A 2019/2020 Summer Studentship research project
Identification of tumours under increased levels of oxidative stress could lead to therapeutic strategies that exacerbate this stress to kill cancer cells.
Student: Christopher Kaldor
Supervisors: Dr Martina Paumann-Page, Professor Mark Hampton
Sponsor: Cancer Society Malvern Group
Introduction
It is commonly stated that cancer cells are under more oxidative stress than normal cells, but this theory has proven difficult to substantiate. We have developed a method to sense subtle levels of oxidative stress in cultured cells and tissue. The method relies on monitoring a family of proteins called peroxiredoxins that convert hydrogen peroxide to water. During the peroxiredoxin catalytic cycle oxidised dimers form that can be visualized by western blotting. In an initial screen of cells within a US National Cancer Institute panel we observed that many of the cancer cells kept their peroxiredoxins reduced. However, metastatic melanoma cells had increased levels of oxidised peroxiredoxin 2.
Aim
The aim of this project is to validate the initial screen using cell lines from a large New Zealand melanoma panel. The genetics and phenotype of these cell lines have been extensively characterised. They have also been cultured under physiological oxygen concentrations since their original isolation, rather than the higher levels of normal cell culture. The amount of oxidised peroxiredoxin 2 will be measured in the melanoma cells, along with the levels of hydrogen peroxide they produce. Activity of the enzyme peroxidasin, which is overexpressed in invasive melanomas and also uses hydrogen peroxide as a substrate, will be measured and compared with peroxiredoxin 2 oxidation.
Method
The student will culture melanoma cells in the laboratory, alongside a selection of normal human melanocytes. Reduced and oxidised peroxiredoxins will be separated by protein electrophoresis and visualized by western blotting. Enzyme assays will be used to measure hydrogen peroxide levels and peroxidasin activity.
Student researcher’s component of the study
The student will be responsible for all of the laboratory work undertaken for this project.
Student Prerequisites
Student studying cell biology or biochemistry
