Cell cycle as a therapeutic target

Question 1

What are cell cycle check points?

Answer 1

For a multicellular organism genetic or chromosome damage can interfere with tissue organisation, embryonic development and the development of tumours as a result human cells have a variety of mechanisms to check for potential genetic chromosomal damage and to eliminate them either by cell death or irreversible cell cycle arrest so that they do not become a problem

Question 2

What is the G0 phase of the cell cycle?

Answer 2

This consists of either quiescent and senescent cells some cells will enter this state of quiescence and can reinitiate growth and division in the presence of growth factors
Upon DNA damage cells that enter the G0 phase are senescent
Some cells however will never enter this phase and continue to divide throughout the organisms life

Question 3

What are post mitotic cells?

Answer 3

This refers to the Non-proliferative cells in multicellular eukaryote such as neurons generally escape the cell cycle forever

Question 4

What is the Gap 1 (G1) phase?

Answer 4

Synthesize mRNA and protein that are required for DNA synthesis
Cells grow in size, increase number of organelles and increase its supply of protein
The duration of this phase is the highly variable and it is at this phase the cell will determine whether it will commit to cell division or leave the cell cycle
It is also the period during which cells respond to external signals like growth factors

Question 5

What is the role of cyclins and CDK in the cell cycle?

Answer 5

These act as a molecular valve preventing the repetition of phases of the cell cycle
Cyclin D is the first cyclin to increase in concentration in the cell and this a result of exogenous signals or environment while all other components of this system

Question 6

How are cyclin dependent kinase regulated?

Answer 6

The level of phosphorylation on particular residues may control their activity
Stimulatory phosphorylation is carried out by CDK-activating-kinase
Inhibitory phosphorylations are removed by a class of phosphatases CDC25A,B and C

Question 7

What is the Synthetic (S) phase?

Answer 7

This follows the G1 phase
DNA elongation and termination occurs in this stage
Usually this stage lasts for about 6-8 hours but is cell dependent
At the end of this stage the cell consists of two sets of chromosomes called chromatids

Question 8

What regulates the G1 phase checkpoint?

Answer 8

Cyclin D binds to cdk4/6 (this may be inhibited by p15,16,18 or 19) if not inhibited this will go on to phosphorylate Rb leading to release of E2F
E2F transcription factors then activate MCM, Cyclin E, Geminin and Cdt1 leading to S phase entry

Question 9

How does the initiation of DNA replication form?

Answer 9

There is a pre-replication complex made of ORC1-6 bound to CDC6 which is associated with DNA
Cdt1 then complexes with MCMs which allows it to act on the pre-replication complex to activate DNA transcription
This is able to occur because cyclin E activates cdk2 which (if not inhibited byp27) will lead to activation of cdc25a phosphatase which allows association of the above mentioned molecules

Question 10

How does the initiation complex transition to the replisome?

Answer 10

ORC1-6 dissociates from the complex via phosphorylation and MCM will bind cdc45which will in turn associate with replication factors

Question 11

What is the S phase checkpoint?

Answer 11

The replication fork stalls and is then bound to by ATR kinase which is associated with replication protein A this will then activate either DNA repair, p53 leading to inhibition of cyclinA/cdk2 resulting in cell cycle arrest and activation of bax to cause apoptosis, ATR can also activate CHK21 kinase which activates CDC25a phosphatase which inhibits cdk2 and cyclin A but leads to cell cycle progression

Question 12

How is cyclin A degraded?

Answer 12

At the end of S phase cyclin A becomes phosphorylated
This then becomes a substrate for the SCF ubiquitin ligase which adds a string of ubiquitin units to cyclin A
This results in proteasomal degradation
This with other mechanisms prevents re-initiation of previously replicated DNA by cdk2

Question 13

What is the G2 phase of the cell cycle?

Answer 13

This where it is determined if the DNA has been damaged since the last divison
Provides an opportunity for DNA repair of damaged cells
The G2 checkpoint helps us to maintain genetic stability making it an important focus in understanding the molecular causes of cancer

Question 14

How does the G2 phase checkpoint occur?

Answer 14

MRE11, NBS1 and RAD50 all associate with the DNA this complex then associates with ATM kinase
This will then activate DNA repair, chromatin changes through histone H2AX phosphorylation and activation of CHK2 kinase
CHK2 kinase may trigger apoptosis (either directly or through p53 activation of Bax) or it may activate cdc25C phosphatase which inihibts cdk1/cyclinB leading to G2 phase arrest
Alternatively p53 may activate p21 which also inhibits cdk1/cycB leading to G2 phase arrest

Question 15

What is Mitosis phase of the cell cycle?

Answer 15

This is where nuclear and cellular division occurs
This phase is complex and tightly regulated
Mitosis is the process through which the cells separate the chromosomes into two nuclei and is followed by cytokinesis which divides the nuclei, cytoplasms, organelles and cell membrane into two cells

Question 16

What cyclin/cdk complex drives mitosis?

Answer 16

Activation of cdk1 by cyclin B followed by activation of this complex by CAK kinases, CdK phosphatase and cdc25c phosphatase
This only occurs if DNA replication is complete

Question 17

What are the functions of cdk1/cycB?

Answer 17

This phosphorylates transcription machinery to turn it off
Phosphorylates lamin in endoplasmic recticulum membrane homogenisation
Phosphorylates tubulin associated proteins allowing formation of the metaphase plate
Activates the anaphase promoting complex
Phosphorylates histones H1/H3
Phosphorylates condensing leading to chromatin condensation

Question 18

What is anaphase promoting complex?

Answer 18

This is APCcdc20 and is a ubiquitin ligase activated during metaphase it activates seperase to break down cohesion allowing cell division
It degrades cyclin B to rest the cell cycle

Question 19

What is the mitotic checkpoint?

Answer 19

Unpaired DNA leads to kinetophores lacking microtubule attachement
Mad2 brings phosphorylated cdc20 protein going to a closed form
The complex inhibits APC ubiquitin kinase and seperase necessary for chromatic separation (BubR1 and Bub3 are also involved in this process)
This leads to the cells arrest in metaphase

Question 20

What is the post mitotic checkpoint?

Answer 20

Some cells escape the mitotic checkpoint but still have DNA or chromosomal damage
This damage is recognized by complex mechanisms leading to failure of cell division known as mitotic slippage
These cells will then have a G1 phase with twice the genetic content where they will be arrested by the G1 phase checkpoint mechanism

Question 21

What are the different roles of each of the checkpoints?

Answer 21

The G1 phase checkpoint or restriction point stops entry into the cell cycle and is controlled externally by the microenvironment
The DNA double strand break checkpoint this can occur at G1, S, G2 phases and is controlled mainly by ATM, p53 and CHK2 pathways
The mitotic checkpoint which is activated by the failure of attachment of the chromatids to the mitotic complex
The post-mitotic checkpoint which is activated by defects in the steps between mitosis and cell division

Question 22

How are the cell cycle checkpoints deregulated in cancers?

Answer 22

Alteration of pRb can occur via inactivating mutations, promoter methylation or sequestering by viral E7 protein
Alterations of cyclins can occur which cycD being over expressed due to gene amplification, promoter hyperactivity or reduced degradation, cycD3 may be overexpressed along with cycE
Alteration of cdks with structural mutatiosn of cdk4
Alteration of cdk inhibitors with deletion of the INK genes/promoter methylation, decreased transcription of the Kip genes or increased degradation

Question 23

How is apoptosis inducted?

Answer 23

This is mediated by the activation of a protease mainly capsase 3 which causes:
Cleavage of key proteins
Condensation of chromatin
Cleavage of a control protein linked to DAN endonuclease allowing internucleosomal breakage of DNA leading to apoptotic ladders
Cleavage of gelsolin which binds to actin filaments causing blebbing of cytoplasm
Polymerisation of cytoplasmic proteins leading to cytoplasmic shrinkage
Fragmentation of the nucleus and disintegration of the cell into apoptotic bodies

Question 24

What is the intrinsic control of the cell for the transition to apoptosis?

Answer 24

The signal may be both intrinsic or extrinsic with the intrinsic mechanism thought to involve a permeability transition in the mitochondrial membrane which releases cytochrome C which activates caspases 9 and 3

Question 25

How can DNA damage induce apoptosis?

Answer 25

The bcl2 family is made up of a group of related proteins which bind to the outer mitochondrial membrane and regulate its permeability, proapoptotic members (Bad and Bax) increase the chance of a permeability transition while anti-apoptotic members (bcl2, bcl-Xl) decrease it
Tumour cells often shown intrinsic DNA damage capable of inducing p53, p38 (stress kinase) and other proteins capable of activating proapoptic members
This causes membrane permeability which triggers apoptosis

Question 26

How might mitotic damage lead to apoptosis?

Answer 26

Damage to the mitotic apparatus can lead to mitotic arrest and phosphorylation of bcl2
Phosphorylated bcl-2 is then removed via proteolysis
Bcl 2 will stabalise the mitochondrial outer membrane leading to increased activity of proapoptotic members which tips the balance in favour of apoptosis

Question 27

What is the extrinsic control of transition to apoptosis?

Answer 27

Death receptors such as the Fas receptor are located on the plasma membrane
FasL is located on the surface of both tumour and host cells
Fas receptor stimulation leads to FADD mediated activation of procaspase 8
This activates the caspase cascade to apoptosis

Question 28

What are the drugs that target the mitotic checkpoint of the cell cycle?

Answer 28

These typically try to induce a spindle defect and as a result antimicrotubule agaents are used including
Vinca alkaloids like
Vincristine (Acute Lymphocytic Leukaemia and Nephroblastoma)
Vinblastine (Hodgkin lymphoma)
And Taxanes like
Paclitaxel (lung, ovarian, breast and head and neck cancer)
Docetaxel (prostate cancer)

Question 29

What are the drugs which target the G2 checkpoint?

Answer 29

These target the Chromosome catenation checkpoint and typically use topoisomerase II inhibitors such as
Etopside (Sarcomas, lymphomas, lungs, testicular, leukaemia and glioblastoma multifrome)
Teniposide in childhood acute lymphocytic leukaemia

Question 30

What drugs work in the S phase of the cell cycle?

Answer 30

These aim to produce replication faults by causing DNA damage and typically use anti-metabolites such as
5-flurouracil (colon cancer)
Gemcitabine (Pancreatic, breast, blaader, ovarian and NSCLC)
Fludarabine (Chronic lymphocytic and B cell leukaemia)
6-Mercaptopurine (acute lymphocytic leukaemia)

Question 31

What drugs target the G1 phase of the cell cycle?

Answer 31

These drugs are typically alkylating agents which induce DNA cross-linking and apoptosis this includes
Cyclophosphamide (lymphomas, leukaemias and solid tumours)
Cisplatin and derivatives (sarcoma, solid tumours)
Dacarbazine (melanoma and lymphoma)

Question 32

What is the effect of cytotoxic therapy on normal tissues?

Answer 32

Most normal tissues only have a small proportion of cycling cells (the exceptions being the skin/hair follicles, Gut epithelium and bone marrow)
Tumours contain high proportions of cycling cells even if the rate of proliferation is not always high allowing a selective effect of anticancer therapies whose selectivity is based off the cell cycle
These agents will however also induce side effects based on damage to rapidly dividing normal cells in the skin hair follicles, gut epithelium and bone marrow