell lines compared to NHA (with a p-value of <0.01), or those which were expressed in U251 but not expressed in NHA

Total RNA was extracted using RNEasy Mini Kits (Qiagen, CA) 500ng of total RNA was used for reverse transcription reactions to generate complementary DNA (cDNA) Complementary DNA was generated using the SuperScript III First-Strand Synthesis System (Invitrogen, CA) for RT-PCR using Oligo dT

primers For Real Time quantitative PCR, 1:100 dilution of cDNA was used to set

up a 25-μl reaction  using  RT2 SYBR green qPCR master mix (Superarray) and around 200nM of each primer The primers for IFI30 were designed so as to amplify a 139 bp fragment in the IFI30 gene β-actin was used as an internal control The primers were custom made from Superarray (SABiosciences, MD)

The  Real  Time  PCR  results  were  normalized  to  the  internal  β-actin control Relative quantification of the mRNA expression was done with respect to the normal human brain The results were expressed in terms of fold change in expression with respect to the control and was calculated using the mathematical expression 2−ΔΔCt as described (Livak et al., 2001)

2.3 Cloning of the IFI30 promoter

2.3.1 PCR amplification of IFI30 promoter from genomic DNA

Genomic DNA was extracted from U87 cells for PCR amplification of putative promoter region U87 cells were cultured (till ~90% confluency) in 10cm dishes (Nunc, Denmark) and Qiagen DNeasy Tissue Kit (Qiagen, Hamburg) was used

to extract the genomic DNA Primer3 program was used to design relevant primers for the PCR amplification The first "ATG" or possible start codon after the transcription start site (TSS) in the 5'-untranslated region was not included in the promoter region to be amplified The AccuPrime Supermix kit (Invitrogen, CA) was used to carry out the PCR and the PCR product was analyzed on a 0.9%

agarose gel containing 0.1µl/ml of SYBR Green The PCR product was purified using the Qiagen PCR Purification Kit

2.3.2 Cloning into TOPO and pGL4.11 vectors

The 2 kb IFI30 promoter region was cloned into the pCR 2.1 TOPO Vector using the AccuPrime Supermix Kit to ensure higher cloning efficiency The promoter region was then amplified from the constructed TOPO vector using PCR Supermix High Fidelity Kit (Invitrogen, CA) and In-Fusion 2.0 Dry-Down PCR Cloning Kit (Clontech, CA) The In-Fusion PCR primers were designed according

to the instructions for the In-Fusion™ PCR Cloning System (Clontech, CA) The PCR product was analyzed on a 0.9% agarose gel and the PCR fragment was treated with Cloning Enhancer according to the In-Fusion Cloning protocol

TOPO cloning primers and conditions:

Forward primer: 5’- CCCACTGCGACGTCATTGTCCCTTCA-3’ 

Reverse Primer: 5’- CGGCTGCCTGGGAAATAAGCGCCTTTAAC-3’ 

Step Conditions Cycles

35 53°C, 30s

68°C, 2 min

Table 2.1- PCR conditions for cloning the IFI30 promoter (2 kb) in TOPO vector

In-Fusion cloning primers and conditions: The in-fusion primer was designed such that it had complementarity to the multiple cloning region of the TOPO vector with a 15 base homology to the linearization site (in this case Bgl II and Xho I) of the destination vector (pGL4.11)

InFusion Forward primer

5-’GCTCGCTAGCCTCGACCACTAGTAACGGCCGCCAGT- 3’ 

InFusion Reverse primer

5’-CGCCGAGGCCAGATCCCAGTGTGATGGATATCTGCA - 3’ 

Step Conditions Cycles

35 60°C, 30s

2.3.3 Cloning of the deletion variants of IFI30

A deletion analysis of the IFI30 2 kb promoter was carried out keeping the 3’ end unchanged and gradually shortening the 5’ end. Promoter regions of length 1.5

kb and 1 kb were cloned The 600 bp promoter was cloned previously in the lab The 3' end IFI30 reverse primer used for the cloning of the 2 kb promoter region was kept the same

2.3.4 Cloning of the 0.68 kb IFI30 promoter into pFastBac Dual vector

The pFastBac Dual (pFBD) vector containing CMV-EGFP and HSV-Tk was kindly provided by Dr Poonam Balani The IFI30 (680 bp) promoter was PCR amplified from the pGL4.11 IFI30 construct and cloned in the pFBD vector upstream of the HSV-Tk gene using BamHI and NotI enzymes Positive clones (pFBD IFI30-HSVTk) were selected on LB-ampicillin plates and confirmed by restriction digestion with BamHI and NotI pFBD IFI30-HSVTk was then used for the generation of recombinant bacmid

IFI30 F ClaBamHI:

5'- ACGTATCGATACTTGGATCCTGGGTACCCTAGAGAAATGA-3’

IFI30 R NotI: 5'- ACTTGCGGCCGCTGCCTGGGAAATAAG-3'

Step Conditions Cycles

30 55°C, 30s