(432e) Human Glioma Cells Exhibit Marked Reductions in 13c NMR-Detected Tca Cycle Activity and Oxygen Consumption during Late-Stage Apoptosis

Introduction: Most cancer chemotherapeutics are
designed to interact with DNA in a manner that eventually induces
apoptosis.  Clinical methods that
specifically detect apoptosis could very valuable for monitoring
chemotherapeutic efficacy.  One of the
early changes that occurs during apoptosis is release of cytochrome-c from the
space between the inner and outer mitochondrial membranes (1). In the
cytoplasm, cytochrome-c serves as a critical signaling molecule in the
apoptotic cascade.  However, it can no longer
perform its normal functions in oxidative phosphorylation.  Other changes are known to occur in
mitochondria during apoptosis, which may also alter aerobic metabolism
(1).  We hypothesized that human glioma
cells treated with temozolomide (TMZ), a new DNA methylating for treatment of
primary brain tumors, would exhibit disrupted oxidative metabolism that could
be detected with ¹³C NMR. To examine this hypothesis, cells were
studied with an artificial tumor method developed in our lab, which allows apoptosis
to be monitored under well-defined conditions (2). 

Materials and Methods: SF188 cells (human glioma
grade 4, Brain Tumor Research Center, UCSF, San Francisco, CA) were grown with
DMEM medium (supplemented with 10% serum and 50 mg/ml gentamicin) in porous
collagen microcarriers (Hyclone, Logan, UT). These microcarriers had a mean
diameter of 200 mm
when fully hydrated and were used in combination with solid polystyrene spheres
(1:1 volume ratio) inside a 20-mm NMR tube (2). The cells were sustained at
physiologic conditions (37 °C, pH = 7.2, dissolved oxygen = 0.2 mM) with a system
constructed in our laboratory (3). NMR spectra were acquired with a 9.4T
spectrometer (Varian, Palo Alto, CA). ³¹P spectral parameters were:
60°
pulse width, 1000 ms repetition time, 4096 points, and 15000 Hz spectral width.
¹³C spectra were acquired with 60° pulses, a repetition time
of 1200 ms, 4096 points, 25000 Hz spectral width and ¹H bi-level
WALTZ-16 decoupling. Cells were initially fed DMEM with 10 mM un-enriched glucose
while background spectra were acquired. Subsequently, the un-enriched medium
was completely replaced with DMEM containing 10 mM [1,6-¹³C₂]
glucose (Cambridge Isotopes, Andover, MA). Absolute intracellular
concentrations were calculated as described previously (3). Oxygen consumption
was determined continuously with polarographic oxygen probes located upstream
and downstream of the tumor (4). ¹³C measurements were performed for
three independently prepared artificial tumors, before and 4 days after TMZ
treatment. In parallel studies, cells grown inside microcarriers were treated
with TMZ and examined with a standard TUNEL assay for apoptosis (BD
Biosciences, Palo Alto, CA).  With this
assay, endonuclease-cleaved DNA was labeled with fluorescein and detected with
fluorescence confocal microscopy (Biorad, Hercules, CA).

Results: Approximately 12 days after microcarrier
inoculation, the total viable cell number (estimated from ³¹P
NMR-detected NTP levels (4)) in the artificial tumor was ~8 x 10⁸. ¹³C
spectra of the tumor exhibited resonances for C-3 of lactate and alanine, C-2,
C-3 and C-4 of glutamate, C-1 and C-6 of fructose-1,6-diphosphate (F-1,6-DP),
and ?CH₂- groups of fatty acids. 
The C-3 glutamate resonance was a triplet due to coupling with C-4 of
glutamate (3), which demonstrates that complete TCA cycle activity is present
in these cells.  Cells in the tumor were
treated twice with TMZ (125 mg/ml with a delay of 24 h between treatments). NTP
levels and oxygen consumption began to decline approximately 12 hours after the
second treatment. The decline in oxygen consumption preceded the decline in
NTP.  Approximately 4 days after the
second TMZ treatment, significant levels of NTP remained and the cells continued
to produce lactate; however, oxygen consumption was undetectable.  The tumor was again perfused with medium
containing 10 mM [1,6-¹³C₂] glucose.  No labeling was detected for any
intracellular resonances except a very small amount at C-4 of glutamate. On an
NTP normalized basis, the rate of incorporation of ¹³C label into
C-4 glutamate by 85%. TUNEL analysis demonstrated that 2 days after the second
treatment, ~10% of cells in the microcarriers contained endonuclease cleaved
DNA. This value increased to ~30% 3 days post-treatment.

Discussion: Glutamate is a key reporter molecule
for TCA cycle labeling (5).  The marked
reduction in labeling for glutamate and the lack of oxygen consumption indicate
that mitochondrial function was disrupted sub-acutely by TMZ. Because
endonuclease cleavage of DNA occurs relatively late in apoptosis, the finding
that only 30% of cells were stained in the TUNEL assay was not surprising. Many
cells may have released their cytochrome-c into the cytoplasm, but had not yet
initiated DNA cleavage. Direct detection of cytochrome-c release will be
studied in future work to elucidate the time course for this important change
following TMZ treatment.

Conclusions: Our results indicate that ¹³C
NMR can be used to detect changes in oxidative metabolism of human glioma cells
following treatment with TMZ. This finding may help to advance the use of ¹³C
MRS methods (e.g. ¹H-detected ¹³C MRS) for clinical
monitoring of apoptosis in human gliomas.

Acknowledgements: This work was supported by NIH
grant R21-CA84380.

References:

(1) Green DR Reed JC Science 281:1309-1312 (1998).

(2) Mancuso A et al. Magn Reson Med in press (2005).

(3) Mancuso A et al. Biotech Bioeng 87:835-848 (2004).

(4) Mancuso A et al. Bio/Technol 8:1282-1285 (1990).

(5) Lewandowski ED et al. MRM 35:149-154 (1996).