S07100901
Molecular Biology of the Cell
Part II
Instructor,温龙平
lpwen@ustc.edu.cn
Topics
Cancer
Stem Cells
Biomembranes
Nanobiology
Grading
Class Attendance & Discussion (20%)
Team Presentation (40%)
Final Exam (40%)
Presentations
5-member team (self-assembled)
Select one paper (Science,Nature,Cell) within the
fields of cancer,stem cell or apoptosis,discuss
extensively by the team,and prepare for a 20-minute
presentation (10-15 slides)
Address the following in your presentation,
1,Scientific question the paper was trying to answer;
2,Experimental approaches taken and main
techniques used;
3,Major findings and conclusions;
4,Significance of the paper;
5,Your ideas to further the research,
Tobacco
&
Cancer
Cancer,The Bottom Line
In animals,cell numbers are under
exquisite control
Loss of this control leads to proliferation of
unwanted cells
Cancer arises when the above situation
reaches a certain degree
Cancer vs Tumor
Cancer cells are malignant tumor (vs,
benign tumor) cells
All cancer cells are eventually capable of
metastasis
Tissue Culture Systems
Primary Cell Cultures
Secondary Cell Strains
Continuous Cell Lines
Transformed Cells
Reduced cell adhesion
Round-up morphology
Reduced serum requirements
Elevated hexose uptake
Anchorage-independent growth (ability to grow
in semi-solid media,soft agar
Loss of density-dependent growth inhibition
(“Contact Inhibition”)
Secretion of plasminogen activator
Capable of forming tumor in appropriete setting
Causes of Cancer
Generalizations
Spontaneous genetic mutations
Inherited cancer susceptibilities
Carcinogenic substances
Viruses
Radiation
Genes,Genes,Genes,Genes,
Genes
All five of the above listed causes act
through genes
Cancer is due to genetic changes in
individual cells which then proliferate and
pass on the mutations
Oncogenes & Protooncogenes
same molecules
Called oncogenes for their role in
promoting neoplasia
Called protooncogenes in the normal cell
Play diverse roles in control of cell growth
– Proliferation
– Differentiation
– Apoptosis
Evidence supporting that
cancer results from genetic
changes
Discussion
Somatic vs Germ-line
Cancer in the individual
Somatic Cell changes
Most cancer genes are these
Heritable
Predisposition to cancers
Germ-line Cells
fewer of the cancer genes are these
Evidence supporting that
cancer results from genetic
changes
in multiple genes
Discussion
1,Epidemiological Studies
2,Intermediate Stage Cancer
Studies (Colon Cancer)
3,Oncogene Overexpression
Studies
Ras
Activated Ras transforms NIH 3T3 cells
but not primary cells
Later studies revealed that NIH 3T3 cells
harbors an inactivation mutation in p16,a
cyclin-kinase inhibitor gene
Transgenic Mice Study
Polygenic Changes
Development of cancer may require
mutations in more than one gene
Cooperativity
Many cell-control functions are redundant
Mutations may affect different aspects of
cellular growth control
Session Summary
Cancer is a fundamental aberration in cellular
behavior,touching on many aspects of molecular
cell biology,Most cell types of the body can give
rise to malignant tumor (cancer) cells,
Cancer cells can multiply in the absence of
growth-promoting factors required for proliferation
of normal cells and are resistant to signals that
normally program cell death (apoptosis),
Cancer cells also invade surrounding tissues,
often breaking through the basal laminas that
define the boundaries of tissues and spreading
through the body to establish secondary areas of
growth,a process called metastasis,Metastatic
tumors often secrete proteases,which degrade the
surrounding extracellular matrix,
Session Summary continued
Certain cultured cells transfected with tumor-cell
DNA undergo transformation,Such transformed
cells share many properties with tumor cells,
The requirement for multiple mutations in cancer
induction is consistent with the observed increase
in the incidence of human cancers with advancing
age,Most such mutations are somatic and are not
carried in the germ-line DNA,
Colon cancer develops through distinct
morphological stages that commonly are
associated with mutations in specific tumor-
suppressor genes and oncogenes,
Cancer Genetics
Increased cell proliferation
Decreased cell death
Control of Cell Proliferation
Cell Cycle
Promoters of the cell cycle =
oncogenes
“accelerators”
Inhibitors of the cell cycle =
suppressors
“brakes”
Cancer Genes
Uncontrolled Accelerators
Oncogenes staying in the
On Position
Defective Brakes
Mutations that cause
suppressors to be non-
functional
Proto-oncogenes,
Seven Categories
Oncogene,On” Mechanism 1,
Promoter/Enhancer Insertion
Oncogene,On” Mechanism 2,
Chromosome Translocation
Oncogene,On” Mechanism 3,
Activating Mutation
Ras
Src
Many Others
Tumor Suppressor Genes
Intracellular proteins,such as the p16 cyclin-
kinase inhibitor,that regulate or inhibit
progression through a specific stage of the cell
cycle
Receptors for secreted hormones (e.g.,
tumorderived growth factor b) that function to
inhibit cell proliferation
Checkpoint-control proteins that arrest the cell
cycle if DNA is damaged or chromosomes are
abnormal
Proteins that promote apoptosis
Enzymes that participate in DNA repair
RB
First identified tumor suppressor gene
Autosomal dominant
Loss of heterozygocity (LOH)
Session Summary
Dominant gain-of-function mutations in protooncogenes and
recessive loss-of-function mutations in tumor-suppressor genes
are oncogenic,
Among the proteins encoded by proto-oncogenes are positive-
acting growth factors and their receptors,signal-transduction
proteins,transcription factors,and cell-cycle control proteins
An activating mutation of one of the two alleles of a proto-
oncogene converts it to an oncogene,which can induce
transformation in cultured cells or cancer in animals,
Activation of a proto-oncogene into an oncogene can occur by
point mutation,gene amplification,and gene translocation,
The first recognized oncogene,v-src,was identified in Rous
sarcoma virus,a cancer-causing retrovirus,Retroviral
oncogenes arose by transduction of cellular proto-oncogenes
into the viral genome and subsequent mutation,
The first human oncogene to be identified encodes a
constitutively active form of Ras,a signal-transduction protein,
Session Summary continued
Slow-acting retroviruses can cause cancer by integrating near
a proto-oncogene in such a way that gene transcription is
activated continuously and inappropriately,
Tumor-suppressor genes encode proteins that slow or inhibit
progression through a specific stage of the cell cycle,
checkpoint-control proteins that arrest the cell cycle if DNA is
damaged or chromosomes are abnormal,receptors for
secreted hormones that function to inhibit cell proliferation,
proteins that promote apoptosis,and DNA repair enzymes,
Inherited mutations causing retinoblastoma led to the
identification of RB,the first tumor-suppressor gene to be
recognized,
Inheritance of a single mutant allele of many tumor-suppressor
genes (e.g.,RB,APC,and BRCA1) increases to almost 100
percent the probability that a specific kind of tumor will develop,
More on Mechanism of Oncogenes
The growth of a cell is controlled by
molecules that impinge on its outer
plasma membrane
Cytokines
Low molecular-weight hormones
Growth factors
Other proteins
But how do these external factors
affect activities inside a cell and,
perhaps,inside the nucleus
Proto-oncogenes,
Seven Categories
Signal Transduction
? Most commonly,receptors in the plasma
membrane bind to specific external
factors and then interact with molecules
on the inner surface of the membrane,
? Some external factors can migrate
across the cell membrane and bind to
ligands in the cytoplasm,
Cytoplasmic Effects
Signal Transduction frequently
1,Activates an enzymatic protein e.g,
protein kinase
Or
2,Enhances nucleotide binding affinity,
e.g,enhanced GTP binding
Features of Signal Transduction
Reliance on multi-protein complexes of
signaling molecules
Generation of second messenger
molecules
Eventual transduction of the signal into the
nucleus
The Destination
The nucleus is the destination for signals,
Those dependent on interaction of plasma
membrane receptors with their ligands
and
Those initiated by the molecules that
migrate into & interact with their ligands
within the cytoplasm
Significance
Remember the properties of transformed
cells include the reduced requirement for
serum,i.e,reduced requirement for
external factors,
This is due to oncogene products that may
replace the normal ligands or otherwise
affect the molecular interactions leading to
behavior modification
Dominant Oncogenes
Their presence or activation cause cells to
proliferate
Acting in the membrane (Growth factors &
receptors)
Acting in the cytoplasm
Acting in the nucleus
Growth Factors
Some cells normally produce mitogens,
e.g.platelet-derived growth factor,
epidermal growth factor,
Genetic changes may enhance this
production or make it continuous,
Differentiation of Blood Cells
Viral gp55 mimics EPO
Growth Factor Receptors
Receptor Genes may be mutated so that
They are over-expressed
Be changed to a continuously active state,
a less common event
Growth Factor Receptor Mutation
ERB-B
Family of
Epidermal Growth Factor Receptors
Amplification is common to
Glioblastomas
Squamous cell carcinomas
Breast & Ovarian Cancers (ERB-B2,also
known as Neu-2)
Translocational Activation
GTP Binding Proteins
Two Broad Groups
– G Proteins
– Ras Superfamily
They are molecular switches that regulate
numerous physiologic processes,
Two states,
– On when bound to GTP
– Off when bound to GDP
Normal Regulation by Ras Proteins
Mitogenic Signaling
Cytoskeleton Organization
Nuclear Functions
Membrane & Protein Trafficking
Cell Transformation by Ras
DNA proliferation
Changes in cell morphology
Ras On or Off
Bound to GDP,Ras proteins are neutral in
regard to mitotis
Bound to GTP,they send mitogenic
signals to the nucleus
Normal Control
Normal Ras quickly cleaves GTP to GDP
by an intrinsic GTPase activity
A protein called GAP assists in this activity,
enhances the digestion by 10,000 times
Ras Mutants
Some mutations cause a loss of the
GTPase activity
Then the protein is constitutively active
because remains bound to GTP
Also inactivation of GAP may leave Ras
constitutively bound to GTP
Ras Superfamily in Cancers
Alleles found in many tumor types
– 50% of colon cancers
– 90% of pancreatic carcinomas
In human cancers,virtually all Ras
mutations are ones that impair GTPase
Neurofibromatosis Type I
Loss of a GAP-like protein,neurofibromin,
predisposes Ras proteins to remain in
their active state,
An autosomal disorder with a wide range
of possible symptoms including enhanced
susceptibility to benign or malignant
tumors
Significance in Research
RAS mutation in human bladder
carcinoma lines was the first identification
of a human oncogene
Ability of RAS proteins to bind guanine
nucleotides was one of the first
biochemical functions ascribed to an
oncogene product
Hope for Therapy
Activation of human RAS genes are the
most common dominant mutations in
human cancer
Function and cellular localization of RAS
proteins require processing steps,
These steps provide targets for
therapeutic interventions
Constitutively Active Cytoplasmic
Protein Kinase v-Src
Transcription Factor Activation
by Growth Factor
Session Summary
Certain virus-encoded proteins can bind to and activate
host-cell receptors for growth factors,thereby stimulating
cell proliferation in the absence of normal signals,
Mutations or chromosomal translocations that permit
growth factor receptor protein-tyrosine kinases to dimerize
lead to constitutive receptor activation in the absence of
their normal ligands,Such activation ultimately induces
changes in gene expression that can transform cells,
Overexpression of growth factor receptors can have the
same effect and lead to abnormal cell proliferation,
Most tumors express constitutively active forms of one or
more intracellular signal-transduction proteins,causing
growth-promoting signaling in the absence of normal
growth factors,
Session Summary continued
A single point mutation in Ras,a key transducing
protein in many signaling pathways,reduces its
GTPase activity,thereby maintaining it in an activated
state,
The activity of Src,a cytosolic signal-transducing
protein-tyrosine kinase,normally is regulated by
reversible phosphorylation and dephosphorylation of a
tyrosine residue near the C-terminus,The unregulated
activity of Src oncoproteins that lack this tyrosine
promotes abnormal proliferation of many cells,
Inappropriate expression of nuclear transcription
factors,such as Fos,Jun,and Myc can induce
transformation
How about factors that Inhibit
Entry into G1
or
Progression Through the Cycle
permanent
Differentiation Inducers
temporary
Antimitogenic factors
Cell-to-Cell Contacts
Anchorage Factors
Depict Cell Proliferation
using the cell cycle
Cell Cycle
New cells formed as a result of mitosis
But first need synthesis of new DNA
Five stages
G0
G1
S
G2
M
G0
Quiescent Stage,
may be permanent or reversible
Due to,
? terminal differentiation
? anti-mitotic agents
? cell contact
G1
R
S
G2
M
Restriction Point,
Once passed,cell
is committed to
pass through S,
even in absence of
mitogenic signals
G1,Transcription of
genes whose
products are
required for DNA
synthesis,
Progression
through G1
dependent on
internal & external
factors
Phases
2 functional
Synthesis and Mitosis
2 preparatory
G1 and G2
What factors push the cell through
the cycle?
External
– Cytokines,growth factors,mitogens,
hormones,nutrients
Internal
– Cyclins,enzymes,telomerase
Controls at every stage
of the Cell Cycle
After synthesizing DNA,cells are
prevented from synthesizing it again until
after mitosis
If there are errors in DNA synthesis,repair
enzymes try to fix them,
If they persist during the cycle,there are
controls that interrupt the cycle,
They may just stop the cycle,or they may
cause cell death - apoptosis
p53
The normal protein p53 acts as a
“checkpoint” in the cell cycle,
If DNA is damaged,p53 causes arrest in
the G1 phase or may cause apoptosis,
P53 Gene Diagram
As An Oncogene
Loss of p53 function results in
uncontrolled cell cycle progression
Increased number of cells,damaged DNA,
decreased apoptosis
Effects of p53 defects
Too many cells
– Due to loss of control in G1
– And to decreased apoptosis
Genetic Instability
– Loss of control over damaged DNA
– Cells can undergo rapid mutation
Loss of p53 Function
How?
Mutation,50% of human tumors have
abnormal p53 genes
Overexpression of the p53 binding protein,
mdm2
Human Papillomavirus protein E6 binds to
p53 and increases its rate of degradation
Another Checkpoint Gene
Rb
Retinoblastoma Susceptibility Gene
The name does not indicate its
protooncogene function
pRb Functions
The protein product of the Rb gene is
expressed in many tissues
Controls cell proliferation in normal
development
Is a tumor-suppressor gene
Loss of its function is associated with
many types of tumors
How Cyclin,CdK and Rb works
pRb
Normal pRb provides another checkpoint in the cell cycle,
holding the cell in G1,
It binds to proteins that are needed for transcription
steps preparatory to DNA synthesis
Loss of pRb function loses this checkpoint
But,conversely,loss of pRb enhances apoptosis
Other genes needed for carcinogenesis
Session Summary
Overexpression of the proto-oncogene encoding cyclin
D1 or loss of the tumor-suppressor genes encoding
p16 and Rb can cause inappropriate,unregulated
passage through the restriction point in late G1,a key
element in cell-cycle control,Such abnormalities are
common in human tumors,
The p53 protein is essential for the checkpoint control
that arrests human cells with damaged DNA in G1,
Replication of such cells would tend to perpetuate
mutations,
p53 functions as a transcription factor to induce
expression of p21,an inhibitor of G1 Cdk-cyclin
complexes.,
Session Summary continued
Mutations in the p53 gene occur in more than 50
percent of human cancers,
Because p53 is a tetramer,a point mutation in one
p53 allele can be sufficient to inhibit all p53 activity,
MDM2,a protein that normally inhibits the ability of
p53 to restrain the cell cycle or kill the cell,is
overexpressed in several cancers,
Defects in cellular DNA-repair processes found in
certain human diseases are associated with an
increased susceptibility for certain cancers,
The
molecular
circuitry
of cancer,
Some Perspectives in
Cancer Therapy
Pharmacology