The cell cycle is the ordered process by which a cell grows and divides into two daughter cells. It consists of mitosis (M phase) and interphase (G1, S, and G2 phases). Cell cycle time is measured between successive mitotic divisions. Cell labeling techniques like autoradiography using tritiated thymidine or bromodeoxyuridine are used to identify cells in the DNA synthesis (S) phase. The cell cycle is regulated by cyclin-dependent kinases whose activity drives cell cycle progression. Radiosensitivity varies through the cell cycle, with cells most resistant in late S phase and most sensitive in G1, G2, and M phases. Fractionating radiation doses takes advantage of reassortment

1. Sneha Susanna George

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

3.  MITOSIS - 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 Pele in 1953
 Autoradiography
1) Tritiated thymidine
2) 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

9. Comparison of Cell Cycle –
Hamster and Hela cells
Mitosis Interphase
M G1 S G2
Total
Hamster 1 1 6 3 11
HeLa 1 11 8 4 24

10. 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

11. 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

12.  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

15. 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)

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

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

18. 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 G1 - survival fraction of 13%

19. 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

20. TIME-SURVIVAL FRACTION FOR
CHINESE HAMSTER CELL

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

22. TIME SURVIVAL FRACTION FOR
HELA CELLS

23. Variation of radiosensitivity with
cell age in the mitotic cycle
1. Cells are most sensitive at or close to mitosis
2. 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

24.  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

25. 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

26. 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”

27.  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

29. 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)

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

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

34. 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

35. 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.