T
he experiment performed by Gamcsik, Millis and Colvin is based
on research showing that an intracellular compound known as
glutathione (GSH) is much more prevalent in cancer cells which
are resistant to treatments such as 4-HC than in native cells
(those which are damaged by such treatments). However, previous
studies which detect the high levels of GSH require that cells
be disrupted, or broken apart. Due to this restriction, it is
not yet clear whether increased GSH in cells is the cause of
resistance to treatment, or simply a byproduct. However, if
GSH could be measured using a noninvasive method, one which
would not involve damaging the cells, it would be possible to
treat the resistant cells in several ways and measure the changes
in GSH levels over a period of time, as the cells would continue
to live.
Gamcsik, Millis and Colvin attempt to do exactly this: measure
intracellular GSH noninvasively, using NMR technology. Since
GSH molecules contain several carbon atoms, it should be possible
to detect its presence using an NMR signal which measures carbon
(specifically, carbon-13 provides a clear NMR signal). Thus,
cells which are resistant to treatment, and therefore contain
higher levels of GSH, should show higher NMR resonances in the
carbon-13 spectrum, than those which are not resistant. In fact,
the results show that resonances of carbon in GSH are clearly
shown (as well as carbon in other intracellular proteins and
lipids), and that, as expected, the GSH resonances are much more
intense in the cell line which is resistant to 4-HC. To further
prove that the resonances are due to elevated GSH levels, the cells
are treated with buthionine sulfoximine (BSO), a known inhibitor
of GSH synthesis. The carbon-13 resonances due to GSH are less
intense after BSO treatment, indicating that GSH levels have been
diminished.
In addition to measuring the carbon-13 spectrum, the
phosphorous-31 NMR spectrum is measured in the same cells. The
purpose for this spectrum is to measure the existence of
phosphorous-containing molecules such as ATP, which would
indicate that the cells are indeed living throughout the
experiment. These spectra were unchanged over the period
meaning the cells were indeed viable.
Group 13 - November 25, 1997