An analysis of cellular bioenergetics in K-ras and p53 mutant colon cancer cells using single cell imaging.pdf (6.9 MB)
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An analysis of cellular bioenergetics in K-ras and p53 mutant colon cancer cells using single cell imaging

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posted on 10.08.2020 by James Kealey
A reprogrammed energy metabolism is an abundant feature in cancer. This
mechanism provides cells with the ability to evade cell death and increases the
ability to proliferate even under adverse conditions. K-ras and p53 are two of the
most commonly mutated genes in colon cancer and many studies have shown
that mutations in these genes affect several cellular metabolic processes including
glycolysis and mitochondrial respiration.
In this project, we employed a single-cell time-lapse imaging approach to analyse
how colon cancer cell lines, with varying K-ras and p53 status, respond to
metabolic stress induced by nutrient deprivation or FCCP exposure in the
presence of two different substrates, glucose and lactate. Fluorescent probes and
biochemical assays were utilised to measure ATP and NADH in the cytosol and
mitochondria, while the membrane-permeant cationic fluorescent probe TMRM
was employed to monitor mitochondrial membrane potential. We have observed
that lactate is an excellent substrate for replenishment of both NADH and ATP
after cells experienced bioenergetic stress induced by nutrient deprivation.
Results demonstrated that p53-deficient cells containing wt K-ras exhibit the
highest concentration of ATP after a period of starvation and display enhanced
ATP generation in the presence of lactate and glucose. Moreover, wt K-ras cells
showed a faster mitochondrial ATP influx/efflux. We have also found that wt p53
and wt K-ras cells present the highest NAD+/NADH ratio following cell starvation,
whilst wt K-ras cells exhibit greater capacity for OXPHOS and cytosolic NADH
oxidation, suggesting superior mitochondrial function.
Furthermore, we developed a microfluidic platform capable of generating nutrient
gradients for simulation of the tumour microenvironment. This platform was
specifically designed to work in conjunction with confocal microscopy as a means
of analysing cellular responses to the gradients produced in a microfluidic device

History

First Supervisor

Prof. Jochen H. M. Prehn

Second Supervisor

Dr. Beatrice D‘Orsi

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2018.

Published Citation

Kealey J. An analysis of cellular bioenergetics in K-ras and p53 mutant colon cancer cells using single cell imaging [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2018.

Degree Name

Doctor of Philosophy (PhD)

Date of award

30/06/2019

Programme

  • Doctor of Philosophy (PhD)

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