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Mechanisms underlying the downregulation of the transporter associated with antigen processing (TAP)-1… Setiadi, Alvernia Francesca 2007

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MECHANISMS U N D E R L Y I N G THE D O W N R E G U L A T I O N OF THE TRANSPORTER ASSOCIATED WITH ANTIGEN PROCESSING (TAP)-l IN CARCINOMAS by A L V E R N I A F R A N C E S C A SETIADI B.Sc., University ofBritish Columbia, 2001 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Zoology) THE UNIVERSITY OF BRITISH C O L U M B I A June 2007 © Alvernia Francesca Setiadi, 2007 11 Abstract Expression of Transporter associated with Antigen Processing (TAP)-l is often lost in metastatic carcinomas, resulting in defective antigen processing and presentation, and escape of the cancer cell from immune surveillance. In this study, the nature of TAP-1 deficiencies in tumors was investigated. By chromatin immunoprecipitation assay, it was shown that the recruitment of R N A polymerase II to the Tap-1 gene was impaired in TAP-deficient, metastatic cells derived from murine melanoma, prostate and lung carcinomas. Levels of TAP-1 promoter activity, as assessed by stable transfections with a reporter construct containing the TAP-1 promoter, were also relatively low in TAP-deficient cells. These suggest that the deficiency in TAP-1 expression resulted—at least partially—from a reduced level of transcriptional activity of the Tap-1 gene. In order to examine genetic heritability of regulators of TAP-1 promoter activity, TAP- and M H C class I-deficient cells of H-2 b origin were fused with wild-type fibroblasts of H-2 k origin. Fusion with TAP-expressing cells complemented the low levels of TAP-1 promoter activity in TAP-deficient cells. However, these fused cells exhibited lower levels of TAP-1 mRNA and H-2 k than unfused fibroblasts. Further analysis showed that TAP-1 mRNA stability was lower in fused carcinoma-fibroblasts than in unfused fibroblasts. Taken together, TAP deficiency in many carcinomas appears to be caused by a decrease in activity/expression of ^ram-acting factors regulating TAP-1 promoter activity, as well as a decrease in TAP-1 mRNA stability. The hypothesis that epigenetic regulation plays a fundamental role in controlling TAP-1 transcription was also tested. Chromatin immunoprecipitation analyses showed that the lack of TAP-1 transcription correlated with low levels of recruitment of a histone Ill acetyltransferase, CBP, and of histone H 3 acetylation at the TAP-1 promoter, leading to a decrease in accessibility of the R N A polymerase II complex to the TAP-1 promoter. These findings lie at the heart of understanding immune escape mechanisms in tumors and suggest that the reversal of epigenetic codes may provide novel immunotherapeutic paradigms for intervention in cancer. IV Table of Contents Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Figures ' viii List of Abbreviations xiv List of Abbreviations xiv Acknowledgments xviii Dedication xx Co-Authorship Statement xxi Chapter 1: General Introduction 1 1.1 Overview of the Immune System 2 1.2 Antigen Presentation 3 1.2.1 Major Histocompatibility Complex (MHC) genetics _3 1.2.2 M H C molecules 5 1.2.3 M H C class I antigen presentation 6 1.2.4 Transporters associated with Antigen Processing (TAP) structure and function 7 1.2.5 M H C class 11 antigen presentation and CD4 + T cells in cancer immunity 8 1.3 Disruption of the MHC Class I Antigen Processing Pathway and the Development of Tumors 11 1.4 TAP-based Immunotherapy 13 1.5 Regulation of TAP-1 Expression 15 1.5.1 Organization of Tap and Lmp genes within the mouse M H C class II locus 15 1.5.2 Possible mechanisms of TAP-1 mRNA downregulation in carcinomas 16 1.5.2.1 Transcriptional mechanisms 16 1.5.2.2 Post-transcriptional mechanisms 16 1.5.2.3 Epigenetic mechanisms 17 1.6 Synopsis of Thesis Objectives, Hypotheses and Results 19 1.7 References 21 Chapter 2 : Identification of Mechanisms Underlying TAP-1 Deficiency in Metastatic Murine Carcinomas 28 2.1 Introduction 28 2.2 Materials and Methods 30 2.2.1 Cell lines 30 2.2.2 Reverse transcription-PCR analysis 30 2.2.3 Chromatin immunoprecipitation (ChIP) assays 33 2.2.4 Cloning of the TAP-1 promoter 33 V 2.2.5 Transfection and selection 34 2.2.6 Generation of pTAP-l-EGFP-transfected clones by FACS . 34 2.2.7 Cell Fusion and FACS analysis 35 2.2.8 Endogenous levels and overexpression of 1RF-1 and IRF-2 in cell lines 35 2.2.9 Luciferase and B-Galactosidase Assays 36 2.2.10 Western Blot 36 2.2.11 Analysis of mRNA stability 37 2.2.12 Real time quantitative PCR analysis 37 2.3 Results 38 2.3.1 Levels of TAP-1 mRNA in CMT.64, L M D , B16, Ltk and R M A cells 38 2.3.2 The recruitment of RNA polymerase II to the Tap-1 gene is lower in the CMT.64, L M D and B16, than in the Ltk and R M A cells 39 2.3.3 Cloning and analysis of the-557 to+1 region of the CMT.64-derived TAP-1 promoter 42 2.3.4 Activity of the -557 to +1 region of the TAP-1 promoter is impaired in TAP-deficient cells 43 2.3.5 Effects of fusions between carcinoma cells and wild type fibroblasts on TAP-1 promoter activity and M H C class 1 expression levels 47 2.3.6 Overexpression of IRF-1 and IRF-2 in CMT.64 cells did not result in significant changes in TAP-1 promoter activity 51 2.3.7 Analysis of TAP-1 expression in unfused and fused cells 54 2.3.8 TAP-1 mRNA stability decreases in carcinoma-fused fibroblasts 56 2.4 Discussion 58 2.5 References 61 Chapter 3 : Epigenetic Control of TAP-1 expression and the Immune Escape Mechanisms in Malignant Carcinomas 65 3.1 Introduction _65 3.2 Materials and Methods _68 3.2.1 Cell lines and reagents ' 68 3.2.2 Reverse transcription-PCR analysis 69 3.2.3 Real-time quantitative PCR analysis 70 3.2.4 Flow cytometry : 71 3.2.5 Chromatin immunoprecipitation assays ; 71 3.2.6 Plasmid construction 71 3.2.7 Transfection and selection 72 3.2.8 Luciferase assays 73 3.2.9 Western Blots 73 3.2.10 Cytotoxicity assays 74 3.2.11 Establishment of HP V-positive cancer xenografts and treatment with Trichostatin A 75 3.3 Results _ 7 6 3.3.1 Chromatin remodeling regulates TAP-1 transcription 76 3.3.2 Histone H3 acetylation within the TAP-1 promoter is low in M H C class I-deficient carcinomas 80 3.3.3 Identification of a region in TAP-1 promoter responsible for the differential activity in TAP-expressing and TAP-deficient cells 82 3.3.4 CBP binding to TAP-1 promoter is impaired in metastatic carcinomas 85 3.3.5 IFN-y treatment increases the level of CBP, acetyl-histone H3 and RNA polymerase II recruitment to the TAP-1 promoter in TAP-deficient metastatic carcinomas 86 3.3.6 Trichostatin A (TSA), a histone deacetylase inhibitor, increases the expression of TAP-1 ; and other antigen-processing machinery (APM) components in metastatic (TAP-deficient) and pre-: metastatic (TAP-expressing) carcinomas 88 vi 3.3.7 T S A treatment increases the expression of TAP-1 and surface M H C class I, resulting in an increased susceptibility of TAP-deficient metastatic carcinomas to C T L killing 91 3.3.8 T S A treatment suppresses the growth of TAP-deficient, metastatic tumors in vivo- 95 3.4 Discussion 98 3.5 References 103 Chapter 4 : Characterization of Novel TAP-1 Regulator Genes 109 4.1 Introduction 109 4.2 Materials and Methods 111 4.2.1 Cell lines 111 4.2.2 Human cDNA library ' 111 4.2.3 Cell Fusion and F A C S analysis T11 4.2.4 Determination of target cell transduction efficiency using the pFB-Luc control viral supernatant ; 112 4.2.5 Transduction of 1E10 cells with ViraPort cDNA library retroviral supernatants 112 4.2.6 Selection of positive transductants 112 4.2.7 Recovery of cDNA clones from positive transductants 113 4.2.8 Sub-cloning of TAP-regulator gene candidates__ 114 4.2.9 Transfection and selection 115 4.2.10 Reverse transcription-PCR analysis _115 4.2.11 Flow cytometry ; 116 4.3 Results 117 4.3.1 Mouse TAP-1 promoter activity is up-regulated in the fused 1E10-A549 cells 117 4.3.2 The transduction efficiency of 1E10 cells is three times lower than that of the NIH/3T3 cells 118 4.3.3 Identification of gene candidates from human lung cDNA library transductants 118 4.3.4 Identification of gene candidates from human spleen cDNA library transductants 121 4.3.5 Cancer-related information about the screened genes 124 4.3.6 Overexpression of several TAP-regulator gene candidates in CMT.64 and L M D cells up-regulates TAP-1 expression, but not H-2K b expression 126 4.4 Discussion 129 4.5 References 133 Chapter 5: General Discussion 137 5.1 Summary and Conclusions 137 5.2 Future Work '• 143 5.3 The Big Picture 147 5.4 References _ _ _ . • 148 Appendix A: Details of Methods 153 A . l Chromatin Immunoprecipitation Assay . .153 A.2 Calculation of copy numbers of pTAP-l-EGFP construct integrated in stable transfectants 154 Appendix B: List of Cloning Vectors . 155 Appendix C: Comparison of TAP-1 Coding Sequence of CMT.64 and Ltk.cells 156 Vll List of Tables Table 2.2.2.1: Primers used for RT-PCR analysis 32 Table 2.2.5.1: G418 doses for selection of transfectants in various cell lines 34 Table 2.3.4.1: Statistical Evaluation of the Transfected pTAP-l -EGFP Copy Numbers Per Cell 44 Table 3.2.2.1: Primers used for RT-PCR and real time PCR analysis 70 Table 3.2.6.1: Primers used for PCR amplifications of full TAP-1 promoter and its truncations 72 Table 4.2.8.1: Primers used for PCR amplification 114 Table 4.3.3.1: Identity of cDNAs recovered from bulk-sorted cells of human lung cDNA library transductants 119 Table 4.3.3.2: Identity of cDNAs recovered from single-cell clones of human lung cDNA library transductants 120 Table 4.3.4.1: Identity of cDNAs recovered from single-cell clones of human spleen cDNA library transductants 123 Table 4.3.5.1: Cancer-related information about the proteins that match the hits obtained from the screenings of human lung and spleen cDNA libraries 125 Vll l List of Figures Figure 1.2.1.1: Genetic organization of human and mouse M H C genes (image reprinted from reference no. [10], by permission) 4 Figure 1.2.3.1: M H C class I antigen presentation pathway (image reprinted from reference no. [7], by permission) 6 Figure 1.2.4.1: A heterodimer of the TAP-1 and TAP-2 molecules (image reprinted from reference no. [7], by permission) 8 Figure 1.2.5.1: M H C class II antigen presentation pathway (image reprinted from reference no. [22], by permission) 9 Figure 1.2.5.2: The complex interaction between a tumor cell, professional antigen-presenting cells, CTLs, Th cells, B cells and other lymphocytes in the tumor microenvironment. A (+) sign indicates an activating signal on the antitumor response while a (-) sign indicates an inhibitory effect (image reprinted from reference no. [24], by permission) 10 Figure 1.5.1.1: Organization of Tap and Lmp genes in the mouse M H C class II locus. The sizes of the coding sequences (CDS) and intergenic regions are shown in the diagram 15 Figure 2.3.1.1: IFN-y-treatment restores TAP-1 expression in TAP-deficient murine lung, prostate and skin carcinoma cells (CMT.64, L M D and B16). Amplification of [3-actin cDNA served as an internal control. RT-PCR analyses were performed at least three times and representative data are shown 38 Figure 2.3.2.1: The recruitment of R N A polymerase II to 3' coding region of the Tap-1 gene in CMT.64, L M D and B16 is impaired in comparison to that in Ltk and R M A . Chromatin immunoprecipitation using anti-RNA polymerase II antibody was performed in each cell line, and the eluted D N A fragments were purified and used as templates for real time PCR analysis using primers specific for the 3' coding region of the Tap-1 gene. Relative R N A polymerase II levels were determined as the ratio of copy numbers of the eluted 3' coding region of the Tap-1 gene and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with TAP-expressing cells (Student's t-test) 40 Figure 2.3.2.2: The recruitment of R N A pol II to the TAP-1 promoter was low in TAP-deficient CMT.64, L M D and B16 cells. Chromatin immunoprecipitation using anti-R N A polymerase II antibody was performed, and the relative levels of R N A polymerase II recruitment to the TAP-1 promoter were assessed as described above using primers specific for the TAP-1 promoter. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with TAP-expressing cells (Student's t-test) : : 41 Figure 2.3.3.1: Nucleotide sequence of the CMT.64-derived TAP-1 promoter region is identical to the corresponding region in murine M H C class II locus. The A T G codon was arbitrarily determined as +1. Motifs located on the sense strand are indicated by a (+), and motifs located on the antisense strand are indicated by a (-) 43 Figure 2.3.4.1: TAP-1 promoter activity is impaired in TAP-deficient CMT.64, L M D and B16 cells. Flow cytometry analysis was performed to measure TAP-1 promoter activity, based on the levels of EGFP expression in pTAP-l-EGFP stable ix transfectants after selection in G418 for 1 month. The IFN-y-treated cells were incubated with 50 ng/ml IFN-y for 48 hours prior to the FACS analysis. Levels of EGFP in the cells transfected the pTAP-l-EGFP were normalized against corresponding values obtained upon transfection with pEGFP-1 vector alone. Data shown are the mean fluorescence intensity ± S E M of three independent experiments. * P < .05 compared with TAP-expressing cells; ** P < .05 compared with untreated cells (Student's t-test) 46 Figure 2.3.5.1: TAP-1 promoter-driven EGFP expression increased in the fused CMT.64-Ltk and LMD-Ltk cells (thick line) in comparison to that in the unfused pTAP-1-EGFP-transfected CMT.64 and L M D cells (thin line). Wild type CMT.64 and L M D were used as negative controls for EGFP expression (shaded area) 48 Figure 2.3.5.2: Surface expression of K b was increased when CMT.64 or L M D (shaded areas) were fused to Ltk cells (broken line). Expression of K b on fused CMT.64-Ltk and fused LMD-Ltk cells are displayed by thick lines. As a positive control, expression of K b was assessed on IFN-y-treated CMT.64 and L M D cells (thin lines). 48 Figure 2.3.5.3: No induction of K b surface expression in the fused CMT.64(pTAP-l-EGFP)-CMT.64(pEYFP-Nl) cells (thick line). CMT.64 cells untreated (shaded area) or treated with IFN-y (thin line) were used as negative or positive control, respectively, for K b expression 49 Figure 2.3.5.4: K k surface expression was reduced in the fused CMT.64-Ltk and L M D -Ltk (thick lines) in comparison to that in the wild type Ltk cells (thin lines). Wild type CMT.64 and L M D cells were used as negative controls for K k expression (shaded area) 50 Figure 2.3.6.1: Endogenous levels of IRF-1 and -2 mRNA did not correlate with TAP levels in the cell lines 51 Figure 2.3.6.2: Overexpression of IRF-1 or -2 in a clone of pTAP-l -EGFP construct-transfected CMT.64 cells did not result in significant changes of the TAP-1 promoter activity. Representative histograms show EGFP expressed by the IRF-1 or IRF-2-overexpressing cells. The shaded areas indicate background green fluorescence of the cells transfected with PBS as negative controls, the thin and thick lines represent EGFP expression in cells overexpressing the IRF that were treated with IFN-y or left untreated, respectively 52 Figure 2.3.6.3: Overexpression of IRF-1 and -2 modulated IFN-(3 promoter activity linked to a luciferase gene. Luciferase activities were normalized against P-galactosidase activities, to correct for variations in transfection efficiencies. Data shown are the average ± S E M of three independent experiments 53 Figure 2.3.7.1: No expression of TAP-1 mRNA from the CMT.64 genome was detectable by RT-PCR in the fused CMT.64-Ltk cells. Amplifications of reverse-transcribed CMT.64 TAP-1 mRNA from IFN-y-treated cells and P-actin mRNA were used as positive controls and loading controls, respectively 55 Figure 2.3.7.2: Total TAP-1 expression was reduced when CMT.64 and L M D cells were fused with wild type Ltk cells. Results from RT-PCR analysis and Western Blot were shown in top and bottom panel, respectively. Levels of P-actin were used as loading controls 56 Figure 2.3.8.1: TAP-1 mRNA is rapidly degraded in fused carcinoma-fibroblast cells. . 57 Figure 2.3.8.2: The stability of S15 and prion mRNA is similar in unfused and fused cells 57 Figure 3.3.1.1: Differences in TAP-1 promoter activity in stable transfectants generally match the TAP-1 expression profiles better than in transient transfectants. Relative luciferase activity (RLA) in transient (12-72 hours post-transfection) and stable (3-4 weeks post-transfection) transfectants. In the transient transfectants, the luciferase unit in each cell line was determined as the ratio of firefly :renila luciferase unit. In the stable transfectants, the luciferase unit in each cell line was determined as the ratio of firefly luciferase:copy number of pTAPl-Luc construct integrated into the genome. Relative luciferase activity (RLA) was determined as the luciferase unit in a particular cell line divided by the lowest value of luciferase unit obtained in that particular group of cells. Columns, average of three to six independent experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test) 77 Figure 3.3.1.2: Analysis of TAP- 1 and surface M H C class I expression by RT-PCR and flow cytometry, respectively. Shaded area, thin and thick lines represent low (A9 or LMD) , medium (Dl 1) and high (TC-1 or PA) levels of M H C class I expression, respectively. Amplification of 13-actin cDNA served as an internal control in the RT-PCR analysis. Data are representatives of three experiments 78 Figure 3.3.1.3: R N A pol II binding to TAP-1 promoter is low in TAP-deficient carcinomas. The levels of R N A pol II in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-RNA pol II Ab. The eluted D N A fragment were purified and used as templates for real-time PCR analysis using primers specific for the 3'-end of the TAP-1 promoter. Relative R N A pol II levels were determined as the ratio of copy numbers of the eluted TAP-1 promoter and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test) 80 Figure 3.3.2.1: Acetyl-histone H3 binding to TAP-1 promoter is low in TAP-deficient carcinomas. The levels of acetyl-histone H3 in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-acetyl-histone H3 Ab. The eluted D N A fragments were purified and used as templates for real-time PCR analysis using primers specific for the 3'-end of the TAP-1 promoter. Relative acetyl-histone H3 levels were determined as the ratio of copy numbers of the eluted TAP-1 promoter and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test) 82 Figure 3.3.3.1: TAP-1 promoter sequence with transcription factor binding motifs and 5' truncation sites. The TAP-1 A T G codon was arbitrarily determined as +1. Truncation sites are indicated by numbered arrows. Motifs located on the sense strand are indicated by a (+) and motifs located on the antisense strand are indicated by a (-) 83 Figure 3.3.3.2: A critical region that is responsible for differential activity of the TAP-1 promoter in TAP-expressing and TAP-deficient cells is found in-between -427 and -xi 401 region of the promoter. R L A was measured in stable transfectants. The largest value in each group of experiments was arbitrarily determined as 1. Columns, average of five experiments; bars, SEM. * P < .05 compared with TAP-expressing cells in the same group (Student's t-test). 84 Figure 3.3.4.1: Expression of CBP proteins is similar in TAP-expressing and in TAP-deficient carcinoma cells 85 Figure 3.3.4.2: CBP binding to TAP-1 promoter is impaired in TAP-deficient, metastatic carcinomas. Chromatin immunoprecipitation using anti-CBP antibody was performed as described earlier. Columns, average of four experiments; bars, S E M . * P < .05 compared with cells that expressed the highest TAP-1 and M H C class I in the same group of cells (Student's t-test) 86 Figure 3.3.5.1: IFN-y treatment improves the recruitment of CBP, acetyl-histone H3 and R N A pol II to TAP-1 promoter, most significatly in TAP-deficient cells. Chromatin immunoprecipitation using A, anti-CBP, B, anti-acetyl-histone H3 or C, anti-RNA pol II antibody was performed as described earlier. Columns, average of three to four experiments; bars, SEM. * P < .05 compared with untreated cells (Student's t-test) ., 87 Figure 3.3.6.1: TSA treatment enhances R N A pol II recruitment to TAP-1 promoter and TAP-1 promoter activity. A, The levels of R N A Pol II in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-RNA pol II. B, TAP-1 promoter activity in stable transfectants was determined by luciferase assay. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with untreated cells (Student's t-test) 89 Figure 3.3.6.2: TSA treatment does not significantly alter the levels of histone H3 acetylation in TAP-1 promoter. The levels of acetyl-histone H3 in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-acetyl-histone H3 antibody. Columns, average of three to six experiments; bars, S E M , . . . 90 Figure 3.3.7.1: TAP-1 expression in all cell lines, particularly in the TAP-deficient carcinomas, was up-regulated with TSA treatment. Up-regulated TAP-1 expression from IFN-y-treated cells was used as a positive control. P-actin expression served as a loading control. Data are representatives of three experiments 91 Figure 3.3.7.2: Expression of LMP-2, TAP^2 and tapasin in all cell lines, particularly in the TAP-deficient carcinomas, was up-regulated with TSA treatment. Amplification of A P M cDNA from IFN-y-treated cells was used as a positive control. P-actin expression served as a loading control. Data are representatives of three experiments 92 Figure 3.3.7.3: Surface H-2K b expression, particularly on M H C class I-deficient cells, was enhanced by TSA treatment. Cells untreated (shaded areas) or treated with 100 ng/ml TSA (thick lines) or 50 ng/ml IFN-y (thin lines) were stained with PE-conjugated anti-H-2Kb mAb. Data are representatives of three experiments 93 Figure 3.3.7.4: TSA treatment improves HPV-positive tumor cell killing by CTLs. Target cells were uninfected or VSV-infected, untreated or treated with TSA or IFN-y for 24 hours before infection with the V S V . CTL assays were performed using effector:target ratio of 0.8:1 to 200:1. Representative data using 22:1 effectontarget ratio are shown in this figure. A l l cells were infected with V S V at a MOI of 7.5 for Xll 16hr. Columns, average of three experiments; bars, SEM. * P < .05 compared with untreated cells (Student's t-test) 94 Figure 3.3.7.5: TSA treatment improves B16F10 tumor cell killing by CTLs. Target cells were uninfected or VSV-infected, untreated or treated with various concentrations of TSA for 24 hours before infection with the V S V . CTL assay was performed using effectontarget ratio of 2.5:1 to 67:1. A l l cells were infected with V S V at a MOI of 7.5 for 16hr 95 Figure 3.3.8.1: TSA treatment suppresses tumor growth in vivo. A9 (MHC class I-deficient cells) tumor growth was suppressed in mice treated with 500 pg/kg of TSA daily compared to in those treated with DMSO vehicle control (n = 8 per treatment group). TC-1 group represented tumor growth in mice injected with high M H C class I-expressing cells (n = 9). Data represent the mean tumor volume ± S E M 96 Figure 3.3.8.2: TAP-1 promoter activity is enhanced in TAP-deficient tumor cells isolated from TSA-treated mice. TAP-1 promoter-driven Luciferase (pTAPl-Luc) expression is higher in A9 cells isolated from TSA-treated mice than in those isolated from DMSO-treated mice (n=4 per treatment group). * P < .05 compared with cells from DMSO-treated mice (Student's t-test) 97 Figure 4.3.1.1: TAP-1 promoter-driven EGFP expression is enhanced in the fused 1E10-A549 cells. The levels of EGFP expression in untransfected CMT.64 cells, 1E10 (pTAPl-EGFP-transfected CMT.64) cells and fused 1E10-A549 cells are represented by a shaded area, a thin line and a thick line, respectively 117 Figure 4.3.3.1: The ORE found in the homologous region of human chromosome 3 clone RP11-27016 map 3p (NCBI accession no. AC090885) and the 1.3 kb sequence obtained from human lung cDNA library screening 119 Figure 4.3.6.1: RT-PCR analysis of the overexpression of the TAP-1 activator candidate genes in CMT.64 and L M D cells. G: cDNA template was derived from pIRES2-gene transfectants; V : cDNA template was derived from pIRES2-EGFP transfectants. Amplification of P-actin cDNA served as a loading control 126 Figure 4.3.6.2: TAP-1 expression was up-regulated in some of the CMT.64 and L M D cells transfected with individual pIRES2-gene constructs. Amplification of cDNA from IFN-y-treated cells was used as a positive control, p-actin expression served as a loading control. Data are representatives of one set of RT-PCR analysis per group of CMT.64 and L M D transfectants 127 Figure 4.3.6.3: No upregulation of H-2K b expression was detected on the surface of L M D cells overexpressing HLA-Cw*04 null allele and HC3 (thick lines). H-2K b expression in pIRES-2-EGFP-transfected L M D cells (shaded areas) was similar to the negative control, the unstained pIRES-2-gene-transfected cells (thin lines). As a positive control, H-2K expression was assessed in IFN-y-treated L M D cells (broken lines) 128 Figure 5.1.1: A proposed model of the mechanisms underlying TAP-1 deficiency in carcinomas and release of the transcriptional repression upon relaxation of chromatin structure around the Tap-1 gene locus. The boxes on the D N A strand represent critical binding sites for various transcription factors that regulate TAP-1 transcription. CBP is a transcriptional co-activator that possesses intrinsic HAT activity. In this model, the recruitment of CBP to TAP-1 promoter facilitates histone acetylation that leads to relaxation of the chromatin structure around the region, Xll l increasing the accessibility of the D N A template by transcription factors (TFs) and R N A pol II complex 140 List of Abbreviations Cr : chromium-51 A B C : ATP-binding cassette A C C R E : autonomous chromatin condensation regulatory element A M P : adenosine monophosphate APC : antigen-presenting cell A P M : antigen processing machinery A R E : adenosine-uracil-rich element ATP : adenosine triphosphate P2m : beta-2 microglobulin bp : base pair CBP : CREB-binding protein cDNA : complementary D N A CDS : coding sequence ChIP : chromatin immunoprecipitation CREB : cyclic A M P (cAMP) response element-binding CTL : cytotoxic T lymphocyte D N A : deoxyribonucleic acid dsDNA : double-stranded D N A EDTA : ethylenediaminetetraacetic acid EGFP : enhanced green fluorescent protein EYFP : enhanced yellow fluorescent protein ER : endoplasmic reticulum XV FACS : fluorescent-activated cell sorting FBS : fetal bovine serum FCS : fetal calf serum FITC : fluorescein isothiocyanate GM-CSF : granulocyte-macrophage colony-stimulating factor HAT : histone acetyltransferase H D A C : histone deacetylase H D A C i : histone deacetylase inhibitor H L A : human leukocyte antigen HPV : human papillomavirus IFN : interferon Ii : invariant chain IL-2 : interleukin-2 i.p. : intraperitoneal IRES : internal ribosome entry site IRF : interferon regulatory factor kb : kilo base pair L M P : low-molecular-weight protein M H C : major histocompatibility complex MIIC : major histocompatibility complex class II compartment MOI : multiplicity of infection M W : molecular weight mRNA : messenger ribonucleic acid N B D : nucleotide-binding domain N K cell : natural killer cell P A G E : polyacrylamide gel electrophoresis PBS : phosphate-buffered saline PCAF : p300/CBP-associated factor PCR : polymerase chain reaction PE : phycoerythrin Poly(A) : poly-adenylated R N A : ribonucleic acid R N A pol II : R N A polymerase II RT-PCR : reverse transcriptase PCR s.c. : subcutaneous SD : standard deviation SDS : sodium dodecyl sulfate S E M : standard error of the mean TAP : transporter associated with antigen processing TCR : T-cell receptor TF : transcription factor TGF-P : transforming growth factor beta T M D : transmembrane domain TNF-a : tumor necrosis factor alpha TSA : trichostatin A TTP : tristetraprolin XVII UTR : untranslated region V S V : vesicular stomatitis virus XV111 Acknowledgments This thesis could not have been written without the invaluable support I received from my thesis committee, family, friends and colleagues. I would like to acknowledge my thesis supervisor, Dr. Wil f Jefferies, for giving me the opportunity to do this graduate work in his laboratory, and for the support and guidance he has provided throughout my studies. I would also like to thank my committee members: Dr. Doug Waterfield, Dr. John Gosline, Dr. Linda Matsuuchi and Dr. Fumio Takei, and my external examiner, Dr. Soldano Ferrone, for discussions, guidance and encouragement throughout my studies, as well as for thorough reading of my thesis and excellent suggestions for improvements. I am deeply grateful to my mentor, Dr. Penny Le Couteur, who always believed in me and encouraged me to fly high. Penny has been an amazing role model as a great scientist, a good mother and a wonderful person. Many thanks to Dr. Muriel David, Susan Chen and Robyn Seipp for their great collaborations, ideas and input. This project would not have been a success without help from hardworking undergraduate project students, Alonso Garduno and Jennifer Hartikainen, as well as excellent technical assistance from Andy Johnson in flow cytometry, Kyung-Bok Choi, Linda L i and Bing Cai in molecular biology and immunology, and Ray Gopaul in in vivo experimentation. I would like to acknowledge the National Cancer Institute of Canada, Canadian Institutes of Health Research, Canadian Network for Vaccines and Immunotherapeutics, Prostate Cancer Research Foundation of Canada and Genemax, Inc. for providing grant support. I am grateful to all my wonderful labmates, especially Cheryl, Robyn, Anna, Kyla, Meimei and Kaan, for discussions and input, for being with me in good and in bad times at the lab, and of xix course for all the good times we had together. I would also like to thank past and present members of the Biomedical Research Centre and Michael Smith Laboratories for their help, friendship and for a wonderful work environment. Finally, I would like to thank my parents, sister and grandparents for their unconditional love and support all these years. Very special thanks to Simon for his love and encouragement all along, especially over the miles in the past 3.5 years. This separation has been very difficult, but will hopefully only make us stronger. At the same time, this gave me an interesting thesis-writing experience up in the sky above every continent in-between Vancouver and Boston. X X Dedication This thesis is dedicated to my mother, Christiana Budiono, for her unconditional love, support and tireless encouragement from 12,783 km away. xxi Co-Authorship Statement Dr. Muriel D. David (Biomedical Research Centre, Vancouver, BC) provided guidance and technical help with promoter analysis, chromatin immunoprecipitation and luciferase assays reported in this thesis. She also performed the P-galactosidase assays and their data analysis reported in Chapter 2. . Susan S. Chen (Department of Zoology, University of British Columbia, Vancouver, BC) trained and helped me to master new laboratory techniques during the early stage of my doctoral research. Dr. John Hiscott (Lady Davis Institute of Medical Research, McGi l l University, Montreal, QC) provided the interferon regulatory factor (IRF) expression vectors and the interferon beta ( I F N - P ) promoter construct reported in Chapter 2. Robyn P. Seipp (Department of Zoology, University of British Columbia, Vancouver, BC) performed the cytotoxicity (CTL) assays and CTL data analysis reported in Chapter 3. Jennifer A . Hartikainen (Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC) provided help with the construction of truncated TAP-1 promoter constructs and the luciferase assays reported in Chapter 3. Rayshad Gopaul (Michael Smith Laboratories, Vancouver, BC) shared the duties with me in injecting mice, recording data and monitoring the animals during the in vivo experiment reported in Chapter 3. I performed all the other experiments and data analyses reported in this thesis. GENERAL INTRODUCTION 1 Chapter 1 : General Introduction Recognition of pre-cancerous and virus-infected cells by our immune system is critical for protecting us against malignant cells and viral infections. The host's immune system has the potential to kill abnormal cells before they become malignant cancers; however, many cancer cells have defects in components of the antigen processing and presentation pathways that allow them to avoid detection by the immune system [1-4]. One of the key components that is often missing in tumors is a heterodimer of the Transporters associated with Antigen Processing (TAP)-l and TAP-2 molecules that normally functions in transporting self-, virus-, or tumor-derived peptides into the endoplasmic reticulum, in which the peptides can be loaded into Major Histocompatibility Complex (MHC) class I molecules. In a functional pathway, the M H C class I - peptide complexes are then transported via the Golgi and further displayed on the cell surface, where they can be recognized by the circulating cytotoxic T lymphocytes (CTLs). The CTLs function to bind to and eventually kill the cancer or virus-infected cells. The importance of TAP-1 function in immunosurveillance has been highlighted in previous studies that demonstrated that by restoring TAP-1 expression in TAP-deficient cancer cells, the entire M H C class I antigen presentation pathway can be reconstituted, leading to the elimination of the cancer cells by CTLs [1, 5-8]. However, the mechanisms underlying TAP-1 downregulation in cancer cells remain poorly understood [7]. The goal of this research is to contribute to a better understanding of the mechanisms that lead to TAP downregulation in malignant cells, as well as to further characterize novel transcriptional regulators of TAP-1. GENERAL INTRODUCTION 2 1.1 Overview of the Immune System The immune system that protects organisms from infections is divided into 2 components: the innate and the adaptive immune system. Innate immune systems, which are found in nearly all living organisms, provide an immediate, non-specific response against all invading pathogens [9, 10]. Jawed vertebrates possess another layer of protection provided by the adaptive immune system that elicits delayed, but specific and long-lasting immune responses against pathogens, as well as pre-cancerous cells that express tumor-specific antigens [10]. Tumor specific antigens can be classified either as self- or non-self antigens, depending on their origin. Tumor-specific antigens in cells transformed by viral infection, such as human papillomaviruses (HPV), the Epstein-Barr virus (EBV)and the human herpesvirus-8 (HHV-8), are mainly derived from viral proteins and can potentially be recognized by the host's immune system as non-self antigens [11]. Alternatively, tumors may develop from spontaneous transformation of normal cells, for example, due to mutations of self molecules [12]. Although T-cell receptors (TCRs) can often discriminate even single amino acid changes in peptides, thus allowing activated T cells to recognize cancer cells that express mutated self peptides bound to M H C class I molecules [12], immunotherapy approaches for these types of tumors are facing great challenges due to low immunogenicity against cells that express self antigens. Additional special strategies, such as vaccination and breaking of tolerance [11, 12], are required to be implemented in parallel with the treatments in order to enhance immune responses against the abnormal cells. GENERAL INTRODUCTION 3 1.2 Antigen Presentation Adaptive immunity relies on the ability of the T-cell receptors (TCRs) to distinguish between self and non-self molecules, bound by M H C molecules and presented on the cell surface. Presentation of peptides derived from self proteins on a cell surface does not normally trigger immune response, since T cells with the ability to recognize self-derived peptides are deleted during T-cell development in the thymus, in a process known as negative selection [10]. In contrast, recognition of cells that express foreign antigens or mutated self antigens by the T cells generally triggers a cascade of activation of components of the immune system, such as B cells, macrophages and other T cells, that leads to the elimination of the invading pathogens and the abnormal cells [10, 12]. 1.2.1 Major Histocompatibility Complex (MHC) genetics The M H C locus that is located on chromosome 6 in humans and chromosome 17 in the mouse, contains the most polymorphic genes known in the living organisms [10]. M H C polymorphism is highly advantageous for the presentation of a wide range of diversity of peptides to T-cell receptors. In humans, the M H C is called the Human Leukocyte Antigen (HLA). In the mouse, the M H C genes are known as the H-2 genes. The cluster of M H C genes is divided into MHC class I, MHC class II and MHC class III genes. The organization of the M H C locus is similar in both species, although the H-2k gene in the mouse is translocated to the edge of the M H C gene cluster, near the Tapbp gene and the centromere (Figure 1.2.1.1). There are three main M H C class I genes in both species: HLA-A, -B and -C in humans, or H-2k, -d and -/ in the mouse. The M H C class II region includes the genes for HLA-DP, -DQ and -DR in humans, or H-2A and -E GENERAL INTRODUCTION 4 in the mouse. M H C class III region includes the genes encoding various other immune function proteins, including the complement proteins C4, C2 and factor B, as well as the tumor necrosis factor (TNF)-a and lymphotoxin. Gen* structure a( tht human PHC DP OH DO DR & C ffl#8P (3 a a « p LMPfW 1 (5 « P p a _ n r\r\  r\jr\t~i MT] n r i .njTJ/i~4TiT^n _ M liii LJI ' ™J L_J i_J Gil L&J KE WSJ ; _JI «J iLJ L«J _r Gene structure ai the rnoiiea MHC H _ 0 M O A E * i * i j r—'—s f—'—t iPK n « || UlrVW P p a p a 1 1 LrUU D L _r^_r ' Janeway C , et al., 2001. Copied under license from Access Copyright. Further reproduction prohibited. Figure 1.2.1.1: Genetic organization of human and mouse MHC genes (image reprinted from reference no. [10], by permission). GENERAL INTRODUCTION 5 1.2.2 MHC molecules An M H C molecule functions by binding peptide fragments and presenting the complex on the cell surface. A c t i v a t i on of immune responses only occurs upon TCR recognition of a complex of a foreign peptide or a mutated self peptide bound to an M H C molecule on the cell surface. The M H C molecules are divided into 2 classes: M H C class I and M H C class II molecules, which differ in their structure, their expression pattern on tissues, and the types and sizes of antigen presented [10]. M H C class I molecules are expressed on the surface of all nucleated cells, whereas M H C class II molecules are normally present only on B cells and on special antigen-presenting cells (APCs): dendritic cells and macrophages. M H C class I molecules generally present short (8-10 amino acids) intracellularly-derived peptides, such as viral antigens and tumor-specific antigens. Recognition of M H C class I - peptide complexes by specific TCRs on CD8 + cytotoxic T lymphocytes (CTLs) triggers the activation of CTL responses that ultimately result in killing of the virus-infected or cancerous cells. M H C class II molecules are capable of presenting longer (i.e., more than 13 amino acids) extracellularly-derived peptides, such as peptides generated from bacterial proteins. Peptides bound by M H C class II molecules can be recognized by CD4 + T cells, also known as T helper (Th) cells, which mainly function to activate other effector cells of the immune system. M H C class II antigen presentation and the importance of Th cells in cancer immunity will be further discussed in section 1.2.5. GENERAL INTRODUCTION 6 1.2.3 MHC class I antigen presentation The work i n this thesis focuses on the presentation o f tumor-specif ic antigens by M H C class I molecu les , since the recognit ion o f neoplastic ce l ls by C D 8 + C T L s has been shown to be one o f the most important events required for an effect ive anti-tumor response [13]. M H C class I molecules general ly present peptides that are generated intracel lu lar ly , hence the name endogenous antigens. V i r a l antigens and tumor-specif ic antigens are examples o f intracel lular ly-derived proteins. In some cases, M H C class I molecu les can present peptides der ived f rom extracel lular proteins, or exogenous antigens, i n a process ca l led cross presentation [14, 15]. Th i s thesis focuses on the study o f tumor antigen presentation v i a the classical M H C class I antigen presentation pathway (F igure 1.2.3.1). Figure 1.2.3.1: MHC class I antigen presentation pathway ( image reprinted f rom reference no. [7], by permission) . GENERAL INTRODUCTION 7 In this pathway, endogenous proteins are degraded into smaller peptides by the action of multicatalytic enzyme subunits of the proteasome. The peptides are then transported by a heterodimer transporter that consists of TAP-1 and TAP-2 subunits into the lumen of endoplasmic reticulum (ER), in which M H C class I heavy chains are synthesized de novo, folded and coupled with beta-2 microglobulin ((32m) molecules in a process assisted by chaperone proteins (BiP, calnexin, calreticulin, ERp57 and tapasin) [16, 17]. In the lumen of ER, the peptides are loaded into an M H C class I heavy chain/(32m molecule, forming a stable ternary complex that can be transported onto the cell surface via the trans-Golgi network. The host's cytotoxic T lymphocytes (CTLs) have the potential to recognize neoplastic cells that express tumor-specific antigens on their surface, and furthermore, to kill the abnormal cells through the release of cytolytic granules: perforin and granzymes [10]. 1.2.4 Transporters associated with Antigen Processing (TAP) structure and function TAP-1 and TAP-2 molecules belong to the superfamily of ATP-binding cassette (ABC) transporters, one of the largest protein families that span bacteria to humans. They are characterized by the presence of two cytoplasmic ATP-binding domains and two hydrophobic transmembrane domains (TMDs) [7, 18] (Figure 1.2.4.1). Most A B C transporters function by transporting a broad range of substrates, including amino acids, lipids, sugars, ions and drugs, across membranes by using the energy obtained from ATP hydrolysis [18, 19]. The TAP transporter, which consists of a heterodimer of TAP-1 and TAP-2 molecules embedded in the membranes of the endoplasmic reticulum (ER) and cz's-Golgi, specializes in transporting endogenous peptides from the cytosol into the GENERAL INTRODUCTION 8 lumen o f E R [7, 20 , 21] . The energy resulted f rom A T P hydro lys i s triggers conformat ional changes i n the nucleotide-binding doma in ( N B D ) and the T M D s , caus ing the b ind ing and movement o f peptides across the E R membrane [21]. TAP1 T A P 2 Figure 1.2.4.1: A heterodimer of the TAP - 1 and T A P - 2 molecules ( image reprinted f rom reference no. [7], by permission) . 1.2.5 MHC class II antigen presentation and CD4* T cells in cancer immunity In the M H C class II antigen presentation pathway (F igure 1.2.5.1), extracel lular proteins are endocytosed into intracel lular vesicles and degraded into peptides by the act ion o f cathepsins i n the vesicles. S im i l a r l y to the M H C class I molecu les , the M H C class II molecu les are synthesized de novo inside the endoplasmic re t i cu lum (ER) . A n invariant cha in (Ii) molecu le binds in the groove o f a new ly synthesized M H C class II molecu le to prevent the b ind ing o f peptides in the E R . The M H C class II - Ii comp lex is then transported f r om the E R into a vesic le , ca l led the M H C class II compartment (M I IC ) , where I i is t r immed into a short f ragment—ca l led the C L I P pept ide—that is st i l l bound to the M H C class II groove. A peptide-loaded ves ic le then fuses w i t h the M I I C , GENERAL INTRODUCTION 9 where CLIP is removed by H L A - D M and antigenic peptides are loaded onto M H C class II molecules. The M H C class II - peptide complexes are then transported to the cell surface for surveillance by CD4 + T-helper (Th) cells. MHC class I (/peptide rtace expression antigen proteolysis class If peptide oading ( ( > < Endoplasmic reticulum MHC class II li MHC HLA-DM Ii complex chain class I) Figure 1.2.5.1: MHC class II antigen presentation pathway (image reprinted from reference no. [22], by permission). The two main subsets of CD4 + Th cells are the Thl and Th2 cells. They are classified based on their distinct cytokine production patterns [23]. The Thl cells are particularly important for antitumor immunity due to their ability to produce interferon gamma (IFN-y), tumor necrosis factor alpha (TNF-a) and interleukin-2 (IL-2), which are important for the development and sustained proliferation of CD8 + CTLs [24, 25]. The Th2 cells are predominantly involved in humoral immune response, producing IL-4 and IL-5 that stimulate antibody production by activated B cells [24]. The presence of a large number of Th2 cells in a tumor microenvironment generally impairs tumor immunity due GENERAL INTRODUCTION 10 to their product ion o f IL-10 and TGF-(3 that act as immunosuppress ive cytokines [26, 27] . However , i n rare cases, Th2 cel ls can produce natural antibodies against tumors [28]. A s imp l i f i ed diagram out l in ing complex interactions between tumor ce l l s , professional antigen-presenting cel ls and various lymphocytes is shown in F igure 1.2.5.2 be low. Figure 1.2.5.2: The complex interaction between a tumor cell, professional antigen-presenting cells, CTLs, Th cells, B cells and other lymphocytes in the tumor microenvironment. A (+) sign indicates an act ivat ing s ignal on the anti tumor response wh i l e a (-) s ign indicates an inh ib i tory effect ( image reprinted f rom reference no. [24], by permiss ion) . GENERAL INTRODUCTION 11 1.3 Disruption of the MHC Class I Antigen Processing Pathway and the Development of Tumors Forty to ninety percent of H L A (or M H C in the mouse) class I-positive precursors of human tumors are found to develop into H L A class I-negative tumors [29]. TAP deficiency is one of the common phenotypes that distinguish malignant transformed cells from their normal precursors [20]. TAP downregulation leads to the disruption of the process by which tumor-specific peptides are transported into the lumen of ER and loaded onto M H C class I - P 2 m complex [1]. As a consequence, no stable complex of tumor peptide - M H C class I can be presented on the cell surface, rendering the abnormal cell to stay undetected by the immune surveillance. Downregulated or total loss of expression of TAP-1 and TAP-2 has been observed in many tumor cell lines and surgically-removed tumor specimens, such as Hodgkin's lymphoma, myeloma, melanoma and many types of carcinomas, such as, colorectal, cervical, breast, lung, prostate, renal cell and hepatocellular carcinomas [1, 2, 4]. Interestingly, the level of TAP-1 downregulation in carcinomas was found to significantly correlate with tumor progression and reduced survival [1, 7, 8, 20]. The downregulation is more often caused by regulatory defects rather than by structural defects due to mutations within the TAP-1 gene itself [2, 4]. Other mechanisms that lead to an impaired M H C class I antigen presentation in tumor cells include the loss of one copy of the chromosome in which M H C class I genes are located, structural alterations, methylation, or deregulation of genes coding for the M H C class I heavy chain and P 2 m , as well as of those coding for antigen processing machinery (APM) components, such as Tapasin, and the proteasome subunits: LMP-2 and LMP-7 [4, 30]. GENERAL INTRODUCTION 12 Based on surface M H C class I expression levels, a major anti-tumor mechanism other than that exerted by the CD8 + CTLs is provided by natural killer (NK) cells [31]. CD8 CTLs recognize and destroy target cells that express the surface M H C class I-tumor antigen complex, whereas N K cell activity is triggered upon recognition of cells that do not express surface M H C class I, since M H C class I molecules generally serve as ligands for N K cell inhibitory receptors [31]. N K cell-mediated cytotoxicity is regulated by a balance of signals transmitted by activating and inhibitory receptors. Unfortunately, N K cell infiltration is generally low in neoplastic tissues [29] and the N K activating signal is often hampered by immunosuppressive cytokines, such as TGF-[3, within the tumor microenvironment [13, 32]. Therefore, N K cell-mediated cytotoxicity is generally not sufficient to provide complete protection against a large proportion of cancer cells with down-regulated expression of M H C class I. Further research is needed in order to create ways to attract more N K cells into neoplastic tissues and to make the N K cell activating signal more favorable within the tumor microenvironment. GENERAL INTRODUCTION 13 1.4 TAP-based Immunotherapy The importance of TAP-1 function in immunosurveillance against cancers has been highlighted in several studies that demonstrated the reconstitution of M H C class I antigen presentation upon restoration of TAP-1 expression in cancer cell lines exhibiting down-regulated expression of several A P M components [27, 33]. This finding is encouraging for the development of a general immunotherapy method for different types of cancers that possess multiple deficiencies of A P M components. Moreover, reconstitution of TAP -1 function in highly metastatic, M H C class I-deficient murine lung carcinoma and melanoma cell lines, CMT.64 and B16, respectively, has been shown to improve immune recognition of the cancer cells in vivo, resulting in a decrease of tumor growth and metastatic ability [5, 20, 34]. Restoration of TAP-1 alone resulted in an improved ability of cancer cells to present viral and/or tumor-specific peptides, yielding similar anti-viral and anti-tumor effects as those resulted from the induction of both TAP-1 and TAP-2, whereas expression of TAP-2 alone did not show the effects [6, 35, 36]. This may be caused by instability of TAP-2 molecule in the absence of pre-existing TAP-1, whereas TAP-1 molecule and assembled TAP heterodimer are highly stable in vivo [5, 6]. Heterodimerization of TAP subunits is required to protect TAP-2 from rapid degradation by a proteasome-dependent pathway [21]. These observations indicate that induction of TAP-1 expression is an important aspect that shall be considered in the development of immunotherapy for various types of cancers. The TAP-based immunotherapy approach possesses several advantages over many other immunotherapy methods. One of the advantages is that TAP is a component of normal cells, and to-date, no toxicity or autoimmunity reaction has been observed in GENERAL INTRODUCTION 14 animal tumor models that were given TAP-based therapy [21]. Therefore, based on the low risk of toxicity, it may not be necessary to target TAP exclusively into the cancer cells. This would make TAP an attractive candidate for cancer gene therapy since the development of methods to deliver an efficient dose of therapeutic gene remains the primary challenge in cancer gene therapy [6]. Another advantage of TAP-based therapy is that it is not restricted by the haplotype of M H C molecules and the type of tumor antigens, since TAP molecules act independently of the two factors [37]. M H C polymorphism and various types of tumor-associated antigens—many of them not yet characterized—remain as challenges in the development of immunotherapy approaches [6, 36]. Finally, recent studies have demonstrated that recombinant TAP could be used as a novel, non-toxic adjuvant that increases vaccine efficiency [38-41]. Adverse responses to standard doses of inocula are common problems encountered in vaccination, due to toxicity of vaccine components [42]. With the use of an adjuvant, immune responses could potentially be triggered by administering lower doses of inocula, thus decreasing the risk of adverse side effects. This finding denotes the importance of TAP, which is to be considered in the development of vaccines against cancers and widespread infectious diseases. GENERAL INTRODUCTION 15 1.5 Regulation of TAP-1 Expression The goal of this thesis is to gain an understanding of how Tap-1 gene expression is impaired in metastatic carcinomas. Initially, this work was focused on transcriptional regulation of TAP-1, since the TAP-1 downregulation observed in many carcinomas occurred at the mRNA level [5, 6, 43]. This work was then expanded to study epigenetic mechanisms that often also play significant roles in the regulation of gene expression [44]. Defects in translational mechanisms can also contribute to the impairment of a gene expression; however, translational regulation of TAP-1 is beyond the scope of this thesis. 1.5.1 Organization of Tap and Lmp genes within the mouse MHC class II locus The genes encoding TAP-1 and TAP-2 molecules are located within the M H C class II region, in close association with the genes encoding two proteasomal subunits: low-molecular-mass polypeptides (LMP)-2 and L M P -7 [7, 10]. Multicatalytic enzyme subunits of the proteasome are capable of degrading intracellular proteins into smaller peptides that can be loaded into the lumen of the endoplasmic reticulum by a heterodimer of TAP-1 and TAP-2 molecules. A diagram outlining the organization of these genes within the mouse M H C class II locus is shown in Figure 1.5.1.1 below. TAP-1 LMP-7 ] TAP-2 L M P - Z •570 bp 1611 bp 3862 bp 660 bp 2175 bp 831 bp 2109 bp Figure 1.5.1.1: Organization of Tap and Lmp genes in the mouse MHC class II locus. The sizes of the coding sequences (CDS) and intergenic regions are shown in the diagram. GENERAL INTRODUCTION 16 In humans, it was found that a bi-directional promoter of LMP-2 and TAP-1 genes is located in the 593 bp region in-between the two genes [45]. This promoter contains GC-rich regions characterizing putative Sp-1 binding sites, but does not contain a T A T A boxT At least 18 transcriptional start sites of TAP-1 gene were found within the promoter. This finding of multiple transcriptional start sites is consistent with observations in other TATA-less, GC-rich promoters [46]. Prior to the work in this thesis, mouse TAP-1 promoter had never been cloned and analysed. This work is described for the first time in Chapter 2. 1.5.2 Possible mechanisms of TAP-1 mRNA downregulation in carcinomas Several mechanisms tested in this thesis include: 1.5.2.1 Transcriptional mechanisms Reduced transcription of Tap-1 gene could be caused by mutations in cw-acting consensus sequences within its promoter. This might lead to the inability of important transcription factors to recognize and to bind to the mutated D N A template in the promoter. Other possible mechanisms include overexpression of transcriptional repressors and/or the lack of ^ ram-activators binding to the TAP-1 promoter. 1.5.2.2 Post-transcriptional mechanisms Degradation of mRNA transcripts is a normal turnover process of coordinated gene expression [44, 47, 48]! Stability of an mRNA depends on its structure and sequence, such as the presence of a cap in its 5' end that protects against 5'-3' exonucleases, the presence of de-stabilizing sequences, for example AU-rich elements GENERAL INTRODUCTION 17 (ARE) in the 3'-untranslated region (3'UTR), and the length of poly-adenylated (poly(A)) sequence in its 3' end that protects against 3'-5' exonucleases [44]. Gene mutation and/or aberrant expression of RNA-binding proteins with the ability to either promote or inhibit mRNA degradation might lead to accelerated rate of mRNA degradation, thus reduced expression of the gene. 1.5.2.3 Epigenetic mechanisms Epigenetic mechanism is described as a heritable change in gene expression that occurs without a change in D N A sequence [49]. Epigenetic modifications affect gene expression through modulation of chromatin structures and/or D N A methylation [44]. Epigenetic mechanisms could provide an extra layer of transcriptional control that regulates how genes are expressed, by modulating the interaction between histone and D N A complexes [49]. This would in turn affect the accessibility of D N A templates by transcriptional machinery. The key event that leads to the modulation of chromatin structures is the modification of N-terminal tails of the histone proteins, particularly of histone H3 and H4 [44]. Known modifications of histone proteins include: acetylation, methylation, phosphorylation, ubiquitination and sumoylation [44, 50-54]. To-date, histone acetylation is the most widely studied mechanism. A high level of acetylated core histones in a chromatin template, particularly in the proximal region of an acetylation-sensitive promoter [55, 56], has been associated with transcriptionally active sites [50, 51, 53, 56, 57]. The acetylation of N-terminal lysines of histones neutralizes positive charges in the proteins, thus loosening histone-DNA contacts and increasing the accessibility of transcription factors to the D N A template [56]. GENERAL INTRODUCTION 18 Epigenetic abnormalities have been found to be responsible for development of several cancers, genetic disorders and autoimmune diseases [49]. In parallel with the investigation of genetic-related mechanisms, epigenetic modifications are clearly important to be considered in the study of gene regulation. GENERAL INTRODUCTION 19 1.6 Synopsis of Thesis Objectives, Hypotheses and Results Chapter 2 focuses on elucidating the mechanisms underlying the transcriptional deficiency of TAP-1 in several metastatic murine carcinoma cell lines. The hypothesis tested was whether the deficiency is caused by mutations in c/s-acting elements of the TAP-1 promoter, the lack of transcription factors binding to TAP-1 promoter, overexpression of transcriptional repressors, and/or post-transcriptional defects. Experimental outcomes showed no mutation in cw-acting elements of the TAP-1 promoter, and that TAP-1 downregulation is caused by the lack of trans-acting factors binding to TAP-1 promoter and accelerated degradation of TAP-1 mRNA. . The work in Chapter 3 aims to study the epigenetic regulation of TAP-1 expression and antigen processing in malignant cells. The hypothesis is that TAP-1 transcription is controlled by epigenetic mechanisms that regulate the permissive/repressive state of nucleosomal structure around the TAP-1 locus, thus controlling the accessibility of the D N A template by RNA pol II complex and general transcription factors. Experimental results revealed the lack of histone H 3 acetylation in TAP-1 promoter as an epigenetic mechanism that is likely to contribute to a condensed nucleosomal structure around the TAP-1 promoter in TAP-deficient cells, thus preventing the binding of transcriptional activators to the promoter. • Chapter 4 describes the characterization of several novel TAP-1 activator gene candidates obtained by cDNA library screenings. With the assumption that TAP-1 regulator genes are present in the highly complex cDNA library used, the hypothesis tested was that expression of genes (cDNAs) that have the ability to up-regulate TAP-GENERAL INTRODUCTION 20 1 promoter activity would enhance the expression of TAP-1 and surface M H C class I in TAP- and M H C class I-deficient carcinoma cells. Several TAP-activator gene candidates were recovered from cells that showed up-regulated TAP-1 promoter activity upon infection with cDNA library retroviral supernatants. The overall objective of this research is to contribute to a better understanding of tumor antigen presentation deficiency and furthermore, to the development of effective immunotherapeutic strategies for the treatment of cancer. GENERAL INTRODUCTION 21 1.7 References 1. Seliger, B., M J . Maeurer, and S. Ferrone. TAP off—tumors on. Immunol Today, 1997. 18:292-9. 2. Seliger, B., M.J. Maeurer, and S. Ferrone. Antigen-processing machinery breakdown and tumor growth. Immunol Today, 2000. 21:455-64. 3. Ritz, U . and B. Seliger. The transporter associated with antigen processing (TAP): structural integrity, expression, function, and its clinical relevance. Mol Med, 2001.7:149-58. 4. Seliger, B., T. Cabrera, F. Garrido, and S. Ferrone. H L A class I antigen abnormalities and immune escape by malignant cells. Semin Cancer Biol, 2002. 12:3-13. 5. Gabathuler, R., G. Reid, G. Kolaitis, J. Driscoll, and W.A. Jefferies. Comparison of cell lines deficient in antigen presentation reveals a functional role for TAP-1 alone in antigen processing. J Exp Med, 1994. 180:1415-25. 6. Alimonti, J., Q.J. Zhang, R. Gabathuler, G. Reid, S.S. Chen, and W.A. Jefferies. TAP expression provides a general method for improving the recognition of malignant cells in vivo. Nat Biotechnol, 2000. 18:515-20. 7. Lankat-Buttgereit, B. and R. Tampe. The transporter associated with antigen processing: function and implications in human diseases. Physiol Rev, 2002. 82:187-204. 8. Kageshita, T., S. Hirai, T. Ono, D.J. Hicklin, and S. Ferrone. Down-regulation of H L A class I antigen-processing, molecules in malignant melanoma: association with disease progression. Am J Pathol, 1999. 154:745-54. GENERAL INTRODUCTION 22 9. L i tman , G.W. , J .P. Cannon , and L.J. D i shaw . Reconstruct ing immune phy logeny : new perspectives. Na t R e v Immuno l , 2005. 5:866-79. 10. Janeway, C , P. Travers, M . Walpor t , and M . Sh lomch ik . Immunob io logy : the immune system in health and disease. 5th ed. 2001 , N e w Y o r k , N Y Gar l and Pub l i sh ing . 11. K l e i n , G . and E. K l e i n . Surve i l lance against tumors—is it ma in l y immuno log i ca l ? Immuno l Lett, 2005. 100:29-33. 12. Houghton , A . N . and J . A . Guevara-Patino. Immune recogni t ion o f se l f i n immun i t y against cancer. J C l i n Invest, 2004. 114:468-71. 13. Tomas i , T .B . , W. J . Magner , and A . N . K h a n . Epigenet ic regulat ion o f immune escape genes i n cancer. Cancer Immuno l Immunother, 2006. 55:1159-84. 14. Rock , K . L . and L. Shen. Cross-presentation: under ly ing mechanisms and role i n immune surveil lance. Immuno l Rev , 2005. 207:166-83. 15. Cresswe l l , P., A . L . A c k e r m a n , A . G i o d i n i , D.R. Peaper, and P.A. Wearsch . Mechan i sms o f M H C class I-restricted antigen processing and cross-presentation. Immuno l Rev , 2005. 207:145-57. 16. Germa in , R . N . and D . H . Margu l i es . The b iochemistry and ce l l b io logy o f antigen processing and presentation. A n n u Rev Immuno l , 1993. 11:403-50. 17. Cresswe l l , P. and J . Howa rd . A n t i g e n recognit ion. Cu r r O p i n Immuno l , 1999. 11:61-3. 18. A l tenberg , G . A . The engine o f A B C proteins. N e w s Phys io l S c i , 2003. 18:191-5. 19. Chang , G . M u l t i d r u g resistance A B C transporters. F E B S Lett, 2003. 555:102-5. GENERAL INTRODUCTION 23 20. Abele, R. and R. Tampe. Modulation of the antigen transport machinery TAP by friends and enemies. FEBS Lett, 2006. 580:1156-63. 21. Keusekotten, K. , R . M . Leonhardt, S. Ehses, and M.R. Knittler. Biogenesis of functional antigenic peptide transporter TAP requires assembly of pre-existing TAP1 with newly synthesized TAP2. J Biol Chem, 2006. 281:17545-51. 22. Kelly, A . MHC class II antigen presentation, [website] 2006 [cited; Available from: http://www.path.cam.ac.uk/pages/kelly/image/. 23. Murakami, H. , H . Ogawara, and H . Hiroshi. Thl/Th2 cells in patients with multiple myeloma. Hematology, 2004. 9:41-5. 24. Knutson, K . L . and M . L . Disis. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother, 2005. 54:721-8. 25. Wang, J.C. and A . M . Livingstone. Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo. J Immunol, 2003. 171:6339-43. 26. Kidd, P. Thl/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev, 2003. 8:223-46. 27. Wahl, S.M., J. Wen, and N . Moutsopoulos. TGF-beta: a mobile purveyor of immune privilege. Immunol Rev, 2006. 213:213-27. 28. Chamuleau, M.E. , G.J. Ossenkoppele, and A . A . van de Loosdrecht. M H C class II molecules in tumour immunology: prognostic marker and target for immune modulation. Immunobiology, 2006. 211:619-25. 29. Bubenik, J. M H C class I down-regulation: tumour escape from immune surveillance? Int J Oncol, 2004. 25:487-91. GENERAL INTRODUCTION 24 30. Meissner, M . , T.E. Reichert, M . Kunkel, W. Gooding, T.L. Whiteside, S. Ferrone, and B. Seliger. Defects in the human leukocyte antigen class I antigen processing machinery in head and neck squamous cell carcinoma: association with clinical outcome. Clin Cancer Res, 2005. 11:2552-60. 31. Garrido, F., F. Ruiz-Cabello, T. Cabrera, J.J. Perez-Villar, M . Lopez-Botet, M . Duggan-Keen, and P.L. Stern. Implications for immunosurveillance of altered H L A class I phenotypes in human tumours. Immunol Today, 1997. 18:89-95. 32. Albertsson, P.A., P.H. Basse, M . Hokland, R.H. Goldfarb, J.F. Nagelkerke, U . Nannmark, and P.J. Kuppen. N K cells and the tumour microenvironment: implications for NK-cell function and anti-tumour activity. Trends Immunol, 2003. 24:603-9. 33. Rook, A . H . , J.H. Kehrl, L . M . Wakefield, A . B . Roberts, M . B . Sporn, D.B. Burlington, H.C. Lane, and A.S. Fauci. Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness. J Immunol, 1986. 136:3916-20. 34. Seliger, B., U . Ritz, R. Abele, M . Bock, R. Tampe, G. Sutter, I. Drexler, C. Huber, and S. Ferrone. Immune escape of melanoma: first evidence of structural alterations in two distinct components of the M H C class I antigen processing pathway. Cancer Res, 2001. 61:8647-50. 35. Agrawal, S., K. Reemtsma, E. Bagiella, S.F. Oluwole, and N.S. Braunstein. Role of TAP-1 and/or TAP-2 antigen presentation defects in tumorigenicity of mouse melanoma. Cell Immunol, 2004. 228:130-7. GENERAL INTRODUCTION 25 36. Lou, Y. , T.Z. Vitalis, G. Basha, B. Cai, S.S. Chen, K . B . Choi, A.P. Jeffries, W . M . Elliott, D. Atkins, B. Seliger, and W.A. Jefferies. Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival. Cancer Res, 2005. 65:7926-33. 37. Scanlon, K.J . Cancer gene therapy: challenges and opportunities. Anticancer Res, 2004.24:501-4. 38. Lazoura, E. and V . Apostolopoulos. Insights into peptide-based vaccine design for cancer immunotherapy. Curr Med Chem, 2005. 12:1481-94. 39. Mocellin, S. Cancer vaccines: the challenge of developing an ideal tumor killing system. Front Biosci, 2005. 10:2285-305. 40. Morris, E., D. Hart, L . Gao, A. Tsallios, S.A. Xue, and H. Stauss. Generation of tumor-specific T-cell therapies. Blood Rev, 2006. 20:61-9. 41. Ruttinger, D., H . Winter, N . K . van den Engel, R.A. Hatz, M . Schlemmer, H. Pohla, S. Grutzner, D.J. Schendel, B.A. Fox, and K.W. Jauch. Immunotherapy of lung cancer: an update. Onkologie, 2006. 29:33-8. 42. Vitalis, T.Z., Q.J. Zhang, J. Alimonti, S.S. Chen, G. Basha, A. Moise, J. Tiong, M . M . Tian, K . B . Choi, D. Waterfield, A . Jeffries, and W.A. Jefferies. Using the TAP component of the antigen-processing machinery as a molecular adjuvant. PLoS Pathog, 2005. I:e36. 43. Seliger, B., D. Atkins, M . Bock, U . Ritz, S. Ferrone, C. Huber, and S. Storkel. Characterization of human lymphocyte antigen class I antigen-processing machinery defects in renal cell carcinoma lesions with special emphasis on GENERAL INTRODUCTION 26 transporter-associated with antigen-processing down-regulation. Clin Cancer Res, 2003. 9:1721-7. 44. Lewin, B. Genes IX, ed. J. Louis C. Bruno. 2008, Sudbury, M A : Jones and Bartlett Publishers. 45. Wright, K . L . , L .C. White, A. Kelly, S. Beck, J. Trowsdale, and J.P. Ting. Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter. J Exp Med, 1995. 181:1459-71. 46. Azizkhan, J.C., D.E. Jensen, A.J . Pierce, and M . Wade. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev Eukaryot Gene Expr, 1993. 3:229-54. 47. Benjamin, D., M . Colombi, G. Stoecklin, and C. Moroni. A GFP-based assay for monitoring post-transcriptional regulation of ARE-mRNA turnover. Mol Biosyst, 2006. 2:561-7. 48. Hau, H.H., R.J. Walsh, R.L. Ogilvie, D.A. Williams, C.S. Reilly, and P.R. Bohjanen. Tristetraprolin recruits functional mRNA decay complexes to A R E sequences. J Cell Biochem, 2006. 49. Rodenhiser, D. and M . Mann. Epigenetics and human disease: translating basic biology into clinical applications. Cmaj, 2006. 174:341-8. 50. Berger, S.L. Histone modifications in transcriptional regulation. Curr Opin Genet Dev, 2002. 12:142-8. 51. Legube, G. and D. Trouche. Regulating histone acetyltransferases and deacetylases. E M B O Rep, 2003. 4:944-7. GENERAL INTRODUCTION 27 52. Mahadevan, L.C. , A . L . Clayton, C A . Hazzalin, and S. Thomson. Phosphorylation and acetylation of histone H3 at inducible genes: two controversies revisited. Novartis Found Symp, 2004. 259:102-11; discussion 111-4, 163-9. 53. Eberharter, A. , R. Ferreira, and P. Becker. Dynamic chromatin: concerted nucleosome remodelling and acetylation. Biol Chem, 2005. 386:745-51. 54. Iniguez-Lluhi, J.A. For a healthy histone code, a little SUMO in the tail keeps the acetyl away. ACS Chem Biol, 2006. 1:204-6. 55. Gregory, P.D., K . Wagner, and W. Horz. Histone acetylation and chromatin remodeling. Exp Cell Res, 2001. 265:195-202. 56. Struhl, K. Histone acetylation and transcriptional regulatory mechanisms. Genes Dev, 1998. 12:599-606. 57. Eberharter, A . and P.B. Becker. Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics. E M B O Rep, 2002. 3:224-9. MECHANISMS UNDERLYING TAP DEFICIENCY 28 Chapter 2: Identification of Mechanisms Underlying TAP-1 Deficiency in Metastatic Murine Carcinomas 2.1 Introduction Antigen processing and presentation play a crucial role in immune surveillance. Peptides derived from self, viral, or tumor-related proteins are generated in the cytoplasm by the action of proteasome. Transporter associated with antigen processing (TAP), a member of the ATP-binding cassette (ABC) transporter family, functions by transporting these peptides from the cytoplasm to the lumen of the endoplasmic reticulum (ER), where each peptide forms a ternary complex with beta-2 microglobulin (p2m) and major histocompatibility complex (MHC) class I heavy chain, a process promoted by chaperone proteins (BiP, calnexin, calreticulin, ERp57 and tapasin) [1, 2]. These complexes are then transported to the cell surface and recognized by cytotoxic T lymphocytes (CTLs), which eventually kill cells that present non-self antigens. Studies have shown that components of the antigen presentation pathway are impaired in the majority of human tumor cells [3, 4], allowing them to evade immune surveillance. In particular, low expression or absence of TAP (TAP-1 and -2) molecules, a feature common to many tumors [5-7], impairs the formation of the ternary complex in the lumen of ER. This results in a lack of M H C class I expression on the cell surface. As A version of this chapter has been published. Setiadi, A.F. , David, M.D., Chen, S.S., Hiscott, J., and Jefferies, W.A. (2005) Identification of Mechanisms Underlying TAP Deficiency in Metastatic Murine Carcinomas. Cancer Research. 65: 7485-92. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 29 a consequence, specific CTLs are unable to recognize and destroy many malignant cells [6]. The importance of TAP-1 function in immunosurveillance has been highlighted in studies using a mouse lung carcinoma cell line, CMT.64 [8]. It was demonstrated that the restoration of TAP-1 expression by introducing exogenous TAP-1 or by up-regulating endogenous TAP-1 expression upon interferon gamma (IFN-y) treatment, could correct the M H C class I deficiency, resulting in recognition of these antigen-presenting cells by CTLs in vitro, as well as in a decrease of tumor growth and incidence in vivo [3, 7, 9]. This finding is encouraging for the development of therapeutic approaches that could restore TAP deficiencies in cancer cells, therefore resurrecting immune recognition of neoplastic cells. However, the mechanisms underlying TAP-1 deficiency in tumor cells remain poorly understood. Previous studies reported that downregulation of TAP-1 expression in many cancer cells likely occurs at the mRNA level [3, 7, 10]; however, these studies did not distinguish between defects in transcription or stability of the RNA. Therefore, in this study, the properties and activities of the TAP- l /LMP-2 bi-directional promoter in TAP-expressing and TAP-deficient cells were investigated in order to provide a better understanding of the transcriptional regulation of TAP-1 mRNA in tumor cells. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 30 2.2 Materials and Methods 2.2.1 Cell lines The CMT.64 cell line established from a spontaneous lung carcinoma of a C57BL/6 (H-2b) mouse [8] and the Ltk fibroblast cell line derived from a C3H/An (H-2k) mouse were grown in D M E M media. The L M D cell line derived from a metastatic prostate carcinoma of a 129/Sv mouse (a kind gift of Dr. T. C. Thompson) [11], as well as the B16F10 (B16) melanoma [12] and R M A lymphoma cell lines, both derived from C57BL/6 mice, were maintained in RPMI 1640 media. RPMI 1640 and D M E M media were supplemented with 10% heat-inactivated FBS, 2 m M L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, and 10 m M HEPES. 2.2.2 Reverse transcription-PCR analysis A l l primers used for PCR amplifications were purchased from Sigma-Genosys, Oakville, ON, and are listed in Table 2.2.2.1. Total cellular R N A were extracted using RNeasy Mini Kit (Qiagen, Mississauga, ON), and contaminating D N A was removed by treating the R N A samples with DNase 1 (Ambion Inc., Austin, TX). Reverse transcription of 1 ug of total cellular R N A was performed using the reverse transcription kit from Invitrogen (Carlsbad, CA), in a total volume of 20 pi. Two microliters aliquots of cDNA were used as a template for PCR in a total of 50 pi reaction mixture containing l x PCR buffer, 250 uM deoxynucleotide triphosphate, 1.5 m M M g C l 2 , 0.2 uM of each primer and 2.5 units Platinum Taq D N A Polymerase. A l l PCR reagents were obtained from Invitrogen. cDNA amplifications were carried out with specific primer sets in a T-© Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 31 gradient thermocycler (Biometra, Goettingen, Germany) with 25-35 cycles of denaturation (1 min, 95°C), annealing (1 min, 54-64°C), and elongation (2 min, 72°C). The cycling was concluded with a final extension at 72°C for 10 min. Twenty microliters of amplified products were analysed on agarose gels, stained with ethidium bromide and photographed under U V light. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 32 Table 2.2.2.1: Primers used for RT-PCR analysis. Ol igonuc leot ide P r imer sequence ( 5 ' - 3 ' ) a Tm( °C ) b p b M o u s e P - a c t i n F: A T G G A T G A C G A T A T C G C T G C R: T T C T C C A G G G A G G A A G A G G A T 54.0 713 TAP-1 5'-end F: A T G G C T G C G C A C G T C T G G R: A C T C A G G C C C A C C A C C C A 63.0 138 TAP-1 3'-end F: T G G C T C G T T G G C A C C C T C A A A R: T C A G T C T G C A G G A G C C G C A A G A 64.0 775 TAP-1 3^-end (last 155 bp) F: T T A T C A C C C A G C A G C T C A G C C T R: T C A G T C T G C A G G A G C C G C A A 61.0 155 TAP-1 promoter 0 F: c g g a a t t c G G C T C G G C T T T C C A A T C A R : g a g g a t c c G A G C G T G A G C T G T C C A G A G T C T 60.0 557 p T A P - l - E G F P F (TAP-1 promoter) : T T C T T C C T C T A A A C G C C A G C A R ( E G F P ) : C T C G C C C T T G C T C A C C A T 61.0 190 C M T . 6 4 TAP-1 F: C T C A C T C T G G T C A C C C T G A T C A A C R: T G G T C C A G A C T T C A G C C A C G 62.0 225 IRF1 F: C A A C T T C C A G G T G T C A C C C A T R: C A C A G G G A A T G G C C T G G A T 54.9 363 I R F 2 F: T G A T G A A G A G A A C G C A G A G G G R: T T A A C A G C T C T T G A C A C G G G C 54.0 328 S15 F: T T C C G C A A G T T C A C C T A C C R: C G G G C C G G C C A T G C T T T A C G 60.0 357 P r i on protein F: A T G G C G A A C C T T G G C T A C T R: C C A A C T A C C A C C A T G A G G T T G 58.0 239 a F: forward pr imer ; R: reverse pr imer. b Length o f the P C R ampl i f i ca t ion product. L Rest r ic t ion enzyme sites are under l ined. © Cancer Research, 2005 , 65 : (16), adapted by permiss ion. MECHANISMS UNDERLYING TAP DEFICIENCY 33 2.2.3 Chromatin immunoprecipitation (ChIP) assays Chromat in immunoprec ip i ta t ion experiments us ing 2 x 10 7 cel ls o f C M T . 6 4 , L M D , B 1 6 , L t k or R M A ce l l l ines were per formed as descr ibed i n A p p e n d i x A . 1. F i ve micrograms o f an t i -RNA polymerase II ant ibody (N-20, sc-899, Santa Cruz ) was used for the immunoprec ip i ta t ion. Leve l s o f mur ine TAP-1 promoter or TAP-1 cod ing region co-immunoprec ip i ta t ing w i t h R N A polymerase II f rom each sample were quant i f ied by real t ime P C R , us ing pr imers specif ic for the TAP-1 promoter or the last 155 bp o f TAP-1 cod ing reg ion (3'end) as l isted i n Table 2.2.2.1. Ser ia l d i lut ions o f genomic D N A or p lasmid conta in ing the murine TAP-1 promoter were used to generate standard curves for real t ime P C R us ing the corresponding pr imer sets. 2.2.4 Cloning of the TAP-1 promoter Sequence o f the murine Tap-I gene region was obtained f rom the Na t iona l Center for B io techno logy Informat ion database (GenBank Access i on N o . A F 0 2 7 8 6 5 ) . In order to predict putative transcr ipt ion factor b ind ing sites, the reg ion in-between Lmp-2 and Tap-I genes was analysed us ing the Mat inspector software f r om Genomat ix website. The predicted mur ine TAP-1 promoter reg ion was then ampl i f i ed by P C R , us ing genomic D N A f rom C M T . 6 4 cel ls as a template and the f o l l o w i n g pr imers (S igma) : 5' c g g a a t t c G G C T C G G C T T T C C A A T C A 3 ' (forward), 5' g a g g a t c c G A G C G T G A G C T G T C C A G A G T C T 3 ' (reverse). A TAP-1 promoter construct ( p T A P - l - E G F P ) was then created by insert ing the P C R product in-between the E c o R I and B a m H I sites o f promoterless pEGFP-1 vector (C lontech, Pa lo A l t o , C A ) (Append ix B ) . © Cancer Research, 2005, 65 : (16), adapted by permiss ion . MECHANISMS UNDERLYING TAP DEFICIENCY 34 2.2.5 Transfection and selection The CMT.64, L M D , B16, Ltk and R M A cells were transfected with the pTAP-1-EGFP construct or the promoterless pEGFP-1 vector using LIPOFECTAMINE PLUS Reagent (Invitrogen). Transfected cells were then selected in the presence of geneticin (G418) (Sigma) for 1 month (Table 2.2.5.1). Levels of EGFP expression in transfectants, treated or untreated with 50 ng/ml IFN-y for 48 hours, were assessed by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA). Table 2.2.5.1: G418 doses for selection of transfectants in various cell lines. Cell line Description G418 dose (mg/ml) CMT.64 TAP-deficient, mouse lung carcinoma 1 L M D TAP-deficient, mouse prostate carcinoma 1 B16 TAP-deficient, mouse melanoma 1 Ltk TAP-expressing, mouse fibroblast 0.8 R M A TAP-expressing, mouse lymphoma 0.5 2.2.6 Generation of pTAP-1-EGFP-transfected clones by FACS The pTAP-l-EGFP-transfected CMT.64, L M D and B16 cells that displayed a small level of EGFP were selected by flow cytometry using a FACSVantage DiVa cytometer (Becton Dickinson), grown in bulk culture, and treated with 50 ng/ml © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 35 recombinant murine IFN-y (R&D Systems, Minneapolis, MM) for 2 days. Cells that express high fluorescence in response to IFN-y were then sorted twice and cloned. 2.2.7 Cell Fusion and FACS analysis Twenty million cells from clones of the TAP- and M H C class I-deficient CMT.64, L M D and B16 cells stably transfected with pTAP-l-EGFP were fused with TAP- and M H C class I-expressing Ltk fibroblasts in a 1:1 ratio, following a polyethylene glycol cell fusion protocol [13]. Cells were then incubated with PE-conjugated anti-Kk mouse monoclonal antibody at 4°C for 30 minutes. The fused cells, which displayed both red (PE-anti-Kk) and green (EGFP) fluorescence, were selected by FACS. Flow cytometry analyses of EGFP, K b and K k expression were performed 1 week after the fusions. PE-conjugated anti-Kb and anti-Kk mouse monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA). Fusion experiments between a clone of pTAP-l-EGFP-transfected CMT.64 cells and a clone of pEYFP-Nl-transfected CMT.64 cells were also performed. The fused cells were then selected based on both yellow (EYFP) and green (EGFP) fluorescence. One week after the fusion, levels of EGFP and K b in the fused cells were analysed by FACS. 2.2.8 Endogenous levels and overexpression of IRF-1 and IRF-2 in cell lines Levels of endogenous IRF-1 and -2 in CMT.64, L M D , B16, Ltk and R M A cells were assessed by RT-PCR using primers specific for IRF-1 and IRF-2, following the conditions described above. In order to investigate the effects of IRF-1 and -2 overexpression on TAP-1 promoter activity in TAP-deficient carcinoma cells, a pTAP-1-© Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 36 EGFP-transfected CMT.64 clone was co-transfected with 0.1 ug of p E Y F P - N l (enhanced yellow fluorescent protein) vector (Clontech) and 1 pg of pCMV/IRF-1 or pCMV/IRF-2 [14] expression vector. Since the pCMV/IRF vectors contained no selection gene, the EYFP served as a marker to select for successfully transfected cells by FACS. 48 hours after transfection, levels of EGFP in the CMT.64 transfectants were analysed by flow cytometry. 2.2.9 Luciferase and ft-Galactosidase Assays In order to demonstrate whether the overexpressed IRF-1 and -2 were functional, cells were co-transfected with the IRF-coding constructs, an IFN-[3 promoter-luciferase construct [14], and a pCMV/B-galactosidase vector (Promega, Madison, WI) used to monitor transfection efficiency. A promoterless pGL3-luciferase vector (Promega) was also used as a background control. 48 hours after transfection, the cells were washed twice with PBS and lysed with Reporter Lysis Buffer (Promega). The luciferase and the B-galactosidase activity were measured using the Luciferase Assay System (Promega), and the B-galactosidase Enzyme Assay System (Promega), respectively. 2.2.10 Western Blot Fifty micrograms proteins per sample were separated through 8% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Bio-rad, Hercules, CA). Blots were blocked with 5% skim milk in PBS, probed with rabbit anti-mouse TAP-1 polyclonal Ab (made by Linda L i in Jefferies Lab), followed by HRP-conjugated goat anti-rabbit secondary Ab (Jackson Immunoresearch Lab., West Grove, PA). The rabbit anti-mouse TAP-1 polyclonal antibody was made by immunizing rabbits with a common © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 31 TAP1 peptide sequence, R G G C Y R A M V E A L A A P A D - C with a cysteine at the C-terminal, linked to keyhole limpet hemocyanin (Pierce Biotechnology, Rockford, IL) [15]. For the loading controls, anti-(3-actin mouse monoclonal Ab (Sigma) was used, followed by HRP-conjugated goat anti-mouse secondary Ab (Pierce Biotechnology). Blots were developed using Lumi-light reagents (Pierce). 2.2.11 Analysis of mRNA stability Unfused Ltk fibroblasts, and fused CMT.64-Ltk, LMD-Ltk and Ltk-Ltk cells were treated with 5 pg/ml actinomycin D (Sigma) for 2, 4 or 8 hours, or left untreated. 4 ug of total cellular R N A were used as templates for reverse transcription and amplification by real time PCR, using TAP-1 (5'-end) or SI5 or prion protein-specific primer sets listed in Table 1. Serial dilutions of RT products were used as templates for PCR to generate the corresponding standard curves. 2.2.12 Real time quantitative PCR analysis In this study, this method was employed for quantification of levels of endogenous TAP-1 promoter or TAP-1 coding region co-precipitating with R N A polymerase II in ChIP assays, quantification of copy number of the pTAP-l -EGFP construct integrated in stably transfected cells, and measurement of TAP-1 mRNA levels in cells upon actinomycin D treatment. cDNAs reverse-transcribed from 1-4 ug R N A and genomic D N A were used as templates for amplifications using 200-500 nM of each primer and 10 ul SYBR Green Taq ReadyMix (Sigma). Thirty five cycles of denaturation (5 seconds, 95oC), annealing (5 seconds, 61-63oC), and elongation (20 seconds, 72oC) were performed using a Roche LightCycler. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 38 2.3 Results 2.3.1 Levels of TAP-1 mRNA in CMT.64, LMD, B16, Ltk and RMA cells To investigate the mechanism underlying the lack of TAP-1 expression in carcinoma cells, CMT.64 (lung), L M D (prostate) and B16 (melanoma) cell lines were used as models for TAP-deficient cells [3, 7, 11, 16], and Ltk and R M A cell lines [7] as models for TAP-expressing cells. RT-PCR analysis confirmed that the levels of TAP-1 mRNA were higher in Ltk and R M A cells than in CMT.64, L M D and B16 cells (Figure 2.3.1.1). Treatment of the TAP-deficient cells with IFN-y increased the TAP-1 mRNA expression to similar levels as in TAP-expressing cells (Figure 2.3.1.1). TAP-1 p-actin + CO CO H t— Q z LL Q I z LL + CO CD r z I z U-4 O O _ l _ J £fl CQ J J CC DC Figure 2.3.1.1: IFN-y-treatment restores TAP-1 expression in TAP-deficient murine lung, prostate and skin carcinoma cells (CMT.64, LMD and B16). Amplification of p-actin cDNA served as an internal control. RT-PCR analyses were performed at least three times and representative data are shown. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 39 2.3.2 The recruitment of RNA polymerase II to the Tap-1 gene is lower in the CMT.64, LMD and B16, than in the Ltk and RMA cells In order to investigate whether the lack of TAP-1 mRNA expression in CMT.64, L M D and B16 cells is due to an impairment of TAP-1 transcription, the levels of R N A pol II within the 3' end of the TAP-1 coding region in TAP-deficient and TAP-expressing cells were compared, by means of chromatin immunoprecipitation assay. The results showed that the levels of RNA pol II within the 3' end of the TAP-1 coding region were lower in CMT.64, L M D and B16 than in Ltk and R M A cells (Figure 2.3.2.1), indicating that deficiency in transcription is, at least partially, underlying TAP deficiency in the carcinoma cells. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 40 9 8 _ 6 = 5 i * 0 CMT.64 LMD B16 Ltk, RMA Cell lines Figure 2.3.2.1: The recruitment of RNA polymerase II to 3' coding region of the Tap-1 gene in CMT.64, LMD and B16 is impaired in comparison to that in Ltk and RMA. Chromatin immunoprecipitation using anti-RNA polymerase II antibody was performed in each cell line, and the eluted D N A fragments were purified and used as templates for real time PCR analysis using primers specific for the 3'coding region of the Tap-1 gene. Relative R N A polymerase II levels were determined as the ratio of copy numbers of the eluted 3' coding region of the Tap-1 gene and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with TAP-expressing cells (Student's t-test). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 41 Using the same approach, but with primers specific for the TAP-1 promoter region, it was found that the recruitment of R N A pol II to the TAP-1 promoter was also relatively lower in TAP-deficient cells (Figure 2.3.2.2). Recruitment of R N A pol II complex to genes' promoters is an important event that supports transcription initiation [17]. Therefore, this result directly supported the notion that initiation of TAP-1 transcription was impaired in TAP-deficient cells. 10 9 .8: vel •7. -_ _ 6 ~ o 5 •: Q. 4 < Z - : 3 2 .1 o -* -i-* * CMT.64 LMD B16 Ltk Cel l l ines RMA Figure 2.3.2.2: The recruitment of RNA pol II to the TAP-1 promoter was low in TAP-deficient CMT.64, LMD and B16 cells. Chromatin immunoprecipitation using anti-RNA polymerase II antibody was performed, and the relative levels of R N A polymerase II recruitment to the TAP-1 promoter were assessed as described above using primers specific for the TAP-1 promoter. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with TAP-expressing cells (Student's t-test). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 42 2.3.3 Cloning and analysis of the -557 to +1 region of the CMT.64-derived TAP-1 promoter One possible explanation for the impairment of transcription of the Tap-I gene in TAP-deficient cells was the presence of mutation(s) in cw-acting elements involved in the regulation of TAP-1 promoter activity. Previous studies have demonstrated that the 593-bp-long region located in-between TAP-I and LMP-2 genes in humans acts as a bi-directional promoter: that drives the transcription of both genes [18]. Analysis of the murine TAP-1 promoter had not been reported in prior work; however, the organization of the murine Lmp-2/Tap-l genes within the M H C class II locus is reminiscent of its human ortholog [19]. Therefore, primers flanking the LMP-2/TAP-1 intergenic region were used to amplify the murine TAP-1 promoter region, using genomic D N A from CMT.64 cells as a template. The resulting PCR product was then cloned and sequenced. Analysis of the CMT.64-derived TAP-1 promoter region revealed the presence of putative binding sites for various transcription factors, including SP1, CREB, AP-1, P U . l , NF-KB and IRF (Figure 2.3.3.1), By alignment, the nucleotide sequence of the CMT.64-derived TAP-1 promoter region was found to be identical to the corresponding region in the murine M H C class II locus (NCBI accession no. AF027865). This result demonstrated that there was no mutation present in the -557 to +1 region of the CMT.64-derived TAP-1 promoter. Additional analysis of TAP-1 promoter sequence amplified from genomic D N A of TAP-deficient L M D cells and TAP-expressing R M A cells also showed that the LMD-derived TAP-1 promoter sequence was identical to the published sequence in NCBI and to that of R M A cells. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 43 I I ,1 C G G C G C T C G G „„„ T__ a T C ,'< •.( ,(.",""" r Crt ' S P - < j t — :t * _ . , v 1 C L ,1 / ' , i ' I* ? _ / I T , ' , i V V > - 1 i (, Cafe/ « -JO t >«< ' A C / & i l f / < . C r OF 7 ' _ ~J -7 . G R E B ( + ) r " ~t\A I G A G A T -- '1 p ; . . n + > T C A T G G A G A A C r t ^ c C " "? - G i " A " C ~ T - C - ~ ? A ? - l ( " \ i " C T C T C A C M O T A G T n- • • • l . l <(;>. . T C r * 1 u . ' l f ' - 1 ' - j « ,. R ' - A 1 ; C i , c -13 o-. • . •' I R F (-)•.• • T C G A G C G G G T TCCfGGGGACT TTACGfGGCAC GCCCTGJGGAC C C G C C C T T C f r T C C I T C C C C A -.?0 C G G A G A C T C C N F - K B (.+).' T G T G C A G C G C - G G A C G l l C G A G - H G C GTCT GGAC .AG -.10 -1 C T C A C G C T C G ATGf TAP-r|=':> Figure 2.3.3.1: Nucleotide sequence of the CMT.64-derived TAP-1 promoter region is identical to the corresponding region in murine MHC class II locus. The A T G codon was arbitrarily determined as +1. Motifs located on the sense strand are indicated by a (+), and motifs located on the antisense strand are indicated by a (-). 2.3.4 Activity of the -557 to +1 region of the TAP-1 promoter is impaired in TAP-deficient cells To demonstrate that this murine LMP-2/TAP-1 intergenic region indeed displays promoter activity, and to assess whether it contains cw-acting elements conferring a differential activity of the TAP-1 promoter between TAP-expressing and TAP-deficient cells, a reporter plasmid (pTAP-l-EGFP) was generated, containing the -557 to +1 region of the murine TAP-1 promoter upstream of the egfp gene in the pEGFP-1 vector. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 44 CMT.64, L M D , B16, Ltk and R M A cells were then transfected with the pTAP-l -EGFP construct, or the pEGFP-1 vector as a control, and cultured for one month under selective pressure to generate stable transfectants. Real time PCR analysis was performed using TAP-1 promoter-specific forward and EGFP-speciflc reverse primers (Table 2.2.2.1), and 100 ng of genomic D N A from the stable pTAP-l -EGFP transfectants as templates. The results showed that, on average, one copy of the pTAP-l-EGFP construct was integrated per cell in all the cell lines used (Table 2.3.4.1). An example of detailed calculations used to determine the copy numbers is outlined in Appendix A.2. Table 2.3.4.1: Statistical Evaluation of the Transfected pTAP-l-EGFP Copy Numbers Per Cell. Transfected cells pTAP-l-EGFP-1 average copy number SD CMT.64 1.59 0.06 L M D 1.49 0.03 B16 1.42 0.01 Ltk 1.51 0.02 R M A 1.41 0.02 Furthermore, EGFP levels in these stable transfectants were assessed by flow cytometry. In all the cell lines, the promoterless pEGFP-1 vector transfectants displayed low levels of background EGFP expression; however, the levels of fluorescence were higher in cells transfected with the pTAP-l -EGFP construct than in cells transfected with the vector alone. In addition, the TAP-expressing Ltk and R M A cells expressed higher levels of EGFP than the TAP-deficient CMT.64, L M D and B16 cells. (Figure 2.3.4.1). This indicated that the -557 to +1 region indeed displayed promoter activity. Finally, treatment © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 45 w i t h IFN-y resulted i n 3- to 6-fold increases i n E G F P expression i n C M T . 6 4 , B 1 6 and L M D cel ls (F igure 2.3.4.1). Th i s treatment elevated E G F P expression o f the T A P -def ic ient cel ls to s imi lar or even higher levels than those i n untreated TAP-express ing cel ls , suggesting that treatment w i th IFN-y was able to overcome the def ic iencies responsible for the l o w act iv i ty o f the TAP-1 promoter i n TAP-def i c ien t cel ls . Taken together, these results indicated that the c loned TAP-1 promoter reg ion possesses fa i thful promoter act iv i ty and contains cw-acting elements conferr ing the re lat ively l o w promoter act iv i ty i n TAP-def i c ien t cel ls . Furthermore, based on the observat ion that the levels o f E G F P tr iggered by the c loned TAP-1 promoter correlated w i th the levels o f recruitment o f R N A P o l II to the endogenous TAP-1. promoter observed by C H I P , these transfected cel ls were proven to be suitable as tools to further investigate the mechanisms under ly ing the di f ferent ia l act ivat ion o f TAP-1 promoter i n TAP-def i c ient and TAP-express ing cel ls . © Cancer Research, 2005 , 65 : (16), adapted by permiss ion. MECHANISMS UNDERLYING TAP DEFICIENCY 46 i 300 250 • - IFN-gamma • + IFN-gamma j Cell lines r-> ••- : •- -• - " ••• : • • ' \ ^ •<•-••••• •. • . •• • --• Figure 2.3.4.1: TAP-1 promoter activity is impaired in TAP-deficient CMT.64, LMD and B16 cells. Flow cytometry analysis was performed to measure TAP-1 promoter activity, based on the levels of EGFP expression in pTAP-l-EGFP stable transfectants after selection in.G418 for 1 month. The IFN-y-treated cells were incubated with 50 ng/ml IFN-y for 48 hours prior to the FACS analysis. Levels of EGFP in the cells transfected the pTAP-l -EGFP were normalized against corresponding values obtained upon transfection with pEGFP-1 vector alone. Data shown are the mean fluorescence intensity ± S E M of three independent experiments. * P < .05 compared with TAP-expressing cells; ** P < .05 compared with untreated cells (Student's t-test). I 200 w . S- 150 a) Q. LL o LU 100 50 ** ** ** 1 -=E-CMT.64 LMD B16 Ltk RMA © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 47 2.3.5 Effects of fusions between carcinoma cells and wild type fibroblasts on TAP-1 promoter activity and MHC class I expression levels The relatively low activity of the -557 to +1 region of the TAP-1 promoter in TAP-deficient cells suggested that these cells might be deficient in positive trans-acting factors that regulate TAP-1 promoter activity, or that they might display an abnormally high level of activity of trans-acting factors that negatively regulate TAP-1 promoter activity. To test these hypotheses, the effects of fusing the TAP- and M H C class I-expressing Ltk cells with TAP- and M H C class I-deficient carcinoma cells (CMT.64, L M D and B16) were investigated. Before fusion, stable pTAP-l -EGFP transfectants of carcinoma cells were sorted into single-cell clones by FACS, and a clone that displayed high induction of TAP-1 promoter activity and M H C class I expression in response to IFN-y treatment was chosen. By flow cytometry, it was found that levels of EGFP were higher in the fused CMT.64-Ltk and LMD-Ltk cells than in the unfused CMT.64 and L M D cells (Figure 2.3.5.1). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 48 CMT.64-Ltk fusion LMD-Ltk fusion EGFP EGFP Figure 2.3.5.1: TAP-1 promoter-driven EGFP expression increased in the fused CMT.64-Ltk and LMD-Ltk cells (thick line) in comparison to that in the unfused pTAP-l-EGFP-transfected CMT.64 and LMD cells (thin line). Wild type CMT.64 and L M D were used as negative controls for EGFP expression (shaded area). Further flow cytometric analysis indicated that K b was expressed on the surface of the fused CMT.64-Ltk and LMD-Ltk cells, while the unfused cells did not express K b (Figure 2.3.5.2). CMT.64-Ltk fusion LMD-Ltk fusion Figure 2.3.5.2: Surface expression of K b was increased when CMT.64 or LMD (shaded areas) were fused to Ltk cells (broken line). Expression of K b on fused CMT.64-Ltk and fused LMD-Ltk cells are displayed by thick lines. As a positive control, expression of K was assessed on IFN-y-treated CMT.64 and L M D cells (thin lines). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 49 As a control, two groups of TAP-deficient cells were fused: the CMT.64 cells stably transfected with the pTAP-l-EGFP construct with another group of CMT.64 cells expressing EYFP. The results showed no induction of K b surface expression in the fused CMT.64(pTAP-l-EGFP)-CMT.64(pEYFP-Nl) cells (Figure 2.3.5.3). Figure 2.3.5.3: No induction of K b surface expression in the fused CMT.64(pTAP-l-EGFP)-CMT.64(pEYFP-Nl) cells (thick line). CMT.64 cells untreated (shaded area) or treated with IFN-y (thin line) were used as negative or positive control, respectively, for K b expression. The increase in EGFP expression of the pTAP-l-EGFP-transfected TAP-deficient carcinoma cells that were fused with TAP-expressing fibroblasts suggests that the TAP-deficient cell lines studied display a relatively low level of activity of trans-dLC\'mg factor(s) positively regulating the TAP-1 promoter activity. This deficiency could, at least partially, be corrected by a fusion with TAP-expressing cells. Furthermore, the expression of M H C class I allotype of the fibroblasts (K k ) in the fused CMT.64-Ltk and © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 50 LMD-Ltk cells were also assessed. The results showed a slight but consistent decrease in the level of K k expressed (Figure 2.3.5.4). CMT.64-Ltk fusion 8. | k 8 : 1 8 . J S A M 8 8 -1: / \ y \ f -LMD-Ltk fusion LA io° io' I O 2 io3 ,o() io1 'to2 ' m3 Figure 2.3.5.4: K k surface expression was reduced in the fused CMT.64-Ltk and LMD-Ltk (thick lines) in comparison to that in the wild type Ltk cells (thin lines). Wild type CMT.64 and L M D cells were used as negative controls for K k expression (shaded area). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 51 2.3.6 Overexpression of IRF-1 and IRF-2 in CMT.64 cells did not result in significant changes in TAP-1 promoter activity IFN-y treatment resulted in high induction of TAP-1 promoter activity and could subsequently overcome TAP and M H C class I deficiencies in carcinoma cells [11, 20, 21]. Therefore, factors that are known to be activated in response to IFN-y are attractive candidates for the future discovery of positive trans-acting factors that are either absent or functionally defective in TAP-deficient cells, thus accounting for the impairment of TAP-1 promoter activity in carcinoma cells. The interferon regulatory factors (IRFs), that binding motif was found in the TAP-1 promoter (Figure 2.3.3.1), were examples of proteins that expression could be regulated by IFN-y [22]. In order to investigate whether TAP downregulation might be caused by abnormally low or high levels of IRF-1 and IRF-2, respectively, endogenous levels of IRF-1 and -2 mRNAs in TAP-expressing and TAP-deficient cells were observed by RT-PCR. The results indicated that both IRF-1 and -2 mRNA levels did not correlate with TAP levels in the cell lines (Figure 2.3.6.1). CD U CO < r_ ^mmmfM g ^ ^ s ^ ^ H m^^&^m. ^ ^ ^ ^ ^ B ? • ^ ^ ^ M P R ^ ^ | j j | j | |g(P : fiiiwir « m » mm® :ms • m s Figure 2.3.6.1: Endogenous levels of IRF-1 and -2 mRNA did not correlate with T A P levels in the cell lines. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 52 In order to further investigate the putative role of IRF-1 and -2 in modulating TAP-1 promoter activity in TAP-deficient cells, human IRF-1 and -2 [14] were overexpressed in a clone of CMT.64 cells containing the pTAP- l -EGFP construct. Flow cytometry analysis showed that the overexpression of the IRFs had no significant effect on EGFP levels in the transfectants (Figure 2.3.6.2). Figure 2.3.6.2: Overexpression of IRF-1 or -2 in a clone of pTAP-l-EGFP construct-transfected CMT.64 cells did not result in significant changes of the TAP-1 promoter activity. Representative histograms show EGFP expressed by the IRF-1 or IRF-2-overexpressing cells. The shaded areas indicate background green fluorescence of the cells transfected with PBS as negative controls, the thin and thick lines represent EGFP expression in cells overexpressing the IRF that were treated with IFN-y or left untreated, respectively. IRF-1 IRF-2 EGFP EGFP © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 53 In order to test whether the transfected IRFs were functional, the cells were co-transfected with either an I F N - P promoter-luciferase construct or a promoterless pGL3-luciferase vector. As expected [14], the overexpression of exogenous IRF-1 was found to increase the basal level of I F N - P promoter activity, whereas the IRF-2 decreased it (Figure 2.3.6.3). This indicated that overexpression of IRF-1 and IRF-2 alone was not sufficient for modulating the TAP-1 promoter activity in CMT.64 cells. Figure 2.3.6.3: Overexpression of IRF-1 and -2 modulated IFN-P promoter activity linked to a luciferase gene. Luciferase activities were normalized against P-galactosidase activities, to correct for variations in transfection efficiencies. Data shown are the average ± S E M of three independent experiments. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 54 2.3.7 Analysis of TAP-1 expression in unfused and fused cells During the course of these studies, it was noted that the TAP-1 promoter displayed a low but detectable activity in TAP-deficient cells, while TAP-1 mRNA was barely detectable in these cells. To investigate whether the low levels of TAP-1 mRNA in TAP-deficient cells resulted solely from the deficiency in TAP-1 promoter activity demonstrated above, the levels of TAP-1 mRNA transcribed from the CMT.64 genome and from the Ltk genome in fused CMT.64-Ltk cells were compared. To distinguish between TAP-1 expressed from the two genomes, analysis of the TAP-1 mRNA polymorphism in both cells was carried out by PCR amplifications of TAP-1 mRNA transcribed in each cell line. A total of 12 bp differences was found between the nucleotide, sequence of CMT.64-derived and Ltk-derived TAP-1 mRNAs (Appendix C). These polymorphisms are most likely strain-specific and are not responsible for TAP-deficiency in CMT.64 cells, since another analysis showed that the CMT.64 TAP-1 sequence was identical to that of TAP-expressing R M A cells. Both CMT.64 and R M A cells originated from C57/BL6 mice, whereas Ltk cells were derived from a C3H/An mouse. Primers specific to CMT.64-derived TAP-1 mRNA were then designed in order to investigate its levels in the fused CMT.64-Ltk cells. Non-polymorphic primers were used for further analysis of total TAP-1 expression in the fused cells. Despite the increase in TAP-1 promoter activity driving the egfp gene in the fused CMT.64-Ltk cells (Figure 2.3.5.1), no TAP-1 mRNA from the CMT.64 genome could be detected in cells that were not treated with IFN-y (Figure 2.3.7.1). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 55 was Figure 2.3.7.1: No expression of TAP-1 mRNA from the CMT.64 genome detectable by RT-PCR in the fused CMT.64-Ltk cells. Amplifications of reverse-transcribed CMT.64 TAP-1 mRNA from IFN-y-treated cells and B-actin mRNA were used as positive controls and loading controls, respectively. In fact, analysis of total TAP-1 expression showed that the fused CMT.64-Ltk and L M D -Ltk cells displayed drastically lower levels of TAP-1 mRNA and protein than the unfused Ltk fibroblasts did (Figure 2.3.7.2). This suggested the existence of post-transcriptional mechanisms that further down-regulate levels of TAP-1 mRNA in TAP-deficient cells. Therefore, the possibility of a difference in TAP-1 mRNA stability between TAP-expressing and TAP-deficient cells was further investigated. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 56 Total TAP-1 Total TAP-1 p-actin p-actin Figure 2.3.7.2: Total TAP-1 expression was reduced when CMT.64 and LMD cells were fused with wild type Ltk cells. Results from RT-PCR analysis and Western Blot were shown in top and bottom panel, respectively. Levels of P-actin were used as loading controls. 2.3.8 TAP-1 mRNA stability decreases in carcinoma-fused fibroblasts To assess the regulation of TAP-1 at the level of mRNA stability, unfused Ltk fibroblasts, as well as fused CMT.64-Ltk, LMD-Ltk and Ltk-Ltk cells were treated with actinomycin D (Act. D) for 2 to 8 hours, in order to block neo-synthesis of mRNA. Residual levels of TAP-1 mRNA after treatment of cells with Act. D were then assessed by real time PCR, using constant amounts of total R N A as templates. Levels of TAP-1, S15 and prion mRNA in actinomycin D-treated cells are expressed as percentages of the levels of the corresponding mRNA in untreated cells. The results showed that TAP-1 mRNA stability decreased when fibroblasts were fused to carcinoma cells (Figure 2.3.8.1). © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 57 0 2 4 8 Hours of Actinomycin D treatment *- Ltk *~ Fused Ltk-Ltk Fused LMD-Ltk Fused CMT.64-Ltk Figure 2.3.8.1: TAP-1 mRNA is rapidly degraded in fused carcinoma-fibroblast cells. As a control, the stability of unrelated mRNA, such as the mRNA coding for S15 and prion protein (Figure 2.3.8.2) was assessed. The results demonstrated that the S15 and prion mRNA stability was unaffected by the cell fusion events, and that the decrease in mRNA stability is fairly specific to TAP-1 in carcinoma-fused cells. These results suggest the existence of factors, which remain to be identified, that enhanced TAP-1 mRNA degradation in carcinoma cells. 120 100 80 60 40 20 0 0 2 4 8 Hours of Ac t inomyc in D treatment - o - Ltk ~ » ~ B 1 6 * Fused LMD-Ltk ~m~ Fused CMT 64-Ltk 140 120 < 100 1 80 £ 60 I i— 0 2 4 8 Hour s of A c t i n o m y c i n D t r ea tment - o - Ltk -*-F u s e d Ltk-Ltk ~m— F u s e d LMD-Ltk - F u s e d CMT-Ltk Figure 2.3.8.2: The stability of S15 and prion mRNA is similar in unfused and fused cells. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 58 2.4 Discussion TAP-1 downregulation in tumors has been reported in many studies [6, 10, 23-25]; however, the molecular mechanisms underlying this defect remain poorly understood. One of the recently proposed mechanisms of A P M downregulation is the overexpression of oncogenes, for example HER-2/neu, that gives rise to the emergence of immune escape phenotype of tumors [26]. Related mechanisms of TAP downregulation may include an impairment of TAP-1 promoter activity (due to mutation in cw-acting elements in the promoter or in distal enhancer/silencer regions, chromatin remodeling at the TAP-1 locus, and/or difference in expression or functionality of trans-acting factors), as well as a relatively low stability of TAP-1 mRNA in these cells. In this study, murine lung, prostate and skin carcinoma cells (CMT.64, L M D and B16) were used as models for TAP- and M H C class I-deficient cancer cells. The results in this study suggested that TAP deficiency in these cells is caused by the lack of activation or expression of TAP-1 transcriptional activators, as well as a decrease in TAP-1 mRNA stability. These results demonstrated that: a) treatment of the TAP-deficient cells with IFN-y increased the TAP-1 mRNA expression to similar levels as in TAP-expressing cells; b) the initiation of TAP-1 transcription was impaired in TAP-deficient cells; c) the relatively low activity of TAP-1 promoter in the carcinoma cells is due to regulatory defects rather than mutations in the TAP-1 promoter; d) low TAP-1 promoter activity and M H C class I deficiency in carcinoma cells could be corrected, at least partially, by fusions with wild type fibroblasts; and e) a decrease in TAP-1 mRNA stability also contributed to TAP-1 deficiency in murine lung carcinoma cells. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 59 Based on the structural and functional analysis of TAP-1 promoter of TAP-deficient cells, as well as results obtained from chromatin immunoprecipitation assays of endogenous TAP-1 promoter in various TAP-expressing and TAP-deficient cells, it was proposed that one mechanism underlying TAP deficiency in these cells is the impairment of the ability of TAP-1 promoter to drive transcription. As no mutation was observed in the CMT.64- and LMD-derived TAP-1 promoter, this deficiency was likely to be caused by the lack of transcriptional activators necessary for optimal binding of the R N A polymerase II complex to the TAP-1 promoter, or conversely, by the presence of transcriptional inhibitors that prevent the binding. FACS analysis of the fusions between TAP- and M H C class I-deficient carcinoma cells (CMT.64, L M D and B16) of H-2 b origin and TAP- and M H C class I-expressing fibroblasts (Ltk) of H-2 k origin showed an increase of TAP-1 promoter activity and some increase in K b expression. However, despite the increase of the promoter activity in the fused CMT.64-Ltk, no TAP-1 mRNA from the CMT.64 genome could be detected by RT-PCR (Figure 2.3.7.1); instead, total levels of TAP-1 mRNA and protein in the fused CMT.64-Ltk and LMD-Ltk cells were lower than in the unfused Ltk cells (Figure 2.3.7.2). This might have accounted for the decrease in surface expression of K k that was observed in the fused cells (Figure 2.3.5.4). These unexpectedly low levels of TAP-1 mRNA and protein in the fused fibroblasts-carcinoma cells had prompted the investigation whether other mechanisms contribute to the TAP-1 deficiency in carcinoma cells, in addition to the impairment of TAP-1 promoter activity that would also account for the disappearance of TAP-1 in the fused cells. It was then found that the stability of TAP-1 mRNA was decreased when fibroblasts were fused with carcinoma cells. This © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 60 result suggests that the stability of TAP-1 mRNA is lower in CMT.64 and L M D cells than in the Ltk cells. Unfortunately, the extremely low levels of TAP-1 mRNA in the carcinoma cells, even in absence of actinomycin D, preclude direct confirmation of this hypothesis. Further studies will be required in order to precisely characterize the positive regulatory factors that are lacking or defective in carcinoma cells, as well as the mechanism that leads to a reduction of TAP-1 mRNA stability in carcinoma cells. These studies are of fundamental importance, as they will significantly contribute to a better understanding of the underlying cause of antigen processing deficiency in many tumor types. This will in turn lead to new approaches to modify the immunogenicity and antigenicity of tumor cells, thereby allowing recognition of tumors by immune surveillance mechanisms. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 61 2.5 References 1. Germain, R.N. and D.H. Margulies. The biochemistry and cell biology of antigen processing and presentation. Annu Rev Immunol, 1993. 11:403-50. 2. Cresswell, P. and J. Howard. Antigen recognition. Curr Opin Immunol, 1999. 11:61-3. 3. Alimonti, J., Q J . Zhang, R. Gabathuler, G. Reid, S.S. Chen, and W.A. Jefferies. TAP expression provides a general method for improving the recognition of malignant cells in vivo. Nat Biotechnol, 2000. 18:515-20. 4. Seliger, B., M.J. Maeurer, and S. Ferrone. Antigen-processing machinery breakdown and tumor growth. Immunol Today, 2000. 21:455-64. 5. Ritz, U . and B. Seliger. The transporter associated with antigen processing (TAP): structural integrity, expression, function, and its clinical relevance. Mol Med, 2001. 7:149-58. 6. Seliger, B., M.J. Maeurer, and S. Ferrone. TAP off—tumors on. Immunol Today, 1997. 18:292-9. 7. Gabathuler, R., G. Reid, G. Kolaitis, J. Driscoll, and W.A. Jefferies. Comparison of cell lines deficient in antigen presentation reveals a functional role for TAP-1 alone in antigen processing. J Exp Med, 1994. 180:1415-25. 8. Franks, L . M . , A.W. Carbonell, V.J . Hemmings, and P.N. Riddle. Metastasizing tumors from serum-supplemented and serum-free cell lines from a C57BL mouse lung tumor. Cancer Res, 1976. 36:1049-55. 9. Jefferies, W.A., G. Kolaitis, and R. Gabathuler. IFN-gamma-induced recognition of the antigen-processing variant CMT.64 by cytolytic T cells can be replaced by © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 62 sequential addition of beta 2 microglobulin and antigenic peptides. J Immunol, 1993.151:2974-85. 10. Seliger, B., D. Atkins, M . Bock, U. Ritz, S. Ferrone, C. Huber, and S. Storkel. Characterization of human lymphocyte antigen class I antigen-processing machinery defects in renal cell carcinoma lesions with special emphasis on transporter-associated with antigen-processing down-regulation. Clin Cancer Res, 2003. 9:1721-7. 11. Lee, H.M., T.L. Timme, and T.C. Thompson. Resistance to lysis by cytotoxic T cells: a dominant effect in metastatic mouse prostate cancer cells. Cancer Res, 2000.60:1927-33. 12. Poste, G., J. Doll, I.R. Hart, and I.J. Fidler. In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties. Cancer Res, 1980. 40:1636-44. 13. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, Production of antibodies, in Current Protocols in Molecular Biology. 2001, John Wiley & Sons, Inc. p. 11.7.1-11.7.3. 14. Lin, R., A. Mustafa, H. Nguyen, D. Gewert, and J. Hiscott. Mutational analysis of interferon (IFN) regulatory factors 1 and 2. Effects on the induction of IFN-beta gene expression. J Biol Chem, 1994. 269:17542-9. 15. Zhang, Q.J., R.P. Seipp, S.S. Chen, T.Z. Vitalis, X .L . Li , K.B. Choi, A. Jeffries, and W.A. Jefferies. TAP expression reduces IL-10 expressing tumor infiltrating lymphocytes and restores immunosurveillance against melanoma. Int J Cancer, 2007. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 63 16. Seliger, B., U . Wollscheid, F. Momburg, T. Blankenstein, and C. Huber. Characterization of the major histocompatibility complex class I deficiencies in B16 melanoma cells. Cancer Res, 2001. 61:1095-9. 17. Burley, S.K. and R.G. Roeder. Biochemistry and structural biology of transcription factor IID (TFIID). Annu Rev Biochem, 1996. 65:769-99. 18. Wright, K . L . , L .C. White, A . Kelly, S. Beck, J. Trowsdale, and J.P. Ting. Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidirectional promoter. J Exp Med, 1995. 181:1459-71. 19. Janeway, C , P. Travers, M . Walport, and M . Shlomchik. Immunobiology: the immune system in health and disease. 5th ed. 2001, New York, N Y Garland Publishing. 20. Matsui, M . , S. Machida, T. Itani-Yohda, and T. Akatsuka. Downregulation of the proteasome subunits, transporter, and antigen presentation in hepatocellular carcinoma, and their restoration by interferon-gamma J Gastroenterol Hepatol, 2002. 17:897-907. 21. Mikyskova, R., J. Bubenik, V . Vonka, M . Smahel, M . Indrova, J. Bieblova, J. Simova, and T. Jandlova. Immune escape phenotype of HPV16-associated tumours: M H C class I expression changes during progression and therapy. Int J Oncol, 2005. 26:521-7. 22. Brucet, M . , L. Marques, C. Sebastian, J. Lloberas, and A . Celada. Regulation of murine Tapl and Lmp2 genes in macrophages by interferon gamma is mediated by STAT1 and IRF-1. Genes Immun, 2004. 5:26-35. © Cancer Research, 2005, 65: (16), adapted by permission. MECHANISMS UNDERLYING TAP DEFICIENCY 64 23. Vitale, M . , R. Rezzani, L. Rodella, G. Zauli, P. Grigolato, M . Cadei, D.J. Hicklin, and S. Ferrone. H L A class I antigen and transporter associated with antigen processing (TAP1 and TAP2) down-regulation in high-grade primary breast carcinoma lesions. Cancer Res, 1998. 58:737-42. 24. Restifo, N.P., F. Esquivel, Y . Kawakami, J.W. Yewdell, J.J. Mule, S.A. Rosenberg, and J.R. Bennink. Identification of human cancers deficient in antigen processing. J Exp Med, 1993. 177:265-72. 25. Murray, P.G., C M . Constandinou, J. Crocker, L.S. Young, and R.F. Ambinder. Analysis of major histocompatibility complex class I, TAP expression, and LMP2 epitope sequence in Epstein-Barr virus-positive Hodgkin's disease. Blood, 1998. 92:2477-83. 26. Herrmann, F., H.A. Lehr, I. Drexler, G. Sutter, J. Hengstler, U . Wollscheid, and B. Seliger. HER-2/neu-mediated regulation of components of the M H C class I antigen-processing pathway. Cancer Res, 2004. 64:215-20. ©Cancer Research, 2005, 65: (16), adapted by permission. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 65 Chapter 3 : Epigenetic Control of TAP-1 expression and the Immune , Escape Mechanisms in Malignant Carcinomas 3.1 Introduction The current paradigm is that emergence of tumors is limited by a robust adaptive immune response that recognizes aberrant expression of tumor associated antigens. This mechanism of immune-surveillance is thought to work efficiently until tumor cells undergo chromosomal alterations that result in phenotypic conversion to a form that is no longer recognizable by the immune system. This conversion closely parallels the emergence of metastatic forms of the tumor cells that, in turn, gain a growth advantage over non-metastatic forms that remain hampered by the fidelity of the immune system. Several immune escape mechanisms that allow the metastatic tumor to go undetected have been observed; however, most tumors down-regulate a cassette of genes involved in antigen processing and presentation [1-3]. These include the genes encoding beta-2 microglobulin (B2m), the transporters associated with antigen processing (TAP) -1 and -2, tapasin, the low molecular weight proteins (LMP)-2 and -7, as well as the proteasome activator PA28 [1, 4, 5]. TAP-1 down-regulation has specifically been attributed to tumor growth and metastatic ability, and is used as a predictor of rapid tumor A version of this chapter has been submitted for publication. Setiadi, A.F. , David, M.D., Seipp, R.P., Hartikainen, J.A., Gopaul, R., and Jefferies, W.A. (2007) Epigenetic Control of the Immune Escape Mechanisms in Malignant Carcinomas. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 66 progression and poor survival rates in humans [1-3, 5-8]. Furthermore, this antigen presentation deficiency can be temporarily reversed in vitro by treatment of the tumor cells with interferon gamma (IFN-y), and can also be genetically complemented in vivo by the sole restoration of TAP-1 expression [1, 8]. Therefore, the presence of TAP-1 improves the ability of the host to mount a therapeutic and protective tumor antigen-specific immune response. This finding is encouraging for the development of therapeutic approaches that can restore TAP deficiency in cancer cells, and thus result in restoration of immune recognition of tumors. However, the actual defect that leads to TAP downregulation and immune escape mechanisms remains undescribed. Previous studies in this thesis concluded that TAP-deficiency in metastatic carcinomas is not caused by mutations within the TAP-1 promoter. Instead, it was found that the. metastatic carcinomas lack positive trans-acting factors that regulate TAP-1 transcription [9]. An intriguing observation at the initiation of the present study was that transient expression of episomal copies of a reporter gene driven by TAP-1 promoter led to high levels of expression even in TAP-deficient carcinomas, while stable transfection and genomic integration of the same plasmid led to transcriptional silencing. This lead to the investigation whether epigenetic mechanisms play a role in the regulation of antigen processing in tumors, and whether the transcriptional activators that are deficient or non-functional in malignant cells are those with intrinsic histone acetyltransferase (HAT) activity. Deregulation of genes involved in the modulation of chromatin structure has been closely linked to immune evasion, uncontrolled cell growth, and development of tumors [10-12]. The study in this chapter will provide fundamental insights into the EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 67 epigenetic mechanisms of tumor immune escape and metastatic diseases, which may help in revising immunotherapeutic methodologies for eradicating cancers. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 68 3.2 Materials and Methods 3.2.1 Cell lines and reagents The TC-1 cell line was developed by transformation of murine primary lung cells with HPV16 E6 and E7 oncogenes and activated H-ras [13]. TC-1 cells display high levels of TAP-1 and M H C class I. TC1-/D11 (= Dll) and TC1-/A9 (= A9) were tumor cell clones that exhibited spontaneous downregulation of M H C class I expression, and were derived from TC-1-inoculated mice [14]. These cell lines were cultured in the presence of 0.4 mg/ml G418. Another model used in this study consists of a murine primary prostate cancer cell line, PA, and its metastatic, TAP- and M H C class I-deficient derivative, L M D , both derived from a 129/Sv mouse [15]. The TAP-deficient CMT.64 cell line was established from a spontaneous lung carcinoma in a C57BL/6 mouse [16]. The TAP-expressing Ltk (=L-M(TK-)) fibroblast cell line was derived from a C3H/An mouse (ATCC, Manassas, V A ) . A l l cell lines above were grown in D M E M . The C57BL/6-derived B16F10 melanoma [17] and R M A lymphoma [1] cell lines were cultured in RPMI 1640 media. RPMI 1640 and D M E M media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 m M L-glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, and 10 mM HEPES. When indicated, cells were treated with 100 ng/ml Trichostatin A (TSA) (Sigma, St. Louis, MO) for 24 hours (CMT.64, B16F10, PA and LMD) or 48 hours (TC-1, D l 1 and A9), or with 50 ng/ml IFN-y for 48 hours. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 69 3.2.2 Reverse transcription-PCR analysis Total cellular R N A was extracted using Trizol Reagent (Invitrogen, Burlington, ON); contaminating D N A was removed by treatment with DNase 1 (Ambion Inc., Austin, TX). Reverse transcription of 1 ug of total cellular RNA was performed using the reverse transcription kit (SSII RT) from Invitrogen, in a total volume of 20 pi. Two microliters aliquots of cDNA were used as a template for PCR in a total of 50 pi reaction mixture containing lx PCR buffer, 250 uM deoxynucleotide triphosphate, 1.5 m M MgCl2, 200 nM of each primer and 2.5 units Taq or Platinum Taq D N A Polymerase. cDNA amplifications were carried out in a T-gradient thermocycler (Biometra, Goettingen, Germany) with 25-35 cycles of denaturation (1 min, 95°C), annealing (1 min, 54-64°C), and elongation (2 min, 72°C). The cycling was concluded with a final extension at 72°C for 10 min. Twenty microliters of amplified products were analyzed on agarose gels, stained with ethidium bromide and photographed under U V light. Primers used for PCR amplifications (Sigma-Genosys, Oakville, ON and Integrated D N A Technologies (IDT), Coralville, IA) are listed in Table 3.2.2.1 below. A l l PCR reagents were obtained from Invitrogen and Fermentas (Burlington, ON). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 70 Table 3.2.2.1: Primers used for R T - P C R and real time PCR analysis. Ol igonuc leot ide P r imer sequence (5'-3') a Tm( °C ) b p b TAP-1 F: T G G C T C G T T G G C A C C C T C A A A R: T C A G T C T G C A G G A G C C G C A A G A 64.0 775 T A P - 2 F: G C T G T G G G G A C T G C T A A A A G R: T A T T G G C A T T G A A A G G G A G C 60.0 665 L M P - 2 F: C G A C A G C C C T T T A C C A T C G R: T C A C T C A T C G T A G A A T T T T G G C A G 56.0 240 Tapas in F: A C G T C A C C C T G G A G G T G G C A R: A C T G G A G T C A T C T G G G C C A G 60.0 166 B 2 M F: A T G G C T C G C T C G G T G A C C R: T C A C A T G T C T C G A T C C C A G T A G A 56.0 360 (3-actin F: A T G G A T G A C G A T A T C G C T G C R: T T C T C C A G G G A G G A A G A G G A T 54.0 713 TAP-1 promoter (5'-end) F: G G C T C G G C T T T C C A A T C A R: G G A T G G G A A A A T T C A C G C A A 60.0 207 TAP-1 promoter (3'-end) F: T T C T T C C T C T A A A C G C C A G C A R: C G A G C G T G A G C T G T C C A G A G T C T 61.0 172 p T A P l - L u c copy number F (TAP-1 promoter) : T T C T T C C T C T A A A C G C C A G C A R ilucl): A G T G G G T A G A A T G G C G C T G 61.0 190 a F: forward pr imer ; R: reverse pr imer. b Length o f the P C R ampl i f i ca t ion product. 0 Restr ic t ion enzyme sites are under l ined. 3.2.3 Real-time quantitative PCR analysis Pur i f i ed genomic D N A was used as template for ampl i f icat ions us ing 200-500 n M o f each pr imer and 1 u l S Y B R Green Taq R e a d y M i x (Roche, M a n n h e i m , Germany) i n a total o f 10 u l reaction mixture. 35-40 cycles o f denaturation (5 seconds, 95°C), EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 71 annealing (5 seconds, 61-63°C), and elongation (20 seconds, 72°C) were performed using a Roche LightCycler. 3.2.4 Flow cytometry Flow cytometric analysis of H-2K b expression was performed using PE-conjugated anti-Kb mouse monoclonal antibody (mAb) (BD Pharmingen, San Diego, CA) and a FACScan cytometer (Becton Dickinson, Mountain View, CA). 3.2.5 Chromatin immunoprecipitation assays Chromatin immunoprecipitation experiments using 7 x 106 cells per sample were done as previously described [18]. Five micrograms of anti-RNA pol II (N-20, sc-899, Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-acetyl-histone H3 (Upstate Biotechnology Inc., Lake Placid, N Y ) or anti-CBP (A-22, sc-369, Santa Cruz Biotechnology) polyclonal antibody (Ab) were used for the immunoprecipitation. Levels of murine TAP-1 promoter co-immunoprecipitating with the antibody from each sample were quantified by real-time PCR using primers specific for the TAP-1 promoter. Primers specific to the 3'-end of the TAP-1 promoter region were used for PCR when the templates were immunoprecipitated using anti-RNA pol II or anti-acetyl-histone H3 antibody, while 5'-end-specific primers were used for templates immunoprecipitated using anti-CBP antibody. Serial dilutions of plasmid containing the murine TAP-1 promoter were amplified following the same protocol to generate a standard curve. 3.2.6 Plasmid construction The plasmid containing an EGFP coding region driven by the TAP-1 promoter (pTAPl-EGFP) was described previously [9]. A similar construct containing a luciferase EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 72 gene dr iven by the TAP-1 promoter region ( p T A P l - L u c ) was created by insert ing the TAP-1 promoter between the Sac I and B g l II sites o f the pGL4 .14 [ luc2/Hygro ] vector (Promega, M a d i s o n , WI ) (Append ix B ) . 5'-end truncations o f the TAP-1 promoter reg ion were also c loned into pGL4 .14 [ luc2/Hygro ] vector. The A T G codon o f the Tap-1 gene was arbitrar i ly numbered as +1, and the truncated promoters were named accord ing to the starting base pos i t ion o f forward pr imers w i th respect to the A T G codon (-427, -401 and -150). Pr imers used for P C R ampl i f icat ions o f the fu l l TAP-1 promoter and its truncations are l isted i n Table 3.2.6.1. Table 3.2.6.1: Primers used for PCR amplifications of full TAP-1 promoter and its truncations. Ol igonuc leot ide P r imer sequence ( 5 ' - 3 ' ) a Tm( °C ) b p b F u l l TAP-1 promoter c F: c g a g a g c t c G G C T C G G C T T T C C A A T C A R: gaagatctG A G C G T G A G C T G T C C A G A G T C T 60.0 557 -427 truncated TAP-1 promoter 0 F: c g a g a g c t c A T C T G C C C A G A G A C A G G T G A R : g a a g a t c t G A G C G T G A G C T G T C C A G A G T C T 55.0 427 -401 truncated TAP-1 promoter c F: c g a g a g c t c A G G G T C C T G C C C T C A A T C R : g a a g a t c t G A G C G T G A G C T G T C C A G A G T C T 55.0 401 -150 truncated TAP-1 promoter 0 F: c g a g a g c t c T T C T A G T C A G C T C C A C C A G C T C R : g a a g a t c t G A G C G T G A G C T G T C C A G A G T C T 60.0 150 a F: forward pr imer ; R: reverse pr imer. b Leng th o f the P C R ampl i f i ca t ion product. 0 Rest r ic t ion enzyme sites are under l ined. 3.2.7 Transfection and selection T C - 1 , D l l , A 9 , P A and L M D cel ls were transfected w i t h the p T A P l - L u c constructs or the promoterless pGL4.14[ luc2/Hygro ] vector us ing E x G e n 500 in vitro EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 73 Transfection Reagent (Fermentas). Transient transfectants were analysed between 12-72 hours after co-transfection of the pTAPl-Luc construct or the promoterless vector with pRL-TK plasmid (Promega) in 10:1 ratio. To obtain stable transfectants, the transfected cells were selected for three weeks in the presence of 200-550 ng/ml Hygromycin B (Sigma). 3.2.8 Luciferase assays Relative luciferase activity (RLA) in transient transfectants was assessed by dual luciferase assay (Promega) 12-72 hours after transfection. Copy numbers of the p T A P l -Luc construct integrated into the genome of the stable transfectants were quantified by real time PCR using a forward primer specific for.the TAP-1 promoter and a reverse primer specific for the luc2 gene. R L A in stable transfectants (3-4 weeks post-transfection) was assessed by Bright-Glo luciferase assay (Promega) using 10,000 cells per sample and was determined by normalizing the luciferase values with copy number of plasmids integrated into the genome of each stable transfectant. 3.2.9 Western Blots Fifty micrograms of protein per sample were separated with 6% (CBP and TAP-1) or 15% (P-actin and acetyl-histone H3) SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Bio-rad, Hercules, CA). Blots were blocked with 5% skim milk in PBS and incubated with the following rabbit polyclonal antibodies: anti-mouse TAP-1 [19]; anti-acetyl-histone H3 (Upstate Biotechnology Inc.); anti-p300 (C-20, sc-585, Santa Cruz); anti-CBP (A-22, sc-369, Santa Cruz). Secondary antibody was an HRP-conjugated goat anti-rabbit polyclonal antibody (Jackson Immunoresearch Lab., EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 74 West Grove, PA). For the loading controls, anti-P-actin mouse monoclonal antibody (Sigma) was used, followed by HRP-conjugated goat anti-mouse secondary antibody (Pierce, Rockford, IL). Blots were visualized using Lumi-light E C L reagents (Pierce). 3.2.10 Cytotoxicity assays CTL effector cells were generated by injecting a C57BL/6 mouse (Charles River, St. Constant, QC) with 107 tissue culture infectious particles (TCIP) of Vesicular Stomatitis Virus (VSV). The spleen was harvested seven days later, homogenized, and cells were incubated for five days in RPMI-1640 containing 10% FBS (Hyclone), 20 m M HEPES, 1% N E A A , 1% sodium pyruvate, 1% L-glutamine, 1% penicillin/streptomycin and 0.1% 2-ME, in the presence of l u M VSV-NP peptide ( R G Y V Y Q G L ) . TC-1, D l l and A9 cells were treated with IFN-y (50 ng/ml) or TSA (100 ng/ml) for 24 hours, or left untreated; V S V at an MOI of 7.5 was then added and the experiment was performed 16 hours later. Cells were washed with PBS and loaded with 5 1 C r by incubating 106 cells with 100 uCi of 5 1 C r sodium chromate (Amersham, Arlington Heights, IL) in 250 ul of culture media for one hour. Following three washes with PBS, the target cells were incubated with the effector cells at the indicated ratios for four hours. One hundred microliters of supernatant from each well were collected and the 5 1 C r release was measured by a y-counter (LKB Instruments, Gaithersburg, MD). The specific 5 l C r release was calculated as follows: ((experimental - media control) / (total - media control)) x 100%. The total release was obtained by lysis of the cells with a 5% Triton X-100 solution. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 75 3.2.11 Establishment of HPV-positive cancer xenografts and treatment with Trichostatin A Four hundred thousand pTAPl-Luc stable transfectants of TC-1 or A9 cells were resuspended in PBS, then injected subcutaneously (s.c.) into seven-week-old female C57BL/6 syngeneic mice. TSA was dissolved in DMSO to a concentration of 0.2 mg/ml. Daily treatment with 50 pi TSA (500 pg/kg) or DMSO (vehicle control) was administered via intraperitoneal (i.p.) injection for 20 days, starting on day seven after injection with tumor cells. The dose of 500 pg/kg TSA per animal per day was chosen as it had been successfully used by others to suppress other types of tumor growth in murine tumor models [20-22]. Mice were assigned to three groups (TC-1 treated with DMSO, A9 treated with DMSO and A9 treated with TSA) of four animals per group. Mice were weighed weekly, and their behavior and food intake were monitored throughout the course of the experiment. Tumors were measured three times a week and tumor volume was calculated using the formula: tumor volume = length x width x height x n/6 [20]. The study period was determined by the size of the tumors in the A9 group treated with DMSO vehicle control. A l l procedures were performed in compliance with the Canadian Council on Animal Care (CCAC) guidelines. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 76 3.3 Results 3.3.1 Chromatin remodeling regulates TAP-1 transcription In order to investigate transcriptional regulation of TAP-1, a luciferase reporter construct was generated by cloning the mouse TAP-1 promoter region upstream of the luc gene in the pGL4.14[luc2/Hygro] vector (pTAPl-Luc) and was transfected into the TAP-expressing (Ltk, R M A ) and TAP-deficient (LMD, CMT.64, B16F10) cells. Consistent with previous results obtained with the TAP-1-promoter-EGFP reporter construct [9], the pattern of expression of luciferase (Luc) in these stable transfectants was found to correlate with the pattern of endogenous TAP-1 expression ([9] and Figure 3.3.1.1). However, when the TAP-expressing and TAP-deficient cells were transiently transfected with the same construct, the pattern of differential expression no longer correlate with endogenous TAP-1 expression. It was conceivable that this lack of correlation may have resulted, at least in part, from the fact that the cell lines used were derived from distinct types of carcinomas, and. therefore were unrelated to each other. To avoid this problem, two other models of TAP-expressing (TC-1 and PA) and TAP-deficient (TC-1/A9 and LMD) cells that were derived from primary tumors and their metastases, respectively, were used in subsequent experiments. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 77 12-72 hrs post-transfection Vector control L* CMT.64 I r f l D11 AS Cell lines 3-4 weeks post-transfection Vector control raba Uk ^fT64 I LB CMT.64 ....Eia... TC-1 D11 A9 TC-1 D11 A9 PA LMD PA LMD Cell lines Figure 3.3.1.1: Differences in TAP-1 promoter activity in stable transfectants generally match the TAP-1 expression profiles better than in transient transfectants. Relative luciferase activity (RLA) in transient (12-72 hours post-transfection) and stable (3-4 weeks post-transfection) transfectants. In the transient transfectants, the luciferase unit in each cell line was determined as the ratio of firefly:renila luciferase unit. In the stable transfectants, the luciferase unit in each cell line was determined as the ratio of firefly luciferasexopy number of pTAPl-Luc construct integrated into the genome. Relative luciferase activity (RLA) was determined as the luciferase unit in a particular cell line divided by the lowest value of luciferase unit obtained in that particular group of cells. Columns, average of three to six independent experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 78 It was confirmed that TAP-1 expression levels in these new models of TAP-expressing and TAP-deficient cells correlated with M H C class I surface expression levels (Figure 3.3.1.2). In the prostate carcinoma model, PA cells that expressed a higher level of TAP-1 than L M D cells also expressed a higher level of surface M H C class I. Similarly, in the HPV-positive carcinoma model, TC-1, D l l and A9 cells that express high, moderate and low levels of TAP-1, respectively, also expressed the same pattern of surface M H C class I. Figure 3.3.1.2: Analysis of TAP- 1 and surface MHC class I expression by RT-PCR and flow cytometry, respectively. Shaded area, thin and thick lines represent low (A9 or LMD), medium ( D l l ) and high (TC-1 or PA) levels of M H C class I expression, respectively. Amplification of P-actin cDNA served as an internal control in the RT-PCR analysis. Data are representatives of three experiments. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 79 These TAP-expressing and TAP-defieient cells were then transfected with the pTAPl-Luc construct. With the TC-1/D11/A9 model, it was found that, again, the relative levels of luciferase expression in stable population of transfectants matched the profiles of endogenous TAP-1 expression better than those in transient transfectants. Taken together with the results obtained with Ltk, R M A , CMT.64 and B16F10 cells, this observation suggests that integration of the reporter construct into the chromatin correlates with the differential activity of the TAP-1-promoter between TAP-expressing and TAP-deficient cells. The prostate carcinoma model was an exception to this trend, since the luciferase expression patterns obtained from stable and transient transfection both correlated with the endogenous TAP-1 expression (Figure 3.3.1.1). As a control, it was verified that luciferase expression in all cell lines transfected with pGL4.14[luc2/Hygro] vector alone was negligible compared to that in cells transfected with the pTAPl-Luc construct. Furthermore, it was found that R N A polymerase II (pol II) binding to the endogenous TAP-1 promoter was lower in cells with low TAP-1 expression levels than that in cells with high TAP-1 expression levels (Figure 3.3.1.3). One possible explanation to account for this phenomenon was that the chromatin structure was forming a physical barrier, reducing the access of R N A pol II complex to the TAP-1 promoter. The L M D cells, the only cells that did not follow the TAP-1 expression trend in stable versus transient transfection, are likely to have additional defects that impair the activity of the TAP-1 promoter in addition to those related to chromatin remodeling. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 80 • 3.5 I ' ft* < • ' ' lllilill * K ..V 0 •TO; ' AS : • CelHsnes .: . - 5 s 4 S 2 11 * • r i RMA CMTS4--• Ceii hues .: :>. Figure 3.3.1.3: R N A pol II binding to TAP-1 promoter is low in T A P -deficient carcinomas. The levels of R N A pol II in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-RNA pol II Ab. The eluted D N A fragment were purified and used as templates for real-time PCR analysis using primers specific for the 3'-end of the TAP-1 promoter. Relative R N A pol II levels were determined as the ratio of copy numbers of the eluted TAP-1 promoter and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test). 3.3.2 Histone H3 acetylation within the TAP-1 promoter is low in MHC class l-deficient carcinomas A well-known epigenetic mechanism that regulates gene expression is the acetylation of histone H3 within a gene locus [23-25], which promotes relaxation of the nucleosome structure and allows the transcriptional machinery to access the promoter [24, 26]. Therefore, the hypothesis tested was that the differential TAP-1 promoter EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 81 activity between TAP-expressing and TAP-deficient cells results from distinct levels of histone H3 acetylation within the TAP-1 promoter. In the HPV-positive carcinoma model, it was found that histone H3 acetylation within TAP-1 promoter was impaired in A9 cells, which express the lowest level of TAP -1. D l 1 cells that express intermediate levels of TAP-1 compared to TC-1 and A9 also had intermediate levels of acetyl-histone H3 within the TAP-1 promoter (Figure 3.3.2.1). In the prostate carcinoma model, the metastatic, TAP-deficient L M D cells also had less acetyl-histone H3 within the TAP-1 promoter than in the non-metastatic, TAP-positive PA cells. In addition, the level of acetyl-histone H3 within the TAP-1 promoter in several other TAP-expressing (Ltk and R M A ) and TAP-deficient (CMT.64 and B16F10) cell lines was also assessed. Acetyl-histone H3 was again found in low levels in the TAP-deficient carcinomas; in fact, it was absent in the highly metastatic CMT.64 cells. Taken together, these results demonstrate a clear correlation between the levels of TAP-1 expression and histone H3 acetylation within the TAP-1 promoter. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 82 _ 2- ? o <» \ Z-rt x 1 2-8 1 s . ;-. . •*• •.. ..Cell lines' Figure 3.3.2.1: Acetyl-histone H3 binding to TAP-1 promoter is low in TAP-deficient carcinomas. The levels of acetyl-histone H3 in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-acetyl-histone H3 Ab. The eluted D N A fragments were purified and used as templates for real-time PCR analysis using primers specific for the 3'-end of the TAP-1 promoter. Relative acetyl-histone H3 levels were determined as the ratio of copy numbers of the eluted TAP-1 promoter and copy numbers of the corresponding inputs. The smallest ratio in each group of experiments was arbitrarily determined as 1. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with cells that expressed higher TAP-1 and M H C class I in the same group of cells (Student's t-test). RMA •• :Cf,fT.64-••. .Gel! >tnes\ 3.3.3 Identification of a region in TAP-1 promoter responsible for the differential activity in TAP-expressing and TAP-deficient cells In order to determine the critical region of the TAP-1 promoter that is responsible for the differential promoter activity in TAP-expressing and TAP-deficient cells, several constructs were made by cloning 5'-end truncations of the TAP-1 promoter into the pGL4.14[luc2/Hygro] vector (constructs -427, -401 and -150 in Figure 3.3.3.1). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 83 -55.01- • . .•' C T T T C C M l ' C AGCGGCTGCG • CGCGGTGCAG' :GCAACTTGCA-; GAC'llGAGGCC C C G C C C C A T J C . ..• SP-1 (-) • -490 . . • f ATCGCGCAAG GGGCGIGC.CG -TTCTACGAGC ' AT-TTGGCGCG CAGAGCRAAC• GTGAGCAGGG -430. ' r**-42?-- • ' F ~ r - 4 0 r .. • • •• CAAATCTGCC CAGAGACAGG,.|TGRCG ACAJGA: GGGTCCTGCC' CTCAA'IJCTGG GGTGGGGCC'XJ • . C R I B Th : V . .. • - '• SP. -K + ) -370' " • ' . :' • " . . '. GGGATGGGAA AATTCACGCA' AGCAAGTTAA, GGGGGCT[GGG G A A G A A J G A G G . AGAATGAGAT -310 a rr A 1.1 i f r r Cjx t If i r C • - : -.. . / AP-1 ( + ) • -250 • • : .. .,: :• • - .• • . . . f r n I i - - i V " L T " " T C T T T L C -190 • • ,. .•,:••!• v..: •• l—*> -15D- : I r II k f I Kfc ( I " f ) ur k ) 1 (. ATG j Figure 3.3.3.1: TAP-1 promoter sequence with transcription factor binding motifs and 5' truncation sites. The TAP-1 A T G codon was arbitrarily determined as +1. Truncation sites are indicated by numbered arrows. Motifs located on the sense strand are indicated by a (+) and motifs located on the antisense strand are indicated by a (-). It was found that progressive truncations of 5'-end of the TAP-1 promoter resulted in a gradual decrease in the promoter's activity (as measured by Luc expression) in the TAP-expressing cells (Figure 3.3.3.2). This decrease in promoter activity indicates that the deleted regions normally participate in TAP-1 promoter activity. Interestingly, the truncations up to -401 did not affect the activity of the TAP-1 promoter in the TAP-deficient cells. This indicates that, in contrast to what was observed in TAP-expressing EPIGENETIC CONTROL OF TUMOR IMMUNOEVASIONMECHANISMS 84 cel ls , the -567 to -401 region o f the TAP-1 promoter does not participate i n the promoter 's act iv i ty i n TAP-def i c ien t cel ls . F ina l l y , it is important to note that the -401 construct y ie lded s imi la r promoter act iv i ty i n TAP-express ing and TAP-def i c ient ce l ls . Taken together, these observations indicate that a l l the cw-acting elements responsible for the differential TAP-1 promoter act iv i ty i n TAP-express ing versus TAP-def i c ien t cel ls are located in the region encompass ing base pairs -567 to -401. Figure 3.3.3.2: A critical region that is responsible for differential activity of the TAP-1 promoter in TAP-expressing and TAP-deficient cells is found in-between -427 and -401 region of the promoter. R L A was measured i n stable transfectants. The largest value in each group o f experiments was arbitrari ly determined as 1. Columns, average o f five exper iments; bars, S E M . * P < .05 compared w i th TAP-express ing cel ls in the same group (Student's t-test). A more detai led analysis revealed that the region encompass ing base pairs -427 to -401 was suff icient to confer the differential TAP-1 promoter act iv i ty observed in T A P -expressing versus TAP-de f i c i en t cel ls. Ana l y s i s o f putative transcr ipt ion factor b i nd ing sites, us ing Tfs i tescan software (www.i f t i .org) , suggested the presence o f a C R E B b ind ing site w i th in this region (Figure 3.3.3.1). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASIONMECHANISMS 85 3.3.4 C B P binding to TAP-1 promoter is impaired in metastatic carcinomas The existence of a putative CREB binding site in the region responsible for the differential activity of the TAP-1 promoter in TAP-expressing versus TAP-deficient cells is particularly interesting, since the CREB-binding protein (CBP) is one of the well-known transcriptional co-activators that possess intrinsic histone acetyltransferase (HAT) activity [27-29]. In addition, CBP is known to acetylate histone H3 [30], and the HAT activity and recruitment of CBP can be stimulated by various transcription factors [27] including SP-1 and AP-1 whose binding sites are also found in the TAP-1 promoter (Figure 3.3.3.1). These notions are consistent with the hypotheses that the differential TAP-1 promoter activity results from differences in chromatin structure at the tap-1 locus, and that the /ram-acting factors deficient or non-functional in TAP-deficient carcinomas can be those with the ability to control chromatin structures. Therefore, whether CBP plays a role in the differential TAP-1 promoter activity in TAP-expressing versus TAP-deficient cells was further analysed. Western blot analysis showed that CBP is not lacking or truncated in TAP-deficient metastatic carcinomas (Figure 3.3.4.1). CBP p-actin Q 0. _ J 1 T— O *- a) H Q < — mm, mm mm mm — mm* mmm mmm mmm Figure 3.3.4.1: Expression of CBP proteins is similar in TAP-expressing and in TAP-deficient carcinoma cells. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 86 However, chromatin immunoprecipitation analysis showed that CBP binding to the TAP-1 promoter was significantly lower in TAP-deficient, metastatic carcinomas compared to TAP-expressing, pre-metastatic cells (Figure 3.3.4.2). These results suggest that, in TAP-deficient cells, the lack of H A T activity normally exerted by CBP in TAP-1 promoter is likely to play a role in the inaccessibility of the promoter to the R N A pol II complex and in the subsequent impairment of TAP-1 transcription. " f+1 0.5 o i I 1 I 1 Figure 3.3.4.2: CBP binding to TAP-1 promoter is impaired in TAP-deficient, metastatic carcinomas. Chromatin immunoprecipitation using anti-CBP antibody was performed as described earlier. Columns, average of four experiments; bars, SEM. * P < .05 compared with cells that expressed the highest TAP-1 and M H C class I in the same group of cells (Student's t-test). 4 j : " 3.5 •] 12! CO 1 5 ' O 1 , 05 ' 0 ' D11 •• • CelHines 3.3.5 IFN-y treatment increases the level of C B P , acetyl-histone H3 and RNA polymerase II recruitment to the TAP-1 promoter in TAP-deficient metastatic carcinomas Since the lack of CBP-mediated acetylation of histone H3 at the TAP-1 promoter appears to contribute to the TAP-1 deficiency in metastatic carcinomas, the hypothesis EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 87 that IFN-y, a we l l -known inducer o f TAP-1 expression [9, 15, 31 , 32] , restored TAP-1 express ion by increas ing histone H 3 acetylation at this locus was tested. The results showed that IFN-y increased the level o f C B P , acetyl-histone H 3 and R N A p o l II recruitment to the TAP-1 promoter (F igure 3.3.5.1 A , B and C , respect ively) . A. B. I 2 5 EL 2 ca i.r, o 1 0.5 0 T rt i Cell lines 4 1 _ 3-5 § 3 a> ~ 2.5 i CO X 2 ! 2-15-0.5 0 r f i D11 Cell lines 12 _ iO B 8 1 6 a. < i : z o: 2 ! o ....! D11 Cell lines : Q untreated £3 + IFN-garrma Q untreated • •HFN-gairirB untreated : m • IFN-gamma 3.5 3 s r> 2 I t 1 < 0.5 90 80 « 70 5 60 = 50 0 40 1 30 2 20 PA LMD Cell lines I PA LMD Cell lines Cell lines Q untreated E3 4- IFTFgarrma [•untreated j 8 + IFN-gamma • untreated E3+ IFN-gamma Figure 3.3.5.1: IFN-y treatment improves the recruitment of CBP, acetyl-histone H3 and RNA pol II to TAP-1 promoter, most significatly in TAP-deficient cells. Ch romat in immunoprec ip i ta t ion using A, an t i-CBP , B, anti-acetyl-histone H 3 or C, a n t i - R N A po l II antibody was performed as descr ibed earlier. Columns, average o f three to four experiments; bars, S E M . * P < .05 compared w i th untreated cel ls (Student's t-test). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASIONMECHANISMS 88 Based on these results, a possible mechanism of TAP-1 induction by IFN-y is proposed to be via the improvement of CBP binding to the TAP-1 promoter, thereby promoting histone H 3 acetylation in the region and leading to relaxation of the surrounding chromatin. This would in turn increase the accessibility of R N A polymerase II complex to the TAP-1 promoter and promote transcription of this gene. 3.3.6 Trichostatin A (TSA), a histone deacetylase inhibitor, increases the expression of TAP-1 and other antigen-processing machinery (APM) components in metastatic (TAP-deficient) and pre-metastatic (TAP-expressing) carcinomas As the acetylation level of histones in the TAP-1 promoter seems to regulate TAP-1 expression in carcinomas, an investigation was performed to test whether inhibition of histone deacetylase (HDAC) activity in TAP-deficient carcinoma cells with TSA would restore TAP-1 expression. TSA is a highly specific, hydroxamic acid-based H D A C inhibitor (HDACi) [33-35]. By chromatin immunoprecipitation assays, it was found that TSA treatment, indeed, enhanced the recruitment of R N A pol II complex to the TAP-1 promoter in most cell lines (Figure 3.3.6.1 A). This phenomenon was particularly prominent in the TAP-deficient cells, in which TSA treatment enhanced the recruitment of R N A pol II to levels similar to those in TAP-expressing cells. In addition, TAP-1 promoter activity also increased significantly in TAP-deficient cells upon treatment with TSA (Figure 3.3.6.IB). The fact that TSA treatment increases TAP-1 promoter activity confirms that chromatin remodeling plays a crucial role in the regulation of TAP-1 promoter activity. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 89 A B 6 _ S > „ i 3-Q. < 2 1 6 5 4 3 3 a 2 1 o 1 P I ^1 &sfci.... D11 Cell lines Vector control 1 TC-1 D11 A9 TC-1 D11 Cell lines A9 | • untreated JB + TSA • untreated S + T S A > „ = 5 a * 2 3 rr 2 1 o 4 • 3 • < a. 2 M S i • untreated H + TSA Cell lines Vector control • untreated! | S + TSA L j _ LMD RA Cell lines Figure 3.3.6.1: TSA treatment enhances R N A pol II recruitment to TAP-1 promoter and TAP-1 promoter activity. A, The levels of R N A Pol II in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-RNA pol II. B, TAP-1 promoter activity in stable transfectants was determined by luciferase assay. Columns, average of three to six experiments; bars, SEM. * P < .05 compared with untreated cells (Student's t-test). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 90 Based on the observations that levels of histone H3 acetylation correlate with TAP-1 expression and that TSA treatment enhances TAP-1-promoter activity, it was tempting to speculate that the effect of TSA on TAP-1 promoter activity could be occurring via an increase in histone H3 acetylation. However, chromatin immunoprecipitation results showed that TSA treatment did not significantly alter the levels of acetyl-histone H3 in TAP-1 promoter in any of cell lines tested (Figure 3.3.6.2). This suggests that the mechanism by which TSA increases the TAP-1 promoter activity does not involve a direct improvement of histone H3 acetylation within the TAP-1 promoter itself. Further investigation will be required to delineate the exact mechanism underlying the effect of TSA on TAP-1 promoter activity. Figure 3.3.6.2: TSA treatment does not significantly alter the levels of histone H3 acetylation in TAP-1 promoter. The levels of acetyl-histone H3 in TAP-1 promoter of each cell line were assessed by chromatin immunoprecipitation using anti-acetyl-histone H3 antibody. Columns, average of three to six experiments; bars, SEM. 2.5 - 3 • untreated Cell lines Cell lines EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 91 3.3.7 TSA treatment increases the expression of TAP-1 and surface MHC class I, resulting in an increased susceptibility of TAP-deficient metastatic carcinomas to CTL killing Consistent w i t h the increase i n TAP-1 promoter act iv i ty , the express ion o f TAP-1 at the m R N A and protein levels (as assessed by R T - P C R and Western B lot ) was enhanced i n response to T S A or IFN-y treatment (F igure 3.3.7.1). Figure 3.3.7.1: TAP-1 expression in all cell lines, particularly in the TAP-deficient carcinomas, was up-regulated with TSA treatment. Up-regulated TAP-1 express ion f rom IFN-y-treated cel ls was used as a posi t ive contro l , p-actin express ion served as a loading control . Data are representatives o f three experiments. R T - P C R analyses were performed to test whether treatment w i th T S A or IFN-y also restored the expression o f L M P - 2 and poss ib ly other A P M components, since the TAP-1 promoter is a bi-direct ional promoter that also controls the express ion o f the LMP-2 gene EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 92 [36]. The results showed that the Lmp-2 and Tap-2 genes have s imi la r patterns o f expression as the Tap-I gene i n T C - 1 , D l l and A 9 cel ls (h igh, moderate and l o w , respectively) (F igure 3.3.7.2). The expression o f L M P - 2 , as w e l l as T A P - 2 and tapasin, was also increased by T S A or IFN-y treatment in the HPV-pos i t i v e ca r c inoma ce l l l ines. LMP-2 TAP-2 Tapasin p-actin Figure 3.3.7.2: Expression of LMP-2, TAP-2 and tapasin in all cell lines, particularly in the TAP-deficient carcinomas, was up-regulated with TSA treatment. A m p l i f i c a t i o n o f A P M c D N A f rom IFN-y-treated ce l ls was used as a posit ive contro l . P-actin expression served as a loading contro l . Data are representatives o f three experiments. F l o w cytometr ic analyses o f surface H-2K b express ion were per formed to test whether T S A treatment w o u l d also result in an increase o f M H C class I antigen presentation in the metastatic carc inoma cel ls, since the express ion o f several antigen processing components increases upon T S A treatment. The results demonstrated that T S A treatment increased the H-2K b surface expression by approx imate ly 10 fo ld i n T A P -EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 93 deficient cel ls , whereas the levels were unchanged in P A and TC-1 cel ls , w h i c h natural ly express h igh levels o f surface H - 2 K b (F igure 3.3.7.3). IFN-y treatment increased the surface H - 2 K b expression i n a l l ce l l l ines. P A L M D D11 Figure 3.3.7.3: Surface H-2K expression, particularly on MHC class I-deficient cells, was enhanced by TSA treatment. Ce l l s untreated (shaded areas) or treated w i th 100 ng/ml T S A (thick lines) or 50 ng/ml IFN-y (thin l ines) were stained w i th PE-conjugated ant i-H-2K b m A b . Data are representatives o f three experiments. Furthermore, cy totox ic i ty ( C T L ) assays were per formed to test whether the increased expression o f H - 2 K b at the ce l l surface after T S A treatment w o u l d improve the recognit ion and the k i l l i n g o f v ira l ly-infected cancer cel ls by virus-specif ic cy totoxic T EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 94 lymphocytes. In this assay, VSV-derived peptides are presented by the infected target cells in the context of H-2K b only i f the cells have functional antigen processing and presenting machinery [1, 8]. As expected, DI 1 and A9 cells, which express lower surface K b than the TC-1, are less susceptible to killing by the CTLs (Figure 3.3.7.4). TSA treatment of the virus-infected DI 1 and A9 cells enhanced CTL killing by approximately 6 and 7 fold, respectively. IFN-y treatment resulted in a drastic increase in the killing of all VSV-infected cell lines. However, L M D cells remained resistant to CTL killing despite a significant induction of A P M and M H C class I expression by IFN-y due to unknown mechanisms independent of M H C class I expression [15]. 70 60 50 Jj 40 5' 30 20 10 0 I—I—I * Cell lines * 7 Figure 3.3.7.4: TSA treatment improves HPV-positive tumor cell killing by CTLs. Target cells were uninfected or VSV-infected, untreated or treated with TSA or IFN-y for 24 hours before infection with the V S V . CTL assays were performed using effector:target ratio of 0.8:1 to 200:1. Representative data using 22:1 effectontarget ratio are shown in this figure. A l l cells were infected with V S V at a MOI of 7.5 for 16hr. Columns, average of three experiments; bars, SEM. * P < .05 compared with untreated cells (Student's t-test). EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 95 An additional experiment using VSV-infected, TAP-deficient B16F10 as target cells also showed a similar improvement in the ability of specific CTLs to kill the cancer cells upon TSA treatment (Figure 3.3.7.5). Taken together, these results indicate that TSA or IFN-y treatment increases TAP-1 expression, and ultimately results in an increase in immune recognition of metastatic carcinoma cells. efrr 22:1 7.4:1 Effector: Target Ratio • uninfected - 0 ng/ml TSA - 20 ng/rrt TSA - 50 ng/rr! TSA -100 ng/ml TSA -* 2.5:1 Figure 3.3.7.5: TSA treatment improves B16F10 tumor cell killing by CTLs. Target cells were uninfected or VSV-infected, untreated or treated with various concentrations of TSA for 24 hours before infection with the V S V . CTL assay was performed using effector:target ratio of 2.5:1 to 67:1. A l l cells were infected with V S V at a MOI of 7.5 for 16hr. 3.3.8 TSA treatment suppresses the growth of TAP-deficient, metastatic tumors in vivo As TSA treatment increased CTL killing of TAP-deficient cells in vitro, it was further investigated whether it would result in a decrease in tumor formation in vivo. The EPIGENETIC CONTROL OF TUMOR IMMUNOEVASION MECHANISMS 96 results indicate that indeed, daily treatment of mice with TSA reduced the growth rate of A9 tumors (Figure 3.3.8.1). In addition, as expected, the TAP-expressing TC-1 cells were significantly less tumorigenic than the TAP-deficient A9 cells. Only four out of nine mice injected subcutaneously with TC-1 cells grew small tumors that only appeared at approximately 3-4 weeks after the injection, as compared to one week in all mice injected with the A9 cells. 700 n 600 500 day 5 day 9 day 13 day 17 day 21 day 26 Days after tumor cells injection Figure 3.3.8.1: T S A treatment suppresses tumor growth in vivo. A9 (MHC class I-deficient cells) tumor growth was suppressed in mice treated with 500 ug/kg of TSA daily compared to in those treated with DMSO vehicle control (n = 8 per treatment group). TC-1 group represented tumor growth in mice injected with high M H C class I-expressing cells (n = 9). Data represent the mean tumor volume ± SEM. EPIGENETIC CONTROL OF TUMOR IMMUNOEVASIONMECHANISMS 97 TAP-1 promoter activity in A9 tumor cells isolated from the TSA-treated mice was found to be significantly higher than in those isolated from the control mice (Figure 3.3.8.2). These observations support the hypothesis that the improvement of TAP-1 expression and M H C class I antigen presentation leads to increased killing of the TAP-deficient metastatic cancer cells, thus suppresses tumor growth in vivo. A9 + DMSO A9 + TSA Cells Figure 3.3.8.2: TAP-1 promoter activity is enhanced in TAP-deficient tumor cells isolated from TSA-treated mice. TAP-1 promoter-driven Luciferase (pTAPl-Luc) expression is higher in A9 cells isolated from TSA-treated mice than in those isolated from DMSO-treated mice (n=4 per treatment group). * P < .05 compared with cells from DMSO-treated mice (Student's t-test). EPIGENETIC CONTROL OF THE IMMUNE ESCAPE MECHANISMS 98 3.4 Discussion The regulation of chromatin structure plays an important role in controlling gene expression. The observations that silencing of the TAP-1 promoter reporter construct occurs exclusively after the construct integrates into the genome, and that levels of R N A pol II bound to the TAP-1 promoter are relatively low in metastatic, TAP-deficient cells suggest that access of the R N A pol II complex to the TAP-1 promoter is limited in the TAP-deficient cells. A compact nucleosome structure around a promoter region can act as a physical barrier that prevents the binding of transcriptional activators to the promoter and consequently, halts the transcription process [37]. Therefore, it was tested i f TAP deficiency in metastatic cancer cells results from a repressive state of chromatin structure. A high level of acetylated core histones in a chromatin template, particularly in the proximal region of an acetylation-sensitive promoter [37, 38], has been associated with active transcription [23, 24, 26, 27, 38]. As the results in this study demonstrate a role for chromatin remodeling in the regulation of TAP-1 expression, the specific players involved were sought to be identified. Although histone H3 is not the only core histone whose modification has been shown to influence gene expression [23, 39], the correlation between the acetylation of histone H3 and activation of several genes has been widely studied and is now well established [23-25, 39]. Therefore, histone H3 acetylation status within TAP-1 promoter was investigated in this study. The results demonstrate that the levels of acetylated histone H3 within the TAP-1 promoter show a trend similar to the levels of R N A pol II binding to TAP-1 promoter, and to TAP-1 expression at both the R N A and the protein levels in all the cell lines tested. These observations suggest that the level of histone H3 acetylation within the TAP-1 promoter plays a role in the regulation EPIGENETIC CONTROL OF THE IMMUNE ESCAPE MECHANISMS 99 of TAP-1 transcription, although it is not likely to be the sole mechanism involved, since the activation of transcription generally involves synergistic actions of several factors [24]. Furthermore, it was found that the region in the TAP-1 promoter that is responsible for the differential activity in the TAP-expressing versus the TAP-deficient carcinoma cells is located between bases -427 and -401, and that this region encompasses a putative CREB binding motif. CBP, a mammalian histone acetyltransferase, is known to associate with CREB [28, 29]. Therefore, the recruitment of CBP to the TAP-1 promoter in TAP-deficient cells was investigated. It was found that, indeed, the levels of CBP binding to TAP-1 promoter in TAP-deficient cells were lower than in cells that express higher levels of TAP-1. CBP is known to acetylate histones around promoter regions, resulting in increased accessibility of the promoters to essential transcriptional regulators [29]. Current findings suggest that in the metastatic cancer cells, the lack of CBP binding to TAP-1 promoter potentially contributes to the TAP-1 deficiency, probably by impairing histone H3 acetylation. In the study described in the previous chapter, it was proposed that the lack of expression or activity of trans-acting factors is one of the mechanisms that contributes to TAP-1 deficiency in malignant cells [9]. CBP is likely to be one of these factors. The exact mechanisms responsible for the decreased recruitment of CBP to the TAP-1 promoter remain to be identified. IFN-y is known to be a potent inducer of TAP-1 and surface M H C class I expression in cancer cells [9, 15, 31, 32]; however, little is known about molecular mechanisms that lead to TAP-1 induction by IFN-y. The findings that the improvement of CBP recruitment to the TAP-1 promoter correlated with higher levels of histone H3 EPIGENETIC CONTROL OF THE IMMUNE ESCAPE MECHANISMS 100 acetylation, and therefore, active transcription of the Tap-I gene, provide one mechanism by which IFN-y increases TAP-1 expression. Furthermore, it was demonstrated for the first time that treatment of TAP-deficient cells with TSA resulted in a significant increase in R N A pol II binding to the TAP-1 promoter and in the promoter's activity. TSA belongs to a group of hydroxamic acid-based histone deacetylase inhibitors (HDACi) that act on selective genes, altering the transcription of only approximately 2% of expressed genes in cultured tumor cells [35]. Consistent with this notion is the fact that histone acetylases and deacetylases act selectively on specific genes, and hence do not universally affect the transcription of all genes [38]. TSA has been shown to confer anti-tumor effects in vitro and in vivo [33-35]. However, the mechanism underlying the effect of TSA in tumor antigen presentation was not fully understood. In addition to the improvement in TAP-1 expression, it was found that treatment with TSA also resulted in the upregulation of several other A P M components, such as TAP-2, LMP-2 and tapasin. Given the proximity of Tap-I, Tap-2 and Lmp-2 genes within the M H C class II locus, and the location of gene encoding tapasin in the same chromosomal region [40], it is likely that these genes are co-regulated at the chromatin level. It has been reported previously that a small region (approximately 300 bp) could control the transcription of a cluster of genes by serving as a binding site for factors involved in chromatin remodeling [41, 42]. Since transcription of a TAP-1 promoter-driven reporter gene in TAP-deficient cells was silenced as the construct integrated into random sites of the genome, and since both the endogenous and the transfected TAP-1 promoter activity improved upon TSA treatment, it is possible that EPIGENETIC CONTROL OF THE IMMUNE ESCAPE MECHANISMS 101 an autonomous chromatin condensation regulatory element (ACCRE) at the M H C class II locus exists within the LMP-2/TAP-1 intergenic region. Despite the efficacy of TSA in improving M H C class I antigen presentation in metastatic cancer cells, it was observed that the levels of induction resulting from TSA treatments were never as strong as the effects generated by IFN-y. The results showed that one of the key differences is their distinct ability to enhance the levels of histone H 3 acetylation in the TAP-1 promoter. IFN-y treatment of TAP-deficient cells enhanced histone H 3 acetylation within the TAP-1 promoter to much higher levels than TSA did, up to similar or even higher levels than in the TAP-expressing cells. It is conceivable that IFN-y treatment results in a maximal state of relaxation of chromatin structures around the TAP-1 locus, thus enabling optimal levels of binding of general transcription factors and R N A pol II to the TAP-1 promoter, sufficient to support high levels of transcription of this gene. Finally, results from in vivo experiments showed that daily treatment with TSA suppressed tumor growth in mice inoculated with A 9 cancer cells. The increase in TAP-1 promoter activity, hence enhanced transcription of the Tap-1 gene and possibly the expression of other A P M components in TAP-deficient A 9 tumor cells of TSA-treated mice, may significantly contribute to the reduction of tumor growth in these mice. These findings are encouraging for the development of therapeutic approaches that aim to increase tumor antigen presentation as a way to improve the recognition and the killing of neoplastic cells by specific CTLs. Nevertheless, further studies are required to improve the in vivo efficiency of natural H D A C i , such as TSA, depudecin, trapoxins, apicidins, sodium butyrate, phenylbutyrate and suberoyl anilide hydroxamic acid (SAHA) as anti-EPIGENETIC CONTROL OF THE IMMUNE ESCAPE MECHANISMS 102 cancer agents, as their effectiveness is greatly impaired by their instability and low retention in vivo [43]. 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CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 109 Chapter 4 : Characterization of Novel TAP-1 Regulator Genes 4.1 Introduction Previous studies have demonstrated that one of the underlying mechanisms contributing to TAP deficiency in metastatic carcinomas is the lack of activation or expression of TAP-1 transcriptional activators [1]. Therefore, expression of the functional activator(s) in TAP-deficient carcinoma cells should lead to increased TAP-1 expression and restoration of tumor antigen presentation. Results from a parallel study provided deeper insight into the characteristics of the unknown transcriptional activators, in that they might play a role in chromatin remodeling within the Tap-1 gene locus (Chapter 3). The present chapter describes the development of a new method and preliminary results obtained in the attempt to characterize novel TAP regulator genes. In this classical complementation study, a stable clone of pTAPl-EGFP-transfected CMT.64 cells [1] was transduced with high complexity, human cDNA library retroviral supernatants derived from normal human lung or spleen specimens (ViraPort; Stratagene). The cDNA-packaging virus possesses vesicular stomatitis virus G (VSV-G) envelope protein, which recognizes common membrane phospholipids, such as phosphatidylserine [2]. Therefore, the VSV-G-coated retrovirus is capable of infecting a wide variety of living cells with enhanced transduction efficiency, unlike viruses with other commonly-used envelope proteins that recognize only specific cell surface receptors [2]. cDNA libraries from human sources were used since at the time of this work, the only VSV-G-packaged mouse cDNA library available was derived from testes, CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 110 which was certainly not a good source of TAP and M H C class I activators, since a testis was an immune privilege site that displayed downregulated expression of M H C class I [3]. Normal lung-derived cDNA library was initially chosen since the CMT.64 target cells were originated from murine lung tumor cells. Expression of TAP-1 activators from normal lungs could potentially restore TAP and M H C class I expression in the metastatic cancer cells. A similar experiment using normal spleen-derived cDNA was performed to observe any similarities in the identity of TAP-activator candidates obtained from the lung cDNA library screening. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 111 4.2 Materials and Methods 4.2.1 Cell lines The C M T . 6 4 ce l l l ine established f r om a spontaneous lung ca rc inoma o f a C 5 7 B L / 6 mouse [4], the L t k and N IH/3T3 mouse f ibroblasts ( A T C C , Manassas, V A ) , and A 5 4 9 human lung carc inoma ce l l l ine ( A T C C ) were grown i n D M E M media . C M T . 6 4 cel ls used for in fect ion targets i n this study were der ived f r om a c lone o f p T A P l - E G F P stable transfectants [1], named 1E10. The L M D ce l l l ine was der ived f r om a metastatic prostate ca rc inoma o f a 129/Sv mouse (a k i n d gift o f D r . T . C . Thompson ) [5] and mainta ined i n R P M I 1640 media . B o t h the R P M I 1640 and the D M E M med ia were supplemented w i t h 1 0 % heat-inactivated F B S or ca l f serum for the N I H / 3 T 3 , 2 m M L-glutamine, 100 U/ml pen i c i l l i n , 100 ug/ml streptomycin, and 10 m M H E P E S . 4.2.2 Human cDNA library V i raPo r t c D N A l ibrary retroviral supernatants f rom different sources, the norma l human lung and human spleen specimens, were purchased f rom Stratagene ( L a Jo l la , C A ) . Ind iv idua l c D N A l ibrary was harbored i n p F B retroviral vector (Stratagene). 4.2.3 Cell Fusion and F A C S analysis A fus ion between human (A549) and mouse (1E10) cel ls was per formed as a pre l iminary experiment to test whether proteins originated f rom human cel ls w o u l d be able to modulate mouse TAP-1 promoter act ivity. The two groups o f ce l l l ines were fused in a 1:1 rat io, f o l l o w i n g a polyethylene g l y co l ce l l fus ion protocol [6]. Ce l l s were then incubated w i t h PE-conjugated a n t i - H L A - A , B, C mouse monoc lona l ant ibody ( B D CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 112 Pharmingen, San Diego, CA) at 4°C for 30 minutes. The fused cells, which displayed both red (PE-anti-HLA) and green (EGFP) fluorescence, were selected by FACS (FACSVantage DiVa, Becton Dickinson, Mountain View, CA). TAP-1 promoter-driven EGFP expression in the fused cells were then analysed by FACScan (Becton Dickinson). 4.2.4 Determination of target cell transduction efficiency using the pFB-Luc control viral supernatant Prior to the actual library screenings, both the 1E10 and the NIH/3T3 cell lines were infected with the pFB-Luc control retroviral supernatant, in order to assess both the quality of the retroviral supernatants and the transduction efficiency of 1 E l 0 as compared to that of the NIH/3T3 cells. This experiment was done according to the manufacturer's protocol. 4.2.5 Transduction of 1E10 cells with ViraPort cDNA library retroviral supernatants The multiplicity of infection (MOI) was adjusted so approximately 20% of the 1E10 cells were transduced, since positive transductants obtained with low MOI screens have higher probability of containing a single cDNA of interest [7]. 4.2.6 Selection of positive transductants Forty eight hours after transduction with the cDNA library retroviral supernatants, human lung cDNA-infected 1E10 cells that displayed up-regulated expression of TAP-1 promoter-driven EGFP were selected by FACS (FACSVantage DiVa). Alternatively, human spleen cDNA-infected 1E10 cells were incubated with PE-conjugated anti-Kb mouse monoclonal antibody (BD Pharmingen) at 4°C for 30 minutes. Then, cells that CHARACTERIZATION OF NOVEL TAP-I REGULATORS 113 d isp layed up-regulated express ion o f both the H - 2 K b and the E G F P were selected by F A C S . Se lect ion and expans ion i n culture was repeated twice before the cel ls were f ina l l y sorted into single-cel l c lones. 4.2.7 Recovery of cDNA clones from positive transductants G e n o m i c D N A f r om the posi t ive transductants, as we l l as f rom the pFB-Luc-infected 1E10 cel ls (control) was extracted and used as a template for P C R recovery o f the c D N A inserts. The pr imers used are speci f ic for regions f l ank ing the mul t ip le c lon ing site ( M C S ) o f the p F B vector (Stratagene): G G C T G C C G A C C C C G G G G G T G G (forward) and C G A A C C C C A G A G T C C C G C T C A (reverse). T w o microl i ters al iquots o f genomic D N A were used as a template i n a total o f 50 p i reaction mixture conta in ing l x P C R buffer, 250 u M deoxynucleot ide triphosphate, 1.5 m M M g C b , 200 n M o f each pr imer and 2.5 units P l a t inum Taq D N A Polymerase. c D N A ampl i f icat ions were carr ied out i n a thermocyc ler (Uno II, B iomet ra , Goett ingen, Germany) w i th 35 cycles o f denaturation (1 m i n , 95°C), anneal ing (4 m i n , 65°C), and elongat ion (2 m i n , 72°C). The c y c l i ng was conc luded w i t h a final extension at 72°C for 10 m i n . Twenty micro l i ters o f ampl i f i ed products were analysed on agarose gels and then pur i f ied. A l l P C R reagents were obtained f rom Invitrogen (Bur l ington, O N ) and Fermentas (Bur l ington, O N ) . The P C R products were sequenced and ident i f ied through a b io informat ics database search (ht tp://www.ncbi .n lm.nih.gov/BLAST/) . B L A S T n was used to compare nucleot ide sequences obtained f rom the screenings w i th k n o w n nucleotide sequences i n the database; B L A S T x was used to compare translated nucleotide sequences w i t h k n o w n amino ac id sequences i n the database. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 114 4.2.8 Sub-cloning of TAP-regulator gene candidates Several gene candidates obtained f rom the screening were PCR-amp l i f i ed f rom either genomic D N A f rom posit ive transductants or c D N A f rom w i l d type ce l ls , us ing gene-specific pr imers (S igma-Genosys, Oakv i l l e , O N ) (Table 4.2.8.1). Gene expression constructs (pIRES2-gene) were created by l igat ing the P C R products into the X h o l and E c o R I sites o f p I R E S 2 - E G F P vector (C lontech, Pa lo A l t o , C A ) (Append ix B ) . Table 4.2.8.1: Primers used for PCR amplification. Oligonucleotide Primer sequence (5'-3') a b P b Template 0 ENO-1 F: c t c g a g A G T G G C T A G A A G T T C A C C A T G T C T A R: g a a t t c T G C C T G C C C A C A G C T T A C T T 1337 Lungs (2 n d sort) genomic D N A SFTPC F: c t c g a g T A G C A C C T G C A G C A A G A T G R: g a a t t c T C C T A G A T G T A G T A G A G C G G C A 612 Clone 2B5 genomic D N A HLA-Cw*04 null allele F: c t c g a g A T G C G G G T C A T G G C G R: g a a t t c T C A G A T G C C T T T G C A G A A A G 1197 Lungs (2nA sort) genomic D N A Human chr. 3 ORF (HC3) F: G A c t c g a g G A A A T G G G A T T T G G C C T C C T R: g a a t t c A A T G G G T C A A A C A G C A G C C 723 Clone 1C6 genomic D N A Mouse P N K P ( M P N K P ) F: c t c g a g G A G G A T G T C A C A G C T T G G A T C R: g a a t t c G C T C A G C C C T C G G A G A A C T 1569 Ltk c D N A Human P N K P (HPNKP ) F: c t c g a g A C A C A A G G A T G C A A A T C C T G A C R: g a a t t c G C T C A G C C C T C G G A G A A C T 1459 Clone P4-B9 genomic D N A 27376 (unknown protein) F: c t c g a g G A A C C A T G G A A A C C C C A G C R: g a a t t c G G C A C T T C T C C C T C T A A C A C T C T 726 Clone P5-E10 genomic D N A a F: forward pr imer ; R: reverse pr imer. Restr ic t ion enzyme sites are under l ined. b Leng th o f the P C R ampl i f i ca t ion product. 0 G e n o m i c D N A f rom 1E10 posi t ive transductants or c D N A f rom w i l d type cel ls . CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 115 4.2.9 Transfection and selection C M T . 6 4 and L M D cel ls were transfected w i th the pIRES2-gene constructs or the p I R E S 2 - E G F P vector us ing E x G e n 500 in vitro Transfect ion Reagent (Fermentas). Because the I R E S sequence enables E G F P to be translated f rom the same m R N A transcript as the c loned gene, EGFP-pos i t i ve cel ls were selected by F A C S . The selected cel ls were then cultured i n 1 mg/ml G418-conta in ing med ia pr ior to further analyses, i n order to obtain a r i ch popula t ion o f cel ls that overexpress the gene o f interest. 4.2.10 Reverse transcription-PCR analysis Tota l ce l lu lar R N A was extracted f rom pIRES2-gene and p I R E S 2 - E G F P (control) stable transfectants us ing T r i z o l Reagent ( Invitrogen); contaminat ing D N A was removed by treatment w i t h D N a s e 1 ( A m b i o n Inc., A u s t i n , T X ) . Reverse transcr ipt ion o f 1 p g o f total cel lu lar R N A was performed us ing the reverse transcript ion k i t (SSII R T ) f rom Invitrogen, i n a total vo lume o f 20 p i . TAP-1 expression was assessed by R T - P C R analysis us ing TAP-1-spec i f i c pr imers (S igma) : T G G C T C G T T G G C A C C C T C A A A (forward) and T C A G T C T G C A G G A G C C G C A A G A (reverse). M o u s e P-actin was ampl i f i ed as an internal control us ing the fo l l ow ing pr imers (S igma) : A T G G A T G A C G A T A T C G C T G C (forward) and T T C T C C A G G G A G G A A G A G G A T (reverse). T w o microl i ters al iquots o f c D N A were used as a template for P C R in a total o f 50 p i reaction mixture conta in ing l x P C R buffer, 250 u M deoxynucleot ide triphosphate, 1.5 m M M g C L : , 200 n M o f each pr imer and 2.5 units Taq or P la t inum Taq D N A Polymerase. c D N A ampl i f i cat ions were carr ied out i n a T-gradient thermocyc ler (B iometra) w i th 25-35 cyc les o f denaturation (1 m i n , 95°C), anneal ing (1 m i n , 54-64°C), and elongat ion (2 m i n , 72°C). The cyc l i ng was conc luded w i th a final extension at 72°C CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 116 for 10 min. Twenty microliters of amplified products were analysed on agarose gels, stained with ethidium bromide and photographed under U V light. 4.2.11 Flow cytometry Flow cytometric analysis of H-2K b expression was performed using PE-conjugated anti-Kb mouse monoclonal antibody (BD Pharmingen) and a FACScan cytometer (Becton Dickinson). CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 117 4.3 Results 4.3.1 Mouse TAP-1 promoter activity is up-regulated in the fused 1E10-A549 cells Since the c D N A l ibraries avai lable were der ived f rom human specimens, a pre l iminary experiment was performed to test whether proteins f r om TAP-express ing human cel ls cou ld up-regulate mouse TAP-1 promoter act iv i ty i n the TAP-def i c ient C M T . 6 4 cel ls . F l o w cytometr ic analysis results showed that mouse TAP-1 promoter-dr iven E G F P expression was indeed up-regulated i n the fused 1E10-A549 cel ls (F igure 4.3.1.1). The extent o f this up-regulation was s imi lar to that obtained f rom the fus ion between murine carc inomas and fibroblasts (F igure 2.3.5.1). Th i s indicates that transducing 1E10 cel ls w i t h normal human c D N A l ibrary retroviral supernatants w o u l d be a va l id method to screen for genes ( c D N A s ) that can enhance TAP-1 promoter act iv i ty i n mouse cel ls . Figure 4.3.1.1: TAP-1 promoter-driven EGFP expression is enhanced in the fused 1E10-A549 cells. The levels o f E G F P expression i n untransfected C M T . 6 4 cel ls , 1E10 (pTAP l-EGFP- t rans fec ted C M T . 6 4 ) cel ls and fused 1E10-A549 ce l ls are represented by a shaded area, a th in l ine and a thick l ine, respectively. EGFP CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 118 4.3.2 The transduction efficiency of 1E10 cells is three times lower than that of the NIH/3T3 cells The luciferase activity displayed by NIH/3T3 cells, 48 hours after transduction with 10-10,000x dilutions of pFB-Luc retroviral supernatant, was on average 3.2±1.2x higher as compared to 1E10 cells. This indicates that the NIH/3T3 cells are transduced approximately three times more effectively than the 1E10 cells. Therefore, three times less 1E10 cells than NIH/3T3 cells were plated one day prior to transduction with the cDNA library retroviral supernatant. 4.3.3 Identification of gene candidates from human lung cDNA library transductants Forty eight hours after infection of the 1E10 cells with the human lung cDNA library retroviral supernatant, 0.12% of the infected cells were found to display a high expression of TAP-1 promoter-driven EGFP. These cells were selected by FACS and expanded in culture. Two weeks later, cells that still expressed high EGFP (8.3%) were re-selected by FACS. Six integrants were recovered from the 1E10 bulk transductants. The results obtained from BLASTx or BLASTn hit are summarized in Table 4.3.3.1 below. A sequence obtained from the 1.3 kb band showed a homology to a region in human chromosome 3; however, no known protein is translated from this region. Interestingly, an open reading frame (ORF) consisting of 218 amino acids was detected, opening a possibility that a novel protein may be translated from this region (Figure 4.3.3.1). B L A T analysis (UCSC Genome Browser, www.genome.ucsc.edu) indicated that this ORF is located in the p25.1 locus of human chromosome 3. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 119 Table 4.3.3.1: Identity of cDNAs recovered from bulk-sorted cells of human lung cDNA library transductants. BLASTn (nucleotide sequence) or BLASTx (protein sequence) hit Amino acid identity (human) Insert size (kb) NCBI accession # Enolase-1 (ENO-1) 182/185 (98%) Total protein: 434 aa 2.0 NP_001419 HLA-Cw*04 null allele 255/291 (87%) Total protein: 367 aa 1.7 CAC05372 Prostaglandin-D2 synthase (PTGDS) 189/190 (99%) Totalprotein: 190 aa 1.6 CAI12758 Human chr.3 clone RP11-27016 map 3p (HC3) 218 aa ORF 1.3 AC090885 Pulmonary surfactant protein C isoform C R A a (SFTPC) 174/191 (91%) Total protein: 240 aa 0.9 EAW63705 Tropomyosin-2 beta 121/160 (75%) Total protein: 284 aa 0.7 AAH11776 atgggat ttggc'ctcct. • 2 05"81. :gggtgacatt ccctcaagca' :c ;cagt.'ctctg ga'gctg'ggtc- agagccatcc t c t g g t t g g a : 2-0 641 g t t c c t g g t t ccaggtcaec •acagtccccg' gggct'ccctg gcccaggcccr 'tgtccetgc'c. .. ;"'20701 t c t c c a t g c c •tggeccctgc' a.ggcttctga g t t t g t t g t c ctca.cttcaa gcctgtggag v ;' 20761 t g ' t c a c a t c t gtcacct'ggg aaatgaggac acgtcaggct a g c c t g c t t t ggctcccaag 20821 tggat.taaag gggctgagaa tge.a'gccctg gagcaggggc tggcgagacc ccctgaaggg. 20881 t a a c c t c t c c c g c a t c c t t t ccaggg'gagc caaggctaca gggaagggag a'ggtg'gccga: 2 0.9:41:-ggctcggace ctggc'aagag c t g ' g g a a g a a ccgctgetgg g'ca'gcgtcc t ctggaaggca ;' 2100T g c a t c c c c t g tcatcct.cca a.tgctgcccc tcaccaccag c c c c t c g c c t c c c c t t c . t t g ' 21061 c t t a c t c t g t ctgggcagtc cc'cacacccg ccatgctggg g g t t g g a t t c cagggttagc • 21121 actgacagct gcagcctctc. acagtgatga gtgcaggccc c c c a c a t c c c aaagcacagc '21181-' tcatcca'gca cagcagggct gtgaggtggc t g c t g t t t g a Figure 4.3.3.1: The ORF found in the homologous region of human chromosome 3 clone RP11-270I6 map 3p (NCBI accession no. AC090885) and the 1.3 kb sequence obtained from human lung cDNA library screening. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 120 Furthermore, the 1E10 bulk transductants were sorted into single-cell clones and expanded in culture. From the 20 clones that were analysed, 10 clones contained single integrants (Table 4.3.3.2). No integrants were detected in the remaining 10 clones. cDNAs that correspond to the SFTPC and the HC3 that were detected in previous screening of bulk transductants were recovered individually from the single-cell clones. A new sequence corresponding to the 3'-untranslated region of the N-deacetylase/N-sulfotransferase (NDST-1) gene, was obtained from two of the clones. No cDNAs that encode for human ENO-1, HLA-Cw*04 null allele, PTGDS and Tropomyosin 2 were recovered from the single-cell clones. Table 4.3.3.2: Identity of cDNAs recovered from single-cell clones of human lung cDNA library transductants. Clone BLASTn (nucleotide sequence) or BLASTx (protein sequence) hit Base pairs or amino acid identity (human) Insert size (kb) NCBI accession # 2B5 Pulmonary surfactant protein C isoform C R A a (SFTPC) 188/194 (96%) Total protein: 240 aa 0.9 EAW63705 1A2 Pulmonary surfactant protein C isoform C R A a (SFTPC) 194/194(100%) Total protein: 240 aa 0.9 EAW63705 1F3 Pulmonary surfactant protein C isoform C R A a (SFTPC) 193/194 (99%) Total protein: 240 aa 0.9 EAW63705 1E3 Pulmonary surfactant protein C ? n/a (bad sequence) 0.9 1C6 Human chr.3 clone RP11-27016 map 3p (HC3) 218 aa ORF 1.3 AC090885 2H1 Human chr.3 clone RP11-27016 map 3p ? n/a (bad sequence) 1.3 2A10 Human chr.3 clone RP11-27016 map 3p ? n/a (bad sequence) 1.3 1H7 ? n/a (bad sequence) 1.6 1C9 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 (NDST1) 583/585 (99%) (outside of CDS) Total gene: 7913 bp 2.0 NM_001543 1E11 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 (NDST1) 534/535 (99%) (outside of CDS) Total gene: 7913 bp 2.0 NM_001543 CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 121 Levels of surface K b expression in the single-cell clones were then analysed by flow cytometry, since the upregulation of TAP-1 promoter activity may lead to enhanced transcription of the gene and reconstitution of M H C class I expression on the surface of 1E10 cells. However, no increase in the surface K b expression on any of the clones was detected. This may be caused by low levels or total loss of protein expression encoded by the cDNA inserts, inability of the foreign proteins to induce M H C class I expression in CMT.64 cells, or false positive signals during the selection process. Indeed, loss of induction of TAP-1 promoter-driven EGFP expression was observed in most cells over the period in culture, indicating the instability of its enhanced state. 4.3.4 Identification of gene candidates from human spleen cDNA library transductants After several TAP-1-activator gene candidates were obtained from the screening of human lung cDNA library, a similar experiment using human cDNA library derived from human spleen specimens was performed. However, in this experiment, the positive transductants were selected based on up-regulated expression of both the TAP-1 promoter-driven EGFP and the H-2K b molecule. Forty eight hours after the infection with human spleen cDNA library retroviral supernatant, 0.1% of the infected 1E10 cells were found to display an up-regulated expression of both the EGFP and H-2K b . After a month of expansion in culture, a population of cells that remained double-positive (0.5%) was re-selected by FACS and sorted into single-cell clones. Unfortunately, similar to the problem encountered during the lung cDNA library screening, most cells were no longer EGFP- and Kb-positive after being expanded in culture. From the 20 clones that were analysed, 5 clones contained CHARACTERIZATION OF NOVEL TAP-I REGULATORS 122 single integrants, 5 clones contained two integrants, and 10 clones contained no integrants (Table 4.3.4.1). No identical hits to those obtained from the lung cDNA library screening were discovered in this screening. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 123 Table 4.3.4.1: Identity of cDNAs recovered from single-cell clones of human spleen cDNA library transductants. Clone BLASTn (nucleotide sequence) or B L A S T x (protein sequence) hit Base pairs or amino acid identity (human) Insert size (kb) NCBI accession # P4-A3 Polynucleotide kinase 3'-phosphatase (PNKP) and R N A binding protein, autoantigenic (hnRNP-associated with lethal yellow homolog (mouse)) (RALY), transcript variant 2, mRNA 81/95 (85%) Total protein: 482 aa (bad sequence) 87/98 (88%) Total CDS: 1541 bp (bad sequence) 1.3 .6 AAH02519.2 NM_007367 P4-B9 Polynucleotide kinase 3'-phosphatase (PNKP) 469/482 (97%) Total protein: 482 aa 1.3 AAH02519.2 P5-A6 Polynucleotide kinase 3'-phosphatase (PNKP) ? and Prosaposin (variant Gaucher disease and variant metachromatic leukodystrophy) or sphingolipid activator proteins 1 and 2 processed mutant n/a (bad sequence) 38/38 (100%) Total protein: 559 aa 1.3 .75 CAI40836 P4-E3 Human chromosome 5 genomic contig and Glucocorticoid receptor-interacting protein GRIP1 associated protein 1 (GRIPAP1) 92/100 (92%) Total: 48999907 bp 115/120 (95%) Total CDS: 3032 bp .8 .4 NW_922784 NM_020137 P5-F9 Human chromosome 5 genomic contig and Glucocorticoid receptor-interacting protein (GR1P)1 associated protein 1 (GRIPAP1) 91/100 (91%) Total: 48999907 bp 106/111(95%) Total CDS: 3032 bp .75 .4 N W 922784 NM_020137 P4-F1 Actin, gamma 1 (ACTG1) 812/931 (87%) Total CDS: 1919 bp 1.2 NM_001614 P5-B3 Unnamed protein product (from M H C 11 alpha domain) 253/254 (99%) Total protein: 254 aa 1.1 CAA24917 P5-E4 Unknown (protein for MGC:27376) ? n/a (bad sequence) 1.2 P5-E10 Unknown (protein for MGC:27376) 210/235 (89%) Total protein: 235 aa 1.2 AAH16380 P5-F10 Immunoglobulin light chain variable region and Unknown (protein for MGC:27376) 112/179 (62%) Total protein: 209 aa 104/165 (63%) Total protein: 235 aa . 1.5 1.4 AAF86917 AAH16380 CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 124 4.3.5 Cancer-related information about the screened genes Aberrant expression o f many o f the genes obtained f rom both screenings has been reported to contribute to development o f var ious cancers (Table 4.3.5.1). Interestingly, some o f these gene products, such as the ENO-1 homo log , P T G D S , P N K P and R A L Y , have been reported to confer tumor-suppressing funct ions [8-12]. However , no cancer-related in format ion related to S F T P C , N D S T 1 , G R I P A P I and A C T G 1 has been reported to date. CHARACTERIZATION OF NOVEL TAP-I REGULATORS 125 Table 4.3.5.1: Cancer-related information about the proteins that match the hits obtained from the screenings of human lung and spleen cDNA libraries. Isolated f rom Prote in name Cancer-related in format ion Lung cDNA library Enolase-1 G l y co l y t i c enzyme, often overexpressed i n metastatic cancers [13-15] Shares a great structural s imi lar i ty w i th c-myc promoter b ind ing prote in ( M B P ) - l , a transcript ional repressor o f c-myc oncogene [12] H L A - C w A l igand for an inh ib i tory N K ce l l receptor [16] . A l l o t ypes are used as prognost ic markers for autologous transplantation i n l y m p h o m a patients [16] • N u l l al leles are not expressed on ce l l surface [17, 18] P T G D S Down-regulated i n bra in tumors and pre-malignant oral lesions [8, 19] • Inhibit oral cancer ce l l pro l i ferat ion in vitro [8] P T G D S metabolites inhib i t prostate cancer ce l l growth v i a a perox isome proliferator-activated receptor gamma (PPARgamma)-dependent mechan ism [10] T ropomyos in 2 Loss o f express ion was reported i n esophageal and lung cancer tissues [20, 21] S F T P C N o record N D S T 1 N o record Spleen cDNA library P N K P • Involved i n base exc i s ion repair as a mechan ism to prevent cancer development due to oxidat ive damage o f D N A [9] R A L Y R N A b ind ing prote in ; mutat ion is associated w i t h lethal y e l l ow h o m o l o g i n mice that phenotypes inc lude a predispos i t ion to tumor growth [11] G R I P A P I p i 6 0 nuclear receptor co-activator • One o f the two autonomous act ivat ion domains o f GR I P1 is mediated by CBP/p300 [22, 23] Increased b ind ing o f G R I P 1 to E R a l p h a promoter mediated growth and surv iva l o f breast cancer cel ls [24] no record about G R I P A P I deregulat ion and development o f cancers A C T G 1 N o record CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 126 4.3.6 Overexpression of several TAP-regulator gene candidates in CMT.64 and LMD cells up-regulates TAP-1 expression, but not H-2Kb expression To overexpress the TAP-regulator gene candidates, individual cDNA was sub-cloned into pIR£S2-EGFP vector. This vector contains a neomycin gene for selection of positive transfectants in G418-containing media. Seven pIRES2-gene constructs: the ENO-1, SFTPC, HLA-Cw*04 null allele, unknown protein (27376), mouse PNKP (MPNKP), human PNKP (HPNKP) and human chromosome 3 ORF (HC3) were completed. CMT.64 and L M D cells were independently transfected with pIRES2-gene constructs or pIRES2-EGFP vector. Overexpression of the transfected genes was confirmed by RT-PCR analysis using gene-specific primers (Figure 4.3.6.1). o c LU O 0-Q. o CD LA- Is-co Nd X CM 2 Z X O X G V G V G V G V G V G V G V 2.0 fcb-1.0 kb-0.5 kb-C M T . 6 4 Genes p-actin L M D Genes p-actin Figure 4.3.6.1: RT-PCR analysis of the overexpression of the TAP-1 activator candidate genes in CMT.64 and LMD cells. G: cDNA template was derived from pIRES2-gene transfectants; V: cDNA template was derived from pIRES2-EGFP transfectants. Amplification of p-actin cDNA served as a loading control. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 127 Overexpression of the TAP-regulator gene candidates appeared to induce TAP-1 expression in TAP-deficient CMT.64 and LMD cells, although the levels of induction were very low compared to those from IFN-y treatment (Figure 4.3.6.2). Most inductions observed in three independent transfection experiments (two groups of CMT.64 cells and one group of L M D cells) were inconsistent, with the exception of HLA-Cw*04 null allele and the unknown protein from human chromosome 3 (HC3). In all the three experiments done, TAP-1 expression was consistently up-regulated in both the CMT.64 and L M D cells that overexpressed the HLA-Cw*04 null allele and the HC3 (Figure 4.3.6.2). TAP-1 p-actin TAP-1 P-actin TAP-1 p-actin MNHf «MM» mmm n—r WMf M M * mmm mmm >mmnt CMT.64 # 1 CMT.64 #2 LMD Figure 4.3.6.2: TAP-1 expression was up-regulated in some of the CMT.64 and LMD cells transfected with individual pIRES2-gene constructs. Amplification of cDNA from IFN-y-treated cells was used as a positive control, p-actin expression served as a loading control. Data are representatives of one set of RT-PCR analysis per group of CMT.64 and LMD transfectants. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 128 Finally, flow cytometric analyses were performed in order to assess induction of surface H-2K b expression in the positive transfectants of CMT.64 and L M D cells. Unfortunately, no improvement in surface H-2K b expression could be detected in any of the cell lines tested. This may due to fairly low levels of induction of TAP-1 expression in the transfectants. Representative data from HLA-Cw*04 null allele- and HC3-overexperessing L M D cells are shown in Figure 4.3.6.3. H L A - C w H C 3 Figure 4.3.6.3: No upregulation of H-2Kb expression was detected on the surface of LMD cells overexpressing HLA-Cw*04 null allele and HC3 (thick lines). H-2K b expression in pIRES-2-EGFP-transfected L M D cells (shaded areas) was similar to the negative control, the unstained pIRES-2-gene-transfected cells (thin lines). As a positive control, H-2K b expression was assessed in IFN-y-treated L M D cells (broken lines). Further research is needed to confirm the ability of the TAP-1 regulator gene candidates, particularly the HLA-Cw*04 null allele and HC3, to up-regulate TAP-1 at the protein level, and possibly surface M H C class I expression in other TAP-deficient cell lines. In addition, ten further candidates obtained from the screenings remain to be sub-cloned and analysed. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 129 4.4 Discussion In order to identify novel TAP-1 activators, cDNA libraries from human lung and spleen specimens were screened for gene products that could up-regulate either TAP-1 promoter activity, or both the promoter's activity and surface M H C class I expression in pTAPl-EGFP stable transfectants of CMT.64 cells. A total of seven and nine candidates were obtained from the lung and the spleen cDNA library screening, respectively. Interestingly, deregulation of many of the genes identified, such as ENO-1, PTGDS, Tropomyosin-2, PNKP, and RALY, had been shown to associate with the development of various types of cancers (Table 4.3.5.1). To-date, only PTGDS and H L A - C have been shown to be involved in antigen processing. PTGDS, an enzyme responsible for the bio-synthesis of prostaglandin D2, is produced by resident macrophages and dendritic cells in spleen, thymus, and Peyer's patch of intestine [25]. Prostaglandin D2 was shown to affect the differentiation and functions of human dendritic cells [26]. In the presence of prostaglandin D2, dendritic cells demonstrated upregulated endocytic and antigen processing activities; however, their ability to stimulate naive T cells was impaired [26]. It has been reported that a variety of alleles in H L A - C locus increases the risk of bone marrow graft rejection in transplantations [27]. This might be caused by presentation of allogeneic, possibly null-derived peptides, by H L A class I molecules on cell surface, thus triggering allospecific T cell responses [18]. The precise role of H L A class I molecules with no cell surface expression, such as the HLA-Cw*04 null allele, in antigen presentation remains to be elucidated [18]. Interestingly, several candidates showed significant similarities to regions of human chromosomes with no known genes or to unnamed protein products. One CHARACTERIZATION OF NOVEL TAP-I REGULATORS 130 sequence, with a similarity to an open reading frame encoding 218 amino acids in human chromosome 3, named HC3, was particularly intriguing. Overexpression of HC3 in three independent groups of transfectants of TAP-deficient CMT.64 and L M D cells consistently showed up-regulated expression of TAP-1, as assessed by RT-PCR using cDNA templates from the transfectants (Figure 4.3.6.2). This result indicates that a novel protein with the ability to modulate TAP-1 expression could be translated from the newly-found ORF (Figure 4.3.3.1). However, TAP-1 upregulation was not consistently observed in CMT.64 and L M D cells that overexpressed most of the other gene candidates. More independent transfections of CMT.64, L M D and possibly other TAP-deficient cells,- RT-PCR and Western Blot analyses are required to confirm the consistency of TAP-1 induction in cells that overexpressed a specific gene of interest. Several problems encountered during the screening process include the acquisition of EGFP and Kb-positive clones with no integrants ("empty clones"), the loss of EGFP and K b expression as the cells were expanded in culture, and the difficulty in obtaining sequencing results from PCR products. The first problem may result from the loss of cDNA inserts from the infected cells during the period in culture, prior to genomic D N A extraction, although this is unlikely as plasmids introduced by retroviral infection generally integrate into the genome of the infected cells. Also, false positive signals during FACS selection could result in unwanted selection of non-infected cells. Nevertheless, one or two inserts were successfully detected in many of the single-cell clones analysed, indicating that the multiplicity of infection (MOI) was correctly adjusted [7]. If the MOI used was too low, very few cells would be transduced. In contrast, transduction at high MOI would yield a higher number of cells transduced; however, the majority of the cells would bear multiple integrants, making it difficult to determine CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 131 which of those was actually responsible for the phenotype. A similar problem was encountered during PCR recovery of cDNA inserts from selected bulk transfectants. Therefore, in future experiments, it is best to first sort positive transductants into single-cell clones. An independent transduction of CMT.64 cells by human cDNA library retroviral supernatant from the same source is necessary to test the reproducibility of the primary screen hits. The absence of EGFP and K b expression in selected cells after a period in culture may result from either false positive signal during the selection or diminishing expression of the inserted gene products. An independent method, such as sub-cloning the cDNA into a different vector and over-expressing the gene product in TAP-deficient cancer cells (Figure 4.3.6.1), must be performed to confirm the validity of the acquired phenotypes. Difficulty in obtaining good quality results from the sequencing of PCR products accounts for errors in the percentage of sequence identity recorded in the data tables (Table 4.3.3.1, Table 4.3.3.2 and Table 4.3.4.1). It may also be one of the reasons many of the recovered cDNAs showed sequence similarities only to a part of specific genes, instead of to full-length cDNAs or protein products. However, some sequences were recovered from bands that were clearly smaller than the known, full-length sequences, for example the R A L Y , prosaposin and GRIP API (Table 4.3.4.1), indicating that sequencing errors were not the only reason for the finding of several incomplete sequences. The sequencing results could generally be improved by sub-cloning all PCR products individually into a TOPO vector (Invitrogen); however, this method is ineffective for screening large numbers of clones due to cost and time demands. Future research should include refinement of the screening method and verification of the results, followed by application of independent methods to confirm the CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 132 validity of the primary screen hits, as discussed in the previous paragraph. Furthermore, a TAP-expressing, human primary cell line and its TAP-deficient, metastatic derivative may be used to assess differential endogenous expression levels and functionality of several strong candidates obtained from the initial screenings. Finally, it is intriguing to investigate whether disruption of a specific gene in TAP-expressing cells would cause detrimental effects on M H C class I antigen presentation. For example, it would be interesting to observe whether deletion of a region within the newly identified "HC3" ORF would transform human cells that displayed a functional antigen presentation pathway into antigen presentation-defective, metastatic cells. This would confirm whether the particular gene product has fundamental roles in tumor antigen processing and human malignancies. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 133 4.5 References 1. Set iadi , A . F . , M . D . D a v i d , S.S. Chen , J . Hiscott , and W . A . Jefferies. Ident i f icat ion o f mechanisms under ly ing transporter associated w i th antigen process ing def ic iency i n metastatic murine carcinomas. Cancer Res, 2005. 65:7485-92. 2. Lee , H . , J.J. Song, E. K i m , C O . Y u n , J . C h o i , B. Lee , J . K i m , J .W. Chang , and J . H . K i m . E f f i c ient gene transfer o f V S V - G pseudotyped retroviral vector to human brain tumor. Gene Ther, 2001 . 8:268-73. 3. Ito, T. , N . Ito, A . Bettermann, Y . Tokura , M . Tak igawa , and R. Paus. Co l l apse and restoration o f M H C class-I-dependent immune pr iv i lege : exp lo i t ing the human hair fo l l i c l e as a model . A m J Pathol , 2004. 164:623-34. 4. Franks, L . M . , A . W . Carbone l l , V . J . Hemmings , and P .N . R idd le . Metastas iz ing tumors f rom serum-supplemented and serum-free ce l l l ines f rom a C 5 7 B L mouse lung tumor. Cancer Res , 1976. 36:1049-55. 5. Lee , H . M . , T . L . T i m m e , and T . C Thompson . Resistance to lys is by cy to tox ic T cel ls : a dominant effect i n metastatic mouse prostate cancer cells. Cancer Res , 2000 .60 :1927-33 . 6. Fu l ler , S.A., M . Takahash i , and J .G .R . Hur re l l , Production of antibodies, i n Current protocols in molecular biology, F . M . Ausube l , et a l . , Edi tors . 2001 , John W i l e y & Sons. p. 11.7.1-11.7.4. 7. O n i s h i , M . , S. K inosh i t a , Y . M o r i k a w a , A . Sh ibuya , J . Ph i l l i ps , L .L . Lan ier , D . M . G o r m a n , G.P. N o l a n , A . M i y a j i m a , and T . K i t amura . App l i ca t ions o f retrovirus-mediated expression c loning. E x p Hemato l , 1996. 24:324-9. CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 134 8. Banerjee, A . G . , I. Bhattacharyya, and J . K . V ishwanatha . Ident i f icat ion o f genes and molecu lar pathways invo l ved i n the progress ion o f premal ignant ora l ep i the l ia M o l Cancer Ther, 2005. 4:865-75. 9. Haz ra , T .K . , A . Das , S. Das , S. Choudhury , Y . W . K o w , and R. Roy . Ox ida t i ve D N A damage repair i n mamma l i an ce l l s : A new perspective. D N A Repa i r (Amst ) , 2006. 10. K i m , J . , P. Y a n g , M . Suraokar, A . L . Sab ich i , N . D . L l ansa , G . M e n d o z a , V . Subbarayan, C.J . Logothet is , R .A . N e w m a n , S . M . L i p p m a n , and D .G . Menter . Suppression o f prostate tumor ce l l g rowth by stromal ce l l prostaglandin D synthase-derived products. Cancer Res, 2005. 65:6189-98. 11. M i c h a u d , E.J., S.J. Bu l tman , M . L . K l e b i g , M. J . van Vug t , L.J. Stubbs, L .B . Russe l l , and R.P. W o y c h i k . A molecu lar mode l for the genetic and phenotypic characteristics o f the mouse lethal y e l l o w (Ay ) mutation. Proc N a t l A c a d Sc i U S A , 1994. 91:2562-6. 12. Onyango , P., B. L u b y o v a , P. Garde l l i n , R. Kurzbauer , and A . We i th . M o l e c u l a r c lon ing and expression analysis o f f i ve nove l genes i n chromosome lp36 . Genomics , 1998. 50:187-98. 13. D o w l i n g , P., P. Me leady , A . D o w d , M . Henry , S. G l y n n , and M . C l ynes . P roteomic analysis o f isolated membrane fract ions f rom superinvasive cancer cells. B i o c h i m B iophys A c t a , 2007. 1774:93-101. 14. Zhang , D., L .K . T a i , L .L . W o n g , L .L . C h i u , S.K. Sethi , and E.S. K o a y . P roteomic study reveals that proteins invo l ved i n metabol ic and detoxi f icat ion pathways are h igh l y expressed i n HER-2/neu-posit ive breast cancer. M o l C e l l P roteomics , 2005 .4 :1686-96 . CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 135 15. A l tenberg , B. and K . O . G reu l i ch . Genes o f g lyco lys is are ubiqui tous ly overexpressed i n 24 cancer classes. Genomics , 2004. 84:1014-20. 16. Skerrett, D., O. Ros ina , C . Bod i an , L. Isqla, O. Gudzowaty , E. Sc ig l i ano , and S. F ruchtman. H u m a n leukocyte antigens ( H L A ) - C w as prognostic indicators i n autologous transplantation for l ymphoma. Cancer Invest, 2001. 19:487-94. 17. W a n g , Z .C . , A . G . Smi th , E.J. Y u n i s , A . Se lvakumar, S. Ferrone, S. M c K i n n e y , J . H . Lee , M . Fernandez-Vina , and J . A . Hansen. Mo l e cu l a r characterization o f the H L A - C w * 0 4 0 9 N allele. H u m Immuno l , 2002. 63:295-300. 18. Ba las , A . , S. Santos, M. J . A v i l e s , F. Garcia-Sanchez, R. L i l l o , A . A l v a r e z , L . M . V i l l a r-Gu imerans , and J . L . V i c a r i o . E longat ion o f the cytoplasmic domain , due to a point delet ion at exon 7, results i n an H L A - C nu l l al lele, C w * 0 4 0 9 N . Tissue Ant igens , 2002. 59:95-100. 19. Saso, L., M . G . Leone , C . Sorrentino, S. G i a c o m e l l i , B. S i lvestr in i , J . G r i m a , J .C . L i , E. Samy, D. M r u k , and C . Y . Cheng . Quant i f i ca t ion o f prostaglandin D synthetase i n cerebrospinal f l u i d : a potential marker for bra in tumor. B i o c h e m M o l B i o l Int, 1998.46 :643-56. 20. J az i i , F.R., Z . Na j a f i , R. Ma l ekzadeh , T .P . Conrads, A . A . Ziaee, C . Abne t , M . Y a z d z n b o d , A . A . Ka rkhane , and G . H . Salekdeh. Identi f icat ion o f squamous ce l l ca rc inoma associated proteins by proteomics and loss o f beta t ropomyos in expression i n esophageal cancer. W o r l d J Gastroenterol, 2006. 12:7104-12. 21 . Pitterle, D . M . , E . M . Jo l icoeur, and G . Bepler . Ho t spots for molecu lar genetic alterations i n lung cancer. In V i v o , 1998. 12:643-58. 22. Huang , S . M . and Y . S . Cheng . Ana l y s i s o f two C B P (cAMP-response-element-b ind ing protein-binding protein) interacting sites i n GR IP1 (glucocort icoid-CHARACTERIZATION OF NOVEL TAP-1 REGULATORS 136 receptor-interacting protein), and their importance for the funct ion o f G R I P 1 . B i o c h e m J , 2004. 382:111-9. 23 . L i u , P.Y., T . Y . Hs i eh , W . Y . C h o u , and S . M . Huang . M o d u l a t i o n o f g lucocor t i co id receptor-interacting protein 1 (GR IP1 ) transactivation and co-activation activit ies through its C-terminal repression and self-association domains. Febs J , 2006. 273:2172-83. 24. W e i , X . , H . X u , and D. K u f e . M U C 1 oncoprote in stabi l izes and activates estrogen receptor a lpha M o l C e l l , 2006. 21:295-305. 25. Urade , Y . , M . U j ihara , Y . Ho r i guch i , K. Ikai , and O. Haya i sh i . The major source o f endogenous prostaglandin D 2 product ion is l i ke l y antigen-presenting ce l ls . Loca l i za t i on o f glutathione-requiring prostaglandin D synthetase i n hist iocytes, dendrit ic , and Kup f f e r cel ls i n var ious rat tissues. J Immuno l , 1989. 143:2982-9. 26. Gosset, P., M . P ichavant, C . Faveeuw, F. Bureau, A . B . Tonne l , and F. Trotte in. Prostaglandin D 2 affects the dif ferent iat ion and funct ions o f human dendri t ic ce l ls : impact on the T ce l l response. Eu r J Immuno l , 2005. 35:1491-500. 27. Petersdorf, E.W., G . M . Long ton , C . Anaset t i , E . M . M i c k e l s o n , S.K. M c K i n n e y , A . G . Smi th , P.J. Ma r t i n , and J . A . Hansen. Assoc i a t i on o f H L A - C dispari ty w i th graft fai lure after mar row transplantation f rom unrelated donors. B l o o d , 1997. 89:1818-23. GENERAL DISCUSSION 137 Chapter 5 : General Discussion 5.1 Summary and Conclusions To-date, l o w immunogen ic response to tumor immunotherapy due to escape mechanisms ut i l i zed by tumor cel ls remains the ma in chal lenge i n c l in i ca l trials [1-4]. Th is is at least part ia l ly caused by the h igh inc idence o f M H C class I downregulat ion i n cancer cel ls [1-3], w h i c h has been attributed to the decreased expression o f several A P M components, i nc lud ing the heterodimer o f TAP-1 and T A P - 2 molecules [5-10]. Interestingly, restoration o f the TAP-1 alone i n cel ls w i th mul t ip le def ic iencies o f other A P M components has been shown to reconstitute M H C class I antigen presentation and k i l l i n g o f the tumor cel ls by speci f ic C T L s [5, 11-13]. These effects can also be attained by treatment o f TAP-def i c ien t cancer cel ls w i t h IFN-y in vitro [5, 6, 11, 14]. Howeve r , the mechan ism through w h i c h this occurs is unknown. In order to develop effect ive immunotherapy approaches that a im to restore TAP-1 expression i n cancer ce l ls , a better understanding o f the mechanisms by w h i c h cancer cel ls down-regulate TAP-1 express ion is essential. Th is thesis contributes to the e luc idat ion o f molecu lar mechanisms by w h i c h metastatic carc inomas down-regulate TAP-1 expression and escape immune survei l lance. It appears that TAP-1 def ic iency i n carc inomas is caused by two ma in mechanisms: lack o f transcriptional activators and rapid TAP-1 m R N A degradation [14]. Studies a imed at ident i fy ing the transcript ional activator(s) indicated that these factors p lay important roles i n chromat in remode l ing w i th in the TAP-1 locus. B y per forming a chromat in immunoprec ip i ta t ion assay, it was found that histone H 3 acetylat ion was def ic ient i n TAP-1 promoter o f TAP-def i c ient cel ls . Th is lack o f histone GENERAL DISCUSSION 138 H 3 acetylat ion l i ke l y leads to a condensed nuc leosomal structure around the TAP-1 promoter, w h i c h prevents the b ind ing o f R N A po l II comp lex and o f other general transcr ipt ion factors to the promoter region. A s a result, further transcr ipt ional act iv i ty is halted. Compar i son o f fu l l TAP-1 promoter act iv i ty and that o f its truncations revealed a reg ion that is responsible for differential act iv i ty o f the promoter i n TAP-express ing and TAP-def i c ien t cel ls. Intr iguingly, this region was f o u n d to contain a C R E B b ind ing site, p rompt ing the speculat ion that C R E B - b i n d i n g protein ( C B P ) , a we l l -known histone acetyltransferase ( H A T ) [15-18], was one o f the possible factors responsible for the dif ferent ia l act iv i ty o f the promoter. It was found that indeed, C B P b ind ing to TAP-1 promoter was impai red i n TAP-def ic ient , metastatic carc inomas, strengthening the hypothesis that the lack o f histone acetyltransferase activit ies around the TAP-1 promoter leads to a repressive structure o f the chromat in that is inaccessible by transcr ipt ional machinery complexes. Furthermore, an interesting l ink was found between TAP-1 induct ion and improvement o f C B P b ind ing to TAP-1 promoter upon treatment o f TAP-def i c i en t cel ls w i t h IFN-y. Th i s effect may result f rom IFN-y-induced associat ion o f S T A T - l a and C B P [19]. Th is i n turn modulates the TAP-1 promoter act iv i ty upon associat ion o f the factors w i th the promoter and improvement o f histone acetylat ion around the reg ion, thus promot ing a permiss ive state o f the chromat in structure. Th is provides a nove l epigenetic mechan ism by w h i c h IFN-y enhances TAP-1 transcript ion. A l te rnat i ve ly , IFN-y may also act through STAT-1 independent pathways, such as through a nove l IFN-y-activated transcript ional element or through immediate early proteins and transcr ipt ion factors [20]. GENERAL DISCUSSION 139 By interacting simultaneously with the basal transcription machinery and with one or more upstream transcription factors, CBP may function as a physical bridge that stabilizes the transcription complex [17]. A l l the described mechanisms are summarized in Figure 5.1.1. GENERAL DISCUSSION 140 Histone acetylation and J, chromatin relaxation Figure 5.1.1: A proposed model of the mechanisms underlying TAP - 1 deficiency in carcinomas and release of the transcriptional repression upon relaxation of chromatin structure around the Tap-I gene locus. The boxes on the D N A strand represent cr i t ica l b ind ing sites for var ious t ranscr ipt ion factors that regulate TAP-1 transcript ion. C B P is a transcript ional co-activator that possesses intr insic H A T activity. In this mode l , the recruitment o f C B P to TAP-1 promoter faci l itates histone acetylat ion that leads to relaxat ion o f the chromat in structure around the reg ion, increasing the accessibi l i ty o f the D N A template by transcr ipt ion factors (TFs ) and R N A pol II complex . GENERAL DISCUSSION 141 In addit ion to the mechanisms described above, this thesis also contributes to the f ind ing that tr ichostatin A ( T S A ) , a histone deacetylase inhib i tor ( H D A C i ) that has been used in c l in i ca l trials for anti-cancer agents [21-23], has the abi l i ty to improve TAP-1 expression, M H C class I antigen presentation and k i l l i n g o f mal ignant cel ls by M H C class I antigen-restricted, tumor antigen-specific cytotoxic T lymphocytes ( C T L s ) . The effects seen were not as strong as those generated by IFN-y treatment; however, this f i nd ing is encouraging for the development o f non-toxic, smal l molecu lar compounds w i th the abi l i ty to improve patients' immunogen ic responses i n cancer therapy. F ina l l y , a new method based on c D N A l ibrary screening was deve loped as an approach to identify other proteins w h i c h may improve TAP-1 expression and M H C class I antigen presentation i n metastatic cancer cel ls . B y screening o f c D N A l ibraries or ig inat ing f rom human lung and spleen specimens, several gene candidates that showed the abi l i ty to activate the Tap-1 gene were discovered. Further research is required to refine the screening methods and to con f i rm the va l id i ty o f the pr imary screen hits. In this thesis, the second mechan ism that was found to cause TAP-1 def ic iency i n metastatic carc inomas, the l o w stabi l i ty o f TAP-1 m R N A [14], has not been further investigated. Acce lera ted degradation o f TAP-1 m R N A due to a mutat ion w i th in the TAP-1 cod ing sequence i n a me lanoma ce l l l ine has been reported [24]. However , the mechanisms remain unknown in the absence o f mutations w i th in the TAP-1 cod ing sequence itself, w h i c h is the case i n most TAP-def i c ient carc inoma lesions [7, 9]. The presence o f a destab i l iz ing A U - r i c h element ( A R E ) i n the 3'-untranslated region ( 3 ' U T R ) found in a large variety o f ce l lu lar transcripts is general ly responsible for m R N A degradation in a normal turnover process o f coordinated gene expression [25, 26]. GENERAL DISCUSSION 142 A R E was or ig ina l l y ident i f ied as a component o f the granulocyte-macrophage colony-st imulat ing factor ( G M - C S F ) m R N A 3 ' -UTR [27]. Identif icat ion o f b ind ing proteins w i th the abi l i ty to either promote or inhibi t ARE-med ia ted m R N A instabi l i ty is o f great interest i n the f i e ld o f post-transcriptional regulat ion o f gene expression [28]. A widely-studied group o f proteins that was found to accelerate m R N A degradation is the tr istetraprolin (TTP ) f am i l y o f C C C H tandem z inc f inger proteins that b inds U U A U U U A U U consensus sequence i n A R E s o f m R N A [26, 28] . T T P recruits m R N A decay complexes to A R E sequences, p romot ing remova l o f the p o l y ( A ) ta i l (deadenylation) and degradation o f the m R N A [26]. Interestingly, the core heptamer o f the ideal T T P b ind ing site, U A U U U A U , that is conserved in m ice and humans [28], is also present i n the 3'-untranslated region (3 '-UTR) o f human and rat TAP-1 m R N A ( N C B I accession no. N M _ 0 0 0 5 9 3 and N M _ 0 3 2 0 5 5 , respect ively) . However , no p o l y A site was speci f ied i n a mouse TAP-1 m R N A sequence prov ided in the N C B I website ( N M O 1 3 6 8 3 ) . Nevertheless, the presence o f T T P b ind ing site i n human and rat TAP-1 m R N A opens the poss ib i l i ty that the TAP-1 m R N A is regulated i n TTP-dependent manner. Therefore, it is int r iguing to study whether deregulat ion o f T T P funct ion i n TAP-def i c ient cel ls is responsible for the accelerated degradation o f TAP-1 m R N A . GENERAL DISCUSSION 143 5.2 Future Work Based on the findings of pre- and post-transcriptional, as well as epigenetic mechanisms underlying TAP-1 deficiencies in carcinomas, the research presented in this thesis could go forward in a number of interesting ways: 1. Characterization of TAP-activating factors. This would require a thorough dissection of specific mechanisms outlined in this thesis. Differential states of core histone modifications in TAP-1 promoter of TAP-expressing and TAP-deficient cells need to be explored in greater details. The strongest correlations of histone modifications and gene transcription observed so far are: methylation of lysine 4, 36 and 79 in histone H3 (H3K4me, H3K36me and H3K79me, respectively), as well as acetylation of lysine 9 and 14 (H3K9ac and H3K14ac) and lysine 16 in histone H4 (H4K16ac) with activation, while methylation of lysine 9 and 27 in histone H3 (H3K9me and H3K27me) and lysine 20 in histone H4 (H4K20me) with repression [4, 29]. The finding of more specific differences will aid the identification of specific factors involved. Exact mechanisms responsible for the impaired recruitment of CBP to TAP-1 promoter in TAP-deficient cancer cells are yet to be determined. It is important to determine whether the CBP itself is dysfunctional in TAP-deficient cancer cells, or whether the impairment is caused by deregulation of one or more upstream factors/co-activators. This study could be initiated by observing the effect of overexpressing normal CBP in TAP-deficient cells. If the results indicated that the defect does not lie in the CBP itself, it would be worth exploring the functionality of several CBP-associated factors, GENERAL DISCUSSION 144 such as p/CIP and P C A F [17, 30, 31]. It is also interesting to test whether the H A T act iv i ty o f C B P is a cr i t ica l factor responsible for histone acetylat ion and act ivat ion o f TAP-1 promoter. Th i s may be tested by overexpression o f dominant-negative mutants o f C B P that lack the H A T domain . The c D N A l ibrary screening method cou ld be ref ined by deve lop ing a V i raPort-l ike (Stratagene) c D N A l ibrary f rom a TAP-express ing , pr imary cancer ce l l l ine. The c D N A l ibrary retroviral supernatant cou ld then be used to transduce its TAP-def i c ien t , metastatic derivatives. Screening o f posi t ive transductants and recovery o f candidate genes ( c D N A s ) cou ld be per formed as descr ibed in Chapter 4. Th is way, the candidates obtained w o u l d l i ke l y to be more speci f ic to the type o f tissues f rom w h i c h the cancers arose. M i c roa r r ay analysis cou ld be per formed to observe differential express ion o f genes i n a TAP-express ing , pr imary cancer ce l l l ine and its TAP-def ic ient , metastatic derivat ive. Aberrant express ion o f genes i n the metastatic cel ls might be responsible for the downregulated express ion o f T A P and immune evasion mechanisms. 2. Invest igat ion o f the mechanisms by w h i c h T S A improved TAP-1 expression and tumor antigen presentation. The studies i n Chapter 3 indicated that unl ike the IFN-y, T S A effects d id not occur through the improvement o f histone H 3 acetylation in TAP-1 promoter. One approach w o u l d be to isolate TSA-respons ive element(s) i n TAP-1 promoter by testing the response o f fu l l TAP-1 promoter and its truncations to T S A treatment. Ident i fy ing the mechan ism by w h i c h T S A up-regulates TAP-1 expression is o f fundamental importance, GENERAL DISCUSSION 145 since any non-toxic compounds w i th the abi l i ty to enhance T A P and M H C class I expression i n cancer cel ls are o f great interest for the development o f cancer immunotherapy approaches. In the future, the ef f icacy o f the treatment may be improved by the invent ion o f synthetic T S A - l i k e molecules w i th a greater abi l i ty to induce T A P and M H C class I expression in cel ls w i th down-regulated express ion o f A P M components. 3. Broader appl icat ions o f the use o f p T A P l - E G F P construct for screening nove l T A P -1-regulator genes. In contrast to the use o f this system to search for TAP-act i va t ing c D N A s i n T A P -defic ient ce l ls , it may also be used to hunt for nove l genes w i th the abi l i ty to repress T A P - 1 promoter act ivity i n TAP-express ing cel ls. TAP-suppress ing compounds may be incorporated i n immunotherapeutic approaches that a im to inhib i t immune responses i n transplant reject ion and autoimmune diseases. 4. Investigation o f the mechanisms responsible for rap id TAP - 1 m R N A degradation i n TAP-def i c ient carc inomas. Th i s invest igat ion may be init iated by mutat ing the TTP-b ind ing sequence i n the 3 ' -UTR region o f TAP - 1 and testing whether this w o u l d increase T A P - 1 m R N A stabil i ty i n TAP-def i c ien t cel ls. Whether T T P plays a role i n regulat ing TAP - 1 m R N A stabil i ty cou ld also be determined by compar ing TAP - 1 m R N A stabil i ty i n cel ls originated f rom normal versus TTP-def ic ient mice [32]. Th i s may be f o l l owed by studies that a im to GENERAL DISCUSSION 146 characterize proteins w i th the abi l i ty to prevent de-adenylation [33], thus protecting T A P -1 m R N A transcripts f r om rapid degradation i n a poly(A)-dependent fashion. GENERAL DISCUSSION 147 5.3 The Big Picture According to Burnet's immune surveillance theory, thousands of tumor cells emerged de novo everyday and the hosts' immune surveillance system was able to kill these antigenic tumor cells [34]. Unfortunately, this perfect scenario does not always happen, since M H C class I antigen presentation is often disrupted in cancer cells, due to downregulation of A P M components, followed by immune selective pressure that works in favor of the outgrowth of cells that are unable to present tumor-specific antigens [3]. These cells can proliferate uncontrollably as they remain invisible by the circulating killer T cells (CTLs). This phenomenon remains a challenge for the development of anti-cancer vaccines and T-cell-based tumor immunotherapy methods that largely rely on the ability of CTLs to recognize abnormal cells expressing tumor-specific antigens. Effective approaches that lead to the induction of immune responses against neoplastic cells are of interest for the advancement of cancer immunotherapy. TAP-1-based therapy is a particularly attractive approach to be developed, since the entire M H C class I antigen presentation pathway in cells with multiple deficiencies of A P M components may be reconstituted by restoring the expression of this particular component. A successful clinical approach would require a combination of fundamental knowledge and application of TAP-1-activating factors and effective delivery methods. GENERAL DISCUSSION 148 5.4 References 1. Buben ik , J . M H C class I down-regulat ion: tumour escape f rom immune survei l lance? Int J O n c o l , 2004. 25:487-91. 2. C a m p o l i , M . , C . C . Chang , and S. Ferrone. H L A class I antigen loss, tumor immune escape and immune selection. Vacc i ne , 2002. 20 Suppl 4 :A40-5. 3. Chang , C . C . and S. Ferrone. Immune selective pressure and H L A class I antigen defects i n mal ignant lesions. Cancer Immuno l Immunother, 2007. 56:227-36. 4. T o m a s i , T .B . , W. J . Magner , and A . N . K h a n . Epigenet ic regulat ion o f immune escape genes i n cancer. Cancer Immuno l Immunother, 2006. 55:1159-84. 5. Abe l e , R. and R. Tampe. M o d u l a t i o n o f the antigen transport machinery T A P by fr iends and enemies. F E B S Lett, 2006. 580:1156-63. 6. Lankat-Buttgereit , B. and R. Tampe. The transporter associated w i th antigen process ing: funct ion and impl icat ions i n human diseases. Phys io l Rev , 2002. 82:187-204. 7. Sel iger, B., T. Cabrera , F. Gar r ido , and S. Ferrone. H L A class I antigen abnormal i t ies and immune escape by mal ignant cells. Semin Cancer B i o l , 2002. 12:3-13. 8. Sel iger, B., M. J . Maeurer , and S. Ferrone. T A P off—tumors on. Immuno l Today , 1997. 18:292-9. 9. Sel iger, B., M. J . Maeurer , and S. Ferrone. Ant igen-process ing machinery breakdown and tumor growth. Immuno l Today , 2000. 21:455-64. GENERAL DISCUSSION 149 10. Rest i fo , N.P., F. E squ i ve l , Y . K a w a k a m i , J .W . Y e w d e l l , J.J. M u l e , S.A. Rosenberg, and J .R. Benn ink . Identif icat ion o f human cancers def ic ient i n antigen processing. J E x p M e d , 1993. 177:265-72. 11. A l i m o n t i , J . , Q.J. Zhang , R. Gabathuler, G . R e i d , S.S. Chen , and W . A . Jefferies. T A P expression provides a general method for improv ing the recognit ion o f mal ignant cel ls i n v ivo . Na t B io techno l , 2000. 18:515-20. 12. Gabathuler, R., G . R e i d , G . Ko l a i t i s , J . D r i s c o l l , and W . A . Jefferies. Compar i son o f ce l l l ines deficient i n antigen presentation reveals a funct ional role for TAP-1 alone i n antigen processing. J E x p M e d , 1994. 180:1415-25. 13. Sel iger, B., U . R i t z , R. Abe l e , M . B o c k , R. Tampe, G . Sutter, I. Drex le r , C . Huber , and S. Ferrone. Immune escape o f me lanoma: first evidence o f structural alterations i n two dist inct components o f the M H C class I antigen process ing pathway. Cancer Res, 2001 . 61:8647-50. 14. Set iadi , A . F . , M . D . D a v i d , S.S. Chen , J . Hiscott , and W . A . Jefferies. Ident i f icat ion o f mechanisms under ly ing transporter associated w i t h antigen process ing def ic iency i n metastatic murine carcinomas. Cancer Res, 2005. 65:7485-92. 15. Dav i e , J .R . and V . A . Spencer. Cont ro l o f histone modif icat ions. J C e l l B i o c h e m , 1999. Supp l 32-33:141-8. 16. G io rdano , A . and M . L . Avantaggiat i . p300 and C B P : partners for l i fe and death. J C e l l Phys i o l , 1999. 181:218-30. 17. K a l k h o v e n , E. C B P and p300 : H A T s for different occasions. B i o c h e m Pharmaco l , 2004 .68 :1145-55 . GENERAL DISCUSSION 150 18. Legube, G . and D. Trouche. Regula t ing histone acetyltransferases and deacetylases. E M B O Rep , 2003. 4:944-7. 19. M a , Z. , M. J . Chang , R .C. Shah, and E .N . Benveniste. Interferon-gamma-activated STAT-1 alpha suppresses M M P - 9 gene transcript ion by sequestration o f the coactivators CBP/p300. J L eukoc B i o l , 2005. 78:515-23. 20. Ramana , C .V . , M .P . G i l , R .D . Schreiber, and G.R. Stark. Stat 1-dependent and -independent pathways i n IFN-gamma-dependent s ignal ing. Trends Immuno l , 2002 .23 :96-101 . 21 . Jung, M . Inhibitors o f histone deacetylase as new anticancer agents. Cu r r M e d C h e m , 2001 . 8:1505-11. 22. Tadde i , A . , D. Roche , W . A . B i c k m o r e , and G . A l m o u z n i . The effects o f histone deacetylase inhibitors on heterochromatin: impl icat ions for anticancer therapy? E M B O Rep, 2005. 6:520-4. 23. Yosh ida , M . , R. Fu ruma i , M . N i s h i y a m a , Y . Koma t su , N . N i s h i n o , and S. Ho r inouch i . H is tone deacetylase as a new target for cancer chemotherapy. Cancer Chemother Pharmaco l , 2001 . 48 Suppl l :S20-6. 24. Y a n g , T., B .A . M c N a l l y , S. Ferrone, Y . L i u , and P. Zheng. A single-nucleotide delet ion leads to rapid degradation o f TAP-1 m R N A in a me lanoma ce l l l ine. J B i o l C h e m , 2003. 278:15291-6. 25. Ben jamin , D., M . C o l o m b i , G . S toeck l in , and C . M o r o n i . A GFP-based assay for moni tor ing post-transcriptional regulat ion o f A R E - m R N A turnover. M o l B iosyst , 2006. 2:561-7. GENERAL DISCUSSION 151 26. H a u , H .H . , R.J. Wa l sh , R.L. Og i l v i e , D . A . W i l l i a m s , C.S . Re i l l y , and P.R. Bohjanen. Tr istetraprol in recruits funct ional m R N A decay complexes to A R E sequences. J C e l l B i o c h e m , 2006. 27. Shaw, G . and R. K a m e n . A conserved A U sequence f rom the 3' untranslated region o f G M - C S F m R N A mediates selective m R N A degradation. C e l l , 1986. 46:659-67. 28. L a i , W.S. , J .S. Parker, S.F. G r i s som, D.J. S tumpo, and P.J. B lackshear . N o v e l m R N A targets for tr istetraprolin (TTP ) ident i f ied by g lobal analysis o f s tab i l ized transcripts i n TTP-def ic ient fibroblasts. M o l C e l l B i o l , 2006. 26:9196-208. 29 . L o y o l a , A . , T. Bona ld i , D. Roche , A . Imhof, and G . A l m o u z n i . P T M s on H 3 variants before chromat in assembly potentiate their f ina l epigenetic state. M o l C e l l , 2006. 24:309-16. 30. Torch ia , J . , D .W. Rose , J . Inostroza, Y . K a m e i , S. Wes t in , C . K . G lass , and M . G . Rosenfe ld . The transcript ional co-activator p/CIP binds C B P and mediates nuclear-receptor funct ion. Nature, 1997. 387:677-84. 31 . Herrera, J .E. , R.L. Sch i l tz , and M . Bus t in . The accessibi l i ty o f histone H 3 tails i n chromat in modulates their acetylat ion by P300/CBP-associated factor. J B i o l C h e m , 2000. 275:12994-9. 32. L a i , W.S. , E. Carba l lo , J . M . Thorn , E .A . Kenn ing ton , and P.J. B lackshear . Interactions o f C C C H z inc f inger proteins w i th m R N A . B i n d i n g o f tristetraprolin-related z inc f inger proteins to Au-r i ch elements and destabi l izat ion o f m R N A . J B i o l C h e m , 2000. 275:17827-37. GENERAL DISCUSSION 152 33. Conrad , N .K . , S. M i l i , E.L. M a r s h a l l , M . D . Shu, and J . A . Steitz. Identif icat ion o f a rapid mamma l i an deadenylation-dependent decay pathway and its inh ib i t ion by a v i ra l R N A element. M o l C e l l , 2006. 24:943-53. 34. Burnet, M . Cancer ; a b io log i ca l approach. I. The processes o f control. B r M e d J , 1957. 1:779-86. APPENDIX 153 Appendix A: Details of Methods A. 1 Chromatin Immunoprecipitation Assay Twenty m i l l i o n cel ls were t ryps in ized, washed twice w i th P B S , pel leted, and incubated for 10 minutes at r o o m temperature in P B S supplemented w i t h a cross-l inker reagent, 1 % formaldehyde (S igma). Cross- l ink ing was stopped by addit ion o f g lyc ine to a f ina l concentrat ion o f 125 m M for 5 m i n . Ce l l s were washed twice w i th ice-cold P B S and centr i fuged for 5 m i n at 4 °C . Ce l l s were then lysed i n 1 m l cytoplasmic lys is buffer (5 m M P ipes , p H 8.0, 85 m M K C 1 , 0 . 5 % NP-40 , and protease inhib i tor (Complete M i n i , Roche) ) , and the pel leted fract ion was then lysed i n and 500 p i o f nuclear lys is buffer ( 1 % S D S , 10 m M E D T A , 50 m M T r i s - H C l , p H 8.0 and protease inhibitor) . B o t h steps o f lys is were conducted for 10 minutes. Then , chromat in was sheared by sonicat ion ( M i c r o s o n X L sonicator, M i s o n i x Inc., Farmingdale , N Y ) (power setting 13 Watts; 3 x 1 5 seconds burst) to obtain 0.5-1 kb fragments. Samples were centr i fuged to pellet debris and di luted 4 t imes i n d i lu t ion buffer ( 1 % Tr i ton X-100 , 2 m M E D T A , 20 m M T r i s - H C l , p H 8.0, 150 m M N a C l and protease inhibi tor ) . T w o micrograms sa lmon sperm D N A (Sigma) was added, and samples were pre-cleared w i th 100 p i slurry o f protein A agarose beads (Protein A Sepharose C L - 4 B , A m e r s h a m B iosc iences , Upps l a , Sweden) for 2 hours at 4 °C . Immunoprecipi tat ions were carr ied out overnight at 4 °C w i th 5 ug o f a n t i - R N A polymerase II ant ibody (N-20, sc-899, Santa Cruz ) . Immunoprecipi tat ions wi thout ant ibody were also inc luded as controls to assess levels o f background signals. Immune complexes were col lected w i th protein A agarose beads for 1 hour and washed successively in l o w salt buffer, h igh salt buffer and L i C l buffer for 10 minutes, then twice APPENDIX 154 i n T E for 2 minutes. The complexes were then eluted i n 1 % S D S , 0.1 M N a H C 0 3 and cross-links were reversed by heating at 65°C for 6 hours to overnight. F o l l o w i n g 1 hour o f proteinase K (Invitrogen) digest ion at 45 °C, D N A was phenol-chloroform extracted and ethanol precipitated. Pel lets were resuspended i n 50 p i m i l l i Q water, and 2 p i o f the immunoprec ip i tated D N A was used i n each P C R sample. A.2 Calculation of copy numbers of pTAP-1-EGFP construct integrated in stable transfectants (6 x 1 0 2 3 [copies/mol] x concentrat ion [g/uf]) / M W [g/mol] = amount [copies/ ul] 1 m o l = molecu lar weight ( M W ) [g] 1 m o l = 6 x 1 0 molecules (= copies) Standard size o f mouse D N A : 2.7 x 1 0 9 b p . As sume this is per ce l l . Standard convers ion o f ug to p m o l : 1 i ig o f a 100 bp d s D N A fragment = (1 ug x 1515) / 100 = 15.2 p m o l Therefore, 100 ng o f 2.7 x 1 0 9 b p d s D N A fragment = (0.1 ug x 1515) / (2.7 x 10 9 ) = 5.61 x 10" 8 p m o l = 5.61 x 10 " 2 0 m o l = 33,660 copies Thus , i n 100 ng mouse D N A , there are 33,660 copies o f genomic D N A . Sample ca lcu lat ion: U s i n g 100 ng o f C M T . 6 4 ( p T A P l - E G F P stable transfectants) genomic D N A as a rea l -t ime P C R template, the average number o f p T A P l - E G F P constructs integrated into the C M T . 6 4 cel ls was determined to be 53,535 copies. Therefore, average number o f p l asmid per ce l l = 53,535 / 33,660 = 1.59 APPENDIX 155 Appendix B: List of Cloning Vectors MCS Stii\ © Clontech Laboratories, Inc. Reprinted by permission. © Clontech Laboratories, Inc. Reprinted by permission. APPENDIX 156 Appendix C: Comparison of TAP-1 Coding Sequence of CMT.64 and Ltk cells B l a c k : L t k sequence; I t a l i c s : CMT.64 sequence; B o l d : p o l y m o r p h i c r e g i o n s . 1 ATGGCTGCGC ACGTCTGGCT GGCGGCCGCC CTGCTCCTTC TGGTGGACTG GCTGCTGCTG 1 '' ATGGCTGCGC ACGTCTGGCT GGCGGCCGCC CTGCCCCTTC TGGTGGACTG GCTGCTGCTG 61 CGGCCCATGC TCCCGGGAAT CTTCTCCCTG TTGGTTCCCG AGGTGCCGCT GCTCCGGGTC 61 CGGCCCATGC TCCCGGGAAT CTTCTCCCTG TTGGTTCCCG AGGTGCCGCT GCTCCGGGTC 121 TGGGTGGTGG GCCTGAGTCG CTGGGCCATC CTAGGACTAG GGGTCCGCGG GGTCCTCGGG 121 TGGGTGGTGG GCCTGAGTCG CTGGGCCATC CTAGGACTAG GGGTCCGCGG GGTCCTCGGG 181 GTCACCGCAG GAGCCCATGG CTGGCTGGCT GCTTTGCAGC CGCTGGTGGC CGCACTGAGT 181 GTCACCGCAG GAGCCCATGG CTGGCTGGCT GCTTTGCAGC CGCTGGTGGC CGCACTGAGT 241 TTGGCCCTGC CTGGACTTGC CTTGTTCCGA GAGCTGGCCG CCTGGGGAAC ACTCCGGGAG 241 TTGGCCCTGC CTGGACTTGC CTTGTTCCGA GAGCTGGCCG CCTGGGGAAC ACTCCGGGAG 301 GGTGACAGCG CTGGATTACT GTACTGGAAC AGTCGTCCAG ATGCCTTCGC TATCAGTTAT 301 GGTGACAGCG CTGGATTACT GTACTGGAAC AGTCGTCCAG ATGCCTTCGC TATCAGTTAT 361 GTGGCAGCAT TGCCCGCAGC CGCCCTGTGG CACAAGTTGG GGAGCCTCTG GGCGCCCAGC 361 GTGGCAGCAT TGCCCGCAGC CGCCCTGTGG CACAAGTTGG GGAGCCTCTG GGCGCCCAGC 421 GGCAACAGGG ACGCTGGAGA CATGCTGTGT CGGATGCTGG GCTTCCTGGG CCCTAAGAAG 421 GGCAACAGGG ACGCTGGAGA CATGCTGTGT CGGATGCTGG GCTTCCTGGG CCCTAAGAAG 481 AGACGTCTCT ACCTGGTTCT GGTTCTCTTG ATTCTCTCTT GCCTTGGGGA AATGGCCATT 481 AGACGTCTCT ACCTGGTTCT GGTTCTCTTG ATTCTCTCTT GCCTTGGGGA AATGGCCATT 541 CCCTTCTTCA CGGGCCGCAT CACTGACTGG ATTCTTCAGG ATAAGACAGT TCCTAGCTTC 541 ' CCCTTCTTCA CGGGCCGCAT CACTGACTGG ATTCTTCAGG ATAAGACAGT TCCTAGCTTC 601 ACCCGCAACA TATGGCTCAT GTCCATTCTC ACCATAGCCA GCACAGCGCT GGAGTTTGCA 601 ACCCGCAACA TATGGCTCAT GTCCATTCTC ACCATAGCCA GCACAGCGCT GGAGTTTGCA 661 AGTGATGGAA TCTACAACAT CACCATGGGA CACATGCACG GCCATGTGCA CAGAGAGGTG 661 AGTGATGGAA TCTACAACAT CACCATGGGA CACATGCACG GCCGTGTGCA CAGAGAGGTG 7 21 TTTCGGGCCG TCCTTCGCCA GGAGACAGGG TTTTTCCTGA AGAACCCAGC AGGTTCCATC 721 TTTCGGGCCG TCCTTCGCCA GGAGACAGGG TTTTTCCTGA AGAACCCAGC AGGTTCCATC 781 ACATCTCGGG TGACTGAGGA CACAGCCAAC GTGTGCGAGT CCATTAGTGG CACGCTGAGC 781 ACATCTCGGG TGACTGAGGA CACAGCCAAC GTGTGCGAGT CCATTAGTGG CACGCTGAGC 841 CTGCTGCTGT GGTACCTGGG GCGAGCCCTG TGTCTCTTGG TGTTCATGTT TTGGGGGTCA 841 CTGCTGCTGT GGTACCTGGG GCGAGCCCTG TGTCTCTTGG TGTTCATGTT TTGGGGGTCA APPENDIX 157 901 CCGTACCTCA CTCTGGTCAC CCTGATCAAT CTGCCCCTGC TTTTTCTTTT GCCTAAGAAG 901 CCGTACCTCA CTCTGGTCAC CCTGATCAAC CTGCCCCTGC TTTTTCTTTT GCCTAAGAAG 961 CTGGGAAAAG TGCACCAGTC ACTGGCAGTG AAGGTGCAGG AGTCTCTAGC AAAGTCCACG 961 CTGGGAAAAG TGCATCAGTC ACTGGCAGTG AAGGTGCAGG AGTCTCTAGC AAAGTCCACG 1021 CAGGTGGCCC TTGAGGCCTT ATCGGCAATG CCTACTGTGC GGAGCTTTGC CAACGAGGAG 1021 CAGGTGGCCC TTGAGGCCTT ATCGGCGATG CCTACTGTGC GGAGCTTTGC CAACGAGGAG 1081 GGTGAGGCCC AGAAGTTCAG GCAGAAGTTG GAAGAAATGA AGACGCTAAA CAAGAAGGAG 1081 GGTGAGGCCC AGAAGTTCAG GCAGAAGTTG GAAGAAATGA AGACTCTAAA CAAGAAGGAG 1141 GCCTTGGCTT ATGTCGCTGA AGTCTGGACC ACGAGTGTCT CGGGAATGCT GCTGAAGGTG 1141 GCCTTGGCTT ACGTGGCTGA AGTCTGGACC ACGAGTGTCT CGGGAATGCT GCTGAAGGTG 12 01 GGAATTCTGT ACCTGGGCGG GCAGCTGGTG ATCAGAGGGA CTGTCAGCAG CGGCAACCTT 1201 GGAATTCTGT ACCTGGGCGG GCAGCTGGTG ATCAGAGGGG CTGTCAGCAG CGGCAACCTT 12 61 GTCTCATTCG TTCTCTACCA GCTTCAGTTC ACCCACGCTG TTCAGGTCCT GCTCTCCCTC 1261 GTCTCATTCG TTCTCTACCA GCTTCAGTTC ACCCAGGCTG TTCAGGTCCT GCTCTCCCTC 1321 TACCCCTCCA TGCAGAAGGC TGTGGGCTCC TCAGAGAAAA TATTCGAATA CTTGGACCGG 1321 TACCCCTCCA TGCAGAAGGC TGTGGGCTCC TCAGAGAAAA TATTCGAATA CTTGGACCGG 1381 ACTCCTTGCT CTCCACTCAG TGGCTCGTTG GCACCCTCAA ACATGAAAGG CCTTGTGGAG 1381 ACTCCTTGCT CTCCACTCAG TGGCTCGTTG GCACCCTCAA ACATGAAAGG CCTTGTGGAG 14 41 TTCCAAGATG TCTCTTTTGC CTACCCAAAC CAGCCCAAAG TCCAGGTGCT TCAGGGGCTG 1441 TTCCAAGATG TCTCTTTTGC CTACCCAAAC CAGCCCAAAG TCCAGGTGCT TCAGGGGCTG 1501 ACGTTCACCC TGCATCCTGG AACGGTGACA GCGTTGGTGG GACCCAATGG ATCAGGGAAG 1501 ACGTTCACCC TGCATCCTGG AACGGTGACA GCGTTGGTGG GACCCAATGG ATCAGGGAAG 15 61 AGCACCGTGG CTGCCCTGCT GCAGAACCTG TACCAGCCCA CCGGGGGCCA GCTGCTGCTG 1561 AGCACCGTGG CTGCCCTGCT GCAGAACCTG TACCAGCCCA CCGGGGGCCA GCTGCTGCTG 1621 GATGGCCAGC GCCTGGTCCA GTATGATCAC CATTACCTGC ACACTCAGGT AGCCGCAGTG 1621 GATGGCCAGT GCCTGGTCCA GTATGATCAC CATTACCTGC ACACTCAGGT GGCCGCAGTG 1681 GGACAAGAGC CGCTGCTATT TGGAAGAAGC TTTCGAGAAA ATATTGCGTA TGGCCTGAAC 1681 GGACAAGAGC CGCTGCTATT TGGAAGAAGC TTTCGAGAAA ATATTGCGTA TGGCCTGAAC 1141 CGGACTCCAA CCATGGAGGA AATCACAGCT GTGGCCGTGG AGTCTGGAGC CCACGATTTC 1741 CGGACTCCAA CCATGGAGGA AATCACAGCT GTGGCCGTGG AGTCTGGAGC CCACGATTTC 18 01 ATCTCTGGGT TCCCTCAGGG CTATGACACA GAGGTAGGTG AGACTGGGAA CCAGCTGTCA 1801 ATCTCTGGGT TCCCTCAGGG CTATGACACA GAGGTAGGTG AGACTGGGAA CCAGCTGTCA 18 61 GGAGGTCAGC GACAGGCAGT GGCCTTGGCC CGAGCCTTGA TCCGGAAGCC ACTCCTGCTT 1861 GGAGGTCAGC GACAGGCAGT GGCCTTGGCC CGAGCCTTGA TCCGGAAGCC ACTCCTGCTT APPENDIX 158 1921 ATCTTGGATG ATGCCACCAG TGCCCTGGAT GCTGGCAACC AGCTACGGGT CCAGCGGCTC 1921 ATCTTGGATG ATGCCACCAG TGCCCTGGAT GCTGGCAACC AGCTACGGGT CCAGCGGCTC 1981 CTGTATGAGA GCCCCAAGCG GGCTTCTCGG ACGGTTCTTC TTATCACCCA GCAGCTCAGC 1981 CTGTATGAGA GCCCCAAGCG GGCTTCTCGG ACGGTTCTTC TTATCACCCA GCAGCTCAGC 2041 CTGGCAGAGC AGGCCCACCA CATCCTCTTT CTCAGAGAAG GCTCTGTCGG CGAGCAGGGC 2041 CTGGCAGAGC AGGCCCACCA CATCCTCTTT CTCAGAGAAG GCTCTGTCGG CGAGCAGGGC 2101 ACCCACCTGC AGCTCATGAA GAGAGGAGGG TGCTACCGGG CCATGGTAGA GGCTCTTGCG 2101 ACCCACCTGC AGCTCATGAA GAGAGGAGGG TGCTACCGGG CCATGGTAGA GGCTCTTGCG 2161 GCTCCTGCAG ACTGA 2161 GCTCCTGCAG ACTGA 

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