LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...
LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...
LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...
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Bystander effect: role of iNOS d S. GHOSH et al. 1569<br />
Fig. 1. Gene expression of inducible nitric oxide synthase (iNOS),<br />
p21, p53, and NF-kB (p65) in EL-4 cells cultured for 2 h in presence<br />
of different conditioned media. Total RNA from EL-4 cells was isolated<br />
and reverse transcribed. Reverse transcriptase-polymerase<br />
chain reaction (PCR) analysis of iNOS, p21, p53, and NF-kB<br />
(p65) genes was carried out as described in materials and methods.<br />
PCR products were resolved on 1.5% agarose gels containing ethidium<br />
bromide. b-Actin gene expression in each group was used as an<br />
internal control. Ratio of intensities of (A) NF-kB (p65), (B) iNOS,<br />
(C) p53, and (D) p21band to that of respective b-actin band as quantified<br />
from gel pictures are shown above each gel picture. Key: Lane<br />
1, control unirradiated EL-4 cells; Lane 2, g irradiated EL-4 cells;<br />
Lane 3, unirradiated EL-4 cells receiving medium from g-irradiated<br />
EL-4 cells. Data represent means SE of three independent experiments;<br />
significantly different from unirradiated controls: *p < 0.05,<br />
**p < 0.01.<br />
There was a clear DNA ladder formation (a hallmark of apoptosis)<br />
in irradiated and bystander EL-4 cells. Apoptosis<br />
was found to be higher in irradiated cells than bystander cells<br />
(Fig. 4, Lanes 2, 3). Apoptosis in irradiated and bystander<br />
EL-4 cells was further confirmed by annexin V/PI assay<br />
(Fig. 5).<br />
EL-4 cells that had received only irradiated medium or<br />
medium from unirradiated control cells did not show any<br />
upregulation of NF-kB and iNOS or NO production or<br />
DNA damage or apoptosis (data not shown).<br />
Bystander effect in dissimilar cells (EL-4 Vs RAW 264.7)<br />
Having confirmed the existence of bystander signaling between<br />
similar cells (EL-4 Vs EL-4), the work was extended to<br />
determine if bystander effects can be demonstrated between<br />
different cell type (RAW 264.7 and EL-4) and whether the<br />
Fig. 2. Nitric oxide (NO) production from EL-4 cells cultured for 24<br />
h in the presence of different conditioned media. The culture supernatant<br />
from each group of EL-4 cells was used for determination of<br />
NO 2 – with Griess reagent. Key: Lane 1, control unirradiated EL-4<br />
cells; Lane 2, g irradiated EL-4 cells; Lane 3, unirradiated EL-4 cells<br />
receiving medium from g irradiated EL-4 cells. Data represent<br />
means SE of three independent experiments; significantly different<br />
from unirradiated controls: *p < 0.05, **p < 0.01.<br />
state of stimulation of macrophages (RAW 264.7) affects bystander<br />
response. The expression of NF-kB was not altered in<br />
EL-4 cells that had received medium from either unstimulated<br />
RAW 264.7 cells or irradiated RAW 264.7 cells, but<br />
the expression of iNOS was downregulated (Fig. 6, Lanes<br />
3, 5). In EL-4 cells that had received medium from LPS stimulated<br />
RAW 264.7 cells there was an upregulation of iNOS<br />
gene (Fig. 6, Lane 4). The expression of both the genes<br />
(NF-kB and iNOS) was significantly upregulated in EL-4<br />
cells that had received medium from LPS stimulated and<br />
irradiated RAW 264.7 cells (Fig. 6, Lane 2).<br />
The EL-4 cells that had received medium from unstimulated<br />
RAW 264.7 cells or LPS-stimulated RAW 264.7 cells<br />
or irradiated RAW 264.7 cells did not show any increase in<br />
DNA damage (Fig. 7, Lanes 3–5). But there was significantly<br />
higher DNA damage in EL-4 cells that had received medium<br />
from LPS stimulated and irradiated RAW 264.7 cells<br />
(p < 0.01) (Fig. 7, Lane, 2).<br />
Effect of L-NAME and cPTIO on Bystander response<br />
Because the expressions of NF-kB and iNOS gene were<br />
significantly upregulated in irradiated and bystander cells<br />
(Fig. 8, Lanes 2, 3), it was of interest to determine what