Cell CyclePrepared by Mostafa A. Askar, Ass.Lec

NCRRT, Cairo, EgyptContinuity of life depends on cells

• Growing• Replicating their genetic material• Dividing

The cell cycle is the process by which cells replicate their DNA. Phases of the cell cycle

G0 phase: or resting phase is a period in the cell cycle in which cells exist in a quiescent state.

Some cells enter the G0 phase semi-permanently e.g., some liver and kidney cells. Many cells do not enter G0 and continue to divide throughout an organism's life, e.g. epithelial cells.

Cells that stopped cycling (nerve cells) enter G0

Interphase1. G1 phase

A period of cellular growth preceding DNA synthesis. Most DNA repair occurs in the G1 phase.

2. S phasePhase of DNA replication

Mitosis (M phase, mitotic phase)

 M phase consists of nuclear division (karyokinesis). It is a relatively short period of the cell cycle. M phase is complex and highly regulated. The sequence of events is divided into phases, known as: prophase, metaphase, anaphase, telophase cytokinesis (cytokinesis is not part of mitosis but is an event that directly follows mitosis in which cytoplasm is

divided into two daughter cells)

Regulation of cell cycle

Figure 1 The four phases of cell cycle progression and their regulationsCdk, cyclin dependent kinase . Rb, retinoblastoma protein.

P21 CIP1 familyp21, p27

M

G2

S

G1

G0

Growth factors, mitogens…

D Cyclins / cdk4, 6

Rb

P-Rb

Cyclin A / cdk2

Cyclin B /cdk 1

Cyclin A /cdk 2

Cyclin E / cdk2

P21 CIP1 familyp21, p27

P16 INK4afamily

G1

cdc25C

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The different cell cycle phases are driven by cyclin dependent kinases (CDKs) identified as being the “cell cycle engine”. CDKs are the catalytic subunits that get activated only when associated with a cyclin, the cdk-cyclin complex then phosphorylates downstream components. Both the CDK and the cyclin protein form the holoenzyme. The orchestrated changes in the level of cyclins throughout the cell cycle control the transmission from one phase of the cycle to the other. For example, cyclin D is synthesized during G1, cyclin E is synthesized in late G1, cyclin A is synthesized during S and G2 phases, and cyclin B is synthesized in G2 and M phases (Fig 1).The cdk activity in G2 phase was found to inhibit initiation of a further S phase. The cell cycle is also controlled by post translational modification of different cell cycle regulators (phosphorylation-dephosphorylation) Cdk1 is inactivated by phosphorylation on tyrosine 15 and threonine14 by wee1, a small kinase. The rate limiting step in triggering the G2M transition is mediated by the dual phosphatase cdc25C which dephosphorylates cdk1 and hence activates the holoenzyme (cdk1-cyclin B). It is itself inhibited by phosphorylation by the Chk1 and Chk2 kinases that become activated in response to DNA damage. Interestingly, cyclin B/ cdk1 active complex in a negative feedback regulatory loop also inhibits cdc25c phosphatase. Phosphorylation of cdc25C at serine 216 forms a docking site for the 14-3-3 protein. The 14-3-3 bound Cdc25C cannot translocate to the nucleus to dephosphorylate cdk1 ending into G2M cell cycle arrest.

Another level of cell cycle regulation is mediated by cyclin dependent kinase inhibitors (CKIs). P21 is a non-specific CKI that can inhibit a wide range of cdks. In response to DNA damage, activated p53 (the genome guardian) directly up-regulates p21. P21 mediates G1 arrest by inhibiting cdk 4 and cdk6. It has also been reported to inhibit cdk1 and mediate a G2M arrest in response to treatment with certain agents in various tumor cell lines. However, other studies demonstrated that the role p21 plays in inducing G2M arrest was dispensable.

The final level of cell cycle regulation is mediated by degradation. CDKs are finally removed by ubiquitin mediated degradation. Degradation of B cyclins is necessary to drive the cell cycle machinery

The key regulators of the checkpoint pathways in the mammalian DNA damage response.ATM (ataxia telangiectasia, mutated) protein kinase ATR (ATM and Rad3-related) protein kinase

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The major downstream target of Cyclin D is the retinoblastoma protein (pRB). The Cyclin D/Cdk4/6 complex directly phosphorylates pRB. This relieves the inhibitory effects of pRB on the transcription factor, E2F resulting in the expression of a large number of cell cycle regulated genes and eventual progression into S-phase.

Checkpoints on the cell cycle

G1 CheckpointCell preparing to enter in cycling (cyclin D,E)

S-phase Checkpoint Monitors cell cycle progression and decreases the rate of DNA synthesis following DNA damage. This pathway is the least understood of the mammalian checkpoints. Cyclones that enter in checkpoint regulation (Chk2, cdc25A, BRCA-1)

G2 Checkpoint Monitors the DNA synthesized correctly, arrest if it not synthesized correctly.

Apoptosis

1- Intrinsic pathway(IP)The intrinsic pathway is initiated from within the cell. This is usually in response to cellular signals resulting from DNA damage, a defective cell cycle. Mostly, the IP trigger by P53

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2- Extrinsic pathwayDue to loss of internal control, the cll cell communication is begin by pro-apoptotic ligands. as Fas/Fas ligand and bind their cognate receptors CD95/Fas.

Assessment Of The Cell Cycle And Apoptosis

Propidium Iodide ( Pi )

A fluorescent dye is bound directly to the DNA in the nucleus of cells.Measuring the fluorescence provides a measure of the amount of dye taken up by the cell and indirectly the amount of DNA content …………………………………………………………………………

Apoptotic cells show a diminished staining below the G0/G1 population of normal diploid cells. The DNA specific fluorochrome PI identified a distinct hypo-diploid cell population.

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ANNEXIN ASSAY

In the early stages of apoptosis changes occur at the cell surface. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the

plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca’+ dependent phospholipid-binding protein with high affinity for PS. Annexin V can be used as a sensitive probe for PS exposure upon the cell membrane.

Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane

integrity.

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