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Abnormal Cell Proliferation,Differentiation
Apoptosis and Related Disease
Department of Pathophysiology
Shanghai Jiao-Tong University School of Medicine
The Cancer Problem
Evan, GI and Vousden, K. (2001) Nature 411:342
Proliferation
Differentiation
Cell
Apoptosis
Abnormal Cell Proliferation and Diseases
【Cell Proliferation】
Cell proliferation is a process of cell division and regeneration,
which results in an increase in the cell number with exact passages
of genetic information to their daughter cells.
Phase of cell cycle
• M-Phase
– Mitosis
– Cytokinesis
• Interphase
– G1 = Gap between M and S
– S = Synthesis (DNA and
centrosomes replicated)
– G2 = Gap between S and M
Mitosis
Interphase
Anaphase
Prophase
Prometaphase
Telophase
Cytokinesis
Metaphase
2001 Nobel Prize
Leland H. Hartwell
1970s
“Checkpoint”
Yeast genetics
~100 CDC genes
Start gene
Paul M. Nurse
1970s
CDKs
yeast
Tim Hunt
1980s
Cyclins
Sea Urchins
Cell Cycle Regulation
• Cyclins
• Cyclin Dependent Kinases (CDKs)
• Cyclin Dependent Kinases Inhibitors (CDKIS)
●Cyclin
【Classification 】
Cyclin B1、Cyclin A、Cyclin E、Cyclin D1,D2,D3. F,G,H,T.
Cyclin Protein
kinase
process regulated
Cyclin D1-3
Cdk 4,6
G1-phase progression
Cyclin E
Cdk2
G1 to S-phase
Cyclin A
Cdk2
S-phase progression
Cyclin A
Cdk1
S through G2
Cyclin B
Cdk1
M-phase
 Cyclic Degradation:
Ubiquitin-mediated breakdown by Proteasome
Experimental Demonstration that Cyclin D is Required for Passage
Through the Restriction Point in the Mammalian Cell
●CDKs
CDKs are group of serine and threonine kinases.
【CDK1 activates cell cycle】
Thr14、Tyr15 dephorsphorylation
Cyclin B binds CDK1
CDK1activation
phosphorylation of CDK1 by CAK
Cell cycle
CDK activation kinase (CAK)
【CDK activation and cell cycle regulation】
▲
CDK2 binds cyclin E， CDK4、CDK6 binds cyclin D1、D2、D3
pRb phosphorylation
▲
E2F release↑
initiate G1 phase
DNA synthesis
initiate S phase
CDK2 binds cyclin E and A recognize chromosome ARS
Accelerate S phase
▲
CDK1 binds cyclin B1
initiate M phase
▲
CDK1 binds cyclin A
initiate G2 phase
●CDI
Ink4：P16INK4a、P15INK4b、P18INK4c和P19INK4d
▲
Cip/Kip：p21Cipl、P27Kip1和P57Kip1
▲
【mechanism of inhibition】
CDIs direct bind the
phosphorylation site of CDKcyclin complex.
▲
P16INK4a binds CDK4, inhibits
its interaction with cyclins.
▲
CDK2
CDK4、CDK6
■Checkpoint
of cell cycle）
DNA damage
Checkpoint
G1/S
DNA replication
S/G2
spindle assembly
G2/M
【Check mechanism】
●P53 ：In response to DNA damage in G1, the protein level of p53 rises
dramatically due to the activation of a checkpoint protein kinase (chk2) that
phosphorylate p53 and make it less susceptible to degradation by the
ubiquitination/proteasome pathway. This enables p53 to activate transcription of the
cdk inhibitor (CKI) p21 which binds to all cdks and inhibits their action and thus
“arrests” the cells until the DNA damage can be repaired.
DNA damage, other insults
Growth Signal
p53
p21
G1 phase
cyclin/CDK
S phase
►Abnormal cell cycle & diseases
Deregulation of cell cycle causes cell proliferation excessive or
insufficency.
【Deregulation of cell cycle】
■loss
■The
of control in driving cell cycle progression(Cyclin,CDK,CDI）
impairment of checkpoint system
Abnormal proliferation differentiation related disease
abnormal proliferation
abnormal differentiation abnormal proliferation
and differentiation
aplastic anemia
leucoderma
obesity
malignant tumor
heritage hemoglobinopathy
prostatic hypertrophy muscular dystrophy
scleratheroma
Familial erythrocytosis
psoriasis
dysembryom
congenital deformity
X-linked hyper IgM syndrome
Malignant tumor
Cancer is a disease where regulation of the cell cycle goes
awry and normal cell growth and behavior is lost .
1． loss of control in driving cell cycle progression
■ Overexpression
of cyclins
【Cyclin D1 overexpression】（oncogene）
▲gene
amplification（major reason）
Observed in breast, gastric, esophageal cancer.
●Chromosome
rearrangement
Cyclin D1 gene under the control of parathyrin promoter
Synthesis of Cyclin D1 increase
●Chromosome
Parathyroid carcinoma
translocation
t(11:14) (q13:q32)
Cyclin D1 gene under the control of Ig heavy chain gene enhancer
Cyclin D1 overexpression
▲gene
mutation
Cyclin D1 T286mutation
Cyclin D1 ubiquitination block
Cyclin D1↑
【mechanism of Cyclin D in tumorgenesis】
Cyclin D overexpression + cytokines
CDKs water fall effect↑
cell over proliferation
susceptible to
cancerization
■
abnormal expression of CDK
Mainly observed in CDK4、CDK6 overexpression.
CDK4↑+ cyclin D binding↑
pRb
CDK4/cyclin D↑
CDKs expression↑
pRbphosphorylation↑ cell over proliferation
E2F ↑
G1/S transition accelerated
■
CDI insufficency and mutation
▲ InK4
inactivation
【causes of p16 InK4 inactivation】
mutation or deletion, chromosome translocation, hypermethylation.
【Mechanism】
p16 InK4 gene expression↓
CDK4/cyclin D binding↓
cell susceptible to proliferation
susceptible to tumorgenesis
▲
Deficient expression of Kip/Cip
P21cipl function
PCNA
cyclins/CDKs activity↓
cell cycle
DNA replication block
【Mechanism】
p53 mutation
P21cip1 transcription↓
DNA damage cell↑
2. Impairment of checkpoint system
main reason：G1/S、G2/M checkpoint dysregulation
consequence：function of detect DNA damage
Table 8.3 The Biology of Cancer (© Garland Science 2007)
Table 8.4 The Biology of Cancer (© Garland Science 2007)
■G1/S
transition dysregualtion
▲P53
enter S phase
DNA damage
G1/S check
successful
G1 arrest
DNA repair
failure
apoptosis
▲P53
mutation or loss
bcl-2
bax
P53
susceptible to cancergenesis
mutagen
checkpoint function↓
genetic instability
replication fidelity↓
Table
human tumor p53 gene mutation hotspot and frequency
tumor
frequency（%） hotspot
tumor
frequency（%） hotspot
Lung cancer
56
157，248，273
prostatic caner
30
unknown
Colon carcinoma
50
175,245,248,273 hepatocellular carcinoma 45
249
Esophageal carcinoma
45
unknown
colloid carcinoma
25
175,248
Ovarian cancer
44
273
breast cancer
22
175,248,273
Pancreatic cancer
44
273
endometrial cancer
22
248
Skin cancer
44
248、278
thyroid cancer
13
248,273
Gastric cancer
41
unknown
leukemia
12
175,248
Head and neck
squamous cell carcinoma 37
48
uterine cervix cancer
7
273
Bladder cancer
34
280
soft tissue sarcoma
31
unknown
DNA virus: SV40、HPV、adenovirus
P53 inactivation
■
impairment of G2/M checkpoint
G2/M transition
DNA double strand break
activation of DNA damage checkpoint
block cell enter M phase
induce repair gene transcription
DNA repair
▲
loss of G2/M checkpoint
chromosome rearrangement, loss
Primary Throbocythemia
symptoms ：is characterized by a platelet count greater
than 600,000/µL, megakaryocytic hyperplasia, splenomegaly,
and a clinical course complicated by thrombotic and/or
hemorrhagic episodes.
■ clinical
■ The cause of ET is not fully understood. About half of
patients with ET have a mutation of the JAK2 (Janus kinase 2)
gene in their blood cells. Whether or not a patient has the
mutation does not appear to affect the nature or course of the
disease. Research is under way to determine the precise role of
JAK2 mutations and to identify other mutations in ET patients.
Apoptosis and Diseases
【Concept of apoptosis】
Apoptosis is a genetically controlled process regulated by
complex molecular signaling systems. It is also known as cellular
self destruction or cell-suicide or programmed cell death (PCD).
【physiology implication】
■ is
involved in tissue development
■ maintain
■is
the homeostasis
involved in defense response
Physiology or Medicine Nobel Prize 2002
1090 –131= 959(cells)
Robert Horvitz
John Sulston
Sydney Brenner
“for their discoveries concerning genetic regulation
of organ development and programmed cell death”
Features of apoptosis cells
1. The morphologic characteristics of apoptosis
■ loss
of contact with neighbering cells
■Blebbing
of cell membrane
■Condensation
■Apoptotic
APL
of chromatin
bodies
Apoptosis
apoptotic cell (SEM)
(TEM)
Morphological features of
apoptosis
Membrane blebbing
2. The biochemical characteristics of apoptosis
1）DNA ladding pattern
activation of endonuclease
cleave DNA at specific sites between nucleosomes
oligonucleosomal fragments （160～200bp intervals）
2）Phosphatidylserine externalization
3）activation of caspase and its function
Caspase (cysteine-containing aspartate-specific protease)
 Stimuli of apoptosis
Table：effects of various factors on apoptosis
Inducers
Inhibitors
Physical and
Irradiation, high
chemical factors temperature,
stress,
chemotherapeuti
c drugs
cytokines
IL-2、NGF
Hormone and
cytokines
Glucocorticoid,
TNF
hormone
ACTH, testosterone, estrogen
Immunity
granzyme
others
Zn2+、agrypnal、cystein
protease inhibitor, EBV,
cowpox, virus, neutral amino
acid
pathogen
HCV, HIV
 Mechanism of apoptosis
 Caspase-dependent apoptosis
（1） Concept, Classification, Construction of Caspases
■ Caspase
belong to cystein proteases
■ Caspase
categories
▲ Caspase
Death receptor pathway
upstream（Caspase-8 , -9, -10） Mitochondrial pathway
（Ced-3 subfamily）downstream（Caspase
■
-2, -3,-6,-7）（common pathway）
Caspase activation
Pro-caspase： NH2 terminal domain、 20KD subunit、 10KD subunit
cleavage
Interaction of large and small subunit （dimer）
caspase activation（tetramer）
1. Extrinsic or cytoplasmic pathway
■Death
receptor
TNFR, FasR, DR3, DR4, DR5,
The DR family is part of the tumor necrosis factor receptor superfamily.
Triggering members of the DR family by death ligands results in the transduction
of either apoptotic or survival signals.
Growth factors, Fas antibody
■pathway
FSAL
FasR
DD
FADD
DED
DED
Pro-caspase-8
Caspase-8
apoptosis
Pro-caspase-3
Caspase-3
FADD（Fas-associated death domain）； DD（death domain）；
DED （death effector domain）。
2. Intrinsic or mitochondrial pathway
Bid
Ca2+
NO
hypoxia
ROS
stress
Caspase-8
MPT open
Apaf-1
Cyt.c +
ATP
MMP↑、swelling、△m↓
AIF
Apoptosome
Cyt.c
caspase-independent
apoptosis
Apaf-1activation
Cyt.c
Apaf-1activation
CARD
+
Pro-caspase-9
Caspase-9
apoptosis
Pro-Caspase-3
Caspase-3
3. Activation of caspase-3 (Effector caspase)
Pro-caspase-3
caspase-9
Caspase-3
Pro-caspase-9
Pro-caspase-6
Caspase-6
caspase-7
Pro-caspase-7
caspase-2
CAD
Cleavage of
structural protein
Pro-caspase-2
ICAD
DNA cleavage
apoptosis
 Non-Caspase dependent apoptosis
■calcium
overload
Activation of Ca/Mg2+dependent endonuclease，
DNA cleavage；activation of transglutaminase, apoptotic
bodies.
■AIF
Mechanism of calcium overload
induced apoptosis
TNF-α、CD3-Ab、TCDD
Plasma [Ca2+] ↑
■activation
of Ca2+/Mg2+dependent endonuclease，DNA cleavage；
■activation
of transglutaminase, wide cross-linking of cytoskeleton.
■transcription
of apoptosis related gene；
■ mitochondrial
dysfunction
apoptosis
 Mechanism of apoptosis
1．Oxidation damage
Oxygen free radicals disrupt the normal redox equilibrium, result in the
injuring of protein,lipid and DNA.
【Mechanism】
Activation of p53, consuming of ATP, membrane lipid peroxidation,
activation of endonuclease, initiating apoptosis.
2．Calcium overload
3．Mitochondria damage
 Regulators of apoptosis
Table 1
apoptosis regulators
function
gene
inhibitors
Bcl-2 Bcl-XL、A1/Bfl-1、Bcl-w、Bcl-G and Mcl-1
activators
P53、Bax、Bad、Bak、Bid、Bim、Bik、Bok、
Bcl-B、Bcl-Xs、Krk、Mtd、Nip3、Nix、Noxa
Dual regulators
c-myc、Bcl-x
1．Bcl-2 Family & regulating functions
■
Bcl-2（Bcl-XL）inhibit MPT open，Cyt.c 、AIF release↓
■
Bcl-2 binds and inactivates Apaf-1，blocks caspase-9 activation
■
Bcl-2 binds cyt C
Bcl-2
MPT open
Apaf-1
Cyt.c +
ATP
MMP↑、swelling、△m↓
AIF
Apoptosome
Cyt.c
caspase-independent
apoptosis
Apaf-1activation
Cyt.c
Apaf-1activation
CARD
+
Pro-caspase-9
Caspase-9
apoptosis
Pro-Caspase-3
Caspase-3
2．p53（tumor suppressor gene）
As a transcription factor, p53 regulates downstream genes
important in cell cycle arrest, DNA repair, and apoptosis. After
DNA damage, p53 holds the cell at a checkpoint until the damage
is repaired. If the damage is irreversible, apoptosis is triggered.
【Mechanism】
■
Downregulates the expression of Bcl-2.
■
P53 induces Bax, NOXA, PUMA expression.
■
P53 induces Fas expression.
■
P53 relocalizes Fas to cell membrane.
Table . Human diseases associated with disordered apoptosis
Decreased Apoptosis
Increased Apoptosis
Epithelial
Tissue
Carcinogenesis
Macrophage
s
Bacillary dysentery
Blood Vessels
Intimal hyperplasia
Myocardium
Peri-infarct border
zones
Lymphocytes
Autoimmune
disorders
Lymphocytes lymphocytes
depletion in HIV
injections and sepsis
Haemopoeitic
systems
Leukemia,
lymphoma
CNS
Neurodegenerative
diseases like
Alzheimer’s and
Parkinson’s disease
 Apoptosis inhibition
1. Tumor
■
bcl-2 overexpression
In follicular lymphoma, a chromosomal translocation commonly occurs between the
fourteenth and the eighteenth chromosomes-t(14;18)-which places the Bcl-2 gene
next to the immunoglobulin heavy chain locus. This fusion gene is deregulated,
leading to the transcription of excessively high levels of bcl-2. This decreases the
propensity of these cells for undergoing apoptosis.
（also observed in breast cancer, lung cancer, malanoma, prostate cancer）
■
p53 deletion or mutation
Abnormal of P53 in stability, localization or activation
p53 expression or function defect
apoptosis rate↓
（ abnormal P53 observed in at least 50% malignant cancer）
tumorgenesis
■virus
oncogene
HBV encoded HBX is inhibitor for Caspase-3, and is associated with
liver cancer.
■fusion
protein
Chronic myeloid leukemia（CML）
t (9；22)( Ph chromosome)
Bcr-abl fusion gene
NF-B activation、inhibit apoptosis↓
p210 Bcr-abl fusion protein
CML cells proliferation
leukemia
2．Autoimmune diseases
A common feature of autoimmune diseases is the breakdown of
tolerance of self antigens, a consequence of which is the
production of autoantibodies reactive with multiple self proteins.
Defect in apoptosis can cause autoimmunity by allowing the
survival of autoreactive T and B cells.
 Apoptosis excess
1. Ischemia reperfusion injury
■characters
▲early
phase apoptosis，late phase necrosis.
▲in
the centre of infarct necrosis，in the peri-infarct zone
apoptosis；
▲mild
ischemia apoptosis，severe ischemia necrosis；
■mechanism
▲oxidation
▲FasR
▲p53
activation
Deregulated cell differentiation and
Disease
►Concept
Cell differentiation is a process in which a generic cell
develops into different kinds cells with the specialized morphology,
metabolism and physiological functions in response to specific
triggers from the body or the cell itself.
Cell differentiation is tightly regulated by a series of regulatory
proteins.
Ey gene in the morphogenesis of eyes
Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
Cell 2007,131,861-872 （Oct3/4, Sox2, Klf4, c-Myc ）

Determination and differentiation
The determination of different cell types (cell fates) involves
progressive restrictions in their developmental potentials.
When a cell “chooses” a particular fate, it is said to be
determined, although it still "looks" just like its undetermined
neighbors. Determination implies a stable change - the fate of
determined cells does not change. Differentiation follows
determination.
In some cases, determination results from the asymmetric
segregation of cellular determinants. However, in most cases,
determination is the result of inductive signaling between
cells.
 The regulation of cell differentiation
1. Regulation on the genomic level）
■ House-keeping
genes
House-keeping genes generally encode proteins that
participate in basic or universal cellular functions essential for
maintaining cell survival.
■tissue
specific gene
2. Regulation on transcription and post- transcription levels）
■transcription
level
■post-transcription
level
3. tranlational and post-translational level
4. Extracellular factors that control differentiation
Hematopoiesis
“Acute promyelocytic leukemia (AML M3) is now the
most frequently curable acute leukaemia in adults if
promptly diagnosed and adequately treated.”
Parmar S, Tallman MS. , 2003
acute promyelocytic leukemia
1988: Breakthrough in treatment
from China
1990: Multiple
groups identify
RARa as t(15; 17)
breakpoint
Drug
Gene
How does ATRA induce remission?
How does PML-RARa induce APL?
Pulmonary fibrosis
Pulmonary vascular lesions
myofibroblast
hypoxia
collagenoblast
gas diffusion disorder
brinase↑↑↑
collagen deposition
alveolar membrane thickening
Morbid obesity
Excess pre-adipocyte differentiate into adipocyte.