cell cycle checkpoints, apoptosis and cancer
TRANSCRIPT
Surender Rawat
M. Sc. Microbial Biotech
Roll no. 1784
CELL CYCLE• Includes 4 coordinated processes-
– Cell growth
– DNA replication
– Disrtribution of chromosomes
– Cytokinesis
In bacteria, cell growth and DNA replication takes place throughout the cell cycle.
In eukaryotes, it consists of four phasesG1 phase – Gap 1Synthesis phase- S phaseG2 phase- Gap 2Mitosis phase – M phase
CELL CYCLE
Different systems-- yeast: cell cycle mutations-- frog: big dividing embryos-- sea urchin & clam: many
embryos
Asynchronously dividing cells DNA/nucleus staining Flow cytometry
Synchronously dividing cells
DAPI stained cells
REGULATION OF CELL CYCLE BY EXTERNAL FACTORS
• Major regulatory point in G1 in Saccharomyces cerevisiae
• Called START
• Once START is passed cells are commited to enter the S phase.
• It is highly regulated and controlled by extracelluar signals.
– Nutrients
– Mating factors
– Cell size
• If signals are absent the cells are arrested at G1.
• This quiescent stage is called G0 in which they are metabolically active but cease growth.
• Eg. Skin fibroblasts, nerve cells
CELL CYCLE CHECKPOINTS
• Prevent entry into next phase of the cell cycle.
• Also called DNA damage checkpoint
MOLECULES OF CELL CYCLE REGULATION
• Three experimental approaches contributed to identification of key molecules responsible for cell cycle regulation
1. Identification of MPF in frog oocytes
2. Identification of cdc molecules in Saccharomyces cerevisiae mutants
3. Identification of cyclins in sea urchin embryos
DISCOVERY OF MPF
YOSHIO MASHUI
IDENTIFICATION OF CDK• Studied cdc mutants of Saccharomyces cerevisiae
• These required Cdc28 to pass START
•Studied cdc mutant of Schizosaccharomyces pombe•Discovred cdc2 which arrest cell cycle at G1 and G2 to M transition
LEE HARTWELL
Cdc28 and cdc2 were homologous and coded for a kinase known as Cdk1
PAUL NURSE
IDENTIFICATION OF CYCLINS• Stuied in sea urchin and calm embryo in
1983
• Accumulation in interphase and degradation in the end of mitosis
• Hunt called these cyclin A and cyclin B
• In 1986, Joan Ruderman showed cyclin A triggers G2 to M transition in frog oocyte
STRUCTURE OF MPF
• Purified in 1988 by James Maller from frog eggs
• MPF is composed of two subunits
– Cdk1 – catalytic subunit
– Cyclin B – regulatory subunit
• Cyclin B is synthesized and form complexes with cdk1 during G2.• Phosphorylation of cdk2 at threonine 161 is required for its activity• Phosphorylation of tyrosine 15 by wee1, inhibits cdk1 activity and
leads to the accumulation of cdk and cyclin B complex.• Activation is by deposphorylation of threonine 14 and tyrosine 15 by
the phosphotase cdc25 for G2 to M transition
MPF Regulation
Mechanism of cdk regulation
Association with cyclins
Phosphorylation at threonine 160 by Cak composed of cdk7 and cyclin H
Inhbitory Phosphorylation at threonine 14 and tyrosine 15
2 families of cdk inhibitors Ink family cip/kip family
•Ink family (p15, p16, p18, p19) cdk4/cdk6 G1
•Cip/kip family (p21, p27, p57) cdk2/cyclin E G2cdk2/cyclin A S
Families of cyclins and cyclindependent kinases
• In eukaryotes
– G1 to S - cdk2, cdk4,cdk6 + cyclin D & E
– G0 – cdk4, cdk6 + cyclin D1,D2 & D3
– Late G1 - cdk2 + cyclin E1 &E2
– Through S – cdk2 + cyclin A1 & A2
– S to G2 – cdk1 + cyclin A1 & A2
– G2 to M – cdk1 + cyclin B1, B2, & B3
GROWTH FCTORS AND REGULATION OF G1 CDKs
• Cyclin D synthesis is induced in response to growth factor stimulation
• If growth factors are removed cyclin D level decreses
• Cdk4, 6/cyclin D complex drive cells through START
• Mutations in continual unregulated expression of cyclin D is associated with many lymphoma and breast cancer.
• Mutations that inactivate cdk4 and cdk6 are found in cancer cells• A substrate of cdk4/6 is Rb protein which is a tumor suppresor• Inactivation of Rb gene leads to cancer• This complex is inhibited by p16
Role of Rb
• When Rb is phosphorylated by cdk4,6/cyclin B then it binds to E2F which transcribes cyclin E
• Prgression through restriction point and entry into S phase is mediated by cdk2/cyclin E complex
• MCM helicase is activated by cdk2/cyclin E complex
• It is degraded by p27
DNA DAMAGE CHECKPOINTS
• These checkpoints are operative in G1, S & G2
• Cell cycle arrest is mediated by 2 proteins
– ATM – ds breaks
– ATR- ss breaks
• These are activated in response to DNA damage and then activate the signalling pathway that leads to cell cycle arrest, activation of DNA repair and apoptosis
ROLE OF p53
• Progression to anaphase is mediated by activation of Anaphase promoting complex/cyclosome (APC/C)
• Unattached kinetochores lead to the assembly and activation of a complex of Mad/Bub proteins that inhibit APC/C binding to cdc20.
SPINDLE ASSEMBLY CHECKPOINT
• Once all chromosomes are aligned on the spindle, the Mad/Bub complex dissociates, relieving inhibition of Cdc20 and leading to APC/C activation.
• APC/C ubiquitinates cyclin B leading to inactivation of cdk1.• In addition, APC/C ubiquitinates securin, leading to activation
of separase.• Separase degrades a subunit of cohesin, breaking the link
between two sister chromatids and initiating anaphase.
SPINDLE ASSEMBLY CHECKPOINT
SPINDLE ASSEMBLY CHECKPOINT
• APC/C degrades cyclin B which is necessary for exiting mitosis and return to interphase
• Inactivation of cdk1 is triggers cytokinesis
• Abnormalities in chromosome segregation is resulting from failure of spindle assembly checkpoint are common in cancer cells and are thought to play an important role in many tumors.
Cdk1/cyclin B
Chromatin condensation
Nuclear envelopebreakdown
Fragmentation of Golgi apparatus
Spindle formation
• Programmed cell death is a active process which usually proceeds with a distinct series of cellular changes known as apoptosis.
– Chromosomal DNA is fragmented
– Chromatin condenses
– Nucleus breaks up
– Cell shrinks into apoptotic bodies
• Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms.
• Cells die in response to a variety of stimuli and during apoptosis they do so in a controlled, regulated fashion.
SIGNIFICANCE
• Balances cell proliferation
• Maintains constant cell numbers in tissues
– 5 × 1011 blood cells eliminated daily in bone marrow
• Provides a defense mechanism
– Eliminates Virus infected cells
– Eliminates DNA damaged cells
• Plays a key role in development
– Elimination of tissues between the digits
– 50% neurons are eliminated by apoptosis
webbed fingers
Apoptosis during the metamorphosis of a tadpole into a frog.
The cells in the tadpole tail are induced to undergo apoptosis stimulated by
the increases in thyroid hormone that occurs during metamorphosis.
The nematode
Caenorhabditis elegans has
also been a very important
model system for studying
apoptosis in development.
Two distinct forms of cell death – apoptosis and necrosis
Programmed cell death eliminates unwanted cells
www.imm.ki.se/ sft/bilder/Image1.jpg
Murder? Suicide?
Classical View of Cell Death: Apoptosis vs Necrosis
Comparison between two forms of cell death, apoptosis and necrosis
Apoptotic cells are biochemically recognizable
Characteristic biochemical changes in cells undergoing apoptosis
1. Cleavage of DNA into fragments at internucleosome site2. Chromatin condensation3. Change in the plasma membrane – phosphatidylserine in the
outer leaflet4. Cytoplasm shrinkage5. Membrane blebbing6. Loss of electrical potential across the inner membrane of the
mitochondria7. Relocation of cytochrome c8. Corpse clearance via phagocytosis
Fragmentation of Golgi bodies
Phagocytosis of apoptotic bodies
Apoptotic
cellNuclear
fragments
HISTORY
•Studied Development of C. elegans in 1986
• Specific 130 out of 1090 cells are eliminated by PCD
• Identified 3 genes by mutagenesis
•Ced 3 - PCD •Ced 4 - PCD•Ced 9 - Regulator
ROBERT HORVITZ
PHOTOGRAPH OF A NORMAL WORM AND A Ced 3 MUTANT
Lodish et al. 6th Figure 21.37 Evolutionary conservation of apoptotic pathways
Caspases : The executioner of Apoptosis
•Caspases involved in inflammation caspases 1 , 4, 5
•Caspases involved in apoptosis Initiator caspases caspases 2, 8, 9, 10 Executioner caspases caspases 3, 6, 7
Cysteine-dependent aspartate specific proteases
APAPTOSOME
Caspase
Inhibitors of DNase
Nuclear lamins
Cytoskeletal proteins
Golgi matrix proteins
CENTRAL REGULATORS OF APOPTOSISBcl2 proteins regulate the intrinsic pathway of apoptosis
The three classes of Bcl2 proteins
inhibit apoptosis
promotes apoptosis
bind and regulate the anti-apoptotic BCL-2 proteins to promote apoptosis
INTRINSIC PATHWAY OF APOPTOSIS
The intrinsic pathway of apoptosis depends on mitochondria
IAPs inhibit caspases
• IAP – inhibitors of apoptosis directly intract with caspases and supress apoptosis by
• inhibiting caspase activity• ubiquitination and degradation of caspases
• Present in Dorsophila and mammals
ROLE OF p53 IN APOPTOSIS
EXTRINSIC PATHWAY
Three ways that extracellular survival factors can inhibit apoptosis
• Uptake of proteins or orgenelles into vesicles (autophagosomes) that fuse with lysozomes.
• Promotes cell survival under starvation
• Does not require caspases
• Characterized by accumulation of lysozomes
• Alternative pathway of cell death when apoptosis is blocked
• Bak/Bax cells die by autophagy
AUTOPHAGY – ALTERNTIVE WAY OF CELL DEATH