Thursday, August 30, 2001
Dr. Ralph Buttyan Jessica L. Fiorelli
Dr. Buttyan began by introducing three genetically regulated cellular pathways:
1. Proliferation – occurs by response of cells to signals from adjacent cells in the environment
2. Differentiation – cells taking on properties that give them distinctive features
3. Programmed death – cells under a “suicide” process when necessary
The idea of programmed death as an important cell action pathway emerged in the scientific literature of the 1930s
and 1940s. Research in five areas established apoptosis as an important contributor to development and
1. Embryonic development
In 1942, Saunders published a study in Science on the development of the limb bud in avian
embryos. He noted that at a certain time in development of the embryo, there was distinct and
extensive pattern of cell death in the limb bud. This cell death was important in the
morphogenesis of the limb bud.
Similarly, in the limb buds of developing humans 4-6 weeks after implantation, regions of
cells between the developing digits begin to die. Within 3 days, all of these inter-digital
mesenchymal cells have died leaving the fetus with 5 distinct digits on each limb bud. This
shaping of tissue (morphogenesis) via cell death is necessary for development of many
morphological features of the adult.
2. Sexual development
Up until the 6th week of prenatal development, fetuses express both male and female
primordial genital tissues (Mullerian ducts which develop into the adult female UG tissue and
Wolfian ducts which develop into the adult male UG tissue). At 6 weeks, one or the other
progenitor sets begins to die via programmed cell death. In the developing male for instance,
Mullerian Inhibiting Substance (MIS) produced by the primitive testes at 6 weeks causes
death of the Mullerian duct and ensures a male only phenotype in the adult.
3. Nervous system development
In the mid 1940s, Hamburger discovered that up to 50% of the neurons present in the prenatal
human brain die shortly after birth. Nerve cells depend upon continuous stimulation by nerve
growth factor (synthesized and secreted by target tissues). Those neurons that do not contact
target tissue, and hence a source of growth factor, die by apoptosis.
4. Immune system development
During the formation of mature T cells, progenitors migrate from the bone marrow to the
thymus (aka “death camp”). 95% or more of the primitive thymocytes that reach the thymus
will die there because they failed one of two tests:
Does the thymocytes react to self Ag? If so, dies.
Is the thymocyte capable of reacting to foreign Ag? In not, dies.
5. Hormone action in sex tissues
Atresia: During each human ovulation cycle, approximately 20 ovum begin the maturation
process. Only one of these ova will survive to full development and be released. The other
19 will die during the maturation process due to an inability to retain sufficient FSH.
The loss of uterine tissue during menstruation is another example of cell death.
In the post lactational breast, one sees cell death as prolactin levels decline.
In males, epithelial cells of the prostate are dependent upon androgens for survival.
Following castration, these cells will die due to the loss of steroid androgen hormones.
Three English pathologists, John Kerr, Andrew Wiley and John Curry are considered to be the founders of the
modern study of apoptosis (programmed cell death). These UK pathologists developed the principles we accept
today regarding the processes of cell death.
First, there are two distinct pathways by which a cell can die:
Programmed cell death Caused by exposure to harsh environment like burning,
freezing or chemical agents
Second, these 2 cell death pathways can be distinguished from one another by a set of characteristics:
1. Cell morphology
2. DNA degradation pattern
3. Inflammatory response
4. Activation of proteases
5. Genetic regulation of the process
1. Cell morphology:
Cell shrinks due to loss of H2O via blebbing, the Cell swells due to influx of H2O
bubbling off of pieces of the outer membrane (blebs) as
vesicles with cytoplasm inside
Membranes and organelles remain intact Outer membrane and organelle membranes rupture
leading to non-specific release of damaging enzymes
like DNAases and proteases
Nucleus condenses Nucleus swells
Nuclear fragmentation where chromatin takes on the Autolysis of nucleus with chromatin disappearance
appearance of a group of dense bodies
Most importantly, in necrosis, the cell’s outer membrane loses its integrity and the nucleus and organelles lose
density, all leading to a loose membrane with little internal structure- like a popped balloon. In apoptosis on the
other hand, the cell shrinks by giving off blebs, the nucleus condenses in a crescent architecture and then fragments,
all the while the outer membrane and inner organelle membranes remain intact – like a bag of garbage, which is then
dumped out into lumen of the organ or phagocytized by neighboring cells.
These concepts can be illustrated with the example of prostatic disease. The prostate gland is a small walnut shaped
organ that sits at the base of the male bladder. The urethra passes through the prostate gland. The prostate
contributes fluids to the ejaculate which enrich the sperm and provide nutrition, however its function is not critical.
The prostate is susceptible to disease with age, both cancer and benign prostatic hyperplasia (BPH). It is estimated
that every male that lives to the age of 115 will develop prostate cancer. BPH describes a condition where
beginning around age 30 or 40 the prostate begins to grow and can continue to grow until it reaches 10x the normal
size. BPH is of unknown cause and if severe can squeeze off the urethra. Development of the prostate is under
control of male steroid androgens. The treatments for prostatic disease often work to decrease these hormones,
causing regression of the prostate.
In the rat, removal of the testicles will cause 85% of the cells in the prostate to die within 2 weeks via apoptosis. If
testosterone is then reintroduced, the prostate will grow back. On histological examination, the normal prostate is
composed of epithelial cells with some smooth muscle and stromal cells. With castration, the epithelial cells will
shrink, their nuclei will become chaotic and condense and dense bodies will appear (apoptotic bodies) which are
remnants of the dead cells. In addition, during apoptosis, cellular junctions between the epithelial cells will break
down, as will the junctions between the epithelial cells and the basement membrane. Once freed, these apoptotic
bodies will be kicked out into the lumen for removal or be phagocytized by neighboring cells.
2. DNA degradation
Internucleosomal DNA degradation Random DNA degradation
Caused by activation of specific nucleases Caused by intracellular enzymes released upon organelle
When DNA is electrophoresed on an agarose gel, one normally sees a single band representative of the entire (large)
DNA molecule. In the case of a necrotic cell, the DNA will show a smear pattern on the gel due to random
degradation into fragments of various sizes. In the apoptotic cell, DNA is degraded preferentially between
nucleosomes (which comprise approximately 180 bp of DNA wrapped around a histone protein core). When this
DNA is run on a gel, a 180 bp “ladder” pattern appears due to cleavage via specific nucleases exclusively between
This DNA fragmentation can be used to identify apoptotic cells in tissue. If a thin section of tissue containing
apoptotic cells is incubated with an enzyme that repairs DNA (DNA polymerase) and chemically tagged
nucleotides, the enzyme attempts to fill in the breaks in the nuclear DNA with the modified nucleotides.
Subsequently, an antibody probe that recognizes the modified nucleotides can be used to specifically immunostain
apoptotic cells so they can be easily counted. This method is called “in situ end labeling” or “TUNEL” staining.
3. Inflammatory response
No inflammatory response because membranes remain Extensive inflammatory response due to release of
intact intracellular material from the necrotic cell
Synthesis of anti-inflammatory agents Inflammation can further damage normal tissue
4. Activation of proteases
Activation of caspases specifically – proteases that Lysosomal rupture leads to release of many proteases
cleave specifically at cysteine when it follows aspartic which cause non-specific destruction of cell proteins
Caspases are critical for apoptosis to occur and inhibitors of caspases suppress apoptosis. Caspases are normally
present in an inactive pro-enzyme form which require cleavage into a smaller active form to start apoptosis. There
are two types of caspases, activated “initiator” caspases cleave “effector” pro-caspases into their active forms. The
effector caspases then cleave proteins involved in three main processes in the apoptotic cell:
morphogenic change – cleavage of nuclear lamins proteins, focal adhesion kinases
regulation – cleavage of regulatory proteins like cyclins and Rb
Nucleic acid metabolism – cleavage of pro-nucleases (leading to inter- nucleosomal DNA
fragmentation), PARP, splicing enzyme
5. Genetic regulation
Under strict genetic control Induced by harsh cellular environment
Robert Horvitz at MIT studied the genetics of programmed cell death in c. Elegans. He found that 12 neurons
formed early in development of c. Elegans die by the time it reaches maturity. Horvitz looked for genetic mutations
whereby these 12 neurons did not die. He found a huge repertoire of mutations and categorized them into those
gene products involved in either commitment to the cell death process or processing and removal of the dead cells.
This classification scheme has been expanded for mammalian cells to categorize gene products involved in
apoptosis as important in either initiation of apoptosis, regulation of initiation, execution of apoptosis (like caspases)
and elimination of dead cells. Each category now has numerous gene products that have been discovered.
As an example of some of the gene products involved in the regulation aspect of apoptosis, the prototypic gene
family of bcl-2 and bax can be examined. These are two homologous proteins that share significant similarity in
amino acid structure. These two proteins have a yin-yang like relationship where bcl-2 is a suppressor of apoptosis
and bax is a promoter of apoptosis. These two proteins can bind to themselves forming homodimers (bcl-2 + bcl-2,
bax + bax) or they can bind to each other to form a heterodimer (bcl-2 + bax). It is this binding pattern that
regulates cellular sensitivity to an apoptotic stimulus. Bcl-2 is an apoptotic suppressor when it binds to bax and
prevents the formation of bax + bax homodimers.
In the rat prostate model, bax RNA expression (and expression of the protein as well) is low in normal prostate
tissue. However, following castration, the levels of bax RNA increase significantly, peaking at two to three days
post-castration, and then decrease by day six. Bcl-2 expression is also low in normal tissue. It too increases
following castration but slowly and continuously. A bax:bcl-2 ratio reaches a peak at 2-3 days following castration,
the time at which most of the cells in the tissue undergo apoptosis. Those prostate cells that survive apoptosis
following castration have a very low bax:bcl-2 ratio, making them insensitive to apoptotic agents.
Bax/bcl-2 = prone to apogen response
Bax/bcl-2 = apogen unresponsive
What is the mechanism of bcl-2 and bax action? Bcl-2 and bax are membrane bound proteins, prominent in the
mitochondrial membrane. When bax binds bax to form a homodimer, that homodimer becomes a pore in the
mitochondrial membrane which allows leakage of cytochrome C into the cytoplasm. Once in the cytoplasm,
cytochrome C will bind a protein referred to as aoptosis activating factor-1 (APAF-1). This dimer will then bind
pro-caspase 9, cleaving this pro-caspase into its active form. Caspase 9 (the initiator) then cleaves pro-caspase 3.
The active form of caspase 3 (the effector) begins the cellular cascade that leads to the death of the cell. When bcl-2
is bound to bax in the mitochondrial membrane (rather then a bax + bax dimer) cytochrome C cannot escape into the
cytoplasm and it is through this mechanism that bcl-2 suppresses apoptosis.
Bcl-2 and bax are prototypic members of a large family of gene products that share this homology and operate in a
similar manner. Half are apoptotic suppressors (bcl-2, bxl-1, A1, Mcl-1) and half are apoptotic promoters (bax, bxl-
2, bak, bik).
This cytochrome C APAF-1 caspase pathway represents the intrinsic path to apoptosis. It is called intrinsic
because it is activated by signals that originate within the cell. There is another pathway, referred to as the extrinsic
pathway, which requires cells to receive a signal from the outside to initiate apoptosis. An example of this is Fas
antigen induced apoptosis. Fas Ag is a transmembrane protein with an external, transmembrane and internal
cytoplasmic (death) domains. Normally these Fas Ags float around individually in the cell membrane. Fas ligand
delivered to the outside of the cell will attach to the extracellular domains of 3 different Fas Ags, bringing them into
juxtaposition on the cell membrane (clustering). Once this happens, the intracellular domains of the cluster begin to
attract cytoplasmic proteins (FADD, RIP and TRAD for example) which bind to the intracellular death domains.
Once this has occurred, the Fas Ag cluster can bind pro-caspase 8, leading to its cleavage to an active form and
subsequent activation of other caspases and apoptosis.
Fas Ag is a member of a large family of molecules like the TNF and CD40 receptors which act similarly. All three
have an internal death domain, a transmembrane domain and an external domain. The TNF and CD40 receptors
also bind specific extracellular ligands which activate their clustering and initiate apoptosis.
We can again consider the yin – yang relationship in the cell death process. For every molecule made by the cell
that acts as an apoptotic stimulator, there is another molecule that can counteract it. The normal cell is an exquisite
balance of these pro-survival and anti-survival factors. To induce cell death, you must induce change in the pro-
apoptotic (survival repressor) factors and some coordinate change in the anti-apoptotic (survival enhancer) factors,
upsetting the balance between proliferation and apoptosis. In the case of growth diseases like BPH, warts and
malignancy, we now know that cell death is an important part of the disease process. Some growth diseases show
no change in the proliferation rate of cells at all, but rather a decrease/dysfunction of the apoptotic pathway.
In summary, necrosis and apoptosis, although described above as very distinct cell death pathways, exist rather a
continuum. For example, some harsh environmental conditions like radiation and hyperthermia will induce necrosis
when experienced at high doses. However, when a cell receives only small doses, it might activate apoptosis
instead. The difference seems to lie in whether the cell has time to sense and respond to the stimulation (when
present at low intensity) and then activate apoptosis. If the stimulus is excessively intense it will damage the cell
membrane and lead to necrosis instead.