Cell death and Cell renewal

Programmed Cell Death● Programmed cell death (apoptosis cell suicide) 기능● Programmed cell death (apoptosis, cell suicide) 기능

Defense – virus-infected cells undergo programmed cell deathcellular insults such as DNA damage induce programmed cell death

Development – elimination of unwanted cells- elimination of larval tissue during morphogenesisg p g- Neuron cells are produced in excess & cells in incorrect connection with target cells are eliminated- Many cells undergo programmed cell death unless they contact with neighboring cells or ECM

correctly or in the absence of appropriate growth factors

Cells that die as a result of acute injury swelland lyse, releasing their contents into theextracellular space and causing inflammation

● Th f A i● The events of Apoptosis

The chromatin decondenses

The nucleus breaks up

Ch l DNA i f t dChromosomal DNA is fragmented

The cell shrinks and breaks up into

membrane-enclosed fragments

(apoptotic bodies)(apoptotic bodies)

Apoptotic cells are efficiently recognized and removed by phagocytic cellsy p g y

The removal of apoptotic cells is mediated by the expression of so-called “eat me” signals on the cell surface

This signal include phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane.

During apoptosis, phosphatidylserine become exposed on the cell surface (outer leaflet of the plasma membrane) by “scramblase” where it is recognized by receptors expressed by phagocytic cells

Caspases – the executers of apoptosis

Molecular mechanism of apoptosis – from studies of the development of nematode (C. elegans)

The nematode consists of 959 somatic cells – during normal nematode development, 131 cells are

eliminated by apoptosiseliminated by apoptosis

Mutants that affect apoptosis in nematode were isolated, ced-3, ced-4, ced-9

Ced-3 was first identified as a protease and is the prototype of a family of more than a dozen

proteases, known as caspases (cystein residues at their active sites – C, cleave after aspartic acid – Asp)

● Key targets of the caspases

a inhibitor of a DNase

nuclear lamins

cytoskeletal proteinscytoskeletal proteins

Caspases – act as either initiators or effectorsCaspases act as either initiators or effectors

Caspases are synthesized as inactive precursors and converted to active

forms by proteolytic cleavage, catalyzed by other caspases

The activation of an initiator caspase starts off a chain reaction leading to

activation of downstream effector caspases

The key initiator caspase – caspase-9

Apoptosome – Apaf-1 (ced-4), caspase-9, and cytochrome c

- cytochrome c is released from mitochondira by stimulicytochrome c is released from mitochondira by stimuli

that trigger apoptosis and activates apoptosome

- Then the activated caspase-9 cleaves and thus activates

d ff ddownstream effector caspases, caspase-3 and caspase-7

Central regulators of Apoptosis – Bcl-2 family

C. elegans Ced-9 = mammal Bcl-2, Bcl-2 was previously identified as an oncogene that contribute to B cell lymphomasBcl-2 was shown to inhibit apoptosis first identified connection between apoptosis and cancer

Bcl 2 family membersBcl-2 family membersAll members share conserved regions called Bcl-2 homology (BH) domain

Some are antiapoptoticOthers are proapoptoticOthers are proapoptotic

In normal cells, multidomain t ti t i l ithproapoptotic proteins complex with

antiapoptotic proteins

In response to apoptotic signals, BH3-l t ti t i ti t donly proapoptotic proteins are activated

and bind to antiapoptotic proteins, releasing multiapoptotic proteins

In mammals, regulatory apoptotic proteins act at mitochondria

1. When activated, Bax and Bak (multiproapoptic proteins)

form oligomers in the mitochondrial outer membrane

(either form pores or interact other mitochondrial outer ( p

membrane proteins), leading to release of cytochrome c

from mitochondria

2. Other mitochondrial proteins that promote apoptosis

Smac/Diablo and Omi/Htr2 are also released via Bax

and Bak oligomers and inhibits IAPs

- IAP (inhibitors of apoptosis): Members of IAP family

directly interact with caspases and suppress apoptosis y p pp p p

either by inhibiting caspase activity or by targeting

caspases for ubiquitination and degradation in the

proteasomeproteasome

Signaling pathways that regulate apoptosis

p53

One important role of apoptosis is the elimination of damaged One important role of apoptosis is the elimination of damaged

cells such as DNA damage by UV light

DNA d i d ATM d CHK t i ki hi h iDNA damage induces ATM and CHK protein kinases, which in

turn phosphorylate and stabilize p53

p53 either arrests cells by activating p21 (CKI) expression or

induces apoptosis

p53 activates the expression of BH3-only proapoptotic p p y p p p

proteins, PUMA or Noxa, leading to apoptosis

Tumor necrosis factor (TNF)

Secreted polypeptides that signal cell death

O f th b t h t i d TNF t i FOne of the best characterized TNF receptors is Fas

TNF-Fas plays important roles in immune system

- Killing target cells such as cancer cells, virus-infected cells, or

excess lymphocytes at the end of an immune response

TNF and related family members contain the cytoplasmic y y p

domain that adaptor molecules bind.

The adaptors recruit and activate caspase-8 (an initiator caspase)

Caspase 8 either activates other caspases ( 3 and 7) or cleaveCaspase-8 either activates other caspases (-3 and-7) or cleave

the proapoptotic BH3-only protein Bid, leading to Bid activation,

permeabilization of mitochondria, and activation of caspase-9

Signaling pathways that promote cell survival

A half of neuron cells die by apoptosis with the survivors having received sufficient amounts of survivalA half of neuron cells die by apoptosis, with the survivors having received sufficient amounts of survival signals from their target cells – NGF (nerve growth factor)

One of the major intracellular signaling pathways for cell survival is initiated by PI 3-kinase, leading to activation of Aktactivation of Akt

- Akt phosphorylates Bad (proapoptotic protein) 14-3-3 chaperone binds and sequester Bad

Akt inhibits FOXO which is a transcription factor to stimulate transcription of proapoptotic genes- Akt inhibits FOXO, which is a transcription factor to stimulate transcription of proapoptotic genes including Bim

Stem cells and the maintenance of adult tissue

Proliferation of differentiated cells

Fully differentiated that remains capable of proliferateFully differentiated that remains capable of proliferate

Skin fibroblast (dispersed in connective tissues where they secrete

collagen)

N ll t d i G- Normally arrested in G0

- rapidly proliferate if needed to repair damage

PDGF from blood platelets activates a receptor protein-tyrosine

kinase, stimulating both their proliferation and migration

The endothelial cells that line blood vessels

-VEGF (vascular endothelial growth factor) triggers endothelial cell

proliferation and the resulting formation of blood capillaries

The prod ction of VEGF is in t rn triggered b a lack of o gen- The production of VEGF is in turn triggered by a lack of oxygen

The epithelial cells of liver and pancreas are able to proliferate to

replace damaged tissue

Stem cells

Most fully differentiated cells in adult animals are no longer capable of cell divisionA subpopulation of less differentiated self-renewing cells called stem cells proliferate and replace the loss of differentiated cells

The key property of stem cells is that they divide to produce one daughter cell that remains a stem cell and one that divides and differentiates

Blood cells – their life spans are limited ranging from less than a day to a few monthsAll are derived from the same population of hematopoietic stem cells in bone marrow

The epithelial cells lining intestine- These cells are exposed to harsh environment and have a lifetime of only a few days

Stem cells reside at the bottom of intestinal crypts- Stem cells reside at the bottom of intestinal crypts- Stem cells give rise to a population of transit-amplifying cells- The transit-amplifying cells proliferate for 3-4 divisions and then differentiate into the three types of the colon surface epithelium (an absorptive epithelial cells and two secretory cells)

The epithelial cells of the skinThese cells are exposed to a harsh external- These cells are exposed to a harsh external

environment - The epidermis turns over every two weeks- The epidermis is a multi-layered epithelium, which p y pis maintained by epidermal stem cells resining in a single basal layer- The epidermal stem cells give rise to transit-amplifying cells, which undergo 3-6 divisions before differentiating and moving outward to the surface of the skin

The satellite cells of the Skeletal muscle cells- Skeletal muscle is composed of large multinucleated cells formed by cell fusion during y gdevelopment- Satellite cells-the stem cells of the muscle-are located beneath the basal lamina of muscle fibers

h ll i d i h- They are normally quiescent, arrested in the G0

phase, but are activated to proliferate in response to injury or exercise- Once activated they give rise to progeny thatOnce activated, they give rise to progeny that undergo several divisions and then differentiate and fuse to form new muscle fibers

Adult stem cells, embryonic stem cells and their medical applications

Adult stem cells (성체줄기세포)

Bone marrow transplantation

- The hematopoietic stem cells are among the most rapidly dividing cells of the body, so the toxic

effects of anticancer drugs on theses cells limit the effectiveness of chemotherapy in cancer treatmenteffects of anticancer drugs on theses cells limit the effectiveness of chemotherapy in cancer treatment

- The patient is treated with high doses of chemotherapy, which kill both tumor cells and the

hematopoietic cells

- This damage is then repaired by transplantation of new hematopoietic stem cells, which are obtainedThis damage is then repaired by transplantation of new hematopoietic stem cells, which are obtained

from the patient himself prior to chemotherapy or from a healthy donor

Epithelial stem cells

- A clinical application in the form of skin grafts that are used to treat patients with burns or woundspp g p

- The epidermal skin cells are in vitro cultured, made to form an epithelial sheet, and then transferred

to the patient

- This eliminates the potential complication of graft rejection by the immune systemp p g j y y

Difficulties in the use of the adult stem cells

- Isolation and culturing the appropriate stem cell populations are difficult

- Stem cells have not yet been identified in many adult tissues

Embryonic stem cells (배아줄기세포)

Embryonic stem cells can be propagate indefinitely in culture

and retain the capacity to develop into all of the different types

of cells

Mouse embryonic stem cells are grown in the presence of a

growth factor called LIF (for leukemia inhibitory factor), which

signals through the JAK/STAT pathway and is required to

maintain these cells in their undifferentiated state.

-If LIF is removed, the cells aggregate into structure that

resemble embryos and then differentiate into a wide range of

cells

- A great deal of current research is focused on the

development of culture conditions to promote the

differentiation of embryonic stem cells along specific pathways,

thereby producing populations of differentiated cells that can

be used for transplantation therapy

Somatic cell nuclear transfer

Cloning of an animal by somatic cell nuclear transfer

In 1997, Dolly arose from the nucleus of a mammary epithelial

cell that was transplanted into an unfertilized egg in place of the

normal egg nucleus – a process called somatic cell nuclear

transfer

(In the 1950s, this type of experiment was first carried out in

frogs)

Si h i i i l f f l i f d l i llSince the initial success, transfer of nuclei from adult somatic cells

into enucleated eggs has been used to create cloned offspring of

a variety of animals

O l 1 2% f b ll i i t li ff i- Only 1-2% of embryos generally give rise to live offspring

Therapeutic cloning (환자맞춤형줄기세포)

- A nucleus from an adult human cell is transferred to an

enucleated egg which is then used to produce an early embryoenucleated egg, which is then used to produce an early embryo

in culture

- Embryonic stem cells are then cultured form the cloned embryo

and used to generate appropriate types of differentiated cells forand used to generate appropriate types of differentiated cells for

transplantation therapy

- The major advantage is that the embryonic stem cells derived

by this procedure would be genetically identical to the recipientby this procedure would be genetically identical to the recipient

of the transplant – this bypasses the barrier of the immune

system in rejecting the transplanted tissue

The application of therapeutic cloning combined with gene transfer – correction of an inherited e app cat o o t e apeut c c o g co b ed t ge e t a s e co ect o o a e ted

immunodeficiency, the genetic defect from the absence of a functional gene encoding the RAG2

N l i f fib bl t f RAG2 d fi i t i t f d t l t d hi hNuclei from fibroblasts of RAG2-deficient mice were transferred to an enucleated egg, which was

used as a source of embryonic stem cells. A functional RAG2 was then introduced into embryonic

stem cells in culture, correcting the genetic defect. These corrected embryonic stem cells were

tehn differentiated into hematopoietic stem cells, and transplanted into RAG2-deficient mice,

resulting in rescue of the immunodeficiency at least partially