Introduction. Recent lines of evidence suggest that retinoids1,2 and RARbeta23 may have both beneficial and harmful effects in cancer. Little is known regarding the specifics of RARbeta2 promoter silencing in cancer, or about the role that RARbeta2 plays in RARbeta2-expressing cancer cells. Objectives. To analyze the patterns and heritability of RARbeta2 promoter methylation in cancer and determine whether it is subject to allelic bias; and to analyze the effects of RARbeta2 knockdown on growth and mRNA expression profiles in cancer. Methods. RARbeta2 promoter methylation was analyzed in 20 parental cancer cell lines and 5 subcloned lines using bisulfate genomic sequencing (>150 sequencings); the proportion of methylated alleles was estimated using methylation-sensitive restriction enzyme digestion followed by PCR and single nucleotide polymorphism identification; 18 antisense oligonucleotides against RARbeta2 were tested in various cancer cell lines using RT-PCR, cell counting and annexin V staining; and gene expression profiles were compared following knockdown versus all trans retinoic acid (ATRA) stimulation using cDNA microarray technology (>14,000 genes), SOURCE and GOMINER databases. Results. Hypo- and hypermethylated alleles frequently co-exist in lines in which RARbeta2 is inactivated (5111, 45%); divergent methylation is heritable in the majority of subclones analyzed (618, 75%); and methylation is subject to allelic bias at a ratio of ~2:1 (3l3 CpG sites, 100%). Cellular proliferation is correlated with RARbeta2 expression levels (p=0.0003); the most effective oligo reduces cellular proliferation by up to 80% in cancer cell lines in which RARbeta2 expression has been retained (313, 100%), but has no apparent effects in lines in which it has been lost (313, 100%); reduction in cell growth following oligo treatment is at least partially due to activation of programmed cell death; and over a dozen pro-carcinogenesis genes are downr / 1Omenn GS, Goodman GE, Thornquist MO, Balmes J, Cullen MR, Glass A, et al. Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial. J Natl Cancer Inst 1996;88:15509. 2Anon., The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. N Engl J Med 1994;330:1029-35. 3Khuri FR, Wu H, Lee JJ, Kemp BL, Lotan R, Lippman SM, et al. Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Olin Cancer Res 2001;7:861-7
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.85948 |
| Date | January 2005 |
| Creators | Pappas, Jane Joanna |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002260927, proquestno: AAINR21685, Theses scanned by UMI/ProQuest. |
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