cyclin-dependant kinases (CDKs)

Question 1

Role of cyclin-dependant kinases (CDKs)

Answer 1

In order to drive the cell cycle forward, a cyclin must activate or inactivate many target proteins inside of the cell. Cyclins drive the events of the cell cycle by partnering with a family of enzymes called the cyclin-dependent kinases (Cdks). A lone Cdk is inactive, but the binding of a cyclin activates it, making it a functional enzyme and allowing it to modify target proteins.

Question 2

Cdk1 is the only on required to drive through the cell stages…

Answer 2

Cyclin B1 and A2 are essential for normal cell cycle.
•At G1 phase: CDK 4 & CDK 6 binds to Cyclin D – Cyclin D levels are influenced by extracellular signals (mitogens, growth factors and survival factors). The main target of CDK 4 & 6 is Retinoblastoma protein (Rb). Dysfunction of the Rb protein (stuck in on position) may lead to uncontrolled proliferation, where mutations are likely.
•G1/S phase: CDK 2 binds to Cyclin E
•S phase: CDK 2 binds to Cyclin A
•M/ G2 phase: CDK 1 (aka Cdc25) binds to Cyclin B1

Question 3

CDKs are activated…

Answer 3

•CDKs are activated by activating phosphorylation of threonine around position 160.
•CDK-inhibitors are proteins that bind to CDK-cyclin complexes and block their activity.
oWee1 kinase inhibits phosphorylation of threonine 14 & tyrosine 15 on CDK 1, inhibiting cell cycle progression
oCDK 1 phosphatase reverses the change of Wee1, leading to active CDK.

Question 4

Cell cycle checkpoints

Answer 4

The availability of cyclins controls the activity of CDKs and promotes cell progression. CDKs become active via cyclin-binding; when complexed with M-phase cyclin, mitosis machinery is triggered, when complexed to S-phase cyclin, DNA replication is triggered.
- There are also several checkpoints which ensure complete genomes are transmitted to daughter cells, and cells with damaged DNA do not replicate.

Question 5

At G1

Answer 5

cell size, growth factors & DNA damage are checked. If the cell doesn’t meet the requirements, it will leave the cell cycle and enter a resting state – G0

Question 6

G0

Answer 6

o DNA damage causes p53 levels to increase, causing transcription of p21
o p21 binds to PNCA – a component of DNA replication machinery – preventing its activity
o By ensuring that cells don’t divide when their DNA is damaged, p53 prevents mutations; when p53 is defective or missing, mutations can accumulate quickly, potentially leading to cancer. Indeed, out of all the entire human genome
o p53 is the single gene most often mutated in cancers.

Question 7

At G2

Answer 7

un-replicated or damaged DNA are checked. If errors or damage are detected, the cell will pause to allow for repairs. Topoisomerase II is responsible for repairing DNA at this stage. If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the damaged DNA. If the damage is irreparable, the cell may undergo apoptosis, or programmed cell death

o A signal is sent to a series of protein kinases, which phosphorylate and inactivate Cdc25
o The dephosphorylation of M-Cdk is blocked, and it does not activate, preventing entry to mitotic phase

Question 8

At M (aka spindle checkpoint)

Answer 8

chromosomal misalignment (i.e. the chromosome is not attached to the spindle) will stop the cycle. If a chromosome is misplaced, the cell will pause mitosis, allowing time for the spindle to capture the stray chromosome

Question 9

PI3K signalling

Answer 9

PI3K signalling is a form of endocrine signalling. The pituitary releases growth hormone, which acts on tyrosine kinase receptors. A kinase is an enzyme that transfers phosphate groups to a protein or other target, and a receptor tyrosine kinase transfers phosphate groups specifically to the amino acid tyrosine, activating it. The signal relay pathway through PI3K, PTEN, AKT, mTOR and S6K1 or 4EBP results in gene expression & cell proliferation.

The PI3K/AKT/mTOR pathway is an intracellular signalling pathway important in regulating the cell cycle. In many cancers, this pathway is overactive, thus reducing apoptosis and allowing proliferation. In many kinds of breast cancer, aberrations in the PI3K/AKT/mTOR pathway are the most common genomic abnormalities (e.g. PIK3CA gene mutation

Question 10

HER-2 signalling

Answer 10

HER-2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. The HER-2 receptor is a transmembrane tyrosine kinase receptor that consists of an extracellular ligand-binding domain, a transmembrane region, and an intracellular or cytoplasmic tyrosine kinase domain.
It is activated by the formation of homodimers or heterodimers with other epidermal growth factor (EGFR) proteins.
Further downstream molecular signalling cascades are activated, such as the Ras/Raf/mitogen-activated protein kinase (MAPK), the phosphoinositide 3-kinase/Akt, and the phospholipase Cγ (PLCγ)/protein kinase C (PKC) pathways that promote cell growth and survival and cell cycle progression.

Question 11

MAPK pathway:

Answer 11

When growth factor ligands bind to their receptors, the receptors pair up and act as kinases. The activated receptors trigger a series of events:
• Ras is activated
o KRAS ( K-ras or Ki-ras) is a gene that acts as an on/off switch in cell signalling. When it functions normally, it controls cell proliferation. When it is mutated, negative signalling is disrupted.
• Guanine nucleotide exchange factors (GEFs) are recruited
• GEF becomes capable of interacting with Ras proteins at the cell membrane to promote a conformational change and the exchange of GDP for GTP.
•Raf is recruited to the cell membrane, and activation stimulates a signalling cascade by phosphorylation of MAPK which successively phosphorylate and activate downstream proteins such as ERK1 and ERK2
•Transcription factors are activated promoting cell growth and division.

Question 12

Over-expression of this oncogene…

Answer 12

Over-expression of this oncogene has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. The protein has become an important biomarker and target of therapy for approximately 30% of breast cancer patients. HER2-positive breast cancer has a faster growth rate than HER2-negative.

Question 13

Pathology

Answer 13

488 (1%) genes are implicated in the development of cancer. Of these, 90% have somatic mutations; 20% have germline mutations (10% have both). Somatic mutations occur in a single cell and are not passed to offspring; germline mutations occur in gametes and may be inherited by the offspring of that cell, affecting many different types of cell.

Cancer cells behave differently than normal cells in the body, related to cell-division. Cancer cells may multiply without any growth factors, or growth-stimulating protein signals.

Question 14

Mutation

Answer 14

Mutations stimulate cell survival and proliferation, and may be:
•Dominant (gain of function) – a single mutation event creates oncogenes - overactive positive cell cycle regulators.
•Recessive (loss of function) – two, or more, mutations eliminate tumour suppression activity - inactive negative regulators.

Question 15

Oncogenes drive abnormal cell proliferation:

Answer 15

They may represent the overactive form of normal cellular genes, called proto-oncogenes or alternatively, they may enter the cell as part of a virus. A proto¬-oncogene may become overactive and be converted into an oncogene due to mutation in coding sequence, gene amplification or chromosomal rearrangement.

Question 16

Tumour suppressors are genes that normally inhibit cell proliferation and tumour development

Answer 16

. In tumour development, tumour suppressors are often lost or inactivated, usually requiring 2 mutational events; inactivation can occur through deletion, point mutation or epigenetic changes (where the gene does not mutate but is inactivated). This may occur due to nondisjunction, giving rise to unequal mitosis; random elimination of a chromosome results in recessive mutation of tumour suppressor.

Question 17

There are 3 types of tumour suppressor:

Answer 17

•Caretakers – promote gene stability & control mutation rate
o Checkpoint genes (p53)
o DNA repair genes (BRCA)
•Gatekeepers – monitor cell division & death
o Cell cycle (Rb)
•Landscapers – control cellular microenvironment

Question 18

Stress hormones

Answer 18

During and after diagnosis and treatment, almost 50% of cancer patients report anxiety and 25% report significant anxiety; 20% experience transient or long-term depression; and 15% are diagnosed with post-traumatic stress disorder.

Question 19

Stress results in the production of the stress response hormones:

Answer 19

Stress results in the production of the stress response hormones: cortisol, (Cort), norepinephrine (NE), and epinephrine (E) at physiological concentrations. Stress hormones can rapidly induce DNA damage and interfere with the DNA-damage repair process in pre-cancerous cells leading to cell transformation and tumorigenicity. They increase levels proteins involved in cell cycle regulation and progression and reduce levels of proteins that cause apoptosis (cell death).

Question 20

Tumour development: e.g. bowel cancer

Answer 20

A cell might first lose activity of a cell cycle inhibitor, an event that would make the cell’s descendants divide a little more rapidly. It’s unlikely that they would be cancerous, but they might form a benign tumour a mass of cells that divides too much. Over time, a mutation might take place in one of the descendant cells, causing increased activity of a positive cell cycle regulator. The mutation might not cause cancer by itself either, but the offspring of this cell would divide even faster, creating a larger pool of cells in which a third mutation could take place. Eventually, one cell might gain enough mutations to take on the characteristics of a cancer cell and give rise to a malignant tumour, a group of cells that divide excessively and can invade other tissues.

Question 21

Mutations:

Answer 21

• PIK3CA mutations most commonly occur concomitantly with loss of adenomatous polyposis coli (APC). APC has many functions, the most prominent is its capacity to regulate beta-catenin-mediated gene transcription in response to Wnt signalling. Unbound ß-catenin binds to Tcf/Lef gene, which transcribes for Cyclin B & D. Loss of APC leads to deregulated beta-catenin and hyperproliferative epithelium.
• Increased genetic instability, and loss of P53 (protein involved in G1 checkpoint preventing damaged DNA from replicating) leads to early adenoma. p53 is the gene most commonly mutated in human cancers, and cancer cells without p53 mutations likely inactivate p53 through other mechanisms
• The Rb protein is implicated in the G1 checkpoint; when Rb is permanently in on mode, G1 phase of cell cycle is continually driven – point of no return.
• HER-2 receptors are upregulated in tumour cells; hyperactivation of this signalling pathway & abnormal cell proliferation is observed
• K-Ras mutation/ activation disrupts negative signalling pathway causing unregulated proliferation into intermediate adenoma (MAPK pathway).