Gene expression of DNA repair proteins in colorectal cancer and medulloblastoma

Odufuwa, K

Abstract

DNA repair plays a critical role in maintaining the integrity of the genome and the dysregulation of key DNA repair genes has been implicated in the development, progression and chemotherapeutic resistance of different cancer types. Consequently, many studies have made attempts to identify and quantify the expression of various DNA repair genes and their products in different cancers. It is on a similar note that this research was conceived, to evaluate the expression patterns of the base excision repair genes Neil1, Neil2, Neil3, Ogg1, and Nthl1, the nucleotide excision repair gene Ercc1 and the mismatch repair gene Mlh1 in colorectal cancer (CRC) tumours and matched normal colon tissue. The project was then extended to analyse a further sixteen colon samples that focused on Neil3, Nthl1 and Ercc1, that encode DNA repair proteins that have been implicated in chemotherapy resitance mechanisms. To learn more about mechanisms of genotoxic agent resistance, Neil3 and Ercc1 were analysed at the transcriptome and proteome level in DAOY medulloblastoma cells and cisplatin – resistant DAOY cells. Additionally, attempts were made to generate mesothelioma-derived cancer stem cells and preliminary gene expression analyses were undertaken on human embryonic stem cells. Thus, RNA was extracted, complementary DNA synthesized, and RT-PCR performed. Gene expression levels were determined by quantitative PCR using the Sybr green method and analysed using the comparative Ct method and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as the standard. Of the matched samples investigated, 75% showed increased expression of one or more of the DNA repair genes analysed, however, there was no clear pattern of expression and a wide range of expression levels observed for individual genes in both normal and tumour tissues. For example, the gene encoding the DNA glycosylase Nthl1 was the most frequently highly expressed in both normal and tumour samples with about 75% showing high expression of the Nthl1 gene with expression levels ranging from 3.3 to over 1400-fold higher than Gapdh. DAOY cells were grown in cisplatin and gene expression of Neil3 and Ercc1 analysed in the resulting cisplatin - resistant cells. Results indicated that the expression of both these genes may be increased in the resistant cell line and that NEIL3 protein was also increased. Cancer stem cells were derived from parental mesothelioma cells but were still too small a fraction of the cell population to be analysed by these methods. The expression of Gapdh, Neil3 and Ercc1 was determined in a series of preliminary experiments on human embryonic stem cells.

Citation

Odufuwa, K. Gene expression of DNA repair proteins in colorectal cancer and medulloblastoma. (Thesis). University of Salford

• Thesis Type: Thesis
• Deposit Date: Nov 12, 2019
• Publicly Available Date: Nov 12, 2019
• Additional Information: Funders : Olabisi Onabanjo University, Ogun State, Nigeria; Projects : Tetfund Scholarship