Radiosensitivity and the Cell Cycle - Chapter 4 jtl

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  • a , Ionizing radiation activates the kinase ATM, which in turn activates the kinase Chk2 .  b , This step appears to affect progression from G1 to the S phase, through phosphorylation (represented by a circled 'P') and stabilization of the p53 protein, which itself enhances the expression of the cell-cycle inhibitor p21 .  c , Activation of Chk2 by ATM also affects progression through the S phase itself, by the phosphorylation of Cdc25A. This protein is more likely to be degraded when phosphorylated . (When unphosphorylated, Cdc25A removes a phosphate group from Cdk2, enabling the initiation of DNA replication, that is, S phase.)  d , Nbs1 is also phosphorylated by ATM and is also involved in the ionizing-radiation-induced inhibition of S-phase progression , although it is not known how it ties in to the pathway described by Falck et al. e , The targets of ATM that control progression from G2 into mitosis (M phase) have not been described.
  • Signal transduction pathways are activated by either ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and RAD3-related (ATR). ATM responds to double strand breaks (DSBs), and evidence suggests an activating role for the NBS1–MRE11–RAD50 (MRN) complex 70 . ATR responds to single stranded regions of DNA, and requires ATR-interacting protein (ATRIP), RPA 71 , the RAD17–RFC2-5 complex, a complex of RAD9–HUS1–RAD1 (the 9–1–1 complex) 72 , TOPBP1 and claspin. The signalling pathways involve the mediator proteins (MDC1, 53BP1, the MRN complex and BRCA1), which amplify the signal, transducer kinases (CHK1 and CHK2) and effector proteins 17 . ATM and ATR show considerable overlap in their phosphorylation substrates but specificity also exists, with CHK1 and CHK2 being specific substrates of ATR and ATM, respectively. Two ATM-dependent G1/S checkpoints have been described. ATM activation by DSBs in G1 leads to CHK2 phosphorylation and subsequent phosphorylation of the phosphatase CDC25A. This increases the ubiquitylation and proteolytic degradation of CDC25A and prevents activating dephosphorylation of CDK2 at Thr14 and Tyr15 59, 73, 74 . As this pathway operates through post-translational modifications, initiation of the G1/S checkpoint occurs soon after damage induction. A second mechanism involves the tumour suppressor p53, which is activated and stabilized by ATM, either directly or indirectly through CHK2, and serves as a transcription factor for the cyclin-dependent kinase (CDK) inhibitor p21 (Refs  49 , 58 , 59 ). This pathway depends on the transcription of p21 and is therefore delayed after ionizing radiation (IR), suggesting that its role lies in the maintenance of the G1/S checkpoint 49, 57, 59, 75 . Collapsed or stalled replication forks in S phase activate ATR, leading to the phosphorylation of CHK1 and the subsequent phosphorylation and proteolysis of CDC25A 59 . This prevents initiation of new replication origins and slows down replication. A second branch of this intra-S phase checkpoint involves the MRN complex, BRCA1, FANCD2 and SMC1 76 (not shown in figure). DSBs in G2 can directly activate ATM, and indirectly, via ATM-dependent strand resection, can lead to ATR activation 64 . Similar to the rapid activation of the G1/S and intra-S phase checkpoints, the G2/M checkpoint is initiated by the phosphorylation of checkpoint kinases (CHK1 and CHK2) and phosphatases (probably CDC25C). This prevents dephosporylation of CDK1–cyclin B, which is required for progression into mitosis 23, 59 . Although p53 has an essential role in p21-dependent G1/S arrest , it promotes but is non-essential for the G2/M checkpoint . Roles include an affect on the maintenance of checkpoint arrest and on CDK1–cyclin B activity through transcriptional activation and regulation of GADD45 and 14-3-3
  • Checkpoint adaptation may lead to IR-induced genomic instability. Most of the cells with unrepairable DNA lesions will die following adaptation to the IR-induced G2 checkpoint. However, some cells might survive and proliferate with DNA lesions, making them prone to development of genomic instability and cancer. Cyclin B/Cdk1 activity is regulated by multiple factors including transcription of cyclin B and the extent of inhibitory phosphorylations on Cdk1, the latter being controlled by Plk1 and Chk1. Similar to adaptation in aphidicolin-treated  Xenopus  extracts, Plk1 may also control Chk1-inactivation during checkpoint adaptation in human cells. DOSE EFFECT: Adaptation likely occurs when the cyclin B/Cdk1 activity accumulates to a level sufficient to trigger mitotic entry. Adaptation may therefore occur owing to accumulation of cyclin B protein level, inactivation of Chk1, accumulation of Plk1 activity, and/or other factors that control cyclin B/Cdk1 activity.
  • Defects in repair and checkpoint arrest significantly affect cell survival following ionizing radiation, but confer only a small (repair defect) or modest (checkpoint loss) increase in chromosome breakage. However, the combined defect results in high levels of chromosome breakage and, by inference, increased chromosome rearrangements and tumour cell develop-ment. DSB, double strand break.
  • Radiosensitivity and the Cell Cycle - Chapter 4 jtl

    1. 1. Chapter 4: Radiosensitivity and Cell Age in the Mitotic Cycle JTL
    2. 2. Cell Cycle <ul><li>Tc = cell cycle time </li></ul><ul><li>Mitosis </li></ul><ul><ul><li>Visible by light microscopy </li></ul></ul><ul><ul><li>Cell rounds up, chromosome condenses, cell divides </li></ul></ul><ul><ul><li>Lasts about 1 hour </li></ul></ul><ul><li>Rest of cell cycle called interphase </li></ul><ul><li>DNA synthesis occurs during S phase of interphase </li></ul>1890 David von Hansemann
    3. 3. The Cell Cycle G1 = 1st Gap S = Synthesis G2 = 2nd Gap M = Mitosis
    4. 4. Lab Techniques: Cell Cycle Analysis <ul><ul><ul><li>Tritiated thymidine </li></ul></ul></ul><ul><ul><ul><li>BrDu (Bromodeoxyuridine) </li></ul></ul></ul><ul><ul><ul><li>Propidium Iodide </li></ul></ul></ul>
    5. 5. Trititiated Thymidine <ul><ul><li>Cells will incorporate labeled nucleotides if in S phase </li></ul></ul><ul><ul><li>Cells in S and G2 will have “grainy appearing” nuclei </li></ul></ul><ul><ul><li>Cells in 1st Mitosis will have stained chromatid </li></ul></ul><ul><ul><li>Cells in 2nd Mitosis will only have one stained chromatid (because DNA was replicated again during 2nd S phase using unlabeled nucleotides) </li></ul></ul><ul><ul><li>Use of BrdU preferred b/c not radioactive and quicker results </li></ul></ul><ul><li>Labelling Index =  (Ts/Tc) Ts = LI (Tc/  ) </li></ul><ul><li>Tc = Cell Cycle Time </li></ul><ul><li>Ts = Time of S Phase </li></ul><ul><li>= Empirically derived </li></ul><ul><ul><li>constant </li></ul></ul>
    6. 6. Propidium Iodide Cell Cycle Analysis <ul><li>Flow Cytometry Based </li></ul><ul><li>PI Labels DNA </li></ul><ul><li>Can estimate Phase of Cell Cycle based on DNA content </li></ul><ul><li>G0 not pictured but % content ~ G1 </li></ul>
    7. 7. <ul><li>BRDU Assay: </li></ul><ul><li>- Measures cells that have synthesized DNA per unit time </li></ul><ul><li>- Compound A ( panels C & D ): Cell Loss > low BRDU Uptake </li></ul><ul><li>- Compound B ( panels E & F ) Cell Loss < low BRDU Uptake </li></ul><ul><li>- 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. </li></ul>Compound A ( panels C & D ) Compound B (panels E & F) DMSO (panels A & B) Hoescht Hoescht Hoescht BRDU BRDU BRDU
    8. 8. Cell Cycle <ul><li>Tc primarily dependent on  ’s in length of G1 </li></ul><ul><li>Regulated by: </li></ul><ul><ul><li>Cyclin Dependent Kinases (CDKs), Cyclins, CDK Inhibitors (CKIs) </li></ul></ul><ul><ul><li>Form complexes based on activation state </li></ul></ul><ul><ul><ul><li>Usually (pS/pT) Ser/Thr PO4 </li></ul></ul></ul><ul><li>Complex inactivation </li></ul><ul><ul><li>Reversible: pY @ ATP binding domain or Cdk inhibitory proteins </li></ul></ul><ul><ul><li>Irreversible: Ubiquitin mediated degradation </li></ul></ul>
    9. 10. G1/S Phase Checkpoint
    10. 11. <ul><li>The central role of the retinoblastoma protein (pRb) in cell-cycle progression is shown. </li></ul><ul><li>S phase entry: </li></ul><ul><li>Rb  pRb by cyclin D1–CDK4/6 & Cyclin E–CDK2 complexes. </li></ul><ul><li>pRb  release & activation of E2F transcription factor </li></ul><ul><li>E2F then activates genes required for cell-cycle progression. </li></ul>G1/S Phase Checkpoint
    11. 15. Cell Cycle Synchronization <ul><li>Mitotic Harvest </li></ul><ul><ul><li>Collect cells which round up off of monolayer as they prepare for M phase </li></ul></ul><ul><li>Flow cytometry can also sort cells based on cell cycle phase </li></ul><ul><li>Hydroxyurea </li></ul><ul><ul><li>Kills S phase cells, block cell cycle at G1 so cells collect in G1 </li></ul></ul><ul><ul><li>Remove hydroxyurea and synchronized cells proceed to S phase </li></ul></ul><ul><li>Radiation treatments synchronize phase </li></ul>
    12. 16. Cell Cycle X-ray Sensitivity <ul><li>Using synchronized cells we can determine sensitivity to XRT during various phases </li></ul><ul><li>Sensitivity: M > G2 > G1> S early > S late </li></ul><ul><ul><li>Most resistant in Late S phase (likely due to homologous recombination b/w sister chromatids to repair damage </li></ul></ul>
    13. 17. Checkpoint Genes <ul><li>G2 checkpoint gene </li></ul><ul><ul><li>Halts cells in G2 to assess and repair DNA damage </li></ul></ul><ul><ul><li>Mutants of this gene proceed to Mitosis without repair & thus are more likely to die from XRT </li></ul></ul><ul><ul><li>Thought to involve Cdk1 </li></ul></ul>Defects in repair and checkpoint arrest significantly affect cell survival following ionizing radiation, but confer only a small (repair defect) or modest (checkpoint loss) increase in chromosome breakage. However, the combined defect results in high levels of chromosome breakage and, by inference, increased chromosome rearrangements and tumour cell develop-ment. DSB, double strand break.
    14. 18. Checkpoints and Repair
    15. 19. O2 and Cell Cycle <ul><li>OER oxygen enhancement ratio </li></ul><ul><ul><li>differences can change radiosensitivity by about the same magnitude as cell cycle differences </li></ul></ul><ul><ul><li>OER = Dose in hypoxia conditions </li></ul></ul><ul><ul><ul><ul><ul><li>Dose in Aerated Conditions </li></ul></ul></ul></ul></ul><ul><li>OER Photons – 2.5 - 3 </li></ul><ul><ul><li>Aerated Photon RT is 2.5-3 x more effective </li></ul></ul><ul><li>OER greatest in S phase > G1 > G2 </li></ul>
    16. 20. OER
    17. 21. Age-Response Function <ul><li>In vivo is similar to in vitro: </li></ul><ul><ul><li>most sensitive at G2/M, most resistant in late S phase </li></ul></ul><ul><li>High LET = Minimal cell cycle variation in sensitivity </li></ul><ul><li>Low LET = Indirect damage by Free Radicals </li></ul><ul><li>Proposed Mechanisms </li></ul><ul><ul><li>In S phase DNA is replicating in M phase it is condensed and separating (i.e. less available for indirect damage) </li></ul></ul><ul><ul><li>Levels of Sulfhydryls (anti-oxidants) vary with cell cycle </li></ul></ul><ul><ul><li>Levels of repair enzyme activity vary with cell cycle </li></ul></ul>
    18. 22. Reassortment <ul><li>For a fractionated regimen cells may again cycle into a radiosensitive part of the cycle by the time the next fraction is given </li></ul><ul><li>Rapidly dividing cells are more likely than late responding normal tissues to undergo this re-assortment resulting in therapeutic gain. </li></ul>In vivo Asynchronous cells Preferentially kills M phase cells Synchronize cells, most residual in S phase D0 XRT kill Re-Cycle
    19. 23. #1 Following exposure to IR, cells that lack functional p53 are most likely to arrest in which phase of the cell cycle? <ul><li>G1 </li></ul><ul><li>S </li></ul><ul><li>G2 </li></ul><ul><li>M </li></ul><ul><li>Will not arrest </li></ul>
    20. 24. <ul><li>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) </li></ul>
    21. 25. #2 Radiation induced G1 arrest involves which sequence of protein changes? <ul><li>FasL binding, caspase 9 activation, mitochondrial breakdown </li></ul><ul><li>ATM autophosphorylation, P53 phosphorylation, p21 upregulation </li></ul><ul><li>Cyt c release, caspase 8 activation, lamin degradation </li></ul><ul><li>PI3K acetylation, P53 ubiquitination, bcl-2 degradation </li></ul><ul><li>NBS1 phos, ATM methylation, Bax upregualtion </li></ul>
    22. 26. <ul><li>ATM autophosphorylation, P53 phosphorylation, p21 upregulation </li></ul><ul><li>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. </li></ul>
    23. 27. #3 Cell cycle phase with greatest IR resistance? <ul><li>M </li></ul><ul><li>G1 </li></ul><ul><li>Early S </li></ul><ul><li>Late S </li></ul><ul><li>G2 </li></ul>
    24. 28. <ul><li>Late S </li></ul>
    25. 29. #4 Proliferating human cells in vivo generally have what Tc <ul><li>6-24 hours </li></ul><ul><li>1-5 days </li></ul><ul><li>5-25 days </li></ul><ul><li>4-8 weeks </li></ul><ul><li>3-6 months </li></ul>
    26. 30. <ul><li>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 </li></ul>
    27. 31. #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 <ul><li>1 hour </li></ul><ul><li>5 hour </li></ul><ul><li>10 hr </li></ul><ul><li>20 hr </li></ul><ul><li>50 hr </li></ul>
    28. 32. Labelling Index =  (Ts/Tc) Ts = LI(Tc/  ) Ts=0.2(72hrs/0.7)=20.6 hrs
    29. 33. #6 Asynchronous population of 2 x 10 7 cells has the following parameters: -About how many cells are in S phase? <ul><li>2.5 x 10 5 </li></ul><ul><li>10 6 </li></ul><ul><li>2.5 x 10 6 </li></ul><ul><li>5 x 10 6 </li></ul><ul><li>10 7 </li></ul>Tm=1hr Tg1=11hr Ts= 5hr Tg2=3hr Growth fraction (% not quiescent (Not G0) = 0.5)
    30. 34. 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 10 7 cells)= 2.5 x 10 6
    31. 35. #7 Which pair is incorrect? <ul><li>G1 - CDK1 </li></ul><ul><li>S - CDK2 </li></ul><ul><li>G1 - CDK4 </li></ul><ul><li>G2 - Cyclin B </li></ul><ul><li>G1 0 Cyclin D </li></ul>
    32. 36. #7 Which pair is incorrect? <ul><li>G1 - CDK1 </li></ul><ul><li>CDK1 is associated with progression from G2 to M </li></ul>
    33. 37. #8 The DNA content of Go cells is equal to that of? <ul><li>S </li></ul><ul><li>G1 </li></ul><ul><li>G2 </li></ul><ul><li>Mitotic cells </li></ul><ul><li>Tetraploid cells </li></ul>
    34. 38. #8 The DNA content of Go cells is equal to that of? <ul><li>G1 cells - Go cells are basically G1 cells that have temporarily or permanently stopped cycling. </li></ul>

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