Functional role of p16INK4A and n myc downstream regulated gene 1 (NDRG1) up regulation in cervical carcinoma

FUNCTIONAL ROLE OF p16INK4A AND N-MYC DOWNSTREAM REGULATED GENE (NDRG1) UP-REGULATION IN CERVICAL CARCINOMA LAU WEN MIN NATIONAL UNIVERSITY OF SINGAPORE 2007 FUNCTIONAL ROLE OF p16INK4A AND N-MYC DOWNSTREAM REGULATED GENE (NDRG1) UP-REGULATION IN CERVICAL CARCINOMA LAU WEN MIN (BBiotech(Hons), Flinders University of South Australia, Australia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements I would like to thank my supervisors, Prof Kam Hui and A/Prof Kanaga Sabapathy for their constant guidance and support I would also like to express my sincere gratitude to Dr Ganesan Gopalan and Dr Michelle Tan for invaluable advice and helpful discussions on many aspects of my project and thesis A special “thank you” to friends and colleagues in NCC and DCR, SGH for their encouragement, and for providing comic relief in the face of seemingly insurmountable experimental woes I would also like to thank the Department of Clinical Research, SGH, for generous technical help and assistance Finally, I would like to thank my parents and family for their constant support and encouragement, and without whom I would most certainly have sustained Permanent Head Damage Lau Wen Min January 2007 i Table of Contents Acknowledgements i Table of Contents ii List of Tables iii List of Figures iii List of Abbreviations vi Summary viii SECTION Chapter Introduction and Literature Review Carcinoma of the cervix SECTION Experimental Procedures Chapter Materials and Methods SECTION Chapter Chapter Chapter 36 Results and Discussion Identification of differentially expressed genes in cervical cancer by microarray analysis of subtracted cDNA libraries 63 p16INK4A silencing augments DNA damage-induced apoptosis in cervical cancer cells 75 N-myc downstream regulated gene (NDRG1) up-regulation contributes to evasion of senescence-like phenotype in cervical carcinoma 98 SECTION References References 125 ii SECTION Appendix I Appendix Publications 141 List of Tables Table 1-1 Staging of cervical cancer according to FIGO 10 Table 1-2 Classification of HPV types by cervical oncogenicity 14 Table 1-3 Induction of NDRG1 expression in various cell types with different treatments 29 Table 2-1 Cervical tissue specimens collected and corresponding stage of Disease 38 Table 2-2 Recipe for SDS-PAGE gels 60 Table 3-1 Twenty-six differentially expressed genes in cervical cancer compared to non-tumourous and normal controls 70 Table 4-1 Affymetrix Genechip analysis of expression changes in UV-induced apoptosis related genes in cells treated with p16 siRNA compared to control siRNA 91 Table 5-1 Affymetrix Genechip analysis of gene expression changes in growthrelated genes in NDRG1-silenced cervical cancer cells 109 List of Figures Figure 1-1 Anatomy of the uterine cervix Figure 1-2 Development of the cervical transformation zone Figure 1-3 Diagram of extent of spread in FIGO staging of cervical cancer 11 Figure 1-4 The human papillomavirus life cycle 15 Figure 1-5 Dual effect of HPV E6 and E7 on the cell cycle 21 Figure 1-6 Alternative transcripts from the p16 gene locus 23 Figure 2-1 Clontech PCR-Select cDNA subtractive hybridization 47 Figure 3-1 Representative microarray image 65 iii Figure 3-2 Hierarchical clustering of microarray gene expression data 67 Figure 4-1 Significant up-regulation of p16 gene expression in cervical cancer 77 Figure 4-2 p16 gene expression in human cancers 78 Figure 4-3 Endogenous p16 mRNA and protein expression level in Cas Ki and SiHa cervical cancer cell lines 79 Figure 4-4 siRNA-mediated silencing of p16 in SiHa cells 80 Figure 4-5 p16 siRNA specifically inhibits expression of p16 but not p14ARF, encoded by the same gene locus as p16 81 Figure 4-6 Silencing of p16 modulates expression of Rb, p53, E6 and E7 in cervical cancer cell lines 83 Figure 4-7 Silencing of p16 has no effect on cell cycle progression in SiHa cells 84 Figure 4-8 Silencing of p16 augments UV- and cisplatin-induced apoptosis in SiHa cervical cancer cells 85 Figure 4-9 TUNEL assay after UV-irradiation of p16-silenced SiHa cells 87 Figure 4-10 Silencing of p16 in SiHa cells enhances p53 phosphorylation under UV- and cisplatin treatment 88 Figure 5-1 Significant up-regulation of NDRG1 gene expression in cervical cancer 100 Figure 5-2 NDRG1 endogenous expression in SiHa and Cas Ki cell lines 101 Figure 5-3 Optimization of siRNA-mediated silencing of NDRG1 protein expression in cervical cancer cells 102 Figure 5-4 NDRG1 siRNA specifically inhibits expression of NDRG1 103 Figure 5-5 Time course of NDRG1 siRNA effects on NDRG1 protein levels 103 Figure 5-6 NDRG1 silencing results in decreased cell proliferation rate in cervical cancer cells and leads to growth arrest 104 Figure 5-7 NDRG1 siRNA-induced growth arrest is related to inhibition of cell proliferation 105 Figure 5-8 Inhibition of cell growth induced by NDRG1 silencing in cervical cancer cells is related to G1 cell cycle arrest 107 Figure 5-9 NDRG1 silencing is associated with senescence-like phenotype in cervical cancer cells 110 Figure 5-10 NDRG1 siRNA-induced senescence-like growth arrest can be restored by up-regulation of endogenous NDRG1 upon cobalt chloride treatment 111 iv Figure 5-11 Senescence-like phenotype mediated by NDRG1 silencing is associated with up-regulation of p53 and p21 and can be attenuated by p53 silencing 113 Figure 5-12 Senescence at day post-transfection induced by NDRG1 silencing is not reversible by subsequent p53 inhibition or release of NDRG1 suppression 114 Figure 5-13 Ectopic over-expression of NDRG1 in SiHa cells transfected with pcDNA3-NDRG1-FLAG or pcDNA3 for 24 hours 116 Figure 5-14 Over-expression of ectopic NDRG1 in SiHa stable cell lines results in increased cell proliferation rate 117 v List of Abbreviations α AIS ANOVA APS β BrdU BSA alpha adenocarcinoma in situ one way analysis of variance ammonium persulphate beta bromodeoxyuridine bovine serum albumin CaCl2 Cdk CDKN2A / p16 CIN Cy3 Cy5 DAPI DMEM DNA / RNA dNTPs EST FIGO g/L HGSIL HIF-1a HIV HPV HSV hTERT J/m2 kb L LB LGSIL LOH LEEP M MES μγ mg calcium chloride cyclin dependent kinase cyclin dependent kinase inhibitor 2A cervical intraepithelial neoplasia cyanine cyanine 4',6-Diamidino-2-phenylindole Dulbecco's modified Eagle's medium Deoxyribonucleic / ribonucleic acid deoxyribonucleotide triphosphates expressed sequence tags International Federation of Obstetrics and Gynaecology grams per litre high grade squamous intraepithelial lesion hypoxia inducible factor 1a human immunodeficiency virus human papillomavirus herpes simplex virus human telomerase reverse transcriptase joules per square metre kilobase litre Luria Bertani / lysogeny broth low grade squamous intraepithelial lesion loss of heterozygosity loop electrosurgical excision procedure molar 2-(N-Morpholino)ethanesulfonic acid, sodium salt microgram milligram MgCl2 magnesium chloride MgSO4 μλ ml μΜ mM MOPS NaCl NaOH NDRG1 ng nM nm magnesium sulphate microlitre millilitre micromolar millimolar 3-(N-morpholino)propanesulfonic acid sodium chloride sodium hydroxide N-myc downstream regulated gene nanogram nanomolar nanometre vi NP-40 ORF Pap smear PBS PCR PI Rb / RB RNAi rpm rRNA SA-β-gal SAPE SDS SDS-PAGE siRNA SSC SSPE TBE TBS TE TUNEL UV VLPs VHL X-gal Nonidet P40 open reading frame Papanicolaou smear phosphate buffered saline polymerase chain reaction propidium iodide retinoblastoma protein/gene RNA interference revolutions per minute ribosomal RNA senescence-associated beta-galactosidase streptavidin-phycoerthythin sodium dodecyl sulphate SDS-polyacrylamide gel electrophoresis short interfering RNA sodium chloride - sodium citrate solution salt sodium phosphate EDTA tris borate EDTA tris buffered saline tris EDTA terminal deoxynucleotide transferase dUTP nick end labeling ultraviolet virus-like particles von-Hippel-Lindau 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside vii Summary Cancer of the cervix is the second most common cancer for women worldwide, with a higher prevalence in developing countries In Singapore, cervical cancer is the fifth most common cancer in females The objective of this study is to employ gene expression profiling of cervical cancer to identify novel differentially regulated genes which may serve as molecular diagnostic markers in cervical cancer, and to characterize their role in cervical carcinogenesis We constructed two reciprocal (forward and reverse) subtracted cDNA libraries from tumourous and non-tumourous cervical tissue taken from a single patient, and 1920 clones obtained from these libraries were used to generate cDNA microarrays which were then employed in the study of patient samples A total of 30 tumour samples, 20 non-tumourous tissues of the same patient and 12 normal cervical tissues from non-cancerous patients were employed in our gene expression studies Amongst the differentially expressed genes, we focused on the study of p16INK4A (p16) and N-myc downstream regulated gene (NDRG1) as these two genes showed the most significant up-regulation in cervical cancer tissues compared to non-cancerous and normal cervical tissues This current work focuses on elucidating the functional roles of p16 and NDRG1 in cervical cancer and our findings suggest that p16 and NDRG1 are able to mediate apoptosis and cell cycle arrest respectively via p53-associated pathways Although p16 has been reported to be up-regulated in cervical cancer, its functional role in cervical carcinogenesis is not well characterized p16 is a bona fide tumour suppressor gene involved in cell cycle regulation, and it is frequently inactivated in other human cancers Interestingly, over-expression of p16 in cervical cancer is seemingly functionally redundant, thus we explored the possible role of p16 upregulation in cervical carcinogenesis We observed that siRNA-mediated silencing of p16 augments DNA damage-induced of apoptosis, and furthermore our results viii Section – References 31 Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L., and Crawford, L Comparison of the in vitro transforming activities of human papillomavirus types Embo J, 7: 18151820, 1988 32 Ward, P., Coleman, D V., and Malcolm, A D Regulatory mechanisms of the papillomaviruses Trends Genet, 5: 97-99, 1989 33 Hudelist, G., Manavi, M., Pischinger, K I., Watkins-Riedel, T., Singer, C F., Kubista, E., and Czerwenka, K F Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade Gynecol Oncol, 92: 873-880, 2004 34 Howley, P M Role of the human papillomaviruses in human cancer Cancer Res, 51: 5019s-5022s, 1991 35 Cripe, T P., Haugen, T H., Turk, J P., Tabatabai, F., Schmid, P G., 3rd, Durst, M., Gissmann, L., Roman, A., and Turek, L P Transcriptional regulation of the human papillomavirus-16 E6-E7 promoter by a keratinocyte-dependent enhancer, and by viral E2 trans-activator and repressor gene products: implications for cervical carcinogenesis Embo J, 6: 3745-3753, 1987 36 Romanczuk, H., Thierry, F., and Howley, P M Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters J Virol, 64: 2849-2859, 1990 37 Thierry, F and Howley, P M Functional analysis of E2-mediated repression of the HPV18 P105 promoter New Biol, 3: 90-100, 1991 38 von Knebel Doeberitz, M New markers for cervical dysplasia to visualise the genomic chaos created by aberrant oncogenic papillomavirus infections Eur J Cancer, 38: 2229-2242, 2002 39 Doorbar, J The papillomavirus life cycle J Clin Virol, 32 Suppl 1: S7-15, 2005 40 Southern, S A and Herrington, C S Molecular events in uterine cervical cancer Sex Transm Infect, 74: 101-109, 1998 41 Ho, G Y., Burk, R D., Klein, S., Kadish, A S., Chang, C J., Palan, P., Basu, J., Tachezy, R., Lewis, R., and Romney, S Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia J Natl Cancer Inst, 87: 1365-1371, 1995 42 Cannistra, S A and Niloff, J M Cancer of the uterine cervix N Engl J Med, 334: 1030-1038, 1996 43 Ault, K A Epidemiology and natural history of human papillomavirus infections in the female genital tract Infect Dis Obstet Gynecol, 14: 40470, 2006 44 Girardi, F., Fuchs, P., and Haas, J Prognostic importance of human papillomavirus type 16 DNA in cervical cancer Cancer, 69: 2502-2504, 1992 45 Burger, R A., Monk, B J., Kurosaki, T., Anton-Culver, H., Vasilev, S A., Berman, M L., and Wilczynski, S P Human papillomavirus type 18: association with poor prognosis in early stage cervical cancer J Natl Cancer Inst, 88: 1361-1368, 1996 127 Section – References 46 Vernon, S D., Unger, E R., Miller, D L., Lee, D R., and Reeves, W C Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer Int J Cancer, 74: 50-56, 1997 47 Kobayashi, Y., Yoshinouchi, M., Tianqi, G., Nakamura, K., Hongo, A., Kamimura, S., Mizutani, Y., Kodama, J., Miyagi, Y., and Kudo, T Presence of human papilloma virus DNA in pelvic lymph nodes can predict unexpected recurrence of cervical cancer in patients with histologically negative lymph nodes Clin Cancer Res, 4: 979983, 1998 48 Herzog, T J New approaches for the management of cervical cancer Gynecol Oncol, 90: S22-27, 2003 49 Kesic, V Management of cervical cancer Eur J Surg Oncol, 32: 832-837, 2006 50 Saxena, A., Yashar, C., Taylor, D D., and Gercel-Taylor, C Cellular response to chemotherapy and radiation in cervical cancer Am J Obstet Gynecol, 192: 13991403, 2005 51 Hockel, M., Schlenger, K., Aral, B., Mitze, M., Schaffer, U., and Vaupel, P Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix Cancer Res, 56: 4509-4515, 1996 52 Sundfor, K., Lyng, H., and Rofstad, E K Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix Br J Cancer, 78: 822-827, 1998 53 Parkin, D M Global cancer statistics in the year 2000 Lancet Oncol, 2: 533-543, 2001 54 Schiller, J T and Lowy, D R Prospects for cervical cancer prevention by human papillomavirus vaccination Cancer Res, 66: 10229-10232, 2006 55 Scheffner, M., Werness, B A., Huibregtse, J M., Levine, A J., and Howley, P M The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 Cell, 63: 1129-1136, 1990 56 Werness, B A., Levine, A J., and Howley, P M Association of human papillomavirus types 16 and 18 E6 proteins with p53 Science, 248: 76-79, 1990 57 Huibregtse, J M., Scheffner, M., and Howley, P M Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53 Mol Cell Biol, 13: 775-784, 1993 58 Hubbert, N L., Sedman, S A., and Schiller, J T Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes J Virol, 66: 6237-6241, 1992 59 Lechner, M S and Laimins, L A Inhibition of p53 DNA binding by human papillomavirus E6 proteins J Virol, 68: 4262-4273, 1994 60 Zimmermann, H., Degenkolbe, R., Bernard, H U., and O'Connor, M J The human papillomavirus type 16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional coactivator CBP/p300 J Virol, 73: 6209-6219, 1999 128 Section – References 61 Thompson, D A., Belinsky, G., Chang, T H., Jones, D L., Schlegel, R., and Munger, K The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints Oncogene, 15: 3025-3035, 1997 62 Kessis, T D., Slebos, R J., Nelson, W G., Kastan, M B., Plunkett, B S., Han, S M., Lorincz, A T., Hedrick, L., and Cho, K R Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage Proc Natl Acad Sci U S A, 90: 3988-3992, 1993 63 Foster, S A., Demers, G W., Etscheid, B G., and Galloway, D A The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest J Virol, 68: 5698-5705, 1994 64 Veldman, T., Horikawa, I., Barrett, J C., and Schlegel, R Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein J Virol, 75: 4467-4472, 2001 65 Shai, A., Brake, T., Somoza, C., and Lambert, P F The human papillomavirus E6 oncogene dysregulates the cell cycle and contributes to cervical carcinogenesis through two independent activities Cancer Res, 67: 1626-1635, 2007 66 Zanier, K., Charbonnier, S., Baltzinger, M., Nomine, Y., Altschuh, D., and Trave, G Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance J Mol Biol, 349: 401-412, 2005 67 Du, J., Chen, G G., Vlantis, A C., Chan, P K., Tsang, R K., and van Hasselt, C A Resistance to apoptosis of HPV 16-infected laryngeal cancer cells is associated with decreased Bak and increased Bcl-2 expression Cancer Lett, 205: 81-88, 2004 68 Dyson, N., Howley, P M., Munger, K., and Harlow, E The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product Science, 243: 934937, 1989 69 Jones, D L and Munger, K Interactions of the human papillomavirus E7 protein with cell cycle regulators Semin Cancer Biol, 7: 327-337, 1996 70 Boyer, S N., Wazer, D E., and Band, V E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway Cancer Res, 56: 4620-4624, 1996 71 Nevins, J R E2F: a link between the Rb tumor suppressor protein and viral oncoproteins Science, 258: 424-429, 1992 72 Munger, K and Phelps, W C The human papillomavirus E7 protein as a transforming and transactivating factor Biochim Biophys Acta, 1155: 111-123, 1993 73 Weinberg, R A The retinoblastoma protein and cell cycle control Cell, 81: 323-330, 1995 74 Vousden, K H., Vojtesek, B., Fisher, C., and Lane, D HPV-16 E7 or adenovirus E1A can overcome the growth arrest of cells immortalized with a temperature-sensitive p53 Oncogene, 8: 1697-1702, 1993 129 Section – References 75 Demers, G W., Foster, S A., Halbert, C L., and Galloway, D A Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7 Proc Natl Acad Sci U S A, 91: 4382-4386, 1994 76 Slebos, R J., Lee, M H., Plunkett, B S., Kessis, T D., Williams, B O., Jacks, T., Hedrick, L., Kastan, M B., and Cho, K R p53-dependent G1 arrest involves pRBrelated proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein Proc Natl Acad Sci U S A, 91: 5320-5324, 1994 77 Hickman, E S., Picksley, S M., and Vousden, K H Cells expressing HPV16 E7 continue cell cycle progression following DNA damage induced p53 activation Oncogene, 9: 2177-2181, 1994 78 Lazo, P A The molecular genetics of cervical carcinoma Br J Cancer, 80: 20082018, 1999 79 Skyldberg, B., Fujioka, K., Hellstrom, A C., Sylven, L., Moberger, B., and Auer, G Human papillomavirus infection, centrosome aberration, and genetic stability in cervical lesions Mod Pathol, 14: 279-284, 2001 80 Hopman, A H., Smedts, F., Dignef, W., Ummelen, M., Sonke, G., Mravunac, M., Vooijs, G P., Speel, E J., and Ramaekers, F C Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalities J Pathol, 202: 23-33, 2004 81 Melsheimer, P., Vinokurova, S., Wentzensen, N., Bastert, G., and von Knebel Doeberitz, M DNA aneuploidy and integration of human papillomavirus type 16 e6/e7 oncogenes in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix uteri Clin Cancer Res, 10: 3059-3063, 2004 82 White, A E., Livanos, E M., and Tlsty, T D Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins Genes Dev, 8: 666-677, 1994 83 Duensing, S., Lee, L Y., Duensing, A., Basile, J., Piboonniyom, S., Gonzalez, S., Crum, C P., and Munger, K The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle Proc Natl Acad Sci U S A, 97: 10002-10007, 2000 84 Pett, M R., Alazawi, W O., Roberts, I., Dowen, S., Smith, D I., Stanley, M A., and Coleman, N Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes Cancer Res, 64: 1359-1368, 2004 85 Atkin, N B and Baker, M C Chromosome in 26 carcinomas of the cervix uteri: structural and numerical changes Cancer, 44: 604-613, 1979 86 Sreekantaiah, C., Bhargava, M K., and Shetty, N J Chromosome abnormalities in cervical carcinoma Cancer, 62: 1317-1324, 1988 87 Park, T W., Fujiwara, H., and Wright, T C Molecular biology of cervical cancer and its precursors Cancer, 76: 1902-1913, 1995 88 Mitra, A B., Murty, V V., Singh, V., Li, R G., Pratap, M., Sodhani, P., Luthra, U K., and Chaganti, R S Genetic alterations at 5p15: a potential marker for progression of precancerous lesions of the uterine cervix J Natl Cancer Inst, 87: 742-745, 1995 130 Section – References 89 Ocadiz, R., Sauceda, R., Cruz, M., Graef, A M., and Gariglio, P High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix Cancer Res, 47: 4173-4177, 1987 90 Riou, G., Barrois, M., Sheng, Z M., Duvillard, P., and Lhomme, C Somatic deletions and mutations of c-Ha-ras gene in human cervical cancers Oncogene, 3: 329-333, 1988 91 Serrano, M., Hannon, G J., and Beach, D A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 Nature, 366: 704-707, 1993 92 Ruas, M and Peters, G The p16INK4a/CDKN2A tumor suppressor and its relatives Biochim Biophys Acta, 1378: F115-177, 1998 93 Jen, J., Harper, J W., Bigner, S H., Bigner, D D., Papadopoulos, N., Markowitz, S., Willson, J K., Kinzler, K W., and Vogelstein, B Deletion of p16 and p15 genes in brain tumors Cancer Res, 54: 6353-6358, 1994 94 Quelle, D E., Ashmun, R A., Hannon, G J., Rehberger, P A., Trono, D., Richter, K H., Walker, C., Beach, D., Sherr, C J., and Serrano, M Cloning and characterization of murine p16INK4a and p15INK4b genes Oncogene, 11: 635-645, 1995 95 Stone, S., Jiang, P., Dayananth, P., Tavtigian, S V., Katcher, H., Parry, D., Peters, G., and Kamb, A Complex structure and regulation of the P16 (MTS1) locus Cancer Res, 55: 2988-2994, 1995 96 Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S., and Peters, G Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence Mol Cell Biol, 16: 859-867, 1996 97 Medema, R H., Herrera, R E., Lam, F., and Weinberg, R A Growth suppression by p16ink4 requires functional retinoblastoma protein Proc Natl Acad Sci U S A, 92: 6289-6293, 1995 98 Okamoto, A., Demetrick, D J., Spillare, E A., Hagiwara, K., Hussain, S P., Bennett, W P., Forrester, K., Gerwin, B., Serrano, M., Beach, D H., and et al Mutations and altered expression of p16INK4 in human cancer Proc Natl Acad Sci U S A, 91: 11045-11049, 1994 99 Sharpless, N E., Bardeesy, N., Lee, K H., Carrasco, D., Castrillon, D H., Aguirre, A J., Wu, E A., Horner, J W., and DePinho, R A Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis Nature, 413: 86-91, 2001 100 Krimpenfort, P., Quon, K C., Mooi, W J., Loonstra, A., and Berns, A Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice Nature, 413: 83-86, 2001 101 Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D., and DePinho, R A Role of the INK4a locus in tumor suppression and cell mortality Cell, 85: 27-37, 1996 102 Campisi, J Cellular senescence as a tumor-suppressor mechanism Trends Cell Biol, 11: S27-31, 2001 103 Brookes, S., Rowe, J., Gutierrez Del Arroyo, A., Bond, J., and Peters, G Contribution of p16(INK4a) to replicative senescence of human fibroblasts Exp Cell Res, 298: 549-559, 2004 131 Section – References 104 Stone, S., Dayananth, P., and Kamb, A Reversible, p16-mediated cell cycle arrest as protection from chemotherapy Cancer Res, 56: 3199-3202, 1996 105 Brookes, S., Rowe, J., Ruas, M., Llanos, S., Clark, P A., Lomax, M., James, M C., Vatcheva, R., Bates, S., Vousden, K H., Parry, D., Gruis, N., Smit, N., Bergman, W., and Peters, G INK4a-deficient human diploid fibroblasts are resistant to RASinduced senescence Embo J, 21: 2936-2945, 2002 106 Maurelli, R., Zambruno, G., Guerra, L., Abbruzzese, C., Dimri, G., Gellini, M., Bondanza, S., and Dellambra, E Inactivation of p16INK4a (inhibitor of cyclindependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment Faseb J, 20: 1516-1518, 2006 107 Nobori, T., Miura, K., Wu, D J., Lois, A., Takabayashi, K., and Carson, D A Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers Nature, 368: 753-756, 1994 108 Parry, D., Bates, S., Mann, D J., and Peters, G Lack of cyclin D-Cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product Embo J, 14: 503-511, 1995 109 Shapiro, G I., Edwards, C D., Kobzik, L., Godleski, J., Richards, W., Sugarbaker, D J., and Rollins, B J Reciprocal Rb inactivation and p16INK4 expression in primary lung cancers and cell lines Cancer Res, 55: 505-509, 1995 110 Li, Y., Nichols, M A., Shay, J W., and Xiong, Y Transcriptional repression of the Dtype cyclin-dependent kinase inhibitor p16 by the retinoblastoma susceptibility gene product pRb Cancer Res, 54: 6078-6082, 1994 111 Tam, S W., Shay, J W., and Pagano, M Differential expression and cell cycle regulation of the cyclin-dependent kinase inhibitor p16Ink4 Cancer Res, 54: 58165820, 1994 112 Lukas, J., Parry, D., Aagaard, L., Mann, D J., Bartkova, J., Strauss, M., Peters, G., and Bartek, J Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16 Nature, 375: 503-506, 1995 113 Khleif, S N., DeGregori, J., Yee, C L., Otterson, G A., Kaye, F J., Nevins, J R., and Howley, P M Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2Fmediated induction of cyclin kinase inhibitor activity Proc Natl Acad Sci U S A, 93: 4350-4354, 1996 114 Sherr, C J The INK4a/ARF network in tumour suppression Nat Rev Mol Cell Biol, 2: 731-737, 2001 115 Volgareva, G., Zavalishina, L., Andreeva, Y., Frank, G., Krutikova, E., Golovina, D., Bliev, A., Spitkovsky, D., Ermilova, V., and Kisseljov, F Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells BMC Cancer, 4: 58, 2004 116 Negri, G., Vittadello, F., Romano, F., Kasal, A., Rivasi, F., Girlando, S., Mian, C., and Egarter-Vigl, E p16INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri Virchows Arch, 445: 616-620, 2004 117 Ishikawa, M., Fujii, T., Saito, M., Nindl, I., Ono, A., Kubushiro, K., Tsukazaki, K., Mukai, M., and Nozawa, S Overexpression of p16 INK4a as an indicator for human 132 Section – References papillomavirus oncogenic activity in cervical squamous neoplasia Int J Gynecol Cancer, 16: 347-353, 2006 118 Queiroz, C., Silva, T C., Alves, V A., Villa, L L., Costa, M C., Travassos, A G., Filho, J B., Studart, E., Cheto, T., and de Freitas, L A P16(INK4a) expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions Pathol Res Pract, 202: 77-83, 2006 119 Murphy, N., Ring, M., Heffron, C C., King, B., Killalea, A G., Hughes, C., Martin, C M., McGuinness, E., Sheils, O., and O'Leary, J J p16INK4A, CDC6, and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer J Clin Pathol, 58: 525-534, 2005 120 Murphy, N., Heffron, C C., King, B., Ganuguapati, U G., Ring, M., McGuinness, E., Sheils, O., and O'Leary, J J p16INK4A positivity in benign, premalignant and malignant cervical glandular lesions: a potential diagnostic problem Virchows Arch, 445: 610-615, 2004 121 van Belzen, N., Dinjens, W N., Diesveld, M P., Groen, N A., van der Made, A C., Nozawa, Y., Vlietstra, R., Trapman, J., and Bosman, F T A novel gene which is upregulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms Lab Invest, 77: 85-92, 1997 122 Zhou, D., Salnikow, K., and Costa, M Cap43, a novel gene specifically induced by Ni2+ compounds Cancer Res, 58: 2182-2189, 1998 123 Kurdistani, S K., Arizti, P., Reimer, C L., Sugrue, M M., Aaronson, S A., and Lee, S W Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage Cancer Res, 58: 4439-4444, 1998 124 Kokame, K., Kato, H., and Miyata, T Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis GRP78/BiP and novel genes J Biol Chem, 271: 29659-29665, 1996 125 Park, H., Adams, M A., Lachat, P., Bosman, F., Pang, S C., and Graham, C H Hypoxia induces the expression of a 43-kDa protein (PROXY-1) in normal and malignant cells Biochem Biophys Res Commun, 276: 321-328, 2000 126 Li, J and Kretzner, L The growth-inhibitory Ndrg1 gene is a Myc negative target in human neuroblastomas and other cell types with overexpressed N- or c-myc Mol Cell Biochem, 250: 91-105, 2003 127 Qu, X., Zhai, Y., Wei, H., Zhang, C., Xing, G., Yu, Y., and He, F Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family Mol Cell Biochem, 229: 35-44, 2002 128 Zhou, R H., Kokame, K., Tsukamoto, Y., Yutani, C., Kato, H., and Miyata, T Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart Genomics, 73: 86-97, 2001 129 Lachat, P., Shaw, P., Gebhard, S., van Belzen, N., Chaubert, P., and Bosman, F T Expression of NDRG1, a differentiation-related gene, in human tissues Histochem Cell Biol, 118: 399-408, 2002 130 Agarwala, K L., Kokame, K., Kato, H., and Miyata, T Phosphorylation of RTP, an ER stress-responsive cytoplasmic protein Biochem Biophys Res Commun, 272: 641-647, 2000 133 Section – References 131 Murray, J T., Campbell, D G., Morrice, N., Auld, G C., Shpiro, N., Marquez, R., Peggie, M., Bain, J., Bloomberg, G B., Grahammer, F., Lang, F., Wulff, P., Kuhl, D., and Cohen, P Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3 Biochem J, 384: 477-488, 2004 132 Wakisaka, Y., Furuta, A., Masuda, K., Morikawa, W., Kuwano, M., and Iwaki, T Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development J Histochem Cytochem, 51: 1515-1525, 2003 133 Okuda, T., Higashi, Y., Kokame, K., Tanaka, C., Kondoh, H., and Miyata, T Ndrg1deficient mice exhibit a progressive demyelinating disorder of peripheral nerves Mol Cell Biol, 24: 3949-3956, 2004 134 Salnikow, K., Kluz, T., and Costa, M Role of Ca(2+) in the regulation of nickelinducible Cap43 gene expression Toxicol Appl Pharmacol, 160: 127-132, 1999 135 Ulrix, W., Swinnen, J V., Heyns, W., and Verhoeven, G The differentiation-related gene 1, Drg1, is markedly upregulated by androgens in LNCaP prostatic adenocarcinoma cells FEBS Lett, 455: 23-26, 1999 136 Cangul, H Hypoxia upregulates the expression of the NDRG1 gene leading to its overexpression in various human cancers BMC Genet, 5: 27, 2004 137 Salnikow, K., Blagosklonny, M V., Ryan, H., Johnson, R., and Costa, M Carcinogenic nickel induces genes involved with hypoxic stress Cancer Res, 60: 3841, 2000 138 Shimono, A., Okuda, T., and Kondoh, H N-myc-dependent repression of ndr1, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant Mech Dev, 83: 39-52, 1999 139 Guan, R J., Ford, H L., Fu, Y., Li, Y., Shaw, L M., and Pardee, A B Drg-1 as a differentiation-related, putative metastatic suppressor gene in human colon cancer Cancer Res, 60: 749-755, 2000 140 Le, N T and Richardson, D R Iron chelators with high antiproliferative activity upregulate the expression of a growth inhibitory and metastasis suppressor gene: a link between iron metabolism and proliferation Blood, 104: 2967-2975, 2004 141 Piquemal, D., Joulia, D., Balaguer, P., Basset, A., Marti, J., and Commes, T Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells Biochim Biophys Acta, 1450: 364-373, 1999 142 Gomez-Casero, E., Navarro, M., Rodriguez-Puebla, M L., Larcher, F., Paramio, J M., Conti, C J., and Jorcano, J L Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis Mol Carcinog, 32: 100-109, 2001 143 Harris, A L Hypoxia a key regulatory factor in tumour growth Nat Rev Cancer, 2: 38-47, 2002 144 Caro, J Hypoxia regulation of gene transcription High Alt Med Biol, 2: 145-154, 2001 145 Greijer, A E., van der Groep, P., Kemming, D., Shvarts, A., Semenza, G L., Meijer, G A., van de Wiel, M A., Belien, J A., van Diest, P J., and van der Wall, E Upregulation of gene expression by hypoxia is mediated predominantly by hypoxiainducible factor (HIF-1) J Pathol, 206: 291-304, 2005 134 Section – References 146 Chen, B., Nelson, D M., and Sadovsky, Y N-myc down-regulated gene modulates the response of term human trophoblasts to hypoxic injury J Biol Chem, 281: 27642772, 2006 147 Bandyopadhyay, S., Pai, S K., Gross, S C., Hirota, S., Hosobe, S., Miura, K., Saito, K., Commes, T., Hayashi, S., Watabe, M., and Watabe, K The Drg-1 gene suppresses tumor metastasis in prostate cancer Cancer Res, 63: 1731-1736, 2003 148 Bandyopadhyay, S., Pai, S K., Hirota, S., Hosobe, S., Takano, Y., Saito, K., Piquemal, D., Commes, T., Watabe, M., Gross, S C., Wang, Y., Ran, S., and Watabe, K Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression Oncogene, 23: 5675-5681, 2004 149 Maruyama, Y., Ono, M., Kawahara, A., Yokoyama, T., Basaki, Y., Kage, M., Aoyagi, S., Kinoshita, H., and Kuwano, M Tumor growth suppression in pancreatic cancer by a putative metastasis suppressor gene Cap43/NDRG1/Drg-1 through modulation of angiogenesis Cancer Res, 66: 6233-6242, 2006 150 Masuda, K., Ono, M., Okamoto, M., Morikawa, W., Otsubo, M., Migita, T., Tsuneyoshi, M., Okuda, H., Shuin, T., Naito, S., and Kuwano, M Downregulation of Cap43 gene by von Hippel-Lindau tumor suppressor protein in human renal cancer cells Int J Cancer, 105: 803-810, 2003 151 Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J M., Lane, W S., and Kaelin, W G., Jr HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing Science, 292: 464-468, 2001 152 Cangul, H., Salnikow, K., Yee, H., Zagzag, D., Commes, T., and Costa, M Enhanced expression of a novel protein in human cancer cells: a potential aid to cancer diagnosis Cell Biol Toxicol, 18: 87-96, 2002 153 Kovacevic, Z and Richardson, D R The metastasis suppressor, Ndrg-1: a new ally in the fight against cancer Carcinogenesis, 2006 154 Bandyopadhyay, S., Pai, S K., Hirota, S., Hosobe, S., Tsukada, T., Miura, K., Takano, Y., Saito, K., Commes, T., Piquemal, D., Watabe, M., Gross, S., Wang, Y., Huggenvik, J., and Watabe, K PTEN up-regulates the tumor metastasis suppressor gene Drg-1 in prostate and breast cancer Cancer Res, 64: 7655-7660, 2004 155 Stein, S., Thomas, E K., Herzog, B., Westfall, M D., Rocheleau, J V., Jackson, R S., 2nd, Wang, M., and Liang, P NDRG1 is necessary for p53-dependent apoptosis J Biol Chem, 279: 48930-48940, 2004 156 Sano, T., Oyama, T., Kashiwabara, K., Fukuda, T., and Nakajima, T Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions Am J Pathol, 153: 1741-1748, 1998 157 Wong, Y F., Cheung, T H., Tsao, G S., Lo, K W., Yim, S F., Wang, V W., Heung, M M., Chan, S C., Chan, L K., Ho, T W., Wong, K W., Li, C., Guo, Y., Chung, T K., and Smith, D I Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray Int J Cancer, 118: 2461-2469, 2006 158 Hayashi, N., Yokoyama, N., Seki, T., Azuma, Y., Ohba, T., and Nishimoto, T RanBP1, a Ras-like nuclear G protein binding to Ran/TC4, inhibits RCC1 via Ran/TC4 Mol Gen Genet, 247: 661-669, 1995 135 Section – References 159 de Silva, H V., Harmony, J A., Stuart, W D., Gil, C M., and Robbins, J Apolipoprotein J: structure and tissue distribution Biochemistry, 29: 5380-5389, 1990 160 Shannan, B., Seifert, M., Leskov, K., Willis, J., Boothman, D., Tilgen, W., and Reichrath, J Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer Cell Death Differ, 13: 12-19, 2006 161 Park, D C., Geun Yeo, S., Young Shin, E., Mok, S C., and Hoon Kim, D Clusterin confers paclitaxel resistance in cervical cancer Gynecol Oncol, 103: 996-1000, 2006 162 Kamura, T., Koepp, D M., Conrad, M N., Skowyra, D., Moreland, R J., Iliopoulos, O., Lane, W S., Kaelin, W G., Jr., Elledge, S J., Conaway, R C., Harper, J W., and Conaway, J W Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase Science, 284: 657-661, 1999 163 Jackson, P K and Eldridge, A G The SCF ubiquitin ligase: an extended look Mol Cell, 9: 923-925, 2002 164 Liu, C., Rangnekar, V M., Adamson, E., and Mercola, D Suppression of growth and transformation and induction of apoptosis by EGR-1 Cancer Gene Ther, 5: 3-28, 1998 165 Baron, V., Adamson, E D., Calogero, A., Ragona, G., and Mercola, D The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin Cancer Gene Ther, 13: 115-124, 2006 166 Virolle, T., Krones-Herzig, A., Baron, V., De Gregorio, G., Adamson, E D., and Mercola, D Egr1 promotes growth and survival of prostate cancer cells Identification of novel Egr1 target genes J Biol Chem, 278: 11802-11810, 2003 167 Adamson, E D and Mercola, D Egr1 transcription factor: multiple roles in prostate tumor cell growth and survival Tumour Biol, 23: 93-102, 2002 168 Karstensen, B., Poppelreuther, S., Bonin, M., Walter, M., Iftner, T., and Stubenrauch, F Gene expression profiles reveal an upregulation of E2F and downregulation of interferon targets by HPV18 but no changes between keratinocytes with integrated or episomal viral genomes Virology, 353: 200-209, 2006 169 Shieh, S Y., Ikeda, M., Taya, Y., and Prives, C DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2 Cell, 91: 325-334, 1997 170 Klaes, R., Friedrich, T., Spitkovsky, D., Ridder, R., Rudy, W., Petry, U., DallenbachHellweg, G., Schmidt, D., and von Knebel Doeberitz, M Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri Int J Cancer, 92: 276-284, 2001 171 Muller, M., Wilder, S., Bannasch, D., Israeli, D., Lehlbach, K., Li-Weber, M., Friedman, S L., Galle, P R., Stremmel, W., Oren, M., and Krammer, P H p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs J Exp Med, 188: 2033-2045, 1998 172 Mihara, M., Erster, S., Zaika, A., Petrenko, O., Chittenden, T., Pancoska, P., and Moll, U M p53 has a direct apoptogenic role at the mitochondria Mol Cell, 11: 577-590, 2003 136 Section – References 173 Jimenez, G S., Khan, S H., Stommel, J M., and Wahl, G M p53 regulation by posttranslational modification and nuclear retention in response to diverse stresses Oncogene, 18: 7656-7665, 1999 174 Bode, A M and Dong, Z Post-translational modification of p53 in tumorigenesis Nat Rev Cancer, 4: 793-805, 2004 175 Oda, K., Arakawa, H., Tanaka, T., Matsuda, K., Tanikawa, C., Mori, T., Nishimori, H., Tamai, K., Tokino, T., Nakamura, Y., and Taya, Y p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53 Cell, 102: 849-862, 2000 176 Al-Mohanna, M A., Manogaran, P S., Al-Mukhalafi, Z., K, A A.-H., and Aboussekhra, A The tumor suppressor p16(INK4a) gene is a regulator of apoptosis induced by ultraviolet light and cisplatin Oncogene, 23: 201-212, 2004 177 Freedman, D A and Levine, A J Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6 Mol Cell Biol, 18: 72887293, 1998 178 Lee, Y S., Wan, J., Kim, B J., Bae, M A., and Song, B J Ubiquitin-dependent degradation of p53 protein despite phosphorylation at its N terminus by acetaminophen J Pharmacol Exp Ther, 317: 202-208, 2006 179 Zhang, J., Pickering, C R., Holst, C R., Gauthier, M L., and Tlsty, T D p16INK4a modulates p53 in primary human mammary epithelial cells Cancer Res, 66: 1032510331, 2006 180 el-Deiry, W S., Harper, J W., O'Connor, P M., Velculescu, V E., Canman, C E., Jackman, J., Pietenpol, J A., Burrell, M., Hill, D E., Wang, Y., and et al WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis Cancer Res, 54: 1169-1174, 1994 181 Haapajarvi, T., Kivinen, L., Heiskanen, A., des Bordes, C., Datto, M B., Wang, X F., and Laiho, M UV radiation is a transcriptional inducer of p21(Cip1/Waf1) cyclinkinase inhibitor in a p53-independent manner Exp Cell Res, 248: 272-279, 1999 182 Hawley-Nelson, P., Vousden, K H., Hubbert, N L., Lowy, D R., and Schiller, J T HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes Embo J, 8: 3905-3910, 1989 183 Jones, D L and Munger, K Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein J Virol, 71: 2905-2912, 1997 184 Putral, L N., Bywater, M J., Gu, W., Saunders, N A., Gabrielli, B G., Leggatt, G R., and McMillan, N A RNA Interference against Human Papillomavirus Oncogenes in Cervical Cancer Cells Results in Increased Sensitivity to Cisplatin Mol Pharmacol, 68: 1311-1319, 2005 185 Scheffner, M., Munger, K., Byrne, J C., and Howley, P M The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines Proc Natl Acad Sci U S A, 88: 5523-5527, 1991 186 Smotkin, D and Wettstein, F O Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein Proc Natl Acad Sci U S A, 83: 4680-4684, 1986 137 Section – References 187 Goodwin, E C and DiMaio, D Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways Proc Natl Acad Sci U S A, 97: 12513-12518, 2000 188 Butz, K., Shahabeddin, L., Geisen, C., Spitkovsky, D., Ullmann, A., and Hoppe-Seyler, F Functional p53 protein in human papillomavirus-positive cancer cells Oncogene, 10: 927-936, 1995 189 Jiang, M and Milner, J Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference Oncogene, 21: 6041-6048, 2002 190 Gu, W., Putral, L., Hengst, K., Minto, K., Saunders, N A., Leggatt, G., and McMillan, N A Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes Cancer Gene Ther, 13: 1023-1032, 2006 191 Goodwin, E C., Yang, E., Lee, C J., Lee, H W., DiMaio, D., and Hwang, E S Rapid induction of senescence in human cervical carcinoma cells Proc Natl Acad Sci U S A, 97: 10978-10983, 2000 192 Roninson, I B Tumor cell senescence in cancer treatment Cancer Res, 63: 27052715, 2003 193 Sharpless, N E and DePinho, R A Telomeres, stem cells, senescence, and cancer J Clin Invest, 113: 160-168, 2004 194 Collado, M and Serrano, M The power and the promise of oncogene-induced senescence markers Nat Rev Cancer, 6: 472-476, 2006 195 Hwang, E S Replicative senescence and senescence-like state induced in cancerderived cells Mech Ageing Dev, 123: 1681-1694, 2002 196 Dimri, G P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E E., Linskens, M., Rubelj, I., Pereira-Smith, O., and et al A biomarker that identifies senescent human cells in culture and in aging skin in vivo Proc Natl Acad Sci U S A, 92: 9363-9367, 1995 197 Hockel, M., Schlenger, K., Hockel, S., and Vaupel, P Hypoxic cervical cancers with low apoptotic index are highly aggressive Cancer Res, 59: 4525-4528, 1999 198 Vaupel, P., Kelleher, D K., and Hockel, M Oxygen status of malignant tumors: pathogenesis of hypoxia and significance for tumor therapy Semin Oncol, 28: 29-35, 2001 199 Brizel, D M., Rosner, G L., Prosnitz, L R., and Dewhirst, M W Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases Int J Radiat Oncol Biol Phys, 32: 1121-1125, 1995 200 Vaupel, P., Kallinowski, F., and Okunieff, P Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review Cancer Res, 49: 64496465, 1989 201 Coleman, C N Hypoxia in tumors: a paradigm for the approach to biochemical and physiologic heterogeneity J Natl Cancer Inst, 80: 310-317, 1988 138 Section – References 202 Bachtiary, B., Schindl, M., Potter, R., Dreier, B., Knocke, T H., Hainfellner, J A., Horvat, R., and Birner, P Overexpression of hypoxia-inducible factor 1alpha indicates diminished response to radiotherapy and unfavorable prognosis in patients receiving radical radiotherapy for cervical cancer Clin Cancer Res, 9: 2234-2240, 2003 203 Haugland, H K., Vukovic, V., Pintilie, M., Fyles, A W., Milosevic, M., Hill, R P., and Hedley, D W Expression of hypoxia-inducible factor-1alpha in cervical carcinomas: correlation with tumor oxygenation Int J Radiat Oncol Biol Phys, 53: 854-861, 2002 204 Birner, P., Schindl, M., Obermair, A., Plank, C., Breitenecker, G., and Oberhuber, G Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer Cancer Res, 60: 4693-4696, 2000 205 Burri, P., Djonov, V., Aebersold, D M., Lindel, K., Studer, U., Altermatt, H J., Mazzucchelli, L., Greiner, R H., and Gruber, G Significant correlation of hypoxiainducible factor-1alpha with treatment outcome in cervical cancer treated with radical radiotherapy Int J Radiat Oncol Biol Phys, 56: 494-501, 2003 206 Fujimoto, J., Alam, S M., Jahan, I., Sato, E., Toyoki, H., Hong, B L., Sakaguchi, H., and Tamaya, T Plausible linkage of hypoxia inducible factor-1alpha in uterine cervical cancer Cancer Sci, 97: 861-867, 2006 207 Hayflick, L and Moorhead, P S The serial cultivation of human diploid cell strains Exp Cell Res, 25: 585-621, 1961 208 Greider, C W Telomeres and senescence: the history, the experiment, the future Curr Biol, 8: R178-181, 1998 209 von Zglinicki, T., Saretzki, G., Ladhoff, J., d'Adda di Fagagna, F., and Jackson, S P Human cell senescence as a DNA damage response Mech Ageing Dev, 126: 111117, 2005 210 Mathon, N F and Lloyd, A C Cell senescence and cancer Nat Rev Cancer, 1: 203213, 2001 211 Serrano, M., Lin, A W., McCurrach, M E., Beach, D., and Lowe, S W Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a Cell, 88: 593-602, 1997 212 Dotto, G P p21(WAF1/Cip1): more than a break to the cell cycle? Biochim Biophys Acta, 1471: M43-56, 2000 213 Alcorta, D A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., and Barrett, J C Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts Proc Natl Acad Sci U S A, 93: 13742-13747, 1996 214 Sugrue, M M., Shin, D Y., Lee, S W., and Aaronson, S A Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53 Proc Natl Acad Sci U S A, 94: 9648-9653, 1997 215 Reddel, R R The role of senescence and immortalization in carcinogenesis Carcinogenesis, 21: 477-484, 2000 139 Section – References 216 Wang, X., Wong, S C., Pan, J., Tsao, S W., Fung, K H., Kwong, D L., Sham, J S., and Nicholls, J M Evidence of cisplatin-induced senescent-like growth arrest in nasopharyngeal carcinoma cells Cancer Res, 58: 5019-5022, 1998 217 Chang, B D., Xuan, Y., Broude, E V., Zhu, H., Schott, B., Fang, J., and Roninson, I B Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs Oncogene, 18: 4808-4818, 1999 218 te Poele, R H., Okorokov, A L., Jardine, L., Cummings, J., and Joel, S P DNA damage is able to induce senescence in tumor cells in vitro and in vivo Cancer Res, 62: 1876-1883, 2002 219 Han, Z., Wei, W., Dunaway, S., Darnowski, J W., Calabresi, P., Sedivy, J., Hendrickson, E A., Balan, K V., Pantazis, P., and Wyche, J H Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin J Biol Chem, 277: 17154-17160, 2002 220 Yu, J., Zhang, L., Hwang, P M., Rago, C., Kinzler, K W., and Vogelstein, B Identification and classification of p53-regulated genes Proc Natl Acad Sci U S A, 96: 14517-14522, 1999 140 Section – Appendix Appendix I – Publications Publications from thesis work Lau, W.M., Ho, T.H and Hui, K.M p16INK4A silencing augments DNA damage-induced apoptosis in cervical cancer cells Oncogene advance online publication, 19 March 2007; doi:10.1038/sj.onc.1210405 Lau, W.M., Ho, T.H and Hui, K.M N-myc downstream regulated gene (NDRG1) upregulation contributes to evasion of senescence-like phenotype in cervical carcinoma Submitted for publication (January 2007) Other publications Cheng Q., Lau, W.M., Tay, S.K., Chew, S.H., Ho, T.H and Hui, K.M Identification and characterization of genes involved in the carcinogenesis of human squamous cell cervical carcinoma Int J Cancer, 98: 419-426, 2002 Cheng Q., Lau, W.M., Chew, S.H., Ho, T.H., Tay, S.K and Hui, K.M Identification of molecular markers for the early detection of human squamous cell carcinoma of the uterine cervix Br J Cancer, 86: 274-281, 2002 Kuss, B.J., Corbo, M., Lau, W.M., Fennell, D.A., Dean, N.M and Cotter, F.E In vitro and in vivo downregulation of MRP1 by antisense oligonucleotides: A potential role in neuroblastoma therapy Int J Cancer, 98: 128-133, 2002 141 ... Section – Introduction and Literature Review 1. 5 A newly identified up -regulated gene in cervical cancer: N- myc downstream regulated gene (NDRG1) 1. 5 .1 The human NDRG1 gene The human NDRG1 gene. .. 1. 5 A newly identified up -regulated gene in cervical cancer: N- myc downstream regulated gene (NDRG1) 28 1. 5 .1 The human NDRG1 gene 28 1. 5.2 Induction of NDRG1 by various agents 29 Section – Introduction.. .FUNCTIONAL ROLE OF p16INK4A AND N- MYC DOWNSTREAM REGULATED GENE (NDRG1) UP- REGULATION IN CERVICAL CARCINOMA LAU WEN MIN (BBiotech(Hons), Flinders University of South Australia,