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Utilization of cancer cell eLF-4E over-expression /over-activation and a prostate specific promoter for… Scott, Christopher Charles 2006

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Utilization of cancer cell eIF4E over-expression / over-activation and a prostate specific promoter for the development of a transcriptionally and translationally regulated gene therapy. by Christopher Charles Scott B.Sc., The University of Victoria, 2003 T H E THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E IN ( P A T H O L O G Y A N D L A B O R A T O R Y M E D I C I N E ) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A A U G U S T 2006 ©Christopher Charles Scott, 2006 Abstract Prostate cancer is the second leading cause of cancer related deaths in males. There is no current cure for advanced and metastatic prostate cancer. The purpose of this research was to design a gene therapy system that w i l l allow for the selective ki l l ing of prostate cancer cells, while sparing normal cells. We have utilized the over-expression of eukaryotic initiation factor 4E (eIF4E) protein in many different cancer types, including prostate cancer. eIF4E is required for the translation of m R N A containing a 5'untranslated regions (5 'UTR) with large degrees of secondary structure, complex 5 'UTRs are most commonly found in growth factors and tumour promoting genes. B y constructing lentiviral vectors with 5 'UTRs inserted upstream of an E G F P reporter gene or herpes simplex virus thymidine kinase (HTK) therapeutic gene we have observed tumour specific expression and ki l l ing. Infection of cancer (LNCaP, PC 3 M , DU145, and M C F 7 cells) and non-cancer cell lines ( B P H 1,267 B l , Plat-E, and Huvec c cells) with lentiviral vectors encoding a CMV-promoter/EGFP-reporter construct resulted in high levels of E G F P expression in all cell lines; however, introduction of a 5 ' U T R restricted high expression levels to cancer cell lines and Plat-E cells, an embryonic cell line that expresses relatively high levels of eIF4E. Cel l lines infected with a 5 ' U T R regulated H T K showed differential levels of ki l l ing with respects to the concentration of gancyclovir (GCV) administered. A t least a 100-fold greater dose of G C V was required for equivalent ki l l ing effects in non-cancer cell lines infected with lentiviral vectors containing an inserted 5 ' U T R than with lentiviral vectors not containing a 5 'UTR. To further achieve prostate cancer specificity, the C M V promoter was exchanged with the prostate-specific A R R 2 P B promoter. Experimental results show that the ki l l ing effects of G C V were restricted to prostate cancer cells and not seen in non-prostate cancer cells. Our results indicate that combined translational regulation, by incorporation of an eIF4E-U T R recognition sequence upstream of a therapeutic gene, together with transcriptional regulation, through using a prostate-specific promoter, provides a mechanism for the selective ki l l ing of prostate cancer cells and the sparing of normal prostate and non-prostate cell lines. i i Table of Contents Abstract P- » Table of Contents P- i i i List o f Tables p. v i i L ist of Figures P- v i i i List of Abbreviations P- x i Acknowledgements p- x i i Chapter 1 Introduction 1 1.1 Overview of Prostate Cancer 1 1.2 Molecular mechanisms for prostate cancer development and 1 progression 1.3 Stages of prostate cancer development ...2 1.4 Prostate Cancer Treatment 5 1. 5 Gene therapy for prostate cancer 6 1.5.1 Lentiviral vectors 9 1.5.2 Adenoviruses and Herpes simplex virus 14 1.5.3 Pro-drug therapies 16 1.6 A R R 2 P B Promoter and Prostate Specificity 17 1.7 Translation and cancer 19 1.7.1 Translation initiation process 19 i i i 1.7.2 Eukaryotic Initiation Factor 4E (eIF4E) 21 1.7.3 Regulation of eIF4E 23 1.7.4 eIF4E Binding Protein (4E-BP) 24 1.7.5 5'-Untranslated Region (5 'UTR) 25 1.8 Mouse models for in vivo testing 29 1.8.1 P T E N Knock Out Mouse 30 1.8.2 P T E N and the PI3K Pathway 31 Objective of study 33 Chapter 2 Materials and Methods 34 2.1 Cel l Lines and Culture Conditions 34 2.2 Lentiviral Plasmid Construction 35 2.3 Lentiviral Preparation 39 2.4 Lentiviral Infection 40 2.5 Determination of eIF4E and H S V - T K expression levels of 41 cell lines by Western blot 2.6 Herpes thymidine kinase (HTK) and ganciclovir (GCV) 42 cytotoxicity and cell viability assay 2.7 Immunohistochemical staining of prostate tissue arrays for eIF4E..44 2.8 R N A isolation and real-time R T - P C R 45 2.9 Quantification of lentiviral transfecting units using R T - P C R 46 iv 2.10 Mouse surgery protocol for direct prostate injection 47 2.11 Genotyping of P T E N K O Mice 48 2.12 Immunohistochemical staining of prostate tissue arrays for eIF4E...50 Chapter 3 Development of a translational regulation for prostate....52 cancer. 3.1 eIF4E Expression levels in Prostate Cancer Tissue Arrays 52 3.2 eIF4E expression levels in cancer and non-cancer cell lines 57 3.3 Determination of eIF4E activity in cancer and non-cancer cell lines60 3.4 R T - P C R determination of translational regulation 64 3.5 Conclusion 65 Chapter 4 Therapeutic effects of 5'UTR insertion 67 4.1 Differential cell ki l l ing using lenitviral vectors expressing 70 herpes thymidine kinase 4.2 Targeting prostate cancer cells using lentiviral vectors 77 expressing thymidine kinase under a prostate specific promoter 4.3 Conclusion 78 Chapter 5 Preparation for in vivo analysis of the gene-therapy 82 model 5.1 eIF4E levels in P T E N K O 83 5.2 Immunohistochemistry for eIF4E 84 v 5.3 In vivo testing of viral infectivity and promoter expression abilities..86 Chapter 6 Conclusion and Future Direction 90 Bibliography 95 v i List of Tables Table 1 - Key proteins required for lentiviral activity 13 Table 2 - Cancer related m R N A with highly secondary structured 27 5 'UTRs. Table 3 - Comparative values for percent distribution of eIF4E 56 immunoreactivity scores and Gleason grades. v i i List of Figures Figure 1 - Three plasmid system for the construction of lentiviral vectors... 12 Figure 2 - Structure of the A R R 2 P B promoter construct 18 Figure 3 - Role of translation initiation factor 4E (eIF4E) in translation 22 Figure 4 - Variations in size and structural complexity of 5 ' U T R 26 Figure 5 - A timeline for the stages of development of prostate cancer 32 in P T E N K O mice Figure 6 - Vector structures for lentiviral transfer plasmids 36 Figure 7 - Representative drawing of the mouse prostate 49 Figure 8 - eIF4E immunohistochemical assay of prostate cancer 54 patient samples Figure 9 - Western blot analysis of eIF4E level in cancer and 59 noncancer cell lines. Figure 10- E G F P expression levels after infection of tumour with 61 lentiviral vectors Figure 11 - E G F P expression levels after infection of non-tumour 62 cell lines with lentiviral vectors. Figure 12 - Percent cell viability of cancer cells treated with G C V 68 vi i i after with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral infected cells. Figure 13 - Bright field view of cancer cells treated with G C V after infection with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors Figure 14 - Expression of H S V - T K after infection with different.... lentiviral vectors Figure 15 - Percent cell viability o f non-cancer cells treated with. . . G C V after with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral infected cells. Figure 16 - Bright field view of non-cancer cells treated with G C V after infection with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors Figure 17 - M T S assay for LNCap and BPH-1 cell lines treated with G C V after with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors Figurel8 - Effects of a prostate-specific promoter on cell ki l l ing using the H S V - T K / G C V system. Figure 19 - Bright field view of a prostate-specific promoter on cell k i l l ing using the H S V - T K / G C V system Figure 20 - Western blot analysis of eIF4E level in P T E N K O 85 mice prostate extracts Figure 21 - Photographic representation showing the distribution 88 of lOOul of Coomassie Blue Dye. Figure 22 - Mouse prostate tissue infected ex vivo with F U G W or 89 C M V - E G F P lentiviral strains. x List of Abbreviations P T E N phosphotase and tensin homolog eIF4E eukaryotic initiation factor 4E PI3K phosphoinositol 3 kinase C M V cytomeaglovirus A R androgen receptor A R E androgen response element G C V ganciclovir F B S fetal bovine serum H S V herpes simplex virus L N C a P Lymph Node (metastases) Prostate Cancer Ce l l Line BPH1 Benign Prostatic Hyperplasia Cel l Line P C 3 M Brain (metastases) Prostate Cancer Cel l Line M C F - 7 Human Breast Carcinoma Cel l Line P B Probasin E G F P enhanced green fluorescent protein x i Acknowledgements and Dedications I would like to acknowledge and thank my supervisor, Dr. Paul Rennie for all his guidance and support. I would also like to thank the following people from the Prostate Centre for their assistance, encouragement and helpful discussion: Dr. Duan Y u , Dr. Kev in Zhang, Maryam Moussavi, Lati f Wafa, Dr. Jason Read, Dr. Ladan Fazl i , Dr. Rob Snoeck, Howard Tearle, Dr. Chris Ong and Robert Bel l . I would also like to thank my supervisory committee: Dr. Mladen Korbelik, Dr. Wi l l iam Jia, Dr. Marcel Bal ly, Dr. Gerald Krystal. Finally, I would like to thank Heidi Barber for all her patience and support. x i i Chapter 1 - Introduction 1.1 Overview of Prostate Cancer Prostate cancer is the most commonly diagnosed cancer in both North American and European men [1]. The probability o f developing prostate cancer during a lifetime is 1 in 9 and prostate cancer is second only to lung cancer in levels of cancer related mortality [1-3]. The treatment of prostate cancer costs the Canadian health care system an estimated $9.75 bi l l ion dollars a year in medical expenses [2, 3]. If caught at an early stage, and i f growth is limited to the prostate capsule, prostate cancer can be treated with radiation therapy or radical prostatectomy, which act to effectively cure the disease. However, there is no effective cure for patients battling advanced stage or metastatic prostate cancer and, in Canada, it is estimated that 20% of new prostate cancer patients present with high-risk disease, which is approximately 4000 new cases each year [4, 5]. The social and economical statistics associated with advanced and metastatic prostate cancer are proof of the need for an effective cure and we believe that the development of our transcriptionally and translationally regulated gene-therapy w i l l provide a possible answer. 1.2 Molecular mechanisms for prostate cancer development and progression Genetic mutation plays a large role in the development and progression of all forms of cancer. Whether through the over-expression, or hyper-activation, of oncogenes (e.g. Ras and FGFR-1 ) or the inactivation, or deletion, of tumour suppressor genes (e.g. P T E N and p53), the overall outcome is genetic instability [6, 7]. These genetic 1 instabilities lead to changes in the normal cellular functions, which allow a cell to proliferate beyond its normal growth limitations [6]. Growth is normally controlled by checks and balances inherent in the cell growth cycle and the extra-cellular environment, genetic instability leads to disregard these controls, which can eventually lead to the development of cancer [6]. Prostate cancer is a disease that develops from the epithelial cell lining of the glandular component of the prostate [8]. One special feature of prostate cancer is the heterogeneity of the disease, where a single diseased prostate may possess multiple nodes, or regions of tumour growth, and it is possible that the genetic mechanism for disease and the phenotype of the disease w i l l vary from node to node [8]. There are a number of over-expressed molecules that are frequently associated with the development of prostate cancer, including; p53, telomerase, interleukin 6 and insulin growth factors [9-12]. Chromosomal loss and gain in prostate cancer are most commonly linked to the epidermal growth factor receptor (EGFR), P T E N , E-cadherin, the androgen receptor, retinoblastoma, and c-myc [13-18] 1.3 Stages of prostate cancer development Prostate cancer develops through a series of defined stages, including; prostatic intraepithelial neoplasia (PIN), prostate cancer in situ, invasive prostate cancer and metastatic prostate cancer [19]. PIN, all though not considered to be cancer, is the first stage in disease progression and it represents an intermediate in the development of a malignant phenotype and normal epithelial cells. During P IN development, variations in 2 biomarker expression levels and oncogene, or tumour suppressor gene, expression levels, both of which are indicative of cancer development, can be observed [20]. P IN is separated into high grade (HGPIN) and low grade (LGPIN) based on structural features observed during needle biopsies [21]. A l l though P IN is not considered to be cancer, the presence of H G P I N is believed to be clinically significant and is con-elated with development of invasive carcinoma [20, 21]. Prostate cancer in situ is the stage in prostate cancer development where malignant growth is limited to epithelial cells of the prostate ducts and has not progressed beyond the basement membrane. A tumour is considered to be an invasive carcinoma i f it has broken through the boundaries of its primary origin and is compromising neighboring tissues. In prostate cancer, invasive tumours most commonly affect surrounding adipose tissue and the smooth muscle of the bladder neck [22]. Prostate cancer is a metastatic cancer. The molecular methods for metastasis are not well understood, it is thought that a multi-step reaction is required for the cell to leave the primary site, enter the blood or lymphatic system, extravasate and develop into a secondary tumour [23]. Extravasation is the process whereby the metastatic tumour cells pass through the walls of blood vessels or lymph vessels, either when leaving the primary tumour site or when entering the secondary tumour site. The most common site for prostate metastasis is the bone marrow [24]. Four methods are commonly used to assess the presence and progression of prostate cancer; digital rectal examinations (DRE), Gleason Grading, measurement of P S A levels and clinical staging. D R E consists of a physical exam performed by a physician to measure growth or enlargement of the prostate. Gleason grading is based on the observation of architectural patterning in tumour biopsies. The biopsy is assessed 3 microscopically and a numerical score is given based on the degree of tumour cell differentiation [25]. Differentiation is the process in which cells develop into normal prostate epithelial cells leading to glandular formation, the greater the degree of differentiation, the more benign a tumour and; therefore, the lower the Gleason grade scored [25, 26]. The lower the degree of differentiation, the more malignant a tumour and the higher the Gleason grade scored [25, 26]. The Gleason grade system has been around for the last 75 years and is the most commonly used grading system in the world [26]. Prostate specific antigen (PSA) is a serine protease, secreted by normal epithelial prostate cells. The production of P S A increases with the development of prostate cancer and the level of P S A has been shown to be a sensitive and specific tumour biomarker for prostate cancer development and subsequent response of the tumour to treatment [27, 28]. Cl in ical staging, or T N M grading system, involves classifying tumours based on specific characteristics. The T refers to specific characteristics of the tumour, including; the location of the tumour in the prostate, whether it is limited to the prostate capsule, how the tumour was discovered and the size of the tumour [29]. A number from 1-4 is associated with the T and indicates the size of tumour, the larger the tumour encapsulated in the prostate, the larger the number. A lettering system is also associated with the T that indicates whether the tumour can be felt by D R E (c) or not usually felt by D R E (a). The N represents invasion of the tumour into neighbouring lymph nodes and M represents the presence of metastasis [29]. The T N M system allows for a thorough means of describing the characteristics of prostate cancer. 4 1.4 Prostate Cancer Treatment The method of prostate cancer treatment is dependent upon the stage of prostate cancer development. For early stage prostate cancer, a common treatment is watchful waiting. Prostate cancer is a relatively slow developing cancer and during the lifetime of the patient, the cancer may not develop to a level of any significant risk. If treatment is required; conventionally, the first options for treatment of prostate cancer are surgery or radiation therapy, or a combination of the two. Surgical treatment of prostate cancer requires the complete removal of the prostate, known as a radical prostatectomy. Surgery does not allow for differentiation of normal and cancer cells and; therefore, the entire prostate must be removed [30]. Surgical methods for prostate cancer treatment are only useful i f the prostate tumour remains localized within the prostate capsule. Drawbacks of surgery include its inability to differentiate between normal and malignant cells, leading to quality of life issues, such as erectile dysfunction and incontinence; invasiveness to the patient; and the inability to treat invasive or metastatic disease. There a two common methods of radiation therapy; external beam radiotherapy and brachytherapy [31]. External beam radiation involves the treatment of the prostate, and a surrounding marginal area, with an external source of radiation [32]. Brachytherapy is the insertion of an external radiation source directly into the afflicted prostate [32]. Both radical prostatectomy and radiotherapy yield 10-year survival rates of 90-95%, 60-70% and 50-60%, in T l , T2 and T3-stages, respectively[33]. Drawbacks of radiotherapy include effects to surrounding tissue and, in the case of external beam radiotherapy, multiple treatments are required [32]. New methodology for radiotherapy 5 that allow for more concentrated and targeted disbursement of radiation are being developed and have shown promising results [34]. Androgen withdrawal therapy is commonly used in combination with surgery and radiation therapy or as a primary treatment for more advanced cases. Androgens, such as dihydrotestosterone (DHT), are male growth hormones required for the development and survival of both normal prostate epithelial cells and early stage cancer cells. Therefore, the removal of androgens from the tumour environment leads to the subsequent apoptosis of malignant cells. Androgen ablative therapy is the blocking of androgen activity. Blateral orchiectomy, which is the removal of the testes, or the introduction of a luteinizing-hormone-releasing hormone (LHRH) agonists, which prevents D H T synthesis and release by the testes, are commonly used treatments for androgen ablation therapy [35]. Androgen ablative therapy allows for stabilization of growth and regression of tumour mass in 80% of prostate tumours [36]. However, in nearly all patients treated with androgen withdrawal therapy, after a period of remission, the development of an androgen independent tumour occurs [37]. It is thought that the removal of androgen dependent cells allows for the development of a phenotypically stronger variety of tumour cells that are not dependent on androgen for growth and survival [36]. The molecular mechanism for progression to androgen independence has yet to be elucidated and no treatments have shown an effective cure for afflicted patients [38]. 1. 5 Gene-therapy for prostate cancer Gene therapy is the insertion of a foreign genetic element into a target cell for the purposes of regulating disease progression. Gene-therapeutic benefits are mediated 6 through the reparation of normal cellular function or through the termination of diseased cells [39]. Gene therapies are currently being tested on a variety of diseases, most successfully in the treatment of rheumatoid arthritis and diabetes [40, 41]. Two possible methods for combating genetic abnormalities are replacement gene therapy and suicide gene therapy. Loss of expression and mutations in tumour suppressor genes, which prevent the protein product from performing tasks required to regulate cellular growth, lead to subsequent uncontrolled cellular growth. The goal of replacement gene therapy is to replace deficient or mutant regulatory genes, thus implementing a method of controlled growth or proper function for diseased cells. Replacement gene therapy is currently being used in A L S research [42]. Suicide gene therapy involves the selective ki l l ing of diseased cells through the introduction of a gene to the host genome that, once expressed, leads to cell death. Suicide gene therapy is being utilized in a variety of gene therapy research projects for cancer treatment, including breast, prostate and lung cancers [43, 44]. Gene therapies are currently being designed and tested as possible treatments for cancers of the prostate, breast, pancreas and many other forms of cancer [45-47]. A s mentioned earlier, a principle factor in tumour development is the presence of abhorrent expression of regulatory genes, including the decreased activity of tumour suppressor genes and over-expression or over-activation of oncogenic growth factors. The first gene therapy using a retroviral vector occurred in 1981, when the herpes thymidine kinase (HTK) gene was introduced into cellular genomes allowing for selective ki l l ing with introduction of gancyclovir (GCV) [48]. A key factor in developing a successful gene therapy is an effective method of transduction or shuttling of genes into target cells. There are two categories of vectors 7 currently being used; non-viral vectors and viral vectors [49]. Examples of non-viral gene transfer systems include; naked D N A delivered through direct tissue injections, liposomes and nanoparticles [50]. Liposomes are cationic l ipid spheres that can pass through the cellular membrane, but are not able to penetrate the nucleus. Nanoparticles are a single D N A molecule encased in a positively charged peptide that is able to penetrate into the nucleus of a cell ; nanoparticles are often transported to the cell within liposomes [51]. Current work is also being completed on magnetic nanopartical technology that allows for targeting through the use of high-field/high-gradient magnets [52]. Non-viral methods provide vector systems with high production levels, minimal toxic or immunological effects; however, they presently offer inefficient and transient gene transfer [39]. V i ra l vectors offer a more efficient means of transport and provide a more sustainable therapeutic effect, due to their ability to incorporate into host genomes [39]. Both R N A and D N A viruses are utilized for gene therapy, including: lentivirus, herpes simplex virus (HSV), adenovirus, retro-viral vectors and adeno-associated vectors. Using a vector that allows for large scale production is important in developing a gene therapy. In order for efficient infection to occur, and thus treatment, a large titre of virus must be produced. There are many obstacles present that can detract from therapeutic loads, such as immune reactions and infections of neighboring cells; therefore, the higher the titre the greater the chance for effective treatment. Currently, viral vectors are being used to deliver wi ld type dystrophin, a structural protein required for muscular contraction, to patients afflicted with Duchenne muscular dystrophy (DMD) , a disease that causes muscular deterioration [53, 54]. V i ra l vectors provide a persistent gene replacement 8 therapy in the majority of muscle fibres of patients, which is required in order to make the gene-therapy clinically beneficial [53]. A second important characteristic of a successful gene-therapy is the incorporation of an effective targeting system. To prevent toxicity of a gene therapy system, it is crucial to have a targeting system that allows treatment to be limited to abberrant cells. Transcriptional regulation can be achieved through the activity of tissue specific promoters. The promoter is placed upstream of the therapeutic gene and requires binding of cell specific elements, such as transcription factors, for activity. The promoter used can be specific for targeted cells (e.g. A R R 2 P B promoter targeted for prostate cancer) or for diseases of interest (Htert promoter allows for cancer specific expression). The underlying principle for a gene-therapy to be successful is to target pathogenic effects to target cells and to minimize adverse effects on surrounding, non-target cells [39]. V i ra l gene therapy for invasive and metastatic prostate cancer is a practical option due to the ability to treat cells beyond the prostate capsule. Surgery and radiation therapy are not effective on metastatic cancers and androgen withdrawal therapy is not effective on androgen independent prostate cancers, whereas gene therapy could provide a possible treatment option. 1.5.1 Lentiviral vectors Lentiviral vectors are currently being used in research to develop stably transfected cell lines for protein over-production and cellular transformation. A modified human immunodeficiency virus (HIV), the lentivirus has a few critical features 9 that make it an advantageous gene delivery system. Lentiviral vectors are replication incompetent, which means they cannot be re-synthesized after initial infection, preventing secondary infections (reviewed in [55]). Lentivirus is highly infectious and able to transduce a wide variety of human cell lines, (reviewed in [55]). Lentiviral vectors are able to deliver genetic material through the nuclear membrane and insert the gene of interest directly into host genome allowing the lentiviral vector to integrate genetic material into non-dividing cell lines and giving lentivirus the ability to infect cells in GO and G l phases (reviewed in [55, 56]. Other commonly used viral vectors, such as adenovirus, are unable to penetrate the nuclear membrane; therefore, they can only insert into the host genome during cellular division, a time of normal nuclear membrane dissolution [56]. Nuclear localization, by the lentiviral vector, is accomplished through a nuclear localization sequences within the gag matrix protein and a 99 nt ' D N A flap that is generated during reverse transcription of a central polypurine tract within the viral genome and acts to improve transduction efficiency [57-59]. The integration of therapeutic genes into the host genome increases the period of therapeutic effectiveness. Experiments have shown that viral vectors inserted into host genomes have consistently expressed transfected E G F P at a high efficiency for three months in vivo and transgenic animals have shown stable integration through out the life of the animal [60]. Prostate cancer is a relatively slow growing disease; therefore, it is beneficial to have a persistent viral therapy to allow cells to cycle to the point where they are most vulnerable to treatment. Finally, since lentiviral vectors do not transfer viral genes, they create a minimal response from host immune systems [61]. This allows for a more effective treatment because fewer vectors w i l l be removed from the dosage by the immune system 10 and therefore, more vectors w i l l reach the target tissue and administer therapy. Having a minimal effect on the immune system also allows for multiple treatments, which can lessen single dose toxicity and allow the same treatment to be utilized for re-current disease. It is important to note that the role of lentivirus is to deliver the appropriate gene therapy and that the lentiviral vector itself is not the therapeutic agent. Oncolytic viruses, discussed later, are viral strains which infect and k i l l tumour cells, without the requirement of a secondary gene-therapy. Currently, lentiviral gene therapy is being used to deliver a number of gene-therapeutics. Painter et al. have utilized lentiviral vectors for delivery of h T E R T promoter regulated H T K [62]. Lentiviral vectors are being used to shuttle interferon-alpha for possible treatment of ovarian cancers [63]. Lentiviral vectors are also being used for developing tumour-specific T-cells through the transduction of CD80 and C D 154 genes to primary myeloma cells [64]. A three plasmid transfection system is used for the production of lentiviral vectors, figure 1. The roles of key individual proteins encoded for by lentiviral vectors are outlined in table 1. The packaging plasmid, p C M V A R 8 . 9 , encodes H I V genes required for production and packaging of the lentiviral particle. The V S V - G , or envelope coding plasmid, contains a sequence for the production of V S V - G envelope protein. V S V - G envelope proteins allow for the infection of a wide range of host cells. The transducing plasmid, containing the gene of interest, is integrated into the host genome through the activity of the long terminal repeats (LTR), which are the essential 11 a) Transfer Vector LTR RRE 1 LTR I 1,1 Transgenic Truncated GAG Promoter b) Envelope Vector CMV VSV-G POLY-A c) R8.9 Packaging Vector CMV pol gag r Hirer »OLY-A Figure 1 - Three plasmid system for the construction of lentiviral vectors [65]. A l l three vectors are co-transfected into 293T cells for lentiviral construction. 12 F u n c t i o n Matrix ( M A ) Matrix Protein (gag gene); lines envelope Capsid (CA) Capsid protein (gag gene); protects core Nucleocapsid (NC) Capsid Protein (gag gene); protects the genome Protease (PR) Gag protein cleavage during maturation Reverse Transcriptase (RT) Transcribes the R N A genome to D N A Integrase (IN) (pol gene); integration of virus into genome Transcriptional activator (Tat) Transcriptional activator - viral replication Table 1 - K e y proteins required for lentiviral activity. The matrix, capsid and nucleocapsid proteins are encoded for in the gag gene and translated together and separated by the protease. The integrase is encoded in the pol gene. 13 sequences required for viral integration are short, allowing for more than 8kb of foreign D N A to be inserted [66]. Being a modified H I V virus bio-safety is a concern when dealing with lentiviral strains. However, H I V virulence genes (vif, vpu, nef, tat and vpr) have been removed or mutated and therefore alleviate some fear of lentiviral pathogenicity. Tat, a transcription factor, and Rev, which is required for nuclear membrane translocation, are two regulatory proteins (reviewed in [55]). Vi f , Vpu, Vpr and Ne f are from the structural and enzymatic portion of H I V [55]. One major drawback of gene therapy using the lentiviral delivery system is the non-directional insertion of therapeutic genes into target cell genomes [62]. Non-directional insertion refers to the inability to target the insertion of vectors into specific regions of the host genome. Inappropriate insertion lead to further problems for the patient, such as the inactivation of tumour suppressor genes or over-activation of oncogenes [62]. Non-directional insertion was observed in the delivery of a wi ld type CD34 receptor gamma chain gene to bone marrow cells of patients suffering from severe combined immunodeficiency. After the initial treatment with the lentiviral based gene-therapy, 17 patients showed immediate improvements in immune system activity [67]. However, over time three patients developed leukemia due to non-directional insertion of the therapeutic gene [67]. 1.5.2 Adenoviruses and Herpes simplex virus Adenovirus is an example of an oncolytic virus. Oncolytic viruses are replication component viruses, that selectively replicate and lyses tumour cells [68]. The advantage to oncolytic viruses is that they are not subject to drug resistance as seen with many other 14 therapeutic agents [53]. Through deletion of one or more early adenoviral genes (eg. E l -E4) the oncolytic effects of adenovirus are removed and the adenovirus can be utilized as a shuttle vector for transfer of gene-therapies [69]. Adenovirus is a double stranded D N A virus that can carry up to 30kb of foreign D N A in their genome. Adenovirus is advantageous as a gene-therapy because of its ability to be produced in large quantities; however, adenovirus is not invisible to the immune system and can i l l icit an immune response, which w i l l eventually decrease effective virion numbers. Adenoviruses can infect dividing and non-dividing cells and gene expression occurs without integration into the host genome, preventing mis-directed insertion [70]. Adenoviruses are advantageous for gene therapies that require only transient effects, once the virus is cleared from the body the gene therapy effects are dismissed [71]. Herpes simplex virus (HSV) is advantageous as a gene therapy vector due to its ability to transport large segments of exogenous D N A , up to 30 kb [72]. H S V has a high efficiency of transduction, due to its high degree of infectivity, and does not insert into the host genome, decreasing opportunity for non-directional insertion [72, 73]. Anti -viral agents, such as gancyclovir and acyclovir, also provide a safe guard for treatment with H S V , should infection get out of control [74]. H S V are made replication defective through the removal or inactivation of immediate early genes, ICPO, ICP4, ICP22 and ICP27, which are required for the expression of late genes required for replication [75]. Unfortunately, H S V display a wide tropism, which leads to infection without specificity, resulting in increased toxicity to neighbouring tissue [76]. Our group is currently utilizing the proof of principle outlined in these experiments to develop tissue and tumour specific H S V . 15 1.5.3 Pro-drug therapies One method for controlling the effects of gene-therapy is through enzyme activated pro-drug therapy. Pro-drug therapies involve a two-step process: first, the insertion of a therapeutic gene into the target genome, and second, the administration of a pro-drug. The therapeutic gene encodes for an enzyme that, when expressed under normal cellular conditions, has no detrimental effect on the cell. Once the pro-drug is administered and enters the cell, the enzyme acts to transform it into its toxic state. If the pro-drug enters a cell that is not expressing the enzyme, there w i l l be no toxic effects to the cell. Herpes thymidine kinase (TK) and gancyclovir (GCV) combination is an example of a commonly used pro-drug therapy. The expression of T K alone is not detrimental to the health of the cells; however, when the cells are treated with G C V , the T K can phosphorylate G C V to produce a G T P analogue which integrates into the genome of dividing cells, ki l l ing the cells during replication [77]. Another available system is cysteine deaminase and 5-flourocystine [78]. Cytosine deaminase is expressed in target cells, converting administered 5-fluorocytosine to toxic 5-fluoruracil [77, 78]. The bystander effect is a common occurrence during treatment of tissue with H T K / G C V pro-drug therapy [79]. During treatment, cells not expressing the H T K are being introduced to the toxic effects of the GTP analogue [79]. It is believed that the G T P analogue is able to migrate from one cell to another through gap junctions located between cells [79]. The presence of the bystander effect allows for greater effective therapeutic effects with each infection. A strong bystander effect was also observed in cells treated with a fusion protein consisting of HSV -1 VP22 and a human papillomavirus 16 (HOV) E2 fusion protein, which acts to cause apoptosis in cells transformed by H P V [80]. The transfer of fusion protein and the apoptotic event has been visualized in a three-dimensional tumour model [80]. 1.6 ARR2PB Promoter and Prostate Specificity Tissue specific promoters allow for restriction of transcription to target cells. The modified probasin promoter, A R R 2 P B , is a common tool for gaining transcriptional specificity in prostate cancer cells. Probasin (prostate basic protein)(PB) is a nuclear basic protein, first discovered in the dorsolateral rat prostate [81]. No equivalent protein has been discovered in humans, and the purpose of the protein in rats has yet to be determined [82]. The A R R 2 P B promoter is composed of two 148bp androgen response regions (ARR) of the probasin promoter and the 314bp proximal promoter [83]. The A R R 2 P B was constructed to optimize prostate specific expression, figure 2 [83]. Previous work by Y u et al. determined that A R R 2 P B is the most proficient version of the probasin promoter in terms of prostate specificity and expression level with lentiviral delivery [84]. The A R R 2 P B plasmid has been used to express Bax in androgen receptor-positive prostate cancer cells [85]. Prostate specific expression of the cell cycle arrest protein p202 has been accomplished utilizing A R R 2 P B specific transcription [86]. The A R R 2 P B has also been used for the development of prostate-specific P T E N knockout transgenic mice that develop prostate cancer upon maturity of the prostate tissue [19]. 17 -244 -96 -244 -96 -286 +28 bp Figure 2 - Structure of the A R R 2 P B promoter construct. Two tandem androgen response regions (-244 to -96 in original P B promoter) up-stream of a minimal probasin promoter (-286 to +28 in original P B promoter)[83]. 18 1.7 Translation and cancer Genomic alterations, transcriptional regulation and post-translational activities are well researched and established as mechanisms for the development of a malignant phenotype [87]. Recently, research has focused on the role of translational processes in cellular transformation and a variety i f translational mechanisms have been outlined, including; the differential expression of oncogenes, localization of oncogene m R N A to polysomes and the over-expression of translational regulatory elements [88-91]. There are three major steps in translation; initiation, elongation and termination. Initiation is the rate determining step in translation and is thought to be the most viable option for malignant occurrences in protein synthesis to occur. To develop a translationally regulate gene-therapy, we intend to exploit the over-expression of the eukaryotic initiation factor 4E (eIF4E), which has been shown to induce differential translation of oncogenes containing highly secondary structured 5 'UTRs. 1.7.1 Translation initiation process Translation is the process in which nucleotide sequences encoded in nuclear D N A are converted to amino acid sequence, eventually forming protein molecules. Figure 3 is a schematic drawing of the steps encompassing the initiation of translation. In the nucleus, D N A sequences are transcribed to intermediary messenger R N A (mRNA) molecules, which undergo post-transcriptional processing and are then shuttled to the cytoplasm to be translated into proteins. The m R N A post-transcriptional events include splicing, addition of a 5'7-methyl guanosine cap and the addition of a 3' poly-adenosine 19 tail. The 3' poly-adenosine tail, synthesized by nuclear localized poly(A) polymerase, acts to stabilize, facilitate localization and enhance translation of m R N A s [92]. The 7-methyl guanosine cap is bound through a 5' -5 ' triphosphate bridge to the 5'terminal nucleoside end, the reaction is catalyzed by a nuclear guanalyl and methyltransferases [93]. The 5'cap acts to target R N A to the cytoplasm, protect m R N A form degradation and facilitate translation initiation through the binding of eukaryotic initiation factor 4F complex (eEF4F) [93]. A 5'untranslated region is located upstream of the start codon on m R N A molecules. The length and complexity of the 5 ' U T R varies between m R N A molecules and its significance to cancer w i l l be discussed later; basically, the 5 ' U T R is a region with varying degrees of secondary structure that plays a role in protection of m R N A and regulation of translation [94]. Genomic translation occurs in the cytoplasm and is mediated by ribosomes, which are protein-RNA complexes responsible for protein synthesis. In order for translation to begin, ribosomes must scan the 5'end of m R N A in search of an A U G start codon. Due to the presence of m R N A secondary structure found in the 5 ' U T R region, the ribosome requires the activity of the eIF4F complex for access to the start codon. The 5'cap acts as an anchoring point for eIF4F construction, which is composed of eIf4E, a 5'cap binding protein that targets the eIF4F complex, eIf4A, an A T P dependent RNA-hel icase, and eIF4G, a scaffold protein that facilitates the formation of the eIF4F complex [95, 96]. Once the eIF4F complex has been formed, the eIF4A subunit unravels the R N A , which opens the secondary structure produced within the 5'untranslated region [95, 97]. eIF4G interacts with eIF3, of the 40S ribosomal subunit, to bind the eIF4F complex to the subunit [97]. The effect of unraveling is to allow access 20 of the 50S subunit of the ribosome to the A U G start codon, where it w i l l jo in the 60S subunit to form the 80S complex and begin translating m R N A [6, 90]. 1.7.2 Eukaryotic Initiation Factor 4E (eIF4E) eIF4E is a 25 K D a , m R N A cap binding phosphoprotein, responsible for binding to the 5' cap of m R N A and acting as a beacon for formation of the eJJF4F complex through direct interaction with eIF4G, see figure 3 [6]. Under normal cellular conditions levels of eIF4E are limited, presenting at 0.01 - 0.2 molecules/ribosome, this compares to 0.5-3 molecules/ribosome for other initiation factors [98]. The low levels, compared to other initiation factors, allow eIF4E to be a rate limiting step during the initiation of protein synthesis [98]. Over-expression of eIF4E has been correlated with T N staging, histological grades, local reoccurrence and metastasis in laryngeal squamous cell carcinoma [99]. Liang et al. believe that the level of eEF4E is correlated with expression of bFGF, an angiogenic factor that plays a role in vascularization of tumour masses during tumour development [99]. In breast cancer, eEF4E was shown to be a predictor of cancer re-occurrence in women with stage I to III disease [100]. Tumour cell lines over-expressing eIF4E have also shown resistance to drug-induced apoptosis through differential translation of anti-apoptotic proteins [101]. eIF4E levels are found to be highest in invasive cancers and have been shown to be localized to areas of tumour vascularization [102, 103]. Over-expression of eIF4E leads to the cellular transformation in normal 21 Role of eIF4E in Translation Figure 3 - Translation initiation factor eIF4E acts as a beacon for the formation of the eIF4F complex. The eIF4F complex is composed of an ATP-dependent helicase, eIF4A, a scaffolding protein, eIF4G, and eIF4E. The eIF4F complex gives the ribosomal sub-units access to the A U G start codon by unwinding the complex hairpin 5 ' - U T R of specific m R N A s . 22 fibroblast cells, indicating eIF4E's strength as a an oncogene [91]. Over-expression of eIF4E in NIH3T and Rat-1 fibroblasts cell lines showed a 30-fold increase in ornithine decarboxylase (ODC) protein levels, a potent oncogene [ 104]. Silencing of eIF4E through anti-sense m R N A led to suppression of O D C expression [104]. eIF4E over-expression was shown to increase levels of cyclin D I , and was also shown to shuttle cyclin D I m R N A from the nucleus [104]. Over-expression of eIF4E in Chinese hamster ovary cell line leads to an increase in c -Myc expression, a known oncogenic protein; however, the cell lines required Max over-expression in order to become tumourgenic [6]. With over-expression of Max, the cells subsequently became highly metastatic [6]. Transformation, tumour growth and metastasis in vivo were observed in C R E F fibroblasts cells over-expressing eIF4E, and the clones expressing the highest levels of 4E showed the most aggressive behavior [105]. Conversely, the reduction in eEF4E, through antisense, in highly metastatic ras-transformed C R E F T24 cell lines decreased eIF4E expression by 60%, this was enough to reverse the transformed phenotype [6]. 1.7.3 Regulation of eIF4E eIF4E activity is regulated through three different methods; transcriptionally, through phosphorylation, and binding protein activity. The method for transcriptional regulation of eEF4E has not yet been completely determined. It has been shown that the eIF4E promoter sequence contains a c -Myc transcription factor binding site and that eIF4E expression is up-regulated in cells over-expressing c-myc [98]. P53 has been shown to repress the expression of eIF4E through binding to c -Myc and thereby preventing the transcription factor from binding to the promoter [106]. Therefore, it is 23 possible that oncogenic events that effect expression levels of P53 or c -Myc may have effects on the levels of eIF4E within the cell, which subsequently may lead to the over-expression of further oncogenic proteins. eIF4E is phosphorylated on Ser-209 by M A P kinase signal integrating kinase (Mnk l ) [107]. Phosphorylation of eIF4E is enhanced in cells transformed though the overexpression of Ras and Src [104]. The effect of eIF4E phosphorylation on translation regulation is currently a point of contention. Initially believed to activiate or enhance translation, Scheper et al. believe that phosphorylation decreases the affinity of eIF4e for the transcript cap, which may play a role in release of the eIF4E molecule from the m R N A for further translational initiation [108]. Other groups have also shown inhibitory effects on translation due to the phosphorylation of eIF4e by M N K 1 [109, 110]. 1.7.4 eIF4E Binding Protein (4E-BP) eIF4E translational activity is regulated through the interaction with the eIF4E-binding proteins (4E-BP), also known as phosphorylated heat and acid soluble protein stimulated by insulin (PHAS-1)[111]. 4E -BP is a family of 3 binding proteins (4E-BP1, 4E -BP2 and 4E -BP3 , with 4E-BP1 being the most prominent), that negatively regulate eIF4E activity through competitively binding to a the same motif as eIF4G ( T y r - X - X - X -X -Leu -0 , where X varies and 0 indicates Leu, Met, Phe)[87]. Inactivation of 4 E - B P occurs through phosphorylation on six serine/threonine sites, two sites (Thr 36 and Thr 47) are confirmed to be phosphorylated by M T O R and the 3 other sites (Ser-60, Thr-70, Ser-83) are thought to be phosphorylated by M T O R or another Ras dependent signal transduction pathways [112, 113]. 4E -BP phosphorylation is induced by extra-cellular 24 signals including growth-factors, amino acids, cytokines and G-protein coupled receptors and the phosphorylation leads to the release and degradation of 4E -BPs (reviewed in [114]). Studies have shown that the over-expression of 4 E - B P in cells transformed through over-expression of eIF4E leads to the reversion of the malignant phenotype [115]. 1.7.5 5'-Untranslated Region (5'UTR) During m R N A post-transcriptional processing, m R N A molecules receive 5' untranslated regions (UTR), which are located upstream of the messenger start codon. 5 'UTR contain a high degree of secondary structuring, such as hair pin loops; caused by areas of high guanine and cytosine content. O f 700 vertebrate m R N A analyzed, 90% contained 5 'UTRs fewer than 200 bps long and the length and the degree of secondary structuring of the 5 'UTR is correlated with the level of translation [89]. A n m R N A with longer and more complex 5 'UTRs are translated less frequently than m R N A with a simple or short 5 ' U T R [88]. Figure 4 gives a representative drawing of the levels of complexity between simple and complex 5 'UTR [6]. Simple 5 'UTRs are encoded to the m R N A of commonly translated proteins, termed competitive m R N A , such as house keeping genes, including p-actin [6]. Complex 5 'UTRs, termed non-competitive m R N A , have been found in a variety of oncogenes, as outlined in this list compiled by Graff et al, table 2 [6]. Under normal cellular conditions, competitive m R N A has an advantage over non-competitive m R N A for access to translational machinery. However, during times of eIF4E over-expression there is a differential increase in the level of non-25 Figure 4 - Variations in size and structural complexity of 5 'UTR. The more highly structured a 5 'UTR, the less competitive an m R N A and the greater the level of differential translation observed with over-expression of eIF4E [6]. 26 PDGF - Autocrine growth stimulation FGF-2 - Angiogenesis 25, 26 TGF-/J 2,3 - Tumor cell viability, autocrine/paracrine interactions IGF-II - Autocrine growth stimulation VEGF - Angiogenesis c-myc - Unregulated tumor growth fos - Transcription factor regulating metastasis-related genes Spi-1/PU.1 - Ets-family transcription factor, subverts cellular differentiation C/EBP - Transcription factor Mdm-2 - Proto-oncogene, interferes with p53 tumor suppressor function Cyclin D1 - Proto-oncogene, drives G1-S transition p27kip1 - Cell cycle regulator, negatively regulated by a translationally controlled proteins NMDA (NR2A subunit) - Cell-extracellular matrix interaction Her2/Neu - Autocrine growth stimulation, kinase IL-1B - Autocrine growth stimulation, tumor invasiveness, growth IL-15 - Autocrine growth stimulation, invasion Ornithine decarboxylase - Proto-oncogene, master regulator of (ODC) polyamine biosynthesis Ornithine amino - Regulation of polyamine levels transferase S-AMDC - Regulation of polyamine levels, polyamine - biosynthesis Lck - Proto-oncogenic kinase Pim-1 - Proto-oncogenic kinase, cell cycle progression Ribonucleotide - DNA synthesis, G1-S transition reductase P23 - Growth-related protein, microtubule interactions MMP-9 - Tumor invasiveness, degradation of extracellular matrix BCL-2 - Anti-apoptotic proto-oncogene BCL-xL - Anti-apoptotic proto-oncogene JunD - Proto-oncogene, transcription factor Tousled-like kinase - Imparts radioresistance Table 2 - Cancer related m R N A with highly secondary structured 5 'UTRs [6] 27 competitive m R N A translation, and no change in the level of competitive m R N A translation [116, 117]. The exact reasoning for the translational discrepancies is unknown. However, it is believed that the competitive m R N A s saturate the translational machinery regardless of eIF4E levels. When levels of eIF4E are increased, there is more available for non-competitive m R N A . Tuxworth et al. altered the length and level of secondary structure in the 5 ' U T R region of a luciferase gene. They then compared luciferase levels in adult cardiocytes that either expressed eJJF4E at normal levels or over-expressed eIF4E. They found that by increasing the G + C content they were able to double the amount of secondary structure, which lowered the level of luciferase expression by roughly 50 % [107]. The effects of 5 ' U T R can also be witnessed in tousled-like kinase I B (TLK1B) , a splice variant of tousled-like kinase, which plays a role related to D N A metabolism [118]. The m R N A of T L K 1 B contains a 1088nt long 5' UTR . O f 87 specimens from cancer patients, Norton et al showed an 9.5-fold elevation of eIF4E and corresponding 9.4-fold increase in the level of T L K 1 B [100]. In our experiment, we have cloned in the fibroblast growth factor 2 (FGF-2) U T R upstream of the herpes thymidine kinase (HTK) gene therapy. FGF -2 , also known as basic fibroblast growth factor (bFGF), is a cytokine shown to play a role in tumour angiogenesis and acts as a growth factor for many mesodermal and ectodermal cells [119, 120]. Research by K e v i l et al. showed an increase in the level of F G F - 2 protein in invasive carcinoma [118]. However, there was only minimal increase in the level of FGF -2 m R N A , indicating that post-transcriptional processes lead to protein over-expression and a subsequent relationship between eIF4E levels and the 5 ' U T R [119]. 28 1.8 Mouse models for in vivo testing Upon completion of successful in vitro analysis, the next step in testing the efficacy of a gene therapy protocol is through in vivo testing. In vivo analysis allows for the testing of a gene therapy in an environment that is more representative of the intended patient than as seen in a tissue culture dish. In vivo analysis allows for measuring dose effects for toxicity levels, the effect of an immune systems response (especially when using a viral gene vector) and the efficiency of the therapy in a more representative cellular environment. Mouse models are commonly used as in vivo models for testing therapeutics destine for human trials. There are number of similarities between mice and humans that allow mice to be good models for future prostate cancer therapeutics, including cellular types and glandular make-up [121]. Xenograft models are commonly used for experimentation on prostate tumours and involve the subcutaneous insertions of malignant cells into severely compromised immuno-deficient (SCID) or athymic nude mice models. For research in prostate cancer, there are multiple transgenic mouse models currently being developed. The three transgenic mice models that are commonly used are T R A M P , L A D Y and P T E N K O mouse model. The T R A M P (Transgenic Adenocarcinoma of the Mouse Prostate) model involves the expression of the large and small SV40 tag and the L A D Y is a grouping of seven mouse models created through the large SV40 tag insertion under probasin promoter control [122, 123]. Both T R A M P and L A D Y models undergo spontaneous adenocarcinoma formation upon prostate maturity [122, 123]. 29 1.8.1 PTEN Knock Out Mouse For determination of the efficacy of our gene therapy, the xenograft model was unsuitable for exploring the representative activities of transcriptional and translational regulation. A xenograft model w i l l not allow for the growth of normal cell lines to use as negative control to test the cancer specificity of the translational regulation. Although the normal prostate can be used in the xenograft model, it is not in the same environment as the subcutaneously inserted tumour. It is also difficult to find a non-malignant prostate cell line for inclusion in a xenograft model that expresses an adequate level of androgen receptor to allow for expression using the A R R 2 P B promoter. The prostate specific P T E N K O mouse model is one possible solution. The P T E N K O mouse model was first published by Wang et al. [19]. However, development was concurrently completed at the Prostate Centre at V G H in the laboratory of Dr. Chris Ong [19]. Early work with the P T E N knockout mouse models have shown the development of a broad spectrum of spontaneous tumours, including large and small intestine, lymphoid, mammary, thyroid, endometrial, and adrenal gland (reviewed in [19]). The P T E N knockout mice are heterozygous P T E N mutants, as homozygotic mutants led to embryonic lethality, and were created using the ere flox system [19]. Cre is a DNase enzyme that specifically cuts at flox sites [124]. The transgenic mice were created with flox sites flanking the P T E N gene and expression of Cre is under the transcriptional control of the A R R 2 P B promoter. At prostate maturity, the androgen receptor is expressed allowing for transcription of prostate specific genes, including Cre under the control of the A R R 2 P B . Once expressed, Cre proteins find flox sites and remove P T E N , creating the P T E N K O prostate and the splice ends are ligated. Due to the 30 specificity of the A R R 2 P B , P T E N K O is limited to prostate, with splicing also being observed in seminal vesicle and testis cells. At 4 weeks of development, hyperplasia can be seen in P T E N K O mouse prostates [19]. At approximately 6 weeks prostatic intraepithelial neoplasia (PIN) and invasive adenocarcinoma can be verified at 9-10 weeks [19]. A t greater than 15 weeks, metastasis can be observed [19]. Figure 5 shows a staging timeline for the development of prostate cancer in P T E N K O mice. A s in human prostate cancer, invasive carcinoma cells respond to androgen ablation therapy, as determined by increased levels of apoptosis [19]. However, sensitivity of P T E N K O prostate cells to androgen withdrawal did not correspond to a decrease in cellular growth rate. Therefore, P T E N K O mouse show development of androgen independent growth [19]. Wang et al. also noted similarities in gene expression changes for P T E N K O mice when compared with both human prostate cancer and other human cancers with metastatic potential [19]. Transgenic mice that do not contain the Cre gene can be utilized as negative controls. 1.8.2 PTEN and the PI3K Pathway P T E N , the phosphotase and tensin homolog deleted from chromosomal region lOq, is one of the most frequently altered tumour suppressors in a variety of cancers [1, 19]. Mutations in P T E N have been found in prostate, breast, brain, lung and bladder cancer [125-127]. Errors in P T E N expression, through deletion or mutation, are found in 30% of primary prostate cancers and 63% of metastatic disease. P T E N is a regulatory 31 0-3 wks ] 1 - Pre-malignancy 4 - 5 wks { i -PIN 6-12 wks • D I I 1 I r i 12-15 wks i | Li 15 +wks-(J - Carcinoma Invasive Carcinoma - Metastasis Figure 5 - A timeline for the stages of development of prostate cancer in P T E N K O mice [19]. 32 protein for the phosphotidylinositol-3 kinase (PI3K) signal transduction pathway (reviewed in [128]). P T E N is a phosphotase that dephosphorylates phosphotidylinositol 3,4,5-triphosphate, (PIP3)(reviewed in [128]). PIP3 is phosphorylated by P I 3 K , allowing for PIP3 to bind A K T , thus activating A K T (reviewed in [128]). P T E N regulates the P I 3 K pathway through its phosphotase activity on PIP3. Alterations in P T E N activity, through mutation or deletion, leads to an increase in PJP3 levels and a constant activation of downstream molecules, including: the phoshotidyl inositol-dependent kinases (PDKs); A K T / P K B ; S6 kinase and m T O R (reviewed in [128]). Objective of Study The objective of this thesis is the development of a viral vector delivery system with a combined transcriptionally and translationally regulated gene-therapy that can selectively k i l l prostate cancer cells while maintaining the integrity of surrounding non-prostate and prostate non-tumour tissue. Transcriptional specificity, in prostate cell lines, has previously been shown through the inclusion of a tissue specific promoter, A R R 2 P B [84]. Translational regulation is attained by utilizing cancer cell e I F 4 E over-expression through the insertion of a highly secondary structured 5 'UTR. Our hypothesis is that a lentiviral vectors incorporating both the prostate specific A R R 2 P B promoter and a highly secondary structured F G F 5 ' U T R element w i l l allow for prostate tumour specific expression. To prove our hypothesis we tested the selectivity and effectiveness of our viral constructs on a variety of cancer and non-cancer cell lines in tissue culture. 33 Chapter 2 Materials and Methods 2.1 Cell Lines and Culture Conditions A variety of cell lines were required to test both tumour specificity and prostate specificity of the viral gene therapy. The more cell lines tested, the greater the measurement of specificity that can be achieved. L N C a P , DU145, M C F - 7 , Huvec-c and 267B1 cell lines were obtained from American Type Culture Collection ( A T C C ) (Manasas, V A , U S A ) . P C - 3 M cells were given by Dr Charles Myers (University of Virginia), BPH-1 cells were donated from the lab of Dr Simon Hayward (Vanderbilt University), and Plat-E cells were provided by Dr T Kitamura (University of Tokyo). 293T, L N C a P , M C F - 7 , and BPH-1 cell lines were maintained in R P M I 1640, supplemented with 5%, or 10%, fetal bovine serum (FBS)(Invitrogen) and 100 units/ml penicillin/streptomycin (Invitrogen). P C - 3 M , Plat-E, and M M 3 M G (TK) cell lines were propogated in D M E M media (Invitrogen) supplemented with 5% F B S and 100 units/ml penicillin/streptomycin. Huvec-c cells, originating from an umbilical chords, were grown in Ham's F12K medium ( A T C C ) supplemented with 2 m M L-glutamine, 0.1 mg/ml heparin, 0.03-0.05 mg/ml endothelial cell growth supplement (ECGS), and 10% FBS . 267B1 cell line was grown in B R F F - H P C 1 medium (AthenaES). A l l cells lines were grown in a 5% C O 2 environment, at 37°C and in order to maintain the integrity of the cell lines, growth conditions were carefully maintained. Overgrowth of cells can lead to alternate proteome expression and cell death. Once cell population reaches 60% confluency with respect to the bottom surface area of either a 10cm or 15cm diameter cell culture dish, the cells require passaging to either continue cell growth for future 34 experiments, alliquot for present experiments or prepare cells for long term storage. A l l three preparations require separation of cells from cell culture dishes through a 2min treatment, at 37°C, with 1ml or 5 ml of trypsin E D T A (Invitrogen), for 10cm or 15cm Plat-Es, respectively. After incubation, cells are collected and pelleted through spinning at 3000rpm for 3min. Cells requiring further propagation are aliqouted to cell culture dishes (10ml of media for 10cm Plat-Es, 25ml of media for 15cm Plat-Es). Cells utilised for titred experiments require quantification prior to allocation. Cel l numbers were determined using a hemocytometer cell counting system. Cells requiring long term storage were resuspended in 7ml of appropriate media, placed on ice for 5min and then treated 1:1 with a 20% D M S O solution (2ml D M S O , 2ml F B S , 6ml 5% F B S D M E M or RPMI) . Cells were then alliquoted to long term storage tubes (1.5ml), placed in -80°C for 24hrs and then stored long term in liquid nitrogen. 2.2 Lentiviral Plasmid Constructions As described in the introduction, a three plasmid system is required for production of lentiviral vectors. In order to take advantage of a lentiviral gene-delivery system, appropriate modifications were made to plasmid p H r ' - C M V - E G F P , also known as the shuttle vector, previously donated from Dr. Baltimore of Caltech University. Figure 6 illustrates the modifications made to the shutle vectors for the purpose to develop transcriptionally and translationally regulated gene-therapy vectors. 35 1.1»CMV-EGI? mvi l i j . til 8CW mv-i LIJ. 2 »CMVUIG lT? T 131 3 1VCMVTK l~l— U T R mviLij. MX j 4I»CMV-UTK HIV-lLIl. UJ! 1 Bi:' Cl£V 5 1> AJUtJB EGFFTAGW H1V1LIL GW5 uz r y t^X mvi LI*. 6 I> AWyB TKTATK BVI UK T mv-i Lit. ? Lv ARRJB -trrKTAUTK mviLij. HZ h " 1 U T R SB mvi LIS. Figure 6 - Vector structure o f the lentiviral transfer plasmid. Seven vectors were constructed and used to determine the efficacy o f our transcriptional and translational regulation. L v - C M V - E G F P ( l ) represents the control vector which acts as the starting point for the construction of the subsequent vectors through insertion of a T K gene to develop L v - C M V - T K ( 3 ) . A 5' -UTR was inserted in upstream of E G F P and T K to develop L v - C M V - U - E G F P ( 2 ) and L v - C M V - U T K (4). A R R 2 P B was inserted in C M V regulated promoters (1), (3), and (4) to create L v - A R R 2 P B - E G F P ( 5 ) , L v - A R R 2 P B -TK(6), and L v - A R R 2 P B - U T K (7), respectively. 36 p H r ' - C M V - U - E G F P - A 5 ' -UTR (619 bp) region from the rat F G F - 2 gene was cleaved from plasmid rFGF(T3) through digestion at Xhol-BamRl sites. 1 jag of D N A was digested for 2 hours at 37°C. Digested fragments were resolved using a 1% agarose gel containing 0.5ug/ml ethidium bromide, for visualization under a U V light source. A ~620bp band of interest was purified from agarose using a Mini -E lute gel extraction kit (QIAGEN) , which melts the gel and isolates the D N A strand through use of a silica membrane and variations in salt concentrations. Ligation was carried out using T7 D N A ligase and cloned into plasmid p E G F P - N l (Clontech) at corresponding sites. The 5 ' U T R containing fragment was then excised from p E G F P - N l using Bglll-BamRl sites and inserted at a single BamRl site of the p H R ' - C M V - E G F P creating L v - C M V - U - E G F P . D N A sequencing was utilized to confirm the correct orientation and positioning of gene. Samples were sent to the Nucleic A c i d / Protein Service Unit (NAPS) of the Michael Smith Laboratory at the University of British Columbia. Sequences were compared to D N A databases to ensure correct cloning. pHr ' - A R R 2 P B - E G F P - A R R 2 P B promoter was isolated from PBluescript-II -SK by digestion with C l a l and B a m H l restriction enzymes. The fragment was isolated as discussed previously, and ligated into corresponding sites of p H R ' - C M V - E G F P . Construction was verified as described previously. pHr ' - A R R 2 P B - U E G F P - all though not utilized for these experiments, plasmid construction was completed using the same cutting and binding sites as with p H r ' - C M V -U E G F P , followed by insertion into pHr ' - A R R 2 P B - E G F P . 37 p H r ' - C M V - T K - Polymerase chain reaction (PCR) was used to create lentiviral vectors expressing the suicide gene H S V thymidine kinase (TK) gene. A ~ 1.2 kb T K was obtained by P C R (94°C for 2 min, 94°C for 30 s, 60°C for 1 min, 72°C for 1 min, 35 cycles from the second step, then 72°C for 7 min) using primers with flanking BamHI andXhoI sites ( 5 ' T C G G G A T C C C G T A T G G C T T C G T A C C and 5 T G T C T C G -A G T G T T T C A G T T A G C C ) , using plasmid B K - T K as the P C R temPlat-E. The P C R product was digested with BamHI and Xhol, purified and inserted into complementary sites of p H R ' - C M V - E G F P . p H r ' - C M V - U - T K - The E G F P gene was removed from plasmid p H R ' - C M V -E G F P at BamRl-Kpnl and replaced by a linker strand of D N A encoding for a multiple cloning sites (-BamRl-XholSpel-Kpnl), creating an intermediary plasmid. A 3.6 kb U T K fragment was released from B K - U T K at XholSpel sites and cloned into the intermediary plasmid at complementary sites to create p H r ' - C M V - U T K . pHr ' -ARR2PB -TK - Plasmid was created using the same method as with A R R 2 P B E G F P ; however, promoter was cloned into p H r ' - C M V - T K . pHr ' -ARR2PB -UTK - Plasmid was created using the same method as with ARR2PB -U-EGFP; however, promoter was cloned into p H r ' - C M V - U - T K . 38 2.3 Lentiviral Preparation The method for lentiviral preparation used in this study was first described in experiments by Naldini et al. [129]. Production of lentiviral particles requires the co-transfection of viral plasmids R8.9, V S V - G and a lentiviral transfer vector into a packaging cell line, 293T was used in this experiment. The transfection process is mediated through a calcium-phosphate precipitation method (Promega Profection Mammalian Transfection Systems). 1 .5xl0 6 293T cells were seeded ontolO-cm Plat-Es 24 hours before transfection in D M E M supplemented with 10% F B S and 100 units/ml penicillin/streptomycin (Invitrogen). 3 hours before transfection the media was replaced with 6ml of unconditioned D M E M . Each 10cm Plat-E of cells was co-transfected with 7.5ug of transfer vector, 7.5u.g of pR8.9, 5ug of p V S V - G , 37ul of 2 M C a C l , 193uJ of d H 2 0 and 250p.l of hepes buffered saline and introduced to the cellular medium for 16-20 h, at 37°C in a 5% C 0 2 environment.. After the transfection period, the medium was replaced with 8ml of D M E M , supplemented with 10% F B S and 100 units/ml penicillin/streptomycin. A t 24hrs intervals after the introduction of the conditioned media, the viral-containing medium was collected and 8ml of condtioned media was replenished. Collected medium is passed through a 0.45 um filter to remove bacteria, yeast, and cellular debris, then ultracentrifuged at 25000RPM for lhr40min, at 4°C, using an SW-28 rotor (Beckman Coulter) The supernatant was removed and the resulting pellet was resuspended in either 60ul of l x P B S or 60ul unconditioned D M E M , which was then aliquoted into 15ul samples and stored at -70°C for future use. Three methods were used for quantification of lentiviral infectious particles: E L I S A analysis, bio-assay and R T - P C R / Q - P C R . Initially, concentrations were determined using a p24 viral protein 39 E L I S A (Vironostika). V i ra l vectors were also measured by counting the number of EGFP-positive cells 72 h post-infection with a serial dilution of prepared virus. Cells were Plat-Ed at a concentration of 2x10 4 L N C a P cells per wel l in 24-well Plat-Es, dilutions were prepared as neat, lOx, lOOx, lOOOx and lOOOOx. Unfortunately, quantification using a bioassay is not an effective means of counting when using viral lines not producing a marker protein, such as E G F P . R T - P C R methodology was adopted and w i l l be discussed later. 2.4 Lentiviral Infection Both tumour and non-tumour cell lines were seeded in 24-well Plat-Es at a density of l x l O 4 cells/well in 0.5 ml of medium or in six-well Plat-Es at a density of l x l O 5 cells/well in 2 ml of medium and incubated for 24h under normal growth conditions. After 24h, viral stocks were added to cell cultures at a multiplicity of infection (MOI) of 30. The M O I refers to the number of transfecting units/cell and the necessary value varies with different viral lines, the larger the M O I , the greater the chance for infection. The media was changed 16h post-infection and the cells were visualized with U V microscopy, and the effectiveness of the transfection procedures was determined through quantification of EGFP-positive cells. 4-days post-infection, a more accurate measurmentf E G F P levels was determined through flow-cytometry. V i ra l ly infected cells and control cells were fixed in preparation for flow-cytometry. Cells were washed with sterile l x PBS , then trypsinized with 1ml of 5% E D T A Trypsin, washed again with P B S and then treated with F I X media (1% formaldehyde in PBS) . Fixed 40 samples were analyzed with a Becton Dickinson FACScal ibur flow cytometer using Cel l Quest software, and data were analyzed with fit mode on W i n M D I version 2.8. 2.5 Determination of eIF4E and HSV-TK expression levels of cell lines by Western blot Western blot analysis was used to determine levels of eIF4E, B-actin and H T K in tumour and non-tumour. Cel l lines were grown to 70% confluency on 10 cm petri-Plat-Es, the medium was removed from cells and cells were rinsed with 5 mis o f ice-cold l x PBS . Cells were lysed with 1.0ml of RIP A buffer (10 m M Tr i s -HC l (pH 8.2), 1% deoxycholate, 0.4% E D T A ) for 30 min on ice, and cell lysate was scraped off and placed in 1.5ml microcentrifuge tubes. Cel l lysate was centrifuged at 12 000 g for 10 min, pelleting cellular debris, the resulting supernatant was removed and assayed for protein concentration using B C A Protein Assays (Pierce). A 20 ug sample of protein lysate was added to 9\xl of 6x sample buffer and brought to a final volume of 54u.l with R J P A buffer. The samples were mixed and heated at 100°C for 3min. 40ul samples were loaded on 12% sodium dodecyl sulfate polyacrylamide gel (SDS -PAGE) , composed of a 5% stacking gel (5ml volume - 0.85ml 30% polyacrylamide, 0.625ml 1 M Tris(pH 6.8), 0.05ml 10%o ammonium persulfate, 0.05ml 10% SDS, 0.005ml T E M E D and 3.4ml of H 2 O ) and a 12%> separating gel (15ml volume - 6.0ml 30% polyacrylamide, 3.75ml 1.5M Tris(pH 8.8), 0.15ml 10% ammonium persulfate, 0.15ml 10% SDS, 0.006ml T E M E D and 4.95ml of H 2 0 ) , and run for 2hrs at 120V. See Blue 2 (Invitrogen) ladder was run with the samples to quantify band weight. Once ample seperation had occurred, protein 41 samples were transferred to Imobilon-P transfer membranes (Millipore), at 300mV for 90min. Membranes were blocked with 5% skim milk in tris-buffered saline (TBS) containing 0.05% tween-20 for lh r at room temperature and then probed with 1:1000 rabbit polyclonal anti-eIF4E antibody (Cell Signalling) or 1:500 mouse monoclonal anti-H T K (a gift from Dr Wi l l iam C Summers, Yale University, overnight at 4°C, followed by 3 x 5min washes with TBS-T . Blots were then treated with a 1:2000 polyclonal goat anti-rabbit antibody solution conjugated to horseradish peroxidase (Bio-Rad) was for anti-eIF4E blots and a 1:2000 polyclonal goat anti-mouse antibody conjugated to horseradish peroxidase for a - H T K blots (Bio-Rad). Bands formed for eIF4E, H T K , and P-actin were detected with 1ml of E C L (Amersham Biosciences) and visualized on K O D A K B ioMax Light f i lm. Band intensities were quantified using Quantity One on the BioRad Versadoc 3000 Imaging Sys-tem. 2.6 Herpes thymidine kinase (HTK) and ganciclovir (GCV) cytotoxicity and cell viability assay Two methods were used to determine cell death in cell lines treated with lentiviral vector encoding H T K : Trypan Blue Exclusion Viabi l i ty assay and M T S cell viability assay. For Hyclone cell viability assay, tumour and nontumour cells were seeded in 24-well Plat-Es at a concentration of 2 x l O 4 cells/well in 0.5 ml of medium. After 24 h, cells were infected with lentiviral vectors C M V - T K / U T K , A R R 2 P B - T K / U T K , and the control vector C M V - E G F P , M O I 30. A t 48 h postinfection, G C V was added at concentrations ranging from 0.1 to 100 u M (Sigma). Cel l viability was determined using the Hyclone 42 cell viability assay, 5 days after addition of G C V . Cells were washed with P B S and then suspended in PBS . The cells were treated with an equal volume of 0.4% Trypan blue was added to cell suspension and incubated for 1-2 min. The cells were observed microscopically and the number of unstained cells and total cells was quantified using a hemocytometer. Stained cells represent dead cells, as l iving cells do not allow the Trypan blue to cross the membrane. The percentage viability was determined by dividing the number of unstained cells by the total number of cells counted and the quotient multiplied by 100%. Each experiment was performed in triplicate and an estimation of the G C V concentration equivalent to 50% cell viability was determined through extrapolation on cell survival curves. To confirm the accuracy of the Hyclone cell viability assay, an M T S assay was completed for L N C a P and BPH-1 cell lines infected with C M V - T K and C M V - U T K . The M T S assay used was the Promega Cel l Titer 96 Aqueous One Solution Ce l l Proliferation Assay. A n M T S assay involves the reduction of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt by N A D P H and N A D H into a coloured formazen in metabolically active cells. The tumour and non-tumour cell lines were seeded in 96-well Plat-Es at a concentration of 5 x l O 3 cells/well in 0.1ml of medium. Cells were infected with lenitviral vectors (MOI 30) and at 48 h postinfection, G C V was added at concentrations ranging from 0.1 to 100 u M (Sigma). 48 hours after addition of G C V , Cel l Titer 96 Aqueous One reagent was added to each wel l and the cells were incubated for 1-4 hours under normal conditions. Absorbance was then measured at 490nm on a 96-well Plat-E reader. Concentration values were determined through comparison to a standard curve of known samples. 43 2.7 Immunohistochemical staining of prostate tissue arrays for eIF4E Prostate tissue samples were obtained from the tissue bank of the Department of Pathology at Vancouver General Hospital. Prostate tissue samples were all obtained from radical prostatectomy specimens and were chosen for their representation of various Gleason grades (G2-G5) . Patient demographics, pathology number, paraffin block label, diagnosis, and clinical correlative data were entered in a central database and appointed to the tissue microarray map. Cancer sites in donor paraffin blocks were identified by a pathologist (Ladan Fazli) using matching H & E reference slides and the tissue microarray was constructed using a tissue puncher (Beecher Instruments, Silver Spring, M D , USA) . A tissue microarray is a tool for visualizing the location of target proteins in isolated parafm embedded tissue samples through treatment with fluorescently labeled antibodies and microscopic imaging. Quadruplicate cores (n=272), each 0.6 mm in diameter, from 68 individual cancer patients were arrayed in recta-linear pattern. Sections were deparaffinized by xylene and rehydrated through 100, 90, and 70% ethanol washes, and then transferred to the 0.02% Triton X for permeabalization. Slides in citrate buffer (pH=6) were heated in the steamer for 30 min. After cooling for 30 min and 3 x5 min wash in P B S , the slides were incubated in 3 % H 2 0 2 for 10 min. Slides were transferred to 3 % B S A for 30 min and then incubated at 4°C overnight with anti-eJT4E antibody (Cell Signaling Technology Inc, Beverly, M A , U S A ) at a working dilution of 1/50 in 1% B S A . The following day unbound primary antibody was washed off extensively with P B S and the L S A B + kit (Dako) was used as the detection system. The Chromogen Nova-red (Laboratories) was applied for 2 min and counterstaining was done with Hematoxylin (Vector Laboratories). After rehydration with ethanol, the slides were sealed with 44 coverslips using a xylene-based mounting media, Cytoseal (Stephen Scientific). Negative control slides were processed in a manner identical to that above, with the substitution of 1% B S A for the primary antiserum. Photomicrographs were taken through a Leica D M L S microscope coupled to a digital camera (Photometries C o o l S N A P , Roper Scientific, Inc.) and corresponding computer software. The staining intensity (0-3) was graded by a pathologist (Ladan Fazli) representing the range from little staining to heavy staining. A l l comparisons of staining intensity and percentages were made at x200 magnification. Mann-Whitney statistical analysis was completed to determine the statistical relationship between eIF4E expression levels and Gleason grading. 2.8 RNA isolation and real-time RT-PCR L N C a P and BPH-1 cells, cultured on 20cm Plat-Es, were infected with lentiviral vectors L v - C M V - E G F P and L v - C M V - U - E G F P . A t 5 days post-infection, cells were washed 3 x with P B S to remove excess medium and the total R N A was isolated from the cells using 1ml of Trizol Reagent (Invitrogen). First-strand c D N A was synthesized using 1 ug of total R N A in a 20 ul reverse transcriptase reaction mixture using Superscript II reverse transcription reagents (Invitrogen). The oligonucleotide primers used for both the E G F P gene ( 5 ' - C A A G G T G A A C T T C A A G A T C C and 5'-C C A G C A G G A C C A T G T G A T C G ) and the P-actin gene were designed for real-time P C R using the Primer Express 2.0 software (Applied Biosystems, Foster City, C A , U S A ) . The P-actin housekeeping gene was converted to c D N A for use as a standard to create relative values. Real-time P C R reactions were performed using the A B I prism 7000 Sequence Detection System (Applied-Biosystems) in the presence of SYBR-green in a 25 u.1 45 mixture containing 1/20 volume of c D N A preparation. The optimization of the real-time P C R reaction was performed according to the manufacturer's instructions and protocols. Real-time quantitations were performed using the fold change method as recommended by the manufacturer o f the A B I prism 7000 Sequence Detection System. Relative quantitation of gene expression was performed using the fold change method as described in Talaat et al. Briefly, the normalized value for all amplification runs was calculated by subtracting threshold cycle (C t) values of the target genes from those of £-actin, as the endogenous control. The standard curve for each gene was constructed from the three serially diluted samples in duplicate, starting with the neat c D N A and diluted to 1:5 and 1:25. The relative levels of transcription were determined by comparison of C t values between the experimental group and the control to calculate fold changes. 2.9 Quantification of lentiviral transfecting units using RT-PCR Lentiviral m R N A extractions were completed using the QIAamp V i ra l R N A Preparation kit (QIAGEN) . Lentiviral preparations, previously frozen at -20°, were thawed and aliquots were diluted 1 in 10. 14ul of diluted and neat lentiviral samples were added to 125u,l o f l x P B S . The R N A was then extracted following instructions outlined in the QIAamp Vira l R N A M i n i Handbook. Upon completion of R N A extraction, R N A was converted to first-strand c D N A using the Superscript II reverse transcription reagents (Invitrogen). The c D N A was synthesized using 5 \ig of total R N A in a 20 ul reverse transcriptase reaction mixture. The R N A was added to 3(0.1 random 46 hexamer (50ng/ul), l u l l O m M dNTP mix and dFLO up to lOul , then incubated at 65°C for 5 min. The reaction master mix containing 2ul lOx R T buffer, 4ul 2 5 m M M g C b , 2ul 0.1M DTT, and 1 p.1 RNAaseOUT was then added and the mixture was incubated at room temperature for 2min. 1 ul of Superscript II was added to each tubs and the reaction was incubated at 42°C for 50min and then heat inactivated at 70°C for 15min. 1 u.1 of Rnase H was added and the reaction was incubated for 20min at 37°C. The oligonucleotide primers for both the GagR primer ( 5 ' - G G T T G T A G C T G T C C C A G T A T T T G T C and 5'-G G A G C T A G A A C G A T T C G C A G T T A ) were designed for real-time P C R by Luke B u at the Laboratory of Dr. Wi l lam Jia, University of British Columbia. Real-time P C R reactions were performed using the A B I prism 7000 Sequence Detection System (Applied-Biosystems) in the presence of SYBR-green in a 25 ul mixture containing 1/20 volume of c D N A preparation. To determine the quantity of the viral particles the following calculation was completed. Values produced by the c D N A lentiviral extracts were standardized based on results dtermined through biological assay, as previously described. 2.10 Mouse surgery protocol for direct prostate injection Surgery was completed on both cre+ and cre black 6 mice to allow for direct prostatic injection of viral constructs. M ice were treated subcutaneously with 0.1 ml of mg/ml bupermorphine, an analgesic, and then initially anesthetized through nasal tube with 5mg/ml isoflurane, which is decreased to 2mg/ml during surgery. The abdominal 47 region was shaved and cleaned using a hand held vacuum and disinfected with 70% ethanol. Pedal reflex tests were completed prior to incision to ensure that the mice were properly anesthetized. A 1.5cm incision was made in the outer dermal layer of the lower abdomen and then the abdominal wal l is opened with 1.5cm incision. The injection was targeted to the capsule of the lateral prostate, see figure 7. 15ul injections were administered using a 27 gauge needle attached to a 1ml syringe. The abdominal wal l was sutured closed with 0.015mm silk sutures, outer dermal layer 1 was closed with 0.35mm sutures. Post surgery mice were placed on a head pad, set at medium heat to simulate body temerature. 2.11 Genotyping of PTEN KO Mice Gentotyping was completed to determine the genetic make-up of the P T E N - K O mice in reference to the presence of ere and pten genes. Genomic D N A was extracted from mice tail clippings. Preparation of the tail clippings involved the anesthetization of the mice with 0.1 ml of mg/ml bupermorphine and the removal of ~0.5cm of the tail with surgical scissors. The tails were then soldered closed, and the clippings were frozen at -20°C. Once thawed, the tails were treated with 300ul of extraction buffer (1 OOmM TRIS, 50mM E D T A and 5% proteinase K ) at 75°C for 4 hours or 70°C overnight. The tail clippings were then treated with 300ul d H 2 0 , 300jal phenol:chloroform:isoamyl alcohol (25:24:1), vortexed for 15 sec and centrifuged at 10000 rpm for 5 min. 500ul of the aqueous layer was removed and added to 1.0 ml of 48 Figure 7 - Representative drawing of the mouse prostate. The large black indicates the ventral prostate, site of viral injection. 49 100% ethanol. The samples were centrifuged at 13000 rpm for 15 min, the supernatant was removed and the pellet was dried at room temperature for 30 min. The dried pellets were re-suspended in lOOul of T E buffer and quantified with the N A N O drop 2000. P C R amplification was carried out individually for each gene of interest, ere genomic inclusion was verified using primer sequences, forward 5 ' -C A T T T C T G G G G A T T G C T T A T A A C A C - 3' and reverse 5 ' -T A T T G A A A C T C C A G C G C G G G C C -3 ' , and P C R protocol (94°C for 3min, 94°C for lm in , 55°C for lm in , 72°C for 2min, 34 cycles from the second step, then 72°C for 7min) . P T E N using primer sequences, F -5 ' - C T C C T C T A C T C C A T T C T T C C C - '3 and R -5 ' - T A T T G A A A C T C C A G C G C G G G C C - '3 and P C R protocol (95°C for 5min, 80°C for 3min, 95°C for lm in , 56.5°C for lmin , 72°C for lmin , 34 cycles from the second step, then 72°C for 5min). P C R product was run on a 1% gel at 90V for 50min, the bands were visualized using ethidium bromide intercalation. Both a positive and negative control was run for each genotyping protocol. 2.12 Immunohistochemical staining of prostate tissue arrays for eIF4E P T E N K O mouse prostate tissue samples were collected from the Transgenic Laboratory of the Prostate Centre at Vancouver General Hospital. Prostate tissue samples were obtained from cre+ and ere' mice representing 9 to 20 weeks of their development. Extracted prostate specimens were parafin embedded and the tissue microarray was constructed using a tissue puncher (Beecher Instruments, Silver Spring, M D , U S A ) . Triplicate cores («=114), each 0.6 mm in diameter, from 38 individual mice were arrayed 50 in recta-linear pattern. Sections were deparaffinized by xylene and rehydrated through 100, 90, and 70% ethanol washes, and then transferred to the 0.02% Triton X for permeabalization. Slides in citrate buffer (pH=6) were heated in the steamer for 30 min. After cooling for 30 min and 3 x5 min wash in PBS , the slides were incubated in 3 % H2O2 for 10 min. Slides were transferred to 3 % B S A for 30 min and then incubated at 4°C overnight with anti-eIF4E antibody (Cell Signaling Technology Inc, Beverly, M A , U S A ) at a working dilution of 1/50 in 1% B S A . The following day unbound primary antibody was washed off extensively with PBS and the L S A B + kit (Dako) was used as the detection system. The Chromogen Nova-red (Laboratories) was applied for 2 min and counterstaining was done with Hematoxylin (Vector Laboratories). After rehydration with ethanol, the slides were sealed with coverslips using a xylene-based mounting media, Cytoseal (Stephen Scientific). Negative control slides were processed in a manner identical to that above, with the substitution of 1% B S A for the primary antiserum. Photomicrographs were taken through a Leica D M L S microscope coupled to a digital camera (Photometries Coo lSNAP , Roper Scientific, Inc.) and corresponding computer software. The staining intensity (0-3) was graded by a pathologist (Ladan Fazli) representing the range from little staining to heavy staining. A l l comparisons of staining intensity and percentages were made at x200 magnification. Student T-test statistical analysis was used to determine whether levels of eIF4E were significantly different in cre+ and ere P T E N K O mice (p<0.05). 51 Chapter 3 - Development of a translational regulation for prostate cancer. In order for a gene therapy to be successful it must have a high degree of specificity and regulation. The objective of this study was to develop a transcriptionally and translationally regulated gene therapy system, which would allow for the systemic treatment of invasive and metastatic prostate cancer. Transcriptional targeting is achieved through the addition of the prostate specific modified probasin promoter, A R R 2 P B . A high degree of prostate specificity has been shown in previous work by Y u et al. and others [84-86]. To attain translational regulation, the tumour specific over-expression of eukaryotic initiation factor 4E (eIF4E), a key regulatory factor in the initiation of translation, and the incorporation of a highly secondary structured 5 ' U T R are to be utilized. Hence, we hypothesize that eEF4E over-expression in prostate cancer, particularly those with high Gleason grade, w i l l allow for the differential expression of m R N A containing highly secondary structured 5 'UTRs. 3.1 eIF4E Expression levels in Prostate Cancer Tissue Arrays Increased levels of eIF4E as been shown in a variety of cancers, including breast, head and neck and lung cancers; however, no published data is available showing levels in prostate cancer [89, 90]. To ensure that levels of eIF4e are elevated in prostate tumours, a tissue microarray analysis was completed. 272 tumour cores, representing quadruplet sampling from 68 patients, were analysed and graded based on the amount of eIF4E expression. The levels of eEF4E expression are based on relative 52 immunohistochemistry staining intensities and graded on a scale ranging from 0 (no staining present) to 3 (intense staining). The assessment and grading of the tumour cores was completed by Dr. Ladan Fazl i , a research pathologist at the Prostate Research Centre, Vancouver General Hospital. Regions of normal prostate cell growth in the cores are designated as negative or non-tumour controls, and their use as controls is due in part to the difficulty in attaining normal prostate samples. Figure 8 is a representative image for immunohistochemistry results and corresponds to staining of a G3-G4 Gleason grade core and is a 3 on the intensity scale. The large and small arrows represent areas of intense and no staining, respectively. eIF4E was found to be generally localized to the cytoplasm with a few incidences of nuclear staining observed, as has been reported in tissue microarrays from other cancer sources [89]. 54% of the tumour cores tested had moderate to strong staining, 43% of the tumour cores tested weakly positive, and 3 % had no visible staining. The greatest differences in levels of staining were found through comparisons of tumour cells to non-tumour cells on the same core sampling. In 82% of patient samples, which show areas of moderate to strong staining, 45% of adjacent non-tumour cells showed no-visible staining and 37% showed weak positive staining. It is important to note that some staining is expected in non-tumour cells due to the requirement of eIF4E for protein production in normal cell house keeping, metabolism and growth. It is also important to note that not all prostate tumours are going to have the same level of protein expression. This is related to the multiple methods of prostate cancer formation and the heterogeneity of prostate cancer. 53 Figure 8 - A 400x magnification of a representative immunohistochemical stain for eIF4E in human prostate cancer microtissue arrays. 272 tumour cores representing 68 patients were analyzed for relative levels of eIF4E expression using antibodies to eIF4E. The thick arrow and thin arrows highlight immunoreactive tumour cells and nonimmunoreactive adjacent normal cells, respectively. 54 Mann-Whitney statistical analysis showed no significant difference in median staining levels of eIF4E expression with increasing Gleason grade, table 3. Currently, P S A levels and Gleason grading are the biomarker for prostate cancer development, further investigation may show eIF4E to be more efficient and less subjective method of screening. These results are indicative of eIF4E over-expression in tumour cells, relative to non-tumour cells, and its validity as a possible target, or tool, in the development of a gene therapy for prostate cancer. 55 <M> Distribution of e IF4E Immunoreactivity Scores Gleason Grade 0 + -H- +++ n 2 0 50.0 20.0 30.0 10 3 0 32.1 25.0 43.7 16 4 0 42.8 23.8 33.4 21 5 0.1 28.6 47.6 14.3 21 Table 3 - Comparative values for percent distribution of eIF4E immunoreactive cells referenced to the Gleason grades of encompassing prostate tissue. 56 3.2 eIF4E expression levels in cancer and non-cancer cell lines After confirming over-expression of eIF4E in prostate tumour samples through tissue microarray, we sought to test the hypothesis that cancer cell lines express higher levels of eIF4E compared to non-cancer cell lines. To determine tumour specificity and verify translational regulation, cell lines chosen must represent both cancer and non-cancer cell lines. As well , to test the prostate specificity and transcriptional regulation, both prostate and non-prostate cell lines must be chosen. Finally, of the cell lines chosen, levels of eIF4E must be verified and the tumour cells chosen must have elevated expression levels. Eight cell lines were chosen for this experiment based on satisfying the above criteria and upon their availability. O f the eight cell lines chosen, three are prostate cancer cell lines (LNCaP, P C 3 - M , DU145), two are non-cancer prostate cell lines ( B P H -1, 267-B1), one is a breast cancer cell line (MCF-7) and two are non-cancer, non-prostate cell lines (Plat-EE, Huvec-c). To measure eIF4E expression levels western blot analysis was conducted, figure 9, on 20u.g of protein extract from each of the cell lines. The western blot was also probed for P-actin to standardize the gels, allowing for relative levels to be determined through density scan, a graphical representation can be seen in figure 9. Western blot results showed increased levels of eIF4E expression in malignant cell lines when compared to non-malignant cell lines. Prostate cancer cell lines; L N C a P , and DU145 show relative eIF4E levels of 0.81 and 0.62, respectively. M C F - 7 , a breast cancer cell line showed and increased level of 0.97. P C 3 M , a prostate cancer cell line, showed minimal expression, 0.37, relative to other prostate cancer cell lines. Non -57 malignant cell lines (BPH-1 , Huvec-c and 267-B1) did not showed non-elevated levels of eIF4e in comparison to the cancer cell lines, 0.17, 0.21 and 0.10, respectively. Plat-E, an embryonic kidney cell , showed a high level of eIF4E expression, 0.95. For Plat -EE, increased level of expression can be accounted for by high protein synthesis in embryonic cell lines. A l l though Western blot analysis is useful in determining total eIF4E levels, the state of the eIF4E, whether active or inactive due to binding with eIF4E binding protein, cannot be directly determined. This is due to the presence of the eIF4e regulatory protein 4E -BP , which binds to prevent eIF4E from joining into the eIF4F complex through direct interactions with eIF4G. 58 f $ $ f t <$ f $ Figure 9 - a) Western blot analysis of eIF4E level in cancer and non-cancer cell lines. Protein extracts from cancer cells (LNCaP, M C F - 7 , P C - 3 M , and DU145) and noncancer cells (BPH-1 , Plat-E, Huvec-c, and 267-B1) were probed with antibodies to eIF4E and (3-actin. b) Relative eIF4E expression levels as determined through ratio of band densities of eIF4E and p-actin (means±s.e.m., n=3). 59 3.3 Determination of eIF4E activity in cancer and non-cancer cell lines Specificity is one of the major goals in the development of a gene therapy. This project is attempting to produce specificity through translation regulation based on over-expression of eIF4E. Previous work has shown a differential level of expression of proteins markers through the incorporation of a 5 ' U T R with a highly secondary structured U T R [116, 117]. We hypothesized that through the incorporation of a 5 ' U T R sequences into a therapeutic m R N A , expression of the therapeutic m R N A , and its subsequent therapeutic protein, would be limited to cancer cells over-expressing eIF4E or cells with hyper active eIF4E. The 5 ' U T R inserted into our viral constructs was originally cloned from F G F - 2 and give to the laboratory by Dr. A.DeBenedetti. To be able to visualize and quantify the specificity of our gene therapy, preliminary in vitro analysis was completed using lentiviral vectors expressing GFP proteins. Since previous work had focused on the prostate specificity of the A R R 2 P B promoter, C M V regulated vectors were utilized for this set of experiments [83]. Ce l l lines were infected with both C M V - E G F P and C M V - U E G F P and depending on the extent of eIF4E expression and activation, a differential level of E G F P expression was expected. Tumour and non-tumour cell lines were grown to 70% confluency and then infected with the appropriate virus and incubated for 4 days. Within 24 hours of incubation E G F P was detectable, reaching a maximum E G F P expression level at 4 days. A t 4 days, infected cells where visualized using flourescent microscopy (figure 10 and 11), and at which time cells were isolated for E G F P expression quantification using flow-cytometry (figure 10 and 11). 60 CMV-EGFP CMV-UEGFP LNCaP MCF-7 PC3M DU145 W 10' 1°' 1° ] lid I i n ' m1 m a in* inH Figure 10- E G F P expression levels after infection of tumour cell lines with C M V - E G F P and C M V - U E G F P lentiviral vectors. E G F P expression levels were observed qualitatively under fluorescence microscopy and quantified using F A C S analysis. Cel l lines (a) L N C a P cells; (b) M C F - 7 cells; (c) DU145 cells; and (d) P C - 3 M cells, were infected (MOI=30) by lentiviral vector L v - C M V - E G F P (left) and L v - C M V - U - E G F P (right). The numbers shown are the means of triplicate experiments. 61 CMV-EGFP CMV-UEGFP Huvec-c PlateE 1 I 1 " i'o ! , Ml Figure 11 - E G F P expression levels after infection of non-tumour cell lines with C M V -E G F P and C M V - U E G F P lentiviral vectors. E G F P expression levels were observed qualitatively under fluorescence microscopy and quantified using F A C S analysis. Cel l lines (a) BPH-1 cells; (b), 267-B1 cells; (c) Huvec-c cells; and (d) Plat-E cells., were infected (MOI=30) by lentiviral vector L v - C M V - E G F P (left) and L v - C M V - U - E G F P (right). The numbers shown are the means of triplicate experiments. 62 Following four days of infection with C M V - E G F P all cell types expressed high levels of E G F P (figure 10 and 11). After four days infection with C M V - U E G F P , only tumour cell lines (LNCaP, M C F - 7 , Du l45) showed no significant difference in levels of E G F P expression. P C 3 M cell line did show a significant difference in E G F P expression with both lentiviral vectors, as determined by a Student T-test (p<0.05). A s shown in figure 11, significantly lower levels of E G F P expression could be seen with non-tumour cell lines (BPH1, Plat-E, Huvec-c, 267-B1). Generally, these results correlate to levels of eIF4E found during western blot analysis. There are a few divergences with these results. P C 3 M showed a significant level of E G F P expression for both C M V - E G F P and C M V - U E G F P infection. However, levels of E G F P are very close to being not significantly different, and are much higher than levels seen in non-tumour cell lines. This result contrasts the results observed with western blot analysis for levels of eIF4E, where P C 3 M expression levels fall in a similar range as non-tumour cell lines. One possible reason is the state of activation of eIF4E. P C 3 M is a P T E N null cell line and, as discussed in the introduction, regulation eIF4E activity is through binding with eIF4E-BP. Binding of eIF4E is negated through phosphorylation by mTOR, which lies down stream of PI3K. P I3K is repressed by P T E N ; therefore, with decreased or no Pten activity, the P I3K pathway is an open flood gate, m T O R can continuously phosphorylate eIF4E-bp, leading to hyper-activation of eIF4E. There may not an elevated level of eIF4E expression; however, hyper-activation may have similar consequences. The development of an assay that measures the activity of the eIF4E would allow for determining whether hyper-activation has the same effects as over-expression with the insertion of a 5 'UTR. 63 Plat-E western results indicated an increased level of eIF4E. This result must be taken into consideration when entering further trials. The presence of stem cell recognition and expression through this method may not matter as much considering the demographic of prostate cancer sufferers. Also our intention is to direct expression solely to the prostate through incorporation of a prostate specific promoter. 3.4 RT-PCR determination of translational regulation One issue of concern is whether differential expression in tumour and non-tumour cell-lines is due to differences in translational control or i f expression levels are related to transcription efficiency of infected cell lines, where it may be assumed that tumour cell lines are more transcriptionally active. One possibility is that decreases in the non-tumour cell line of E G F P expression, with infection of C M V - U E G F P , are due to transcriptional changes presented through introduction of a 5 'UTR. In this situation, targeting of eIF4E over-expression through 5 ' U T R insertion may have no effect on the level of E G F P activity. To determine that translational control has occurred, quantitative R T - P C R was completed for L N C a P and BPH-1 cell lines infected with C M V - E G F P and C M V - U E G F P , results shown in table 2. Values for each cell line, with both infections, were normalized to B-actin m R N A expression, producing relative values for comparison. E G F P m R N A levels in L N C a P cells infected with C M V - E G F P and C M V - U E G F P produced a mean ratio of 2.17±0.32, when comparing E G F P m R N A levels for C M V -U E G F P to C M V - E G F P . In B P H - 1 , the mean ration for E G F P levels was 17.2±0.32 in BPH-1 infected cell lines. These results show that the level of E G F P transcription in 64 C M V - U E G F P infected cell lines was actually higher than in C M V - E G F P . Thus, showing that levels of E G F P activity were due to differences at a translational level and not a transcriptional one. 3.5 Conclusion Our hypothesis stated that by taking advantage of cellular over-expression of eIF4E we would be able to create a translationally regulated, virally delivered gene therapy for the specific treatment of prostate cancer. eIF4E over-expression has been documented in a variety of tumour cell lines, in order for the translational regulation to be applicable, levels of eIF4E expression were determined for prostate cancer [99-103]. The tissue micro-array results showed a moderate to high level of eIF4E expression in more than 90% of prostate cancer tumour cores. A l l though there was no significant relationship between eIF4E levels of over-expression and Gleason grade, there was a significant difference between tumour and the neighbouring non-tumour cells observed. Infection of tumour and non-tumour cell lines with lentiviral vectors expressing C M V - E G F P or C M V - U E G F P indicates that incorporation of a 5 'UTR, w i l l limit expression of reporter genes to cell lines over-expressing eIF4E. Plat-E E and P C 3 - M cell lines are anomalous to theory. In the case of P C 3 - M , no over-expression of the eIF4E was observed; however, expression levels of E G F P when infected with the C M V -U E G F P , although not statistically significant, were closer to levels seen with C M V -E G F P than with non-cancer cell lines that expressed comparable levels of eIF4E. These results are most l ikely due to a hyper-activation of the eIF4E through the phosphorylation 65 of the regulatory protein, 4E -BP . The basis for eIF4E utilization is the availability of free eIF4E for binding to the 5 ' m R N A cap of 5 'UTR, and hyper-activated eIF4E could have the same effects as an over-expression of eIF4E. Plat-E, embryonic kidney cell line, showed elevated levels of eIF4E expression and a significant difference in E G F P expression when comparing infections with C M V - E G F P to C M V - U E G F P . eIF4E is required for normal growth and development of cells, which may explain the high levels seen in Plat-E cells. It is possible that the lower level of E G F P expression is due to eIF4E being in an inactivated state, due to binding with the regulatory protein, which would have the opposite effect as seen with the P C 3 - M cell line. Completion of the R T - P C R study showed that differences in E G F P expression are due to non-transcriptional activities, such as hypothesized with the incorporation of the 5 ' U T R and cellular over-expression of eIF4E. These results support the insertion of a 5 ' U T R as a method of translation regulation and tumour specific expression. 66 Chapter 4 Therapeutic effects of 5'UTR insertion Differential levels of E G F P expression were observed in cancer and non-cancer cell lines infected with lentiviral vectors containing C M V - E G F P or C M V - U E G F P constructs, relative to the level of eIF4E expression. These results give indication that the over-expression of eIF4E is a good cellular tool for developing a novel and effective cancer gene therapy that w i l l specifically k i l l cancer cells while sparing most non cancer cells. Previous work carried out in breast cancer, and breast non-cancer cell-lines, showed that inserting a 5 ' U T R upstream of herpes thymidine kinase (HTK) could provide a means for differential ki l l ing of cells that over-express eIF4E [116, 117, 129]. There are also reports that delivery of H T K through a viral vector is suitable for treatment of a variety of cancer cell-lines [130]. Viral-mediated transfer o f H S V - T K gene has been shown by several investigators to confer sensitivity to nucleoside analogs such as G C V in a variety of tumour cells [131]. Our hypothesis is that by marrying a highly secondary structured 5 'UTR with the effective delivery system of the lentiviral vector and the therapeutic abilities of H T K we w i l l develop a translationally regulated, systemic approach to treatment of advanced and metastatic prostate cancers. 67 LNCaP \ \ c \ 1 1 i *• C M V - E G F P - C M V - T K C M V - U T K i 3 8 0 % I 1 6 0 % l 4 0 % 2 0 % 0 % 0 . 1 1 G C V ( U M ) PC-3M - f ~ - - - ^ > —•— C M V - E G F P » C M V - T K • C M V - U T K 0.01 0.1 1 G C V ( H M ) 10 100 MCF-7 0.1 1 GCV(uM) Figure 12 - Percent cell viability o f cancer cells treated with G C V after with L v - C M V -T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors. After infection with L v - C M V -T K , L v - C M V - U T K , or L v - C M V - E G F P (control), cells were treated with different concentrations of G C V (0.01-100 ^ M ) 48 h later. Percent cell viability was measured using dye exclusion assay at day 5 after G C V was added. Assay was completed in triplicate and results represent the mean±s.e.m. 68 CMV-EGFP CMV-TK CMV-UTK Figure 13 - Bright field view of cancer cells treated with G C V after infection with L v -C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors. After infection with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P (control), cells were treated with different concentrations of G C V (0.01-100 uM) 48 h later. 6 9 4.1 Differential cell killing using lenitviral vectors expressing herpes thymidine kinase To determine the effectiveness of the viral vector as a gene therapy, E G F P genes were replaced with H T K , creating L v - C M V - T K from L v - C M V - E G F P and L v - C M V -U T K from L v - C M V - E G F P . H T K containing viral vectors, along with a L v - C M V - E G F P control, were used to infect a panel of both tumour and non-tumour cell-lines (MOI = 30), as in the previous E G F P expression experiments. Cel l lines were infected and treated as above. A cell death/cell viability assay was completed a varying concentrations of gancyclovir (GCV) to determine the effectiveness of the viral gene therapy. The dose response curves to G C V after infections with L v -C M V - T K , L v - C M V - U T K or L v - C M V - E G F P are shown in figure 12. L v - C M V - E G F P is used as a control to determine background toxic effects of G C V treatment. Figure 13 represents bright field images of cancer cell cultures, at 400x magnification. The concentration at which the cell death is most apparent is indicative of the relative level of H T K expression. For example, cell death at a lower concentration indicates a higher level of H T K expression available to react with the limited G C V . For each assay, a G C V concentration representing 50% viability, or 50 % cell death, was calculated and used to reference cell lines, the apparent IC50. The apparent IC50 for L N C a P prostate cancer cells was seen at a G C V concentration of -0.01 u M after infection with either L v - C M V - T K or L v - C M V - U T K , figure 12. This is the expected result with L N C a P cel l lines due to the over-expression of eIF4E. L N C a P infection with the E G F P control virus did not show a high level of G C V toxicity at any concentration (eg. only - 2 5 % cell k i l l at 100 u M G C V ) . Depending on the cell line concentrations of G C V can be toxic, regardless of 70 Figure 14 - Expression of H S V - T K after infection with different lentiviral vectors, (a) Western blot for H S V - T K in cancer (LNCaP, M C F - 7 , and P C - 3 M ) and noncancer (Plat-E, B P H - 1 , 267-B1 and Huvec-c) cells infected with L v - C M V - T K (right band) and control L v - C M V - E G F P (left band). Protein extract from M M 3 M G ( t k ) cells were a positive control, (b) Western blot for H S V - T K in the same cell lines after infection with L v - C M V - U T K . (c) Densitometer scans (means±s.e.m., n=3) H S V - T K / p - a c t i n expression in cancer and noncancer cells infected with L v - C M V - T K and L v - C M V - U T K . 71 BPH-1 120% | 0% 4- 1 , , , 1 0 0.01 0.1 1 10 100 GCV(uM) 267-B1 1 2 0 % , GCV(uM) Figure 15 - Percent cell viability of non-cancer cells treated with G C V after with L v -C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors. After infection with L v -C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P (control), cells were treated with different concentrations o f G C V (0.01-100 uM) 48 h later. Percent cell viability was measured using dye exclusion assay at day 5 after G C V was added. Assay was completed in triplicate and results represent the mean^s.e.m. 72 CMV-EGFP CMV-TK CMV-UTK BPH-1 G C V I u M 267-B1 G C V I u M Huvec-c G C V I u M t 1 Figure 16 - Bright field view of non-cancer cells treated with G C V after infection with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors. After infection with L v - C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P (control), cells were treated with different concentrations of G C V (0.01-100 uM) 48 h later. 73 H T K expression, using the C M V - E G F P control allows the ability to discern between intentional toxicity and inherent toxic effects due to the G C V [117, 132]. These results also correlate to the level of H T K expression seen through Western blot analysis, figure 14. Proteins extracts were prepared at 4 days post-infection and analyzed with Western blotting for expression of H T K , expression values were determined relative to (3-actin levels. As can be seen in figure 14, all of the cells infected with L v - C M V - T K express high levels of H S V - T K relative to M M 3 M G ( t k ) cells, a breast cell line that stably transfected with H T K [133]. B y comparison, after infection with L v - C M V - U T K , only cell lines expressing a higher level of eJT4E exhibited visible levels of H T K expression, figure 14. In these cells, the amount of H T K relative to (3-actin was slightly less after infection with L v - C M V - U T K compared with L v - C M V - T K , figure 14. The relative level of H T K protein did not directly correlate with the amount of H T K m R N A measured by real time PCR. For example, L N C a P prostate cancer cells infected with L v - C M V - U T K expressed only 24% (average fold induction, 33.9) as much H T K m R N A as the same cell line infected with L v - C M V - T K (average fold induction, 138.3). The reasoning for the differences in m R N A expression is unknown and is a point for further study. However, regardless of the differences in m R N A levels, translation regulation still occurred limiting the H T K expression, in cell infected with C M V - U T K , to cell lines over-expressing eIF4e. P C 3 M prostate cancer cells were less sensitive to the drug, showing an apparent I C 5 0 at 0.1-1 u M G C V ; however, the viability curves for both L v - C M V - T K and L v -C M V - U T K infections were similar to those seen with LNCaP . A s discussed previously, eIF4E levels are not elevated, but thought to be over-activated in P C 3 M cells. M C F - 7 breast cancer cells were generally less sensitive to the ki l l ing effects of G S V , apparent 74 0 0.01 0.1 1 10 100 GCV Cone (uM) Figure 17 - M T S assay for L N C a P and BPH-1 cell lines treated with G C V after with L v -C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P lentiviral vectors. After infection with L v -C M V - T K , L v - C M V - U T K , or L v - C M V - E G F P (control), cells were treated with different concentrations of G C V (0.01-100 uM) 48 h later. M T S assay was completed at day 4 after G C V was added. Assay was completed in triplicate and results represent the mean± s.e.m. Values for C M V - T K and C M V - U T K were calculated by dividing their respective absorbance with absorbance values for C M V - E G F P infection at the same G C V concentrations. 75 IC50 at 5-7 u M G S V , regardless of the lentiviral vector used in the experiments. Since infected M C F - 7 cells expressed high levels of T K (figure 14), the difference in sensitivity is l ikely not due to transcriptional or translational efficiencies. Previous work has shown a decreased sensitivity of the M C F - 7 cell line to H T K / G C V therapy [134]. Non-cancer prostate cell lines, BPH-1 and 267-B1, and Huvec-c cell lines displayed similar sensitivity to G C V , apparent I C 5 0 at 0.1 u M G C V , when infected with L v - C M V - T K (figures 15 and 16). However, cell death levels were similar between the EGFP-control and L v - C M V - U T K vector, which is an expected result due to the lack of eIF4E over-expression. Plat-E embryonic kidney cells, which express relatively high levels of eIF4E and were more sensitive to G C V (<1 uM) than was expected. This result indicates that other factors may play a role in the sensitivity of a cell line to H T K / G C V treatment. Figure 17 shows an M T S assay that was completed on L N C a P and BPH-1 cell lines to verify that results found with the hyclone cell viability assay. As seen with the hyclone assay, the level of cell death observed in L N C a P cells treated with both C M V -T K and C M V - U T K is similar, due to the high level of eIF4E expression. BPH-1 cells infected with C M V - U T K showed no death effects greater than that seen with the C M V -E G F P control and the C M V - T K gene did show cell death after treatment with G C V . 76 4.2 Targeting prostate cancer cells using lentiviral vectors expressing thymidine kinase under a prostate specific promoter To combine all factors explored in this project and develop the ultimate therapy for treatment of prostate cancer, the probasin based promoter ARR2PB was inserted upstream o f the H T K gene, both with and without the 5 'UTR. L N C a P and M C F - 7 cell lines were used to test the efficiency of the transcriptionally and translationally targeted system. N o non-malignant prostate cell lines were available as controls for this experiment. In order for a cell line to be considered a non-malignant control it must have a high level o f A R expression to take advantage o f the prostate specific A R R 2 P B promoter. Non-tumour prostate cell lines investigated did not allow for the transcriptional expression levels seen in the malignant cell lines. Previous work has shown that prostate specific transcriptional regulation can be achieved with lentiviral vectors through the insertion of probasin-derived promoter, A R R 2 P B , upstream of the therapeutic and reporter genes [83, 84]. It was important for our present research to determine whether insertion of a 5 ' U T R has any effect on the transcriptional specificity and efficiency of the A R R 2 P B promoter. L N C a P prostate cancer cells and M C F - 7 breast cancer cells were infected with L v - A R R 2 P B - T K , L v -A R R 2 P B - U T K or with an L v - A R R 2 P B - E G F P control and tested for the level of cell death after treatment with G C V . Figure 18 shows the survival curve for L N C a P and M C F - 7 cell lines infected with prostate specific lentiviral vectors. Ce l l death was observed in L N C a P cells infected with either L v - A R R 2 P B - T K or L v - A R R 2 P B - U T K at a concentration of 0.1 u M G C V . A n apparent IC50 was observed at a G C V concentration of 77 -0 .5 u M for L N C a P cells after infection with either L v - A R R 2 P B - T K or L v - A R R 2 P B -U T K . A minimal level o f cell death was observed in cells treated with the L v - A R R 2 P B -E G F P control virus indicating that cells are largely resistant to the toxic effects of G C V at the concentrations used. A decrease in G C V sensitivity is observed through the utilization of A R R 2 P B promoter compared with C M V controlled expression. The decreased GCV-sensit ivity in L N C a P reflects the relative promoter strengths of A R R 2 P B and C M V [135]. However, there was no evidence o f decreased e IF4E /UTR related expression due to the change in expression levels thorough insertion of the prostate-specific promoter. Under identical experimental conditions, M C F - 7 cells, figure 18, show obvious difference in G C V dose response using any of the A r r 2 P B regulate lentiviral vectors, which is to be expected when using a prostate specific promoter. These results highlight that there is no effect on the transcription of U T R containing m R N A with change to a prostate specific promote. 4.3 Conclusion Insertion of the H T K gene-therapy into the lentiviral vector containing a 5 'UTR has shown to be an effective method for the specific treatment of cancer cells. The levels of T K expression were analogous to those observed with E G F P expression, including a high level of expression in prostate cancer cell lines. The levels of H T K protein seen Western blot analysis correlated with the results seen in the cell-death assay, where both cancer and non-cancer cell lines expressed high levels of H T K when infected with C M V -T K , but only cancer cell lines expressed high levels of H T K when infected with C M V -U T K . The concentration of G C V required for treatment of non-cancer cells with C M V -78 U T K was more than a 100-fold higher than required for cells infected with C M V - T K . In fact, cell death levels seen with infection of non-cancer cells with C M V - U T K were comparable to levels with infection of C M V - E G F P control. L N C a P cells were the most sensitive to infection with C M V - U T K , having an apparent IC50 of -0.1 u M . In contrast, 2 non-cancer prostate cell lines, BPH-1 and 267-B1, infected with C M V - U T K had apparent IC50 values greater than 10uM. These results indicate that translational regulation can be achieved through the insertion of a 5 'UTR for treatment of tumour cells over-expressing eIF4E. Transcriptional control using the A R R 2 P B promoter has been proven in the past facilitate prostate specificity [83, 84]. Our work has shown that incorporating the A R R 2 P B promoter upstream of the H T K gene-therapy does not hinder H T K activity, regardless of the presence of a 5 'UTR. In order to further validate both the transcriptional and translational specificity, we must find a system that w i l l allow us to test non-tumour prostate cells, which express a high enough level of androgen receptor to allow for transcription. The bulk of this data has been published by Y u et al. in 2006 [45] 79 LNCaP « 60% o § 40% u £ a. 20% 0% J , r — , 1 0 0.01 0.1 1 10 100 GCV(uM) F igu re l8 - Effects of a prostate-specific promoter on cell k i l l ing using the H S V -T K / G C V system. L N C a P and M C F - 7 cells were infected with L v - A R R 2 P B - T K , L v -A R R 2 P B - U T K , or L v - A R R 2 P B - E G F P (control) and treated with different concentrations of G C V (0.01-100 uM) 48 h later. The percent cell viability was measured using dye exclusion assay at day 5 after G C V was added. Results are shown as mean±s.e.m. (n=3). 80 Arr 2 PB-TK Arr 2 PB-UTK LNCap G C V I L I M MCF-7 G C V I L I M Figure 19 - Bright field view of a prostate-specific promoter on cel l k i l l ing using the H S V - T K / G C V system. L N C a P and M C F - 7 cells were infected with L v - A R R 2 P B - T K , L v - A R R 2 P B - U T K , or L v - A R R 2 P B - E G F P (control) and treated with different concentrations of G C V (0.01-100 uM) 48 h later. 81 Chapter 5 Preparation for in vivo analysis of the gene-therapy model The purpose of the research discussed in this chapter is to lay groundwork for future in vivo experiments that w i l l be completed by our research group. The availability of an in vivo model for testing the efficacy of a novel gene-therapy, or any proposed treatment, is crucial to the overall success of any biomedical research project. A n in vivo model allows for testing in an environment that more closely represents the eventual targeted population, i.e. the human body. Furthermore, an in vivo model allows for determination of toxic effects, immune system interference and the best mode of deliverance, which are factors in deciding treatment properties to be applied. For many experiments, the next step would be to use a xenograft mouse model, which is a fairly cost effective and accessible option. However, xenografts w i l l not be a viable option for this study. In our research, a xenograft does not allow for the determination of tumour specificity because we are unable to develop a non-tumour environment. Therefore, we have turned to the P T E N K O mouse as a possible model for determining the efficacy of our gene therapy. Unl ike the xenograft model, a negative control, or non-tumour prostate, is available though the use of ere mice. As explained in the introduction, the ere' mouse line is a transgenic mouse that contains the P T E N flanked by flox sites; however, the ere gene has not been successfully introduced into their genome and excision of the P T E N gene cannot occur. The P T E N K O mouse also provides tumour progression and development similar to human prostate cancer and a functional immune system, which w i l l allow for measurement of the immune systems impact upon the administered viral vectors efficiency. Our hypothesis is that the prostate-specific P T E N 82 K O mouse w i l l provide a suitable in vivo model for testing our transcriptionally and translationally regulated gene-therapy. The next section describes experiments completed to determine the suitability of the P T E N K O mouse model for our in vivo experiments. A benefit of the P T E N K O mouse is the availability of a cell line derived from a lymph node metastasis, the L N M e t cell line, which is currently available for use as an in vitro model of the P T E N K O mouse prostate tumour. 5.1 eIF4E levels in PTEN KO One important characteristic for our in vivo model to possess is the over-expression of eIF4E with tumour development. Figure 20 shows a Western blot analysis for eIF4E levels of cre+ and ere- P T E N K O mice. Prostate tumour protein extracts were collected from cre+ and ere- P T E N K O mice ranging in age from 12 to 14 weeks, malignant growth is considered to be in the invasive carcinoma stage of tumour progression. The relative intensities of the bands are determined through comparisons with fj-actin levels. These results show an increased level o f eIF4E expression in the four cre+ P T E N K O mice tested compared to ere- P T E N K O mice, which show a lower level of eIF4E expression in three of four mice assayed. It is unknown why there is over-expression of eIF4E in the single ere' mouse, it may be due to spontaneous events or, more likely, due to an error during genotyping. It appears the mice are too similar in age to produce any significant difference in the level of eIF4E expression. Only a small representative sample was analyzed with 83 Western blot technique, a greater selection is verified in immunohistochemistry experiments. 5.2 Immunohistochemistry for eIF4E Tissue micro-array analysis was completed on prostates of P T E N K O cre+ and ere mice ranging in age from 9 to 20 weeks. The arrays were blotted with a-eIF4E antibody to determine the expression levels of eIF4E over a broader range of the P T E N K O mouse population than was observed with the Western blot analysis. 38 individual mouse prostates were isolated and three separate core samples were completed for each (n=l 14). Immunohistochemistry results were scored form 1 to 3 based on the relative intensity of the eIF4E associated staining. The assessment and grading of the tumour cores was completed by Dr. Ladan Fazli , a research pathologist at the Prostate Research Centre, Vancouver General Hospital. P T E N K O ere' negative controls for each time frame were included in the micro-array analysis and used to determine the presence of eIF4E over-expression. The data from the immunohistochemical staining indicates that there was a significant difference (p<0.05) in the expression levels of eIF4E in cre+ compared to ere' P T E N K O mice at 12weeks. However, tumour extracts at 9-weeks and 15-weeks showed no significant difference between the two genotypes. The results seen at 12 weeks support our earlier results with Western blot analysis. It is unknown why there are differences in the results of the Western blot and 84 elF4E Figure 20 - a) Western blot analysis of eIF4E level in P T E N K O mice prostate extracts. Protein extracts were probed with antibodies to eIF4E and p-actin. b) Relative eIF4E expression levels as determined through ratio of band densities of eIF4E and p-actin (means ±s.e.m., n=3). 85 the tissue micro-array, as seen at 9 and 15 weeks. The same antibody was used for both the Western blot analysis and the immunohistochemistry. 5.3 In vivo testing of viral infectivity and promoter expression abilities In vivo experiments were completed infecting both cre+ and cre P T E N K O mice with C M V - E G F P and C M V - U E G F P ; however, no positive results were obtained. V i ra l infection was attempted through delivery of the virus directly into the ventral prostate capsule (figure 2, page 18), which is a common point of injection for mouse prostate cancer surgery (personal communications with Mary Bowdon and Hoard Tearle). The viral strains were titred using P24 E L I S A and E G F P expression levels were verified through in vitro assays. The final concentration of virus was l x l 0 6 I U / m l , which is a value used by research groups in previous experiments [66, 84, 129, 136, 137]. There are at least two possible avenues of error to take into consideration; inefficiencies in the infectivity of the lentiviral vector in the mouse prostate cells and/or an inability for a high level of molecular expression of the E G F P reporter gene due to the construct of the viral vector. To determine whether the cause is due to an inability o f the lentiviral vector to infect the mouse prostate cells, we must look at the administration of the virus, the preparation of the virus and the suitability of the lentivirus in general for infection of mouse prostates. Figure 21 shows the uptake of blue coomassie stain into the glandular structure of prostate, which was injected in the same manner as done with the viral infections. A n even distribution of the blue coomassie dye was seen throughout the 86 prostate capsule of the ventral prostate. This indicates that the mode of entry was suitable, as has been reported that this mode of infection is a common technique used in mouse prostate experimentation [138, 139]. The next factor is the suitability of the lentiviral vector for targeting to prostate tissue. The lentiviral vector is pseudotyped with V S V - G envelope proteins, which do not provide specificity for any given tissue, giving equal entry to all cell types. The ability for the lentiviral vector to express the reporter gene in the P T E N K O mouse was also taken into question. Previous work published by Chung et al. has shown that mouse cells have an ability to shut down the C M V promoter [140]. A n alternative lentiviral backbone containing an ubiquitin promoter was offered, by the laboratory of Dr C. Ong, to replace the C M V promoter containing backbone. Ubiquitin is a highly conserved protein found in all eukaryotic cells [141]. Previous work has shown the ubiquitin promoter to be highly proficient in mouse cell lines [142, 143]. To determine the efficacy of viral infection prior to further in vivo experiments, work with isolated mouse prostates tissue from the P T E N K O mouse were infected with C M V - E G F P and F U G W . The isolated tissue was harvested and injected with either the C M V - E G F P or F U G W viral constructs; viral titres were equal to l x l 0 6 T U / m l . Qualitative results, shown in figure 22, indicate a larger level of E G F P expression from the lentiviral vectors being controlled by the ubiquitin promoter. This indicates that the ubiquitin promoter is a more suitable option for utilization of the mouse in vivo model. 87 Ventral Figure 21 - Photographic representation showing the distribution of 100u.l of Coomassie Blue Dye. a) Abdominal area of mice after injection after injection of coomassie blue dye, b) Anterior, lateral, dorsal and ventral lobes of an isolated mouse prostate after injection of coomassie blue dye into the ventral lobe. 88 FUG CMV-Figure 22 - Mouse prostate tissue infected ex vivo with F U G W or C M V - E G F P lentiviral strains. Prostate tissue was infected with l x l O 6 T U / m l of the respective virus. Image taken with a fluorescent microscope, 3 days post-infection. 89 Chapter 6 Conclusion and Future Direction Prostate cancer is the most commonly diagnosed cancer in both North American and European men and the second leading cause of cancer related mortality [1]. There is no curative treatment for advanced and metastatic prostate cancer, which makes the development of a novel gene-therapy imperative to patient survival. In the present study we developed a lentiviral based, transcriptionally and translationally regulated gene therapy for treatment of advanced and metastatic prostate cancer. While current therapies are limited in their ability to treat advanced and metastatic prostate cancer, a virally transported gene-therapy system gives the ability for systemic delivery, allowing for targeting outside of the prostate capsule. Furthermore, the addition of transcriptional and translational regulatory controls allow for increased specificity and minimization of toxic effects. The mechanisms of regulation we have incorporated limit the expression of therapeutic gene to prostate tumour tissue, sparing non-prostate and prostate non-tumour cells. Eukaryotic initiation factor 4E (eIF4E) is the rate determining factor in translational initiation and is often over-expressed in tumour cells. eIF4E is a component of the eIF4F translational initiation complex, which acts to unwind m R N A 5 'UTRs, allowing ribosome's access to the translational start codon [6]. Normally low cellular levels of eIF4E create a competition for translation among m R N A with differing lengths of G /C rich 5 'UTRs that form into complex secondary structures. m R N A with shorter less complex 5 'UTRs are more readily translated compared to m R N A with longer more complex 5 'UTRs. Increase in the level of eIF4E allow for a differential increase in the 90 level of complex 5 ' U T R translation compared with less complex more competitive m R N A [89, 90, 144, 145]. Based on previous work by Defatta et al, we have show that the incorporation of a highly secondary structured 5 'UTR, from basic fibroblast growth factor, upstream of herpes thymidine kinase, limits gene-therapy expression to cells over-expressing active eIF4E [116, 117]. Through tissue micro-array analysis, we have been able to observe over-expression of eIF4E in prostate cancer patient biopsies, making prostate cancer a suitable target for our gene-therapy. We have furthered previous work by introducing prostate specific transcription through the insertion of the ARR2PB promoter, upstream of the H T K therapeutic gene, limiting expression of reporter and therapeutic genes to prostate cancer cell lines. The next step w i l l be to show the prostate tumour specificity in an in vivo model. Initial results of eIF4E levels completed on the P T E N K O mice using Western blot analysis indicated that P T E N K O mice would be an ideal in vivo model to test the efficacy of the our gene-therapy. However, further results attained through immunohistochemistry, representing a broader range of both cre+ and cre' were not as conclusive in defining over-expression of eIF4E in P T E N K O mice. Alternatively, rather than over-expression of eIF4E in the P T E N K O mouse, there may be over-activation. The P T E N K O mouse would most likely closely mimic the P T E N null cell line, P C 3 - M , which showed no over-expression in eJT4E and, E G F P expression levels were significantly different when comparing effects from C M V - E G F P and C M V - U E G F P , the level of E G F P expression was much higher than seen with non-cancer cell lines. The P T E N K O mouse may have a hyper-activated eIF4E levels sue to the lack of P T E N , which, as explained previously, could lead to the hyper-phosphorylation and subsequent 91 degradation of the eIF4E inhibitory protein, 4E -BP. A s mentioned earlier, advancement in studies of eIF4E would be aided with the development of an assay that allows for the measurement of eIF4E activity in tumour cells. Insertion of a 5 ' U T R may still allow for the same therapeutic effects as seen with over-expression. When initially collected, the 5 'UTR of F G F was sequenced to ensure that cloning results were correct. After unsuccessful attempts at in vivo assays it was determined that the FGF -2 U T R region should be re-sequenced to ensure the correct sequence was present. These results indicate an incorporation of a 60nt N-terminal external leader sequence from the original F G F sequence, the role of which is to tag the expressed protein for outside of the cell. It is difficult to determine how the incorporation of the N -terminal tag w i l l affect the expression of E G F P protein in vivo. It is possible that either the E G F P may have been exported out of the infected cel l ; however, any effect this had on results would have been relative through out the experiments. Therefore, the addition of the leader sequence should have no effect on the validity of our in vitro results. However, for future experiments, where dosage levels are a major concern, removal of the leader sequence may be necessary. The end result is a need to re-clone the 5 'UTR for insertion upstream of either E G F P or H T K . There are two options; firstly, re-clone the FGF -2 5 'UTR, negating inclusion of the 5' leader sequence, or, secondly, find another 5 'UTR which may provide an even greater degree of specificity for tumour cells over-expressing, or over-activating, eIF4E. To enhance the therapeutic effect, with respects translational regulation, various 5 'UTR sequences can be varied in length and complexity to produce higher levels of tumour specific regulation. A vary diverse range of size and structural complexity in 5 'UTRs is available. It may be possible that a certain 92 combination of size and structure w i l l produce a more intense mechanism for translational control. Recent work has shown that though incorporating a higher degree of G /C content a more complex and less competitive m R N A molecule can be synthesized [107]. The system developed lends itself to treatments of other cancers through the adoption of cell specificity by addition of other tissue specific promoters. For example, utilization of eIF4E over-expression is currently being developed for treatment of melanoma through the insertion of the promoter tyrosinase, laboratory of Dr. Gang L i . There are further uses for the prostate tumour specificity of an A n ^ P b - U - T K viral vector beyond gene therapy. B y replacing the T K with E G F P our research group w i l l be able to take advantage of in vivo imaging that allows for the monitoring of E G F P activity without sacrifice o f the test subject. Through systemic infection o f P T E N K O cre+ mice we wi l l be able to monitor satellite tumour growth to localize areas of l ikely metastasis. The same technology can be used in the development of a reporter/therapeutic combination system. Upon infection, E G F P levels can be observed to determine the viral infection level. During treatment, images can be taken to show the effectiveness of the gene-therapy, as wel l as to determine the prevalence and duration of lentiviral infection. This combination is especially important for the infection of non-tumour cell lines to ensure that lentiviral vectors have infected the cells and that they are transcriptionally active. A s stated earlier, a major factor in the efficiency of a viral gene therapy is the ability to transport a suitable quantity of the genetic element into diseased cells [146]. The use of the lentiviral vector in these experiments was for proof of principal purpose, to 93 show that translational and transcriptional regulation can be achieved and that the gene-therapy could be transported through a viral vector. The results attained here are going to be applied to both the herpes simplex virus and the V S V - G virus. Both these viral strains are preferred when it comes to possible gene therapies. Current work in our research group is focused on the development of prostate specific targeting through the incorporation of monoclonal antibodies to the viral envelope of the V S V - G oncolytic virus. The monoclonal antibodies w i l l be targeted to outer membrane proteins, such as the E G F R , that are over-expressed on prostate cancer cell membranes. A 5 ' U T R w i l l be encoded into early growth genes, which w i l l allow for the selective replication and lysis of cells over-expressing eIF4E. The results observed in this project indicate that insertion of a 5 'UTR region upstream of a therapeutic-gene w i l l allow for translation to be limited to cells over-expressing eIF4E. We have completed the groundwork for the development of a viral based, transcriptionally and translationally regulated gene therapy for treatment of advanced and metastatic prostate cancer. Through the incorporation of a 5 ' U T R and the prostate specific ARR2PB promoter, we have shown the ability to limit treatment to prostate cancer cell lines. 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