Tumor cell reassortment within the cell cycle (including checkpoints and cell-cycle arrest)

Transcription

1 Tumor cell reassortment within the cell cycle (including checkpoints and cell-cycle arrest) Carsten Herskind Dept. of Radiation Oncology. Universitätsmedizin Mannheim Medical Faculty Mannheim, Heidelberg University Workshop Biological basis of RT, Stockholm

2 Outline Split-dose recovery and radiosensitivity in cell cycle Cell-cycle checkpoints: G1/S, intra-s, G2/M Efficiency and resolution of checkpoint arrest Potential for manipulation; Influence of fraction size (novel fractionation schemes)

3 Radiosensitivity changes with time interval between split-dose fractions CHO cells 18h interval SLD repair Reassortment: partly synchronous progression of cell-cycle Elkind & Sutton, Nature 4695 (1959) Complete recovery: curve shape repeated. asynchr. cohort?

4 Synchronized cells: radioresistant in late S radiosensitive in G2/M and early S SF HeLa Sinclair & Morton, Radiat Res 29 (1966) Sinclair, Radiat Res 33 (1968) in Denekamp, Int J Radiat Biol 49 (1986) Time after synchronization (h)

5 Steep increase of radioresistance through M to G1 Fucci staining: G1: red S-G2-M: green Enrichment of Fr.1 after mitotic shake-off G1 l-m e-m late M early M Fr.1: M G1 fraction G1 l-m e-m Nakayama et al, Radiat Res 176 (2011)

6 Aims of the DNA damage response (DDR) Immediate : survive next cell division block replication of damaged DNA preserve integrity of DNA; repair damage prevent cell division with broken chromosomes Longer-term: maintain genomic stability

7 Regulation of cell-cycle progression Cyclins (CCN) and cyclindependent kinases (Cdk) Hall, Giaccia, Radiobiology for the Radiologist. 7th ed. (2012)

8 Textbook view of cell-cycle checkpoints DSB ATM Hall, Giaccia, Radiobiology for the Radiologist. 7th ed. (2012) G1/S S S G2/M Cdk2 G1/S: p53 p21 S: Chk2 Cdc25A MRN SMC G2/M: Chk1 Cdc25C

9 Role of ATR in S-phase slow-down DSB ATM Single-stranded DNA Stalled replication forks ATR DSB MRN complex CHK2 CHK1 SMC? CDC25A Degradation Wee1 CDK2 CCNA Degradation Falck et al., Nature 410 (2001), Nat Genet 30 (2002) Replication Initiation S-phase

10 Cell-cycle analysis: block G2 M G1 Cell-cycle distribution (DNA content) arrest progression in all phases: no change block and progression: accumulation (slow) S G2-marker CENP M-Marker HistH3 Inhibitor of mitosis G2 M G1 BrdU-pos in G2 G2 M G1 S BrdUlabeling of S S BrdU-neg in S

11 G2/M: two distinct checkpoints early Lymphoblastoid cell lines Dose: 6 Gy late Xu et al., Mol Cell Biol 22 (2002)

12 G2/M checkpoint characteristics Early G2/M checkpoint: Blocks entry into M (~1h) of cells irradiated in G2 - is observed early after irrad. when cells stop entering M Late G2/M checkpoint: Accumulation in G2 (12-24h) of cells irradiated in S (6 Gy) - is observed late after irrad. when cells progress to G2 ATM dependent Dose independent (>0.4 Gy) ATM independent Duration is dose dependent Xu et al., Mol Cell Biol 22 (2002)

13 G2/M checkpoint pathways Late (after irrad.) Early (after irrad.) Single-stranded DNA Stalled replication forks Resected DSB DSB ATR ATM CHK1 Plk1 CHK2 Plk1 CDC25A CDC25A/C CDK1 CCNB CDK1 CCNB Centrosome maturation S progression into G2-phase mitosis

14 A third G2/M checkpoint mechanism Early arrest: ATM late arrest: CHK RehTP53+ U698 TP53- ATM inhibitor: KU CHK1 inhibitor: caffeine Intermediate arrest (2-10h after 4 Gy) depends on p53 (TP53) (Reh) (Reh shrna p53-knockdown) 4Gy Caffeine + KU Gy Caffeine 0Gy control 4Gy 4Gy KU Landsverk et al., Cell Cycle 10 (2011)

15 G2/M checkpoint pathways Late (after irrad.) ssdna Intermediate Qi, Martinez, Rad Res 160 (2003) Early (after irrad.) DSB ATR p53 ATM CHK1 Gadd45 CHK2 Sequestration out of nucleus CDC25A Degradation CDC25A/C CDK1 CCNB CDK1 CCNB S progression into G2-phase

16 Low-dose hyper-radiosensitivity (HRS): Induced radioresistance (IRR) is associated with induction of early G2 checkpoint Very early G2 arrest 0.5h after 0.2 Gy (i.e. <0.4Gy): Marples et al., Radiat Res 161 (2004) Martin et al., Cancer Lett 349 (2014) absent in HRS pos T98G present in HRS neg U373 T98G (HRS pos ) U373 (HRS neg ) Very early, low-dose G2 arrest is independent of ATM (not shown) Fernet et al., DNA Repair 9 (2010)

17 G2/M checkpoint adaptation? Late mitotic entry with unrepaired DSB Mitotic cells with foci U2OS cells Syljuåsen et al., Cancer Res 66 (2006)

18 Renewed expression of Plk1 can mediate mitotic entry Syljuåsen et al., Cancer Res 66 (2006) Syljuåsen, Oncogene 26 (2007)

19 Threshold model: DSB appears to be required: insensitive G2 checkpoint induction and release? Lines: mitotic cells Bars: γh2ax foci per cell Time of release depends on number of residual DSB Deckbar et al. J Cell Biol 176 (2007)

20 Cell-cycle progression of BrdU-labelled cells after irradiation in S-phase Artemis and AT cells both have a DSB repair defect but only AT cells have a checkpoint defect. Similar kinetics of release from G2 DSB threshold Pulse-label, detect BrdU+ cells in G2 Foci per BrdU+ cells in G2 1Gy in S AT Artemis wt Krempler et al., Cell Cycle 6 (2007)

21 G2/M adaptation or threshold? Activation of the G2/M checkpoint by Gy Gy corresponds to less than DSB Number of mitotic cells relative to 0 Gy Foci per mitotic cell γh2ax foci in mitotic cells escaped from G2/M arrest at 75 min (<10-15 in 90%) Tkacz-Stachowska et al., Radiother Oncol 101 (2011)

22 G2 adaptation or threshold? Mitotic entry at 30h with <10-15 DSB 75% of cells contain <5-8 DSB (mean ~2 DSB/mitotic cell) A rigid threshold of DSB unlikely? But: counting γh2ax foci is critical Tkacz-Stachowska et al., Radiother Oncol 101 (2011)

23 Summary: G2/M checkpoint arrest 3 (perhaps 4?) different checkpoints / pathways Very early (?): ATM indep. (0.2 Gy), absent in HRS pos cells Early: ATM dep., arrests cells irradiated in G2 ( 0.4 Gy) Intermediate: p53 dep., ATM indep. Late (dose-dep. accum. in G2): ATR/CHK1 dep.; ATM indep. Induction and release Insensitive (threshold of DSB) Release with unrepaired DSB Adaptation (premature release) or Threshold?

24 CHK1 network Scope for manipulation by inhibitors Dai & Grant, Clin Cancer Res 16 (2010)

25 G1 checkpoint and its limitations p53 stabilisation Transcriptional activation (p21) Phosphorylation of CHK2 Phosphorylation of CDC25A inactivation and degradation Arrest in late G1 Deckbar et al., Crit Rev Biochem Mol Biol 46 (2011) G1 phase

26 Fast G1 checkpoint arrest (1-4h) is sensitive but not immediately effective Microscopy: Cells found in S-phase after irrad. In G1 Fast arrest depends on ATM (no arrest in AT cells) and on CHK2 (sirna knockdown) but not on p53 Double-labelling technique : Entry of unlabelled fibroblasts irradiated in G1 into S-phase Deckbar et al., Cancer Res 70 (2010) Irrad. of synchronized G1 cells 8.5h after release from G 0 (1.5h before S)

27 Slow G1/S checkpoint arrest (~3-10h) Very sensitive (~1 DSB), dose-dependent ( 0.1 Gy) Slow arrest depends on ATM (no arrest in AT cells) and on p53 (sirna knockdown) but not on CHK2 Fibroblasts synchronized by release from density arrest. Irradiation 1.5h before S-entry Deckbar et al., Cancer Res 70 (2010)

28 Leaky late G1/S checkpoint after high doses (irradiation of synchronized cells in G1) Pct. G2-phase cells Cells progress to G2 with DSB after irradiation in G1 Deckbar et al., Cancer Res 70 (2010)

29 Summary of G1/S checkpoint arrest Fast G1/S arrest takes several hours to become fully effective sensitive depends on ATM-CHK2 but not on p53 Slow G1/S arrest requires transcriptional activation and translation very sensitive depends on ATM-p53 but not on CHK2 leaky after moderate-high doses

30 Synthetic lethality of ATM & p53 abrogation Doxorubicin-induced DSB in cancer cells arrest apoptosis HR NHEJ ATM+/+ p53 +/+ (baseline) + (+) + + ATM+/+ p53 --/-- resistant ATM suppr. p53 +/+ resistant ATM suppr. p53 --/-- sensitive - mitotic death - + ATM-CHK2 mediates cell-cycle arrest p53 mediates apoptosis (via ATM-CHK2 or ATR-CHK1) p53 mediates cell-cycle arrest (indep. of ATM-CHK2) Jiang et al., Genes Dev 23 (2009) Genetic background may be important (cf. AT cells)

31 Inhibition of ATM radiosensitizes glioblastoma cells U1242: p53 mut U87 p53 wt p53 mut U87: p53 wt P53 wt ATM inhib p53 mut ATM inhib Biddlestone-Thorpe et al.,clin Cancer Res 19 (2013)

32 ATM Inhibition radiosensitizes p53-mutant tumours Human U87 tumours (nude mice) Radiosensitization only of cells expressing p53 mut p53 mut p53 wt Biddlestone-Thorpe et al., Clin Cancer Res 19 (2013)

33 ATM network Shilo & Ziv, Nat Rev Mol Cell Biol 14 (2013)

34 Summary of cell-cycle checkpoints >10-20 DSB? ATM-CHK2-CDC25 Early (1-2h) Dose-indep. ( 0.4 Gy) p53-gadd45-cdc25 Intermediate (2-10h) G2 M G1 ATM-CHK2-CDC25 Fast (1-4h) (>~1 Gy) ATM-p53-p21 Slow (3-10h) (>~0.1 Gy) Transient or permanent dep. on dose? ATR-CHK1-CDC25 S & late G2/M ( 10h) Dose-dep. duration S MRN-SMC 1 DSB?

35 CONCLUSIONS Different sensitivities, dose dependence, kinetics Heterogeneity of reassortment ATM: independent of dose at low-moderate doses (threshold) ATR, p53: dependent on dose more important at higher doses/fx Checkpoints are imperfect Delayed induction, premature release Escape with unrepaired DSB encounter next checkpoint Different pathways Potential for manipulation (modulates repair, mode of cell death) p53: also apoptosis & permanent G1 arrest (genomic stability; p21) 35

36 Acknowledgements Department of Radiation Oncology, UMM, Medical Faculty Mannheim Cellular and Molecular Radiation Oncology Lab Clinic Heidelberg University Miriam Bierbaum Juliane Bradl Linda Hartman, PhD Anne Kirchner Junqi Liu Xiaolei Liu Patrick Maier, PhD Marlon R. Veldwijk, PhD Frank A. Giordano, MD Frederik Wenz, MD (Director) 36