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Slide 1
Diseases assotiated with abnomal signal transduction and
cellular proliferation and apoptosis
Slide 2
Cell proliferation Cell entered the cell cycle sequentially
under the accurate regulation. Significance of cell proliferation
1. Cell renewing 2. Providing resource for cell differentiation 3.
Supplement of dead cells Basal concept
Slide 3
Cell apoptosis Apoptosis is a form of programmed cell death in
multicellular organisms triggered by external and internal signals
under some physio- or pathological conditions. Significance of
apoptosis 1. Maintaining homeostasis of the body 2. Facilitating
development of organs 3. Removing the damaged cell
Slide 4
One Cell proliferation and apoptosis are regulated by genes.
Two Regulation is implemented by gene products of oncogenes and
anti-oncogenes. Article 1 Regulation of cell proliferation and
apoptosis
Slide 5
1 cell proliferation and its regulation 1. cell cycle
definition: a period from the end of one division to the beginning
of next division of a proliferative cell. G 0 G 1 SG 2 M 2. Check
point of cell cycle G 0 / G 1 G 1 / S G 2 / M 3. Major cell cycle
regulatory proteins Cyclin, subtype: A-I, K T Cyclin-dependent
kinase, CDK 1-10 CDK inhibitor, CDKI One Gene regulation of cell
proliferation and apoptosis INK4 family: P15/16/18/19 CIP/KIP
family: P21/27/57
Slide 6
Expression of human cyclins through the cell cycle
Slide 7
Role of CDK in cell cycle cyclin Activity form
Slide 8
2 Proteins involved in cell apoptosis 1. Caspase family 2.
Bcl-2 family anti-apoptotic Bcl-2 pro-apoptotic Bax Bak/Bid Bad, et
al. 3. Tumor necrosis factor receptor TNFR Fas CD95 TNF-R1 DR4
DR5
Slide 9
Two Regulation of onco-gene and anti-oncogene by signal
transduction Normal cells Reproduce themselves exactly, stop
reproducing at the right time, stick together in the right place,
self destruct if they are damaged or become specialized or mature.
Cancer Is a class of diseases in which a group of cells display
uncontrolled growth, invasion, and sometimes metastasis resulted
from the disorder of cell proliferation, differentiation, aging and
death. Mechanisms mutations of onco- and anti-oncogenes caused by
mutagens and unstability of genes Onco-gene and anti-oncogene Main
genes of regulating cell proliferation and differentiation
Slide 10
DNA mutation causes abnormal cell proliferation and apoptosis
Normal cell DNA damage Somatic cell gene mutation Tumor suppressor
inactivation Quantitative and qualitative changes of gene products
Unbalance in cell proliferation and apoptosis Autoimmune disease
(insufficient apoptosis) tumor Degenerative disease in nervous
system (over apoptosis) Genetic faults DNA repair gene inactivation
Change of appoptosis regulatory gene expression Oncogene activation
DNA damage factor Successful DNA repairing DNA repairing
failure
Slide 11
Article 2 Oncogene, anti-oncogene and signal transduction
mediated by onco- and anti- oncogenes Oncogene Signal transduction
associated with cell growth Anti-oncogene (tumor suppressor gene
)
Slide 12
One oncogene, onc Basal concept These genes code for proteins
that are capable of stimulating cell growth and division. In normal
tissues and organisms, such growth-stimulating proteins are
regulated, so that growth is appropriately limited. However,
changes/mutation in these genes may result in loss of growth
regulation, leading to uncontrolled cell proliferation and tumor
development. These changed genes are known as oncogenes, because
they induce the oncogenic state cancer. Oncogenes are dominant,
because a change/mutation of only one allele of that gene can lead
to tumor formation.
Slide 13
1 Oncogene 1. Virus oncogene, v-onc These genes are in viruses,
may lead to uncontrolled cell proliferation and tumor development.
Virus oncogenes are homolog with that corresponding cellular
oncogenes. No intron Mutation pattern
Slide 14
Retrovirus Chronic transforming retrovirus wild type) RNA
virus: containing gene coding for transcriptase Basic structure and
function of retrovirus Genome: Two copies of double strand RNA, 3
9Kb Three structural genes (5-gag--pol--env--3) : gag--- core
protein pol---transcriptase env---coat protein Long terminal
repeats, LTR promoter enhancer adding signal sequence of polyA
Slide 15
General structure of retrovirus LTR
Slide 16
pol gag env
Slide 17
provirus Infectious course of retrovirus
Slide 18
Jumping replication
Slide 19
Provirus A provirus is a DNA mediate with LTR at both ends that
has integrated itself into the DNA of a host cell.
Provirusexpressionpackaged into new viral articlebudding outside
cell surface Resource of v-onc Definite DNA sequence captured from
host cell example: src ALV(no v-src)--------------ASV(containing
v-src) ( avian sarcoma virus, ASV )
Slide 20
Obtaining of v-onc Provirus DNA Cell genome DNA Virus DNA DNA
Integrating Transcripting, splicing Packaged into virus
article
Slide 21
Acute transforming retrovirus Structure features Having
different types of defects in structural genes, replication defect,
non-infective virus Inserting definite sequence-oncogene Only
containing one sort of oncogene Infection features Short latent
period Having ability to transforming the corresponding target
cells in vitro into malignant cells Exceptions Avian sarcoma virus,
ASV Oncogene locates in downstream of structural gene and does not
breakdown structural gene of virus. Avian erythroblastosis virus,
AEV Containing two sorts of oncogenes: erb-A and erb-B
Slide 22
ORF
Slide 23
2. cellular oncogene, C-onc Real copy of v-onc in eukaryotic
cell. An cellular oncogene is an activated form of pro-oncogene. A
proto- oncogene is a normal gene that can become an oncogene due to
mutations or increased expression. Proto- oncogenes code for
proteins that help to regulate cell growth and
differentiationhouse-keeping genes. Upon activation, a
proto-oncogene (or its product) becomes a tumor-inducing agent, an
oncogene. Proto-onc In normal cells, c-onc exists in non-activated
form.
Slide 24
Features of c-onc (1) Structral features with the general
structure of eukaryotic genes ; Different creatures: introns with
greater difference exons conservative sequence (2) Widespread in
living nature (3) Highly conservative in gene sequence (4)
Executing function by products of protein ; (5) Upon activation (
mutations or increased expression ), becomes a tumor-inducing
agent, an oncogene.
Slide 25
Differences between v-onc and c-onc V-onc often loses some
sequences at both ends No introns in v-onc V-onc is the mutated
form of c-onc
Slide 26
Classification of c-onc There are several systems for
classifying oncogenes, but there is not yet a widely accepted
standard. They are sometimes grouped both spatially (moving from
outside the cell inwards) and chronologically (parallelling the
"normal" process of signal transduction). Pro-onc mains codes for
key molecules involved in signal transduction and gene
transcription.
Slide 27
There are several categories that are commonly used: Category
Example Growth factors or mitogens c-sis Receptor tyrosine kinases
epidermal growth factor receptor(EGFR), platelet-derived growth
factor receptor(PDGFR), and vascular endothelial growth factor
receptor VEGFR Cytoplasmic tyrosine kinases src-family, syk-ZAP-70
family, and BTK family of tyrosine kinases, the Abl gene.
Cytoplasmic serine/threonine raf kinases, and cyclin-dependent
kinases. kinases Regulatory GTPases ras protein Transcription
factors myc, myb
Slide 28
1. Growth factors sis gene Homologous with B chain of PDGF
int-2, hst Homologous with fibroblast growth factor ( FGF 2. Growth
factor receptors C-erb-B EGFR C-fms Colony stimulating factor
receptor CSF-1 bit PDGFR ros insulin receptor mas angiotonin AGT
receptor or receptor TPK
Slide 29
3. Non-receptor tyrosine protein kinases src abl 4. GTP binding
protein H-ras K-ras N-ras ( 21KD- small G protein ) 5. Intranuclear
DNA binding protein Fos-Jun transcription factor AP-1 TRE TPA
reaction element CREB-JunCRE C-myc basic amio acid single/double
strand DNA Rel NF-B associated protein
Slide 30
2 Activation mechanisms of pro-onc 1 1 Insertion of regulatory
sequence ( promoter, enhancer) ALV with v-onc induces B cell
lymphoma Experiment of Leder group trans-oncogene animal model LTR
of mouse mammary tumor virus ( MTV ) C-myc MTV/myc 2. Gene mutation
Gene mutation within key modulin of cell carcinoma of bladder T24
cell C ras H T35 --- Val 12 normal G35 --- Gly 12 3 3 Gene
rearrangement Burkitt lymphoma 8q24(c-myc) 14q32 close to immune
globulin There is a strong enhancer in heavy chain region of immune
globulin, and causes overexpression of C-myc. Philadelphia
chromosome ( balanced translocation ) 9q34 22q11 C-abl
interchanging with bcr
Slide 31
LTR inserting near c-myc inserting
Slide 32
TCR T cell receptor Gene translocation Burkitt lymphoma
Slide 33
fusion gene of bcr-abl in CML Gene translocation Philadelphia
chromosome Chromosome 22-bcr geneChromosome 9-c-abl gene Breakpoint
in ALL Breakpoint in CML(bcr=5.8kb) Breakpoint in AML and CML 160KD
of Bcr protein140KD of Abl protein fusion gene of bcr-abl in ALL
18.5KD of fusion protein 210KD of fusion protein ALL: Acute
lymphoblastic leukemia ; CML: Chronic myelogenous leukemia
Slide 34
4. Gene amplification Homogeneously staining regions, HSR
Homogeneously staining regions except normal staining regions
Double minutes, DM Punctiform pseudochromosome 5 Gene couple Under
certain conditions, some pro-oncs are sequentially activated due to
activation of other pro-oncs. Products of oncogene promoting
immortalization of cell locate in nucleus. C-myc Products of
oncogene promoting proliferation of cell locate in cytoplasm. C-ras
6 Elimination of intergenic suppression Special fragment regulating
gene expression No.1 exon of C-myc gene dose not encode protein,
and may have inhibitory action,if loss, causes activation of c-myc.
Methylation of DNA increases stability of double helix.
Slide 35
3 Function of oncogene Immortalization of cell Blocking cell
differentiation retrieving cell apoptosis, often locating in
nucleus. Transformation of cell Decreasing dependence of cell
proliferation on growth factors often locating in cytoplasm or cell
membrane.
Slide 36
Extracellular signal: EGF PDGF and so on. GRB 2 SOS Raf
Regulating activity of other proteins MAPKK MAPK P P P fos jun myc
Regulating gene expression (cyclinD) P P Ras- GTP MAPKKK MEK ERK
PI3K PIP3 Akt Anti-apoptosis promoting proliferation PTEN - Two
Main signal transduction pathway associated with growth Having
mutation in 90% malignant tumor Ovarian cancer Pate cancer Breast
cancer Cell membrane Neuclus
Slide 37
Three Tumor suppressor gene Concept A tumor suppressor gene, or
anti-oncogene, is a gene that protects a cell from one step on the
path to cancer. Tumor-suppressor genes, or more precisely, the
proteins for which they code, either have a dampening or repressive
effect on the regulation of the cell cycle or promote apoptosis,
and sometimes do both. 1. Mostly are recessive genes and not easy
to be discovered. 2. Theoretically, the number of oncogenes matches
that of tumor suppressor genes. 3. The position of oncogene and
tumor suppressor gene may be equally important.
Slide 38
Discovery of tumor suppressor gene: 1. Analysis of cell
genetics 70s KundsonPredisposing gene of retinal glioblastoma
Retinoblastoma Rb deletion Existence of tumor suppressor genes 2.
Experiment of cell hybridization 70s HarrisHybridoma experiment
Proving the exsitence of tumor suppressor gene Cognitive process
oncoprotein oncogene tumor suppressor gene
Slide 39
Experiment of cell hybridization Normal cell Tumor cell
Nontumorous hybrid cell Cell fusion Tumor cell 1 Tumor cell 2 Cell
fusion Nontumorous hybrid cell Normal cell Loss of some genes Tumor
cell
Slide 40
p53 Predisposing gene of many kinds of tumors P53 So far, the
most relevant gene with human tumor. Mutation form 1. Complete loss
of p53 gene 2. p53 gene mutation : Two allelic inactivation of p53
One allele for p53 gene inactivation,molecules of p53 with mutation
dimerise with wild-type p53 and prevent them from executing
functions.
Slide 41
1. Gene localization 17p13.1 2. Gene structure 11 exons No.1
does not code for protein. 33. Protein structure 53kDa ( 7 domains
Th 20min ) p53 Acid regionCore regionBasic acid N- -C Easy to be
hydrolyzed by protease Phosphorylation site associated with protein
stabibility. DNA binding region Important functional position
Homo-oligomerisation ( tetramerization ) domain:many modification
sites associated with regulating DNA binding activity.
Slide 42
p53 pathway Activation form stability Transcription
activity
Slide 43
Slide 44
Cdk1
Slide 45
Mechanism of Growth arrest induced by P53 P53 protein Unwinding
enzyme Replication facor A P21protein Holding the cell cycle at G 1
phase Cytocidal DNA damage P21gene P53 protein Replication failure
Successful repair suppression
Slide 46
Slide 47
Retinoblastoma Rb 1. Gene localization 13q14 2. Gene structure
27 exons. 3. Protein structure 105kDa P105RB 4. Protein function
DNA binding protein binding E2F at phase G0 and G1; Inhibiting
transcription activity of E2F; Inhibiting expression of many
oncoproteins; Regulating cell cycle. 5. Activity regulation:
Phosphorylation decreasing its activity; Dephosphorylation
activation. 6. Regulatory factor: Cyclin-CDK Oligomerisation region
Binding region of E2F and viral oncoprotein DNA binding region
Slide 48
Mechanism of action of Rb gene G 0, G 1 phase Rb protein E-2F S
phase E-2F DNA mRNA DNA P Rb protein
Slide 49
cyclinE A and so on
Slide 50
Kundson Tow-hit hypothesis An inherited, germ-line mutation in
a tumor suppressor gene (Rb) would only cause cancer if another
mutation event occurred later in the organismss life, inactivating
the other allele of that tumor suppressor gene. * It shows for the
first time that disease of dominant heredity in family has
recessive mutant tumor suppressors alleles. * Mutation form of Rb:
Gene deletion and gene mutation; Also associated with osteogenic
sarcoma, prostatic carcinoma, small cell lung cancer and so
on.
Slide 51
Regulatory of RB and P53 on cell cycle
Slide 52
Function of tumor suppressor gene Inducing terminal
differentiation Triggering aging, inducing programed cell death,
maintaining gene stabilization Regulating cell growth negative
signal transduction Inhibiting activity of protease Changing
activity of DNA methylase. Regulating histocompatibility antigen
Regulating angiogenesis Promoting interconnection between
cells
Slide 53
Inactivation of anti-onc and tumorigenesis 1. gene deletion: A
pair of Rb alleles are missing or inactivated before onset 2. gene
mutation (1) Loss of Single or both p53 sites reduces concentration
of tetramers. (2) Nonsense mutation of P53 caused interruption of
translation (3) Loss of acid dmoain in C-terminal affects formation
of tetramers; (4) The most common missense mutation: wild-type and
mutant to form a more stable tetramer, the loss of normal function.
3. Excessive phosphorylation : 4. Binding with oncoprotein
Rb--SV40T antigen adenovirus E1A protein Human papilloma virus E7
protein. P53--SV40T antigen adenovirusE1B protein Human papilloma
virusE6 protein. Cause viral DNA replication protein to lose
activity. Cause Rb and P53 to lose negative regulatory function of
DNA replication.
Slide 54
Other anti-oncs participating in regulating signal transduction
APC SMAD4/DPC4 PTEN ( Phosphatase and tensin homolog ) PTEN PTCH (
Patched homolog ) Functions Regulating signal transduction
associated with embryonic development and cellular
proliferation.
Slide 55
APC ( Adenomatosis polyposis coli ) binding - catenins in the
cytoplasm and targeting -catenin for ubuiquitination and
degradation by cellular proteosomes ( Wnt/wingless pathway ).
SMAD4/DPC4:( Mothers against decapentaplegic homolog 4 ) It belongs
to the proteins which modulate members of the TGF protein
superfamily. It acts as a tumor suppressor that functions in the
regulation of the TGF- signal transduction pathway, which
negatively regulates growth of epithelial cells and the
extracellular matrix (ECM).
Slide 56
Related genes with DNA reparation MSH2 MLH1 BRCA1 BRCA2 and so
on: DNA reparation MSH2 MLH1: DNA mismatch repair
Slide 57
Four Regulation of cells is realized by the coordination of
multiple genes Coordination of pro-onc and anti-onc affects
cellular proliferation and apoptosis. Malignant cell proliferation
is a breach of the normal regulation of cell growth caused by
signal transduction.
Slide 58
Five Other diseases associated with cellular proliferation and
apoptosis Deficient apoptosis of cells leading to autoimmune
disease Excessive apoptosis of cells leading to neural degenerative
disease
Slide 59
Six Growth Factor and Receptor 1 Growth Factor 1.Concept A
growth factor is a naturally occurring substance capable of
stimulating cellular growth, proliferation and cellular
differentiation. Usually it is a protein or a steroid hormone.
Growth factors are important for regulating a variety of cellular
processes. 2. characteristics Microamount active material produced
by living cell, not nutritional ingredient. Is commonly a
polypeptide of 5-80kD, vulnerable to the effects of variability of
physical and chemical factors. Mediates biological effects by
binding to specific receptors on the surface of their target cells.
Has bidirectional biological effects.
Slide 60
Bone morphogenetic proteins (BMPs) Epidermal growth factor
(EGF) Erythropoietin (EPO) Fibroblast growth factor (FGF)
Granulocyte-colony stimulating factor (G-CSF)
Granulocyte-macrophage colony stimulating factor (GM-CSF) Growth
differentiation factor-9 (GDF9) Hepatocyte growth factor (HGF)
(HDGF) Insulin-like growth factor (IGF) Myostatin (GDF-8) Nerve
growth factor (NGF) and other neurotrophins Platelet-derived growth
factor (PDGF) Thrombopoietin (TPO) Transforming growth factor
alpha(TGF-) Transforming growth factor beta (TGF-) Vascular
endothelial growth factor (VEGF) Individual growth factor proteins
tend to occur as members of larger families of structurally and
evolutionarily related proteins. There are many families which are
listed below Diversity Origin Target cells Biological effects
Slide 61
2 Growth Factor Receptors 1.Concept Locat on the cell surface
or sub-cells, the protein can specifically recognize and bind its
ligand growth factors, and transduct growth factor signals into
intracellular, thus activat a series of biological reactions. 2.
Construction features N- end rich in Cys binding ligand
participating in transducting signals Transmembrane domain rich in
hydrophobic amino acid anchoring ligand be fluid C- end key
position of transducting singals