Apoptosis / Department of Pathological Physiology1
Apoptosis
and Clearance of Apoptotic Cells
Apoptosis / Department of Pathological Physiology2
Apoptosis – Programmed Cell Death
The human body generates 10–100 billion cells every day, and
the same number of cells die to maintain homeostasis in our
body.
Cells infected by bacteria or viruses also die.
The cell death that occurs under physiological conditions
mainly proceeds by apoptosis, which is a noninflammatory, or
silent, process, while pathogen infection induces necroptosis
or pyroptosis, which activates the immune system and causes
inflammation.
Dead cells generated by apoptosis are quickly engulfed by
macrophages for degradation. HNSCC cells dying by apoptosis, QPI, 10x mag.
Nagata, S., Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333–340 (2017).
https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology3
Apoptosis – Programmed Cell Death?
Apoptosis occurs in developing embryos or in cells that die under physiological conditions,
“programmed cell death” and “apoptosis” are often used synonymously.
When apoptosis was discovered to be mediated by gene products, it was regarded as being
programmed.
The term programmed cell death has been used, confusingly, with two different meanings:
the cell death programmed into animal development, and the cellular death process elicited
by a molecular mechanism.
Cell death with a necrotic morphology that occurs during inflammation or infection was also
found to be programmed or regulated by gene products and was categorized as necroptosis
and pyroptosis.
In addition, nonapoptotic cell death was observed during Caenorhabditis elegans
development and Drosophila metamorphosis, indicating that cell death in animal development
can occur by a nonapoptotic mechanism.
Programmed cell death should not be used as a synonym for apoptosis; it should be reserved
for the cell death that takes place in animal development.
Apoptosis / Department of Pathological Physiology4
Mechanism: The Mitochondrial Apoptosis Pathway
Singh, R., Letai, A. & Sarosiek, K. Regulation of apoptosis in health and disease: the balancing act of BCL-2
family proteins. Nat Rev Mol Cell Biol 20, 175–193 (2019). https://doi.org/10.1038/s41580-018-0089-8
Apoptosis / Department of Pathological Physiology5
Mechanism: The Bcl-2 protein superfamily
Apoptosis / Department of Pathological Physiology6
BH3 Mimetics
The recent development of novel small-molecule inhibitors of
pro-survival proteins from the BCL-2 family, called BH3
mimetics, enables the direct and selective activation of
mitochondrial apoptosis in cells that are highly dependent on
one or more pro-survival proteins.
The US Food and Drug Administration approved the BCL-2
inhibitor venetoclax (ABT-199) for use in chronic lymphocytic
leukaemia on the basis of its excellent clinical activity,
including in patients who have relapsed after multiple rounds
of therapy and those with mutated p53.
Agents targeting other major pro-survival proteins BCL-XL
and MCL are also undergoing clinical evaluation.
Although great potential in haematological cancers, their
deployment in solid cancers and non-malignant diseases has
been challenging owing to an insufficient understanding of
apoptotic dependencies and how to identify and exploit
them safely and effectively in the clinic.
The development of novel small-molecule inhibitors of pro-survival proteins
from the BCL-2 family, called BH3 mimetics.Singh, R., Letai, A. & Sarosiek, K. Regulation of apoptosis in health and disease: the balancing act of BCL-2
family proteins. Nat Rev Mol Cell Biol 20, 175–193 (2019). https://doi.org/10.1038/s41580-018-0089-8
Apoptosis / Department of Pathological Physiology7
Mechanism: The Receptor Mediated Apoptosis Pathway
Pro-apoptotic death receptors:
• Fas (whose physiological ligand is FasL)
• the tumour necrosis factor (TNF) receptors
TNFR1 and TNFR2,
• the TNF-related apoptosis-inducing ligand
(TRAIL) receptors DR4 and DR5
The intracellular domains of the pro-apoptotic
death receptors include a conserved protein–
protein interaction domain referred to as the
death domain.
Singh, R., Letai, A. & Sarosiek, K. Regulation of apoptosis in health and disease: the balancing act of BCL-2
family proteins. Nat Rev Mol Cell Biol 20, 175–193 (2019). https://doi.org/10.1038/s41580-018-0089-8
Apoptosis / Department of Pathological Physiology8
The Receptor Mediated Apoptosis Pathway
Immune mechanisms can activate cell death through the
extrinsic pathway, for example, through TRAIL produced by
natural killer cells in response to interferons or through the
blockade of cell death.
FAS-mediated extrinsic apoptosis pathway is responsible for the
deletion of peripheral T cells (patients with autoimmune
lymphoproliferative syndrome carry somatic or germline
mutations in the genes that encode FAS or FASL).
FASL expression is restricted to specific lymphocyte populations,
such as CTLs, T helper 1 (TH1) cells and natural killer cells. By
contrast, FAS is widely expressed by most cell types in various
tissues.
Nagata, S., Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333–340 (2017). https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology9
Morphological Hallmarks of Apoptosis
Cells undergoing apoptosis shrink.
Their plasma membrane is apparently intact, but it undergoes blebbing.
The fragmentation of chromosomal DNA into nucleosomal units, exposure
of phosphatidylserine (PtdSer) on the cell surface, and loss of mitochondrial
potential are biochemical hallmarks of apoptosis.
These features disappear when apoptosis is induced in the presence of
caspase inhibitors, indicating that they are all mediated by activated
caspases.
During the apoptosis of a human cell, nearly 1,300 different proteins are
cleaved by caspases at more than 1,700 sites.
Different caspases have preferred substrates. Since caspase 3 is at the end
of the caspase cascade and is activated by both the intrinsic and extrinsic
death pathways, it is conceivable that the general characteristics of
apoptosis are mediated by the caspase 3 substrates. In fact, several
apoptotic characteristics, including membrane blebbing, DNA
fragmentation, and PtdSer exposure are mediated by caspase 3 targets.
The PtdSer exposure is essential for apoptotic cells to be eaten.
Head and neck squamous cell carcinoma FaDu
cell undergoing apoptosis. 20x mag. obj.,
Quantitative Phase Imaging (QPI, Q-PHASE).
Apoptosis / Department of Pathological Physiology10
ASYMMETRICAL DISTRIBUTION OF PHOSPHOLIPIDS
Biological membranes consist of two layers, outer and inner leaflets, between which lipids are asymmetrically distributed.
In the plasma membrane of eukaryotic cells, amine-containing or anionic phospholipids [PtdSer, phosphatidylethanolamine (PtdEtn),
phosphoinositides (PtdIns), phosphatidic acids (PtdOH)] are predominantly or exclusively localized to the inner leaflet, whereas the outer
leaflet is enriched in choline-containing phospholipids [phosphatidylcholine (PtdCho) and sphingomyelin (Sph)] and glycosphingolipids.
Three types of lipid transporters have been proposed, based on the direction of transport, substrate specificity, and ATP requirement:
Using the energy of ATP, flippases and floppases translocate specific lipids from the outer to inner leaflet or from the inner to outer leaflet,
respectively, against a concentration gradient, whereas scramblases, driven by the existing lipid gradient, nonspecifically and bidirectionally
transport lipids between the inner and outer leaflets.
Cells undergoing apoptosis expose PtdSer on their surface in a caspase-dependent manner. The cellular protein annexin V specifically binds to
PtdSer, and fluorescently labeled annexin V is widely used to detect apoptotic cells.
Flippase cleavage by caspase 3 inactivates their flippase activity. However, flippase inactivation alone is insufficient to quickly expose PtdSer on
the cell surface .Thus, once the asymmetrical distribution of phospholipids is established, the rate of their spontaneous translocation or
scrambling is extremely low. Thus, an enzyme(s) or scramblase(s) is needed to perform this job.
Macrophages engulf apoptotic or senescent cells but not healthy cells, suggesting that the engulfed cells present a determinant(s) or eat me
signal on their cell surface.
Nagata S. Apoptosis and Clearance of Apoptotic Cells. Annu Rev Immunol. 2018 Apr 26;36:489-517. doi: 10.1146/annurev-immunol-042617-053010. Epub 2018 Feb 5. PMID: 29400998.
Apoptosis / Department of Pathological Physiology11
ASYMMETRICAL DISTRIBUTION OF PHOSPHOLIPIDS
Apoptosis / Department of Pathological Physiology12
Engulfment of Apoptotic Cell
deCathelineau & Henson coined efferocytosis, Latin
for “carry to grave,” to describe the engulfment of
apoptotic cells.
PtdSer exposure is necessary and sufficient as
an eat me signal.
Molecules that recognize PtdSer:
• secreted soluble proteins, including MFG-E8,
Gas6, and Protein S (PROS),
• type I membrane proteins expressed in
phagocytes, including TIM1, TIM4, and
CD300.
When apoptotic cells are recognized by macrophages, various signaling molecules are activated in the macrophages that
lead to phagosome formation to encapsulate the apoptotic cells. The phagosomes are transported into lysosomes, where
the components of dead cells are degraded into their building units.
Nagata S. Apoptosis and Clearance of Apoptotic Cells. Annu Rev Immunol. 2018 Apr 26;36:489-517. doi: 10.1146/annurev-immunol-042617-053010. Epub 2018 Feb 5. PMID: 29400998.
Apoptosis / Department of Pathological Physiology13
Engulfment of Apoptotic Cell
Efferocytosis often has a defect in patients with SLE. Thus, it was
thought that rapid efferocytosis prevents secondary necrotic cells
from releasing DAMP, which promotes SLE development.
Apoptotic cells are engulfed by macrophages via efferocytosis, in
which the macrophages engulfing apoptotic cells produce antiinflammatory
molecules such as IL-10, TGF-β, and PGE2.
When the efferocytosis does not occur efficiently, the apoptotic
cells undergo secondary necrosis. Chromatin DNA of necrotic cells
is cleaved by DNase 1 or DNase1L3. Other remnants of dead cells
are recognized and cleared by scavenger receptors (Marco and
MSR1) This step may not be inflammatory.
On the other hand, when chromatin DNA of necrotic cells is not
degraded, DNA activates macrophages and dendritic cells (DCs) via
TLR9 to produce inflammatory cytokines such as TNF-α and IFN-α.
It may also promote the aggregation of immune complex, which
can be strongly immunogenic.
Nagata S. Apoptosis and Clearance of Apoptotic Cells. Annu Rev Immunol. 2018 Apr 26;36:489-517. doi:
10.1146/annurev-immunol-042617-053010. Epub 2018 Feb 5. PMID: 29400998.
Apoptosis / Department of Pathological Physiology14
Apoptosis Pathway in Physiology
An important mechanism that ensures organismal homeostasis is the removal of damaged or
superfluous cells via apoptosis, and in the former case, their replacement from stem-cellderived
progeny.
For example neutrophils — the most common nucleated cell type in peripheral blood — have a lifespan of only
5.4 days, meaning that roughly 20% of neutrophils die via apoptosis each day in an adult human. (The short half-life of
these cells is likely due to their prominent role as phagocytes for invading pathogens; mammals have presumably evolved to allow these cells (together with their
microbial load) to self-destruct via apoptosis and be replaced instead of using a detoxification strategy).
Heightened apoptotic sensitivity is a hallmark of developing tissues
Perinatal brain development is associated with massive proliferation of neural progenitors, followed by a wave
of apoptosis that eliminates approximately half of cells that are either excessive or have not established
appropriate synaptic connectivity.
The heart, kidneys and liver are more sensitive to pro-apoptotic stimuli at the perinatal and early postnatal
stages than at the adult stage. Importantly, this heightened apoptotic sensitivity is frequently associated with
active proliferation of tissues and expression of the growth- and division-supporting transcription factor MYC,
suggesting that active proliferation is tightly coupled to expression of the machinery necessary for apoptosis
induction.
Nagata, S., Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333–340 (2017). https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology15
Apoptosis Pathway in Physiology
Apoptosis is dynamically regulated across the
mammalian lifespan.
Tissues that are highly proliferative are typically primed
for apoptosis (red). High apoptotic priming in these
tissues makes them highly sensitive to various insults.
Tissues that are largely postmitotic are apoptosis
refractory (green).
Nagata, S., Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333–340 (2017). https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology16
Apoptosis and Immune System
Around 1990, apoptosis or cell death was reported to have important roles in the adaptive immune system in the deletion
of thymocytes that express autoreactive or non-reactive T cell receptors (TCRs), and in the deletion of autoreactive
immature B cells.
Cell death is involved in various immunological processes:
• During lymphocyte development, many lymphocyte progenitors that express antigen receptors with a high affinity for self-antigens or
that cannot respond to antigens are eliminated by apoptosis. The lymphocytes that survive this stage of development remain in the
periphery and form the naive T cell and naive B cell compartments.
• When naive lymphocytes encounter pathogens, they proliferate and are activated to combat the pathogens. These activated
lymphocytes will subsequently die after the pathogens have been removed. A similar situation can be found with neutrophils during
inflammation. When our body is infected by bacteria, neutrophil populations expand, and neutrophils are activated to phagocytose
bacteria, but they quickly undergo apoptosis after the infection has been cleared.
• Cytotoxic T lymphocytes and natural killer cells recognize virus-infected, bacteria-infected and transformed cancer cells, and induce
these cells to die by apoptosis.
• Bacteria-infected cells, particularly phagocytes, often undergo necrosis (pyroptosis or necroptosis) to prevent bacteria from
proliferating further inside the cell. Unlike apoptosis, necrosis is an inflammatory form of cell death and can lead to further tissue
inflammation. Nagata, S., Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333–340 (2017). https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology17
Apoptosis and Immune System
Nagata, S., Tanaka, M. Programmed cell death and the
immune system. Nat Rev Immunol 17, 333–340
(2017). https://doi.org/10.1038/nri.2016.153
Apoptosis / Department of Pathological Physiology18
Insufficient apoptosis and the development of autoimmune diseases.
Beyond cancer, the most compelling evidence of insufficient apoptosis contributing to disease is seen in
autoimmune disorders.
Defects may involve predominantly perturbations to the extrinsic pathway, but the intrinsic pathway is also
implicated.
In humans, abnormally high expression of BCL-2 in peripheral blood B and T lymphocytes has been observed in
patients diagnosed with systemic lupus erythematosus (SLE) — a prototypical autoimmune disease
characterized by inflammation and organ damage in association with the expression of autoantibodies against
double-stranded DNA. Such high BCL-2 levels may be responsible for the abnormal survival of self-reactive
lymphocytes driving SLE.
Autoimmune lymphoproliferative syndrome (ALPS) is a recently characterized disorder caused predominantly
by inherited mutations in the gene that encodes tumour necrosis factor receptor superfamily member 6 (FAS),
leading to reduction in apoptosis potential and expansion of double-negative T cells (DNTCs) — a hallmark of
ALPS.
Gastrointestinal autoimmune diseases such as Crohn’s disease and ulcerative colitis are believed to occur as a
consequence of chronic inflammation and consequent death of intestinal epithelial cells. This inflammation is
established by hyperproliferating T cells that normally would be eliminated by cell death. However, in these
conditions, an increase in pro-survival BCL-2 prevents T cell apoptosis.
Apoptosis / Department of Pathological Physiology19
Apoptosis and Cancer
In cancer, apoptosis is a well-established tumor suppressor mechanism, and
loss of apoptotic control allows cancer cells to survive longer thus acquiring
additional oncogenic hits.
Tumor cells evolve a variety of strategies to limit cell death:
• loss of p53
• increased expression of antiapoptotic regulators (Bcl-2, Bcl-xL) and
survival signals (insulin-like growth factors; Igf1/2)
• downregulating of proapoptotic factors (Bax, Bim, Puma)
• opportunistic modes of behavior (cell fusion)
Anoikis is a form of programmed cell death that occurs in anchoragedependent
cells when they detach from the surrounding extracellular matrix.
• barrier to metastasis
• circulating tumor cells are anoikis resistant
• TrkB (neurotrophic receptor) overexpression protects disseminated,
circulating tumor cells from undergoing anoikis.
Apoptosis / Department of Pathological Physiology20
Evading apoptosis as a hallmark of cancer
There is little evidence that cancer cells are
more resistant to apoptosis than normal
cells are.
The presence of a therapeutic index for
conventional chemotherapy in most
cancers relies on the increased sensitivity to
apoptosis in cancer cells than in normal
cells .
Apoptosis can facilitate the carcinogenic
action of other oncogenes, but it is by itself
only weakly oncogenic.
When cancer cells are subjected to
chemotherapy, there is selection for
reduced sensitivity to apoptosis, likely an
important contributor to the pan-resistant
phenotype of many relapsed tumors.
Chromosomal translocation associated with B-cell lymphomas. The Bcl-2 gene is translocated
behind a potent immunoglobulin gene promoter. Increased expression of Bcl-2 gene is associated with
inhibition of apoptosis.
Anthony Letai Annual Review of Cancer Biology 2017 1:1, 275-294
Apoptosis / Department of Pathological Physiology21
Apoptosis and Cancer
During the process of neoplastic transformation, oncogene-driven abnormal growth signals
and cell-cycle checkpoint violation lead to cellular stress and upregulation of pro-apoptotic
proteins.
This upregulation results in higher apoptotic priming of malignant cells at the basal state than
normal cells. Because healthy adult tissues are mostly refractory to apoptosis or unprimed, this
increase in apoptotic priming of aberrant cells can be exploited therapeutically using BH3
mimetics, particularly when combined with standard anticancer therapies such as radiation or
chemotherapy.
Haematopoietic cells are naturally highly primed for apoptosis. Hence, oncogenic stress and the
resulting upregulation of pro-apoptotic factors frequently result in the removal of pre-malignant
cells derived from this lineage (middle arrow). Several mechanisms associated with BCL-2
protein deregulation, including BCL-2-interacting mediator of cell death (BIM) mutations (step
1), downregulation of BCL-2-associated X protein (BAX) (step 2) or BIM (step 3) and upregulation
of BCL-2 or myeloid cell leukaemia 1 (MCL1) (step 4) support the emergence of haematopoietic
cancers, which depend on these mechanisms for their survival. Notably, these mechanisms are
mainly employed to keep malignant cells alive, and haematopoietic cancers typically remain
primed for apoptosis and hence are susceptible to therapy and respond well to treatment with
BH3 mimetics. However, certain mechanisms, including mutations of sensitizer proteins (step 1)
and downregulation of mitochondrial outer membrane permeabilization (MOMP) pore-forming
components (step 2) yield cells that are unprimed or even apoptosis refractory and hence
resistant to therapy. Solid tumours can employ similar mechanisms to boost their survival, but
these dependencies are much less pronounced than in haematopoietic cancers. Nevertheless,
these mechanisms may underlie the development of resistance to treatment and disease
relapse. BAK, BCL-2 antagonist/killer; BCL-W, B cell lymphoma W; BCL-XL, B cell lymphoma extra
large; BFL1, BCL-2-related isolated from fetal liver 1; BID, BH3-interacting domain death agonist;
PUMA, p53-upregulated modulator of apoptosis.
Singh, R., Letai, A. & Sarosiek, K. Regulation of apoptosis in health and disease: the balancing act of BCL-2
family proteins. Nat Rev Mol Cell Biol 20, 175–193 (2019). https://doi.org/10.1038/s41580-018-0089-8
Apoptosis / Department of Pathological Physiology22
Pro-oncogenic roles of apoptotic signaling
(A) In dying tumor cells, activated caspases activate iPLA2 leading to generation of arachidonic acid.
Arachidonic acid can be converted into PGE2 dependent on COX-1/2 function.
PGE2 can act through its receptors to exert pro-proliferative effect on surrounding cells. This process is termed apoptosisinduced
proliferation (AiP).
(B) Apoptotic tumor cells not only can induce the anti-inflammatory M2 TAM differentiation that inhibits antitumor
immunity, but also secrete a variety of cytokines/chemokines that promote angiogenesis and metastasis. Some types of
tumor cells can also express death ligands (FASL and TRAIL) and inhibitory B7 family members (PD-L1 and CTLA-4) that can
induce T-cell death, leading to immune escape.
(C) Nonlethal stress can lead to MOMP in a minority of mitochondria, called miMOMP. Following miMOMP, caspases are
activated similar to a sublethal level, which leads to the activation of CAD. CAD activation results in DNA damage and
genetic instability and can lead to cell transformation. The endonuclease Endo G can also be released upon MOMP, leading
to DNA cleavage. Endo G-mediated dsDNA breaks also activate ATM, leading to NF-κB and STAT3 activation and subsequent
tumor growth.
Apoptosis / Department of Pathological Physiology23
Pro-oncogenic roles of apoptotic signaling
Dr. Martina Raudenská
Doc. Michal Masařík
Thanks for your
attention.