Cell cycle Flashcards
What stage of the cell cycle are most of the adult cells in the body?
G0
some reenter in response to stimuli others are terminally differentiated
what are the two main concepts that the cell cycle adheres to?
in normal cells
Alternation - cell cycle evenets always occur in the right order
Completion - one thing must finish before the next one starts
Describe the early fusion experiments that gave 2 clear clues about the cell cycle
explain the results
- Separate cells based on cell cycle state (mitotic cells roll up from plate, can be easily collected) (polyethylene glycol used to merge membrane of cells, fuse them together so there’s two nuclei)
o Fuse a mitotic cell with any other, it tells the other nucleus to go into mitosis – dominant
o Merge S with G1 you end up with the s phase signal being dominant (later stage is always dominant)
o Merge S and G2 cells and fuse – you get a signal from the s phase cell and you get a g2 delay
(Do not want cell to progress from g2 into mitosis if the dna isn’t replicated)
Diffusible factors in S or M ensure that the cell cycle is driven forward.
Diffusible factors in G2 ensure that cells do not enter mitosis until DNA replication is completed
what ensures alternation in the cell cycle
alternation - cell cycle events laways occur in the right order
cyclins and CDKs
Mphase cyclin/cdk trigger mitosis
Sphase cyclin/cdk trigger DNA synthesi
what regulated CDK activity in the cell cycle?
- cycling dependent kinase inhibitors (cdkis)
- oscilltions in cyclin protein levels
(which is regulated by phosphorylation and transcirptional repressors)
what is the key regulatory enzyme in the cell cycle?
cdc25
what occurs as a result of cyclin binding at the molecular level?
activation and inhibition
Cyclin binding allows phosphorylation of cdk at T160 by cdk activating kinase (CAK)
CDKs are inhibited by phosphorylation at T14 and Y15 (by wee1 and mik1 kinases) - these sites are dephosphorylated by cdc25, a promoter of mitosis
what are the two families of cdkis
(cyclin dependent kinase inhibitors)
give three examples of each and one function
- CDKN2 gene family (A-D) (also called INK4) (related to genes that encode the proteins)
– Proteins p16INK4A, p15INK4B, p18INK4C, p19INK4D
– Bind and inhibit cdk4 and cdk6 - CDKN1 gene family (A-C) (encode kip or cip proteins)(these interact more with the cyclin)
– Proteins p21Cip1/WAF1, p27Kip1, p57Kip2
– Inhibit E- and A- dependent cdks
breifly describe the cyclin/cdk cycle that occurs through the cell cycle from G1-M
Simulated by growth factors
First seen is an increase in the activity of cdk4/6, kinase activity increases because cell starts making Cyclin D
Gets the cell to the g1/s boundary but not past it
That trigger comes when cell makes Cyclin E, binds with cdk2, there is a spike of activity
Through S and G2, Cyclin A is made by the cell instead of e which binds to cdk2, taking the cell all the way through G2 to M
Sharp drop of activity
Cdc25 phosphatse removes residues 14/15 from cdk1 to allow activity
Very rapid increase in activity in Cyclin B-cdk1 to mitosis and the sharp drop
Loss of activity in this is the trigger for anaphase
what complex activates the anaphase promoting complex (APC)?
Cyclin B/ CDK1
describe how cyclin b/cdk1 and APC are involved in the cell cycle/ mitosis
1 Synthesis of Cyclin B in interphase
2 binding of cyclin B to cdk1 – as a complex in this context its known as a maturation promoting factor (MPF); high cyclin b so high MPF activity
3 this complex phosphorylates inactive APC to produce active APC
4 APC ubiquitinates the MPF complex targeting it for degradation by the proteosome triggering anaphase
5 Now there are low cyclin b and so low MPF activity in telophase
Until cyclin B is produced in interphase to start the cycle over again
what is the MPF in the cell cycle?
Maturation-promoting factor (abbreviated MPF, also called mitosis-promoting factor or M-Phase-promoting factor)
Cyclin B/ CDK1 complex
what are the 5 main cell cycle checkpoints?
where do they occur in the cell cycle?
G0/G1 restriction point
G1 DNA damage checkpoint
G2 DNA damage checkpoint
G2 decatenation checkpoint
spindle assembly checkpoint (end of metaphase)
what is the trigger for anaphase?
destruction of cyclin b
what is required for passage through the restriction checkpoint in G1?
Growth factor signalling
Breifly decribe the molecular activity that makes up the G1 (R) checkpoint
- Growth factors stimulate Ras
Ras stimulates the cell to produce Cyclin D
Cyclin D binds to Cdk4/6 - This Cyclin D-Cdk4/6 complex phosphorylates Rb
Rb is sitting on promoter of s phase genes. (Is a transcription repressor, when phosphorylated it partially reduces activity allowing transcription of certain s phase genes, like Cyclin E.
Cyclin E bind to cdk2. - p27kip binds to it and keeps the complex shut down.
However there is more cdk2 than kip1, so some activity can occur. (Linear progression not an on off switch.) Pool of the complex ready to go. - Phosphorylation of p27kips1 by the Cyclin E-Cdk2, brings in skp2 to bind, which attracts SCF to tag (ubiquitinate) p27kip1 for destruction. – a self reinforcing on/off switch.
- Leading to degradation of p27kip1 by the proteosome.
The Cyclin E-cdk2 complex phosphorylates other Rb enabling more cyclin e/cdk2 produced
what hallmarks of cancer are involved with cell cycle aberrations
- self sufficiency to growth signals
- insensitivity to anti-proliferation signals
cell cycle checkpoint defects at what stages cause cancer
growth factors
p53
what two defects allow cancer cells to pass through the G1 (R) checkpoint in the absence of external growth factors?
- consituitively active growth factor signalling
- R point defects
what oncogenic growth factors defects occur in cancer
contextx of cell cycle and protein activity
- Continuous exposure to growth factors
- Over-expression of growth factor receptors
- Activating mutations in growth factor receptors
- Activating mutations in growth factor signalling molecules e.g. ras
why did 60% of people present during the cehrnobyll disaster get thyroid cancer?
Fusion translocation in the Ret receptor (expressed in the thyroid) due to radioactive iodine gas concentrated in the thyroid.
why did 60% of people present during the cehrnobyll disaster get thyroid cancer?
Fusion translocation in the Ret receptor (expressed in the thyroid) due to radioactive iodine gas concentrated in the thyroid.
how do oncogenic Ras mutations promote cancer?
in the context of the cell cycle
because the protein cascade will be disrupted causes faults in the transcription factors sitting on the gene contorlling cyclin D - altering cyclin D activity/levels
activating mutations in the oncogenic Ras gene are present in what percentage or pancreatic, colon and all cancers
Pancreatic (95%)
Colon (50%)
All cancer (25%)
What restriction point defects occur in cancer
3 oncogenes and 3 tumour suppressor genes
- cyclin D1 (o)
- CDK4 & CDK6 (o)
- CDKN2A & CDKN2B (tsg)
- Rb (tsg)
- CDKN1B (tsg)
- SKP2 (o)
gene rearrangement of what protein is present in 100% of mantle cell lymphoma
cyclin D1 gene rearrangment
what checkpoint defects occur to cause insensitivity to antigrowth factors?
inhibitory growth factors (eg TGFbeta)
DNA damage checkpoints
cancer associated mutations shift the TGFbeta response from _ to _.
from growth inhibition to growth promotion
in what percentage of cancers in the p53 gene mutated
50%
in what percentage of cancers in the p53 gene mutated
50%
what types of stress does the p53 checkpoint stop the cell cycle for
DNA damaging agents
Hypoxia
Oncogenic stimuli
Ribonucleotide depletion
Senescence
how does p53 stop the cell cycle when the cell is faced with stress
transcription of a cdki (p21^CIP1/WAF1)
What protein leads to apoptosis at the p53 checkpoint?
bax
what type of cancerous gene is p53 and how does it act?
why?
p53 is a dominant negative tumour suppressor gene
P53 is a transcription factor which binds DNA as a tetramer, one mutant p53 molecule out of the four inactivates the transcription factor, therefore a mutation in one p53 allele is sufficient to promote cancer
what is unique about p53 that means it so readily leads to cancer
- p53 is a transcription factor which binds DNA as a tetramer
One mutant p53 molecule in the tetramer inactivates the transcription factor
Therefore a mutation in one p53 allele in is sufficient to promote cancer
more dna mutations occur when it is inactive because cell can progress
what is the major regulator of p53 activity in cells and what does it do
Mdm2
it directs ubiquitilation and degredation of p53
what inhibits p53 degredation?
mdm2 inhibitor p14-ARF
- p14-ARF prevents ubiquitination of p53 by mdm2
oncogenic stimuli induces expression of what protein
involved in the p53 checkpoint
p14-ARF
p14-ARF prevents ubiquitinatioin of p53 by mdm2
When bound to mdm2, it stabilises p53 promoting oncogene induced apoptosis.
what happens when p53 is less active and why?
breifly
When p53 is less active then more dna mutations occur because the cell is allowed to continue through the cell cycle when it shouldn’t
give three mechanisms of p53 inactivation in human cancers
- p53 point mutation and / or gene loss (TSG)
- Loss of p14-ARF expression (TSG)
- mdm2 gene amplification (Oncogene)
what gene defect is seen in about 100% of mantle cell lymphomas?
gene rearrangments of cyclin D1
due translocation of the Cyclin D1 encoding gene (CCD1) being translocated to the Immunoglobulin heavy chain (IgH) resulting in overexpression of the cyclin d1 protein.
give three cancers Rb is mutated in
retinoblastoma, osteosarcoma and small cell lung cancer
describe the G1 dna damage checkpoint
Following DNA damage, p53 is phosphorylated at serine-15 and serine-20 by ATM and Chk2, respectively. This results in the displacement of Mdm2 and mediates both the stabilisation and accumulation of p53. p53 is therefore free to the activate transcription of its target genes. This includes p21, which accumulates in the cell binding both cyclinE/Cdk2 and cyclinA/Cdk2 complexes, inhibiting them. This accumulates in cell cycle arrest and prevents DNA replication
describe the g2 dna damage checkpoint
this checkpoint ensures that the cells don’t initate mitosis until damage is repaired. In response to DNA damage, the ATM/ATR signalling pathway is activated which leads to phosphorylation and activation of Chk1 and Chk2 the subsequent phosphorylation of Cdc25. Phosphorylation of Cdc25 is seqeuestered into the cytoplasm (by 14-3-3) preventing activation of cyclinB/CDK1 resulting in G2 arrest. Activated ATM/ATR also activates p53 dependent singalling to maintain the G2 arrest. P53 induces the transactivation of p21 (a CDN1 CDKi) that binds and inhibits cyclinB/Cdk1 complexes (among others)
breifly describe the G2 decatenation checkpoint
Sister chromatids become entangled as a consequence of DNA replication which can result in gain or loss of whole chromosomes or fragments if they enter mitosis catenated. The G2 decatenation checkpoint delays entry into mitosis until topoisomerase 2 can untangle the chromosomes. Topo2 enzymes carry out this function by passing one double helix through a transient double strand break in another helix and then resealing the break
describe the spindle assembly checkpoint
Unattached kinetochore generate a STOP signal which blocks activity of the anaphase promoting complex/cyclosome (APC/C) – an E3 type ubiquitin ligase. This signal consists of a complex of four proteins: BubR1, Bub3, Mad2 and Cdc20, called the mitotic checkpoint complex (MCC) which binds directly to APC/C and inhibits it. When all the kinetochore are properly attached to microtubules, the MCC is no longer produced and the APC/C becomes active. This means that it is now able to tag cyclin b1 and securin for degradation and allow activity of cdk1 and separase for mitotic exit and chromosome segregation respectively to occur. This is the third checkpoint in the cell cycle. SAC activity gives a cell more time to establish proper MT-KT attachments. Without this extra time cells may proceed into anaphase before these attachments are made.
