LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11
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cell cycle. In case of charged particle irradiation a prolonged to the primary beam, after the gold foil, served as a monitor
cell cycle arrest (19) and a slower rejoining of DSB have been detector. The ratio of the monitor detector counts to that of the
reported to occur (20).
flux measured using SSB detector at the sample position was
Apart from DNA repair pathways, ionizing radiation leads to measured by multiple trials and the calibration factor was obtained.
Monitor detector counts and the measured ratio were
activation of intracellular signaling pathways that play an important
role in cellular decisions of death or survival. Although used for delivering the required fluence to the samples. Flux
a fair amount of literature exists for the activation of signaling was calculated to be 10 8 and the fluence was kept such that each
pathway after charged particle irradiation, there has been no cell is hit by at least 100 proton particles ensuring that bystander
systematic comparison of photon and proton beam irradiation. effect is kept to minimum. Fluence was then used to calculate
In this study, A549 cells were irradiated with either 2 Gy dose with the help of specific ionization curve constructed for
proton beam- or 2 Gy γ -radiation and ensuing activation of few the given energy distribution and composition of the cell line.
important components involved in DNA repair, cell cycle arrest, The average energy of the proton beam used in this study was
and apoptosis pathways were followed to elucidate the mechanisms
behind observed cellular response. Despite the variability 12.5 keV/µm within the cell layer. Initial portion of Bragg’s
measured to be 3.2 MeV and corresponding estimated LET is
in effectiveness of these two radiations, equal doses of each were curve was used for dose estimation since the cell layer used for
chosen to ensure that each cell is hit by proton beam and no bystander
effects are manifested. Essentially all biological differ-
10 µm, which helped to avoid steep increase in LET within the
irradiation was in monolayer geometry with thickness less than
ences between proton and γ -radiations arise from differences sample.
in their ionization tracks. Equitoxic doses were not preferred The cells were grown in monolayer geometry on 35 mm
because the probability of unhit cells would increase. How this petri dishes. Media was removed and petri dishes were mounted
qualitative difference of radiation is translated into activation of on the exit window vertically for the irradiation, after which
crucial intracellular pathways was the focus of the study. Only they were resupplemented with the medium. On an average
one cell line was chosen in this study because the response of samples were irradiated for 1 min to get the required fluence. The
different cell lines is different to proton irradiation (21). cells were exposed to 2 Gy dose with the help of monitor detector
counts.
MATERIALS AND METHODS
Cell culture
Clonogenic cell survival assay
After treatment, cells were seeded out in appropriate dilutions
(500 cells/60 mm petriplate) to form colonies in 14 days.
Human A549 cells were cultured in Dulbecco’s modified eagles
medium (Sigma) supplemented with 10% fetal calf serum
(Sigma). Cells were maintained at 37 ◦ Colonies were fixed with glutaraldehyde (6.0% v/v), stained
C in humidified atmosphere
with 5% CO 2 . Cells were seeded at density of 5 × 10 5 with crystal violet (0.5% w/v) and counted using a stereomicroscope.
Colonies containing more than 50 cells were scored as
cells in small 35 mm petri dishes 24 hr before the irradiation.
survivors. Absolute plating efficiency at 0 Gy (sham irradiated
control) was 32.3 ± 2.4%
Treatment of cells
Irradiation
Semiquantitative reverse transcriptional
For γ -irradiation, cells were exposed to 2 Gy of 60 Co γ -
polymerase chain reaction (RT-PCR)
radiation in in-house facility Gamma Cell 220 at dose rate of
3 Gy/min.
Total RNA from A549 cells (1 × 10 6 cells) was extracted after
Proton beam irradiation was carried out using the Radiation required time periods of irradiation and RT-PCR was carried out
Biology beam line of in-house 4 MeV Folded Tandem Ion Accelerator
(FOTIA) facility. The primary proton beam from the was extracted after required time periods of 1, 2, 3, and 4 hr of
as described earlier (22). Breifly, total RNA from A549 cells
FOTIA was collimated using an adjustable slit to reduce the irradiation and eluted in 30 µL RNAase free water using RNA
fluence and then diffused using a gold foil. The diffused beam tissue kit (Roche). Equal amount of RNA in each group was
was channeled to the exit window made of 20 µm titanium foil reverse transcribed using cMaster RT kit (Eppendorf). Equal
of 3-cm diameter to get uniformly distributed irradiation area. amount (2 µL) of cDNA in each group was used for specific
The samples were thus irradiated under normal atmospheric amplification of β-actin, ATM, DNA-PK, ATR, Chk1, Chk2,
pressure at 24 ◦ C. The target to be irradiated was positioned at a Bcl-2, or Bax gene using gene specific primers (Table 1). The
distance of 11 mm from the exit window, that being the closest PCR condition were 94 ◦ C, 5 min initial denaturation followed
possible place to mount the target. Before the irradiation, a silicon
surface barrier (SSB) detector was positioned at the same annealing temperature of specific primers given in Table 1),
by 30 cycles of 94 ◦ C 45 s, 55–64 ◦ C 45 s (depending on the
position where samples were irradiated and the beam energy 72 ◦ C 45 s and final extension at 72 ◦ C for 10 min. Equal amount
as well as the uniformity was measured. Another SSB detector of each PCR product (10 µL) was run on 1.5% agarose gel
placed inside the scattering chamber at a forward angle of 40 ◦ containing ethidium bromide in tris borate EDTA buffer at 60 V.
616 S. Ghosh et al.