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NITRIC OXIDE. Aseem Hussain. NITRIC OXIDE. 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. - PowerPoint PPT Presentation
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Page 1: NITRIC OXIDE

NITRIC OXIDENITRIC OXIDE

Aseem HussainAseem Hussain

Page 2: NITRIC OXIDE

NITRIC OXIDENITRIC OXIDE

Nitric oxide is a gas. It is highly reactive; Nitric oxide is a gas. It is highly reactive; and is one of the products in automobile and is one of the products in automobile exhaust and plays a major role in exhaust and plays a major role in atmospheric pollution.atmospheric pollution.

Surprisingly, it was found that it has Surprisingly, it was found that it has important physiological functions.important physiological functions.

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NITRIC OXIDENITRIC OXIDE

This was the first discovery that a gas can act as This was the first discovery that a gas can act as a signal molecule in the organism. a signal molecule in the organism.

A highly reactive compound, it only exists for six A highly reactive compound, it only exists for six to ten seconds inside the body, then it is to ten seconds inside the body, then it is converted, by oxygen, into other compounds of converted, by oxygen, into other compounds of nitrogen called nitrites. nitrogen called nitrites.

Uniquely, it is one of the few compounds with an Uniquely, it is one of the few compounds with an odd number of electrons thereby making it a odd number of electrons thereby making it a ‘free-radical' prone to ionizations. ‘free-radical' prone to ionizations.

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NITRIC OXIDENITRIC OXIDE

NO is synthesized within cells by an NO is synthesized within cells by an enzyme enzyme NO synthaseNO synthase ( (NOSNOS). ).

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Sub-types of NOSSub-types of NOS

nNOSnNOS : found in : found in nneurons eurons iNOSiNOS : is : is iinducible and found in nducible and found in

macrophages. Whereas the levels of macrophages. Whereas the levels of nNOS and eNOS are relatively steady, nNOS and eNOS are relatively steady, expression of iNOS genes awaits an expression of iNOS genes awaits an appropriate stimulus (e.g., ingestion of a appropriate stimulus (e.g., ingestion of a parasite). parasite).

eNOSeNOS : found in the : found in the eendothelial cells that ndothelial cells that line the lumen of blood vessels. line the lumen of blood vessels.

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All types of NOS All types of NOS produce NO from produce NO from arginine with the aid arginine with the aid of molecular oxygen of molecular oxygen and NADPH. and NADPH.

NITRIC OXIDENITRIC OXIDE

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NO diffuses freely across cell membranes. NO diffuses freely across cell membranes. There are so many other molecules with There are so many other molecules with

which it can interact, that it is quickly which it can interact, that it is quickly consumed close to where it is synthesized. consumed close to where it is synthesized.

Thus NO affects only cells near its point of Thus NO affects only cells near its point of synthesis. synthesis.

NITRIC OXIDENITRIC OXIDE

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NITRIC OXIDE FUNCTIONSNITRIC OXIDE FUNCTIONS

Blood FlowBlood Flow NO relaxes the smooth muscle in the walls of NO relaxes the smooth muscle in the walls of

the arterioles. the arterioles. Mice whose genes for the NO synthase found in Mice whose genes for the NO synthase found in

endothelial cells (eNOS) has been “knocked out" endothelial cells (eNOS) has been “knocked out" suffer from hypertension. suffer from hypertension.

Nitroglycerine, which is often prescribed to Nitroglycerine, which is often prescribed to reduce the pain of angina, does so by reduce the pain of angina, does so by generating nitric oxide, which relaxes the walls generating nitric oxide, which relaxes the walls of the coronary arteries and arterioles. of the coronary arteries and arterioles.

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Platelet aggregationPlatelet aggregation

NO also inhibits the aggregation of NO also inhibits the aggregation of platelets and thus keeps inappropriate platelets and thus keeps inappropriate clotting from interfering with blood flow clotting from interfering with blood flow

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Kidney FunctionKidney Function

Release of NO around the glomeruli of the Release of NO around the glomeruli of the kidneys increases blood flow through them kidneys increases blood flow through them thus increasing the rate of filtration and thus increasing the rate of filtration and urine formation. urine formation.

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Other functionsOther functions

Penile Erection Penile Erection Intestinal peristalsisIntestinal peristalsis Contractility of the smooth muscle wall of Contractility of the smooth muscle wall of

the uterus during laborthe uterus during labor NO stimulates secretion from several NO stimulates secretion from several

endocrine glands. endocrine glands. NeurotransmitterNeurotransmitter Memory and learning. Memory and learning.

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NITRIC OXIDENITRIC OXIDE

NO aids in the killing of engulfed pathogens like NO aids in the killing of engulfed pathogens like bacteria within the lysosomes of macrophages bacteria within the lysosomes of macrophages

Harmless bacteria, living as commensals at the Harmless bacteria, living as commensals at the rear of our throat, convert nitrates in our food rear of our throat, convert nitrates in our food into nitrites. When these reach the stomach, the into nitrites. When these reach the stomach, the acidic gastric juice (pH ~1.4) generates NO from acidic gastric juice (pH ~1.4) generates NO from them. This NO kills almost all the bacteria that them. This NO kills almost all the bacteria that have been swallowed in our food. have been swallowed in our food.

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Mechanisms of NO ActionMechanisms of NO Action

The signaling functions of NO begin with its The signaling functions of NO begin with its binding to protein receptors on or in the cell. The binding to protein receptors on or in the cell. The binding sites can be either: binding sites can be either: a metal ion in the protein or a metal ion in the protein or one of its Sulfur atoms e.g., on cysteine. one of its Sulfur atoms e.g., on cysteine.

In either case, binding triggers a change in the In either case, binding triggers a change in the protein which, in turn, triggers the formation of a protein which, in turn, triggers the formation of a "second messenger" within the cell. The most "second messenger" within the cell. The most common protein target for NO seems to be common protein target for NO seems to be guanylyl cyclaseguanylyl cyclase, the enzyme that generates , the enzyme that generates the second messenger the second messenger cyclic GMPcyclic GMP ( (cGMPcGMP). ).

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NITRIC OXIDENITRIC OXIDE

The discovery of the biological functions of nitric The discovery of the biological functions of nitric oxide in the 1980s came as a complete surprise oxide in the 1980s came as a complete surprise Nitric oxide was named "Molecule of the Year" in Nitric oxide was named "Molecule of the Year" in 1992 by the journal Science, 1992 by the journal Science,

a Nitric Oxide Society was founded, and a a Nitric Oxide Society was founded, and a scientific journal devoted entirely to nitric oxide scientific journal devoted entirely to nitric oxide was created. was created.

The Nobel prize in Medicine in 1998 was The Nobel prize in Medicine in 1998 was awarded for the discovery of the signaling awarded for the discovery of the signaling properties of nitric oxide. properties of nitric oxide.

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NITRIC OXIDENITRIC OXIDE

It is estimated that yearly about 3,000 It is estimated that yearly about 3,000 scientific articles about the biological roles scientific articles about the biological roles of nitric oxide are published.of nitric oxide are published.

The following article is one of these…The following article is one of these…

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Nitric oxide-induced cellular Nitric oxide-induced cellular stress and p53 activation in stress and p53 activation in

chronic inflammationchronic inflammation

This article was published in “The Proceedings of This article was published in “The Proceedings of the National Academy of Sciences of United the National Academy of Sciences of United States of America” on January 7, 2003.States of America” on January 7, 2003.

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Introduction: NO as a free radicalIntroduction: NO as a free radical

Free radicals perform beneficial tasks Free radicals perform beneficial tasks such as aiding in the destruction of such as aiding in the destruction of microorganisms and cancer cells.microorganisms and cancer cells.

Excessive production of free radicals Excessive production of free radicals however can lead to damage of cellular however can lead to damage of cellular structure and enzymes. structure and enzymes.

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IntroductionIntroduction

Inflammation is the body's response to Inflammation is the body's response to injury injury

The inflammatory response includes The inflammatory response includes redness, swelling and an increased local redness, swelling and an increased local supply of white blood cells. These supply of white blood cells. These changes are an attempt to ward off changes are an attempt to ward off infections and to help repair damaged infections and to help repair damaged tissue. tissue.

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however, the inflammatory response may however, the inflammatory response may be excessive and result in untoward be excessive and result in untoward consequences like cancer and worsening consequences like cancer and worsening of the disease.of the disease.

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An increased cancer risk occurs in tissues An increased cancer risk occurs in tissues with chronic inflammation with chronic inflammation

There are multiple free radicals generated There are multiple free radicals generated by chronic inflammation and they target by chronic inflammation and they target various genes and proteins to cause various genes and proteins to cause cancercancer

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In this article, NO and its actions on p53 In this article, NO and its actions on p53 tumor suppressor gene and how this tumor suppressor gene and how this results in cancer is studied.results in cancer is studied.

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p53 tumor suppressor genep53 tumor suppressor gene The product of the tumor suppressor gene The product of the tumor suppressor gene p53 p53

(chromosome 17)(chromosome 17) is a protein that prevents a is a protein that prevents a cell from completing the cell cycle if cell from completing the cell cycle if

its DNA is damaged or its DNA is damaged or the cell has suffered other types of damage.the cell has suffered other types of damage. If the damage is minor, p53 halts the cell cycle If the damage is minor, p53 halts the cell cycle

— hence cell division — until the damage is — hence cell division — until the damage is repaired or repaired or

if the damage is major and cannot be repaired, if the damage is major and cannot be repaired, p53 triggers the cell to commit suicide by p53 triggers the cell to commit suicide by apoptosis.apoptosis.

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p53 tumor suppressor genep53 tumor suppressor gene

These functions make These functions make p53p53 a key a key player in protecting us against cancer; player in protecting us against cancer; that is, an important tumor suppressor that is, an important tumor suppressor gene. gene.

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There is increased p53 mutation in There is increased p53 mutation in inflamed colon tissue from patients with inflamed colon tissue from patients with ulcerative colitis, a cancer-prone ulcerative colitis, a cancer-prone inflammatory bowel disease, along with inflammatory bowel disease, along with elevated nitric oxide synthase (iNOS) elevated nitric oxide synthase (iNOS) levels levels

The mutated p53 cannot bind to its The mutated p53 cannot bind to its transport protein and accumulates in cellstransport protein and accumulates in cells

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p53 mutations result in genomic instability p53 mutations result in genomic instability because of diminished regulation of cell cycle because of diminished regulation of cell cycle checkpoints, DNA repair, and apoptosis. checkpoints, DNA repair, and apoptosis.

In this article, several mechanisms are explained In this article, several mechanisms are explained through which NO induces p53 mutation through which NO induces p53 mutation in vitroin vitro and inand in

cells exposed to NO-generating drugs and NO-cells exposed to NO-generating drugs and NO-releasing macrophages and releasing macrophages and

Ulcerative colitis. Ulcerative colitis.

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Methods used in this studyMethods used in this study

Comet assayComet assay Cell cultureCell culture Co-cultureCo-culture ImmunoprecipitationImmunoprecipitation Western Blot analysisWestern Blot analysis Mitotic index assaysMitotic index assays ImmunohistochemistryImmunohistochemistry Nitrate and nitrite assayNitrate and nitrite assay

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MethodsMethods

Comet AssayComet Assay the Comet assay is a simple, rapid and the Comet assay is a simple, rapid and

sensitive technique for analyzing and sensitive technique for analyzing and quantifying DNA damage in individual quantifying DNA damage in individual cells. cells.

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Comet AssayComet Assay Cells are embedded in a thin agarose Cells are embedded in a thin agarose

gel on a microscope slide. gel on a microscope slide. The cells are lysed to remove all The cells are lysed to remove all

cellular proteins and the DNA cellular proteins and the DNA subsequently allowed unwinding subsequently allowed unwinding under alkaline/neutral conditions. under alkaline/neutral conditions. Following unwinding the DNA is Following unwinding the DNA is electrophoresed and DNA stained with electrophoresed and DNA stained with a fluorescent dye. a fluorescent dye.

During electrophoresis, broken DNA During electrophoresis, broken DNA fragments (damaged DNA) or relaxed fragments (damaged DNA) or relaxed chromatin migrates away from the chromatin migrates away from the nucleus. nucleus.

The extent of DNA liberated from the The extent of DNA liberated from the head of the comet was directly head of the comet was directly proportional to the DNA damage. proportional to the DNA damage.

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Mitotic indexMitotic index

The percentage of The percentage of cells actively dividing cells actively dividing are calculatedare calculated

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Cell cultureCell culture

The important cell lines used wereThe important cell lines used were

The MCF-7 human cancer cell line The MCF-7 human cancer cell line macrophage cell line, ANA-1 macrophage cell line, ANA-1 colon carcinoma cells (HCT) colon carcinoma cells (HCT)

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Nitrate and nitrite assayNitrate and nitrite assay

Nitrite and nitrate are the stabile end Nitrite and nitrate are the stabile end products of NO metabolism and were products of NO metabolism and were measured in culture media with a measured in culture media with a fluorometric assay kit. fluorometric assay kit.

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HypothesisHypothesis

First, to identify whether NO damages First, to identify whether NO damages p53, MCF-7 cells (human cancer cell line p53, MCF-7 cells (human cancer cell line containing p53) were exposed to NO containing p53) were exposed to NO donors donors SS--nitrosoglutathionenitrosoglutathione or or spermine spermine NONOate .NONOate .

These chemicals result in NO induced These chemicals result in NO induced DNA damage by p53 phosphorylation DNA damage by p53 phosphorylation (serines 15, 20, 33, 46, 315, and 392), and (serines 15, 20, 33, 46, 315, and 392), and acetylation (lysine 382), leading to p53 acetylation (lysine 382), leading to p53 mutation and accumulationmutation and accumulation

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Figure 1a Figure 1a

DNA damage is induced DNA damage is induced in MCF-7 cells after in MCF-7 cells after exposure to 0.5 mM exposure to 0.5 mM SPER/NO. Cells were SPER/NO. Cells were exposed for 4 h, then exposed for 4 h, then processed for the processed for the alkaline comet assay. alkaline comet assay.

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Figure 1bFigure 1b Section ‘b’ and ‘c’ are western Section ‘b’ and ‘c’ are western

blotsblots Increase in p53 posttranslational Increase in p53 posttranslational

modifications and p53 levels after modifications and p53 levels after exposure to 0.5 mM SPER/NO exposure to 0.5 mM SPER/NO recorded by Western blot assays. recorded by Western blot assays. were performed.were performed.

UV treatment (known to cause UV treatment (known to cause maximal damage) was used as a maximal damage) was used as a positive control. positive control.

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Figure 1cFigure 1c Next, MCF-7 cells were co-Next, MCF-7 cells were co-

cultures with NO releasing cultures with NO releasing macrophages and the macrophages and the abnormal p53 accumulation abnormal p53 accumulation and phosphorylation of p53 and phosphorylation of p53 was noted using western blot was noted using western blot analysis.analysis.

After 8 h of coincubation, cells After 8 h of coincubation, cells were lysed, and Western blot were lysed, and Western blot assays were performed. assays were performed.

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Lane 1, MCF-7 cells only Lane 1, MCF-7 cells only lane 2, MCF-7 cells + unstimulated lane 2, MCF-7 cells + unstimulated

macrophagesmacrophages lane 3, MCF-7 cells + stimulated lane 3, MCF-7 cells + stimulated

macrophagesmacrophages lane 4, unstimulated macrophages only lane 4, unstimulated macrophages only lane 5, stimulated macrophages only lane 5, stimulated macrophages only lane 6, MCF-7 cells + cytokine stimulationlane 6, MCF-7 cells + cytokine stimulation lane 7, MCF-7 cells + cytokine lane 7, MCF-7 cells + cytokine

stimulation + L-NMMAstimulation + L-NMMA lane 8, MCF-7 cells + stimulated lane 8, MCF-7 cells + stimulated

macrophages  + L-NMMAmacrophages  + L-NMMA lane 9, HCT 116 p53 / cells lane 9, HCT 116 p53 / cells lane 10, MCF-7 cells + UV. lane 10, MCF-7 cells + UV.

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p53 protein was isolated by using double p53 protein was isolated by using double immunoprecipitation with mouse immunoprecipitation with mouse monoclonal anti-p53 antibodies monoclonal anti-p53 antibodies

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Antibodies used in western blotAntibodies used in western blot

The following primary antibodies were The following primary antibodies were used for protein analysis by Western used for protein analysis by Western blotting procedures: blotting procedures:

anti-human p53 phosphoserine anti-human p53 phosphoserine 15 antibody15 antibody

acetylated lysine 382 antibodyacetylated lysine 382 antibody Other antibodies used were anti-human Other antibodies used were anti-human

p21antibodies, anti-human iNOS p21antibodies, anti-human iNOS antibodiesantibodies

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NO-Induced Phosphorylation of NO-Induced Phosphorylation of p53 at Serine 15p53 at Serine 15

The serine 15 phosphorylation on p53 is a principal The serine 15 phosphorylation on p53 is a principal residue modified residue modified in vitro. in vitro. This modification mediates p53 This modification mediates p53 accumulation and activation.accumulation and activation.

The investigators focused on this residue. The investigators focused on this residue. Cell lines with and without p53 were exposed to NO Cell lines with and without p53 were exposed to NO

donors and donors and Phosphorylation of p53Phosphorylation of p53 at serine 15 was at serine 15 was studied. It was noted that when compared to ‘knockouts’, studied. It was noted that when compared to ‘knockouts’, p53 phosphorylation at 15 was increased when the cell p53 phosphorylation at 15 was increased when the cell lines were exposed to nitric oxide donorslines were exposed to nitric oxide donors

Thus serine 15 p53 phosphorylation is an important step Thus serine 15 p53 phosphorylation is an important step in carcinogenesis.in carcinogenesis.

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NO-Induced p53 Phosphorylation NO-Induced p53 Phosphorylation Activates p53 Targets and Engages Activates p53 Targets and Engages

a G2/M Checkpoint.a G2/M Checkpoint.

This was intended to show that NO also This was intended to show that NO also has some protective effects in preventing has some protective effects in preventing cancercancer

NO reduces the number of cells that are NO reduces the number of cells that are actively dividing by causing a shift in cell actively dividing by causing a shift in cell cyclecycle

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at G1 checkpoint, an early decrease in the percentage of cells in active S-phase, andat G2/M checkpoint

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Changes in cell cycle by NOChanges in cell cycle by NO

HCT 116 (colon carcinoma cells) with HCT 116 (colon carcinoma cells) with p53 and HCT 116 without p53 cells were p53 and HCT 116 without p53 cells were exposed to the 0.5 mM SPER/NO for exposed to the 0.5 mM SPER/NO for indicated time points (hr), then lysed. indicated time points (hr), then lysed. Western blot assays were performed. Western blot assays were performed.

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There is a p53- and p21-dependent G2/M There is a p53- and p21-dependent G2/M arrest in colon cancer cells exposed to arrest in colon cancer cells exposed to NO. NO.

Thus, NO caused a shift in cell cycle by Thus, NO caused a shift in cell cycle by preventing active cell division, increasing preventing active cell division, increasing cells in G2 phase and decreases mitotic cells in G2 phase and decreases mitotic indexindex

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p53 Is Phosphorylated, p53 Is Phosphorylated, Accumulates, and Is Active in Accumulates, and Is Active in

ulcerative colitis, a Chronic ulcerative colitis, a Chronic Inflammatory Disease.Inflammatory Disease.

Ulcerative colitis is a chronic inflammatory Ulcerative colitis is a chronic inflammatory condition of the colon and it can eventually condition of the colon and it can eventually lead to colon cancerlead to colon cancer

In this study tissue samples from both normal In this study tissue samples from both normal colons and colons with ulcerative colons colons and colons with ulcerative colons were obtained.were obtained.

The investigators wanted to prove that there The investigators wanted to prove that there is increased NO production and hence is increased NO production and hence increased mutation and accumulation of p53 increased mutation and accumulation of p53 in ulcerative colitisin ulcerative colitis

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Ulcerative colitisUlcerative colitis

Nine of eleven UC cases had detectable Nine of eleven UC cases had detectable levels of nitric oxide synthase (iNOS) levels of nitric oxide synthase (iNOS) protein. In contrast, nitric oxide synthase protein. In contrast, nitric oxide synthase levels were undetectable in normal colon levels were undetectable in normal colon tissues from non-UC donors. tissues from non-UC donors.

significant increase (significant increase (PP < 0.05) in nitric  < 0.05) in nitric oxide synthase levels with increasing oxide synthase levels with increasing degree of inflammation was noted.degree of inflammation was noted.

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Ulcerative colitisUlcerative colitis

p53 protein levels and posttranslational p53 protein levels and posttranslational modifications also were undetectable in modifications also were undetectable in normal colon tissues normal colon tissues

In contrast, 9 of 11 UC patients had In contrast, 9 of 11 UC patients had detectable P-Ser-15 levels, and all 11 UC detectable P-Ser-15 levels, and all 11 UC patients had detectable p53. There also patients had detectable p53. There also was a significant increase in P-Ser-15 was a significant increase in P-Ser-15 levels (levels (PP < 0.05) with increasing degree of  < 0.05) with increasing degree of inflammation.inflammation.

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Ulcerative colitisUlcerative colitis

There is a immediate rise in P-Ser-15 There is a immediate rise in P-Ser-15 levels with minimal inflammation and this levels with minimal inflammation and this finding is consistent with the hypothesis finding is consistent with the hypothesis that P-Ser-15 is a sensitive biomarker of that P-Ser-15 is a sensitive biomarker of inflammation. inflammation.

There also were detectable levels of There also were detectable levels of acetylated lysine 382 acetylated lysine 382 

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Ulcerative colitisUlcerative colitis

Highly significant positive correlations Highly significant positive correlations were notedwere noted

between nitric oxide synthase and P-Ser-between nitric oxide synthase and P-Ser-15 levels 15 levels

and between P-Ser-15 and p53 levels, and between P-Ser-15 and p53 levels, consistent with the hypothesis that P-Ser-consistent with the hypothesis that P-Ser-

15 is a modification is an early and 15 is a modification is an early and important step in free radical damage by important step in free radical damage by NONO

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Figure 4 aFigure 4 a

iNOS, P-Ser-15, iNOS, P-Ser-15, and p53 protein and p53 protein levels are elevated levels are elevated in colon tissues in colon tissues from UC patients. from UC patients.

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Figure 4bFigure 4b

iNOS and P-Ser-15 levels, iNOS and P-Ser-15 levels, and P-Ser-15 and p53, are and P-Ser-15 and p53, are correlated in UC colon correlated in UC colon tissues. tissues.

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iNOS, P-Ser-15, and p53 iNOS, P-Ser-15, and p53 protein levels are protein levels are elevated in UC patients. elevated in UC patients. Serial tangential sections Serial tangential sections of crypts of Leiberkuhn of crypts of Leiberkuhn were exposed to iNOS, were exposed to iNOS, antiphosphoserine-15, antiphosphoserine-15, anti- p21WAF1, anti-anti- p21WAF1, anti-HDM-2 antibodies.HDM-2 antibodies.

Fig 4c

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Ulcerative colitisUlcerative colitis P-Ser-15 immunostaining was P-Ser-15 immunostaining was

undetectable in the normal colon. In undetectable in the normal colon. In UC, staining occurred in both UC, staining occurred in both epithelial (E) and stromal (S) cells. epithelial (E) and stromal (S) cells.

In UC, but not in normal tissue, both In UC, but not in normal tissue, both epithelial and stromal cells were epithelial and stromal cells were positively stained after incubation positively stained after incubation with the anti-iNOS antibody. with the anti-iNOS antibody.

Although not detectable in normal Although not detectable in normal colon tissue, p53 transcriptional colon tissue, p53 transcriptional targets such as p21WAF1 and targets such as p21WAF1 and HDM-2 were positive in UC colon, HDM-2 were positive in UC colon, consistent with p53 activation. consistent with p53 activation.

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DiscussionDiscussion

Exposure to exogenous -irradiation, UV, and Exposure to exogenous -irradiation, UV, and some chemotherapeutic agents activates a DNA some chemotherapeutic agents activates a DNA damage-response pathway, resulting in damage-response pathway, resulting in phosphorylation, acetylation, and activation of phosphorylation, acetylation, and activation of p53 p53

The authors have demonstrated that this critical The authors have demonstrated that this critical DNA damage-response pathway is induced by DNA damage-response pathway is induced by NO in cell culture and in ulcerative colitis, a NO in cell culture and in ulcerative colitis, a colon cancer-prone chronic inflammatory colon cancer-prone chronic inflammatory disease. disease.

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DiscussionDiscussion

Cell culture data showed that there is a Cell culture data showed that there is a positive correlation between inflammatory positive correlation between inflammatory index, nitric oxide synthase, and P-Ser-15 index, nitric oxide synthase, and P-Ser-15 levels, and in turn, P-Ser-15 and p53 levels, and in turn, P-Ser-15 and p53 levels. levels.

P-Ser-15 is a critical event in NO-induced P-Ser-15 is a critical event in NO-induced p53 activation p53 activation

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DiscussionDiscussion

P-Ser-15 p53 also has been shown to P-Ser-15 p53 also has been shown to have a reduced binding affinity for HDM-2 have a reduced binding affinity for HDM-2 Because HDM-2 targets p53 shuttles it to Because HDM-2 targets p53 shuttles it to the cytoplasm for degradation, this the cytoplasm for degradation, this reduced binding results in elevated p53 reduced binding results in elevated p53 accumulation and transcriptional activity. accumulation and transcriptional activity.

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DiscussionDiscussion

Similarly, P-Ser-15 has been shown to Similarly, P-Ser-15 has been shown to inhibit the nuclear export of p53, leading to inhibit the nuclear export of p53, leading to p53 nuclear accumulation p53 nuclear accumulation

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DiscussionDiscussion

Although the study established NO as Although the study established NO as causing phosphorylation and alteration of causing phosphorylation and alteration of p53, other free radicals may also lead to p53, other free radicals may also lead to p53 activation in chronic inflammation. p53 activation in chronic inflammation.

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DiscussionDiscussion

On the basis of this and other studies the On the basis of this and other studies the authors proposed a model of colon authors proposed a model of colon carcinogenesis in ulcerative colitis patients carcinogenesis in ulcerative colitis patients that highlights a paradoxical role of NO in that highlights a paradoxical role of NO in this process. this process.

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DiscussionDiscussion

During chronic inflammation, NO and other free During chronic inflammation, NO and other free radicals are involved in repeated genomic insult. radicals are involved in repeated genomic insult.

NO can also, paradoxically protect from cellular NO can also, paradoxically protect from cellular damage by altering cell cycle checkpoints and damage by altering cell cycle checkpoints and decreasing dell division and multiplication. This decreasing dell division and multiplication. This transient cell cycle arrest would allow DNA transient cell cycle arrest would allow DNA repair of free radical-induced DNA damage. repair of free radical-induced DNA damage.

But this protective mechanism is overpowered But this protective mechanism is overpowered by other actions of NOby other actions of NO

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DiscussionDiscussion

Normal p53 causes apoptosis (sudden Normal p53 causes apoptosis (sudden death) of abnormal cells. Apoptosis is also death) of abnormal cells. Apoptosis is also a cellular defense to DNA damage. a cellular defense to DNA damage.

NO inhibits apoptosis by phosphorylating NO inhibits apoptosis by phosphorylating p53.p53.

NO can further prevent apoptosis by other NO can further prevent apoptosis by other mechanismsmechanisms

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DiscussionDiscussion

these p53 mutant cells are now resistant to free these p53 mutant cells are now resistant to free radical-induced growth arrest and apoptosis radical-induced growth arrest and apoptosis

clonal selection and expansion of these p53 clonal selection and expansion of these p53 mutant cell occursmutant cell occurs

This uncontrolled growth leads detectable tumor.This uncontrolled growth leads detectable tumor. Thus, the authors propose a model of ulcerative Thus, the authors propose a model of ulcerative

colitis colon carcinogenesis that may also apply colitis colon carcinogenesis that may also apply to other chronic inflammatory conditions.to other chronic inflammatory conditions.

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Nitric oxideNitric oxidesynthasesynthase

NitriNitric c oxidoxidee

Phosph-Phosph-orylationorylationof p53of p53

ApoptosisApoptosis

NormalNormalp53p53

Mutated Mutated p53p53

CancerCancerNo apoptosisNo apoptosis

When DNA is When DNA is damaged..damaged..

When DNA isWhen DNA isdamaged…damaged…

Uncontrolled growthUncontrolled growth

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Thank you!Thank you!


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