CALGB Grant uri icon

abstract

  • Cytosine methylation is an epigenetic process typically involved in normal cellular regulatory processes. However, unregulated control can lead to hypermethylation that effectively acts to silence gene transcription.' From a cancer etiology standpoint, hypermethylation acts to gradually turn off regulatory genes that code for repair enzymes and other cellular signaling elements. In this fashion, hypermethylation is understood to be a key initial step in cellular carcinogenesis.' Determining genetic cytosine methylation status, therefore, represent a promising biomarker detection strategy to identify and diagnose cancer at very early stages of the disease. One such area where a methylation detection strategy holds promise in the diagnosis and treatment of estrogen receptor (ER-?) negative (-) breast cancers. An overall poorer prognosis from this type of breast cancer is based on treatment complexities associated with the lack of endocrine therapy receptors. ER-? (-) cancers are thought to not express ERs due in part to hypermethylation of the estrogen receptor gene ESR1 promoter region Therefore, early detection and treatment of hypermethylation in the ESR1 promoter region may play an important role in improving the diagnosis of ER-? negative breast cancers. Furthermore, hypermethylation is reversible in theory, and early detection could lead to enhanced and directed therapy designed to rescue the cell from a cancerous fate. In order to detect genetic DNA methylation status, a sample is typically treated with bisulfite converting unmethylated cytosines to uracil. The original methylation status is determined by counting the number of remaining cytosines in a PCR amplicon of interest. These methods produce the desired information, but can suffer from bisuffite induced false positives, sample pre-handling complexities, and throughput limits based on well-plate density. To circumvent these limitations, we propose to develop a rapid and inexpensive quantitative electrochemical (echem) assay designed to determine the cytosine methylation status in key ESR1 promoter region segments. Electrochemical methods offer several advantages primarily including instrumentation and material cost, sample amounts, sample workup time, label-free detection, and throughput advantages7 that have yet to be realized for DNA methylation detection. Furthermore, the proposed sensor will offer improved sequence level resolution by providing methylation status information on short 10-25 bp segments of the ESR1 gene. Overall, we are proposing the groundwork for the eventual development of a high throughput methylation status detection device that can report simultaneously on several key cancer related genetic segments. Aim #1: Establish optimal e-chem methylation sensor conditions employing model DNA systems. The e-chem sensor will consist of an immobilized 10-25 bp probe of known sequence that can bind its target complement. Sequence acquisition detection will take place employing a redox active diviologen (CI2Viologen, vide infra) via dual wave voltammetric analysis. Initially, model oligonucleotides 5'-mCpG-3' (m = potential methylation site) and specific ESR1 promoter sequences will be employed. Captured target methylation level will be electrochemically ascertained by assaying the B to Z-form helix transition kinetics upon exposure to moderately increased ionic strength. Unmethylated samples will not undergo this transition, offering the means to sensitively and selectively detect methylation status on short ESR1 segments. Aim #2: Detect ESR1 promoter methylation levels in MCF-7, MDA-MB-231, and MCF-10A breast cancer cell lines. ESR1 promoter methylation status will optimized in plasmid samples and eventually be assayed in breast cancer cell lines exhibiting ER-a positive (MCF-7), negative (MDA-MB-231), or normal epithelial tissue (MCF-10A) character

date/time interval

  • January 2008 - August 2023