LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

Bystander effect: role of iNOS d S. GHOSH et al. 1571
Fig. 5. Apoptosis assay of control, irradiated, and bystander EL-4 cells by Annexin-PI staining. EL-4 cells cultured for 18 h
in the presence of different conditioned media. The EL-4 cells were stained using an annexin V/PI-staining kit. Characterization
and quantification of apoptosis was done by confocal microscopy. Key: (A) Annexin-V; (B) PI; (C) DAPI; (D)
Merge image of annexin-v, PI, and DAPI. Data represent means SE of three independent experiments; significantly
different from unirradiated controls: **p < 0.01.
There are numerous reports on the activation of signaling
factors in the bystanding cells (1,2). However, not much is
known about the genes that are activated in the bystanding
cells in response to these signals. In our earlier work, we
had shown that NF-kB and p21 levels were elevated in
K562 cells (16). The present study was carried out to investigate
which genes are activated in the bystanding cell and
whether the state of activation of the irradiated cell alters
the bystander response. The study was restricted only to the
genes because, from our experience, even the change of irradiated
medium leads to an activation of NF-kB (16). Additionally,
minor stresses are known to alter the state of
phosphorylation of signaling proteins. Our present results revealed
a significant increase in DNA damage in the bystander
cells, but only in those cells that had received medium from
irradiated cells (Fig. 3). Because the cells treated only with
irradiated medium did not show any DNA damage or NO
production (results not shown), the signals that caused the
damage in the bystander cells must be coming from the irradiated
cells themselves. The expression of cell cycle–related
genes (p53 and p21) was significantly upregulated in bystander
similar cells (Fig. 1C, D, Lane 3). Our results are in
agreement with the work on bystander signaling (1,2). The
first report describing the induction of sister chromatid exchanges
was examined in Chinese hamster ovary cells. Because
Chinese hamster ovary cells contain mutant p53, p53
could not be involved in the bystander induction of sister
chromatid exchanges (17). If the p53 of the bystander cell
is mutated, there should be less DNA repair and in fact
more bystander effect can be seen when assessed in terms
of DNA damage.
A clear cause and effect relationship was established between
DNA damage, gene expression, and cell survival and
was evident in the form of increase apoptosis in bystander
cells (Fig. 4, Lane 3, Fig. 5).
An increased expression of NF-kB gene (p65) could lead
to the activation of iNOS gene because the promoter of the
iNOS gene contains binding sites for NF-kB. NF-kB is
known to be activated by several oxidative stresses including
ionization radiation (18–23). As a consequence of iNOS gene