Mitotic cell cycle and radio sensitivity
- 1. THE CELL CYCLE By Dr prathibha First year resident Radiation oncology
- 2. CELL CYCLE • Cells divide into new daughter cells through a series of events that takes place in steps. • Acell cycle is thus a sequence of events that cell goes through as it grows and divides to produce new cells. • Therefore it can be called the life cycle of a cell.
- 3. THE CELL CYCLE TIME • Cells propagate and proliferate by the process of mitosis. • When the cell divides two progeny are produced which carries a chromosome identical to the parent cell. • The progeny cells undergo further division after a interval of time. • The time between the successive division is known as the cell cycle time Or mitotic cycle time.
- 4. CELL CYCLE
- 5. PHASES OF CELL CYCLE • The description of phases of the cell cycle ( M, G1, S, G2) are given by Howard and pelc in 1953. • A cell cycle consists of • 1) Interphase • 2) mitotic phase • 3) G0 phase.
- 6. INTERPHASE • Recognized by the growth period where the chromosome gets duplicated as the cell prepares for division. • It is the longest part of the cell cycle. • It involves three sub phases • G1 phase • S phase • G2 phase.
- 7. • Also known as first gap phase • During this period • Cells grows and increase in size • Synthesize cell organelles and other macromolecules such as proteins that serve as the building block of the cell. • Cells also accumulate suuficient energy required for division. G1 PHASE
- 8. S PHASE • The process of DNA synthesis and replicaation taakes place. • The centrosome is duplicated during this phase and give rise to spindle fibres.
- 9. G2 PHASE • This is the second gap phase. • The cells grow further in size making more proteins and organelles. • Preparation for mitosis gets completed before the cells enters the mitotic phase.
- 10. INTERPHASE
- 11. MITOTIC (M) PHASE • This period also known as the cell division phase. • It occurs just after the G2 phase. • The cells divide it’s genetic material DNA and cytoplasm to form two new cells. • The M phase involves • Karyokinesis • Cytokinesis.
- 12. KARYOKINESIS • The nuclear division consists of four phases • Prophase • Metaphase • Anaphase • Telophase • During this period the cell divides the nucleus and gets separated into two daughter cells where each daughter cell recieves a complete set of chromosomes.
- 13. PROPHASE • In prophase the chromosome thickens in the shape of a dumbbell with a constriction at centre called centromere. • The nuclear membrane breaks open leading to mixture of cytoplasm and nuclear material. • And spindles made of fibres are formed extending from one end of the cell to the other.
- 14. METAPHASE • The chromosome move to the centre or at the equatorial plane of the cell. • Microtubules are formed around centrioles. • Centrioles which are present on either ends of the cell
- 15. ANAPHASE • Two chromatids move to the two poles of the cell.
- 16. TELOPHASE • It involves the deconvolution of the chromosomes leading to the regeneration of the nuclear membrane and nucleoi around both poles. • Division of cytoplasm sets in and ultimately two daughter cells are formed.
- 18. G0 PHASE • Some cells do not immediately enter another round of preparatory phase or interphase following the division or mitosis. • Instead they exit the G1 growth phase and enter a resting stage called G0 phase. • Thus the G0 phase is also called the alternative phase of the cell cycle. • Some cells enter the G0 phase temporarily until an outside signal triggers the onset of G1.
- 19. EXTRACELLULAR SIGNALS STIMULATE A CELL TO ENTER THE CYCLE FROM DORMANT PHASE.
- 20. CELL CYCLE REGULATION • Regulation occurs by activation of different members of the cyclin dependent kinase (cdk) family. • In it’s active form each cdk is complexed with a particular cyclin. • Different cdk cyclin complexes are required to phosphorylate protein subtrates that drive cell cycle evnts as
- 21. CELL CYCLE REGULATION • 1) The initiation of DNA replication. • 2) The onset of mitosis. • 3) preventing the initiation of a cell cycle event at wrong time.
- 22. CELL CYCLE REGULATION • Cyclin D , cyclin E - G1 • Cyclin A -S, G2 • Cyclin B - G2, M
- 24. CHECKPOINT PATHWAYS • Cell cycle must take place in a specific order. • Check points are present • 1) To ensure initiation of late events is delayed until earlier events are completed.
- 25. G1 CHECK POINT • Also known as G1 restriction point. • Normally G1 prepare the cell for DNA synthesis. • There is a stage known as G1 restriction point after which cells are committed to enter S phase. • No longer respond to growth conditions.
- 26. G1 CHECKPOINT • Prior to G1 restriction point cell may progress, differentiate or die depending on external signals. • Key players in G1 restriction point • 1) protein of retinoblastoma (Rb gene) • 2) D type cyclins • Cdk4 and cdk6 • Cdk inhibitors.
- 27. S CHECKPOINT • If there is any mistake in the synthesis and replication of DNA during S phase of the cell cycle. • S checkpoint will ensure to occir the defect during S phase. • Later on the remaining phases of cell cycle will occur.
- 28. G2/M CHECKPOINT • Any defect during the processing of G2 phase of cell cycle • G2 checkpoint will ensure to correct the defect. • Then the later phase mitosis will occur only after the correction of any defects during G2 phase.
- 30. RADIATION DAMAGE TO DNA MOLECULE • It can be due to • 1) loss of base • 2) cleavage of the hydrogen bond between bases. • 3) Breakage of single strand of DNA molecule. • 4) Breakage of both strands of the DNA molecule. • Double strand break occurs the odds to repair decrease.
- 31. RB GENE • Acts on G1S checkpoint • Normal rb gene is active, hypophosphorate, closes G1S gate. • Cyclin D and cyclin E form complexes with rb gene • Where the rb gene will Become inactive, hyerphosphorylate, opens G1s gate. • Rb gene mutation leads to loss of function • G1S gate will be always open.
- 32. CONGENITAL RB SYNDROME • Bilateral more common. • Trilateral RB • Bilateral in both eyes • Pineal gland tumor
- 33. P53 GENE • Also known as guardian angel of genome. • Most common mutated in human cancers. • P53 gene detects any mutation in genome • Stops the cell cycle at G1S Or G2M • If the defect gets repaired cell cycle will continue. • If the defect is not repaired proapoptotic genes will get activated leading to apoptosis.
- 35. AUTORADIOGRAPHY • TECHNIQUE WITH THE USE OF PHOTOGRAPHIC EMULSIONS TO STUDY OCCURRENCE AND DISTRIBUTION OF RADIOACTIVE SUBSTANCES. • IN OUR CASE, THE PRINCIPAL IS TO FEED THE CELLS WITH THYMIDINE (FOR DNA SYNTHESIS), WHICH IS LABELLED WITH RADIOACTIVE TRITIUM. • THE CELLS ACTIVELY SYNTHESIZE NEW DNA, AS A PART OF CHROMOSOME REPLICATION PROCESS, INCORPORATES THE RADIOACTIVE THYMIDINE. • THESE CELLS ARE FIXED, STAINED AND COATED WITH PHOTOGRAPHIC EMULSION, AND LEFT FOR SEVERAL WEEKS IN REFRIGERATOR. • BETA PARTICLES FROM TRITIATED THYMIDINE, PASSES THROUGH THE EMULSION AND FORM IMAGES, WHICH APPEAR AS BLACK GRAINS, INDICATING THAT THEY WERE ACTIVELY SYNTHESIZING DNA WHEN RADIOACTIVE THYMIDINE WAS AVAILABLE.
- 36. 5-BROMODEOXYURIDINE • 5-BROMODEOXYURIDINE, is used as DNA precursor and incorporated in the ‘S phase’ of the cycle. • It can be recognised either by using a Geimsa stain (purple) or with a Monoclonal Antibody to bromodeoxyuridine, which will be tagged with fluorescein dye, which shows up bright green under fluorescent microscope.
- 37. 5-BROMODEOXYURIDINE • CELLS WERE GROWN IN THE PRESENCE OF BROMODEOXYURIDINE AND THEN FIXED AND STAINED 20 HOURS LATER. • INCORPORATED BROMODEOXYURIDINE STAINS PURPLE. THE PURPLE-STAINED INTERPHASE CELL WAS IN S PHASE DURING THE TIME THE BROMODEOXYURIDINE WAS AVAILABLE.
- 38. CELLS LABELLED AND UNLABELLED WITH BROMDEOXYURIDINE
- 39. RADIOSENSITIVITY • The study about the variation of Radiosensitivity with age of the cell cycle was possible by development of techniques to produce synchronously dividing cell cultures. • A population of cells where all the cells occupy same phase of cell cycle at a given time. • 2 techniques were devised: • Mitotic Harvest Technique by Terasima and Tolmach. • Using drugs like HU
- 40. MITOTIC HARVEST TECHNIQUE • Can be used only for cultures that grow in monolayers, attached to the vessel surface. • It exploits the fact that if cells are close to mitosis, they round up and become loosely attached to the surface. • If the growth medium over the cells is subjected to gentle motion (by shaking), mitotic cells detach from the surface and float in the medium. • This medium is removed and plated into new petri dishes where the population consists almost entirely of mitotic cells. • By delivering a dose of radiation at various times after the initial harvesting of mitotic cells, one can irradiate cells at various phases of the cell cycle.
- 41. HYDROXYUREA EXPERIMENT • Alternate method for synchronizing cells, which is applicable to cells in a tissue as well as cells grown in culture, involves the use of a drug, HYDROXYUREA. • First, all cells that are synthesizing DNA (S phase) take up the drug and are killed. • Second, the drug imposes a block at the end of the G1 period; cells that occupy the G2, M, and G1 compartments when the drug is added progress through the cell cycle and accumulate at this block.
- 42. EFFECT OF X-RAYS ON SYNCHRONOUSLY DIVIDING CELL CULTURES • An experiment was conducted in mammalian cells, which were harvested at mitosis, and irradiated with a single dose of 6.6 Gy at different phases of the cell cycle. • 1 hour after the mitotic cells are seeded into the petri dishes, when the cells are in G1, a dose of 6.6 Gy results in a surviving fraction of about 13%. • The proportion of cells that survive the dose increases rapidly as the cells move into S phase; by the time the cells near the end of S phase, 42% of the cells survive this same dose. • When the cells move out of S phase into G2 phase and subsequently to a second mitosis, the proportion of surviving cells falls again.
- 43. THANK YOU