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Molecular mechanisms of differentiation and programmed cell death

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Molecular mechanisms of
differentiation and programmed cell
death – relation to cell pathologies
AleЕЎ Hampl
Tissues
Organs
Individuals
(animals & plants)
Cells that serve
various functions
(supporting stucture, pЕ™ijГ­mГЎnГ­
Еѕivin, locomotion, sensing and
transfer of signals,…)
Characteristic for:
complex multicellular
organisms
Ontogenesis of a complex multicellular organism
Zygote
Multicellular
embryo
Dynamic
multicellular
organism
How does multicellular organism develop from one cell ?
= one of the most critical question in biology
How does this question can be answered?
Ontogenesis of
multicellular
organism in terms of
CLASSICAL
EMBRYOLOGY
=
Changes in numbers,
localisation, and
shape of cells that
take place during
ontogenesis.
Ontogenesis of multicellular
organism in terms of MODERN
DEVELOPMENTAL BIOLOGY
X
=
Processes that drive a development
of various cell types and their
organisation to functional
structures of living organism.
Experiments that lead to
understanding causative relations.
This is only allowed by current approaches
of molecular genetics and biology !
Even recent technologies of molecular biology do not make
understanding regulation of development processes a trivial task !!!
… because
Linear
information
stored in genes
?
Developmental
processes occuring in
four dimensions:
Space (x,y,z) + Time
Zygote
Cell division
Cell division
&
Cell specialisation
Cell division
Cell division
&
Cell specialisation
Multicellular
embryo
Dynamic
multicellular
organism
= =
Cell multiplication
= =
Structural and
functional
specialisation of cells
Proliferation
&
Differentiation
Development
&
= =
Cell death in
predictable place and
time
= =
Tridimensional
organsiation of cells
into fuctional units
Programmed
cell death
&
Morphogenesis
Proliferation
&
Differentiation
Development
&
Programmed
cell death
&
Morphogenesis
Maintenance
of tissue
functions
• „health“ of tissues
• adaptation to
environment
• repair of injured
tissues
Proliferation
&
Differentiation
&
Programmed
cell death
&
Morphogenesis
Outcomes of
cell signaling
Differentiation
Differentiation is continuos
(multistep) process
Increase of cell differentiation
Decrease of developmental capacity - determination
Hematopoiesis
Stem cell
D I F F E R E N T I A T I O N
(example)
Precursors
(myeloid + lymphoid)
Progenitors
Terminally
differentiated cells
Once cells enter a given differentiation pathway
(lineage), they don`t change it.
(for example cells of erythroid lienage never „jump“ to myeloid lineage)
Cancer cells may be the exemption from this rule
• in cancer, cells of a given lineage may begin to express signs
of cells of a different lineage
• in cancer, it is often hard to determine from which cell lineage
cancer cells are derived
Dediferentiation – loss of differentiated phenotype
• typically occurs in cells explanted to in vitro
conditions (represents adaptation to culture conditions)
• existence of this phenomen in vivo is still questionable
and is beiing a matter of current research (similarly to
transdiferentiation)
Multicellular
embryo
Dynamic
multicellular
organism
D I F F E R E N T I A T I O N
Zygote
STABLE GENOME
Genomic equivalence
(= equal amount of DNA and the
same nucletide sequence in all
cells of a organism – cloning)
X
VARIABLE
TRANSCRiPTOME
Transcription Regulators
GENERAL PRINCIPLE
In every organism, all levels of organisation are typical
by the dynamics of elements, from which they are made
Genetic program
= Genes
g
a
b
j
c
i
e
f
A
Genetic information is
used stepwise based
on the activity of
small number of
functional modules,
which are
hierarchically ordered
with increasing level of
complexity.
Proteins
C
F
B
D
Cells
Tissues
Organs
G
Every level contains
information that is
required for building
higher level.
E
Functional networks
made of given elements
develop at every level..
One part of such
networks affects a
lower level, the other
part of such networks
serves as a basis for
formation of elements
of higher level.
Factors (signals) that regulate differentiation
Soluble regulators
• Hormones (glucagon, hydrocortison, tyroxin,…)
• Growth factors (TGF-transforming growth factor,
FGF-fibroblast growth factor, interleukins, …)
Vitamins (D, …)
•
• Ions (Ca++, …)
Cell-to-cell interactions
(gap junctions)
Interaction of cells with extracellular matrix
(colagen, laminin, …)
Polarity and shape of cells
Physical parameters of environment
(temperature, tension of O2, …)
These signals are processed by molecular regulatory mechanisms and networks
Universal molecular basis of regulatory mechanisms and networks
• There is a relatively small number of molecular networks
• These molecular networks are evolutionary highly conserved
• The same molecular networks are employed in different parts
of organism to drive different processes (one molecular network interprets
signal based on the actual molecular status of a given cell)
Fibroblast growth factor - FGF
one
Abnormality in differentiation as a cause of disease
Example
Leukemic SC
Hematopoietic SC
Mutation
Abnormality in
differentiation
Normal growth
and differentiation
Leukemic blasts
Normal blood cells
Is there any use for our understanding of
differentiation-regulating molecular mechanisms?
Differentiation of cells in vivo
(treatment of diseases = differentiation
therapy – cancer, …)
Differentiation of cells in vitro
(manipulation with stem cells = stem cell
therapy)
Programmed cell
death
A natural process for removing unwanted cells.
(cells that are not needed in further development, cells with genetic abnormalities, infected cells, …)
Programmed
cell death
A term originally used to describe
cells that die at predictable place
and time during development
Nearly all programmed
cell death is apoptotic
Apoptosis
A morphological description
of dying cells which contrasts
with necrosis
Terms are used
interchangeably
APOPTOSIS
The stimulus of death activates
a cascade of events that
orchestrate the destruction
of the cell
• Chromatin condensation
• Cell shrinkage
• Preservation of organelles
and cell membranes
• Rapid engulfment by neighboring
cells preventning inflamation
• DNA fragmentation - HALLMARK
Physiologic
(part of development)
Aberrant
(in diseases)
X
NECROSIS
Mechanism
The stimulus of death (e.g. ischemia)
is itself a direct cause of the
demise of the cell
Histologic
and
biochemical
signatures
• Nuclear swelling
• Cell swelling
• Disruption of organelles
and cell membranes
• Rupture of cell and release
of cellular contents
• Inflamatory response
Pathologic process
Apoptosis and necrosis in disease
Morphological features of apoptosis
• Chromatin condensation
• Blebbing of cell membrane
Picture in transmission electron microscope
Picture in scanning electron microscope
Fragmentation of genomic DNA
Internucleosomal
fragments (180 bp)
Electrophoretic separation of DNA in agarose gel
1972
The concept of programmed cell death first established.
(Kerr, Wilie, Currie – Apoptosis: a basic biological phenomenon with wide-ranging
implications in tissue kinetics. Br. J. Cancer)
2002
Nobel Prize in Physiology or Medicine
„Genetic regulation of organ development and
programmed cell death“
Robert Horvitz (1947)
USA
John Sulston (1942)
UK
Sydney Brenner (1927)
UK
What molecular mechanisms are
involved in driving apoptosis?
The last decade of 20ties century
Key findings were obtained using Caenorhabditis elegans
as a model organism (Robert Horvitz lab)
C. elegans
• organims built of exactly 1090 cells
• 131 cells undergoes apoptosis during
ontogenesis
• fate of all cells is well described
• can be chemically mutagenized
Four genes that are
indispensable for execution
of apoptosis during
ontogensis of C. elegans
Homologs in
higher metazoa
ced-3
caspases
ced-4
Apaf-1
egl-1
Bcl-2
ced-9
BH3-only
genes
Caspases – key executioners of apoptosis
Caspases = cysteinyl aspartate proteases
• Proteases dependent on cysteine, they cleave after aspartic AA
• Regulated primarily by posttranslational cleavage (in cells they are
present as inactive enzymes – procaspases = zymogens)
• Regulated also transcriptionally (some neurodegenerative diseases are typical
upregulated expression of caspases)
by
• They have many substrates including themselves – this allows for
amplification of signal in cascades)
• Currently 14 members of caspase family is known, 11 of which are
found in man
• Not all caspases are involved in the process of programmed cell death
• Caspases that participate in programmed cell death are categorized
to „initiating“ and „executing“ caspases
Proteins of BCL2 family – key
regulators of apoptosis
• They contain at least one region of homology with Bcl2
(BCL2 homology region – BH)
• According to their activity they divide to „antiapoptotic“ a „proapoptotic“
• BCL2 – the first identified member of this family = antiapoptotic
• BAX – discovered based on its asociation with BCL2 = proapoptotic
BH4
BH3 BH1
BH2
Number of AA
antiapoptotic
proapoptotic
BH3-only
Two pathways initiating and executing apoptosis
Intrinsic
Extrinsic
• It is activated as a reaction on various stimuli that are generated • It is initiated by binding of secreted
ligand (e.g.FasL) to „death receptor“
in cell (DNA damage, oncogene activation, oxidative stres…)
that is a member of TNFR (tumor
• It is mediated by mitochondria, which as a reaction on stress
necrosis factor rc) family (e.g. Fas)
release proteins from their intermembrane space (cytochrome c,
• Association with other proteins
SMAC, AIF, endoG…)
then leads to a formation of DISC
• Cytochrome c, for example, then binds APAF1 and ATP, thus
leading to conversion of initiation procaspase-9 to active caspase-9 (death-inducing signalling complex)
Diseases & Apoptosis
Abnormalities in apoptosis are involved in many different diseases !
Cancer
Autoimunity diseases
Diabetes
Neurodegenerative diseases
Infertility
Hepatitis
Sepsis
Viral infections
+ many others
Molecules reguluting apoptosis are very attractive
targets for pharmacological intervention !!!
Thank you for your attention !
Questions and comments at:
ahampl@med.muni.cz
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