Radiosensitivity and the Cell Cycle - Chapter 4 jtl

Chapter 4: Radiosensitivity and Cell Age in the Mitotic

Cycle

JTL

Cell Cycle

• Tc = cell cycle time• Mitosis

– Visible by light microscopy– Cell rounds up, chromosome condenses,

cell divides– Lasts about 1 hour

• Rest of cell cycle called interphase• DNA synthesis occurs during S phase of

interphase

1890 David von Hansemann

The Cell Cycle

G1 = 1st Gap

S = Synthesis

G2 = 2nd Gap

M = Mitosis

Lab Techniques: Cell Cycle Analysis

• Tritiated thymidine• BrDu (Bromodeoxyuridine)• Propidium Iodide

Trititiated Thymidine– Cells will incorporate labeled

nucleotides if in S phase– Cells in S and G2 will have

“grainy appearing” nuclei– Cells in 1st Mitosis will have

stained chromatid– Cells in 2nd Mitosis will only

have one stained chromatid (because DNA was replicated again during 2nd S phase using unlabeled nucleotides)

– Use of BrdU preferred b/c not radioactive and quicker results

Labelling Index = (Ts/Tc)Ts = LI (Tc/)

Tc = Cell Cycle TimeTs = Time of S Phase= Empirically derived

constant

Propidium Iodide Cell Cycle Analysis

• Flow Cytometry Based• PI Labels DNA• Can estimate Phase of Cell

Cycle based on DNA content

• G0 not pictured but % content ~ G1

BRDU Assay:- Measures cells that have

synthesized DNA per unit time- Compound A (panels C & D): Cell

Loss > low BRDU Uptake- Compound B (panels E & F) Cell

Loss < low BRDU Uptake- The blue circles in the nuclear

images are the ArrayScan ROI overlays and indicate which cells the computer has identified as BRDU positive from the corresponding BRDU images.

Compound A (panels C & D) Compound B (panels E & F)DMSO (panels A & B)

Hoescht Hoescht HoeschtBRDU BRDU BRDU

Cell Cycle• Tc primarily dependent on ’s in length of G1• Regulated by:

– Cyclin Dependent Kinases (CDKs), Cyclins, CDK Inhibitors (CKIs)

– Form complexes based on activation state• Usually (pS/pT) Ser/Thr PO4

• Complex inactivation– Reversible: pY @ ATP binding domain or Cdk

inhibitory proteins– Irreversible: Ubiquitin mediated degradation

G1/S Phase Checkpoint

• The central role of the retinoblastoma protein (pRb) in cell-cycle progression is shown.

• S phase entry: • Rb pRb by cyclin D1–CDK4/6 & Cyclin E–CDK2 complexes.• pRb release & activation of E2F transcription factor• E2F then activates genes required for cell-cycle progression.

G1/S Phase Checkpoint

Cell Cycle Synchronization• Mitotic Harvest

– Collect cells which round up off of monolayer as they prepare for M phase

• Flow cytometry can also sort cells based on cell cycle phase

• Hydroxyurea– Kills S phase cells, block cell cycle at G1 so cells

collect in G1– Remove hydroxyurea and synchronized cells

proceed to S phase• Radiation treatments synchronize phase

Cell Cycle X-ray Sensitivity• Using synchronized cells we can determine

sensitivity to XRT during various phases• Sensitivity: M > G2 > G1> S early > S late

– Most resistant in Late S phase (likely due to homologous recombination b/w sister chromatids to repair damage

Checkpoint Genes• G2 checkpoint gene

– Halts cells in G2 to assess and repair DNA damage– Mutants of this gene proceed to Mitosis without

repair & thus are more likely to die from XRT– Thought to involve Cdk1

Checkpoints and Repair

O2 and Cell Cycle• OER oxygen enhancement ratio

– differences can change radiosensitivity by about the same magnitude as cell cycle differences

– OER = Dose in hypoxia conditions Dose in Aerated Conditions

• OER Photons – 2.5 - 3– Aerated Photon RT is 2.5-3 x more effective

• OER greatest in S phase > G1 > G2

OER

Age-Response Function• In vivo is similar to in vitro:

– most sensitive at G2/M, most resistant in late S phase• High LET = Minimal cell cycle variation in sensitivity• Low LET = Indirect damage by Free Radicals• Proposed Mechanisms

– In S phase DNA is replicating in M phase it is condensed and separating (i.e. less available for indirect damage)

– Levels of Sulfhydryls (anti-oxidants) vary with cell cycle– Levels of repair enzyme activity vary with cell cycle

Reassortment

• For a fractionated regimen cells may again cycle into a radiosensitive part of the cycle by the time the next fraction is given

• Rapidly dividing cells are more likely than late responding normal tissues to undergo this re-assortment resulting in therapeutic gain.

In vivo Asynchronous cells

Preferentially kills M phase cells

Synchronize cells, most residual in S phase

D0 XRT kill

Re-Cycle

#1 Following exposure to IR, cells that lack functional p53 are most

likely to arrest in which phase of the cell cycle?

• G1• S• G2• M• Will not arrest

• G2 -after XRT all cells regardless of P53 status will halt in G2. With functional P53 there may also be some cells that halt at G1 and S but the majority will still be at G2 (remember the G2 cell cycle checkpoint)

#2 Radiation induced G1 arrest involves which sequence of protein

changes?

• FasL binding, caspase 9 activation, mitochondrial breakdown

• ATM autophosphorylation, P53 phosphorylation, p21 upregulation

• Cyt c release, caspase 8 activation, lamin degradation

• PI3K acetylation, P53 ubiquitination, bcl-2 degradation

• NBS1 phos, ATM methylation, Bax upregualtion

• ATM autophosphorylation, P53 phosphorylation, p21 upregulation

• In response to IR induced DNA damages, principally DNA DSB’s, ATM undergoes conformational changes, and is then autophosphorylated to activate it. Activated ATM phosphorylated P53 which acts as a trxpn factor for P21. P21 is a cdk inhibitor and thus causes cell cycle inhibition.

#3 Cell cycle phase with greatest IR resistance?

• M• G1• Early S• Late S• G2

• Late S

#4 Proliferating human cells in vivo generally have what Tc

• 6-24 hours• 1-5 days• 5-25 days• 4-8 weeks• 3-6 months

• 1-5 days, tumors don’t double in volume this quickly however b/c not all cells are proliferating and there is a high rate of cell death

#5 Assuming that all cells are proliferating, if the cell cycle time is 3

days, the labeling index is 0.2 and is 0.7 what is the duration of S

phase• 1 hour• 5 hour• 10 hr• 20 hr• 50 hr

Labelling Index = (Ts/Tc)Ts = LI(Tc/)

Ts=0.2(72hrs/0.7)=20.6 hrs

#6 Asynchronous population of 2 x 107 cells has the following

parameters:

-About how many cells are in S phase?• 2.5 x 105

• 106

• 2.5 x 106

• 5 x 106

• 107

Tm=1hrTg1=11hrTs= 5hrTg2=3hrGrowth fraction (% not quiescent (Not G0) = 0.5)

Total Tc = 20hrs, Ts=5hrs so roughly 1/4 of dividing cells are in S phase. Growth Fraction is 0.5 so about half of cells are dividing so 1/8 of all cells are in S phase. 1/8 x (2 x 107 cells)=

2.5 x 106

#7 Which pair is incorrect?

• G1 - CDK1• S - CDK2• G1 - CDK4• G2 - Cyclin B• G1 0 Cyclin D

#7 Which pair is incorrect?

• G1 - CDK1

• CDK1 is associated with progression from G2 to M

#8 The DNA content of Go cells is equal to that of?

• S• G1• G2• Mitotic cells• Tetraploid cells

#8 The DNA content of Go cells is equal to that of?

• G1 cells - Go cells are basically G1 cells that have temporarily or permanently stopped cycling.