Radiosensitivity and cell age in the mitotic cycle

RADIOSENSITIVITY

1. RADIOSENSITIVITY AND CELL AGE IN THE MITOTIC CYCLE Sneha Susanna George

2. THE CELL CYCLE Ordered process by which a cell grows and divides into 2 progeny daughter cells

3. M ITOSIS - visible by light microscopy - 1 hour S phase - longest phase - DNA synthesis G1 phase - varies for different cell lines G2 phase - prepares for cell division

4. CELL CYCLE TIME/MITOTIC CYCLE TIME Time between 2 successive mitotic divisions

5. CELL LABELLING TECHNIQUES For cell cycle analysis Markers of DNA synthesis Introduced by Howard and P ele in 1953 Autoradiography Tritiated thymidine 5-Bromodeoxyuridine

6. Tritiated thymidine (3H-TdR) incorporated into chromosomes S phase cells take up 3H-TdR Cells are fixed and stained Covered with a nuclear(photographic) emulsion Left in refrigerator for 1 month Form latent images that appear as black grains

7. 5 bromodeoxyuridine More convenient 1) no radioactive material 2) shorter time to results Presence detected by an appropriate stain (bright green ) To identify cells in S phase – Fluorochrome tagged antibody is used against the bromodeoxyuridine substituted DNA which fluoresces brightly under the microscope

8. Comparison of Cell Cycle – Hamster and Hela cells

9. REGULATION OF THE CELL CYCLE By periodic activation of cyclin dependent kinase family( Cdk ) Cdk + cyclin Phosphorylation of key threonine residue Activated Cdk – cycline complex - Drives cell cycle events - Prevents initiation of a cell cycle event at the wrong time

10. REVERSIBLE IRREVERSIBLE INACTIVATION INACTIVATION By phosphorylation - By ubiquitin on a tyrosine residue mediated degradation located in the ATP of the cyclin subunit binding domain By assoc with Cdk inhibitory proteins

11. Each cyclin protein is synthesized at a discrete phase of the cycle Transitions in the cell cycle occurs if the given kinase activates the proteins required for progression Tumour suppressor genes( p53, Rb ) can block cell division if DNA is damaged

12. SYNCHRONOUSLY DIVIDING CELL CULTURES Populations of cells in which all of the cells occupy the same phase of the cell cycle at a given time By 2 techniques Mitotic harvest Use of a drug( eg : Hydroxyurea )

13. MITOTIC HARVEST TECHNIQUE First described by Terasima and Tolmach Physical separation of cells preparing for mitosis Works on monolayer cell cultures

14. Use of a drug Hydroxyurea is used 1) Kills S phase cells 2) Blocks the cell cycle at G1

15. The effect of X-rays on synchronously dividing cultures Dose used 6.6Gy (660 rad ) Chinese hamster cells were subjected to this at various phases of the cell cycle Proportion of cells that survive the dose - Survival fraction Cells in G 1 - survival fraction of 13%

16. The effect of Xrays on synchronously dividing cell cultures Survival fraction increases rapidly with time as cell enters S phase The proportion of surviving cells fall as the cells move out of S and into G2 This pattern characteristic for Chinese hamster cells

17. TIME-SURVIVAL FRACTION FOR CHINESE HAMSTER CELL

18. CELL SURVIVAL CURVES FOR CHINESE HAMSTER CELLS AT VARIOUS CELL CYCLE STAGES

19. TIME SURVIVAL FRACTION FOR HELA CELLS

20. Variation of radiosensitivity with cell age in the mitotic cycle Cells are most sensitive at or close to mitosis Resistance is usually greatest in late S phase. The increased resistance is thought to be caused by homologous combination repair between sister chromatids that is more likely to occur after the DNA has replicated

21. If G1 phase has an appreciable length, a resistant period is evident early in G1 followed by a sensitive period towards the end of G1 G2 phase is usually sensitive, perhaps as sensitive as the M phase

22. RETROACTIVE SYNCHRONISATION Greater resolution for studying G2 sensitivity Early G2 = Late S Late G2 = M Xray transition point is the checkpoint where sharp transition in radiosensitivity occurs for G2 cell cycle decay

23. MOLECULAR CHECK POINT GENES Family of genes that control cell cycle progression Mammalian cells exposed to radiation - Block in the G2 In several strains of yeast , mutants have been isolated that are more sensitive than the wild type to both ionising radiation and UV light by a factor between 10 and 100 The mutant gene has been cloned and sequenced and found to be a “ G2 molecular checkpoint gene”

24. Mutant cells that lose this G2 checkpoint gene function move directly into mitosis with damaged chromosomes They are at a higher risk of dying – hence their greater sensitivity to radiation and other DNA damaging agents Cells that survive mitosis are likely to give rise to errors in chromosome segregation – hence more prone to carcinogenesis

25. Effect of O2 at various phases of the cell cycle Characterised by Oxygen enhancement ratio(OER) OER = Dose in hypoxic conditions Dose in aerated conditions Greatest in S (2.8-2.9)> G1> G2(2.3-2.4)

26. AGE RESPONSE CURVE FOR A TISSUE IN VIVO Epithelial lining of mouse jejunum Intraperitoneal inj. Hydroxy urea Q1H*5 Single dose 11 Gy ϒ rays at various times Examined sectioned jejunum

27. High LET radiation decreases the variation of radiosensitivity through the cell cycle At v.high LET – Age response function almost straight line

28. MECHANISMS FOR AGE RESPONSE FUNCTION The patterns of radiosensitivity and radio-resistance correlate with the mechanism of repair of DNA DSB’s Radiosensitivity correlates with non homologous end joining, which dominates early in the cell cycle and is error prone Radioresistance correlates with homologous recombination of DSB’s

29. IMPLICATIONS IN RADIOTHERAPY Since general population of cells in tissues is asynchronous , cells in more sensitive phases of the cycle are preferentially killed Variations in sensitivity through the cell cycle may be important in radiation therapy because they lead to sensitization resulting from reassortment in a fractionated regimen.