cell cycle and dna damage

1. CELL CYCLE,
APPLICATIONS,
DNA DAMAGE AND
REPAIR
By Dr. Shounak J. Kamat
1st year Resident
DNB Radiation Oncology
HCG Cancer Centre- Borivali-
Mumbai
Guide:- Dr. Trinanjan Basu

2. Overview
 Cell Cycle
 Regulation of Cell Cycle
 Effect of Radiation on Cell Cycle
 DNA Damage
 DNA Repair Mechanisms

3. Cell Cycle
 A cell cycle is a precisely programmed series of
events which enables a cell to duplicate its
contents and divide into 2 daughter cells
 It has the following phases:-
1] G1 phase
2] S phase Interphase
3] G2 phase
4] M phase or the Mitosis phase
5] G0 or the resting state of cells that
have withdrawn from active cell cycle

4. Interphase
It is the phase in which cells spend most of their lives preparing for mitosis.
It is divided into 3 stages:-
1] G1 phase – phase of cell growth and protein synthesis
2] S phase – phase where DNA and centrosomes are replicated
3] G2 phase – phase where energy replenishment, mitotic specific protein
synthesis, cytoskeleton dismantling and additional growth take
place.

6. Mitosis Phase

7. Prophase
 In this phase the chromosomes which
were invisible microscopically during
interphase begin to condense and
become visible
 Also centrosomes begin to assemble at
the poles of cells

8. Metaphase
 In this phase the chromosomes align
along a plane that bisects the cell and
become attached to microtubule fibres
of mitotic spindle
 Also the nuclear membrane
disappears at this time

9. Anaphase
 During this phase the chromatids are
pulled apart by the mitotic spindle to the
opposite poles of the cell.

10. Telophase and Cytokinesis
 The chromatids cluster into two sets ,
they de-condense and a new nuclear
membrane forms around each set of
chromatids
 During this time the cytoplasm of the cell
also divides into two thus yielding two
daughter cells

11. Regulation of Cell Cycle

12. Cyclin Dependent Kinases
They are serine/threonine kinases that sequentially regulate progression of cell
through the cycle via phosphorylation.
They do this via 4 mechanisms
 Association with cyclins
 Assosiation with CDK inhibitors
 Addition of phosphate groups to activate CDK activity
 Deletion of phosphate groups to inhibit CDK activity

13. Association with Cyclins

14. Association with Inhibitors
 2 Families of inhibitors are involved in regulating cyclin–cdk activity:
- p16 INK 4a family
- p21 Cip/Kip family
 INK Protein binds to cdk 4/6 and interferes with its binding to cyclin D
 Cip/Kip family of inhibitors interact with both cyclins and their associated cdks
(mainly with cdk2 and cyclin E) and disable kinase activity
 ubiquitin-mediated degradation of inhibitors ensures that the inhibitors are
present during a specific period of time during the cell cycle.

15. Regulation by Phosphorylation
 This involves both activation and inhibition
 Two steps are required for cdks to become active:
1] Dephosphorylation of the inhibitory phosphate groups by cdc25 phosphatases
2] Phosphorylation of a central threonine residue- Thr161 by cdk-activating kinase
(CAK).

16. Cell Cycle Checkpoints
 Signaling pathways that sense and
induce a cellular response to DNA
damage.
 The components are DNA damage
sensors, signal transducers, or
effectors.
 Disruption of checkpoint function
leads to genomic and chromosomal
instability leading to mutations that
can induce carcinogenesis

19. Role of p53 Gene
 p53 is a tumor suppressor gene that is critical in the pathway that arrests the G1
checkpoint.
 In response to DNA damage, ataxia telengectasia mutated (ATM)
autophosphorylates and releases active monomer which phosphorylates p53 and
activates it.
 Activated p53 enhances p21 gene expression which results in sustained inhibition
of G1 cyclin and Cdks.
 This in turn inhibits Rb phosphorylation and progression from G1 to S.
 Mutation in this gene compromises this checkpoint function and results in
damaged dna replication leading to carcinogenesis.

20. Effect of Radiation on the Cell Cycle Phases

21. In Chinese hamster cells
 Most sensitive cells to Radiation are the ones
in M and G2 [ steep curve, no shoulder]
 Most radioresistent cells are in late S phase
[less steep curve but very broad shoulder]
 Other phases G1 and Early S are intermediate
between the two extremes.

22. In HeLa Cells
Similar to the hamster cells in most
aspects
Major difference is length of G1 phase in
HeLa cells is appreciably long and at the
beginning of G1 there is a peak of
radioresistance followed by a trough of
radiosensitivity towards end of G1.

24. CELL CYCLE SPECIFIC DRUGS
During M Phase:-
Taxanes and Vinca Alkaloids cause a synergistic action by acting on DNA and causing its damage.
During S Phase:-
Drugs such as Capecitabine, Gemcitabine and 5-fluorouracil inhibit nucleotid formation and DNA replication.
Since this phase is radioresistant, they cause a synergistic action with radiation.
During G2-M phase:-
Drugs such as Topoisomerase Inhibitors arrest the cells in G2M phase which is a radiosensitive phase thus
causing increase in the effect caused by radiation.

25. EFFECT OF OXYGEN ON CELL CYCLE
 OXYGEN ENHANCEMENT RATIO (OER)
Ratio of doses administered under hypoxic to aerated conditions needed to
achieve same biologic effect.
OER for Xrays and Gamma rays has a value between 2.5 to 3.5.
 With respect to cell cycle the OER in the G2 and M phase and also in the G1
phase is lower than in S phase because of the radiosensitivity of these phases.
 Thus maintaining an aerated state helps in increasing the efficacy of the
radiation treatment.

26. DNA Damage

27. Structure of DNA
DNA is made up of 2 strands and
arranged in a helical pattern.
The strands are formed of a Deoxy
ribose sugars and phosphate
molecules.
On those strands are the purine and
pyrimidine molecules which are
bound by Hydrogen bonds.

28. Effect of Radiation on DNA
Direct Effect Indirect Effect
There are 2 effects :-

29. Process of free radical formation

30. Types of DNA damage
 Single strand breaks
 Double strand breaks
 DNA crosslink formations
 Radiation induced chromosome and chromatid aberrations.

31. Single strand breaks
This occurs when one strand of DNA is
damaged or broken.
More common type of defect
Is readily repaired because of availability
of template on intact opposite strand.
Of lesser biologic significance
Approx. 1000 SSBs per cell after 1 -2 Gy

32. Double strand break
This occurs when both the strands of
DNA are damaged.
Less common defect
Has more significance since this takes
time to be repaired and can result in
mutations, carcinogenesis and cell
death
Approx. 40 DSBs after 1-2 Gy

33. Measuring DNA Strand Breaks
 Pulsed Field Gel Electrophoresis (PFGE)
 Single-cell Gel Electrophoresis (also known as the comet assay).
 DNA damage induced nuclear foci assay

34. DNA crosslink formations
This occurs in oxidative stress when O2 free radicals form intermediaries which then
react with DNA nucleotides and form covalent links between the nucleotides.
They can be of the following types
 Intrastrand crosslinks- when crosslinking occurs within same strand
 Interstand crosslinks- when crosslinking occurs between opposite strands of DNA
 DNA Protein crosslink- between DNA and an oxidised protein

36. Radiation Induced Chromosome Aberrations
Occurs when cells are irradiated in the early interphase stage before the chromosome
has been duplicated.
Can be of the following types
1] Dicentric chromosome 2] Ring aberration

37. Radiation Induced Chromatid Aberration
Occurs when the cell is irradiated in the late interphase after the chromosome has
duplicated
Anaphase Bridge

38. Non Lethal Chromosome Lesions
Translocations Deletions

39. DNA Damage Repair Mechanisms

40. Base Excision Repair
 Removal of the defective base by a
Glycosylase/DNA lyase
 Followed by removal of the sugar
residue by an apurinic endonuclease
1 (APE1)
 then replacement with the correct
nucleotide by DNApolymerase
 completed by DNA ligase
 the complex of replication factor C
(RFC)/proliferating cell nuclear
antigen (PCNA)/DNApolymerase /
performs the repair synthesis,
 the overhanging flap structure is
removed by the flap endonuclease 1
(FEN1)
 DNA strands are sealed by ligase I
Single Base Defect Multiple Base Defect

42. Nucleotide Excision Repair
 Nucleotide excision repair (NER) removes bulky adducts in the DNA such as pyrimidine
dimers.
 Subdivided in 2 pathways:-
1] Global genome repair:- to repair DNA that encodes or does not encode for genes.
2] Transcription coupled repair:- to repair DNA strands of actively transcribed genes
 Post DNA damage, RNA polymerase blocks access to the site of damage. The TC-NER
prevents this blockade by removing RNA polymerase from the site.

43.  The 2 mechanisms differ only in detection
of lesion, remaining pathway is same.
 The essential steps in this pathway are:-
(1) damage recognition
(2) DNA incisions that bracket the lesion
usually 24 to 32 nucleotides in length.
(3) removal of the region containing the
adducts
(4) repair synthesis to fill in the gap
region
(5) DNA ligation

44. DNA Double-Strand Break Repair
Homologous Recombination
Repair [HRR]
This requires an undamaged DNA
strand as a participant in repair as a
template
Error free process since repair is
performed by copying information from
undamaged homologoes chromatid.
Primararily occurs in late S/G2 phase
when undamaged sister chromatid is
available to act as template
Non Homologous End Joining
[NHEJ]
This mediates end to end joining
Error prone and accounts for many
premutagenic lesions induced in DNA by
ionizing radiation
Occurs in G1 phase when template
doesn’t exist..

45. NHEJ
 NHEJ can be divided into five steps:
(1) end recognition by Ku binding
(2) recruitment of DNA-dependent protein
kinase catalytic subunit (DNA-PKcs)
(3) end processing
(4) fill-in synthesis or end bridging
(5) ligation

46. HRR
 In this, two of the protiens
used are encoded by genes
BRCA 1 and 2
 Accessory factors such as
Rad54, Rad 54B and Rdh54
help recognize and invade
the homologous region.
 After D-loop formation,
DNA polymerase is involved
to elongate the invading
strand.

47. Cross Linking Repair
Steps
Signals for repair is stalling of the DNA replication fork.
The crosslink is removed in a multistep process:-
1] Crosslink is removed from one strand by a NER, resulting in a
strand break and a DNA adduct.
2] The strand break requires HRR for restitution.
3] Finally, the adduct that remains is removed by NER

48. Miss Match Repair
 The mismatch repair (MMR) pathway removes
base–base and small insertion mismatches that
occur during replication.
 Steps
1] The mismatch must be identified by
sensors that transduce a signal of a
mismatched base repair
2] MMR factors are recruited
3] The newly synthesized strand harboring
the mismatch is identified and the
incorrect/ altered nucleotides are excised
4] Resynthesis and Ligation of the DNA.