molecular biology of cancer yuki juan ntust jun 9, 2003

Molecular Biology of Cancer

Yuki JuanNTUST

Jun 9, 2003

Outline The cell cycle and its control What is the molecular nature of cance

r How do oncogenes cause cancer What are the roles of tumour suppress

or genes

Terms Telomeres Telomerase Benign tumor Metastasis Leukaemias

Growth Characterisitics of Normal and Cancerous Animals Cells In Culture



or genes

Cell Cycle Check Points

Control Mechanism



or genes

Terms Carcinogenic Phorbol ester and diacylglycerol (DA

G)

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 infec

tion Normal genes involved in control may

mutate and become oncogenic Oncogen and protooncogene



or 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



or 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 mi

ce inoculated with different cancer cells Bioinformatics

Peptide Mass Integrator (PMI) Biomic study of human myeloid leukemia cell dif

ferentiated 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




Conclusion Outlook

Our Study

Serum from cancer nude mice

Tumor markers

Proteomics

Cancer cells

Serum

Control

Protein ID by MS/MS

Nude mice

PBS Treatment

Control

12

34

56 7

8

91415 13

12

10 11

pH 3 10

C1

C2 C3C4

C5C6 C7

C8

H1H2

J1J2

J3 J4J5

S1

S2S3

S4

S5S6

S7

S8

S9S10

S11

S12 S13

pH 3 10

97.0

66.0

45.0

30.0

20.1

14.4

97.0

66.0

45.0

30.0

20.1

14.4

97.0

66.0

45.0

30.0

20.1

14.4

97.0

66.0

45.0

30.0

20.1

14.4

97.0

66.0

45.0

30.0

20.1

14.4

CCM1

GBM HONE

J5 SCM1

G1G2

G3 G4

G5

G6

97.0

66.0

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






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% 44573

5~8 Apolipoprotein A-I MALDI gi|231557 1.01E+05 13 46% 30588

9 Prealbumin MALDI gi|136465 1.14E+03 8 71% 15776

10,11 serum albumin MALDI gi|3647327 2.97E+04 13 27% 68693

12 alpha-fetoprotein MALDI gi|416601 2.39E+04 9 26% 47226

13 hemoglobin beta-1 chain MALDI gi|50150 1.34E+04 8 53% 15840

14,15 Immunoglobulin light chain LC/MS/MS gi|1002392 145 2 28% 12170

C1~C4 Apolipoprotein A-IV MALDI gi|109575 6.46E+03 14 28% 44573

C5~C8 Apolipoprotein A-I MALDI gi|111022 3.80E+04 12 44% 30588

G1,G2 Serum albumin MALDI gi|3647327 9.57E+09 24 45% 68693

G3,G4 alpha-fetoprotein MALDI gi|416601 4.46E+04 16 37% 47226

G5 apolipoproein A-I MALDI gi|6753096 343 5 24% 30588

G6 Macrophage inflammatory LC/MS/MS gi|127086 34 1 10% 11250

protein-2-beta precursor

G7 hemoglobin beta-1 chain MALDI gi|122513 664 10 79% 15840

H1 Hemoglobin, beta adult major LC/MS/MS gi|12846963 65 1 8% 15103

chain~putative

H2 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% 15840

S1~S6 Haptoglobin MALDI gi|193754 9.42E+03 11 29% 38753

S7 Haptoglobin LC/MS/MS gi|8850219 83 2 7% 10K / 38727b

S8 Serum amyloid P component MALDI gi|134198 119 6 26% 26247

S8 Serum amyloid P component LC/MS/MS gi|7363449 52 2 9% 26329

S9 Serum amyloid A-2 MALDI gi|6755394 140 6 43% 13622

S9 Serum amyloid A-2 LC/MS/MS gi|6755394 111 3 26% 13622

S10 Serum amyloid A-1 MALDI gi|6677843 403 7 56% 13770

S10 Serum amyloid A-1 LC/MS/MS gi|6677843 242 5 37% 13770

S11 Hemopexin MALDI gi|1881768 1.96E+05 14 29% 50546

S12,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 129.07 315.15 414.21 528.26 629.3 728.37 799.41 856.43 985.47

Gln 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 respon

ses 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



Peptide Mass Integrator (PMI) Biomic study of human myeloid leukemia cell

differentiated to macrophage Bioinformatics


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

150

kDa

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 TPA

1

1223

4 5

6

7

8 9

10 11

13

14

18

1521

20

16

17

19

1

2

3

45

67

89

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

32

38

34

37

35

36

31

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






Conclusion Outlook

The functions of the differential expressed proteins

Classification of these proteins

Searching Protein Functions by BGSS

http://www.sinica.edu.tw:8900/perl/genequery2.plhttp://servx8.sinica.edu.tw/bgss-cgi-bin/protein.pl

Outline






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

databaseInput: 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

KEGG

http://www.genome.ad.jp/kegg/


Point construction (KEGG)

proteinscorresponding tothe pathway

EC number, proteinnames andpathways

catalogs name,URL, pathwaysname and URLcorresponding tothe catalog.

Root Page

Pathway Page

Protein Page


Point 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






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






Conclusion Outlook

Outlook

Identify Proteins

Drug Discovery

Structures

Protein-Protein Interactions

Pathways

Protein Functions

Clinical Diagnosis of disease

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 p

hase 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

molecular biology of cancer yuki juan ntust jun 9, 2003

Documents

active slide

culture slide

checkpoint slide

protooncogene slide

control mechanism slide

oncogenes cause cancer

molecular nature of

diacylglycerol dag slide