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Cell Death and Diseases:
Although, programmed cell death is a physiologically essential mechanism for normal
development and tissue homeostasis in multi-cellular organisms, it is also involved in
many diseases where it is deregulated. Following are the major diseases involving excess
cell death:
1. Neurodegenerative diseases: Alzheimer’s, Parkinson’s disease and stroke.
2. AIDS
3. Auto-immune diseases.
Disease with defective cell death mechanisms:
1. Cancers
2. Auto-immune diseases.
Neurodegenerative diseases:
Alzheimer’s Diseases
AD is a multifactorial syndrome characterized by presence of senile plaques and
neurofibrillary tangles and progressive degeneration of nerve cells. These symptoms lead
to memory loss, dementia and eventually death of the patient. Close to 5 million people
suffer from Ad in US. Biochemical and cellular mechanisms involved in this diseases are
under intense investigation throughout the world, but much of it remains to be
understood. Studies with the patients with familial AD, have given the indications that
amyloid beta peptide plays central role in the pathology of AD.
To date four genetic loci have been associated with Alzheimer's disease (AD):
Amyloid precursor protein (APP) on chromosome 21 (Goate et al, 1991),
Apolipoprotein E (ApoE) on chromosome 19 (Corder et al, 1993), and
Presenilin 1 and 2 (PS1 and PS2) on chromosomes 14 and 1 respectively (Sherrington et
al, 1995 and Levy-Lahad et al, 1995).
Of these four genes and their protein products, three are associated with highly
aggressive, early onset familial forms of Alzheimer's Disease (APP, PS1 and PS2) and
represent a very small subset of AD patients.
Borchelt DR., et al. Accelerated amyloid deposition in the brains of transgenic mice
coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron. 19. 939-945.
1997 Oct 1. Abstract.
Corder EH., et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's
Disease in late onset families. Science 261. 921-923. 1993 Aug 13. Abstract.
Goate AM., et al. Segregation of a missense mutation in the amyloid precursor protein
gene with familial Alzheimer's Disease. Nature 349. 704-706. 1991 Feb 21. Abstract.
Levy-Lahad E., et al. Candidate gene for the chromosome 1 familial Alzheimer's Disease
locus. Science 269. 973-977. 1995 Aug 18. Abstract.
Mesulam MM. and Geula C., (1994) Butyrylcholinesterase reactivity differentiates the
amyloid plaques of aging from those of dementia. Annals of Neurology 36. 722-727.
1994 Nov 1. Abstract.
Perry EK., (1980) The cholinergic system in old age and Alzheimer's Disease. Age
Ageing. 9. 1-8.
Roses AD. Apolipoprotein E alleles as risk factors in Alzheimer's Disease. Annual
Review of Medicine 47. 387-400. 1996 Jan 1. Abstract.
Amyloid precurser protein (APP): Although missence mutations in APP
account for only 0.1% of all AD cases, a proteolytic fragment of this protein
called amyloid is found in senile plaques and seem to be playing a causal
in the development of this disease.
APP is expressed ubiquitously in multiple isoforms in neuronal as well as nonneuronal cells. Neurons express 695-residue isoform abundantly.
751/770 isoforms of APP are present in human platelets and serve as inhibitor of
factor Xia (a serine inhibitor).
APP has a secretory signal sequence at amino terminal and a trans-membrane
domain.
During normal processing Cleavage of APP with -secretase yields a secreted
derivative called sAPPwhich has been shown to function as neuro-protective
growth promoting in cell culture studies. The corboxy terminal peptide is cleaved
by -secretase producing p3 peptide.
In the event of missence mutation at the -secretase site the APP is cleaved by
-secretase resulting n-terminal sAPP and the C-terminal peptide is cleaved by
secretase to produce toxic A peptide. Alternatively, missence mutations at
the beta and gamma sites may enhance the proteolytic activities resulting in
increased production of  peptide. All eight reported missense mutations linked
to AD are clustered around -secretase cleaving site.
Apeptidehas been shown to cause cell death in cultured neurons. This
peptide has capability of producing oxidative free radicals, it can associate with
each other and can cause fibrillation. Fibrilar and oligomeric Ab peptide and
diffusible plaques have been thought to be associated with activation of microglia
and release of cytokines and a multifaceted inflammatory response.
Down’s Syndrome and AD: Tri-somy of chromosome 21 which contains APP
gene is the cause of Down’s syndrome (DS). Due to the trisomy 21, there is
overproduction of APP and excess of APP results into production of Ab very
early in the life of DS patients. Indeed, there is appearance of diffusible Ab
plaques as early as 12 yrs of age and invariably all DS patients develop full
blown AD pathology by their forties or fifties.
Apoptosis and AD:
The question if neuronal cell loss in AD is due to apoptotic cell death is still
controversial. Evidence of DNA fragmentation in the postmortem tissues have
been reported which indicate the apoptotic mode of cell death. Furthermore, the
A has been shown to induce apoptosis in neuronal cells in culture. But the
extremely slow and gradual demise of neurons in AD suggest that there might be
slow functional demise of cells. Involvement of caspases and their presence in
A plaques suggest strongly for apoptotic cell death (see below).
Caspases and AD:
Recent breakthrough have been reported by Canadian researchers (Don
Nicolason’s group, Montreal) have shown that caspases are involved in the
production ofFurthermorewas found to activate caspase-3. It has been
hypothesized that activation of caspases by injury etc may result in the begening
of a viscous cycle of Ab production and caspase activation and thus pre-dispose
the individual to AD like pathology later in life.
Presnillins:
Presenillin mutations result in the early onset of AD. Presenilins 1 and 2 (PS1
and PS2) are homologous membrane proteins localized in ER and Golgi
membranes.
They play critical but unidentified role during development as deletion of PS1 in
transgenic mice is embryonic lethal. However, the mutations observed in AD do
not affect development.
Role of PS1 and PS2 in AD pathology is still not known. Mutations in PS have
been linked with increased production of A and subsequent plaque formation. It
has been hypothesized that PS may modulate activities of scretases and
mutations result in facilitating the cleavage to produce A. Some researchers
believe that PS1 and PS2 might themselves be gamma secretase. Mutant PS2
has been associated with enhanced apoptosis in cell culture studies. But its
mechanism of inducing apoptosis is still not known.
ApoE4:
Apolipoprotein E (ApoE) performs various functions as a protein constituent of
plasma lipoproteins, including its role in cholesterol metabolism. It was first
identified as a constituent of liver-synthesized very low density lipoproteins which
function in the transport of triglycerides from the liver to peripheral tissues.
There are three major isoforms of ApoE, referred to as ApoE2, ApoE3 and
ApoE4 which are products of three alleles at a single gene locus. Three
homozygous phenotypes (Apo-E2/2, E3/3, and E4/4) and three heterozygous
phenotypes (ApoE3/2, E4/3 and E4/2) arise from the expression of any two of the
three alleles. The most common phenotype is ApoE3/3 and the most common
allele is E3. See Mahley, R. W., Science 240:622-630 (1988).
ApoE4 polymorphism results in the increased A production and involved in late
onset of AD. Subjects with the ApoE4/4 genotype are as much as eight times as
likely to be affected by Alzheimer's disease as subjects with the ApoE2/3 or
ApoE3/3 genotypes. Further, the average age of onset of Alzheimer's disease
and the average age of survival is younger for those having one ApoE4 allele,
and youngest for those having two ApoE4 alleles. However, the mechanism of
action of ApoE4 in A plaque formation and Ad pathology is still not clear.
Summary: Although most of the questions regarding the biochemical
mechanisms of the development of AD pathology are still not answered, but with
the help of transgenic mice and studies of disease progression in Down’
syndrome subjects have indicated that accumulation of A as an early event in
all forms of AD. Therapeutic interventions at this early step might be to stop
production of A, or to interfere with its neuro-toxic actions are being investigated
through out the globe.
Therapeutic opportunities:
1. Inhibition of Ab production
2. Inhibition of Ab oligomerization or fibrilization
3. Anti-inflammatory drugs that could interfere with the microglial and
astrocytes response
4. Antioxidants and free radical scavengers
5. Ca channel blockers, modulators of signal transduction
6. Neurorestorative factors or Survival factors: neurotrophins, FGF, BDNF,
NGF etc.
New Hope:
1. Vaccine to fight AD: Recently, researchers have demonstrated the use
of immune system to inactivate Ab peptide. Scientists at the University of
Toronto have developed potent vaccine against Ab peptiede. Antibodies
raised against Ab in transgenic AD mice, were capable of inhibiting the
plaque formation, clearing the senile plaques as well as learning
impairment. (See Nature December 21-28, 2000 issue). Clinical trial for
this vaccine are to start as soon as next year.
2. Apoplipoprotein A-1 (APO-A): Scientists at University of Pittsburgh
reported for the first time that a naturally occurring protein Apo-A which
also clears cholesterol throughout the body including brain, binds to Ab.
This protein is present normally in the body, but it’s level were found to be
decreased in AD patients. The authors believe that increasing the level of
Apo-A, could remove Ab from brain and offer protection against AD
pathology.
3. Anti-oxidants: Dr. Allan Butterfield, a chemistry professor at the
University of Kentucky, have reported that the intrinsic ability of Ab peptide
to produce ROS might be the main cause of brain damage and cell death.
He proposed that the anti-oxidants such as Vitamine-E should be used for
AD patients.
Pharmaceutical Companies active in developing therapeutic modulators foe
neurodegenerative diseases:
Cambridge Neuroscience (Cambridge MA): Developing neurotrophic
factors and NMDA antagonists for therapeutic use.
Cephalon (West Chester, PA): Developing Calpain inhibitors and free
radical scavengers as well as neurotrophic factors. One of the product IGF-1
is in clinical trial for ALS.
Genentech (SF, CA): Neurotrophic factors
Eli Lilly (Indianapolis, In): developing the bezylamine antioxidants for
therapeutic use.
Merck Frost (Montreal, Canada): Caspase inhibitors
References:
Dannis J. Selkoe (1999) Nature, Vol. 399: A23-A31
Gervais et al. (1999) Cell, 97: 395-406.
Marcia Barinaga (1998) Science 281: 1305-1306
Parkinson’s disease (PD):
PD is caused by the gradual death of dopaminergic cells of substantia nigra
and decrease of dopamine production. This results in the loss of muscle
control, rigidity and tremors. PD symptoms become apparent due to gradual
decrease in dopamine content neurons in basal ganglia with age when it
approaches 70-80% decrease in dopamine and about 50% neurons are lost.
PD is mostly sporadic but the chances of developing PD several fold in the
individuals related to PD family. Environmental factors and trauma have been
thought to be the main factors for the development of the disease. Far
example 1-methyl-4-phenylpyridinium (MPTP), a heroin analogue and toxin
has been shown to cause PD. MPTP was found to be inhibiting the
mitochondrial electron transfer chain complex-1, indicating mitochondrial
involvement in PD.
Studies with genetic PD have revealed the genes which are mutated in PD
patient but the Mechanisms of action of the theproteins encoded by these
genes are still not clear. One of the mutations commonly found in inherited
PD patients is in -synuclein. This protein was found to be the prominent
constituent of the Lewy bodies.
A pathological feature of the disease is the presence of cytoplasmic
inclusions called Lewy bodies mostly in substancia nigra region of the brain.
Lewy bodies are spherical, dense, eosinophilic structures with 5-25 micron
diameter.
utatedas well as normal-synuclein is capable of forming self aggregates
and plaques.
The mechanism of action of -synuclein in inducing cell death is not known.
Following are the possible biochemical mechanism which may play critical
role in the development of PD.
Oxidative stress
Mitochondrial dysfunction
Apoptotic cell death
Therapeutic approach:
L-DOPA , a precursor of dopamine
Antioxidants (Vit. E, deprenyl, glutathione enhancing reagents)
Neurotrophic and anti-apoptotic factors (BDNF, GDNF, FGF)
Cell replacement therapies
Gene therapy
Huntington’s Disease (HD):
This is a fatal neurodegenerative genetic disease. It is caused by expanded
tri-nucleotide repeats (coding for long stretches of glutamine) in specific
genes. This disease strikes people in their 30s and 40s gradually destroying
brain cells which results into mental deterioration, memory loss, slurred
speech and personality change.
The biochemical details of development of this disease are not known. Due
to the poor understanding of the mechanism of brain cell death in HD, there is
no therapeutic intervention available.
Amyotrophic Lateral Sclerosis (ALS):
ALS (also called Lau Gehrig’s disease) is a degenerative disease of motor
neurons. Mutations in the gene superoxide dismutase (SOD1) have been
linked to ALS, indicating the involvement of ROS in the death of motor
neurons. It would be interesting to find out as why only motor neurons are so
sensitive to SOD1 mutations. There is still no effective treatment for this
disease.
Multiple Sclerosis (MS):
MS is a autoimmune disease in which T cells start reacting against myelin
(the lipid containing coating on the nerve fibers in brain and spinal cord). The
progressive loss of this insulation and protective sheath results in blocked or
inhibited conduction of nerve impulses and neurological damage.
Ischemia and Stroke:
Refer to the reference: Lee et al (1999) Nature, 399: A7-A14.
Cancer:
Cancer is a disease caused by the deregulation of cell division and/or cell
death mechanisms. At the molecular level, most or all of the cancers arise
from the changes in the genome of cells. Genetic instability in a cell in an
organism increases with age. The genetic instability can be caused by
several factors including environmental mutagens, viral infections and failure
of gene repair and cell death mechanisms.
Two heritable properties needed for a cell to become tumorogenic or
malignat;
1. Uncontrolled cell division
2. Invade and colonize territories normally reserved for other cells.
Benign Tumors: uncontrolled cell growth, neoplasm, a relentlessly growing
mass of abnormal cells. As long as the neoplastic cells remain clustered
together in a single mass, the tumor is called benign and it is completely
cured by surgery.
Malignant Tumors: tumor cells break loose, invade other areas or enter
blood stream or lymphatic vessels and form secondary tumors or metastases.
These become difficult to treat
Cancer Classifications: Cancers are classified according to their original cell
types
Carcinomas: all cancers originating from epithelial cells
Sarcomas: all cancers originating from connective tissues or muscle cells
Lukemias: cancer arising from hemopoietic cells
Other specific cancers like Gliomas originating from glial cells, adenoma
originating from epithelial cells are benign tumors
Carcinoma added to these names indicates that they are of malignant nature,
e.g. gliocarcinomas, adenocarcinomas.
About 90% of all cancers arise from epithelial cells: why?
1. Most of the cell proliferation occurs in epithelia and
2. Epithelial tissues are more frequently exposed to physical and chemical
damages which favor the development of cancer.
Depending on the origin of cell type, tumors can be more prone to be
malignant, e. g.
skin cancers: basal cell carcinoma is derived from keratinocytes stem cells
and is only invasive rarely forms metastases
but the melanomas originating from pigment cell in the skin is much more
malignant and rapidly gives rise to many metastases.
Most cancers derive from a single abnormal cell
Mutagenesis: Production of change in DNA sequence
Chemical Carcinogesis: caused by single local change in the nucleotide
sequence
Ionizing radiations: cause typically cause chromosome breaks and
translocations
Free radicals: cause DNA modifications similar to chemical carcinogens
Mechanism of tumogenesis and cancer development:
As we have discussed before, genetic mutations are the sole reason for cells to
loose control over proliferation and become malignant.
With a intense and relentless research in molecular biology of cancer
development have now revealed the sequence of molecular events during cancer
development. Cancer is no more mystery for scientists, but still it is a very
complicated biological phenomenon, and prevention and treatments for cancer
are still limited.
I will just summarize the recent finding from various groups leading to the
understandsing of tumor development.
Following are the sequential requirements for tumor developmens;
1. Uncontrolled cell division: can be caused by
a) mutation leading to activation or overproduction of oncogenes (cell
division promoting signaling molecules. And/or
b) by mutation leading to inactivation or underproduction of cell cycle
blocking genes or anti-oncogemnes or tumor suppressor genes.
2. Blockage of cell death mechanisms caused by DNA abnormalities, leading
to the growth of cells with mutated genes, thus encouraging more
mutations. This is achieved by mutation in p53 genes, over production of
Bcl2 genes etc.
3. Acquiring immortality: all mammalian cells can divide to a limited number
of population doublings, after which they become senescent. Recent work
suggests that a check on the number of cell division is achieved by
telomere length at the end of chromosomes that keeps getting shorter in
each replication cycle. In many cancers there is production of an enzyme
called telomerase (a reverse transcriptase). This enzyme keeps the
telomere length constant and cells become virtually immortal.
4. Induction of blood capillary formation or angiogenesis.
5. Detachment from the tissue, invasion of other tissues.
Studies on the presence of polyps in colon of patients with genetic predisposition, and their development to colon cancer have established that
there is a sequential mutations which lead to full blown cancers.
APC gene mutation: Adenoma polyposis coli: found mutated or truncated in
all polyps and advance colon cancers.
Oncogenic mutations of a Ras gene, followed by loss of p53 and DCC are
present in successive stages of colon carcinoma.
In parallel to colon cancer studies,
Taken from Scientific American, September, 1996,
3.
AIDS and Apoptosis:
Acquired immunodeficiency syndrome (AIDS) is caused by infection with human
immunodeficiency virus (HIV).
The disease is characterized by the following;
1. Progressive loss of CD4 T-cell number
2. Immunodeficiency and susceptibility to opportunistic infections and
malignancies.
3. It has been established that increased apoptosis of CD4 and CD8 T-cells
is the reason of their decrease in HIV-infected patients.
Deregulation of apoptosis in T cells:
Only a minor fraction of apoptotic lymphocytes are physically infected with HIV
thus indicating that uninfected cells become susceptible to apoptosis in these
patients.
Chronic uncontrolled infections provide continuous antigenic stimulations which
and cause persistent immune activation and apoptosis.
HIV infection can induce lymphocyte apoptosis by direct or indirect mechanisms
other than activation induced cell death of immune cells.
HIV encoded proteins can induce apoptosis of infected or uninfected cells.
Unlike other viruses, which have antiapoptotic mechanisms, HIV does not
have anti-apoptotic proteins.
Four ligand/receptor systems which induce apoptosis in lymphocytes
Fas L/Fas
TNF/TNFR (p55)
TRAIL/APO 2-L receptor 1 and 2.
Aopoptosis inhibitory molecules:
FLICE-like inhibitory protein (FLIP): binds to FADD and inhibit apoptosis
Inhibitor of apoptosis protein (IAP)
Bcl-2 family of anti-apoptotic proteins
HIV-mediated alterations in the apoptosis related molecules:
Increased membrane expression of Fas and FasL in response to HIV infection (in
U937 cells).
Down-regulation of Bcl-2 and up-regulation of Bax.
T cells from HIV-infected patients:
Show increased Fas receptor expression and enhanced susceptibility to Fasmediated cell death.
Plasma level of soluble FasL is increased in HIV-infected patient and correlates
with HIV RNA burden.
Decrease in cFLIP expression resulting enhanced susceptibility of Fas-mediated
apoptosis.
Elavated level of TNF is also observed in symptomatic AIDS patients. Role of
TNF-mediated apoptosis in HIV-infected patients is still not clear.
Gp120-induced apoptosis:
Gp120 is an HIV viral envelope protein, that can bind to and cross-link
CD4receptor. This results in enhanced susceptibility to Fas-mediated killing.
Gp120 can induce apoptosis in previously activated cells.
Tat-induced apoptosis
Tat is readily secreted by infected cells, and it causes Fas-dependent apoptosis
of uninfected cells.
Nef may enhance apoptosis of t cells in HIV-infected patientds.
Indirect mechanisms of HIV-associated apoptosis:
Activation-induced cell death:
T cells from HIV-infected cells undergo spontaneous apoptosis with a faster rate
that normal individuals. This phenomenon is called activation-induced cell
death(AICD) and it occurs only in those cells that have been previously activated.
Naïve T cells form normal subjects undergo proliferation and cytokine secretion
when stimulated through T cell receptor. Subsequent stimulation results in AICD
by the de novo production of Fas L which mediates autocrine and paracrine
apoptosis.
Retinoic acid which inhibits expression of FasL can delay the Fas-mediated
apoptosis of CD4 T-cells and it has been used in therapy.
Autologous infected cell-mediated killing:
Macrophages, monocytes, peripheral blood mononuclear cells, CD4 T-cells and
CD8 T-cells derived from HIV-infected subjects may induce death of un-infected
CD4 T lymphocytes. Autologous infected cell mediated killing may involve gp120
interactions or Fas/FasL system or both.
FasL may be the mediator of uninfected CD4 Tcell death by monocytes and
macrophages.
Death of CD8 T-cells: There is increased CD8 T-cell apoptosis in HIV-infected
patients but the number of CD8 cell count does not change significantly. This
might be due to fast recovery of CD8 cells.
Protease Inhibitor therapy:
Protease inhibitor therapy results in decrease in apoptosis in lymph nodes, rectal
mucosa and peripheral blood lymphocytes in the HIV infected patients within 4 to
14 day of treatment. Protease inhibitors are capable of inhibiting spontaneous
apoptosis, as well as apoptosis induced by Fas, AICD. But there is not much
effect on viral replication.
Effects of Cytokines on HIV-associated apoptosis:
As discussed above one of the hall mark of HIV infection is the dysfunction of
helper T cells.
Helper T cells: Th1 cytokines (IL-2 and IFN g) ----enhance cellular immunity
Th2 cytokines (IL-4, 5, 6, and 10) --------enhance humoral responses.
In HIV-infected patients there is shift from Th 1 cytokines to Th2 cytokines.
Treatment with Th1 cytokines like IL-2 or with antagonists to Th2 cytokines
results in restoring the proliferative capability of T cells. But IL-2 treatment also
results in HIV replication.
IL-2 promotes survival of CD4 T-cells directly by an antiapoptotic mechanisms.
IL-15, a T cell growth factor is capable of enhancing the production of Th1
cytokine, CD8 Tcell activation, inhibiting spontaneous apoptosis possibly by
increasing Bcl-2 expression. IL-15 does not increase the HIV replication.
Highly active antiviral therapy: Protease inhibitor plus viral inhibitor therapy.
Infected T-cells which are rescued from apoptosis by protease inhibitor may act
as reservoirs of the virus. Antiviral therapy is essential in conjugation of antiapoptotic therapy. However, the main obstacle is in the eradication of the HIV
virus is the presence of chronically infected latent reservoir cells such as
macrophages and CD4 T cells. Latently infected CD4 T-cells have remarkable
long half life (6 months) and inhibitors of viral replications alone would not be
able to eliminate the virus. An estimation based on the half life of infected latent
cells predicts that 60 years of viral suppression would be needed to eliminate
viral reservoirs.
The strategy to eliminate viral reservoirs is to target the latently infected
macrophages and CD4 T-cells to induce apoptosis after infection.
References:
Badley A. D. (2000) Blood, 96: 2951-2964
Azad, A. A. (2000) Biochem. Biophys. Res. Communications, 267: 677-685.
“Commercial Opportunities in Apoptosis” CTB international Publisheing Inc, P.O.
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