CELL SENESCENCE, APOPTOSIS, ANGIOGENESIS, AND METASTASIS
CELL SENESCENCE AND APOPTOSIS
GENERAL OVERVIEW
SENESCENCE AND QUIESCENCE
The limitless replicative potential is one of the hallmarks of cancer cells. This hallmark can be arrested
or eliminated via cellular senescence or apoptosis.
The normal cells have a limited replicative potential, which is related
to replicative senescence. This senescence was described for the first
time Leonard Hayflick. The senescence differs from quiescence,
which is reverse by stimulation with mitogenic factors. Conversely,
the senescence is difficulty reverted.
The Hayflick limit refers to the maximum number of divisions that a
cell can sustain. Beyond that number cells can enter in irreversible
growth arrest and acquire the phenotypical characteristic of
senescent cells.
Therefore, quiescence is a transient resting condition that can be reverted into proliferative state,
while senescence is a permanent state in which cells, after several cell divisions, cannot any longer
divide.
SENESCENCE MARKERS
The cellular senescence present specific phenotypic and genotypic features. These cells express some
specific proteins, which can be used as markers for senescence. An example is the senescence-
associated -galactosidase (SA--Gal), which is a lysosomal enzyme that
is used in immunohistochemistry to detect senescent cells. Other
examples are p16, p15, p53, ARF, and p21; these markers can be detected
with both immunohistochemistry and Western blot.
Furthermore, even senescence-associated heterochromatin foci (SAHFs)
are used to determine the senescent status. This marker is used in
immunofluorescence to evaluate the heterochromatin state of cells.
Note that a cell that is undergoing senescence is metabolically active, but
it cannot divide.
TELOMERE AND TELOMERASE
TELOMERASE
The replicative senescence is associated to the progressive shortening of telomeres associated to DNA
replication. This process is called telomere erosion or shortening. A total lifetime loss of 2-4kb in
average telomere length has been observed in human cells
(average length is of 8-12kb).
The telomere replication cannot be performed by the
canonical DNA polymerase (problems of linear
chromosome), and for that reason a specific complex is
used, which is the telomerase. The telomerase is a
ribonucleoprotein complex that is involved in the
replication of telomeres, which cannot be replicated by
canonical DNA polymerases. It extends telomeres by
adding TTAGGG repeats, thereby preventing telomere
erosion. The telomerase complex is made of two components, which are:
• Human telomerase reverse transcriptase (hTERT): it is the catalytic subunit of the complex.
• Human telomere DNA template (hTR): it is used as RNA template to extend telomeres.
The telomerase complex is present in germ cells and stem cells, but not in differentiated cells.
However, this complex is reactivated in more than 80% of malignant cancers. The transduction of
,normal cells with hTERT leads to cell immortalisation, thus favouring the emergence of transformed
cells.
TELOMERASE REGULATION AND ALT
The gene that is involved in the expression of hTERT can be regulated by several TFs, such as c-myc,
EGF, and other growth factors, which promote gene expression. On the contrary, p53 and Menin
repress the hTERT expression. Therefore,
oncogenes promote the hTERT expression,
whereas tumour suppressor genes inhibit its
expression. This means that the telomerase
activity can be seen as an oncogenic function,
which promotes the cell immortalisation.
Moreover, hTERT promoter polymorphisms are
associated with 2-4-fold increased expression.
The telomerase activity may also affect the
prognosis of cancers. In the Kaplan-Meier curve
of neuroblastoma it is possible to observe that the survival rate in telomerase negative patients is
higher respect to telomerase positive. The same is observed in Ewing’s sarcoma patients that present
a low telomerase activity. The telomerase
reactivation and the NMYC amplification are linked
to prognostic factors of tumours.
The telomere length can be maintained integrated
through an hTERT-independent mechanism, that is
called alternative lengthening of telomeres (ALT).
In this mechanism the telomere of a chromosome
is used as template for the replication of the
telomere of another chromosome. The ALT
mechanism is used by some cancers to maintain
resistance, such as osteosarcomas (50%) and
neuroblastomas (25%), but rarely in carcinomas.
Note that his mechanism involved recombination.
The ALT is problematic in case of utilisation of telomerase inhibitors since these drugs will be
ineffective against cancer cells.
TELOMERE PROTECTION
The telomeres do not express any proteins (only some ncRNA); their main function seems to be
protective for chromosomes. Typically, the telomeric ends are not symmetric, but they present some
overhangs. These are used to allow the binding by a protein complex,
called shelterin. The shelterin is involved in the creation of a closed
structure (i. e. loop) in the telomeric end, which stabilise the
chromosome and avoids possible integrations.
The ss overhang invade the double stranded region of the telomere to
form a t loop, while a single stranded displacement will from a D loop.
The t loop sequesters the 3’-end regions of telomere, thus preventing the
recognition as DSBs.
TELOMERE AND SENESCENCE
TELOMERASE EROSION AND SENESCENCE
The shelterin complex contains proteins, which are POTI and TRF2, can inhibit proteins involved in
DNA repair systems. In particular, POTI is involved in the d loop, and it inhibits the ATR activity (SSBs
repair), while TRF2 is involved in the t loop, and it inhibits the ATM (DSBs repair).
,Since these two kinases are inactive, the telomere is maintained integrated. Conversely, the erosions
of telomere will induce the activation of DNA damage response. The cells begin to repair the telomere
erosion via ATM and ATR activation since it is seen as a DNA damage.
Then it will induce cell senescence. If the erosion is continuous and DNA
repair cannot stop it, cell death via apoptosis will occur.
The telomere dysfunction can activate two pathways that are involved
in cell senescence, which are:
• p53 pathway: it is activated via phosphorylation by ATM, ATR,
Chk1, and Chk2, which in turn are activated in case of DNA
damages; the p53 expression and stabilisation will increase the
expression of p21, which will promote cell senescence; this pathway cannot be reversed by
physiological mitogens, but it is reversible upon inactivation of p53.
• RB pathway: it is not yet understood the origin of this pathway, but it
is related to the inhibition of Cdk4 and Cdk6 by INK4A (i. e. p15 and
p16); their inactivation will cause the increase in activity of RB, thus
leading to senescence; the RB can be also activated by p21; the RB
pathway cannot be reversed by inactivating p53 and RB; however, cells
silencing p16 are insensitive to this pathway.
The RB-mediated senescence is an irreversible senescence that is associated
with the recruitment of the SUV39. The SUV39 is a histone methyltransferase
that trimethylates Lys9 in
histone 3 (H3K9me3).
Then, heterochromatin protein 1 (HP1) is
recruited, thus leading to the compaction of
chromatin (i.
e. decrease gene expression). The H3K9me3 is a specific marker of
senescence, and it can be visualised by the appearance of SAHFs.
As already observed, p16, p15, and p14ARF can stimulate the
expression of p53 and RB. The p16 and p15 inhibit the CyclinD-
Cdk4 and Cdk6 activity, thus promoting RB activity. The ARF
instead inhibits the activity of Mdm2, which allows the
stabilisation of p53.
PATHWAY TYPE OF SENESCENCE FACTORS INVOLVED FEATURES
Senescence is
ATM, ATR, Chk1, Chk2,
p53 Reversible reversed by mitogenic
p21 (last effector)
signals
Trimethylation of
INK4A (p15, p16), RB, histones,
RB Irreversible
SUV39, H3K9me3 heterochromatin
formation (SAHFs)
TELOMERE SHORTENING AND CANCER DEVELOPMENT
The telomere length should be longer in cancer cells since they present an intrinsic telomerase
activity. However, it was observed that normal differentiated cells present a telomere length that is
longer respect to telomeres in cancer cells. This seems to be associated to stimuli (e. g. GFs) that are
released by the surrounding environment. The telomere length related to cell doublings shows three
main checkpoints, which are:
• Reversible arrest: it occurs in differentiated cells, in which if GFs are not produced, the
telomere length is maintained constant since cells are well-differentiated and they do not
, divide; when the cell divides due to the presence of mitogenic signals, a shortening of
telomere length occurs; but the shortening will not affect the cell.
• Replicative senescence: it occurs
when the telomere erosion affects
the cell (telomere length 5kbp); the
replicative senescence occurs when
the shortest telomere becomes
dysfunctional, and it triggers p53-
dependent arrest.
• Telomere crisis: it occurs when
telomeres are critically shortened
(telomere length 1-3kbp) due to the
bypassing of cell arrest via
mutations; in cancer cells that are
highly stimulated, the cell division
continues, thus causing a telomere
erosion; once the telomere erosion is extreme the telomere are seen as DNA damages; this
increases the number of mutations due to an excessive recruitment of DNA repair system in
the telomere and not in other chromosomal region; moreover, the extensive telomer
shortening will cause the activation of the telomerase, which will provide the final cell
immortalisation and telomere stabilisation.
Therefore, the telomere erosion is a barrier to cancer in the presence of intact checkpoints, and a
facilitator in their absences.
Furthermore, telomere erosion generates chromosomal abnormalities that can be repaired by NHEJ
or give rise to chromosomal fusion (i. e. breakage-fusion-bridge, BFB, cycle).
To summarise, the telomeres act as tumour suppressor mechanisms that prevent cancer formation.
However, in case of excessive telomere erosion, accompanied with excessive DNA mutations, they
may turn in stimulating factors for cancer cells to produce more mutations and chromosomal
abnormalities.
ONOGENE-INDUCED SENESCENCE (OIS)
ONCOGENES AND SENESCENCE
The cell senescence is not induced only by the telomere erosion, but also other factors, such as
cytotoxic drugs, DNA damages, and oxidative stress can induce it. Moreover, another process that
may induce senescence is the oncogene-
induced senescence (OIS). This is a
phenomenon in which a mutated oncogene
cause senescence. It is quite peculiar since
instead of causing cancer, the oncogene will
prevent the proliferation of damaged cells and
establish senescence.
For instance, HRasG12V mutation causes an
initial hyperproliferation of cells due to Ras
pathway activation; this is followed by a period of senescence. This seems to be related to activation
of DNA damage response (DDR), which is common to both replicative and oncogene-induced
senescence.
EXCESSIVE REPLICATION CAUSES OIS
The DNA replication in eukaryotes begins from several origin of replication (Ori), which are scattered
along chromosomes. It is observed that Ori are activated only once per time, thus explaining why the
S phase in eukaryotic cells is long. This ensure that chromosomal DNA will be precisely duplicated.
CELL SENESCENCE AND APOPTOSIS
GENERAL OVERVIEW
SENESCENCE AND QUIESCENCE
The limitless replicative potential is one of the hallmarks of cancer cells. This hallmark can be arrested
or eliminated via cellular senescence or apoptosis.
The normal cells have a limited replicative potential, which is related
to replicative senescence. This senescence was described for the first
time Leonard Hayflick. The senescence differs from quiescence,
which is reverse by stimulation with mitogenic factors. Conversely,
the senescence is difficulty reverted.
The Hayflick limit refers to the maximum number of divisions that a
cell can sustain. Beyond that number cells can enter in irreversible
growth arrest and acquire the phenotypical characteristic of
senescent cells.
Therefore, quiescence is a transient resting condition that can be reverted into proliferative state,
while senescence is a permanent state in which cells, after several cell divisions, cannot any longer
divide.
SENESCENCE MARKERS
The cellular senescence present specific phenotypic and genotypic features. These cells express some
specific proteins, which can be used as markers for senescence. An example is the senescence-
associated -galactosidase (SA--Gal), which is a lysosomal enzyme that
is used in immunohistochemistry to detect senescent cells. Other
examples are p16, p15, p53, ARF, and p21; these markers can be detected
with both immunohistochemistry and Western blot.
Furthermore, even senescence-associated heterochromatin foci (SAHFs)
are used to determine the senescent status. This marker is used in
immunofluorescence to evaluate the heterochromatin state of cells.
Note that a cell that is undergoing senescence is metabolically active, but
it cannot divide.
TELOMERE AND TELOMERASE
TELOMERASE
The replicative senescence is associated to the progressive shortening of telomeres associated to DNA
replication. This process is called telomere erosion or shortening. A total lifetime loss of 2-4kb in
average telomere length has been observed in human cells
(average length is of 8-12kb).
The telomere replication cannot be performed by the
canonical DNA polymerase (problems of linear
chromosome), and for that reason a specific complex is
used, which is the telomerase. The telomerase is a
ribonucleoprotein complex that is involved in the
replication of telomeres, which cannot be replicated by
canonical DNA polymerases. It extends telomeres by
adding TTAGGG repeats, thereby preventing telomere
erosion. The telomerase complex is made of two components, which are:
• Human telomerase reverse transcriptase (hTERT): it is the catalytic subunit of the complex.
• Human telomere DNA template (hTR): it is used as RNA template to extend telomeres.
The telomerase complex is present in germ cells and stem cells, but not in differentiated cells.
However, this complex is reactivated in more than 80% of malignant cancers. The transduction of
,normal cells with hTERT leads to cell immortalisation, thus favouring the emergence of transformed
cells.
TELOMERASE REGULATION AND ALT
The gene that is involved in the expression of hTERT can be regulated by several TFs, such as c-myc,
EGF, and other growth factors, which promote gene expression. On the contrary, p53 and Menin
repress the hTERT expression. Therefore,
oncogenes promote the hTERT expression,
whereas tumour suppressor genes inhibit its
expression. This means that the telomerase
activity can be seen as an oncogenic function,
which promotes the cell immortalisation.
Moreover, hTERT promoter polymorphisms are
associated with 2-4-fold increased expression.
The telomerase activity may also affect the
prognosis of cancers. In the Kaplan-Meier curve
of neuroblastoma it is possible to observe that the survival rate in telomerase negative patients is
higher respect to telomerase positive. The same is observed in Ewing’s sarcoma patients that present
a low telomerase activity. The telomerase
reactivation and the NMYC amplification are linked
to prognostic factors of tumours.
The telomere length can be maintained integrated
through an hTERT-independent mechanism, that is
called alternative lengthening of telomeres (ALT).
In this mechanism the telomere of a chromosome
is used as template for the replication of the
telomere of another chromosome. The ALT
mechanism is used by some cancers to maintain
resistance, such as osteosarcomas (50%) and
neuroblastomas (25%), but rarely in carcinomas.
Note that his mechanism involved recombination.
The ALT is problematic in case of utilisation of telomerase inhibitors since these drugs will be
ineffective against cancer cells.
TELOMERE PROTECTION
The telomeres do not express any proteins (only some ncRNA); their main function seems to be
protective for chromosomes. Typically, the telomeric ends are not symmetric, but they present some
overhangs. These are used to allow the binding by a protein complex,
called shelterin. The shelterin is involved in the creation of a closed
structure (i. e. loop) in the telomeric end, which stabilise the
chromosome and avoids possible integrations.
The ss overhang invade the double stranded region of the telomere to
form a t loop, while a single stranded displacement will from a D loop.
The t loop sequesters the 3’-end regions of telomere, thus preventing the
recognition as DSBs.
TELOMERE AND SENESCENCE
TELOMERASE EROSION AND SENESCENCE
The shelterin complex contains proteins, which are POTI and TRF2, can inhibit proteins involved in
DNA repair systems. In particular, POTI is involved in the d loop, and it inhibits the ATR activity (SSBs
repair), while TRF2 is involved in the t loop, and it inhibits the ATM (DSBs repair).
,Since these two kinases are inactive, the telomere is maintained integrated. Conversely, the erosions
of telomere will induce the activation of DNA damage response. The cells begin to repair the telomere
erosion via ATM and ATR activation since it is seen as a DNA damage.
Then it will induce cell senescence. If the erosion is continuous and DNA
repair cannot stop it, cell death via apoptosis will occur.
The telomere dysfunction can activate two pathways that are involved
in cell senescence, which are:
• p53 pathway: it is activated via phosphorylation by ATM, ATR,
Chk1, and Chk2, which in turn are activated in case of DNA
damages; the p53 expression and stabilisation will increase the
expression of p21, which will promote cell senescence; this pathway cannot be reversed by
physiological mitogens, but it is reversible upon inactivation of p53.
• RB pathway: it is not yet understood the origin of this pathway, but it
is related to the inhibition of Cdk4 and Cdk6 by INK4A (i. e. p15 and
p16); their inactivation will cause the increase in activity of RB, thus
leading to senescence; the RB can be also activated by p21; the RB
pathway cannot be reversed by inactivating p53 and RB; however, cells
silencing p16 are insensitive to this pathway.
The RB-mediated senescence is an irreversible senescence that is associated
with the recruitment of the SUV39. The SUV39 is a histone methyltransferase
that trimethylates Lys9 in
histone 3 (H3K9me3).
Then, heterochromatin protein 1 (HP1) is
recruited, thus leading to the compaction of
chromatin (i.
e. decrease gene expression). The H3K9me3 is a specific marker of
senescence, and it can be visualised by the appearance of SAHFs.
As already observed, p16, p15, and p14ARF can stimulate the
expression of p53 and RB. The p16 and p15 inhibit the CyclinD-
Cdk4 and Cdk6 activity, thus promoting RB activity. The ARF
instead inhibits the activity of Mdm2, which allows the
stabilisation of p53.
PATHWAY TYPE OF SENESCENCE FACTORS INVOLVED FEATURES
Senescence is
ATM, ATR, Chk1, Chk2,
p53 Reversible reversed by mitogenic
p21 (last effector)
signals
Trimethylation of
INK4A (p15, p16), RB, histones,
RB Irreversible
SUV39, H3K9me3 heterochromatin
formation (SAHFs)
TELOMERE SHORTENING AND CANCER DEVELOPMENT
The telomere length should be longer in cancer cells since they present an intrinsic telomerase
activity. However, it was observed that normal differentiated cells present a telomere length that is
longer respect to telomeres in cancer cells. This seems to be associated to stimuli (e. g. GFs) that are
released by the surrounding environment. The telomere length related to cell doublings shows three
main checkpoints, which are:
• Reversible arrest: it occurs in differentiated cells, in which if GFs are not produced, the
telomere length is maintained constant since cells are well-differentiated and they do not
, divide; when the cell divides due to the presence of mitogenic signals, a shortening of
telomere length occurs; but the shortening will not affect the cell.
• Replicative senescence: it occurs
when the telomere erosion affects
the cell (telomere length 5kbp); the
replicative senescence occurs when
the shortest telomere becomes
dysfunctional, and it triggers p53-
dependent arrest.
• Telomere crisis: it occurs when
telomeres are critically shortened
(telomere length 1-3kbp) due to the
bypassing of cell arrest via
mutations; in cancer cells that are
highly stimulated, the cell division
continues, thus causing a telomere
erosion; once the telomere erosion is extreme the telomere are seen as DNA damages; this
increases the number of mutations due to an excessive recruitment of DNA repair system in
the telomere and not in other chromosomal region; moreover, the extensive telomer
shortening will cause the activation of the telomerase, which will provide the final cell
immortalisation and telomere stabilisation.
Therefore, the telomere erosion is a barrier to cancer in the presence of intact checkpoints, and a
facilitator in their absences.
Furthermore, telomere erosion generates chromosomal abnormalities that can be repaired by NHEJ
or give rise to chromosomal fusion (i. e. breakage-fusion-bridge, BFB, cycle).
To summarise, the telomeres act as tumour suppressor mechanisms that prevent cancer formation.
However, in case of excessive telomere erosion, accompanied with excessive DNA mutations, they
may turn in stimulating factors for cancer cells to produce more mutations and chromosomal
abnormalities.
ONOGENE-INDUCED SENESCENCE (OIS)
ONCOGENES AND SENESCENCE
The cell senescence is not induced only by the telomere erosion, but also other factors, such as
cytotoxic drugs, DNA damages, and oxidative stress can induce it. Moreover, another process that
may induce senescence is the oncogene-
induced senescence (OIS). This is a
phenomenon in which a mutated oncogene
cause senescence. It is quite peculiar since
instead of causing cancer, the oncogene will
prevent the proliferation of damaged cells and
establish senescence.
For instance, HRasG12V mutation causes an
initial hyperproliferation of cells due to Ras
pathway activation; this is followed by a period of senescence. This seems to be related to activation
of DNA damage response (DDR), which is common to both replicative and oncogene-induced
senescence.
EXCESSIVE REPLICATION CAUSES OIS
The DNA replication in eukaryotes begins from several origin of replication (Ori), which are scattered
along chromosomes. It is observed that Ori are activated only once per time, thus explaining why the
S phase in eukaryotic cells is long. This ensure that chromosomal DNA will be precisely duplicated.
