Reports of research work funded by grants prior to 2015
Victoria University of Wellington
Inter-cellular Mitochondrial Transfer – Does Injury Alter Mitochondrial Transfer Between Cells?
School of Biological Sciences
The aim of this project is to determine the ability of chemotherapy and radiation to injure mitochondria in the cancer glioblastoma multiforme (GBM), and to examine how this impacts on the direction and rate of mitochondrial transfer between GBM cells.
1. Mitochondrial injury
We have successfully established the LORD-Q assay to quantify mitochondrial DNA damage. This assay requires the amplification of a very long (3 kb) and a very short (65 bp) PCR product and detection of each by real-time quantitative PCR. As real-time PCR is most efficient with short products, the efficiency of amplification of the long product is not particularly high. We have established a reproducible efficiency of amplification, which is critical for the success of the LORD-Q assay.
The positive controls for mitochondrial injury, and mitochondrial DNA damage, have been set up, and the experiments to look at damage after therapeutically relevant doses of chemotherapy and radiation are in progress.
2. Mitochondrial transfer
Mitochondrial transfer has been observed between the human GBM cell lines LN18 and U87MG using fluorescent dye labelling of the mitochondria. However, we have discovered some technical difficulties with the fluorescent dyes, and have moved to develop a genetic model in parallel.
The GBM cells have been stably transfected with either (i) a plasmid expressing COX4-BFP fusion protein, which labels the mitochondria of transfected cells blue or (ii) a GFP expressing plasmid. The COX4-BFP mitochondrial donor cells are co-cultured with the GFP recipient cells, and transfer of blue mitochondria to green cells monitored by confocal microscopy.
To confirm the fluorescent mitochondrial transfer, we will use a sequencing approach. A polymorphic region of the mitochondrial genome has been sequenced in the LN18 and U87-MG cell lines, and several SNP identified. LN18-GFP cells will be co-cultured with non-fluorescent U87-MG cells, then total mitochondrial DNA sequenced from the LN18 cells. These data confirmed that a low level of mitochondrial transfer occurs between the two cell types.
The final experiments will be to examine the effect of injury (objective 1) on transfer (objective 2) and we are on track to complete these experiments by the end of this academic year, as planned.