Doctoral Dissertations

Date of Award

5-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Hildegard M. Schuller

Abstract

Identification of the full spectrum of gene promoters methylated in cancer, or the cancer methylome, would greatly advance our understanding of gene regulatory networks involved in tumorigenesis. A robust approach was developed that couples genome-wide probabilistic search algorithms with an established pharmacologic unmasking strategy for unbiased and precise global localization of tumor-specific methylated genes. Through this approach, a set of 175 novel candidate genes was identified that cluster throughout the genome and may harbor cancer-specific promoter methylation. In this study, over sixty genes were tested in bladder and prostate cancer by candidate gene approach to evaluate new promising tumor suppressor genes in genitourinary cancer. An initial screen of the methylation status of the promoter region was conducted by bisulfite genomic sequencing in a panel of over sixty genes in seven bladder cancer and four prostate cancer cell lines.Based on the bisulfite sequencing data, we selected nine genes (camk4, fkbp4, hoxB5, krt14, LPAR2, mal, rgs4, vgf and ZMYM2) where aberrant methylation was detected in both bladder and prostate cancer cell lines. The expression of eight genes in carcinoma cell lines was analyzed by semiquantitative reverse transcription-PCR (RT-PCR) after 5-aza-2'-deoxycytidine treatment, alone or in combination with Trichostatin A. Methylation was detected by bisulfite sequencing in these genes at the following frequencies in bladder cancer cell lines: camk4 (16.6%), fkbp4 (71.4%), hoxB5 (42.9%), krt14 (86%), LPAR2 (28.6%), mal (71.4%), rgs4 (33.3%), vgf (57.1%), and ZMYM2 (57.1%). Methylation frequencies in prostate cancer cell lines were: camk4 (50%), fkbp4 (50%), hoxB5 (25%), krt14 (75%), LPAR2 (50%), mal (75%), rgs4 (50%), vgf (75%), and ZMYM2 (75%).RT-PCR analysis revealed re-expression of camk4, hoxB5, LPAR2, mal, rgs4, vgf, and ZMYM2 after 5-aza-2'-deoxycytidine treatment at least in one bladder cancer cell line and camk4, hoxB5, LPAR2, mal, rgs4, vgf, and ZMYM2 at least in one prostate cancer cell line. Previously unreported epigenetically altered genes in genitourinary cancer cell lines were identified. While further analysis is warranted in primary tissues, these results identify novel candidate tumor suppressor genes in bladder and prostate cancer.

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