Radiosensitivity and cell age in the mitotic cycle

RADIOSENSITIVITY AND CELL AGE IN THE MITOTIC CYCLE

RADIOSENSITIVITY AND CELL AGE IN THE MITOTIC CYCLE

Sneha Susanna George

THE CELL CYCLEOrdered process by which a cell grows and divides into 2 progeny daughter cells

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

CELL CYCLE TIME/MITOTIC CYCLE TIME

Time between 2 successive mitotic divisions

CELL LABELLING TECHNIQUES For cell cycle analysis Markers of DNA synthesis Introduced by Howard and Pele in 1953

Autoradiography

Tritiated thymidine5-Bromodeoxyuridine

Tritiated thymidine(3H-TdR) incorporated into chromosomes S phase cells take up 3H-TdRCells are fixed and stainedCovered with a nuclear(photographic) emulsionLeft in refrigerator for 1 monthForm latent images that appear as black grains

5 bromodeoxyuridine

More convenient1) no radioactive material2) 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

Comparison of Cell Cycle Hamster and Hela cells

REGULATION OF THE CELL CYCLEBy 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

REVERSIBLE IRREVERSIBLE INACTIVATION INACTIVATIONBy 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

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

SYNCHRONOUSLY DIVIDING CELL CULTURESPopulations 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)

MITOTIC HARVEST TECHNIQUEFirst described by Terasima and Tolmach

Physical separation of cells preparing for mitosis

Works on monolayer cell cultures

Use of a drugHydroxyurea is used1) Kills S phase cells2)Blocks the cell cycle at G1

The effect of X-rays on synchronously dividing culturesDose used 6.6Gy(660 rad) Chinese hamster cells were subjected to this at various phases of the cell cycleProportion of cells that survive the dose - Survival fractionCells in G1 - survival fraction of 13%

The effect of Xrays on synchronously dividing cell culturesSurvival fraction increases rapidly with time as cell enters S phaseThe proportion of surviving cells fall as the cells move out of S and into G2This pattern characteristic for Chinese hamster cells

TIME-SURVIVAL FRACTION FOR CHINESE HAMSTER CELL

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

TIME SURVIVAL FRACTION FOR HELA CELLS

Variation of radiosensitivity with cell age in the mitotic cycleCells are most sensitive at or close to mitosisResistance 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

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

RETROACTIVE SYNCHRONISATIONGreater resolution for studying G2 sensitivityEarly G2 = Late SLate G2 = MXray transition point is the checkpoint where sharp transition in radiosensitivity occurs for G2 cell cycle decay

MOLECULAR CHECK POINT GENESFamily of genes that control cell cycle progressionMammalian 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 100The mutant gene has been cloned and sequenced and found to be a G2 molecular checkpoint gene

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

Effect of O2 at various phases of the cell cycleCharacterised 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)

AGE RESPONSE CURVE FOR A TISSUE IN VIVO Epithelial lining of mouse jejunum

Intraperitoneal inj. Hydroxy urea Q1H*5

Single dose 11 Gy rays at various times

Examined sectioned jejunum

High LET radiation decreases the variation of radiosensitivity through the cell cycle

At v.high LET Age response function almost straight line

MECHANISMS FOR AGE RESPONSE FUNCTIONThe patterns of radiosensitivity and radio-resistance correlate with the mechanism of repair of DNA DSBsRadiosensitivity correlates with non homologous end joining, which dominates early in the cell cycle and is error proneRadioresistance correlates with homologous recombination of DSBs

IMPLICATIONS IN RADIOTHERAPYSince 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.

MitosisInterphaseTotal

MG1SG2

Hamster116311

HeLa1118424