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MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation...

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MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS R A D I A T I O N Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal Differentiation Defects in Growth Control Resistance to Cytotoxicity Defects in Programmed Cell Death Genetic Change Selective Clonal Expansion Genetic Change Genetic Change INITIATED CELL PRE- NEOPLASTIC LESION MALIGNANT TUMOR CLINICAL CANCER CANCER METASTASIS Genetic Change Activation of Proto-Oncogenes Inactivation of Tumor Suppressor Genes Inactivation of Genomic Stability Genes Nucleus
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Page 1: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

MOLECULAR CARCINOGENESIS

1044-CH

CHEMICAL

VIRUS

RADIATION

Body Surface

Deactivation Excretion

Activation

NORMALCELL

Inhibition

• Defects in Terminal Differentiation• Defects in Growth Control• Resistance to Cytotoxicity• Defects in Programmed Cell Death

GeneticChange

SelectiveClonal

ExpansionGeneticChange

GeneticChange

INITIATEDCELL

PRE-NEOPLASTIC

LESIONMALIGNANT

TUMORCLINICALCANCER

CANCERMETASTASIS

GeneticChange

• Activation of Proto-Oncogenes• Inactivation of Tumor Suppressor Genes• Inactivation of Genomic Stability Genes

Nucleus

Page 2: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

AcquiredDisease Tumor Site Risk

Viral

Hepatitis B Liver 88Hepatitis C Liver 3

Bacterial

Helicobacter Pylori Gastric 11PID Ovary 3

Parasitic

S. hematobium Urinary Bladder 2-14S. japonicum Colon 2-6Liver Fluke Liver 14

Chemical/ Physical

Acid reflex Esophagus 50-100

Metabolic Disease Obesity Colon 1.5

CHRONIC INFLAMMATION AND CANCER

InheritedDisease Tumor Site RiskHemochromatosis Liver 219Hereditary Pancreatitis Pancreas 120 Crohn’s Disease Colon 3Ulcerative Colitis Colon 6

“Chronic infection and associated inflammation contribute to about 1/3 of cancers worldwide”

-B.N. Ames, PNAS, 1995“18% of human cancers, i.e., 1.6 million per year, are related toinfection.”

- B. Stewart and P. KleihuesWorld Cancer Report, IARC Press, p. 57, 2003

Page 3: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

3000-CH

CANCERS ASSOCIATED WITH OBESITY

• Breast (postmenopausal)• Endometrium• Cervical• Ovarian• Colorectal• Kidney• Liver/ Gall Bladder • Pancreatic• Esophageal• Hematopoietic

• Prostate• Colorectal• Kidney• Liver/Gall Bladder• Pancreatic• Esophageal• Hematopoietic

In MenIn Women

Calle, E et al., NEJM 348:1625-38, 2003

Page 4: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

2786*-CH

REACTIVE NITROGEN AND OXYGEN SPECIES DERIVED FROM INFLAMMATORY CELLS

Myeloperoxidase

NO2•

H2O2HOClHOBr

Oxidation & Halogenation

Oxidation & Nitration

Neutrophil

NO2-

+Cl-/Br-

NO•

O2•

N2O3 Deamination

Macrophage

ONOOS

OD

H2O2

ONOOCO2-

Nitrosoperoxycarbonate

Nitrous Anhydride

NO2•

CO3•

OH •iNOS

-

O2

CO2

-Oxidation & Nitration

Of DNA and Proteins

Page 5: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

1760-CH

FREE RADIALS AND INFLAMMATION

ROS •OH O2- •(Hydroxyl (Superoxide) radical)

RNS NO • ONOO- N2O3(Nitric Oxide) (Peroxynitrite) (Nitroxyl Radical)

MDA(malondialdehyde)

4HNE(4-hydroxynonenal)

DNA Damageand Mutation

Nitrosamines/Deamination8--oxo-dG8-nitroguanineEtheno AdductsM1G AdductS-nitrosothiolSSB’sDSB’s

Lipid Peroxidation

Arachidonic AcidCascade

Eicosenoids

Cell Proliferation

Protein Damage (DNA Repair Enzymes, Caspases)

Page 6: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

CaM

CaM

CaM

Ca+++2

L-ARGININE

NO

NO

CaM

CaM

InactivecNOS

InactivecNOS

ActivecNOS

iNOSAlways active

Billiar

NITRIC OXIDE SYNTHASE

CaM

Citrulline

Page 7: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

p53 MODIFICATIONS

p53

P-Ser-15

P-Ser-20

P-Ser-33

P-Ser-46

P-Ser-315

P-Ser-392

K-Lys-382

2016*-CH

NITRIC OXIDE DAMAGES DNA AND ACTIVATES p53 IN MCF-7 CELLS

DNA DAMAGE

SPER/NO

MCF-7 Cells

Page 8: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

% Cells in G2/M

Mitotic Index

2122A*-CH

DOWNSTREAM PROTEINS

NO-INDUCED p53 PHOSPHORYLATIONTRANSACTIVATES DOWNSTREAM PROTEINS

AND ENGAGES A G2/M ARREST

Page 9: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

INDUCIBLE NITRIC OXIDE SYNTHASE (NOS2)AND CYCLOOXYGENASE-2 (COX2)

INTERACTIONS IN HUMAN CARCINOGENESIS

Hypoxia

1079A*-CH

Cytokines

NOS2

NO

Genomicinstability

Apoptosis

Mutantp53

• Selective Clonal Expansion• DNA damage

Lipid Peroxidation

p53

HIF1

e.g., IL-1TNF-

COX2 Prostaglandins (e.g., PGE2)

p53NFB

Hypoxia

K-ras

Page 10: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

1909-CH

Page 11: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

2978-CH

VENN DIAGRAM OF 1396 “p53-DEPENDENT” GENES MODULATED BY

CELLULAR STRESS

NO H2O2

HU Hypoxia

140 genes

139 genes

666 genes

225 genes7

5

29

40 14

4

1433

11

Hypoxia

35

HU

34

(T-test at p<0.001 for each treatment and time point)

Page 12: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

EXAMPLES OF CHRONIC INFLAMMATORY CONDITIONS ASSOCIATED WITH

INCREASED p53 MUTATION LOAD

• ULCERATIVE COLITIS

• HEMOCHROMATOSIS

• WILSON DISEASE

• VIRAL HEPATITIS

Page 13: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

p53 MUTATION LOAD IS INCREASED IN ULCERATIVE COLITIS

054-PH

UC vs. Non-UC (p < 0.001)

UC vs. Non-UC ( p < 0.001)

ULCERATIVE COLITIS

NORMALCONTROL

G TO A (CpG SITE OF CODON 248 )

C TO T (CODON 247)

0

10

20

30

Ab

solu

te M

uta

tio

n f

req

uen

cy x

10

-7

Page 14: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

1908-CH

Page 15: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

509B*-CH

• DNA Repair • Homologous

Recombination• Chromosomal

Segregation

Transcription

Senescence

Programmed Cell Death

p53(1979)

Development

Cell CycleCheckpoints

Page 16: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

p21WA F1

14-3-3 Gadd45

GADD45, p48, p53R2APE1, Pol

PUMA, NOXA, BAX, Apaf1,

XPB, XPD, WRN, BLM

p21WAF1

Others

NOS2

ATM, ATR, CHK2 p14ARF

mdm2

E2F

1306I*-CH

DNA RepairDNA Repair

p53

ApoptosisApoptosis

DNA Damage Oncogene ActivationHypoxia

p53 IS AT THE CROSSROADS OF CELLULAR STRESS RESPONSE PATHWAYS

Cell CycleCell CycleCheckpointsCheckpoints SenescenceSenescence

Page 17: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

EXAMPLES OF p53 NEGATIVE FEEDBACK LOOPS

• Posttranslational Modification and Proteolytic Cleavage

Oliver et al., Nature 362: 857, 1993Wu et al., Genes Dev. 7: 1126, 1993

• ATM-Dependent DNA Damage Pathway

Matsui et al., J. Biol. Chem. in press, 2004

• Nitric Oxide Pathway

2900-CH

Ubiquitination

Inducible Nitric Oxide Synthase DNA Damage

p53 MDM2

CHK2 Kinase p53 PhosphorylationTransrepression

p53Transrepression

Forrester et al., PNAS 93: 2442, 1996Ambs et al., PNAS 95: 8823, 1998

Page 18: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

Cytochrome C+

ProCaspase 9+

APAF1

APOPTOSOME

EXECUTIONERCASPASES

Lipid Peroxidation

Fe2+

1996A*-CH

p53

MitochondrialDepolarization

PUMA, NOXA, BAX, p53AIP1PIG3Ferredoxin reductase

O2 H2O2 H2O + O2

MnSOD GPX1

CAT•

MODEL OF CELLULAR STRESS INDUCED p53 ACTIVATION AND APOPTOSIS

Page 19: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

Caspase 3

APOPTOSIS

PUMA, NOXA, p53AIP1, Bax

Mitochondrial depolarization

Cytochrome C

1917*-CH

Apoptotic stimuli

RE

p53

Apaf-1Pro-caspase 9Apoptosome

APAF-1 IS A TRANSCRIPTIONAL TARGET OF p53 IN DNA DAMAGE-INDUCED APOPTOSIS

Page 20: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

CELL DEATH

1935-CH

Programmed Non-programmed

Page 21: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

MUTATIONAL SPECTRA OF THE p53, APC, ATM AND BRCA-1 GENES IN ALL HUMAN CANCERS

797B-CH

Missense 75%

Frameshift 9%

p53 (n=15,122)

ATM (n=617)

Nonsense 7%

Splice site 2%

Frameshift56%

Nonsense 14%

Missense 28%

In Frame Del/Ins. 2%Silent 5%

APC (n=1,451)

Nonsense 32%

Missense 4%

Splice site 4%

Nonsense 11%

Missense 30%

Splice site 5%

BRCA-1 (n=3,703)

Frameshift 54%

Silent 9%

Frameshift51%

In Frame Del/Ins. 2%

Page 22: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

249

157

Sequence-Specific DNA Binding Domain

N C

EVOLUTIONARILY CONSERVED17-29 97 292 324 352

Missense

Transactivation Domain

Oligomerization andNuclear Localizationand Export Domains

0

567L-CH

Tobacco SmokingLung ,Codon 157

G:C to T:A 78%

EXAMPLES OF p53 MUTATION HOTSPOTS ASSOCIATED WITH CARCINOGEN EXPOSURE

Aflatoxin B1 and HBVLiver, Codon 249

G:C to T:A 98%

281

SunlightSkin, Codon 281

CC to TT 100%

400

200

100

300

HemochromatosisLiver, Codon 220

220

A:T to G:C 100%

Page 23: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

HYPOTHESIS:

• p53 mutation hotspots in clonally derived human cancers reflect the preferential:• sites of carcinogen-DNA adduct formation in

the gene• sites of slow repair of DNA damage• mutagenic potential of certain carcinogen-DNA

adducts• pathobiological effects of the p53 mutant

leading to a selective clonal expansion advantage, including “gain of function” or an increase in genomic instability

1156-CH

Page 24: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

< 3.3

< 5.6

< 9.0

< 15.0

< 98.9

North AmericaN=15 Western Europe

N=82

AfricaN=28

JapanN=242

ChinaN=171

TaiwanN=113

G:C to C:G

G:C to T:A

G:C to A:TCpG

G:C to A:T Non-CpG

A:T to T:A

A:T to G:C

A:T to C:G

Del + ins.p53 MUTATION DIAGRAM

Incidence of HCC per 100,000

2115-CH

WORLDWIDE p53 MUTATIONAL SPECTRA IN HCC FROM DIFFERENT GEOGRAPHICAL AREAS

Page 25: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

• STRENGTH OF ASSOCIATION• Consistency

• Positive correlation in 3 different ethnic populations on 3 continents

• Temporality• 249ser p53 mutant cells observed in non-tumorous liver

in high HCC incidence geographic areas• Specificity

• 249ser p53 mutations are uncommon in other cancer types

• 249ser p53 mutation in serum DNA is a biomarker of liver cancer risk

ASSESSMENT OF CAUSATION BY THE BRADFORD-HILL CRITERIA

From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998 926C-CH

HYPOTHESIS: Dietary aflatoxin B1 exposure can produce 249ser (AGG->AGT) p53 mutations during human liver carcinogenesis

Page 26: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

• BIOLOGIC PLAUSIBILITY

• AFB1 is a potent carcinogen in rodents, monkeys and humans

• AFB1 is enzymatically activated by human hepatocytes to 8,9-AFB1 oxide that binds to DNA, including the 3rd base (G) at codon 249

• AFB1 exposure to human liver cells in vitro produces codon 249ser p53 mutations

• 249ser p53 expression inhibits apoptosis and p53-mediated transcription and enhances liver cell growth rates in vitro

ASSESSMENT OF CAUSATION BY THE BRADFORD-HILL CRITERIA

From: Hussain and Harris, Cancer Res. 58: 4023-37, 1998 926D-CH

HYPOTHESIS: Dietary aflatoxin B1 exposure can produce 249ser (AGG->AGT) p53 mutations during human liver carcinogenesis

Page 27: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

p53 CODON 249ser MUTANT IN SERUM DNA AND SERUM HBVSAg ARE

BIOMARKERS OF LIVER CANCER RISK• HBSAg/249p53 mutant RR(95%CI)

minus/minus 1

plus/minus 10(5-20)

minus/plus 13(5-35)

plus/plus 399(49-3272)Kirk, GD et al., Proc. 11th Int. Symposium on Viral Hepatitis and

Liver Diseases, Sydney, 2003.

FORTY PERCENT OF LIVER CANCER IN QIDONG, PRC IS ATTRIBUTABLE TO AFLATOXIN DIETARY EXPOSURE

Ming, l et al., Hepatology 36: 1214-20, 2002.

Page 28: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.
Page 29: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

COLORECTAL CARCINOGENESIS

• SPORADIC:

Normal Adenomatous Mucosa Polyps Carcinoma

• ULCERATIVE COLITIS ASSOCIATED:

Ulcerative Colitis Dysplasia Carcinoma

885-CH

Mutation

K-ras APC -catenin p53~45% 85% ~10% ~55%

~15% 6% ?% ~55%

Page 30: MOLECULAR CARCINOGENESIS 1044-CH CHEMICAL VIRUS RADIATIONRADIATION Body Surface Deactivation Excretion Activation NORMAL CELL Inhibition Defects in Terminal.

EXAMPLES OF GENETIC LESIONSIN BRONCHIAL DYSPLASIA,

CARCINOMA-IN-SITU AND LUNG CARCINOMALesion Dysplasia CIS Carcinoma• LOH• 3p12, 14, 21• 9p21• 17p13

• p53 (p17p13)• p16 (9p21)• Telomerase• Ki-ras• FHIT (3p14)• Rb

1049-CH


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