Molecular Biology of Cancer
description
Transcript of Molecular Biology of Cancer
Molecular Biology of Cancer
Yuki JuanNTUST
Jun 9, 2003
Outline The cell cycle and its control What is the molecular nature of cancer How do oncogenes cause cancer What are the roles of tumour suppressor genes
Terms Telomeres Telomerase Benign tumor Metastasis Leukaemias
Growth Characterisitics of Normal and Cancerous Animals Cells In Culture
Outline The cell cycle and its control What is the molecular nature of cancer How do oncogenes cause cancer What are the roles of tumour suppressor genes
Cell Cycle Check Points
Control Mechanism
Outline The cell cycle and its control What is the molecular nature of cancer How do oncogenes cause cancer What are the roles of tumour suppressor genes
Terms Carcinogenic Phorbol ester and diacylglycerol (DAG)
What Types of Genetic Changes Are Involved In Generation Of The Cancerous State Oncogenes Tumor suppressor genes
Retinoblastoma genes p53 genes
DNA repair genes
How Are Oncogenes Acquired Thay may be introduced by viral infection Normal genes involved in control may mutate and become oncogenic Oncogen and protooncogene
Outline The cell cycle and its control What is the molecular nature of cancer How do oncogenes cause cancer What are the roles of tumour suppressor genes
Oncogenicity May Include One or More of the Following Signal transduction pathways leading
to formation of active transcription factors may be inappropriately activated.
There may excessive amounts of particular transcription factors.
There may be abnormal transcription factors which are inappropriately active.
Oncogen Products As Components of Signalling Pathways Normal receptor-not activated
Normal receptor-activated
Permanently active truncated EGF receptor
Normal receptor-pathway component permanently active
Outline The cell cycle and its control What is the molecular nature of cancer How do oncogenes cause cancer What are the roles of tumour suppressor genes
Tumour Suppressor Genes P53 genes
P53 protein: a transcription factor known to have several roles in cellular control P53 p21 inactives the G1-specific Cdk activity Apoptosis
Retinoblastoma genes Retinoblastoma protein blocks the cycle at the G1 checkpoint
My Study
Yuki Juan
Hsueh-Fen Juan ( 阮雪芬 )Nov 8, 2002
Proteomics and Bioinformatics on
Biomedical Sciences
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Biochemical Context of Genomics and Proteomics
DNA
mRNA
Proteins
Cell functions
Genome “Genomics”
Proteome“Proteomics”
The Information Stored in Genes Is Expressed by a Multistage Process
Eur J Nucl Med 2002, 29, 115-32
Nature 2000, 405, 837-846
Two-dimensional Gel Approach
Standard Proteome Analysis by 2DE-MS
Current Opinion in Chemical Biology 2000, 4:489–494
Mass Fingerprint Searching in http://www.expasych/tools/peptident.html
Proteomics Applied to Drug-discovery
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Our StudySerum from cancer nude mice
Tumor markers
Proteomics
Cancer cells
Serum
Control
Protein ID by MS/MS
Nude mice
PBS Treatment
Control
1 23
4
5 6 78
91415 13
12
10 11
pH 3 10
C1
C2 C3C4
C5 C6 C7
C8
H1H2
J1 J2
J3 J4 J5
S1
S2S3 S4
S5S6
S7
S8
S9 S10
S11
S12 S13
pH 3 10
97.066.0
45.0
30.0
20.1
14.4
97.066.0
45.0
30.0
20.1
14.4
97.066.0
45.0
30.0
20.1
14.4
97.066.0
45.0
30.0
20.1
14.4
97.066.0
45.0
30.0
20.1
14.4
CCM1
GBM HONE
J5 SCM1
G1 G2
G3 G4
G5
G6
97.066.0
45.0
30.0
20.1
14.4
Peptide Mass Fingerprinting
http://us.expasy.org/tools/peptident.html
Integrated Protein Identification Tools
MALDI Peptide Map and Identification of a Protein
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Create Summary ReportUsing NCBI nr database
to perform query
Get pI value of the first data
Get pI value of the second data
Get pI value of the third data
Create summary report of NCBI nr query
PMIhttp://www.taai.org.tw/MassData/Progress Rate
Query Result
PMI Query Result
a. MALDI: LC/MS/MS: The LC/MS/MS total score for a match contains contributions from the peptide mass matches, ions scores (from queries with MS/MS data), and amino acid sequence or composition qualifiers (sequence queries). Score is -10Log(P), where P is the probability that the observed match is a random event. b. Molecular weight observed on 2D gel / Theoretical molecular weight
Spot. Protein identified as Method NCBI protein Scorea Number of %Coverage Molecular No. accession peptides weight
number matched
1~4 Apolipoprotein A-IV MALDI gi|109575 7.85E+05 15 34% 445735~8 Apolipoprotein A-I MALDI gi|231557 1.01E+05 13 46% 305889 Prealbumin MALDI gi|136465 1.14E+03 8 71% 1577610,11 serum albumin MALDI gi|3647327 2.97E+04 13 27% 6869312 alpha-fetoprotein MALDI gi|416601 2.39E+04 9 26% 4722613 hemoglobin beta-1 chain MALDI gi|50150 1.34E+04 8 53% 1584014,15 Immunoglobulin light chain LC/MS/MS gi|1002392 145 2 28% 12170C1~C4 Apolipoprotein A-IV MALDI gi|109575 6.46E+03 14 28% 44573C5~C8 Apolipoprotein A-I MALDI gi|111022 3.80E+04 12 44% 30588G1,G2 Serum albumin MALDI gi|3647327 9.57E+09 24 45% 68693G3,G4 alpha-fetoprotein MALDI gi|416601 4.46E+04 16 37% 47226G5 apolipoproein A-I MALDI gi|6753096 343 5 24% 30588G6 Macrophage inflammatory LC/MS/MS gi|127086 34 1 10% 11250
protein-2-beta precursorG7 hemoglobin beta-1 chain MALDI gi|122513 664 10 79% 15840H1 Hemoglobin, beta adult major LC/MS/MS gi|12846963 65 1 8% 15103
chain~putativeH2 Haptoglobin LC/MS/MS gi|8850219 57 1 3% 10K / 38727b
J1~J4 Serum albumin variant LC/MS/MS gi|20835629 98 2 3% 27K / 68648b
J5 Hemoglobin beta-1 chain MALDI gi|122513 1.03E+03 8 65% 15840S1~S6 Haptoglobin MALDI gi|193754 9.42E+03 11 29% 38753S7 Haptoglobin LC/MS/MS gi|8850219 83 2 7% 10K / 38727b
S8 Serum amyloid P component MALDI gi|134198 119 6 26% 26247S8 Serum amyloid P component LC/MS/MS gi|7363449 52 2 9% 26329S9 Serum amyloid A-2 MALDI gi|6755394 140 6 43% 13622S9 Serum amyloid A-2 LC/MS/MS gi|6755394 111 3 26% 13622S10 Serum amyloid A-1 MALDI gi|6677843 403 7 56% 13770S10 Serum amyloid A-1 LC/MS/MS gi|6677843 242 5 37% 13770S11 Hemopexin MALDI gi|1881768 1.96E+05 14 29% 50546S12,S13 Apolipopretein A-I MALDI gi|231557 9.66E+17 14 38% 30588
Protein Identification
Acute Phase Proteins in Nude Mice Inoculated with Different Cancer Cells
Markers CCM1 GBM HONE J5 SC-M1
Haptoglobin
+ + + + +
Serum amyloid A
- - - - +
97
66
45
30
14.4
20.1
5 6
S9 S10
MW
Identification of Serum Amyloid A in Serum of Nude Mice Inoculated with Cell Line of Stomach Adenocarcinoma SC-M1
Quantification of S9 and S10
S9( 2 fold) S10 (+)Control Control (-)
Analysis by Porgenesis
100 200 300 400 500 600 700 800 900 1000 1100
175.14
232.17 375.29
526.34 655.4
783.46
930.56 1017.48
R G F F E Q F S yn
m / z m / z
100 200 300 400 500 600 700 800 900 1000 1100
175.14
232.17
375.29
526.34
655.40
783.46
930.56
1001.61
R G F F E Q F A yn
S9: Serum Amyloid A-2 S10: Serum Amyloid A-1
Nanoelectrospray Mass Spectrometry
1 M R A L G A V V T L L L W G Q L F A V E L G N D A M D F E D D S C P K P P E I A N G Y V E H L V R Y
51 R C R Q F Y R L R A E G D G V Y T L N D E K Q W V N T V A G E K L P E C E A B C G K P K H P V D Q V
101 Q R I I G G S M D A K G S F P W Q A KM I S R H G L T T G A T L I S D Q W L L T T A K N L F L N H S
151 E T A S A K D I T P T L T L Y V G K N Q L V E I E K V V L H P N H S V V D I G L I K L K Q R V L V T
201 E R V M P I C L P S K D Y I A P G R V G Y V S G W G R N A N F R F T D R L K Y V M L P V A D Q D K C
02-051197000
66000
45000
30000
14400
20100
5 6
Control
S7
Haptoglobin ( α form ) in Serum of Nude Mice Inoculated with Cell Line of Stomach Adenocarcinoma SC-M1
b 129.07 315.15 414.21 528.26 629.3 728.37 799.41 856.43 985.47Gln Trp Val Asn Thr Val Ala Gly Glu Lys
y 1003.5 817.44 718.37 604.33 503.28 404.21 333.18 276.16 147.11
m / z
100 200 300 400 500 600 700 800 900 1000 1100
147.13 276.20
333.22
404.26
503.35
604.39
718.45
817.53
1003.60
K E G A V T N V W yn
02-051197000
66000
45000
30000
14400
20100
5 6
SCM1-1( 胃癌 )
Haptoglobin(β form )
Acute Phase Response In mice, Haptoglobin and hemopexin The early stages of inflammatory responses are characterized by the rapid synthesis of a heterogenous group of plasma proteins known as acute phase reactants (proteins). In man, C-reactive protein (CRP) or serum amyloid A component (SAA) 1000-fold
Clin Exp Immunol 1986, 66(2):331-9Scand J Immunol 1984, 19: 193-198
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Human myeloid leukemia cells
Macrophages
Genomic Study
Proteomics
Bioinformatics
Our Study
Macrophage
12-O-tetradecanoyl-phorbol-13-acetate
The Structure of TPA
Myeloblast Macrophage
Proteins ?Proteins ?
HL-60 CellHL-60 Cell
HL-60 Leukemia Cell Differentiate to Macrophage
Control Treated with TPA
HL-60 Leukemia Cell Differentiate to Macrophage
3.5 1010
42
6070
150kDa
pH
Increase of 50%
Decrease of 50%
Unmatched spots
Matched spots
Image Matching
kDa150
70
60
42
10
3.5 10 3.5 10pH pH
Control Treated with TPA1
1223
4 5
67
8 9
10 11
13
1418
1521
20
16
1719
12
3
45
678
9
10 1112
13
14
15
16
17
18
19
20
21
2323
22 22
24
25
26
27
2829
30
2425
26
27
28 29
30
35
31
36
33
3238
34
37
35
3631
33
32
38
34
37
2D Electrophoresis
Guanine nucleotide-binding protein G (k), alpha subunit G-rich sequence factor-1 (GRSF-1) Myosin light chain alkali, non-muscle isoform Phosphatidylinositol-4-phosphate 5-kinase type II alpha Myelin basic protein (MBP) Smooth muscle cell LIM protein (cystein-rich protein 2) Histidyl-tRNA synthetase homolog Transforming growth factor beta 4 precursor Visinin-like protein 3 Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial
precursor Amiloride-sensitive brain sodium channel BNaC1 KIAA0413 Alpha enolase Antileukoproteinase 1 precursor Angiotensionogen precursor
Up-regulated Proteins
Calreticulin precursor PINCH protein Alpha enolase Retinoic acid receptor RXR-alpha Alpha-fetoprotein precursor 47k protein T-complex protein 1, zeta subunit Protein kinase C, delta type Norrin Ubiquitously expressed transcript protein 11-cis retinol dehydrogenase Heterogeneous nuclear ribonucleoproteins A2/B1 Heterogeneous nuclear ribonucleoprotein A1
Down-regulated Proteins
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
The functions of the differential expressed proteins
Classification of these proteins
Searching Protein Functions by BGSShttp://www.sinica.edu.tw:8900/perl/
genequery2.plhttp://servx8.sinica.edu.tw/bgss-cgi-bin/protein.pl
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Pathway Database ConstructionBioMiner
Pathway Database Pathway sites available now
KEGG BioCarta
How to build up the DB Data Source
Site Name Web LinkKEGG http://www.genome.ad.jp/kegg/metabolism.html
BioCarta http://www.biocarta.com/genes/index.asp
SwissProt http://www.expasy.ch/sprot/
UniGene http://www.ncbi.nlm.nih.gov/UniGene/
BioMiner Integrate KEGG and Biocarta pathway database
Input: AC no from microarray or proteomics data
KEGG or Biocarta
Count how many genes or proteins are changed in these pathways
BiocartaBiocarta: http://www.biocarta.com/
caspase
Biocarta
KEGGhttp://www.genome.ad.jp/kegg/
KEGG
How to build up the DBPoint construction
(KEGG)
proteinscorresponding tothe pathway
EC number, proteinnames andpathways
catalogs name,URL, pathwaysname and URLcorresponding tothe catalog.
Root Page
Pathway Page
Protein Page
How to build up the DBPoint construction (BioCarta)
Swissprot id,Unigene id, andnames from both
pathwaycorresponding tothe catalog
Protein-pathwaymapping
catalog name, URL
Root Page
Catalog-PathwayPage
Pathway page
Protein page
Protein name,SWISSPROT link,Unigene link
Link page
Service File upload Batch query
http://uranus.csie.ntu.edu.tw:4051/
Service
Cell to Cell Adhesion Signal
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
Using proteomics and bioinformatics, we identified Cancer-specific protein marker---SAA Expressed proteins, functions and related
pathways during myeloid differentiation. Using proteomics and bioinformatics,
we can do: Clinical Diagnosis of disease Drug-discovery
Conclusion
Outline Introduction Identification of acute phase proteins in nude mice inoculated with different cancer cells
Bioinformatics Peptide Mass Integrator (PMI)
Biomic study of human myeloid leukemia cell differentiated to macrophage Bioinformatics
Bulk Gene Search System (BGSS) Pathway Database Construction (BioMiner)
Conclusion Outlook
OutlookIdentify Proteins
Drug Discovery
Structures
Protein-Protein Interactions
Pathways
Protein Functions
Clinical Diagnosis of disease
Protein-protein Interaction Prediction Biological Experiments
Protein-protein Interaction Prediction Biological Experiments
SchemeSwiss-Prot entries Protein Names
Get Medline abstracts
Design Keywords for P-P Interactions{Interact, couple, phosphorylate, etc.}
a1 K1b2
a1K2
b2
a1 and b2 have interactions
Proposed Results Regulate Activate
p107
DRTF1/E2F
Protein-protein Interaction Prediction Biological Experiments
Yeast Two-hybrid System Useful in the study of various interactions The technology was originally developed
during the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature).
Yeast Two-hybrid Assay
GAL4 DNA-binding domain
GAL4 DNA-activation domain
Nature, 2000
Yeast Two-hybrid Assay Library-based
yeast two-hybrid screening method
Nature, 2000
The Study of Protein-protein Interaction by Mass Spectrometry
bait
S14????
* ***
SDS- PAGE
MASS
Peptide Mass Fingerprinting
Protein-protein Interactions on the Web
Yeast http://depts.washington.edu/sfields/yplm/data/index.html http://portal.curagen.com http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://www.pnas.org/cgi/content/full/97/3/1143/DC1 http://dip.doe-mbi.ucla.edu/ http://genome.c.kanazawa-u.ac.jp/Y2H C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html
ACKNOWLEDGEMENTS Identification of acute phase proteins in nude mice inoculated with different cancer cells
Proteomics 陳水田 研究員 陳振漢 醫師 陳玉如 博士
PMI 許聞廉 研究員
Biomic study of human myeloid leukemia cell differentiated to macrophage Proteomics
陳水田 研究員 曾銘仁 教授 張文輝
BGSS 高成炎 教授 黃宣誠 博士
BioMiner 歐陽彥正 教授