A medical perspective

Lecture 11:Cell biology 2014 (12/2 -14)

Definitions of disease:• When something is wrong with a bodily

function• A state that places individuals at increased risk of adverse consequences• Etcetera

2

The cause of different diseases

Infectious diseases

CancerDiabetes

Heart disease AllergyMendelian/Genetic

diseases

100% genetics 100% environment

Complex multi-factorial diseases

3

Three cytoskeleton systems: distinct but overlapping functions

- Cell shape and integrity

- Intracellular organization

- Motility of the whole cell or cellular appendages

Actin filaments

Intermediate filaments

Microtubules

4Intermediate filaments and epithelia blisteringCytosolic intermediate filaments support:

1) Cell-cell contacts (desmosomes: cadherins)

2) Cell-ECM contacts (hemidesmosomes: integrins)

Mutations in keratin 5 or 14 cause Epidermolysis bullosa simplex (1/40 000), a disease manifested as blistering of the epidermis.

Alb

ert e

t al F

ig. 1

6-21

2.

1.5 + 14

1 + 10

5

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.

6

The actin cytoskeleton – an overview

• Support of the plasma membrane

• Cell migration

• Contraction

CytokinesisMuscle contraction

7

Dysfunctional actin regulation in Wiskott-Aldrich syndrome

Manifested by:• Thrombocytopenia

• Eczema (skin blushing)

• Immunodeficiency syndrome

- Wiskott-Aldich syndrome (1/150.000) is caused by mutations in the Wiskott-Aldrich syndrome protein (WASP)

Deficient migratory and phagocytotic capacity of immune cells

- Can only be cured by a hematopoietic stem cell transplant

Underdeveloped cortical actin results in defective platelets

WASP Arp 2/3

WASP Arp 2/3 ZZZZ

8

Pathogenic E. coli: actin dependent colonization

Enteropathogenic E. coli induce actincontaining pedestals in intestinal epithelia

Virulence factorsthat activate N-WASP

Activation of Arp 2/3

Loss of absorptive surface is onecause for the associated diarrhea

9

1.

2.

3.

4.

5.

1-3) Phagocytosis and escape from phagosome

ActAActAActA

Arp 2/3

4) Bacterial multiplication

5) Penetration of a neighboring cell through actin based motility

Listeria: actin dependent motility

video 24.3-listeria_parasites

10

The microtubule system - an overview

- Intracellular organization

Chromosome segregation

- Cell motility

Cell polarisationand transport

Movement of cellular appendages

Organelle positioning

Golgi

ER

Chem

otactic agent

11

Lissencephaly: defective neuron migration

Normal brain Lissencephaly brain

mutated in many cases loss of dynein ( )dependent centrosome reorientation defective cell polarization

- Lissencephaly ("smooth brain," 1/30.000) is a disorder characterized by the lack of normal convolutions (folds) in the brain

- 1/30 000 births, early death in severe cases

Lis1

12

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

NormalPatient with

Kartagener syndrome

Bacteria is not cleareddue to defective cilia

Bacteria is caught in mucus and cleared bya cilia mediated flow

Goblet cell

13

Microtubule-poisoning drug: Taxol

• Alkaloid ester isolated from the bark of Taxus brevifolia (Pacific yew)

• Stabilization of microtubules

Treatment of breast, lung and ovarian cancer

Prevention of restenosis of coronary stents(Surface coating of stents local action)

Therapeutic uses:

Major side-effects: Bone marrow suppression, gastro-intestinal upset and peripheral neuropathy

14

Microtubule-poisoning drug: Vinca alkaloids

• Isolated from Catharanthus roseus

• Sequesters tubulin

Treatment of leukemia, lymphoma, breast,lung, prostate, skin and testicular cancer

Therapeutic use:

Major side-effects: Bone marrow suppression, gastro-intestinal upset and peripheral neuropathy

• Named: Vinblastine, Vincristine, Vindesine and Vinorelbine

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X X

X X

X X

X X

X X

X X

Progression towards malignancy involves : i) uncontrolled proliferation, ii) resistance to apoptosis, iii) cell migration, iv) tissue invasion

Metastasis

An oncology perspective on signal transduction, cell growth, checkpoints, apoptosis and the cytoskeletons

animation 20.2 -contact_inhibitionvideo 20.1 -breast_cancer_cells

Clonal evolutionSelection of malignant clones

16Two distinct types of ”cancer genes”

Gene YGene Y

Tumor suppressors

Two independent events

Loss-of-function

Gene XGene X

Oncogenes

A single genetic change

Gain-of-function

On

OffOn

Off

On

OffOn

On

Dominant phenotype

Definitions: oncogenes and tumor suppressors

Ras

p53Rb

An oncogene is a gene that when mutated, or overexpressed, contributes to converting a normal cell intoa tumor cell (constitutive activity dominant phenotype)

A tumor suppressor-gene is a gene whose loss, or inactivation, contributes to converting a normal cell into a tumor cell (recessive phenotype)

Bcl-2

CKI

point mutation overexpression

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

Cell type A Cell type B Cell type C

RTK Wnt Hedgehog

Cells from different tissues express distinct sets of growth factor receptors and signaling proteins

Alterationsin tumors: RTK signals Wnt signals Hedgehog signals

Major mitogensignaling pathway:

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G1

myc

Ras

Raf

Erk

XGF Wnt

Dishevelled

b-catenin

G1 myc

Frizzled

G1 myc

RTK

Gli

Fused

Patched

Smoothened

Gli

SuFu

Hedgehog

GSK-3b AxinAPC

Aberrant proliferative signals in tumors 19

The

retin

obla

stom

a pa

thw

ay

Cdk G1

Cdk S

Rb

DNAreplisome ORC

Cdc6PP

p15Mitogen signaling

E2F

HPV E7

p21p16

Insensitivity of tumors to anti-growth signals

TGF-b

viral

20 = germ linemutations identified

Survival factor signaling

Caspase 9 Caspase 3

Apoptosis

BH3only

Caspase 8

Adaptor

Deathreceptor

Ligand

BaxBcl-2

Evading cell death (apoptosis)

p53

Cyt. C

21

22

Randomly acquired oncogenic mutations drives tumor progression

1. Self-sufficiency in proliferative signals

3. Evading cell death (apoptosis)

2. Insensitivity to anti-growth signals

4. Limitless replicative potential

5. Sustained angiogenesis

6. Metastasis capability

1

2

3

4

5

6

Mutation

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Same “diagnosis” but different set of mutations

1 2 3 4 5 6

X XX1 2 3 4 5 6

X X

X

Patient A with diagnosis X

Patient B with diagnosis X

X

1013

Controlled and co-ordinated divisions

Cell death and replacement risk for mutations & chromosomal instability

How many somatic mutations during a life time?

Uncontrolled divisions Tumors

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– 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)

Year 1-15

25

Other genesCancer related genes

Time

Normal cells have a very low rate of mutations

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”)

Random mutation

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Genomic instability: Two distinct levels

1. Defective DNA repair (MIN)

2. Chromosome segregation errors (CIN)

X

Mutation in a gene encoding some enzyme required for DNA-repair

No repair many mutations accelerated tumor progression

X

Mis-segregation due to a defective a gene that encodes some protein essential for high fidelity chromosome segregation

27

Other genesCancer related genes

Time

MIN reflects an escalated mutation rate

DNA repair (TS)

?? Often uncertain which ones of all the mutations that contribute to tumor progression

1 2 3 4 5 6

Genetic alteration

MIN: mini-satellite DNA instability (due to defective DNA repair)

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CIN through excessive centrosomesTwo centromes

More than two centrosomes

miss-segregation aneuploidyCIN: Chromosomal instability

Kinetochore attachments satisfy the spindle checkpoint

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Centrosome

CIN through loss of APC

Kinetochore

Centromere

The tumor suppressor gene productAPC functions as a MT plus-end stabilizing protein ( ) that facilitatesstable MT-kinetochore connections

AC

APC

CIN: Chromosomal instability

Satisfied spindle checkpoint

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1) A normal cell

2) A tumor cell with a (partially) defective spindle checkpoint

CIN through a defective spindle checkpoint

Delayed anaphase until all kinetochores are attached

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Genomic instability and tumor progression

xx

”Selection barriers”

Too much genetic

instability

”Optimal” genetic instability

A stable genome

32

Principles of cancer treatment

Surgery-

Chemotherapy-

Radiation-

Impossible to remove all cancer cells

Targets both cancer- and normal cells

Side-effects

General chemotherapy: drugs that interferes with: i) DNA-replicationii) DNA structureiii) The function of the microtubule-system

Chemotherapy may also include cell type specific drugs. E.g. inhibition of hormone dependent tumor growth

33

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!