LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

SYNOPSIS
microscope (Axioskop). The images (40–50 cells/slide) were captured with highperformance
GANZ (model: ZCY11PH 4) color video camera. The integral frame
grabber used in this system (Cvfbo1p) is a PC based card and it accepts color composite
video output of the camera. The quantification of the DNA strand breaks of the stored
images was done using the CASP software by which %DNA in tail, tail length, tail
moment and Olive tail moment could be obtained directly [12].
2.10 Measurement of NO production in EL-4 cells
Control unirradiated cells, cells exposed to radiation and the cells exposed to different
conditioned media as mentioned above were cultured for 24 h at 37 0 C in 5%CO 2 / 95%
air. The production of NO in the culture supernatants was measured by assaying NO 2
-
using colorimetric Griess reaction [13].
In brief, 100 µl of culture supernatants was incubated with an equal volume of Griess
reagent [1% sulfanilamide and 0.1% N-(1-naphthyl-ethylenediamine) in 2.5% phosphoric
acid; Sigma, USA] at room temperature for 10 min in a 96 well plate. The absorbance at
550 nm was measured using a microplate reader (Fluostar Optima, Biotron Healthcare).
Absorbance measurements were converted to µ moles of NO 2
-
using a standard curve of
NaNO 2.
2.11 Measurement of Apoptosis by DNA fragmentation
Apoptosis in control unirradiated cells, cells exposed to radiation and the cells exposed to
different conditioned media as mentioned above was measured by DNA fragmentation
assay [14]. After transfer of medium from irradiated cells, all the above mentioned
groups of EL-4 cells were cultured for 24 h at 37 0 C in 5%CO 2 , 95% air. The cells were
harvested and lysed in lysis buffer (10 mM EDTA, 50 mM Tris–HCl, pH 8.0, 0.5%
sodium lauryl sarcosine) containing the 100 mg/ml proteinase K at 55 o C for 2 h. The
DNA was extracted with phenol/chloroform and precipitated with ethanol. The RNA was
removed by RNAse A treatment and DNA was electrophorased on agarose gel to
visualize the fragmented DNA.
CHAPTER 3: RESULTS
In this study the relative radioresistance of various cell lines (MCF-7, INT 407
and A 549) was first established. Then the differences in the signaling pattern were
established. The variance in signaling with the change in LET was investigated. Finally,
the contribution of the bystanding cell, which had not been exposed to irradiation, to the
cell survival was also looked at. The results obtained in the study are divided into the
following subsections.
3.1 Fractionated irradiation induced signaling and repair in mammalian cells.
Three human cell lines viz. MCF-7, INT 407 and A 549 were exposed to different
doses of γ irradiation; the first set was irradiated with acute dose of 2 Gy or 10 Gy. The
second set was exposed to 5 fraction of 2 Gy each. Among the three cell line the lung
carcinoma cell line A 549 was found to be relatively more radioresistant if the 10 Gy
dose was delivered as a fractionated regimen. The MCF-7 cell line was found to be
relatively more radiosensitive (Fig. 3.1A). This was further confirmed by analysis of
6