The aim of this lesson is to give a concise, but detailed overview of how the cell cycle is regulated. Most importantly, it outlines the roles of the two most important genes that are known to be key to its regulation: TP53 and Rb. Further, the lesson focuses on the mechanisms implicated in apoptosis – the process of programmed cell death, whose dysregulation is important in the pathogenesis of cancer.

Some hints and tips on how to approach this topic can be found at the end of the lesson.

Two main tumour suppressor genes
P105-Rb retinoblastoma protein
Pocket protein that binds and inactivates the E2Fs transcription factors that drive the expression of cell cycle genes
It provides the classical model for a recessive tumour suppressor gene in that both paternal and maternal copies of the gene must be inactivated for the tumour to develop
This is called the loss of heterozygosity (LOH) and can occur in inherited retinoblastoma  heterozygous Rb alleles exhibit a high frequency of LOH
Mechanisms for exchange of genetic information between the paired homologous chromosomes in Rb heterozygote are mitotic recombination, chromosomal non-dysjunction, gene conversion
Individuals with inherited retinoblastoma are also susceptible to malignant tumours of mesenchymal tissues
Inactive Rb alleles are common in small lung cell carcinoma and occur in non-small cell lung, bladder, breast and pancreatic carcinomas
Cell cycle regulation

Rb normally inhibits E2F TFs required for expression of genes in DNA replication
P53 tumour suppressor (TP53 gene)
Transcription factor that responds to stress/damage/oncogenic signalling and induces growth arrest and cell death
It is also sometimes called the guardian of the genome as it ensures that if there is damage to DNA, the cell is arrested and may be diverted into the apoptotic pathway
Mutations in p53 causing loss of function occur in over 70% of human cancers
The rare, autosomal dominant Li-Fraumeni syndrome arises from p53 mutations inherited through the germ line
50% develop diverse cancers by 30 years of age, compared with 1% rate in the general population
In general, p53 mutations are somatic and occur with high frequency in all types of lung cancer, in over 60% of breast tumours and in about 40% of brain tumours (astrocytomas), frequently in combination with the activation of oncogenes

Mechanism of action:
Acts as a transcription factor for genes that cause cell cycle arrest in G1 or those that cause apoptosis

Unlike in Rb, loss of function of p53 is not always dependent on inactivation of both alleles
Since p53 binds DNA  mutations in the DNA binding region will be enough to stop it from performing its normal tumour suppressing action

active p53 binds DNA as a homo-tetramer

The survival plot shows the importance of p53 in cancer outcome
In addition to DNA mutations, epigenetic events can contribute to tumour development
For example, tumour cell DNA is generally hypomethylated (active chromatin conformation) compared with normal cells that are methylated – apart from hypermethylated regions that are silenced in tumours (often tumour suppressor genes)
Importance of understanding cell proliferation and cell death
Cell division is essential for growth of tissues and organisms -> cells must duplicate their essential components before undergoing cell division to produce identical daughter cells
If this is not controlled properly, tumours may develop -> uncontrolled proliferation of cells is one feature of cancer cells
Therefore, in order to understand cancer pathogenesis, we need to understand how the cycle is initiated and regulated and which components are mutated and dysregulated in cancers
Many essential regulatory components of the cell cycle prove to be good targets for therapy
Cell death is essential for sculpting of tissues and removal of damaged cells
Cancer is also a disease of resistance to cell death
Thus, there needs to be a balance between cell proliferation and cell death
If mitosis proceeded without cell death, an 80yo person would have about 2km2 of skin
Phases of the cell cycle
The purpose of the cell cycle is to produce two genetically identical daughter cells
First, the cell grows and the genetic information replicates via DNA replication mechanisms
Then, chromosome segregate whilst they are preparing for cell division
The last step is cell division into two daughter cells that both share the same genetic information

Cell division is required to produce organs
After fertilisation, most cell differentiate from pluripotent stem cells that come from the inner mass of the blastocyst
After differentiation into their specific cell type, the cells become unipotent

Stages of the cell cycle
M phase (mitotic segregation)
In this stage, the cell undergoes mitosis (nuclear division) and undergoes cytokinesis (cytoplasmic division)
G1 phase
The first gap phase can be divided into an early and a late stage, which is separated by restriction point
Early G1 stage is mitogen-dependent and requires extrinsic growth factors or hormones (mitogens) which provide the stimulatory signal to proceed forward
The R point is kind of point of no return
After this point, the cell is committed to progressing to the next phase
Hyperphosphorylation of Rb by CDK4/cyclin D complex is important in passing through the R point (as hyperphosphorylation does not inhibit e2F)
Proteins called cyclins and cyclin-dependent kinases (CDKs) control progression in the cell cycle by phosphorylation of regulatory proteins
An example is the Rb (retinoblastoma tumour suppressor protein)
Unphosphorylated Rb binds to and inhibits e2F, the activation of which will drive gene transcription and cause progression to late G1
CDK4 binds with cyclin D to phosphorylate Rb which allows progression through the R point
The point of this stage is for the cell to grow in size and to synthesise RNAs and proteins required for DNA synthesis (replication)
S phase
Active replication of chromosomes and centromeres takes place (DNA replication)
G2 phase
Phase that precedes mitosis
Cells increase in size further -> DNA synthesis completion is checked
G0 phase
Phase that non-dividing cells are at; also called quiescence

Alternating and completing S and M phases sequentially is critical to ensure maintenance of ploidy
Gap phases (G1 and G2) separate S and M phase in somatic cell cycles

Dysregulation of the cell cycle in cancer

Hallmarks of cancer cells
Growth signal autonomy
Resistance to inhibitory growth signals
Resistance to apoptosis
Unlimited replicative capacity
These first four relate to cell cycle and cell death deregulation
Sustained angiogenesis
Tissue invasion and metastasis
Apoptosis
One of the most noticeable features of apoptosis is condensation of the nucleus and its fragmentation into smaller pieces, a highly distinctive event that is not seen under any other circumstances
Nuclear DNA is hydrolysed into numerous fragments, often in mutliples of 200bp
The Golgi, ER and mitochondrial networks also undergo pronounced fragmentation during apoptosis, and numerous proteins are released from the mitochondrial intermembrane space
Mainly, cytochrome c
The release of cytochrome c drives the assembly of a caspase-activating complex that contains caspase 9 and Apaf-1 (called the apoptosome) on release into the cytosol
Hundreds of proteins undergo restricted proteolysis during apoptosis mainly by caspases
Thus, in mammals, the apoptotic signalling was found to be:

Dysregulation of cell death in cancer
Apoptotic cell clearance
The terminal event of the caspase-mediated demolition phase of apoptosis and removal and consumption of the dead cell by phagocytes
This is a critical step in the pathway as it means that dead cells are removed with their plasma membranes intact
The generation of signals such as ‘eat me’ and binding sites for phagocytes and the release of chemoattractant molecules represent the last acts of the dying cell
Phagocytes are equipped with receptors that specifically detect engulfment signals on the apoptotic cell
Every cell in a multicellular organism has the potential to die by apoptosis
However, tumour cells often have faulty apoptotic pathways
These defects not only increase tumour mass, but also render the tumour resistant to therapy
P53 is a key player in apoptosis induction in tumour cells
P53 is inhibited by MDM2, a ubiquitin ligase, that targets p53 for destruction by the proteasome
MDM2 is inactivated by binding to ARF
Cellular stress, including that induced by chemotherapy or irradiation activates p53 either directly, by inhibition of MDM2, or indirectly by activation of ARF (ARF binds to MDM2 and removes it thus relieves p53 from inhibition)
ARF can also be produced by proliferative oncogenes such as Ras
Active p53 transactivates pro-apoptotic genes such as Bax, Noxa, CD95 and Trail-R1 to promote apoptosis

The Bcl-2 family of proteins
This family of proteins are critical regulators of apoptosis through their ability to control cytochrome c release from mitochondria
The Bcl2 family is divided into three groups based on their BCL-2 homology (BH) domain organisation
Anti-apoptotic Bcl2 proteins
Bcl2 and its close relatives Bcl-XL, MCL1, BCL2a1 and Bcl-W all have BCL-2 homology (BH) domains and all block apoptosis
They do that by preventing BH3-only protein-induced oligomerisation of the pro-apoptotic Bcl2 family members
Pro-apoptotic Bcl2 proteins
These are proteins such as BAX and/or BAK in mitochondrial outer membranes
Their protein-induced oligomerisation would lead to the efflux of cytochrome c
The anti-apoptotic Bcl2 proteins differentially bind to the BH3-only proteins
BID and BIM from the pro-apoptotic BH3-only group interact with all anti-apoptotic Bcl2 proteins, whereas NOXA and PUMA interact with only certain anti-apoptotic members
BH3-only proteins
Comprise 8 members (BID, BAD, BIM, BIK, BMF, NOXA, PUMA and HRK), all of which promote apoptosis when overexpressed
These share only a little sequence homology apart from the BH3 motif and they are regulated in distinct ways
NOXA and PUMA are up-regulated by p53
BID is activated through proteolysis by caspase-8 to generate tBID

Pro-survival (Anti-apoptotic) Bcl2 proteins and pro-apoptotic proteins (such as Bax or Bak) control pore formation
Pore formation and leakage of cytochrome c results in loss of mitochondrial outer membrane potential (MOMP)
Bcl2 proteins are over-expressed in various types of non-Hodgkin’s lymphoma
Apoptosis inhibition via this pathway, or others, is a feature of cancerous cells
Elevated levels of Bcl-2 allow cells to ignore signals to undergo apoptosis
Thus, Bcl2 is an oncogene
The Bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18) found in approximately 90% of human follicular lymphoma cases
The translocation of the Bcl2 gene on the chromosome band 18q21.3 results in consistent expression of the Bcl2 protein
This Bcl2 fusion is an oncogene and plays a crucial role in follicular lymphomagenesis

Hints & Tips on how to study cell cycle and apoptosis

The study of cell cycle can be rather daunting for students, as it appears to involve many genes implicated in its regulation. The key to the cell cycle is to understand the role of cyclins, cyclin-dependent kinases (CDKs) and the different ‘checkpoints’.

Cell cycle can be thought of just like any process. Take for example the manufacturing of a single coke bottle: a highly automated process that has a few ‘checkpoints’ to make sure that the manufacturing line is progressing as expected. This is exactly what happens during the cell cycle; however, this time, the ‘checkpoints’ are biochemical i.e. made up of proteins and enzymes that co-ordinate the process in a complex fashion. For example, the key player in the initiation of the G1 phase is the checkpoint controlled by the Rb protein that keeps the elongation factor E2F in check, thus preventing progression in the cycle. Only when there is enough Cyclin D to bind the kinases CDK4 or CDK6, the kinases become active and hyperphosphorylate Rb, thus removing the inhibition on E2F, allowing the progression of the cycle past this checkpoint. Other signalling molecules control cyclin D, thus providing various levels of cell cycle control.

Indeed, any student who is studying cell cycle should be focussing mainly on these checkpoints: the R point, the G1/S checkpoint and the G2/M checkpoint and know what Cyclins and CDKs are associated with these. Knowing these molecules will enable the student to then recognise what will happen should any of these proteins become dysregulated. For example, it is know clear that if there are mutations in the Rb gene, there will be an inadequate inhibition at the R point and the cell cycle will thus be overactive. In patients who have Rb mutations, retinoblastomas i.e. cancers of the retina, occur.

Every cell has a limit of divisions it can perform – its so-called Hayflick limit. This limit is determined by telomere shortening with every cell division. Once telomere shortens beyond that limit, the cell undergoes programmed cell death – apoptosis in order to prevent the increased risk of any genetic mutations. Apoptosis is therefore a key mechanism that our cells use to control the three-dimensional architecture of tissues. It is therefore not surprising that any dysregulation in apoptosis, or its evasion, may lead to tumour growth. For this reason, it is important to know the key players in this process: the pro- and anti-apoptotic proteins our cells encode to strike the right balance between survival and death, in both cases with the aim of providing a beneficial outcome to tissue architecture. Cell stress therefore induces pro-apoptotic proteins as many mechanisms within the cell become dysregulated, and thus their potential to grow out of control increases. The Bcl-2 family of proteins are the key to apoptosis, and need to be known by any student attempting to understand this process. In order to facilitate the best understanding of this, it is advisable to separate this family into groups of pro- and anti-apoptotic proteins and know how they are linked to those molecules involved in the cell cycle.

Cancer biochemistry is indeed a complex subject and does require memorisation of rather difficult names for one to do well in exams. However, I believe the important message here is to practice layered learning: learn the concepts first and then attach names to outlined processes. My students also frequently benefit from interactive methods, such as online quizzes, where I test their knowledge in simple MCQ, or extended-matching question formats.

Additional resources:

Oxbridge Interview: Biological Sciences
To See Again: The Bionic Eye
Why Study Biology