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Examining the negative impacts of bioflavonoids on the DNA damage response and DNA repair mechanisms in mouse embryonic stem cells

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Abstract

Bioflavonoids are dietary agents found in everyday food items such as fruits, vegetables, and legumes. It has been suggested that bioflavonoids be used with chemotherapeutics as an anti-cancer agent due to their anti-inflammatory capabilities and their ability to inhibit the DNA damage response. However most drugs that inhibit the DNA damage response can have secondary effects on normal cells. For instance, chemotherapeutics, such as etoposide, can cause DNA translocations leading to malignant transformations of normal cells to cancer cells. Studies have shown that etoposides and other chemotherapeutics increase the risk of leukemia. Therefore, it is our hypothesis that bioflavonoids inhibit the DNA damage response in normal cells, leading to improper repair of DNA damage, such as chromosomal translocation that can lead to leukemia. To study this we will use mouse embryonic stem cells, to model both acute high-dose exposure and chronic low-dose exposure to bioflavonoids such as genistein, quercetin, luteolin, myricetin etc. After we expose cells to these bioflavonoids, we will be examining them for DNA damage using Immunohistochemistry (IHC) to identify phosphorylation histone variant 2AX, a well-studied marker of DNA damage, with confocal microscopy. We will also be using a translocation reporter construct, which causes cells to produce green fluorescent protein when a translocation has occurred. We have integrated this construct into a mouse embryonic stem cell line, to measure incidences of DNA translocation by flow cytometry. Finally, we will be collecting DNA from these cells for PCR analysis and DNA sequencing to verify any DNA translocations that have occured.


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Examining the negative impacts of bioflavonoids on the DNA damage response and DNA repair mechanisms in mouse embryonic stem cells



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