Transcript Aseem-NITRIC OXIDE

NITRIC OXIDE
Aseem Hussain
NITRIC OXIDE
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Nitric oxide is a gas. It is highly reactive;
and is one of the products in automobile
exhaust and plays a major role in
atmospheric pollution.
Surprisingly, it was found that it has
important physiological functions.
NITRIC OXIDE
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This was the first discovery that a gas can act as
a signal molecule in the organism.
A highly reactive compound, it only exists for six
to ten seconds inside the body, then it is
converted, by oxygen, into other compounds of
nitrogen called nitrites.
Uniquely, it is one of the few compounds with an
odd number of electrons thereby making it a
‘free-radical' prone to ionizations.
NITRIC OXIDE
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NO is synthesized within cells by an
enzyme NO synthase (NOS).
Sub-types of NOS
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nNOS : found in neurons
iNOS : is inducible and found in
macrophages. Whereas the levels of
nNOS and eNOS are relatively steady,
expression of iNOS genes awaits an
appropriate stimulus (e.g., ingestion of a
parasite).
eNOS : found in the endothelial cells that
line the lumen of blood vessels.
NITRIC OXIDE
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All types of NOS
produce NO from
arginine with the aid
of molecular oxygen
and NADPH.
NITRIC OXIDE
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NO diffuses freely across cell membranes.
There are so many other molecules with
which it can interact, that it is quickly
consumed close to where it is synthesized.
Thus NO affects only cells near its point of
synthesis.
NITRIC OXIDE FUNCTIONS
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Blood Flow
NO relaxes the smooth muscle in the walls of
the arterioles.
Mice whose genes for the NO synthase found in
endothelial cells (eNOS) has been “knocked out"
suffer from hypertension.
Nitroglycerine, which is often prescribed to
reduce the pain of angina, does so by
generating nitric oxide, which relaxes the walls
of the coronary arteries and arterioles.
Platelet aggregation
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NO also inhibits the aggregation of
platelets and thus keeps inappropriate
clotting from interfering with blood flow
Kidney Function
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Release of NO around the glomeruli of the
kidneys increases blood flow through them
thus increasing the rate of filtration and
urine formation.
Other functions
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Penile Erection
Intestinal peristalsis
Contractility of the smooth muscle wall of
the uterus during labor
NO stimulates secretion from several
endocrine glands.
Neurotransmitter
Memory and learning.
NITRIC OXIDE
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NO aids in the killing of engulfed pathogens like
bacteria within the lysosomes of macrophages
Harmless bacteria, living as commensals at the
rear of our throat, convert nitrates in our food
into nitrites. When these reach the stomach, the
acidic gastric juice (pH ~1.4) generates NO from
them. This NO kills almost all the bacteria that
have been swallowed in our food.
Mechanisms of NO Action
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The signaling functions of NO begin with its
binding to protein receptors on or in the cell. The
binding sites can be either:
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a metal ion in the protein or
one of its Sulfur atoms e.g., on cysteine.
In either case, binding triggers a change in the
protein which, in turn, triggers the formation of a
"second messenger" within the cell. The most
common protein target for NO seems to be
guanylyl cyclase, the enzyme that generates
the second messenger cyclic GMP (cGMP).
NITRIC OXIDE
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The discovery of the biological functions of nitric
oxide in the 1980s came as a complete surprise
Nitric oxide was named "Molecule of the Year" in
1992 by the journal Science,
a Nitric Oxide Society was founded, and a
scientific journal devoted entirely to nitric oxide
was created.
The Nobel prize in Medicine in 1998 was
awarded for the discovery of the signaling
properties of nitric oxide.
NITRIC OXIDE
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It is estimated that yearly about 3,000
scientific articles about the biological roles
of nitric oxide are published.
The following article is one of these…
Nitric oxide-induced cellular
stress and p53 activation in
chronic inflammation
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This article was published in “The Proceedings of
the National Academy of Sciences of United
States of America” on January 7, 2003.
Introduction: NO as a free radical
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Free radicals perform beneficial tasks
such as aiding in the destruction of
microorganisms and cancer cells.
Excessive production of free radicals
however can lead to damage of cellular
structure and enzymes.
Introduction
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Inflammation is the body's response to
injury
The inflammatory response includes
redness, swelling and an increased local
supply of white blood cells. These
changes are an attempt to ward off
infections and to help repair damaged
tissue.
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however, the inflammatory response may
be excessive and result in untoward
consequences like cancer and worsening
of the disease.
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An increased cancer risk occurs in tissues
with chronic inflammation
There are multiple free radicals generated
by chronic inflammation and they target
various genes and proteins to cause
cancer
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In this article, NO and its actions on p53
tumor suppressor gene and how this
results in cancer is studied.
p53 tumor suppressor gene
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The product of the tumor suppressor gene p53
(chromosome 17) is a protein that prevents a
cell from completing the cell cycle if
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its DNA is damaged or
the cell has suffered other types of damage.
If the damage is minor, p53 halts the cell cycle
— hence cell division — until the damage is
repaired or
if the damage is major and cannot be repaired,
p53 triggers the cell to commit suicide by
apoptosis.
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p53 tumor suppressor gene
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These functions make p53 a key
player in protecting us against cancer;
that is, an important tumor suppressor
gene.
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There is increased p53 mutation in
inflamed colon tissue from patients with
ulcerative colitis, a cancer-prone
inflammatory bowel disease, along with
elevated nitric oxide synthase (iNOS)
levels
The mutated p53 cannot bind to its
transport protein and accumulates in cells
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p53 mutations result in genomic instability
because of diminished regulation of cell cycle
checkpoints, DNA repair, and apoptosis.
In this article, several mechanisms are explained
through which NO induces p53 mutation in vitro
and in
cells exposed to NO-generating drugs and NOreleasing macrophages and
Ulcerative colitis.
Methods used in this study
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Comet assay
Cell culture
Co-culture
Immunoprecipitation
Western Blot analysis
Mitotic index assays
Immunohistochemistry
Nitrate and nitrite assay
Methods
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Comet Assay
the Comet assay is a simple, rapid and
sensitive technique for analyzing and
quantifying DNA damage in individual
cells.
Comet Assay
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Cells are embedded in a thin
agarose gel on a microscope slide.
The cells are lysed to remove all
cellular proteins and the DNA
subsequently allowed unwinding
under alkaline/neutral conditions.
Following unwinding the DNA is
electrophoresed and DNA stained
with a fluorescent dye.
During electrophoresis, broken
DNA fragments (damaged DNA) or
relaxed chromatin migrates away
from the nucleus.
The extent of DNA liberated from
the head of the comet was directly
proportional to the DNA damage.
Mitotic index
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The percentage of
cells actively dividing
are calculated
Cell culture
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The important cell lines used were
The MCF-7 human cancer cell line
 macrophage cell line, ANA-1
 colon carcinoma cells (HCT)
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Nitrate and nitrite assay
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Nitrite and nitrate are the stabile end
products of NO metabolism and were
measured in culture media with a
fluorometric assay kit.
Hypothesis
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First, to identify whether NO damages
p53, MCF-7 cells (human cancer cell line
containing p53) were exposed to NO
donors S-nitrosoglutathione or spermine
NONOate .
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These chemicals result in NO induced
DNA damage by p53 phosphorylation
(serines 15, 20, 33, 46, 315, and 392), and
acetylation (lysine 382), leading to p53
mutation and accumulation
Figure 1a
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DNA damage is induced
in MCF-7 cells after
exposure to 0.5 mM
SPER/NO. Cells were
exposed for 4 h, then
processed for the
alkaline comet assay.
Figure 1b
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Section ‘b’ and ‘c’ are western
blots
Increase in p53
posttranslational modifications
and p53 levels after exposure
to 0.5 mM SPER/NO recorded
by Western blot assays. were
performed.
UV treatment (known to cause
maximal damage) was used
as a positive control.
Figure 1c
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Next, MCF-7 cells were
co-cultures with NO
releasing macrophages
and the abnormal p53
accumulation and
phosphorylation of p53
was noted using western
blot analysis.
After 8 h of coincubation,
cells were lysed, and
Western blot assays were
performed.
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Lane 1, MCF-7 cells only
lane 2, MCF-7 cells + unstimulated
macrophages
lane 3, MCF-7 cells + stimulated
macrophages
lane 4, unstimulated macrophages only
lane 5, stimulated macrophages only
lane 6, MCF-7 cells + cytokine stimulation
lane 7, MCF-7 cells + cytokine
stimulation + L-NMMA
lane 8, MCF-7 cells + stimulated
macrophages + L-NMMA
lane 9, HCT 116 p53 / cells
lane 10, MCF-7 cells + UV.
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p53 protein was isolated by using double
immunoprecipitation with mouse
monoclonal anti-p53 antibodies
Antibodies used in western blot
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The following primary antibodies were
used for protein analysis by Western
blotting procedures:
anti-human p53 phosphoserine
15 antibody
acetylated lysine 382 antibody
Other antibodies used were anti-human
p21antibodies, anti-human iNOS
antibodies
NO-Induced Phosphorylation of
p53 at Serine 15
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The serine 15 phosphorylation on p53 is a principal
residue modified in vitro. This modification mediates p53
accumulation and activation.
The investigators focused on this residue.
Cell lines with and without p53 were exposed to NO
donors and Phosphorylation of p53 at serine 15 was
studied. It was noted that when compared to ‘knockouts’,
p53 phosphorylation at 15 was increased when the cell
lines were exposed to nitric oxide donors
Thus serine 15 p53 phosphorylation is an important step
in carcinogenesis.
NO-Induced p53 Phosphorylation
Activates p53 Targets and Engages
a G2/M Checkpoint.
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This was intended to show that NO also
has some protective effects in preventing
cancer
NO reduces the number of cells that are
actively dividing by causing a shift in cell
cycle
at G1
checkpoint,
an early
decrease in
the
percentage of
cells in active
S-phase, and
at G2/M
checkpoint
Changes in cell cycle by NO
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HCT 116 (colon carcinoma cells) with
p53 and HCT 116 without p53 cells were
exposed to the 0.5 mM SPER/NO for
indicated time points (hr), then lysed.
Western blot assays were performed.
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There is a p53- and p21-dependent G2/M
arrest in colon cancer cells exposed to
NO.
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Thus, NO caused a shift in cell cycle by
preventing active cell division, increasing
cells in G2 phase and decreases mitotic
index
p53 Is Phosphorylated,
Accumulates, and Is Active in
ulcerative colitis, a Chronic
Inflammatory Disease.
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Ulcerative colitis is a chronic inflammatory
condition of the colon and it can eventually
lead to colon cancer
In this study tissue samples from both normal
colons and colons with ulcerative colons
were obtained.
The investigators wanted to prove that there
is increased NO production and hence
increased mutation and accumulation of p53
in ulcerative colitis
Ulcerative colitis
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Nine of eleven UC cases had detectable
levels of nitric oxide synthase (iNOS)
protein. In contrast, nitric oxide synthase
levels were undetectable in normal colon
tissues from non-UC donors.
significant increase (P < 0.05) in nitric
oxide synthase levels with increasing
degree of inflammation was noted.
Ulcerative colitis
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p53 protein levels and posttranslational
modifications also were undetectable in
normal colon tissues
In contrast, 9 of 11 UC patients had
detectable P-Ser-15 levels, and all 11 UC
patients had detectable p53. There also
was a significant increase in P-Ser-15
levels (P < 0.05) with increasing degree of
inflammation.
Ulcerative colitis
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There is a immediate rise in P-Ser-15
levels with minimal inflammation and this
finding is consistent with the hypothesis
that P-Ser-15 is a sensitive biomarker of
inflammation.
There also were detectable levels of
acetylated lysine 382
Ulcerative colitis
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Highly significant positive correlations
were noted
between nitric oxide synthase and P-Ser15 levels
and between P-Ser-15 and p53 levels,
consistent with the hypothesis that P-Ser15 is a modification is an early and
important step in free radical damage by
NO
Figure 4 a
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iNOS, P-Ser-15,
and p53 protein
levels are elevated
in colon tissues
from UC patients.
Figure 4b
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iNOS and P-Ser-15 levels,
and P-Ser-15 and p53, are
correlated in UC colon
tissues.
Fig 4c
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iNOS, P-Ser-15, and p53
protein levels are
elevated in UC patients.
Serial tangential sections
of crypts of Leiberkuhn
were exposed to iNOS,
antiphosphoserine-15,
anti- p21WAF1, antiHDM-2 antibodies.
Ulcerative colitis
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P-Ser-15 immunostaining was
undetectable in the normal colon. In
UC, staining occurred in both
epithelial (E) and stromal (S) cells.
In UC, but not in normal tissue, both
epithelial and stromal cells were
positively stained after incubation
with the anti-iNOS antibody.
Although not detectable in normal
colon tissue, p53 transcriptional
targets such as p21WAF1 and
HDM-2 were positive in UC colon,
consistent with p53 activation.
Discussion
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Exposure to exogenous -irradiation, UV, and
some chemotherapeutic agents activates a DNA
damage-response pathway, resulting in
phosphorylation, acetylation, and activation of
p53
The authors have demonstrated that this critical
DNA damage-response pathway is induced by
NO in cell culture and in ulcerative colitis, a
colon cancer-prone chronic inflammatory
disease.
Discussion
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Cell culture data showed that there is a
positive correlation between inflammatory
index, nitric oxide synthase, and P-Ser-15
levels, and in turn, P-Ser-15 and p53
levels.
P-Ser-15 is a critical event in NO-induced
p53 activation
Discussion
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P-Ser-15 p53 also has been shown to
have a reduced binding affinity for HDM-2
Because HDM-2 targets p53 shuttles it to
the cytoplasm for degradation, this
reduced binding results in elevated p53
accumulation and transcriptional activity.
Discussion
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Similarly, P-Ser-15 has been shown to
inhibit the nuclear export of p53, leading to
p53 nuclear accumulation
Discussion
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Although the study established NO as
causing phosphorylation and alteration of
p53, other free radicals may also lead to
p53 activation in chronic inflammation.
Discussion
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On the basis of this and other studies the
authors proposed a model of colon
carcinogenesis in ulcerative colitis patients
that highlights a paradoxical role of NO in
this process.
Discussion
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During chronic inflammation, NO and other free
radicals are involved in repeated genomic insult.
NO can also, paradoxically protect from cellular
damage by altering cell cycle checkpoints and
decreasing dell division and multiplication. This
transient cell cycle arrest would allow DNA repair
of free radical-induced DNA damage.
But this protective mechanism is overpowered
by other actions of NO
Discussion
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Normal p53 causes apoptosis (sudden
death) of abnormal cells. Apoptosis is also
a cellular defense to DNA damage.
NO inhibits apoptosis by phosphorylating
p53.
NO can further prevent apoptosis by other
mechanisms
Discussion
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these p53 mutant cells are now resistant to free
radical-induced growth arrest and apoptosis
clonal selection and expansion of these p53
mutant cell occurs
This uncontrolled growth leads detectable tumor.
Thus, the authors propose a model of ulcerative
colitis colon carcinogenesis that may also apply
to other chronic inflammatory conditions.
Normal
p53 When DNA is
damaged..
Apoptosis
Phosphorylation
of p53
Nitric
oxide
Nitric oxide
synthase
Mutated
p53
When DNA is
damaged…
No apoptosis
Uncontrolled growth
Cancer
Thank you!