Mechanisms of disease 1: cell growth and cell differentiation

Question 1

Explain the cell cycle, its checkpoints and its regulation by both mitogenic and growth inhibitory factors

Answer 1

The cell cycle:

The cell cycle is the process of growing and dividing into two identical daughter cells. It consists of several stages, including the G1 phase (gap 1), S phase (synthesis), G2 phase (gap 2), and M phase (mitosis).

1) G1 Phase::

• cell grows and prepares for DNA replication
• If conditions are not favourable for cell division, the cell may enter a quiescent phase known as G0, where it can remain indefinitely until conditions improve

2) S Phase:

• During this phase, the cell synthesises a complete copy of its DNA (i.e., it replicates its genome)

3) G2 Phase:

• cell continues to grow and prepares for mitosis and cytokinesis
• The cell also checks to ensure that all DNA has been accurately replicated and that no damage has occurred

4) M Phase:

• two main processes: mitosis, during which the chromosomes are separated into two identical sets, and cytokinesis, during which the cytoplasm is divided to create two daughter cell

Cell Cycle Checkpoints:

1) G1 Checkpoint:

• monitors cell size, nutrient availability, growth factors, and DNA integrity
• If conditions are favourable and the DNA is undamaged, the cell is committed to cell division and moves into the S phase

2) G2/M Checkpoint:

• ensures that DNA replication in the S phase has been completed correctly before the cell enters mitosis
• If there is any DNA damage or incomplete replication, the cell cycle is halted for repair

3) Metaphase/Spindle Checkpoint:

• ensure that all the chromosomes are properly attached to the spindle fibres before they are separated
• This prevents the mis-segregation of chromosomes, which can lead to aneuploidy

Cell Cycle Regulation:

• The cell cycle is regulated by a series of proteins including cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors
• Cyclin binds to Cdk, activating the kinase. The active cyclin-Cdk complex phosphorylates target proteins to coordinate the various processes of the cell cycle
• Mitogenic factors (growth factors) promote cell division by triggering intracellular signalling pathways that lead to the production of cyclins
• Growth inhibitory factors halt the cell cycle by activating Cdk inhibitors (CKIs) that bind to and inhibit the cyclin-Cdk complexes
  Mitogenic factors stimulate cell proliferation and survival, while growth inhibitory factors suppress it.

Question 2

Define drugs acting on the cell cycle

Answer 2

Chemotherapy drugs that act on the cell cycle are classified as cell cycle-specific or cell cycle-nonspecific. Cell cycle-specific drugs target cells during specific phases of the cell cycle, such as the S or M phase.

S-phase drugs cause DNA damage, e.g. 5-fluorouracil (prevents synthesis of thymidine) and Cisplatin (binds to DNA causing damage and blocking repair)

M phase drugs targets mitotic spindles. Examples include Vinca alkaloids (stabilise free tubulin thus preventing microtubule polyerisation) and Palitaxel/Taxol (stablises microtubules, preventing de-polymerisation)

Cell cycle-nonspecific drugs target cells in any phase of the cell cycle. Examples include alkylating agents, which interfere with DNA replication, and anthracyclines, which inhibit DNA synthesis.

Question 3

Explain key molecular events in the regulation of the cell cycle and apoptosis

Answer 3

Cyclin-dependent kinases (CDKs) and cyclins are two important classes of proteins that regulate the cell cycle.

CDKs are activated by cyclins and the combined complex phosphorylate target proteins, promoting progression through the cell cycle.

Levels of cyclin present for the complex are determined by gene expression

CDKs-CDK activity is regulated by:

• Cycles of degradation (by proteasome) and synthesis (gene expression of cyclin)
• Post translational modification by phosphorylation (resulting in activation, inhibition or destruction)
• Dephosphorylation
• Binding of CDK inhibitors (CKDIs)

Apoptosis is a programmed cell death mechanism that occurs in response to certain signals, such as DNA damage or the activation of tumor suppressor genes. It is regulated by several key proteins, notably TP53.

Question 4

Define the role of RB and outline the sequence of events triggered by growth factors involving RB

Answer 4

Retinoblastoma protein (RB) is a key substrate of G1 and G1/S cyclin-dependent kinases which is ordinarily is unphosphorylated and binds to E2F transcription factor preventing its stimulation of S-phase protein expression but in presents of Cyclin D-CKD4 & Cycline E-CDK2 complexes it is phosphorylated and E2F is dissociated to stimulate the expression of more Cyclin E and S-phase proteins to start DNA replication

1) Growth factors / Mitogens in G1 phase activates ‘early gene’ expression

2) Early gene expresses transcription factors that stimulates the expression of ‘delayed gene’ (including Cyclin D, CDK2/4 and E2F transcription factors)

3) E2F is inhibited by binding to unphosphorylated RB

4) G1 cyclin-CDK complexes (Cyclin D-CDK4/6) hypophosphorylate RB, releasing E2F

5) E2F stimulates expression of more Cyclin E (Cyclin E-CDK2 hyperphosphorylates RB) and S-phase proteins (for DNA replication) via E2F Responsive genes

6) S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis

7) PP1 mediates the dephosphorylation of RB

Question 5

Describe mechanisms by which growth control can be disrupted in neoplasia

Answer 5

Growth control can be disrupted in neoplasia by several mechanisms, including mutations in genes that regulate the cell cycle, such as CDKs and cyclins, and mutations in genes that regulate apoptosis, such as TP53.

TP53 also regulates CDKIs

Question 6

Define the role of TP53 and its response to DNA damage

Answer 6

TP53 ordinarily is destroyed by proteasomes

When DNA is damaged by mutagens, resulting in the activation of kinases and the phosphorylation of TP53

TP53 will - drive the expression of CDKIs causing cell cycle arrest

• activate DNA repair e.g. excision repair or mismatch repair
• apoptosis if repair is not possible

Loss of function of TP53 is frequent in cancer where the prevention of DNA repair could lead to more mutations causing cancer progression