The Cell Cycle and its implications in diseases

Hansjörg Hauser Dept. of Gene Regulation and Differentiation Molecular Biotechnology HZI, Braunschweig

Cell division is a prerequisite for life

•Microorganisms reproduce by cell division •Mammals need cell division during embryogenesis and for tissue homeostasis Example: Adult humans produce several milions of new cells per second (more than 10 11 per day – about 100 grams)

Cell division can be fast or slow

•Microorganisms > 20 min per division •Multicellular organisms: 8 min and several weeks per division •All species can halt cell division

Neben einem zum Vergleich dargestellten Zellkern in der Interphase sind verschiedene Stadien der Mitose gezeigt (entsprechend der deutschen Literatur, daher ohne Prometaphase).

Schematische Darstellung des Zellzyklus. Zur besseren Veranschaulichung sind Chromosomen hier auch in Interphase so gezeichnet, wie sie in der Mitose aussehen. Dies entspricht jedoch nicht der Wirklichkeit.

The Cell Cycle

Schematic of the cell cycle. Outer ring: I= Interphase , M= Mitosis ; Inner ring: M= Mitosis , G 1 = Gap 1 , G 2 = Gap 2 , S= Synthesis ; Not in ring: G 0 = Gap 0/Resting .

Cell cycle control was studied in early embryogenesis of frogs, yeasts and mammalian cells.

The mechanisms and involved molecules are highly conserved The names are sometimes confusing!

Cell division and duplication of cellular constituents

•DNA •Proteins, Lipids, Carbohydrates •Organelles, Membranes...

Standard versus early embryonic cell cycle

•in early embryonic cells DNA replication is uncoupled from other synthesis •The eggs contain more cytoplasm than normal cells: Stocks •Some organisms have eggs with 100.000times more cytoplasm than normal cells •16 –17 divisions are possible without significant protein production. •These divisions are running without feedback control

Methods to measure cell division

•Counting •Amount of DNA •Enzymatic activities •Incorporation of labeled DNA precursors •Cell cycle analysis (FACS) •Dilution of dyes (CFSE) •Time lapse microscopy

CFSE

( Carboxyfluorescein succinimidyl ester ) is a fluorescent cell staining dye. CFSE is commonly confused with CFDA-SE, although they are not strictly the same molecule; CFDA-SE is cell permeable, while CFSE is not. As CFDA-SE enters the cell cytoplasm, intracellular esterases convert the molecule to the fluorescent ester, CFSE, which is retained within cells.

CFSE is a simple and sensitive technique for analysis of multiple parameters of cells. This method allows us to examine specific populations of proliferating cells and identify 7–10 successive cell generations, which has first been employed to detect proliferation of T cells in experimental animals.

[1] CFSE consists of a fluorescent molecule containing a succinimydyl ester functional group and two acetate moieties. CFSE diffuses freely inside the cells and intracellular esterases cleave the acetate groups converting it to a fluorescent, membrane impermeable dye. This dye is not transferred to adjacent cells. CFSE is retained by the cell in the cytoplasm and does not adversely affect cellular function. During each round of cell division, relative fluorescence intensity of the dye is decreased by half. In addition, unlike other methods, CFSE-labeled viable cells can be recovered for further analysis.

FACS profiles of resting and growing cells

G1 82% S 12% G2 6% G1 33% S 47% G2 19%

Kinases control the progression through the cell cycle Example MPF (maturation promoting factor) MPF is composed of a cyclin and a cyclin dependent protein kinase (cdk)

While cdks are constitutively expressed the appearance of cyclins in the cell cycle is transient – they cycle The presence of cyclins regulates the activity of the cdks

Yeasts only have one Cyclin kinase (cdk1)

Cyclic activity of Cyclin kinases

Temporal control of the animal cell cycle. The cyclin-E-, cyclin-A- and cyclin-B-dependent kinases are active at different times in the cell cycle. On this basis, cyclin E–Cdk2 appears to have a role in promoting S phase, cyclin A–Cdk2 in S phase and at G2-to-M phase, and cyclin B–Cdk1 during mitosis. Cyclin B1–Cdk1 is activated at the end of G2 phase by the phosphatase Cdc25.

Cyclin kinases

Targets of cyclin kinases: the G2 Kinase complex (MPF)

The kinase activity of cdc-cyclin compexes is regulated by phosphorylation and dephosphorylation Example MO16 is an activating kinase Wee1 is an inhibitory kinase cdc25 is a phosphatase that removes the inhibitory phosphate from the cdk

Regulation of cyclin-dependent kinases. Arrowheads represent activating events and perpendicular ends represent inhibitory events. Genes known to perform the indicated functions are listed below. Both cyclins and some CKIs (Cdk inhibitors) are regulated by synthesis and ubiquitin-mediated proteolysis. Checkpoint pathways could act to promote inhibitory pathways or inhibit activating pathways to cause cell cycle arrest

The progression through the cell cycle underlies many controls: Example DNA replication A re-replication block ensures that no segment of DNA is replicated more than once Passage through mitosis removes the re replication block Feedback controls generally depend on inhibitory signals

Checkpoint pathways

(A) A genetic pathway illustrating intrinsic and extrinsic checkpoint mechanisms. Letters represent cell cycle processes. The pathway shown as red symbols indicates an intrinsic checkpoint mechanism that operates to ensure that event C is completed before event E. After event B is completed, an inhibitory signal is activated that blocks completion of event E. After event C is completed, a signal is sent to turn off the inhibitory signal from B, thereby allowing completion of E. The blue symbols represent an extrinsic mechanism that is activated when defects such as DNA damage or spindle errors are detected. It is arbitrarily located on the D to E pathway but could also function by inhibiting a later step in the B to C pathway. In that case, the extrinsic pathway would utilize the intrinsic mechanism for cell cycle arrest.

Mutations in any of the red or blue symbols would result in a checkpoint-effective phenotype.

Checkpoint pathways

(B) Schematic representation of several cell cycle checkpoints. The colored arrows depict complex signaling pathways that operate in G1 to transmit information regarding cell proliferation. The red lines connecting particular events and cell cycle transitions represent the inhibitory signals generated by checkpoint pathways in response to those events. The points of contact of the negative growth factor and contact inhibition pathways with the cell cycle are arbitrary and meant to indicate arrest in G1.

http://www.1lec.com/Genetics/Cell%20Growth /index.html

Activation of DNA replication through gene expression in G1

G0 Activation of early response genes: fos, jun,..

G1 delayed response genes: E2F Cyclins E, D S DNA synthesis genes

Resting cells: The retinoblastoma protein Rb blocks cell cycle progression in G1 by binding to and sequestering E2F

Rb-

P

Phosphorylation causes Inactivation of Rb Rb + E2F Rb : E2F

Cell cycle progression by growth factors

Phosphorylation causes Inactivation of Rb Rb-

P

CyclinD.cdk4

Rb CyclinD MAPK pathway E2F Proliferation Rb: E2F Rb captures E2F: E2F cannot activate proproliferative genes Ras EGF

Proliferation

Cell cycle progression

Growth block Phosphorylation causes Inactivation of Rb Rb-

P

CyclinD.cdk4

Rb Rb captures E2F, so that it cannot activate proproliferative genes + E2F CyclinD MAPK pathway P16 Ink4A Ras Rb: E2F

p53

p53 is a transcriptional activator: One of the genes induced by p53 is p21, an inhibitor of the cdk4 kinase activity

p27 Rb-

P

CyclinE.cdk2

CyclinD.cdk4

Rb Rb captures E2F, so that it cannot activate proproliferative genes + E2F p21 CyclinD Rb: E2F p16 Ink4A MAPK pathway Ras

P53 is a general gatekeeper for the G1 checkpoint

P53 is a DNA binding protein

DNA damage leads to a block in cell cycle progression Replication of damaged DNA would fix mutations for all daughter cells

Possible biochemical function of the Rad24 group of checkpoint proteins. Rad24, together with the four small subunits of RFC, is a component of a pentameric complex. By analogy with RFC, this complex might recognise the transition between ssDNA and dsDNA. Such a structure is produced by many repair pathways but the Rad24 complex may only efficiently recognise it in the context of repair complexes (not shown here). Once the Rad24 complex is bound, it then functions to recruit the ‘PCNA-like’ Rad17/Mec3/Ddc1 complex to the DNA, followed by additional recruitment of checkpoint proteins involved in signal transduction (e.g. Mec1 and Rad53)

Organisation of the DNA-damage-dependent checkpoint pathways of budding yeast. Two distinct types of DNA damage, in the context of repair complexes, are represented in the schematic. Some of the components of the NER complex, specific for UV photoproducts, are indicated. The pointers indicate the incisions generated by the structure-specific endonucleases ERCC1/XPF and XPG. The Rad50/Mre11/Xrs2 complex is involved in DSB repair. RAD9 and RAD24 define upstream ‘sensor’ branches of the pathway and seem to respond, primarily outside of S phase, to multiple types of DNA damage. Currently, no other members of the RAD9 branch have been identified but the RAD24 branch can be ordered genetically as shown although strict linearity of function within this branch

Mammalian cells: The protein p53 is sensing DNA damage P53 has a very high turn-over DNA damage: p53 becomes phosphorylated and stabilized

The Events in p53 Activation DNA damage (indicated by the break in the double line at the top) is recognized by a "sensor" molecule that identifies a specific type of lesion and possibly by the p53 protein, using its C-terminal domain. The sensor modifies p53 (by phosphorylation) when both molecules correctly determine that there is damage. A modified p53 is more stable (enhanced half-life), and a steric or allosteric change in p53 permits DNA binding to a specific DNA sequence regulating several downstream genes (p21, MDM2, GADD45, Bax, IGF-BP, and cyclin G). Two modes of signaling for cellular apoptosis are possible: one requiring transcription and one involving direct signaling with no transcription of downstream genes required.

Cellular products influencing the cell cycle

Viral and cellular proteins influencing p53 activity

Cell cycle control in mono- versus multicellular systems

•Monocellular systems: Unlimited proliferation Control by size, nutrients and sex •Multicellular systems: Proliferation is limited to specific regions and circumstances: Growth factors, cell:cell-interactions, In mammals growth and proliferation are independently regulated

Influence of cellular and viral proteins in the cell cycle machinery

Growth factor stimulation through membrane receptors EGF receptor FGF receptor Extracellular ligand binding domain Transmembrane domain Tyrosine kinase domain

Growth factor stimulation through a membrane receptor EGF receptor Tyrosine kinase Phosphorylation causes Inactivation of Rb Rb-

P

CyclinD.cdk4

CyclinD MAPK pathway Ras EGF Rb E2F Rb: E2F Rb captures E2F: E2F cannot activate proproliferative genes

Cell growth inhibitors that act through a membrane receptor Anti-Growth Factors e.g. TGFß p16 Cycl D:CDK4 RB E2Fs Changes in Gene Expression p15 p27 Smads Cycl E:CDK4 p21 Cell Proliferation (Cell Cycle)

Cancer and the cell cycle

Introduction

Current view: • accumulation of multiple mutations within genes of a single cell • mutations confer a competitive advantage for cell growth and (de-) differentiation • mutations lead to initiation and progression of malignancies

Proto-oncogenes

• control cell proliferation and differentiation • are expressed in all subcellular compartments (nucleus, cytoplasm, cell surface) • act as protein kinases, growth factors, growth factor receptors, or membrane associated signal transducers

Oncogenes

• Mutations in proto-oncogenes alter the normal structure and/or expression pattern • Act in a dominant fashion



gain of function

Mechanisms of oncogene action

Biochemically, there are three known mechanisms by which these genes act: • phosphorylation of proteins, with serine, threonine and tyrosine as substrates • signal transmission by GTPases • regulation of DNA transcription

Tumor Suppressor Genes

• Have normal, diverse functions to regulate cell growth in a negative fashion (restrain neoplastic growth; act as cellular “brakes”) • physical or functional loss of both alleles frees the cell from constraints imposed by their protein products 

loss of function

What causes cancer?

• Chemical Carcinogenes – Aflatoxin B • Radiation: • Viruses machinery  1, , Vinylchloride, β-Propiolacton Dimethylsulfate ...

– UV, X-Ray, α,-β,-γ-radiation – RNA-viruses, DNA-viruses • Spontaneoud mutations Loss of DNA-repair p53

„... manifestation of six essential alterations in cell physiology that collectively dictate malignant growth.“ Cell, Vol. 100, 2000

1. Self-Sufficiency in Growth Signals 2. Insensitivity to Antigrowth Signals 3. Evading Apoptosis 4. Limitless Replicative Potential 5. Sustained Angiogenesis 6. Tissue Invasion and Metastasis

1. Self-Sufficiency in Growth Signals

• Cancer cell strategies: – Alteration of extracellular growth signals – Alteration of transcellular transducers of growth signals – Alteration of intracellular circuits that translate growth signals – Synthesis of „own“ GS and sarcomas  autocrine stimulation e.g. production of PDGF, TGFα by glioblastomas

1. Self-Sufficiency in Growth Signals growth signals (PDGF, TGFα) --> autocrine stimulation overexpression or mutation of receptors (EGF-R, HER2) disruption of intracellular circuits (SOS-Ras-Raf -Map-Kinase)

1. Self-Sufficiency in Growth Signals

EGF receptor Tyrosine kinase

2. Insensitivity to Antigrowth Signals

Anti-Growth Factors e.g. TGFß p16 Cycl D:CDK4 RB E2Fs Changes in Gene Expression p15 p27 Smads Cycl E:CDK4 p21 Cell Proliferation (Cell Cycle)

2. Insensitivity to Antigrowth Signals Disruption of Rb-pathway, downregulation of death receptors

3. Evading Apoptosis

Apoptotic machinery: sensors and effectors Survival signals: IGF-1/IGF-2  Receptor: IGF-1R IL-3 Death signals:  Receptor: IL-3R FAS TNFα  Receptor: FAS-R  Receptor: TNF-R1

3. Evading Apoptosis

Intracellular signals: • There are both proapoptotic genes (cell death agonists such as Bax, Bak, Bid, Bim) and antiapoptotic genes (cell death antagonists such as Bcl-2, Bcl-xL) • The prototypic gene in this category is Bcl-2

3. Evading Apoptosis Loss of proapoptotic regulators (p53), nonsignaling deathreceptors (FAS)

CELL DEATH

3. Evading Apoptosis

Death Factor Caspase 9 Cytochrom C Bid Caspase 8 MITO Bim, etc.

FADD Bax p53 Abnormality sensor DNA damage sensor

4.Limitless Replicative Potential

• With each cell division, there is a shortening of specific tracts of DNA at the ends of chromosomes (50 – 100 bp) • These tracts are called telomeres and are composed of repetitive DNA sequences • Once shortened beyond a certain point, cells die • Telomere shortening, therefore, acts as a clock that counts cell divisions

4.Limitless Replicative Potential

• In germ cells, telomere shortening is prevented by the enzyme complex telomerase • Telomerase adds back any repetitive telomere sequences lost after a cell division • • Most somatic cells lack telomerase • For a cell to divide indefinitely, it must prevent telomere shortening

Tumor cells do this by activating telomerase

5. Sustained Angiogenesis

• Angiogenesis (growth of new blood vessels) • Cells reside within 100 µm of a blood vessel (nutrients/oxygen supply) • Regulating signals may stimulate/block angiogenesis • Initiating signals: e.g. VEGF/ bFGF • Inhibitor signals: e.g. Thrombospondin-1

5. Sustained Angiogenesis Increased expression of angiogen. inducers (VEGF, bFGF) loss of p53 -->downregulation of inhibitors (thrombospondin-1)

5. Sustained Angiogenesis

• Tumor cells change the Inducer/Inhibitor balance • Possibilities: – Increased expression of VEGF/FGF – Downregulation of Thrombospondine-1, or ß-interferon • Loss of p53  Thrombospondine-1 • Activation of

ras

 Increased expression of VEGF • Proteases release bFGF stored in the ECM

6. Tissue Invasion and Metastasis

6. Tissue Invasion and Metastasis

• Affected proteins: – cell-cell-adhesion molecules (CAMs)  e.g. Ca-dependent cadherin families – Integrins  responsible for linking cells to extra cellular matrix (ECM)

6. Tissue Invasion and Metastasis

Actin, Myosin II, Tropomyosin Α-Actinin, Vinculin α β Integrin Fibronectin

6. Tissue Invasion and Metastasis

Signal transmission E-cadherin Ca 2+ Cell-to-Cell coupling by E-cadherin Signal transmission

6. Tissue Invasion and Metastasis

• Cancer cells use extracellular proteases to facilitate invasion into: nearby stroma, across blood vessel walls, „normal“ epithelian cells • Upregulation of protease genes, downregulation of inhibitor genes • Inactive zymogen forms of proteases are converted into active enzymes • Often cancer cell contacted stromal and inflammatory cells deliver the proteases

6. Tissue Invasion and Metastasis „Out-of-order“ CAMs (E-cadherin), changing integrin expression pattern, overexpression of extracellular proteases, downregulation of protease inhibitor genes

Summary

„... manifestation of

six

essential alterations in cell physiology that collectively dictate malignant growth.“

Cell, Vol. 100, 2000