Cellular Senescence and Death

Junfeng Ji (纪俊峰), PhD

Laboratory of Somatic Reprogramming and Stem Cell AgingProfessor

Laboratory of Somatic Reprogramming and Stem Cell AgingCenter for Stem Cell and Tissue Engineering

School of Medicine

E il jij f @ j d

Zhejiang University 

Email: jijunfeng@zju.edu.cn

Bi thBirth

DeathAging

Cell DeathCell Division Cell Senescence Cell DeathCell Division Cell Senescence

I. Cellular Senescence

What does a senescent cell look like?

Primary mouse embryonic fibroblasts (MEFs)

Before senescence Senescent after passagesSpindle-shaped Larger, flatten shape and express

senescence-associated β-galactosidasea maker of cellular senescence

Modified from Wikipedia

Classification of Cellular SenescenceClassification of Cellular Senescence

Replicative cellular senescence

ll l Premature cellular senescence

1 Stress induced senescence1. Stress‐induced senescence

2. Oncogene‐induced senescenceg

3. Tumor suppressor loss‐induced senescence

Replicative cellular senescence

“Hayflick phenomenon” or the Hayflick limit in honour of Dr Leonard Hayflick Hayflick phenomenon , or the Hayflick limit in honour of Dr. Leonard Hayflick, co-author with Paul Moorhead, of the first paper describing it in 1961.

Hayflick in 1961 described that the proliferative capacity of primary cells Hayflick in 1961 described that the proliferative capacity of primary cells consistently displays three phases: phase I, a period of little proliferation before the first passage; phase II, rapid proliferation; and phase III, cells gradually stop to proliferate (Hayflick and Moorhead 1961).proliferate (Hayflick and Moorhead 1961).

Commenting on the possible causes of the transition to phase III, Hayflick (1965) hypothesized that ‘‘The finite lifetime of diploid cell strains in vitro may be ( ) yp p yan expression of aging or senescence at the cellular level.’’ The term cellular senescence therefore denotes a stable and long-term loss of proliferative capacity, despite continued viability and metabolic activity.y y

Mechanisms of Replicative cellular senescence

Telomeres act as a molecular clock, reflecting the replicative history of a primary cell (Harley et al. 1990).

TelomereA telomere is a region of repetitive nucleotide sequences at each end of a chromosome. It protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes.

Telomere shortens as cells divide 

Copied from A Genetic Solution to Slowing Aging and Preventing Disease

Why?y

DNA “end‐replication problem” 

When telomeres reach a critical minimal length, their protective structure is disrupted. This triggers a DNA damage response (DDR)gg g ( )

Copied from Dr Forsburg's all-purpose Cell Cycle Lecture Notes

RB and p16 are involved in replicative senescence

Telomerase

Telomerase is a ribonucleoprotein that is an enzyme which adds DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3‘ end of DNA strands in the telomere regionstelomere regions.

Telomerase was discovered by Carol W Greider and Elizabeth Blackburn in 1984Telomerase was discovered by Carol W. Greider and Elizabeth Blackburn in 1984 in the ciliate Tetrahymena.

Human telomerase consists of two molecules each of human telomerase reverse transcriptase (TERT), telomerase RNA (TR or TERC), and dyskerin (DKC1).

TERT is a reverse transcriptase, which is a class of enzyme that creates single-stranded DNA using single-stranded RNA as a template.

Telomerase extends the length of a telomere

St C ll I f ti Th N ti l I tit t f H lth f t llStem Cell Information, The National Institutes of Health resource for stem cell research, Appendix C: Human Embryonic Stem Cells and Human Embryonic Germ Cells.

Premature cellular senescence

Senescence can be induced also in the absence of any detectable telomere loss or dysfunction, by a variety of conditions.

This type of senescence has been termed premature, since it arises prior to the stageat which it is induced by telomere shortening.

In recent years, evidence for the existence of premature senescence in vivo has beenaccumulating rapidly and points to a critical role in tumor suppression.

1. Stress‐induced senescence

2. Oncogene‐induced senescenceg

3. Tumor suppressor loss‐induced senescencepp

Stress‐induced senescence in vitro

In vitro, premature senescence can result from inadequate culturing conditions.

This type of cell cycle arrest is independent of telomere length.

For example, mouse embryonic fibroblasts (MEFs) undergo senescence after y ( ) ga limited number of passages in culture, despite their retaining of long telomeres.

Senescence of MEFs can be bypassed also by inactivation of p53 or simultaneous ablation of RB family genes

Oncogene‐induced senescence in vitro

Early studies on mutant HRAS (HRASV12) led to the discovery that it inducesEarly studies on mutant HRAS (HRASV12) led to the discovery that it induces cell cycle arrest when it is introduced alone into primary cells.

Serrano et al (1997) noted the striking phenotypic resemblance of suchSerrano et al. (1997) noted the striking phenotypic resemblance of such nonproliferating cells to those in replicative senescence, and this phenomenonhas eventually come to be known as OIS.

Unlike replicative senescence, OIS cannot be bypassed by expression of hTERT, confirming its independence from telomere attrition (Wei and Sedivy 1999).)

Like replicative senescence, the p53 and p16INK4A–RB pathways are critically involved in OIS, at least in certain settings.g

Tumor suppressor loss‐induced senescence in vitro

Similar to oncogene mutation or overexpression, loss of a tumor suppressor can also trigger senescence in mouse and human cells. This was first illustrated for PTEN and NF1.for PTEN and NF1.

Biomarkers of cellular senescence

Cell cycle arrest Cell cycle arrestLong-term exit from the cell cycle is the central and only indispensable marker for the identification of all types of cellular senescence both in vitroand in vivo. However, cell cycle arrest is not unique to senescence.

Cellular senescence is largely irreversible. However, there are multiple g y , pways to reverse the arrest, allowing cells to re-enter the cell cycle.For example, inactivation of the p53 pathway permits senescence reversal.

Morphological transformationCell senescence is generally accompanied by morphological changes andsenescent cells can become large, flat,and multinucleated, or rather refractile depending on the trigger.p g gg

Activation of tumor suppressor networksThe p53 and p16INK4A RB signal transduction cascades commonly mediateThe p53 and p16INK4A–RB signal transduction cascades commonly mediatethe activation of the senescence program . Other proteins in the p16INK4A–RB and p53 pathways, notably p21CIP1 and15INK4B l ft l t i t ll d h b dp15INK4B, also often accumulate in senescent cells, and have been used

as markers reflecting the activation of these pathways in senescence

Induction of SA-b-GAL activitySA-b-GAL is a commonly used senescence biomarker.Its increased activity in senescent cells derives from lysosomalIts increased activity in senescent cells derives from lysosomal b-D-galactosidase, which is encoded by the GLB1gene.

Senescence-associated heterochromatic foci (SAHF)Cellular senescence can be associated with an altered chromatin structureCellular senescence can be associated with an altered chromatin structure, at least in vitro. While DNA dyes display overall homogenous staining patterns in cycling or quiescent human cells, senescent cells often showstrikingly different punctate staining patternsstrikingly different punctate staining patterns.

These DNA SAHF (Narita et al. 2003) are specifically enriched in methylated L 9 f hi t H3 ( difi ti t l db th hi t th lt fLys 9 of histone H3 (a modification catalyzedby the histone methyltransferase Suv39h1)

Senescent cells display increased binding of heterochromatin-associated proteins in the promoters of several E2F target genes. SAHF formation is circumvented by interference with p16INK4A–RB pathway y p p ysignaling, correlating with bypass of senescence.

Cell. 2003. 113(6): 703-716.

Formation of Senescence-associated heterochromatic foci (SAHF)( )

Gene. 2007. 397: 84-93.

Senescence-associated secretory phenotype (SASP)Cells undergoing senescence whether in response to telomereCells undergoing senescence—whether in response to telomere malfunction, DNA damage, or oncogenic alterations—exhibit profound changes in their transcriptomes. A major consequence of this is the

ti f d f f t i l di t ki dsecretion of many dozens of factors, including cytokines and chemokines (Campisi 2005).

Nature Reviews Cancer 2009. 9:81‐94. 

Signal transduction pathways (partially) used by SASP

Nature Reviews Cancer 2009. 9:81‐94. 

Cellular senescence in tissue aging and tumorigenesis

Aging 2009, 1(5): 438‐441.

Remaining questions

1. Whether SASP contributes to organismal aging?

2. Which element of SASP is involved in organismal aging?

3 Whether it’s possible to prolong our life-span by targeting3. Whether it s possible to prolong our life-span by targetingSASP components?

II. Cell Death

Cell DeathCell Death

Apoptosis Apoptosis

Necrosis Necrosis

Necrosis(cell swells)

Cell becomes leakyblebbing Cellular and nuclear lysis( ) Cellular and nuclear lysis

causes inflammation

Apoptosis(cell shrinks,

Chromatin condenses)“Budding”

Apoptotic bodiesare phagozytosed,

No inflammation

Modified from the review composed by Andreas Gewies in 2003

Discovery of apoptosisy p p

1962 1972 1972 1970s Mid-1980s

Kerr, Wyllie K d S l

1962-1972 1972

Brenner, Horvitz

Mid 1980s

Horvitz

Formulation of apoptosis

and CurrieObservation of shrinkagenecrosis distinct fromclassical necrosis during

Kerr, and Searle and Sulston

Discovery of cell death during the development

Identified the first "celldeath" genes, called ced-3

d d 4 Th i i ip p

conceptclassical necrosis duringhistological studies ofischaemic liver injury andmalignant tumours

during the development of C. elegans and ced-4. Their activation

is essential for cell death.

19921988 1993 1994

Hengartner and Horvitz

Vaux Shaham and Horvitz

Hengartner and Horvitz

Discovery of ced-9 thatprotects against cell deathby regulating both ced3and ced4

Discovery ofhuman bcl-2’santi-apoptotic andtumorigenic role

Discovery ofinterleukin-1-beta-converting enzyme(ICE), a protein

Showing that ced9has similarsequence to humanbcl-2.and ced4tumorigenic role. similar to CED-3.bcl 2.

19971997

Xiaodong Wangg g

Discovery of Apaf-1, a protein similarto CED 4to CED-4.

Unveiled a surprising roleUnveiled a surprising role of mitochondria in apoptosis

Apoptosis is the process of programmed cell death that is evolutionally conserved

Alexei Degterev and Junying Yuan. Nature Reviews Molecular Cell Biology 2008.

The Nobel Prize in Physiology or Medicine 2002

Sydney Brenner H. Robert Horvitz John E. Sulston

"for their discoveries concerning genetic regulation of organ development and d ll d th'"programmed cell death'".

Dr. Xiaodong WangDr. Junying Yuan

Regulation of Apoptosis

Morita & Tilly 1999

Pathways of Apoptosis The intrinsic pathway: BCL-2-regulated or mitochondrial pathway.

(1) A ti t d b i d l t l l i l i f ti DNA d(1): Activated by various developmental clues, viral infection, DNA damageand growth-factor deprivation

(2): Strictly controlled by BCL-2 family proteins(3): This pathway predominantly activates caspase 9, but in certain cell types

it can proceed in the absence of caspase 9 or its activator Apaf1.

The extrinsic pathway: Death receptor pathway.(1): Triggered by ligation of death receptors (members of the tumour necrosis

factor receptor family, such as Fas or TNF receptor-1 that contains anfactor receptor family, such as Fas or TNF receptor 1 that contains anintracellular death domain.

(2): The death domain can recruit and activate caspase 8 through the adaptorprotein Fas-associated death domain (FADD) at the cell surfaceprotein Fas-associated death domain (FADD) at the cell surface.

(3): This recruitment causes subsequent activation of downstream caspasessuch as caspase-3, -6, or -7 without involvement of the BCL-2 family.

(4): In some cells most notably hepatocytes the extrinsic pathway can(4): In some cells, most notably hepatocytes, the extrinsic pathway can intersect intrinsic pathway through caspase 8 cleavage-mediated activation of BH3-only protein BID. The C-terminal truncated form of BID ( ) f(t-BID) translocates to mitochondria and promotes further caspase activation (caspase 9 and effector caspases 3, 6 and 7) through intrinsic pathway.

Intrinsic and extrinsic pathways of Apoptosis

Youle and Strasser. Nature Reviews Molecular Cell Biology 2008.

The BCl-2 family

Youle and Strasser. Nature Reviews Molecular Cell Biology 2008.

Possible interactions between anti-apoptotic (white) and pro-apoptotic (black) members of the Bcl-2 family at the outer mitochondrial membrane(black) members of the Bcl 2 family at the outer mitochondrial membrane

Conformational changes in BCL-2 family membersDuring apoptosisDuring apoptosis

Youle and Strasser. Nature Reviews Molecular Cell Biology 2008.

The interplay between anti-apoptotic and pro-apoptotic Bcl-2 family factors determine the susceptibility to apoptosis

1 Apoptotic signals relocate Bax from the cytoplasm to the mitochondrion

factors determine the susceptibility to apoptosis

1. Apoptotic signals relocate Bax from the cytoplasm to the mitochondrion.

2. Anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL can2. Anti apoptotic members of the Bcl 2 family, such as Bcl 2 and Bcl XL can block the pro-apoptotic effect of Bax by binding it and forming heterodimers.

3. However, other pro-apoptotic Bcl-2 proteins, e.g. BAD and BID, can interactwith Bcl-2 and Bcl-XL to prevent their anti-apoptotic function.

4. Eventually, the relationship between pro-apoptotic and anti-apoptotic factorsdetermine the susceptibility to apoptosis. If there are more pro-apoptotic factors,the mitochondrion subsequently loses its member potential and a number ofapoptosis-promoting molecules, such as cytochrome C and and apoptosis-inducing factor (AIF) are released to the cytoplasm.

Caspases: the central executors of apoptosisp p p

Discovery of Caspasesy p

Caspase: Cysteine-dependent aspartate specific protease.

Represent a family of cysteine proteases that cleave after an Asp residue in their substrates, hence the name caspase.

Caspases are all expressed as proenzymes (zymogens) which contain at least three domain, and activation of caspases involves proteolytic processing between domainsbetween domains.

CED-3 is similar to mammalian interleukin-1-converting enzyme (ICE-1 or caspase 1).p )

First implicated in apoptosis with the discovery that CED-3, which is a product of a gene required for cell death in C. elegans.g q g

Classification of Caspases

DED: Death Effector Domain

CARD: Caspase Activation andpRecruitment Domain

Annu. Rev. Cell Dev. Biol. 2005. 21:35–56.

Initiator vs Effector caspases

Caspases-2, -8, -9 and -10Initiator caspases:

Containing an extended N-termianl pro-domain (﹥90 amino acids) important for its functionimportant for its function.

Are auto-activated under apoptotic conditions.

Caspases-3, -6, and -7Effector caspases:

Containing a short N-terminal pro-domain (only 20-30 amino acids.

Are activated by initiator caspases.

The prodomains of caspases

DED (Death Effector Domain): Caspase 8 and 10 contain a DED

The DED is required for association with the death domain of death receptor and subsequent activation of the caspasesdeath receptor and subsequent activation of the caspases

CARD: Several caspases contain a caspase activation and recruitmentCARD: Several caspases contain a caspase activation and recruitment domain (CARD)

The CARD domains are found in caspases involved in both inflammation and apoptosis, so its role can be varied from caspase to caspase.p

The structural features of Caspases

M l C ll 2002 9 459

1. Four protruding loops L1, L2, L3 and L4 form a substrate binding groove and shape the active site

Mol. Cell 2002. 9:459.

shape the active site.

2. Loop L1 constitutes one side of the groove whereas L4 represents the other side.

3. Loop L2 harbors the catalytic residue Cys. Loop L3 serves as the base of the active site.

4. Loop L2 stabilizes the L2 and L4 loops

Activation of Caspase

1 Long prodomain caspases contains protein1. Long prodomain caspases contains protein-protein interaction module, which allows it to bind to and associate with its regulators.

2. The first activation cleavage between the large and small subunits.

3. Four surface loop structures form the catalytic groove.

4. Removal of the pro-domain is unnecessary for itscatalytic activity.

Thornberry and Lazebnik. Science. 1998. 281:1312-1316.

How do caspases kill a cell?

Modified from Thornberry and Lazebnik. Science. 1998. 281:1312-1316.

1. Caspases inactivate proteins that protect cells from apoptosis: The cleavage of ICAD,an inhibitor of nuclease CAD (caspase activated deoxyribonuclease) responsible foran inhibitor of nuclease CAD (caspase activated deoxyribonuclease) responsible forDNA fragmentation.

2. Caspases cleaves Bcl-2 proteins, the negative regulator of apoptosis. p p g g p p

How do caspases kill a cell?

Modified from Thornberry and Lazebnik. Science. 1998. 281:1312-1316.

1. Caspases cleaves nuclear lamins , causes lamina to collapse and contribute tochromatin condensation.

2. Caspases recognize cell structure indirectly by cleaving several proteins involved in cytoskeleton regulation, including gelsolin, focal adhesion kinase (FAK), and p21 activated kinase 2(PAK-2).

How do caspases kill a cell?

Modified from Thornberry and Lazebnik. Science. 1998. 281:1312-1316.

Caspases disregulate proteins by separating regulatory and catalytic domains resultingCaspases disregulate proteins by separating regulatory and catalytic domains, resultingin loss of function or gain of function. For example, they inactivate or deregulate proteinsinvolved in DNA repair (DNA-PKcs), mRNA splicing (U1-70K), and DNA replication (such as replication factor C). It’s likely that disabling of critical homeostatic and repair functionsreplication factor C). It s likely that disabling of critical homeostatic and repair functions facilitate the cellular disassembly.

Necrosis

Programmed Necrosis (Necroptosis)g ( )

Physiological necrosis in vivo was also observed around a decade ago undervarious physiological settings including mammalian development and tissuevarious physiological settings including mammalian development and tissue homeostasis (Roach et al, 2000. J. Bone Joint Surg. Br. 82, 601–613; Barkla et al,1999, Pathology 31, 230–238).

The understanding of a specific cellular program for necrosis (necroptosis) was initiated by the discovery that the serine-threonine kinase RIP1 (RIPK1) is required for the necrosis of Jurkat human T lymphocyte cells triggered by the death receptorfor the necrosis of Jurkat human T lymphocyte cells triggered by the death receptor Fas (CD95) and the necrosis of mouse embryonic fibroblasts (MEFs) induced by TNF (Holler et al. 2000. Nat. Immunol. 1, 489–495; Lin et al. 2004. J. Biol. Chem. 279, 10822–10828)., )

The RIP1 family member RIP3 has been identified as a key mediator of caspase-independent cell death (Cho et al. Cell 2009.137, 1112–1123; He et al. Cell 2009.p (137, 1100–1111; Zhang et al. Science 2009. 325, 332–336).

Therefore, necroptosis is controlled by an intrinsic cellular program.

Necroptosis is induced by various stimuli

Han et al. 2011 Nature Immunology.

Necroptosis is induced by various stimuli

Han et al. 2011 Nature Immunology.

Difference between necrosis and apoptosis

Roche Applied Science

Methods to detect and discriminate apoptosis and necrosisapoptosis and necrosis

1. Transmission electron microscopy

Normal cell Apoptotic cell Necrotic cell

Krysko et al. Methods. 2008. 44: 205-211.

2. Flow cytometry

1. Phosphatidylserine (PS) is an aminophospholipid that resides in the inner leaflet of the plasmathat resides in the inner leaflet of the plasma membrane of living cells.

2. During apoptotic cell death, PS is actively g p p , yexternalized to the outer surface of the plasma membrane, where its presence is required for recognition and engulfment of dying cells.g g y g

3. PS is an early marker of apoptotic cells death. It can be detected by using annexin V, a Ca2+-dependent phospholipid-biding protein.

4. During apoptosis, there is a lag period between PS i i i d PI i i i hil i iPS positivity and PI positivity, while in necrotic cells both events coincide.

Krysko et al. Methods. 2008. 44: 205-211.

3. Western blot analysis on caspase 3 activation

Copied from Active Motif website

4. Immunostaining on caspase 3 activation

Taranukhin et al, J. Biomed. Sci. (2010)

5. TUNEL assayTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a

h d f d i DNA f i b l b li h i l d f l imethod for detecting DNA fragmentation by labeling the terminal end of nucleic acids.Assay principle: TUNEL assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. It may also label cells that have suffered severe DNA damage.

Griffin et al. Cancer Cell International 2007 7:10

Copied from R&D System website

6. Agarose gel electrophoresis analysis of apoptotic DNA fragments

Modified from ABD Serotec website

Yu et al. J Investigative Dermatology (2002) 119, 812–819.