Cell biology 2014 (12/2 -14)
Lecture 11:
A medical perspective
Definitions of disease:
•When something is wrong with a bodily function
• A state that places individuals at increased
risk of adverse consequences
• Etcetera
The cause of different diseases
Mendelian/Genetic
diseases
Heart disease
Allergy
100% genetics
100% environment
Diabetes
Cancer
Infectious
diseases
Complex multi-factorial diseases
2
Three cytoskeleton systems:
distinct but overlapping functions
Intermediate filaments
Actin filaments
Microtubules
- Cell shape and integrity
- Motility of the whole cell or cellular appendages
- Intracellular organization
3
Intermediate filaments and epithelia blistering
4
Cytosolic intermediate filaments support:
1) Cell-cell contacts
(desmosomes: cadherins)
2) Cell-ECM contacts
(hemidesmosomes: integrins)
1 + 10
1.
5 + 14
2.
Albert et al Fig. 16-21
Mutations in keratin 5 or 14 cause Epidermolysis bullosa simplex
(1/40 000), a disease manifested as blistering of the epidermis.
Consequences of dysfunctional nuclear lamina
Lamin A, B & C: intermediate filament
proteins stabilize the nuclear envelope
Laminopathies are genetic diseases manifested as either:
I. Dystrophy of skeletal and/or heart muscles, caused by mutations
affecting Lamin A/B or proteins attaching lamins to the nuclear envelope
II. Progeria, caused by mutations in the lamin A gene, or
in a lamin A processing enzyme. This result in excessive
farnesylation. A farnesyltransferase inhibitor, initially
developed to target oncogenic Ras, delays progression.
5
The actin cytoskeleton – an overview
• Support of the plasma membrane
• Cell migration
• Contraction
Muscle contraction
Cytokinesis
6
Dysfunctional actin regulation in Wiskott-Aldrich syndrome
WASP
Arp 2/3
- Wiskott-Aldich syndrome (1/150.000) is caused by mutations
in the Wiskott-Aldrich syndrome protein (WASP)
WASP
Arp 2/3
ZZZZ
Manifested by:
• Thrombocytopenia
Underdeveloped cortical actin
results in defective platelets
• Eczema (skin blushing)
• Immunodeficiency syndrome
Deficient migratory and
phagocytotic capacity of
immune cells
- Can only be cured by a hematopoietic stem cell transplant
7
Pathogenic E. coli: actin dependent colonization
Enteropathogenic E. coli induce actin
containing pedestals in intestinal epithelia
Virulence factors
that activate N-WASP
Activation of Arp 2/3
Loss of absorptive surface is one
cause for the associated diarrhea
8
Listeria: actin dependent motility
1-3) Phagocytosis and escape
from phagosome
1.
2.
4) Bacterial multiplication
Arp 2/3
5) Penetration of a neighboring
cell through actin based motility
ActA
ActA
3.
ActA
4.
5.
video 24.3-listeria_parasites
9
The microtubule system - an overview
10
ER
- Intracellular
organization
Golgi
Organelle
positioning
Chromosome
segregation
Chemotactic agent
- Cell motility
Movement of
cellular appendages
Cell polarisation
and transport
Lissencephaly: defective neuron migration
- Lissencephaly ("smooth brain," 1/30.000) is a disorder
characterized by the lack of normal convolutions (folds) in the brain
Normal brain
Lissencephaly brain
mutated in many cases  loss of dynein ( )
dependent centrosome reorientation  defective cell polarization
Lis1
- 1/30 000 births, early death in severe cases
11
Non-functional cilia in Kartagener syndrome
- Kartagener syndrome (1/20.000) is caused by mutations
affecting cilia specific dynein
- Manifested by respiratory infections, infertility and situs inversus
Upper respiratory epithelia
Patient with
Normal
Kartagener syndrome
Goblet
cell
Bacteria is caught in
mucus and cleared by
a cilia mediated flow
Bacteria is not cleared
due to defective cilia
12
Microtubule-poisoning drug: Taxol
• Alkaloid ester isolated from the bark of
Taxus brevifolia (Pacific yew)
• Stabilization of microtubules
Therapeutic uses:
Treatment of breast, lung and ovarian cancer
Prevention of restenosis of coronary stents
(Surface coating of stents  local action)
Major side-effects: Bone marrow suppression, gastro-intestinal
upset and peripheral neuropathy
13
Microtubule-poisoning drug: Vinca alkaloids
• Isolated from Catharanthus roseus
• Named: Vinblastine, Vincristine, Vindesine
and Vinorelbine
• Sequesters tubulin
Therapeutic use:
Treatment of leukemia, lymphoma, breast,
lung, prostate, skin and testicular cancer
Major side-effects: Bone marrow suppression, gastro-intestinal
upset and peripheral neuropathy
14
An oncology perspective on signal transduction, cell
growth, checkpoints, apoptosis and the cytoskeletons
Progression towards malignancy involves : i) uncontrolled proliferation,
ii) resistance to apoptosis, iii) cell migration, iv) tissue invasion
X X
X X
X X
X X
X X
Clonal evolution
Selection of
malignant clones
animation 20.2 -contact_inhibition
video 20.1 -breast_cancer_cells
X X
Metastasis
15
Two distinct types of ”cancer genes”
Oncogenes
Gene X
Tumor suppressors
On
On
Off
Gene X
Off
Gene Y
Gene Y
On
Off
On
On
Gain-of-function
A single genetic change
Dominant phenotype
Loss-of-function
Two independent events
16
Definitions: oncogenes and tumor suppressors
An oncogene is a gene that when mutated, or
overexpressed, contributes to converting a normal cell into
a tumor cell (constitutive activity dominant phenotype)
Ras
point mutation
Bcl-2
overexpression
A tumor suppressor-gene is a gene whose loss, or
inactivation, contributes to converting a normal cell into
a tumor cell (recessive phenotype)
CKI
p53
Rb
Inactivating point mutations or loss of the entire gene
(germ line mutation in one allele and/or acquired somatic mutations)
17
Cell type specific proliferative signals
18
Cells from different tissues express distinct sets of
growth factor receptors and signaling proteins
Cell type B
Cell type C
Cell type A
Major mitogen
signaling pathway: RTK
Alterations
in tumors:
RTK signals
Wnt
Hedgehog
Wnt signals
Hedgehog signals
Aberrant proliferative signals in tumors
Wnt
XGF
RTK
Ras
Hedgehog
Frizzled
Patched
Dishevelled
Smoothened
Raf
GSK-3b
Erk
b-catenin
myc
G1
19
myc
Fused
Axin
SuFu
G1
Gli
Gli
myc
G1
Insensitivity of tumors to anti-growth signals
The retinoblastoma pathway
Mitogen signaling
p15
p21
= germ line
mutations identified
Cdk
G1
TGF-b
p16
HPV E7 viral
Rb
E2F
Cdk
P
Cdc6
P
ORC
S
20
Evading cell death (apoptosis)
Survival factor signaling
BH3
only
p53
Ligand
Death
receptor
Adaptor
Bcl-2
Bax
Cyt. C
Caspase 9
Caspase 8
Caspase 3
21
Apoptosis
Randomly acquired oncogenic mutations drives tumor progression
1 Self-sufficiency in proliferative signals
1.
Mutation
2 Insensitivity to anti-growth signals
2.
3.
3 Evading cell death (apoptosis)
4.
4 Limitless replicative potential
5 Sustained angiogenesis
5.
6 Metastasis capability
6.
22
Same “diagnosis” but different set of mutations
4
3
4
5
6
X
3
X
2
X
1
X
Patient A with diagnosis X
2
5
6
X
X
1
X
Patient B with diagnosis X
23
How many somatic mutations during a life time?
Cell death and
replacement
 risk for mutations
& chromosomal
instability
Year 1-15
1013
Controlled and coordinated divisions
Uncontrolled divisions
 Tumors
– Human diploid genome: ~6 x109 bp
– Only some few errors per replication cycle
– Average t½ of cells is 7 years (range: 24h to >100 years)
24
25
Normal cells have a very low rate of mutations
Cancer related genes
Other genes
Random mutation
Time
Due to the low normal mutation frequency, progression
to a fully malignant tumor is statistically improbable
How come that malignant tumors have either a lot of
mutations (~10 %) or chromosomal aberrations (90 %)?
(~400 genes are frequently altered in tumors, 6 to 80 genes per “patient”)
Genomic instability: Two distinct levels
1. Defective DNA repair (MIN)
Mutation in a gene encoding some
enzyme required for DNA-repair
X
X
No repair  many mutations 
accelerated tumor progression
2. Chromosome segregation errors (CIN)
Mis-segregation due to a defective a gene that encodes some
protein essential for high fidelity chromosome segregation
26
MIN reflects an escalated mutation rate
Cancer related genes
DNA repair (TS)
Other genes
Genetic alteration
Time
?? Often uncertain which ones of all the mutations that
contribute to tumor progression
1
2
3
4
5
6
MIN: mini-satellite DNA instability (due to defective DNA repair)
27
CIN through excessive centrosomes
Two centromes
More than two centrosomes
Kinetochore attachments
satisfy the spindle checkpoint
CIN: Chromosomal instability
miss-segregation
 aneuploidy
28
CIN through loss of APC
The tumor suppressor gene product
APC functions as a MT plus-end
stabilizing protein (
) that facilitates
stable MT-kinetochore connections
Centromere
Centrosome
AC
APC
Kinetochore
CIN: Chromosomal instability
Satisfied
spindle
checkpoint
29
CIN through a defective spindle checkpoint
1) A normal cell
Delayed anaphase until all
kinetochores are attached
2) A tumor cell with a (partially) defective spindle checkpoint
30
Genomic instability and tumor progression
A stable genome
x
x
Too much
genetic
instability
”Optimal” genetic
instability
”Selection barriers”
31
Principles of cancer treatment
32
Surgery- Impossible to remove all cancer cells
Radiation-
Chemotherapy-
Targets both cancerand normal cells
Side-effects
General chemotherapy: drugs that interferes with:
i) DNA-replication
ii) DNA structure
iii) The function of the microtubule-system
Chemotherapy may also include cell type specific drugs.
E.g. inhibition of hormone dependent tumor growth
Selective killing of tumor cells by chemotherapy
x
x
Mutations that inactivate various checkpoints are common
in malignant tumors
 no cell cycle arrest in patients treated by chemotherapy!
33