The cell cycle consists of four main phases - G1, S, G2, and M phase. The cell cycle is tightly regulated by cyclins and cyclin-dependent kinases (CDKs) that drive progression between phases. CDK activity is controlled by association with cyclins, phosphorylation, and degradation. Checkpoints between phases like G1/S and G2/M ensure DNA replication fidelity before division. Apoptosis, or programmed cell death, can be triggered intrinsically via DNA damage or extrinsically through ligand-receptor interactions and leads to cell death. The cell cycle and apoptosis are assessed using techniques like propidium iodide staining to measure DNA content and annexin assays to detect early apoptotic

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Cell Cycle
Prepared by Mostafa A. Askar, Ass.Lec
NCRRT, Cairo, Egypt
Continuity 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.
 Cellsthatstoppedcycling(nervecells) enterG0
 Interphase
1. G1 phase
 A periodof cellulargrowthprecedingDNA synthesis.
 Most DNA repairoccurs inthe G1 phase.
2. S phase
Phase 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

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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
Figure 1 The four phases of cell cycle progression and their regulations
Cdk, cyclin dependent kinase . Rb, retinoblastoma protein.
P21 CIP1 family
p21, 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 family
p21, p27
P16 INK4afamily
G1
cdc25C
13

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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 Checkpoint
Cell 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.

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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
2- Extrinsic pathway
Due 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.
AssessmentOfThe Cell Cycle And Apoptosis
 PropidiumIodide ( Pi )
A fluorescentdye isbounddirectlytothe DNA inthe nucleusof cells.
Measuringthe fluorescenceprovidesameasure of the amountof dye takenup bythe cell andindirectlythe amountof DNA
content
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 Apoptoticcellsshowadiminishedstainingbelow the G0/G1populationof normal diploidcells.
 The DNA specificfluorochrome PIidentifiedadistincthypo-diploidcell population.
 ANNEXINASSAY
 In the earlystagesof apoptosischangesoccurat the cell surface.
 One of these plasmamembrane alterationsisthe translocationof phosphatidylserine (PS)fromthe inner side of the
plasmamembrane tothe outerlayer,bywhichPS becomesexposedatthe external surface of the cell.
 AnnexinV isaCa’+ dependentphospholipid-bindingproteinwithhighaffinityforPS.
 AnnexinV canbe usedas a sensitive probe forPSexposureuponthe cell membrane.
 AnnexinV assayissensitive andeasytoperform.
 The AnnexinV assayoffersthe possibilityof detectingearlyphasesof apoptosisbefore the lossof cell membrane
integrity.

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