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The Implications of Cell Cycle Deregulation
and Apoptosis in Malignancy
Dr Samantha Potgieter
Dr Mike Webb
Introduction
A tight balance between cell proliferation on the one side, and cell death on the other, is required to
maintain homeostasis in cellular compartments – particularly those with a high intrinsic rate of cell
turnover such as the haematological system. When these two critical mechanisms i.e. - cell cycle
regulation and apoptosis - are disrupted cancers occur.
Two characteristic features define a cancer: Unregulated cell growth and tissue invasion or metastasis.
We know that cancer development results from the accumulation of genetic mutations that lead to 1.
Deregulation of the cell cycle and therefore uncontrolled and unchecked proliferation of cells, as well
as 2. Resistance to apoptosis, that allows these cells to become immortalized and capable of invading
other tissues.
Historical Perspective
The Egyptians described the first recorded cases of cancer in1600BC on what is known as the Edwin
Smith Papyrus. This is a copy of part of an ancient Egyptian textbook on trauma surgery and it
describes 8 cases of tumours or ulcers of the breast that were treated by cauterisation, with a tool called
the fire drill. The writing says about the disease: "There is no treatment."
These ancient Egyptians blamed cancer on the Gods, and over the next three and a half thousand years
humans endeavoured to understand the exact cause.
The Humoral Theory
Born in 460BC, Hippocrates believed that the body had 4 humours or body fluids-- blood, phlegm,
yellow bile, and black bile. He described a humoral theory in which he postulated that an excess of black
bile in various body sites was the cause cancer. This idea remained the unchallenged standard through
the Middle Ages for over 1300 years.
The Lymph Theory
In the 1600’s Stahl and Hoffman theorized that cancer was composed of fermenting and degenerating
lymph varying in density, acidity, and alkalinity. This lymph theory gained rapid support. And in the
1700’s the eminent surgeon John Hunter agreed that tumours grow from lymph constantly thrown out by
the blood.
The Blastema Theory
In 1838, German pathologist Johannes Muller demonstrated that cancer is made up of cells and not
lymph, but he believed that cancer cells did not arise from normal cells. Muller proposed that cancer
cells arose from budding elements (blastema) between normal tissues. In the late 1800’s his student,
Rudolph Virchow the famous German pathologist, determined that all cells, including cancer
cells, are derived from other cells.
The Biology of Cancer - What We Know Today
The second half of the 20th century saw rapid progress in our understanding of the origins of cancer.
After the 1962 Nobel prize winners James Watson and Francis Crick discovered the exact chemical
structure of DNA and its importance as the basis of the genetic code, scientists were able to understand
how genes worked and how they could be damaged to form mutations
It was already known that cancer could be caused by chemicals, radiation, and viruses, and that
sometimes cancer seemed to run in families. But as the understanding of DNA and genes increased, we
learned that it was the damage to DNA by chemicals and radiation, or the introduction of new DNA
sequences by viruses that often led to the development of cancer. It became possible to pinpoint the
exact site of the damage to a specific gene. It was recognised that DNA damage or mutations lead to
abnormal groups of cells or clones. These mutant clones evolved to even more malignant clones over
time, and the cancer progressed.
The Importance of Cell Cycle Regulation and Apoptosis
Central to this understanding of the biology of cancer is the fact that normal cells with damaged DNA
die, whilst cancer cells with damaged DNA do not. It is this critical difference that we will discuss today.
Normal cells have two important mechanisms to guard against unregulated growth or unchecked
proliferation – the hallmark of cancer. These are firstly, the tightly ordered regulation of the cell cycle and
secondly, apoptosis or programmed cell death.
The Implication of Cell Cycle Deregulation in
Malignancy
The Normal Cell Cycle
The cell cycle is a highly ordered process that results in the duplication and subsequent transfer of the
genetic code from one cell generation to the next. It involves the accurate replication of DNA to form 2
chromosomal copies and results in the subsequent formation of 2 identical daughter cells
The process of cell division was initially divided into two stages, namely mitosis (M phase) – the process
of nuclear division, and interphase – the interlude between to M phases. During interphase, observed
under the microscope, cells merely grow in size, but in 1992 Norbury and Nurse revealed that
interphase consist of 3 discrete phases. S phase during which DNA replication occurs, and two Gap
phases, labelled G1 and G2. G1 precedes S phase and is a period of time during which the cell is
preparing for DNA synthesis. G2 occurs after S phase prior to M phase and is a period during which the
cell prepares for Mitosis. So the 4 traditional subdivisions of the cell cycle are G1, S, G2 and M phase.
Prior to entry into S phase and commitment to DNA replication, cells in G1 can enter a rest state called
G0. Cells in G0 account for the majority of non-growing, non-proliferating cells in the body.
Figure 1. Stages of the cell cycle
Regulation of the Cell Cycle
The CDKs and Cyclins
The entire process of the cell cycle is propelled by a number of different cellular proteins called Cyclin
Dependent Kinases (CDK’s). When bound to their respective activating cyclin, the activated CDK/cyclin
complex phosphorylates a number of specific downstream protein substrates to propel the cell through
the cell cycle. Each CDK acts at a specific stage of the cell cycle. These CDK protein levels remain
constant throughout the cell cycle whereas in contrast, their activating proteins the cyclins rise and fall in
concentration during the cell cycle as they are synthesized and then again degraded. It is in this way
that they periodically activate their respective CDK.
Up to now, nine CDKs have been identified – five of which are active during the cell cycle. These are
CDK 4, CDK 6 and CDK 2 – active during G1. CDK2 is again active during S phase. Then CDK1 active
during both G2 and M phase.’
Sixteen different cyclins have been identified, but like the CDK’s, not all are cell cycle related. Like their
respective protein kinases, the cyclins act at very specific points in the cell cycle. The D cyclins D1 D2 &
D3 bind to CDK4 and CDK6 and it is this step that is crucial for entry into G1. CyclinD is unique in that it
is not synthesized periodically, as are the other cyclins, but rather its synthesis is prompted by
stimulation of various growth factors. As the cell progresses through late G1 there is increased
expression of CyclinE. The cyclinE-CDK2 complex is required for progression from G1 to S phase.
Increased CyclinA expression occurs at the G1/S transition and persist throughout S phase. Binding of
Cyclin A to CDK2 allows DNA synthesis to proceed. In M phase the binding of CyclinsA and B to CDK1
propel the cell through mitosis. So throughout the cell cycle, the CDKs, activated by the cyclins,
phosphorylate dowstream substrates that are necessary for the ordered progression through the various
phases of the cell cycle. It is the periodicity of the cyclins, mediated by their synthesis and subsequent
degradation that ensures the ordered and well-delineated transition between cell cycle stages.
Figure 2. Regulation of the cell cycle by Cyclin Dependent Kinases
CDK Inhibitors
CDK activity can be countered by cell cycle inhibitory proteins called CDK Inhibitors (CKI’s). These
inhibitory proteins bind to CDK’s alone or the CDK-cyclin comlex and regulate CDK activity. Two distinct
families of CKI’s have been discovered- the Cip/Kip family and the INK4 family. The INK4 family
includes p15(INK4b), p16(INK4a), p18(INK4c) and p19(INK4d). This INK4 family of CDK Inhibitors
specifically inactivates the G1 CDKs - CDK4 and CDK6. This is achieved by the binding of an INK4
inhibitor to the CDK enzyme before cyclin binding. The CDK-CKI forms a stable complex preventing the
CDK from binding with CyclinD
The second family of CKIs is the Cip/Kip family. These are p21(Cip1) p27(Cip2) and p57(Cip3). These
inhibitors inactivate the CDK-cyclin complex. They inhibit the G1 CDK4/6-CyclinD complex and to a
lesser extent the CDK1-Cyclin B complex.
Table 1. Cyclin Dependent Kinases (CKI) bind to CDK alone or CDK-Cyclin complex and regulate CKD activity
CDK Substrates
When a CDK is activated by its respective cyclin, downstream target proteins become phosphorylated –
this results in changes that are physiologically important for cell cycle progression.
The most frequently studied target is the substrate of CDK4/6-CyclinD1 in early G phase. The binding of
CyclinD1 to CDK4/6 results in the phosphorylation of retinoblastoma(Rb) protein. Retinoblastoma
protein family members are called “pocket proteins” because the unphosphorylated Rb protein tightly
binds, and thereby inhibits, transcription factor E2F. Once phosphorylated by the active CDK4/6 Rb
protein dissociates form E2F – allowing E2F to transcribe a number of responder genes that are
necessary for the cell to progress though G1 and into S phase. These include genes that code for
CyclinA and CyclinE.
Figure 4. Binding of cyclin D to CDK4/6 phosphorylates the substrate Retinoblastoma protein
Checkpoints and Restriction Points
The regulation of the cell cycle must ensure that transition from each phase to the next occurs in an
orderly fashion and that the events in each phase are accurately completed before moving onto the next
phase. Because errors encoded in the genome would result in abnormal clones if allowed to proliferate,
the cell cycle is closely monitored for abnormal programming. There are 2 strategically placed
checkpoints, one late in G1 prior to entry into S phase, and another at the G2/M interface, prior to entry
into M phase. These checkpoints monitor the integrity of DNA and prevent the propagation of damaged
or mutated cells.
1. The restriction point (R) is the most important and the best defined checkpoint in the cell cycle and
occurs in the latter third of G1.
2. An S phase DNA-damage checkpoint exists but its mechanisms are poorly understood.
3. When DNA damage occurs in G2 cells are again able to initiate cell cycle arrest and entry into mitosis
is prevented.
Restriction Point (R)
In response to growth or mitotic signalling the cell moves out of G0 and into G1. At the beginning of G1,
increased expression of CyclinD (D1,2 and 3) occurs. The D cyclins associate with CDK4 and CDK6.
The activated complexes then phosphorylate retinoblastoma protein as described above. That in turn
releases E2F transcription factor, which induces transcription of responder genes necessary for
progression through the restriction point. Critical to this step is P53 tumour suppressor gene. In the
presence of genomic damage P53 interrupts the cell cycle to allow time for DNA repair. This is
accomplished by the inhibition of phosphorylation of Rb protein by P53.
Normally in replicating cells the level of P53 is low or undetectable and is maintained as such that
replication can continue unimpeded. Murine double minute 2 (MDM2) protein negatively regulates P53.
MDM2 functions at two levels. Firstly at genomic level – it inhibits the transcription of P53. Secondly it
binds to P53, inhibiting its activity and increasing its proteosomal degradation.
In the presence of DNA damage P53 binds to a sequence specific DNA site, and gene induction results
in increased P53 protein synthesis with subsequent increased P53 phosphorylation. With P53
phosphorylation two things occur – the protein becomes more active and its binding to MDM2 is
decreased. Remembering that MDM2 causes P53 degradation as well as decreased P53 gene
transcription, one can understand that a decreased binding to MDM2 can increase P53 activity up to
100 fold in response to DNA damage. P53 up-regulates the CDK inhibitor P21. This inhibits the CyclinDCDK4/6 complex thus preventing the phosphorylation of Rb protein. This causes the
hypophosphorylated Rb protein to tightly hold E2F transcription factor thus inhibiting transcription of the
proteins necessary for entry into S phase. This holds the cell in G1 phase and allows time for DNA
repair. If DNA damage is severe and exceeds the cells capability for DNA repair then P53 guides the
cell into apoptosis – a process we will discuss later.
Figure 5. DNA damage causes P53 phosphorylation and inhibits P53 binding to MDM. The result is decreased
proteasomal degradation of P53 as well as increased P53 gene transcription. The net result is marked increase in
P53 activity.
The Importance of Cell Cycle deregulation in the Development
of Cancer
As we have mentioned – the hallmark of cancer cells is that they stay in cycle, escaping both internal
and external control. Mutations can occur in genes encoding for CDKs, cyclins, CDK substrates, CDK
inhibitors and checkpoint proteins and can cause deregulation of the finely controlled cycle described
above.
Commonly Observed Mutations
1. P53
As one can see, a blunted P53 response would result in decreased P21 expression and the unopposed
binding of CyclinD to CDK4/6, despite the presence of DNA damage. The damage cell, instead of
halting in G1 to either repair itself or to be directed into apoptosis, would therefore be able to continue
through the restriction point into S phase. The result would be the uncontrolled proliferation of a
defective cell or clone. P53 is a protein encoded for by the gene TP53 on the short arm of chromosome
17. It gets its name from its molecular mass - being a 53kDa protein. It is the most commonly mutated
gene in human cancer.
Point and missense mutations lead to a conformational shape change in the transcribed protein – this
renders it unable to bind to its target DNA sequence – the result of which is inactivation of the protein.
A rare inherited disorder – the Li-Fraumeni syndrome is an autosomal dominant disorder involving germline mutations of TP53. Affected individuals inherit only one functional copy of P53. This results in the
development of multiple tumours in early adulthood. These patients have a high occurrence of breast
cancer, brain tumors, acute leukaemia, soft tissue and bone Sarcomas and adrenal cortical adenoma.
More common in clinical practice are acquired mutations of P53. P53 is thought to be damaged by a
variety of mutagens, including chemicals, radiation and viruses. P53 mutations are in fact, thought to be
found in more than 50% of human cancers.
Amplification of MDM2, described above as a P53 inhibitor, is seen in a number of lymphomas and soft
tissue sarcomas.
Patients with P53 mutation not only have an increase in tumour incidence, but they also have a poorer
prognosis and decreased response to ionizing radiation and chemotherapy – which would trigger
apoptosis in normal cells, but which fails to do so in the absence of functional P53. An example of this is
in CLL – patients with Chromosome 17p mutations (P53 located on short arm of chromosome 17)
have a notoriously poor response to chemotherapy. This is one group of CLL patients in whom one
should consider allograft BMT. This is a great example of how understanding the underlying
cytogenetics of a cancer can guide one clinically.
2. Retinoblastoma
Deletion and missence mutations may result in truncated and non-functional Rb protein or complete
absence of the protein. In addition, binding of certain virus proteins such as HPV E7, adenovirus and
SV40 can result in the inactivation of Rb protein. Whatever the cause, decreased activity or absence of
Rb protein results in unrestrained cell cycle progression and is common in ALL, human retinoblastoma
and types of lung cancer.
Figure 6. Binding of HPV viral protein E7 inactivates Rb – releasing E2F and allowing transcription of responder genes
that propel the cell through G1 into S phase regardless of the presence of DNA damage
3. INK4A
The loss of INK4a has been documented in familial malignant melanoma. In addition deletion of
P16INK4a is seen in up to 50% of T cell ALL and 20% of B cell ALL. Loss of INK4A results in decreased
expression of P16 and thus unopposed binding of Cyclin D to CDK4/6. This in turn causes unregulated
or unchecked progression of the cell through G1 into S phase regardless of the presence of DNA
damage. Loss of INK4A also has a less direct effect by removing the negative effect of p19 on MDM2.
This results in increased expression of MDM2, which as described above is a P53 inhibitor. This again
result in decreased P53 activity even in the face of DNA damage – the result of which is the unchecked
progress though the cell cycle of potentially malignant clones.
Figure 7. INK4a mutations result in decreased p16 inhibition of cyclinD-CDK4/6 binding, as well as decreased P53 inhibition
by MDM2 (via p19) The net result is increased prosphorylation of Rb and propulsion through the retstriction point. This is
common in T and B cell ALL
4. Cyclin D1
Mantle Cell Lymphoma (MCL) is one of the subtypes of NHL. The t(11;14) is a characteristic alteration
in MCL. In this translocation the BCl 1 gene, on chromosome 11, encoding for cyclin D1, is positioned
adjacent to the enhancer region of the immunoglobulin heavy-chain gene on chromosome 14, resulting
in upregulation of the Bcl-1 gene and overexpression of cyclin D1. Cyclin D1 protein expression is
universal in MCL and can be detected by immunohistochemical staining, polymerase chain reaction
(PCR) analysis, or flow cytometry. Translocation (11;14) is detected in 65% of MCL cases by classic
cytogenetic analysis, and in nearly all cases, by fluorescent in situ hybridization (FISH). Cyclin D overexpression is also seen in breast, oesophageal, bladder and lung cancer.
Figure 8. The t(11;14)translocation is characteristic of MCL
5. Virus associated tumours
The association of HPV serotypes16 and 18 with carcinoma of the cervix is well known. HPV 16 and 18
produce the gene products E6 and E7 respectively. E6 binds to and inactivates P53 whilst E7 causes
the proteasomal degradation of Rb protein. This releases the cell form cell cycle regulation and is critical
in the development of Ca cervix.
HHV8 – the causative agent in Kaposi’s sarcoma - produces a viral cyclin similar to CyclinD1. This binds
to CDK6 and forms a complex that is resistant to p16INK4a and P21. This results in unchecked
progression through the restriction point and unrestricted cell proliferation.
Therapeutic Targets
A better understanding of the cell cycle and the recognition that Cyclin Dependant Kinases, their
regulators and their substrates are targets of genetic alteration in different types of human cancer, has
lead to a dramatic change in strategy for cancer drug development. The focus has turned from drugs
that are intended to kill tumour cells, towards a more mechanistic approach that is aimed at molecular
targets that regulate the cell cycle and cell proliferation.
There are two strategies for modulating CDK activity – a direct strategy in which the catalytic activity of
the CDKs is directly inhibited, and an indirect strategy in which the major regulators of the CDKs are
targeted.
Indirect Strategies for Modulating CDK Activity in Cancer Therapeutics
Approaches include over expression of CKD inhibitors(CKIs), the synthesis of peptides that mimick the
effects of CKIs , decrease of cyclin levels and modulation of the phosphorylated state of CDKs.
Direct Strategies for Modulating CDK Activity
Direct inhibition of CDK activity has been a more successful strategy for the development of cell cycle
inhibitors. The main mechanism of action is by competitive inhibition of ATP at its binding site to CDK.
ATP occupies a small, defined pocket on the CDK and the potential to disrupt this is much greater than
disrupting a large protein-protein interface such as the cyclin-CDK binding surface. A number of
inhibitors have been described. The most studied include:
1.
Purine analogues
Olomoucine is highly a CDK specific inhibitor and has been found to inhibit cell proliferation
and induce apoptosis in tumour cells.
Roscovitine, also a purine analogue has been shown to be a potent inhibitor of CDK1 and has
been shown to have anti-proliferative effects in vitro, in leukaemic cell lines, primary myeloid
cells and on lymohocytes.
2.
Butyrolactone
Butyrolactone is isolated from aspergillus strain F-25799 and has been found to inhibit CDK1
and CDK2. It has been shown to inhibit the phosphorylation of Rb protein and inhibits G1/S and
G2/M transition as well as DNA synthesis in human fibroblasts. It has also been proven to
inhibit in vitro proliferation of lung, colon and pancreatic cell lines.
3.
Flavanoids
Flavopiridol was first discovered as a potent inhibitor of several breast and lung cancer cell
lines and has since been shown to inhibit a broad range of human tumours, lymphomas and
leukaemias.
The Implication of Apoptosis in Malignancy
Apoptosis vs Necrosis
Cells die by two main mechanisms – Necrosis and Apoptosis. Necrosis generally follows a major
pathological event or injury such as hypothermia, hypoxia, ischaemia, viral invasion, toxin exposure or
complement attack. With necrosis, mitochondrial swelling, dysfunction of the plasma membrane, cell
swelling and cell rupture is seen. The resultant release of cellular contents, including proteases and
lysozymes, causes local macrophage cytokine release and an inflammatory reaction.
Mitochondrial swelling and failure
Dysfunction of the plasma membrane
and cell swelling
Cell rupture, release of cell contents ,local
inflammatory reaction
Figure 9. The morphology of necrosis
Apoptosis on the other hand is the major physiological mechanism for cell removal. Eloquently named –
apoptosis is a descriptive Greek term for falling leaves or petals. This is a “silent” mechanism for
removing cells are damaged, are no longer needed, or that have reached the end of their lifespan. This
is an important defence against tumorigenesis, as cells that contain DNA damage that is beyond repair
are removed from the cell cycle.
Cells shrink and lose contact with
neighbouring cells
Chromatin condenses and marginates
at the nuclear membrane
Blebbing/budding of plasma
membrane
Fragmentation into apoptotic bodies
Figure 10. The morphology of apoptosis
In cells undergoing apoptosis there is a condensation of cytoplasm and nuclear membranes and
subsequently an aggregation of nuclear chromatin. Mitochondria retain their structure and part of their
function. There is disruption of the cytoskeleton, the cell shrinks and then fragments into a clusters of
membrane enclosed apoptotic vesicles that are rapidly ingested by surrounding macrophages. The
apoptotic bodies cause no cytokine release and no inflammatory response. Apoptosis may be set into
motion by genes responding to DNA damage, death signals from the cell membrane surface and
proteolytic enzymes entering the cell.
Physiological Importance of Apoptosis
Apoptosis in Development and Morphogenesis
During limb formation the death of inter-digital mesechymal tissue results in the formation of separate
digits of the hands and feet
Apoptosis is important in the formation and sculpturing of hollow structures in the body
Apoptosis is of critical importance in the genesis of the male and female genital tract. The Mullerian duct
develops into the female reproductive organs. Apoptosis results in the dissapearance of the mullerian
duct in the male. Conversely, the Wolffian duct will develop into the make sex organs, where as lack of
exposure to testosterone will result in apoptosis and regression of Wolffian duct in females.
Apoptosis in the Haematological System
Apoptosis plays an important role in the regulation of the red blood cell population. The maintenance of
erythropoetic stem cells relies on the presence of EPO. When erythropoeitin is withdrawn theses cells
undergo apoptosis.
Apoptosis in the Immune System
Apoptosis plays a role in the deletion of neutrophils and T cells at the end of an immune
response,resulting in the removal of the effectors of inflammation once they are no longer required.
Apoptosis in the Prevention of Malignancy
Apoptosis results in the removal of cells with severely damaged as we will discuss below.
Regulation of Apoptosis:
There are two major apoptosis signalling pathways, The cytotoxic pathway and the genotoxic pathway.
The genotoxic pathway is triggered by DNA damage and regulated by P53. The cyctotoxic pathway
consists of a secretory pathway as well as a death receptor pathway.
The Genotoxic/Mitochondrial Pathway
As we saw above, DNA damage activates a cascade beginning with the DNA binding transcription factor
- P53. This either induces cell cycle arrest, allowing time for genomic repair in the manner explained
above, or in the case of severe damage, guides the cell into apoptosis. The major downstream
regulators of this genotoxic induced apoptotic signal is the BCl2/Bax family of proteins. There are 16
members of this family identified so far. The pro-apoptotic members a.k.a. apoptosis promoters, are
Bax, Bad and Bid, whilst BCl2 and BClX are anti-apoptotic. The BCl2 protein members are found at the
outer mitochondrial membrane and govern the transition pores within the membrane. The Bax
monomers are found in the cytosol of the cell.
Figure 11. Anti-apoptotic Bcl-2 members govern transition pores of the outer mitochondrial
envelope. Pro-apoptotic Bax members are found in the cyctoplasm
Areas of commonality of structure enable these proteins to homo and heretrodimerize to form Bax/Bax,
Bax/BCl2 and BCl2/BCl2 dimers. It is likely that P53 regulates the ratios of these dimers in favour of the
pro-apoptotic Bax family.
Upon receipt of the apoptotic signal form P53 the Bax members form dimers and oligomers that bind to
the mitochondrial membrane permeability transition pore. This alters the permeability of the
mitochondrial membrane. As a result the contents of the intermembranous space of the mitochondria is
released into the cytosol – including Cytochrome C and Apoptosis Inducing Factor (AIF).
Figure 12. The mitochondrial pathway for apoptosis
AIF moves to the nucleus where it causes chromosomal condensation and nuclear fragmentation.
Cytochrome C acts in the cytosol where it combinds with Apaf1 (Apoptotic protease activating factor-1)
and procaspase 9 which, in the presence of ATP, forms a complex called the apoptosome. Formation of
the apoptosome results in the activation of procaspase 9. Caspase 9 in turn activates down stream
caspases, including procaspase 3, which results in the cytological changes characteristic of apoptosis.
The caspases are proteases that are present in the cytoplasm of the cell in an inactive from –
procaspases. When activated they in turn activate downstream caspases in a sequential cascade
reminiscent of the completement system. Activity of the caspases results in cleavage of cytoskeletal
proteins, disruption of the nuclear membrane, disruption of cell-cell contact and freeing of DNA
nucleases that are responsible for DNA fragmentation.
A family of caspase inhibitors called IAPs (Inhibitors of apoptosis) inhibit the effector caspases blocking
the apoptotic process. Smac/DIABLO is a mitochondrial protein that binds to and inactivates IAPs.
Implication for Malignancy
Inhibitors of Apoptosis(IAP)
One can imagine that IAP proteins may have a role in oncogenesis by their ability to inhibit apoptosis.
Strong evidence for this theory is seen with the identification of the IAP Survivin. Survivn is not found in
adult differentiated tissue yet is present in most transformed cell lines and cancers. Survivin has been
shown to inhibit caspase 3 directly and apoptosis in general. Survivin protein levels have been found to
correlate inversely with five year survival rates in colorectal cancer. Similarly high levels of survivin were
found to predict early relapse in children with pre B ALL.
Targets for Intervention:
A number of small molecules that bind to the Smac/DIABLO binding site on various IAPs, thus
mimicking the inhibitory effect of smac/DIABLO on the IAP and removing its antiapoptotic effect, have
been investigated. This seems to be a promising target to either directly induce apoptosis in tumour cells
or to sensitise malignant cells to apoptosis induced by cytotoxic agents or radiotherapy.
Figure 13. smac/DIABLO mimetics and IAP antagonists
remove the anti-apoptotic effect of the IAP proteins and are
promising targets to restore apoptosis in cancer.
BCl2/Bax
Over expression of Bcl 2 or under expression of Bax also inhibits apoptosis and therefore plays a role in
tumourigenesis. Overexppression of BCl2 occurs with the translocation of the BCl2 gene from
chromosome 18 into the Ig locus on chromosome 14. This is associated with low-grade follicular non
Hodgkins lymphoma. Further, the Bcl-2 gene has been implicated in a number of cancers including
melanoma, breast, prostate and lung carcinoma. There is also evidence to suggest that a high
expression of anti-apoptotic BCl2 family member proteins is associated with resistance to chemo and
radiotherapy by blocking the mitochondrial pathway of apoptosis. This is particularly true of NHL and
CLL – where high levels of BCl2 confer drug resistance.
Targets for Intervention
An attempt to target the protein-protein interaction site between anti-apoptotic Bcl2 and proapoptotic
Bax, has resulted in the generation of a small molecule antagonist ABT-737. This prevents the binding
of anti-apoptotic BCl2 to Bax, freeing Bax to engange in the mitochondrial pathway of apoptosis. This
has been reported to directly trigger apoptosis especially in malignancies that depend on anti-apoptotic
BCl2 proteins for survival.
Oral forms of BCl2 family inhibitors (ABT 263) have recently entered into clinical trials and have shown
promising results in paediatric and adult lymphoid malignancies.
Obatoclax is another small molecule that has been reported to antagonize members of the antiapoptotic BCl2 family. It has demonstrated antileukaemic activity against several adult haematological
maliganancies including AML, CLL, mantle cell lymphoma and multiple myeloma.
Figure 14. Obatoclax is one of the small molecules that bind to and inhibit BCl 2 – thus
inducing apoptosis
The Cytotoxic/Death Receptor Pathway
The death receptor pathway is mediated by cell surface receptors of the TNF receptor superfamily.
These include CD95(Fas) and TNFR-1.
Fas- Fas Ligand
Fas/CD95 is a cell surface receptor found on the surface of a number of somatic and tumour cells. Fas
ligand is expressed on the surface of cytotoxic T lymphocytes as well as NK cells. Binding of Fas ligand
to Fas results in the interaction of the cytoplasmic tail of Fas which contains a death domain, with FADD
which is Fas associated protein with Death Domain
TNF-TNFReceptor
Similarly TNF binds to the TNF receptor. The cytoplasmic tail of the TNF receptor contains a death
domain and interacts with a cytoplasmic adaptor protein - TRADD (TNF receptor associated Death
domain protein)
Figure 15. The death receptor pathway
Subsequent to the binding of their respective ligands, both the interaction of the TNF receptor with
TRADD and the Fas receptor with FADD results in the activation of procaspase 8.Caspase 8
subsequently activates downstream effector caspases including procaspase3 – resulting in the
cytological changes characteristic of apoptosis. A second ligand – TRAIL (TNF related apoptosis
inducing ligand) also signals via FADD to activate procaspase 8.
Implications for malignancy
Mutations of CD95/Fas receptor have been implicated in childhood T-ALL and Hodgkins Lymphoma.
Targets for Intervention
Among the death receptor ligands TRAIL is a prime target for apoptosis inducing therapies. TRAIL
predominantly kills tumour cells whilst sparing normal cells. The basis for this tumour selectivity is not
yet fully understood. Several strategies have been investigated to target TRAIL receptors therapeutically
using either agonistic monoclonal antibodies to TRAIL receptors or recombinand soluable TRAIL. These
agents have entered into early clinical trials and little data regarding in vivo efficacy is available to date.
Methods of Disruption of Apoptosis and Cell
Cycle Regulation
It is clear from the above that disruption of cell cycle regulation and of apoptosis plays a major role in the
development of cancer. A number of mechanisms may contribute to the disruption of these normally
ordered processes.
Oncogenes and Cancer
Proto-oncogenes are normal genes that encode for various proteins that are involved in the regulation of
cell growth, proliferation and differentiation. Proto-oncogenes may be activated to form oncogenes.
Oncogenes are abnormal genes that encode for various products that are associated with cellular
transformation and the uncontrolled proliferation that is characteristic of cancer cells.
Methods of activation of oncogenes
1. Mutations
When an oncogene is activated by mutation, the structure of the protein for which it encodes is changed
and this can cause an increase in protein/enzymatic activity. An example of this is the Ras oncogene.
Ras sub family is a protein sub family of small GTPases that are involved in cellular signal transduction
– transmitting signals from the cellular membrane to the nucleus. Ras is activated by various growth
factors such as endothelial derived growth factor (EGF) and transforming growth factor alpha
(TGFalpha). Ras activates several intracellular pathways, of which the mitogen-activated protein (MAP)
kinase cascade has been the most studied. This cascade transmits signals downstream and results in
the transcription of genes involved in cell growth and proliferation Mutations in Ras oncogene can
permanently activate Ras inducing downstream signal transduction even in the absence of extracellular
growth factor stimuli. This results in disordered cell replication and inappropriate cell growth during G1
phase of the cell cycle. Ras mutations are found in adenocarcinomas of the pancreas, the colon and the
lung, as well as in thyroid tumors and in myeloid leukaemia.
2. Gene Amplification
The second mechanism of activation of oncogenes is via gene amplification. An example of this is the
t(11;14) translocation which, as mentioned earlier, juxtaposed the Bcl-1 gene (encoding for cyclin D1) on
chromosome 11 and the immunoglobulin heavy chain enhancer elements on chromosome 14. This
results in the amplification of Cyclin D1. This causes unchecked cell proliferation and is characteristic of
mantle cell lymphoma. Bcl-1 gene amplification is also seen with breast , oesophageal and
hepatocellular carcinoma.
3. Chromosomal Rearrangements
The third mechanism of activation of oncogenes involves chromosomal re-arrangements which may
include inversions or translocations. A well studied example of this is the activation of the myc oncogene
that is seen in Burkitts lymphoma. The myc gene encodes for a transcription factor that binds to the
DNA of other genes and regulates their transcription. In burkitts lymphoma the myc gene on
chromosome 8, is juxtaposed to immunoglobulin genes that are transcriptionally highly active in B cells t(8;14) translocation. This causes myc to be persistently expressed. Myc drives cell proliferation by up-
regulating the cyclins and down-regulating P21. Myc also regulates apoptosis by down-regulating Bcl-2
expression.
Conclusion
I think you will agree that we have come a long way since the Ancient Egyptians in our understanding of
the biology of cancer. We recognise the critical importance of cell cycle regulation and apoptosis in
guarding against cancer. Likewise we realize the implication of a de-regulated cell cycle or impaired
apoptosis in tumourigenesis. This puts us in an exciting era of cancer drug development. The new
paradigm is to develop agents that target the precise molecular pathology driving the progression of
cancer – that is increased cell proliferation and decreased cell death. I think the future of cancer
therapeutics will be interesting.
.
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