The Biology of Apoptosis

Fouad Boulos, MD
August 2010

I. Definition:

Apoptosis takes its name from the Greek “Falling off” used for falling
dead leaves or petals. It describes a tightly regulated intracellular
pathway of cell death involving a cascade of internal enzymes resulting
in the elimination of unwanted cells without inflammation or injury to
the surrounding tissue. It is opposed to (but may coexist with) necrosis,
which results in rupture of the cell membrane, enzymatic spillage and
inflammation. Apoptosis occurs in physiologic states but can also occur
in pathologic states, and its dysregulation can be the cause of disease.

II. Apoptosis in Physiology:

a. Programmed cell death during all stages of embryogenesis: from

placental implantation to organogenesis (pronephros, mesonephros) to
developmental involution (yolk sac, cells between fingers).
b. Hormone dependent involution in the adult: endometrial breakdown,
ovarian follicular atresia at menopause, regression of lactating breast,
prostatic atrophy after castration.
c. Cell deletion in constantly proliferating cell populations such as the
gastrointestinal tract.
d. Death of inflammatory cells during or at the end of an immune
reaction (neutrophils, lymphocytes).
e. Elimination of self-reactive lymphocytes.

III. Apoptosis in Pathologic conditions:

a. Atrophy in parenchymal organs following duct obstruction.

b. Cell death in tumors (regression or growth).
c. Virally induced cell death (hepatitis).
d. Cell death secondary to injurious stimuli such as heat, hypoxia, and
more importantly radiation and cytotoxic cancer drugs which result in
irreversible DNA damage and cell suicide.
e. Graft vs. Host Disease, where cytotoxic T-cells from the graft induce
apoptosis in “foreign” host cells through perforin and Granzyme B.

IV. Cell Morphology in Apoptosis:

a. Cell shrinkage, cytoplasmic condensation with packed organelles.

b. Chromatin condensation under nuclear membrane, followed by
nuclear fragmentation.
c. Cytoplasmic blebs and apoptotic bodies that are phagocytosed by
macrophages mostly.
d. Apoptosis affects single cells or cell groups and is not easily seen in
tissue sections due to the rapidity of the process. Apoptotic cells have a
densely eosinophilic cytoplasm and very dark fragmented nucleus.

V. Biochemistry of Apoptosis:

• Protein cleavage: activation of cysteine proteases “caspases” that

cleave the nuclear scaffold and cytoskeletal proteins and induce
endonucleases.
• DNA breakdown: into large 50-300 kb fragments then multiples of
180-200 bp through internucleosomal cleavage by Ca++ and Mg++-
dependent endonucleases  DNA ladder on agarose gel
electrophoresis, characteristic but not specific for apoptosis; and DNA
smear in late autolytic stage.
• Phagocytic recognition: through expression of phosphatidylserine
and thrombospondin on outer surface of plasma membrane;
opsonization by proteins secreted by phagocytes that recognize dying
not live cells, allowing recognition by phagocytic cells without release of
pro-inflammatory factors.

VI. Mechanisms of Apoptosis:

a. Extrinsic Pathway:
Death receptors on the surface of cells, mostly a family of TNFR (tumor
necrosis factor receptor) including Fas and TNFR1 with cytoplasmic
“death domains”, interact with soluble ligands and initiate the cascade:
Fas + Ligand  FasL  Fas receptors polymerize  cytoplasmic death
domain  binding of cytoplasmic “adapter protein” with its own death
domain (FADD)  binding of caspase-8  autocatalytic activation 
execution phase of apoptosis.

b. Intrinsic Pathway:
Mitochondrial permeability determined by ratio of pro- and anti-
apoptotic factors. Apoptotic signal  increased mitochondrial
permeability with pores opened in inner membrane  reduction of
membrane potential and swelling of mitochondria  increased
permeability of outer mitochondrial membrane  release of
cytochrome c into cytosol  Apoptotic morphologic changes occur
immediately thereafter. The main mitochondrial regulators belong to
the Bcl-2 family including Bcl-2 and Bcl-x (antiapoptotic) and Bak, Bax
and Bim (proapoptotic).
•Bcl-2 prevents cytochrome c release.
•If Bcl-2 is low  Cytochrome c is released from mitochondria by
death signals, and activates Apaf-1 (apoptosis activating factor).
•Apaf-1 usually activates initiator caspases by cleavage.
•Bcl-2 also binds Apaf-1 and inhibits its catalytic function.

c. Execution Phase:
Final & common pathway for different and heterogeneous signaling and
regulatory mechanisms, it is mediated by a family of cysteine-aspartic
acid proteases called “caspases” which are mammalian homologs of Ced
genes. They are divided into 2 groups: initiators & executioners.
Caspases exist in inactive form and are activated by cleavage. Cleavage
sites are hydrolyzed by other caspases and autocatalytically.
Initiator caspases trigger enzymatic death program, executioner
caspases cleave cytoskeletal and nuclear matrix proteins and disrupt
cytoskeleton and nucleus.

d. Removal of dead cells:

The dying cell releases soluble substances that attract histiocytes, and
apoptotic bodies express surface molecules that allow opsonization and
phagocytosis by macropahges.

VII. Apoptosis dysregulation and Disease:

• Defective apoptosis and increased cell survival:
 a. Seen in tumors with p53 mutation and hormone-dependent
tumors (breast cancer, prostate cancer, ovarian cancer).
b. Also seen in autoimmune disorders where auto-reactive
lymphocytes are not eliminated after encounter with self-
antigen.
 Increased apoptosis and excess cell death:
a. Neurodegenerative disorders
b. Ischemic myocardial injury
c. Viral infection (hepatitis, aplastic anemia)