(317g) Human Glioma Cells Undergoing Chemotherapy-Induced Apoptosis Exhibit Marked Reductions in Intracellular Phosphocholine and Phosphocreatine

Introduction:  In current clinical practice, the response of human brain tumors
to therapy is generally monitored with magnetic resonance imaging (MRI) methods
that detect changes in tumor morphology or blood flow.  These changes generally occur slowly and
often many weeks can pass before they are detectable.  Without a reliable means for detecting response early during the
course of treatment, patients can be subjected to harsh, ineffective
therapeutics for an unnecessarily long period of time. 

³¹P NMR spectroscopy can detect
biochemical changes in treated tumors that often precede morphological changes
(1).  Rapidly proliferating tumor cells
frequently contain high levels of free phosphomonoesters such as phospho-choline
and phospho-ethanolamine.  These compounds
are precursors for phosphoglycerides, which are a major subclass of
phospholipid found in cellular membranes. 
Proliferating tumor cells also frequently contain elevated levels of
phosphocreatine, which is used for storage of metabolic energy.  In response to successful chemotherapy,
tumor cells generally undergo apoptosis. 
This process produces marked changes in cellular energetics and membrane
structure as the cytoplasm and nucleus condense into apoptotic bodies.  We hypothesized that human glioma cells
undergoing apoptosis would exhibit marked, selective changes in
phosphomonoester and phosphocreatine levels. 

To examine this hypothesis, metabolically active
human glioma cells were studied non-invasively with ³¹P NMR
spectroscopy ex vivo.  The cells
were grown in a perfused artificial tumor, constructed of porous collagen and
non-porous polystyrene microspheres. 
The tumor was specifically designed to retain apoptotic cells, which are
often lost in perfusion experiments as they detach from their growth
surfaces.  The results may be useful for
the design of new clinical methods that are much more sensitive to early
therapeutic response than current MRI methods.

Materials and Methods: SF188 cells (human grade 4
glioma) were obtained from the UCSF Brain Tumor Research Center (San Francisco,
CA).  They were grown in Dulbecco's
Modified Eagles Medium (DMEM) that contained 25 mM glucose, 6 mM glutamine, 50 mg/ml
gentamicin sulfate and 10% fetal bovine serum. 
Pre-sterilized porous collagen microcarriers (200 mm
diameter, Hyclone, Logan, UT) were inoculated with freshly trypsinized cells at
a ratio of ~10⁷ cells/hydrated gram of collagen.  The cells were allowed to grow inside the
microcarriers for 9 to 11 days.  To
construct the artificial tumor, the microcarriers were mixed at a 1:1 volume
ratio with solid polystyrene micro-spheres and tightly packed into a 20-mm NMR
tube (2). The polystyrene helped to limited compression of the porous collagen
during perfusion (12 ml/min).  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.
Oxygen consumption was determined continuously with polarographic oxygen probes
located upstream and downstream of the tumor. 
Experiments were conducted with either one 160 mg/ml (N=1) or two 125 mg/ml (N=4)
doses of TMZ (with a 24-h delay between treatments).  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). Endonuclease-cleaved DNA was labeled with
fluorescein and detected within the microcarriers with fluorescence confocal
microscopy (Biorad, Hercules, CA).

Results: Compounds detected with high signal to noise
included phosphocholine (PCh), inorganic phosphate, gycerophospho-choline (GPC),
phosphocreatine (PCr), nucleoside triphosphates (NTP, the sum of ATP, CTP, GTP,
TTP and UTP) and diphosphodiesters (DPDE, which are the sum of NAD(H) and
uridine diphospho-sugars).  The total
number of viable cells in the tumor was ~4 x 10⁸ prior to treatment
(estimated from the NTP level).  In
response to a single dose of TMZ (160 mg /ml), NTP and PCr levels
continued to increase for an additional 40 hours and subsequently declined
linearly.  PCr reached undetectable
levels ~85 h after treatment, which coincided with cessation of oxygen
consumption.  In contrast, PCh levels
began to decline within 25 hours of treatment and were nearly undetectable 65
hours later.  The GPC and DPDE levels
were the slowest to respond to therapy. 
The percentage reductions were largest for PCr (100%) and PCh (87%);
smaller reductions were observed for GPC (39%) and DPDE (57%). 

TMZ has a very short half-life in blood and culture medium (~1.5
hr) and is normally administered once every 24 hours for five successive
days.  To examine the effects of
repeated doses, an artificial tumor containing a much higher level of cells, ~9
x 10⁸, was treated with two doses of TMZ (125 mg/ml, 24 h between treatments).  The results were qualitatively similar to
those described above.  The percentage
reductions were largest for PCr (100%), and PCh (83%); significantly smaller
reductions were observed for GPC (58%) and DPDE (54%). 

Confocal
microscopy results indicated that for the tumor treated with 160 mg/ml TMZ, no apoptotic cells were detected
over the first 24 hours, but nearly half were apoptotic by 72 hours.  For the tumor containing a much higher cell
number and treated with twice with 125 mg/ml,
apoptosis developed more slowly; approximately 20% of cells were apoptotic 72
hours after the second treatment.

Discussion: The reduction in PCh is consistent with
the findings for other tumor types, including breast and prostate, in response
to therapy (1,5).  However, the
reduction was not associated with an increase in GPC levels as had been
reported with anti-mitotic agents (6). 
Although changes in PCr are less commonly reported in tumors, this
metabolite was the most sensitive indicator of response for SF188 cells.  Complete depletion occurred when oxygen
consumption ceased, while significant levels of NTP were still present.  These findings are consistent with the
belief that PCr is used to store excess metabolic energy. 

Conclusions: The selective reductions in PCh and
PCr observed in response to therapy may be useful for the design of clinical
methods for detecting apoptosis.  In
future work, we will conduct more detailed studies to quantify the relationship
between reductions in the levels of these two compounds and specific events in
the apoptotic cascade.

References:

(1) Evelhoch JL et al., Neoplasia 2:152-165 (2000).

(2) Mancuso A et al., Magnetic Resonance in Medicine, in
press (2005).

(3) Mancuso A et al., Biotechnology and Bioengineering
87:835:848 (2004).

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

(5) Podo F, NMR Biomedicine 12:413-439 (1999).

(6) Sterin M et al. Cancer Research 61:7536-7543 (2001).

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