LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

1570 I. J. Radiation Oncology d Biology d Physics Volume 72, Number 5, 2008
Fig. 3. DNA damage in EL-4 cells cultured for 2 h in presence of
different conditioned media was measured using single-cell gel electrophoresis
(‘‘comet assay’’). (A) Tail length, (B) % DNA in tail, (C)
olive tail moment, and (D) tail moment. Key: Lane 1, control unirradiated
EL-4 cells; Lane 2, g-irradiated EL-4 cells; Lane 3, unirradiated
EL-4 cells receiving medium from g-irradiated EL-4 cells. Data
represent means SE of three independent experiments; significantly
different from unirradiated controls: *p < 0.05, **p < 0.01.
Fig. 4. DNA fragmentation assay of EL-4 cells. EL-4 cells cultured
for 24 h in the presence of different conditioned media. DNAs of
each group of cells were isolated as described in Methods and Materials
section and electrophoresed on 1.8% agarose gel containing
ethidium bromide. Key: Lane M, DNA molecular weight marker;
Lane 1, control unirradiated EL-4 cells; Lane 2, g irradiated EL-4
cells; Lane 3, unirradiated EL-4 cells receiving medium from g-
irradiated EL-4 cells. Data shown from representative experiment
of three independent experiments.
transmits the signal to the bystanding cells. The response was
looked at in the presence of cPTIO or L-NAME. L-NAME
was washed out before irradiation so that the bystander cells
are not exposed to L-NAME, and iNOS is inhibited only in
the irradiated cells. The addition of cPTIO, which scavenges
the NO released in the medium, did not affect the expression
of iNOS gene if the bystanding cell was of same origin, although
NF-kB was somewhat inhibited (Fig. 8, compare
Lanes 5 and 3). The addition of L-NAME, which inhibits
the iNOS in the irradiated cells, completely inhibited the bystander
response in the EL-4 cells (Fig. 8, compare Lanes 4
and 3).
However, both L-NAME and cPTIO inhibited the expression
of NF-kB and iNOS in the bystander EL-4 cells if the
medium was from LPS stimulated and irradiated RAW
264.7 cells (Fig. 8, Lanes 6, 7) (i.e., the cell type was different),
indicating that the bystander response was dependent on
the cells type and the state of stimulation of the irradiated
cells.
DNA damage was significantly higher in irradiated and
bystander cells (Fig. 9, Lanes 2, 3). However, the EL-4 cells
that had received medium either from L-NAME–treated and
irradiated EL-4 cells or from LPS-stimulated, L-NAMEtreated
and irradiated RAW 264.7 cells did not show any increase
in DNA damage (Fig. 9, Lanes 4, 6). Similarly, the EL-
4 cells that had received medium either from cPTIO-treated
and irradiated EL-4 cells or from LPS-stimulated, cPTIOtreated
and irradiated RAW 264.7 cells did not show any increase
in DNA damage (Fig. 9, Lanes 5, 7). Neither L-NAME
nor cPTIO itself had any effect on DNA damage in EL-4 cells
(Fig. 9, Lanes 8, 9).
DISCUSSION
In an attempt to understand bystander signaling, several
studies have been made between the same cell lines (1, 2,
16). However, it is not known whether bystander effect is
extendable to dissimilar cells, particularly those cells that
communicate with each other under physiologic situations
like the macrophages and lymphocytes. In immunology,
there is an immense cross-talk between the lymphocytes
and the macrophages. This could make them more prone to
bystander signaling.