145 to overexpress PRAP1 before subjecting them to 5-FU treatment. Lysates were harvested and immunoprecipitation were carried out using antibody against Hsp 70. The immunoprecipitated proteins were then analyzed by western blotting. As shown in Figure 3.72, PRAP1 and Hsp 70 coprecipitated. PRAP1 was detected in Hsp 70 precipitates. These data demonstrated that Hsp 70 interacted with PRAP1 and were present in the same complex upon 5-FU treatment. 3.12 Role of PRAP1 in apoptotic cells In our study, we observed that PRAP1 was highly expressed in both differentiated cells as well as stress-induced apoptotic cells. In addition, the protein PRAP1 was found to bind to the surface of bacteria cells. We hypothesized that PRAP1 may also bind to the surface of apoptotic cells and may play a role in the clearance of apoptotic cells. 3.12.1 Induction of PRAP1 expression in apoptotic cells To further validate the expression of PRAP1 in apoptotic cells, we used HT 29 cells which have high basal levels of PRAP1 protein expression. Briefly, HT 29 cells were treated with 5mM sodium butyrate for 48 hours to induce apoptosis. Cell culture medium containing dead cells that were detached from the culture flask were harvested. The live cells that remained attached to the culture flask were harvested by trypsinization. Cells were stained with propidium iodide and subjected to flow cytometry for analysis of DNA content. Our results showed that the population of the attached cells in sub-G1 was comparable to that of the untreated control (Figure 3.72). On the other hand, the floating cells showed a significant increase in the sub-G1 population. This indicated that the floating cells were composed of mainly dead cells. 146 Figure 3.72 Dying cells in floating population Representative histogram showing HT 29 cells untreated (control) or treated with 5mM sodium butyrate for 48 hours. Floating cells (Floating) from the treated sample were harvested separately from the adherent cells (Adherent). All cells were fixed and stained with propidium iodide and DNA content was analyzed by flow cytometry. Box indicates cells at Sub-G1 (dying cell population). 147 To further validate the apoptotic nature of these floating cells, we employed the annexin V-FITC staining assay. Annexin V is a phospholipid binding protein that has a high binding affinity for phosphatidylserine (PS) that translocates from inner to outer leaflet of plasma membrane during early apoptosis. As shown in Figure 3.73, there was a shift of the fluorescent peak to the right in the floating cells as compared to that of the adherent cells. There was minimal annexin V staining in the adherent cells, as indicated by a lack of shift in the fluorescent peak when compared to that of the untreated control. The population of cells with positive annexin V staining was calculated by setting the gating at R1 as shown in Figure 3.73. In the floating cells, 83% of cells were positive for annexin V expression, while only 14% of adherent cells and 12% of the untreated control were positive. These results demonstrated that the majority of the floating cells were apoptotic cells, suggesting that we have successfully obtained two distinct populations of cells, the live cells (attached cells) and the apoptotic cells (floating cells). The expression of PRAP1 in these two populations was analyzed. Our results showed that PRAP1 mRNA was highly induced in the apoptotic cells as compared to the adherent cells (Figure 3.74-A). Consistent with an increase at the mRNA level, PRAP1 protein was also increased in the apoptotic cells (Figure 3.74-B). These results showed that PRAP1 expression was highly induced in apoptotic cells compared to the live cells. 3.12.2 PRAP1 binds to the surface of apoptotic cells As PRAP1 is a secreted protein, we investigated whether PRAP1 binds to the surface of the apoptotic cells. Apoptotic cells were harvested and either labeled with anti-PRAP1 antibody directly or fixed with 4% paraformaldehyde, 148 Figure 3.73 Apoptotic cells in floating population Representative histogram showing annexin-V staining in HT 29 cells after treatment with 5mM sodium butyrate for 48 hours or untreated (blue). Floating cells (green) and attached cells (red) from the treated sample were harvested separately. All cells were fixed and stained with annexin-V conjugated to FITC and analyzed by flow cytometry. R1 gates the population of cells with positive annexin-V staining. Figure 3.74 PRAP1 expression is increased in apoptotic cells A: Representative figure showing the relative expression level of PRAP1 mRNA level in the adherent (control) and floating (Apoptotic cells) of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours. B: Representative western blot of PRAP1 and GAPDH (loading control) of the adherent (control) and floating (Apoptotic) population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours. proteins were detected on the surface of both unfixed and fixed apoptotic cells (Figure 3.75). The staining was not homogenous, with some cells showing weak detection of PRAP1 whereas others with strong detection of PRAP1. This 149 followed by immunofluorescence staining with PRAP1 antibody. Our results showed that PRAP1 observation was confirmed using flow cytometry. As shown in Figure 3.76, there were two populations of cells as indicated by two peaks (shaded in red). Those cells with higher PRAP1 (black arrow) on the surface were detected by a right shift as compared to the IgG control. There was a population with no or little detection of PRAP1 (red arrow). This observation was further confirmed to be not cell line specific. We repeated the experiment in another cell line, HCT 116. Briefly, HCT 116 was treated with 3mM sodium butyrate and apoptotic cells were harvested after 48 hours for immunofluorescence staining. Consistent with our previous finding, PRAP1 was detected on the surface of apoptotic cells (Figure 3.77). Together, our results showed that PRAP1 was induced in the apoptotic cells and bound onto the surface of the apoptotic cell. The binding of PRAP1 on the surface of apoptotic cells were confirmed using two additional methods: direct immunofluorescence and transmission electron microscopy. In the direct immunofluorescence assay, apoptotic cells were fixed and incubated with recombinant HisPRAP1 protein conjugated to Alexa fluor dye. Our results showed that exogenous PRAP1 was detected on the surface of apoptotic cells (Figure 3.78). Both free dye and BSA conjugated to Alex fluor dye were not detected on the surface of apoptotic cells. In the transmission electron microscopy (TEM) assay, apoptotic cells were fixed, stained with PRAP1-specific antibody and detected using secondary antibody conjugated to gold particles. Cells were processed for TEM and pictures were taken. As shown in Figure 3.79, there were gold particles, as indicated by the red arrows, detected on the 150 Figure 3.75 Immunofluorescence images showing the binding of PRAP1 to the surface of apoptotic cells Representative immunofluorescence images of PRAP1 binding to the floating population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hr. Floating cells were either unfixed or fixed in 4% paraformaldehyde and immunofluorescence was performed by incubating with anti-PRAP1 antibody (1:200) for hour, followed by secondary anti-rabbit antibody conjugated to Alexa Fluor 488 dye (Green). Figure 3.76 Histogram showing PRAP1 binds to the surface of apoptotic cells Representative histogram of PRAP1 (shaded in red) and rabbit IgG isotype (unshaded) staining in floating population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours. Cells were either fixed or unfixed in 4% paraformaldehyde. Red arrow indicates the fluorescence peak with unstained population. Black arrow indicates fluorescence peak with positive staining. 151 Figure 3.77 PRAP1 binds on the surface of HCT 116 apoptotic cells Representative immunofluorescence images of PRAP1 binding to apoptotic HCT 116 cells after treatment with 5mM sodium butyrate for 48 hours. Figure 3.78 Alexa fluor labeled HisPRAP1 binds directly to the surface of apoptotic cells Representative immunofluorescence images of floating population of HT 29 cells obtained after treatment with 5mM sodium butyrate for 48 hours incubated with alexa fluor labeled HisPRAP1 (PRAP1) or free alexa fluor dye. Images were taken using Confocal microscope. 152 Figure 3.79 Transmission electron microscopy of PRAP1 on apoptotic cells Representative transmission electron microscopy (TEM) images of apoptotic HT 29 cells stained with PRAP1 antibody. PRAP1 protein is indicated by red arrows and apoptotic bleb is indicated by black arrow. 153 outer surface of the membrane. One gold particle was detected on the surface of a bleb (as indicated by a black arrow). These data showed for the first time that PRAP1 binds on the outer surface of cell membrane of apoptotic cells. 3.12.3 PRAP1 enhanced the phagocytosis of beads As we have shown previously that PRAP1 binds to the surface of bacteria, we investigated whether the presence of PRAP1 on the surface of apoptotic cells would affect their clearance by phagocytosis. To achieve that, we used FluoSpheres polystyrene with yellow-green fluorescence. Briefly, these microspheres were either coated with HisPRAP1, BSA or uncoated. These coated beads were then incubated with a monocytic cell line, U937. Microspheres that were not ingested by U937 cells were quenched. Population of cells with ingested beads was measured by flow cytometry. Figure 3.80-A showed the representative pictures of scatter-histogram plot analysis of phagocytosis of beads. Cells with ingested beads are gated (as indicated by the circle) and percentage of cells with ingested beads was calculated and summarized in Figure 3.80-B. Coating of PRAP1 onto the surface of microspheres significantly enhanced their uptake by U937. At as early as 15 min, coating of PRAP1 resulted in more than 3-fold increase in the ingestion of the microspheres as compared to the BSA coated microspheres control (16% vs 4%). After hour, microspheres coated with PRAP1 were ingested by about 50% of the cells while only 13% of cells had ingested the BSA coated microspheres. To mimic the in vivo system, we differentiated the monocytic cell line, U937 into a macrophage lineage by treating it with dibutyryl cyclic adenosine monophosphate (dcAMP). As shown in Figure 3.81, these macrophages were 154 Figure 3.80 Flow cytometry analysis of phagocytosis of beads by U937 Representative scatter plot of phagocytosis of beads (A). Fluorescent beads uncoated or coated with BSA or HisPRAP1 were incubated with U937 cells and phagocytosis were analyzed with flow cytometry. U937 cells with ingested fluorescent beads were positive with fluorescent and gated (circle) and expressed as a percentage of total cells (B). Number on top of column indicates the mean. Columns, mean of replicates; Bars, SE. 204 Friedberg, E. C., G. C. Walker, et al. (1995). DNA repair and mutagenesis. Washington, D.C., ASM. Fujii, S., M. Fukushima, et al. (1989). "Pharmacokinetic modulation of plasma 5fluorouracil concentrations to potentiate the antitumor activity of continuous venous infusion of 5-fluorouracil." Jpn J Cancer Res 80(6): 509-12. Ganz, T. (2003). "Defensins: antimicrobial peptides of innate immunity." Nat Rev Immunol 3(9): 710-20. Ganz, T., M. E. Selsted, et al. (1985). "Defensins. Natural peptide antibiotics of human neutrophils." J Clin Invest 76(4): 1427-35. Giacchetti, S., B. Perpoint, et al. (2000). "Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer." J Clin Oncol 18(1): 136-47. Gill, S., C. L. Loprinzi, et al. (2004). "Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: who benefits and by how much?" J Clin Oncol 22(10): 1797-806. Groden, J., A. Thliveris, et al. (1991). "Identification and characterization of the familial adenomatous polyposis coli gene." Cell 66(3): 589-600. Halliwell, B. (1987). "Oxidants and human disease: some new concepts." Faseb J 1(5): 358-64. Hanayama, R., M. Tanaka, et al. (2002). "Identification of a factor that links apoptotic cells to phagocytes." Nature 417(6885): 182-7. Hartwell, L. H. and M. B. Kastan (1994). "Cell cycle control and cancer." Science 266(5192): 1821-8. Harwig, S. S., L. Tan, et al. (1995). "Bactericidal properties of murine intestinal phospholipase A2." J Clin Invest 95(2): 603-10. Heliez, C., L. Baricault, et al. (2003). "Paclitaxel increases p21 synthesis and accumulation of its AKT-phosphorylated form in the cytoplasm of cancer cells." Oncogene 22(21): 3260-8. Herrmann, M., R. E. Voll, et al. (1998). "Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus." Arthritis Rheum 41(7): 1241-50. Hess, M. T., U. Schwitter, et al. (1997). "Bipartite substrate discrimination by human nucleotide excision repair." Proc Natl Acad Sci U S A 94(13): 6664-9. Hoeijmakers, J. H. (2001). "Genome maintenance mechanisms for preventing cancer." Nature 411(6835): 366-74. 205 Hofmann, K., P. Bucher, et al. (1997). "The CARD domain: a new apoptotic signalling motif." Trends Biochem Sci 22(5): 155-6. Hoh, J., S. Jin, et al. (2002). "The p53MH algorithm and its application in detecting p53-responsive genes." Proc Natl Acad Sci U S A 99(13): 8467-72. Hollstein, M., K. Rice, et al. (1994). "Database of p53 gene somatic mutations in human tumors and cell lines." Nucleic Acids Res 22(17): 3551-5. Houlston, R. S. and I. P. Tomlinson (1997). "Genetic prognostic markers in colorectal cancer." Mol Pathol 50(6): 281-8. Hurwitz, H., L. Fehrenbacher, et al. (2004). "Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer." N Engl J Med 350(23): 2335-42. Iliakis, G., Y. Wang, et al. (2003). "DNA damage checkpoint control in cells exposed to ionizing radiation." Oncogene 22(37): 5834-47. Irmler, M., M. Thome, et al. (1997). "Inhibition of death receptor signals by cellular FLIP." Nature 388(6638): 190-5. Ishimoto, Y., K. Ohashi, et al. (2000). "Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6." J Biochem 127(3): 411-7. Johnson, K. R., L. Wang, et al. (1997). "5-Fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells." Clin Cancer Res 3(10): 1739-45. Johnston, P. G. and S. Kaye (2001). "Capecitabine: a novel agent for the treatment of solid tumors." Anticancer Drugs 12(8): 639-46. Kabbinavar, F., H. I. Hurwitz, et al. (2003). "Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer." J Clin Oncol 21(1): 60-5. Kabbinavar, F. F., J. Hambleton, et al. (2005). "Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer." J Clin Oncol 23(16): 3706-12. Kabbinavar, F. F., J. Schulz, et al. (2005). "Addition of bevacizumab to bolus fluorouracil and leucovorin in first-line metastatic colorectal cancer: results of a randomized phase II trial." J Clin Oncol 23(16): 3697-705. Kasik, J. and E. Rice (1997). "A novel complementary deoxyribonucleic acid is abundantly and specifically expressed in the uterus during pregnancy." Am J Obstet Gynecol 176(2): 452-6. Kerr, J. F., A. H. Wyllie, et al. (1972). "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics." Br J Cancer 26(4): 239-57. 206 Kinzler, K. W. and B. Vogelstein (1996). "Lessons from hereditary colorectal cancer." Cell 87(2): 159-70. Klausner, R. D., J. G. Donaldson, et al. (1992). "Brefeldin A: insights into the control of membrane traffic and organelle structure." J Cell Biol 116(5): 1071-80. Koepp, D. M., J. W. Harper, et al. (1999). "How the cyclin became a cyclin: regulated proteolysis in the cell cycle." Cell 97(4): 431-4. Koffler, D., P. H. Schur, et al. (1967). "Immunological studies concerning the nephritis of systemic lupus erythematosus." J Exp Med 126(4): 607-24. Korb, L. C. and J. M. Ahearn (1997). "C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited." J Immunol 158(10): 4525-8. Korinek, V., N. Barker, et al. (1998). "Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4." Nat Genet 19(4): 37983. Korinek, V., N. Barker, et al. (1997). "Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma." Science 275(5307): 17847. Kotzin, B. L. (1996). "Systemic lupus erythematosus." Cell 85(3): 303-6. Lai, K. N., J. C. Leung, et al. (1996). "Increased release of von Willebrand factor antigen from endothelial cells by anti-DNA autoantibodies." Ann Rheum Dis 55(1): 57-62. Lambert, P. H. and F. J. Dixon (1968). "Pathogenesis of the glomerulonephritis of NZB/W mice." J Exp Med 127(3): 507-22. Lanemo Myhrinder, A., E. Hellqvist, et al. (2008). "A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies." Blood 111(7): 3838-48. Lanemo Myhrinder, A., E. Hellqvist, et al. (2008). "A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies." Blood 111(7): 3838-48. Lee, H. K. and S. Jeong (2006). "Beta-Catenin stabilizes cyclooxygenase-2 mRNA by interacting with AU-rich elements of 3'-UTR." Nucleic Acids Res 34(19): 5705-14. Lee, I. H., Y. Cho, et al. (1997). "Effects of pH and salinity on the antimicrobial properties of clavanins." Infect Immun 65(7): 2898-903. 207 Lepourcelet, M., L. Tou, et al. (2005). "Insights into developmental mechanisms and cancers in the mammalian intestine derived from serial analysis of gene expression and study of the hepatoma-derived growth factor (HDGF)." Development 132(2): 415-27. Leverrier, Y. and A. J. Ridley (2001). "Requirement for Rho GTPases and PI 3kinases during apoptotic cell phagocytosis by macrophages." Curr Biol 11(3): 195-9. Levine, A. J. (1997). "p53, the cellular gatekeeper for growth and division." Cell 88(3): 323-31. Licht, R., J. W. Dieker, et al. (2004). "Decreased phagocytosis of apoptotic cells in diseased SLE mice." J Autoimmun 22(2): 139-45. Liu, J. J., B. H. Huang, et al. (2006). "Repression of HIP/RPL29 expression induces differentiation in colon cancer cells." J Cell Physiol 207(2): 287-92. Liu, J. J., J. Y. Wang, et al. (2000). "Activation of neutral sphingomyelinase participates in ethanol-induced apoptosis in Hep G2 cells." Alcohol Alcohol 35(6): 569-73. Liu, X., P. Yue, et al. (2004). "p53 upregulates death receptor expression through an intronic p53 binding site." Cancer Res 64(15): 5078-83. Longley, D. B., D. P. Harkin, et al. (2003). "5-fluorouracil: mechanisms of action and clinical strategies." Nat Rev Cancer 3(5): 330-8. Mason, D. Y. and C. R. Taylor (1975). "The distribution of muramidase (lysozyme) in human tissues." J Clin Pathol 28(2): 124-32. Memisoglu, A. and L. Samson (2000). "Base excision repair in yeast and mammals." Mutat Res 451(1-2): 39-51. Mevorach, D. (2000). "Opsonization of apoptotic cells. Implications for uptake and autoimmunity." Ann N Y Acad Sci 926: 226-35. Moertel, C. G., T. R. Fleming, et al. (1995). "Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: a final report." Ann Intern Med 122(5): 321-6. Morin, P. J., A. B. Sparks, et al. (1997). "Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC." Science 275(5307): 178790. Mosser, D. D., A. W. Caron, et al. (1997). "Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis." Mol Cell Biol 17(9): 531727. 208 Mountz, J. D., J. Wu, et al. (1994). "Autoimmune disease. A problem of defective apoptosis." Arthritis Rheum 37(10): 1415-20. Nakamura, Y. (2004). "Isolation of p53-target genes and their functional analysis." Cancer Sci 95(1): 7-11. Navratil, J. S., S. C. Watkins, et al. (2001). "The globular heads of C1q specifically recognize surface blebs of apoptotic vascular endothelial cells." J Immunol 166(5): 3231-9. Neng Lai, K., J. C. Leung, et al. (1996). "Anti-DNA autoantibodies stimulate the release of interleukin-1 and interleukin-6 from endothelial cells." J Pathol 178(4): 451-7. Nowell, P. C. (1976). "The clonal evolution of tumor cell populations." Science 194(4260): 23-8. Ochsenbein, A. F., T. Fehr, et al. (1999). "Control of early viral and bacterial distribution and disease by natural antibodies." Science 286(5447): 2156-9. Oda, K., H. Arakawa, et al. (2000). "p53AIP1, a potential mediator of p53dependent apoptosis, and its regulation by Ser-46-phosphorylated p53." Cell 102(6): 849-62. Ogden, C. A., A. deCathelineau, et al. (2001). "C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells." J Exp Med 194(6): 781-95. Ouellette, A. J. and M. E. Selsted (1996). "Paneth cell defensins: endogenous peptide components of intestinal host defense." Faseb J 10(11): 1280-9. Parlato, S., A. M. Giammarioli, et al. (2000). "CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway." Embo J 19(19): 5123-34. Paulovich, A. G., D. P. Toczyski, et al. (1997). "When checkpoints fail." Cell 88(3): 315-21. Peifer, M. (2002). "Developmental biology: colon construction." Nature 420(6913): 274-5, 277. Pelham, H. R. (1991). "Multiple targets for brefeldin A." Cell 67(3): 449-51. Peltomaki, P. (2001). "DNA mismatch repair and cancer." Mutat Res 488(1): 7785. Pereira, H. A., I. Erdem, et al. (1993). "Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes." Proc Natl Acad Sci U S A 90(10): 4733-7. 209 Pinto, D. and H. Clevers (2005). "Wnt control of stem cells and differentiation in the intestinal epithelium." Exp Cell Res 306(2): 357-63. Platt, N., H. Suzuki, et al. (1996). "Role for the class A macrophage scavenger receptor in the phagocytosis of apoptotic thymocytes in vitro." Proc Natl Acad Sci U S A 93(22): 12456-60. Podda, E., M. Benincasa, et al. (2006). "Dual mode of action of Bac7, a prolinerich antibacterial peptide." Biochim Biophys Acta 1760(11): 1732-40. Poon, M. A., M. J. O'Connell, et al. (1989). "Biochemical modulation of fluorouracil: evidence of significant improvement of survival and quality of life in patients with advanced colorectal carcinoma." J Clin Oncol 7(10): 1407-18. Potten, C. S. and M. Loeffler (1990). "Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt." Development 110(4): 1001-20. Radtke, F. and H. Clevers (2005). "Self-renewal and cancer of the gut: two sides of a coin." Science 307(5717): 1904-9. Radtke, F., H. Clevers, et al. (2006). "From gut homeostasis to cancer." Curr Mol Med 6(3): 275-89. Richman, S. and J. Adlard (2002). "Left and right sided large bowel cancer." Bmj 324(7343): 931-2. Rubin, R. L. (1999). "Etiology and mechanisms of drug-induced lupus." Curr Opin Rheumatol 11(5): 357-63. Rubinfeld, B., P. Robbins, et al. (1997). "Stabilization of beta-catenin by genetic defects in melanoma cell lines." Science 275(5307): 1790-2. Ruiz-Irastorza, G., M. A. Khamashta, et al. (2001). "Systemic lupus erythematosus." Lancet 357(9261): 1027-32. Saltz, L. B., J. V. Cox, et al. (2000). "Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group." N Engl J Med 343(13): 905-14. Saltz, L. B., J. V. Cox, et al. (2000). "Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group." N Engl J Med 343(13): 905-14. Satoh, S., Y. Daigo, et al. (2000). "AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1." Nat Genet 24(3): 245-50. Sattler, M., H. Liang, et al. (1997). "Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis." Science 275(5302): 983-6. 210 Schroder, J. M. and J. Harder (1999). "Human beta-defensin-2." Int J Biochem Cell Biol 31(6): 645-51. Scott, R. S., E. J. McMahon, et al. (2001). "Phagocytosis and clearance of apoptotic cells is mediated by MER." Nature 411(6834): 207-11. Seoane, J., H. V. Le, et al. (2002). "Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage." Nature 419(6908): 729-34. Shafer, W. M., M. E. Shepherd, et al. (1993). "Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity." Infect Immun 61(5): 1900-8. Shapiro, G. I., C. D. Edwards, et al. (2000). "The physiology of p16(INK4A)mediated G1 proliferative arrest." Cell Biochem Biophys 33(2): 189-97. Shaw, P. X., S. Horkko, et al. (2000). "Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity." J Clin Invest 105(12): 1731-40. Sherr, C. J. (1993). "Mammalian G1 cyclins." Cell 73(6): 1059-65. Sherr, C. J. and J. M. Roberts (1999). "CDK inhibitors: positive and negative regulators of G1-phase progression." Genes Dev 13(12): 1501-12. Shibata, M. A., K. Yoshidome, et al. (2001). "Suppression of mammary carcinoma growth in vitro and in vivo by inducible expression of the Cdk inhibitor p21." Cancer Gene Ther 8(1): 23-35. Shiloh, Y. (2001). "ATM and ATR: networking cellular responses to DNA damage." Curr Opin Genet Dev 11(1): 71-7. Sionov, R. V. and Y. Haupt (1999). "The cellular response to p53: the decision between life and death." Oncogene 18(45): 6145-57. Skeel, R. T. (2003). Handbook of cancer chemotherapy. Philadelphia, Lippincott Williams & Wilkins. Slee, E. A., C. Adrain, et al. (2001). "Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis." J Biol Chem 276(10): 7320-6. Smits, V. A. and R. H. Medema (2001). "Checking out the G(2)/M transition." Biochim Biophys Acta 1519(1-2): 1-12. Stankiewicz, A. R., G. Lachapelle, et al. (2005). "Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation." J Biol Chem 280(46): 38729-39. 211 Stewart, Z. A. and J. A. Pietenpol (2001). "p53 Signaling and cell cycle checkpoints." Chem Res Toxicol 14(3): 243-63. Stewart, Z. A., M. D. Westfall, et al. (2003). "Cell-cycle dysregulation and anticancer therapy." Trends Pharmacol Sci 24(3): 139-45. Sun, K. H., C. L. Yu, et al. (2000). "Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha from normal human mononuclear cells involving in the lupus pathogenesis." Immunology 99(3): 352-60. Suzuki, A., Y. Tsutomi, et al. (1999). "Mitochondrial regulation of cell death: mitochondria are essential for procaspase 3-p21 complex formation to resist Fasmediated cell death." Mol Cell Biol 19(5): 3842-7. Takizawa, F., S. Tsuji, et al. (1996). "Enhancement of macrophage phagocytosis upon iC3b deposition on apoptotic cells." FEBS Lett 397(2-3): 269-72. Taylor, P. R., A. Carugati, et al. (2000). "A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo." J Exp Med 192(3): 359-66. Tou, L., Q. Liu, et al. (2004). "Regulation of mammalian epithelial differentiation and intestine development by class I histone deacetylases." Mol Cell Biol 24(8): 3132-9. Troughton, W. D. and J. S. Trier (1969). "Paneth and goblet cell renewal in mouse duodenal crypts." J Cell Biol 41(1): 251-68. Tsao, Y. P., S. J. Huang, et al. (1999). "Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines." J Virol 73(6): 4983-90. van de Wetering, M., W. de Lau, et al. (2002). "WNT signaling and lymphocyte development." Cell 109 Suppl: S13-9. van de Wetering, M., E. Sancho, et al. (2002). "The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells." Cell 111(2): 241-50. Vazquez-Doval, J. and A. Sanchez-Ibarrola (1993). "Defective mononuclear phagocyte function in systemic lupus erythematosus: relationship of FcRII (CD32) with intermediate cytoskeletal filaments." J Investig Allergol Clin Immunol 3(2): 86-91. Vousden, K. H. and X. Lu (2002). "Live or let die: the cell's response to p53." Nat Rev Cancer 2(8): 594-604. 212 Wu, G. S. and Z. Ding (2002). "Caspase is required for p53-dependent apoptosis and chemosensitivity in a human ovarian cancer cell line." Oncogene 21(1): 1-8. Yang, D., O. Chertov, et al. (1999). "Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6." Science 286(5439): 525-8. Yu, J., L. Zhang, et al. (1999). "Identification and classification of p53-regulated genes." Proc Natl Acad Sci U S A 96(25): 14517-22. Zhang, J., N. Rajkumar, et al. (2000). "Characterization and expression of the mouse pregnant specific uterus protein gene and its rat homologue in the intestine and uterus." Biochim Biophys Acta 1492(2-3): 526-30. Zhang, J., H. Wong, et al. (2003). "The proline-rich acidic protein is epigenetically regulated and inhibits growth of cancer cell lines." Cancer Res 63(20): 6658-65. Zhou, B. B. and S. J. Elledge (2000). "The DNA damage response: putting checkpoints in perspective." Nature 408(6811): 433-9. Zhou, B. P., Y. Liao, et al. (2001). "Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells." Nat Cell Biol 3(3): 245-52. Zou, X. H., W. C. Foong, et al. (2004). "Chondroitin sulfate in palatal wound healing." J Dent Res 83(11): 880-5. Zurier, R. B. (1976). "Reduction of phagocytosis and lysosomal enzyme release from human leukocytes by serum from patients with systemic lupus erythematosus." Arthritis Rheum 19(1): 73-8. Zweibaum, A., M. Pinto, et al. (1985). "Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose." J Cell Physiol 122(1): 21-9. 213 APPENDIX I 214 General reagents dd water Distilled, deionized water was used in all experiments except otherwise stated. . Autoclaving All autoclaving was carried out at 15 lb/sq inch for 20 minutes. Apparatus used for RNA work were autoclaved for hour. 1X PBS (Phosphate buffered saline), pH 7.4 (per liter) To 800 ml of dd water, add: 8g 0.2 g 0.24 g 1.44 g NaCl KCl KH2PO4 Na2HPO4 Adjust pH to 7.0 with HCl and add dd water to liter. Tris-EDTA (TE) buffer, pH 8.3 (per liter) 10 mM 0.1 mM Tris-HCl EDTA Dissolve 1.2 g of Tris Base, 37 mg EDTA in 900 ml of dd water. Adjust pH to 8.3 with N HCl and top up to L with dd water. RNA quantification Dilute µL of RNA in 99 µL of dd water. Adjust the spectrophotometer to an absorbance wavelength of 260 nm. dd water was used as a blank. Calculate the concentration of RNA according to this formula: OD260 = 40 µg/ml. DNA quantification Dilute µl of DNA in 99 µl of distilled deionized water. Adjust the spectrophotometer to an absorbance wavelength of 260 nm. dd water was used as a blank. Calculate the concentration of DNA according to this formula: OD260 = µg/ml. 215 Reagents for DNA gel electrophoresis 1% Agarose gel Dissolve 0.5 g DNase-free agarose in 50 ml 0.5X TBE. Allow to cool and add µl of 10 mg/ml ethidium bromide. 10X Tris-Borate EDTA (TBE) buffer, pH 8.2 (per liter) 0.89 M 0.89 M 0.01 mM Tris base Boric acid EDTA Reagents used for transformation Luria Bertani (LB) agar medium (per liter) To 950 ml of water, add: 10 g 5g 10 g 20 g Bacto-trypton Bacto-yeast extract NaCl Agar Dissolve solutes. Adjust pH to 7.0 with N NaOH. Adjust volume to liter with dd water. Sterilize by autoclaving. Cool to 50 oC, then add antibiotics to desired concentration and pour onto petri dishes. Allow to solidify at room temperature and store plates at 4oC. Luria Bertani (LB) Broth (per liter) To 950 ml of water, add: 10 g 5g 10 g Bacto-trypton Bacto-yeast extract NaCl Dissolve solutes. Adjust pH to 7.0 with N NaOH. Adjust volume to l liter with dd water. Sterilize by autoclaving. Store at 4oC SOC medium (per liter) To 950 ml of water, add: 20 g 5g 0.5 g Bacto-trypton Bacto-yeast extract NaCl Dissolve solutes. Add 10 ml of 250 mM KCl. Adjust pH to 7.0 with N NaOH. Adjust volume to 980 ml with dd water. Sterilize by autoclaving. Allow to cool to 60oC or less, then add 20 ml of sterile M glucose. Add ml of sterile M MgCl2 just prior to use. Store at 4oC. 216 Reagents for Western blots RIPA extraction buffer (1 ml) 0.5 % 1% 0.1 % mM mg/ml 10 mg/ml 10 mg/ml Sodium deoxycholate NP-40 SDS Pefabloc Pepstatin A Leupeptin Aprotinin Top up with 1X PBS to ml (protease inhibitors added just prior to use). 12 % SDS-PAGE stacking gel (10 ml) ml 2.5 ml 3.3 ml 100 µl 100 µl µl 30% Bis/Acrylamide 1.5 M Tris-HCl, pH 8.8 dd water 10 % SDS 10 % APS Temed % SDS-PAGE stacking gel (5 ml) 0.53 ml 0.49 ml 2.86 ml 40 µl 40 µl µl 30% Bis/Acrylamide 0.5 M Tris-HCl, pH 6.8 dd water 10 % SDS 10 % APS Temed 5X SDS / Glycine Buffer (per liter) 15.1 g 72 g 5g Tris base Glycine SDS 2X sample loading buffer (per 10 ml) 2.5 ml 3.3 ml ml 0.31 g 100 µl 0.5 M Tris-HCl (pH 6.8) 60 % glycerol 10 % SDS DTT powder Bromophenol Blue (1mg/ml) 217 Towbin transfer buffer 25 mM 192 mM 20 % (v/v) Tris Glycine Absolute ethanol Reagents for Immunohistochemistry of paraffin-embedded sections Dewaxing 1. Xylene, x 2. Absolute EtOH, 10 dips x 3. 95% EtOH, 10 dips 4. 70% EtOH, 10 dips 5. dd water Antigen retrieval 1. Heat approximately 750 ml Antigen Unmasking Solution in a L beaker containing a glass slide rack, in the microwave oven to at least 95°C. 2. Immerse slides in preheated Antigen Unmasking Solution and slow cook for 10 in the microwave oven. 3. Allow slides to cool in solution for 20 min. 218 APPENDIX II 219 I. Publications: 1. Jian-Jun Liu, Baohua Huang, Shing Chuan Hooi. Acetyl-keto-bboswellic acid inhibits cellular proliferation through a p21-dependent pathway in colon cancer cells. Br J Pharmacol. 2006 Aug. 148(8):1099107. 2. Liu JJ, Huang BH, Zhang J, Carson DD, Hooi SC. Repression of HIP/RPL29 expression induces differentiation in colon cancer cells. J Cell Physiol. J Cell Physiol. 2006 May. 207(2):287-92. 3. Huang BH, Laban M, Leung CH, Lee L, Lee CK, Salto-Tellez M, Raju GC, Hooi SC. Inhibition of histone deacetylase increases apoptosis and p21Cip1/WAF1 expression, independent of histone deacetylase 1. Cell Death Differ. 2005 Apr. 12(4):395-404. 4. Huang BH, Hooi SC. PRAP1, a modulator of cell fate in response to genotoxic stress. (in preparation) II. Communications published in abstract form: 1. Bao Hua Huang, Shing Chuan Hooi. The novel protein Proline Rich Acidic Protein is regulated by p53 and has anti-apoptotic functions. 97th American Association for Cancer Research Annual Meeting. Washington, DC. 1-5 April 2006. 2. BH Huang, HS Howe, Ko Kong, BP Leung, SC Hooi. Detection of a novel protein, proline rich acidic protein, in patients with systemic lupus Erythematosus. Annals of the Academy of Medicine. 2005 Oct. Vol. 34 (Suppl) No. 9: S230. 3. Bao H. Huang, Mirtha L., Manuel S.T., Raju G.C., Shing C. Hooi. Role of HDAC2 in apoptosis via p21. Hong Kong-Taiwan Joint Physiology Symposium. Hong Kong. 8-9 Dec 2003. 4. Bao H. Huang, Anil Kumar, Hong M. Ni, Shing C. Hooi. Identification of genes regulated in colon cancer metastasis. International Journal of Molecular Medicine. 2003 Vol. 12 (Suppl): 286. [...]... Failure to induce PRAP1 expression may be one of the factors that accounts for the impaired clearance of apoptotic cells in SLE 17 2 CHAPTER FOUR DISCUSSION 17 3 4 .1 Role of PRAP1 in differentiated epithelial cells 4 .1. 1 Regulation of PRAP1 by differentiation 4 .1. 1 .1 Expression of PRAP1 in intestinal epithelium The proline- rich acidic protein (PRAP1) gene was first identified by Kasik et al In the article,... Ts/U/P 200 10 0 50 10 1 Ts Ts/U Ts/U/P Ts/U/P Ts/U/P Ts/U/P Ts/U/P 200 10 0 50 10 1 Sample Name IL-6 production 16 00 14 00 IL-6 conc (pg) 12 00 10 00 800 600 400 200 0 M U U/P 200 U/P 10 0 U/P 50 U/P 10 U/P 1 Tc Tc/U Tc/U/P Tc/U/P Tc/U/P Tc/U/P Tc/U/P 200 10 0 50 10 1 Sample Name IL-1B production 18 00 16 00 IL-1B conc (pg) 14 00 12 00 10 00 800 600 400 200 Ts Ts /U Ts /U /P 20 Ts 0 /U /P 10 Ts 0 /U /P 10 0 Ts /U... HL-60 and THP -1) and T-cell (Jurkat) lines were treated with camptothecin (CPT), etoposide (Eto) and 5-Fluorouracil (5-FU) for 48 hours to induce cell death 5-FU was the most potent in inducing PRAP1 expression in U937, THP -1 and Jurkat (Figure 3.90) We failed to detect any significant induction of PRAP1 by 5-FU in HL-60 cells PRAP1 expression was also induced by CPT and Eto in all the four cell lines... PRAP1 cDNA encoded a putative protein of 15 1 amino acids and shared 50% homology to that of rat and mouse The NH2 terminus was highly conserved among human and rodent, and was predicted to be a signal peptide In addition, the COOH terminus comprising amino acids from position 13 5 to 14 9 was 80% conserved between human and rodent The predicted cleavage site was between amino acid 20 and 21 PRAP1 was... secreted protein (Zhang, Wong et al 2003) The human PRAP1 gene was found to be expressed in the epithelial cells of gastrointestinal tract, kidney and liver 17 4 The PRAP1 gene was commonly expressed in the epithelial cells of the gastrointestinal tract of both human and rodent, suggesting a conserved function of PRAP1 in the intestine during development Interestingly, the expression of PRAP1 gene was... expression in the proximal gastrointestinal tract These suggest that PRAP1 may be involved in the differentiation of epithelial cells during development and/ or in the constitutive cell renewal program that maintains the gastrointestinal epithelium In this study, the expression of PRAP1 in the intestinal epithelium was further elucidated Our results showed that PRAP1 protein was expressed abundantly in the... The PRAP1 protein is neither homologous nor similar to other antibacterial peptides identified In the human gastrointestinal tract, antimicrobial peptides such as α-defensins (HD-5 in small intestine) and β-defensin (hBD-3 in stomach and colon) are the major players These defensins are small (15 -20 residues for α-defensin and 38-47 residues for β-defensin), cysteine -rich cationic peptides In contrast,... PRAP1 protein in the serum of normal individuals and SLE patients was assayed by ELISA HisPRAP1 was used as a standard to calculate the amount of PRAP1 protein present in the serum B: Summary of the ELISA data showing two populations of SLE patients expressing lower or higher PRAP1 than the normal individual Column, mean; Bar, SE * p . Figure 3.82 PRAP1 protein was detected in the serum of both normal and SLE patients A: Scatter plot showing the distribution of PRAP1 protein level in serum. Level of PRAP1 protein in the serum. IL-1B production 0 200 400 600 800 10 00 12 00 14 00 16 00 18 00 M U U/P 200 U/P 10 0 U/P 50 U/P 10 U/P 1 Tc Tc/U Tc/U/P 200 Tc/U/P 10 0 Tc/U/P 50 Tc/U/P 10 Tc/U/P 1 Ts Ts/U Ts/U/P 200 Ts/U/P 10 0 Ts/U/P. patients. 3 .13 .3 PRAP1 and proinflammatory cytokines As PRAP1 autoantigens and autoantibodies were detected in SLE patients, we investigated whether PRAP1 plays a role in immune cells interaction.