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MCB2023S > Cell Cycle > Flashcards

Cell Cycle Flashcards

1
Q

What are the phases of the cell cycle?

A

G1 phase: Immediately after mitosis

S phase: DNA Replication

G2 Phase: Before Mitosis

M Phase: Mitosis

G0 Phase: After mitosis if cell leaves cycles and stops dividing

Interphase: G1+S+G2

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2
Q

How was MPF discovered?

A
  1. Fully grown Xenoopus oocytes treated with progesterone
  2. Oocytes mature by going through meiosis I and II
  3. Some cytoplasm removed from 2, and injected into fresh oocyte
  4. Recipient oocytes mature as if triggered by progesterone
  5. Some cytoplasm removed from 4, and injected into fresh oocyte. Oocytes immediately undergo meiosis I and meoisis II
    6 Conclusion:
    o Factor present in
    cytoplasm called
    MPF
  6. Then added protein blocker to 4 and oocyte maturation was still induced when cytoplasm was injected into G2-arrested oocyte
    - Conclusion:
    o Immature oocytes
    contain pre-MPF proteins
    that are converted to
    activate MPF by post-
    translational reactions
  7. Also found that other organisms cytoplasm going through mitosis injected into a G2-arrested frog oocyte still stimulated meiosis I and II
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3
Q

What does cdc2 encode?

A

A protein kinase

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4
Q

What is MPF?

A

cdc2 & cyclin

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5
Q

Dosage effect of wee1 and cdc25 on the cell cycle

A

Wee1: ‘break’ in the cell cycle (slows it down. Inactive wee1 = small cells that divide early)

cdc25: Accelerator of the cell cycle (Inactive cdc25 = long cells)

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6
Q

What is the role of Cyclin B in the cell cycle?

A
  • Accumulation of Cyclin B required for MPF activity and entry into mitosis
  • Destruction of cyclin B by ubiquitination destroys MPF activity and is required for exit from mitosis
  • Cyclin B ubiquitination is tightly regulated by the Anaphase promoting complex (APC)
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7
Q

How is cyclinB destruction regulated?

A
  • Deletion analysis showed N-terminus 90 amino acids required for destruction
  • Identified small conserved sequence in amino-terminus called destruction box.
  • Destruction box has conserved motif recognized by ubiquitin ligase
  • Ubiquitination is prominent degradation pathway
  • When proteins are multi-ubiquitinated at a single site they become targets for degradation
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8
Q

How is the G2/M Checkpoint Regulated?

A
  1. As mitotic cyclins accumulates -> forms complex with CDK1 (cdc2)
    a. CDK potentially active
    once it reacts with cyclin
    so ->inactivated by
    phosphorylation to make
    sure all checkpoints are
    met before mitosis
    happens
  2. Wee1 & Mik1 phosphorylate Tyrosine 15 (Y15)
  3. CAK (Cyclin-dependant kinase) phosphorylates T161
  4. When both aa residues are phosphorylated -> if all checkpoints & controls have been activated -> cdc25 dephosphorylates Y15 -> active MPF
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9
Q

How did they test whether the phosphorylation status of Cdc2 changed during the G2/M transition?

A
  • Used cdc25ts mutants (arrest in late G2- premitosis) and cdc13ts mutants (arrest mid mitosis)
  • Added 32P-orthophosphate to the medium to visualise phosphorylation
  • Assay of immunoppt of cdc2 from equivalent amounts of 32P-labeled cdc25ts and cdc13ts mutants arrested at 36 degC
    o Found much more phosphorylation on cdc25ts (late G2)
    o Therefore, cells more phosphorylated in late G2 than mid-mitosis
  • Assay of immunoppt of cdc2 from equal numbers of cdc25ts cells shifted to permissive temp for different times
    o Shows that as cells progress from G2 into mitosis the phosphorylation of cdc2 reduced
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10
Q

Which amino acids in cdc2 are phosphorylated during G2?

A

Tyrosine 15

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11
Q

Why does phosphorylation of Y15 stop progress into mitosis?

A

o Y15 residue is located in ATP binding site of cdc2

o If Y15 is phosphorylated then cdk can’t transfer gamma P from ATP to substrate.

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12
Q

What protein phosphorylates Y15 on cdc2?

A

Wee1 & Mik1

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13
Q

Which compound dephosphorylates Y15?

A

cdc25

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14
Q

What are the targets of active MPF?

A
  • It phosphorylates all the proteins required for mitosis
  • First targets the lamins (A, B & C) in the nucleus resulting in nuclear membrane breakdown
  • Also phosphorylates the histones
  • And microtubule associated proteins
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15
Q

Genes active in G1/S Checkpoint

A
  • cdk2 in mammals
    o CDC28/cdc2
  • CyclinE
  • P21: cdk2,4 inhibitor (active at G1/S)

[G0->G1]

  • CyclinD
  • cdk4,6
  • P16: cdk inhibitor of cdk4
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16
Q

Assay to prove CyclinD activity in G0/G1 transition

A
  • Growth factors signal G0 -> G1 transition
  • BrdU (T analogue, incorporates opposite Adenine) will be incorporated into the cell; if cell progresses into S phase, BrdU will be incorporated as thymidine analogue during DNA replication
  • Anti-cyclin D antibody will bind to cyclin D; so if it is required for G0-G1 (and therefore S) transition, then that will be blocked
  • Therefore BrdU won’t be incorporated because DNA replication won’t occur

Findings:
- Mammalian cells arrested in G0 respond to growth factors by entering G1 phase, passing start and replicating their DNA.
- They continue to replicate their DNA (BrdU incorporation) and divide in presence of growth factors
- If cyclin D is inhibited by the injection of anti-cyclin D antibodies (<14hr after growth factor addition) cells do not incorporate BrdU (Cell Cycle stopped)
- BUT: after 14 hrs, cells have passed start already and cyclin D is not required – so incorporation of anti-cyclinD antibody has no effect
- Conclusion:
o CyclinD is required for G0 -G1 transition, but is only active for a particular amount of time after Growth Factor signalling (before cell enters G1)

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17
Q

What is a Proto-oncogene?

A

o Regulates cell cycle. Responds to growth factor signalling
o Are genes that cause cancer if they acquire a gain-of-function mutation that leads to their over-expression or constitutive activation.
o A single mutation in one copy of a proto-oncogene can lead to the loss of cell cycle control, and these are thus considered to be dominant mutations.
o Gain-of-function mutations include:
- Point mutations that change the activity of a protein
- Chromosomal translocations that fuse two genes to make a chimeric protein
- Chromosomal translocation to bring a gene under the control of a different promoter
- Amplification leading to numerous copies of a gene and overproduction of the encoded protein

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18
Q

What is a Tumour Suppressor Gene?

A

o ‘guardians of cell cycle’
o Wild type function is to prevent uncontrolled cell growth
o Recessive mutations (i.e. mutation of 1 copy doesn’t affect cell cycle)
o If both gene copies are mutated = cancerous (uncontrolled cell growth)

E.g.:

  • cdk inhibitors p16, p21, p27, p53
  • Rb
  • ATM
  • Chk2
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19
Q

Action of p16

A
  • Potent inhibitor of cdk4/cyclin D (inhibits ability to phosphorylate)
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20
Q

Action of p21 & p27

A

Inhibits both cdk4/cyclin D & cdk2/CyclinE

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21
Q

What is the action of the Rb protein?

A
  1. Rb protein shuttles between being hyperphosphorylated, and hypophosphorylated after mitosis in G0 (zero phosphate groups associated with it)
  2. E2F transcription factors: very active from G1 to S
    a. Responsible for activity CDK2/Cyclin E (required for transition from G1 to S)
    b. Mid G1 (G0): Rb (hypophosphorylated) binds very tightly to E2F – recruits histone de-acetylases which actively repress transcription of cyclin E/Cdk2
    c. G0: p16 associated with cdk4/cyclinD = inactive kinase
    d. p16 dissociates in response to growth factors, cdk4/cyclinD will phosphorylate Rb -> it can no longer bind to E2F -> frees E2F from Histone Deacetylases -> E2F then targets directly promoters for CyclinE/cdk2 and its own transcription.
    e. i.e. as soon as Rb is phosphorylated = rapid increase in E2F conc. and CyclinE/cdk2 conc.
    f. once cyclinE & Cdk2 levels increase, can now go through START into replication
  3. Important guardian of cell cycle, because after Mitosis it stops progress into G1 by mopping up all the E2F transcription factors until its phosphorylated.
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22
Q

What is the wild-type function of p53?

A

o All normal cells have low levels of p53
o It is a transcription factor which associates as a tetramer
o It regulates the cell’s response to DNA damage
o In absence of DNA damage it associates with protein MDM2 which ubiquitinates it and sends it to proteosome for degradation
o In response to irradiation causing DNA damage – p53 is phosphorylated meaning it no longer associates with MDM2 & is no longer ubiquitinated so its conc stabilises
o It is a transcription factor that regulates itself, so its conc quickly rises as its transcription is increased
o. It also targets genes involved in growth arrest (p21) & DNA repair and apoptosis (leads to implosion of cell)

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23
Q

DNA Damage response genes

A
  • Rad3 (S. pombe)/ATM (human homologue): First activation when DNA is Damaged by ionising radiation.
  • Chk1 at G2/M: Phosphorylated by Rad3/ATM
  • Chk2 at G1/S: Phosphorylated by Rad3/ATM
  • Rad24: 14-3-3 proteins, bind to signalling molecules
  • ATR (paralogue of ATM): activated in mammalian cells in response to DNA damage caused by UV irradiation.
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24
Q

DNA Damage control at G2/M checkpoint

A
  1. spRad3 (S. Pombi) or hATR (humanATM)/hATR activated in response to damage detection
  2. Phosphorylates Chk1
  3. Chk1 phosphorylates Serine residue on Cdc25
  4. P-Cdc25 associates with 14-3-3 proteins (Rad24)
    o Rad24 associates with nuclear export factor crm1
    o Crm1 exported out of nucleus, taking with it Rad24 and P-Cdc25 by association
  5. Inactivated Cdc25 can’t dephosphorylate the Tyrosine on Cdc2
  6. Cell remains in G2 arrest
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25
Q

Where is cdc25 distributed within the cell during the cell cycle?

A

o Looked at the intracellular distribution of Cdc25 in S. pombe during the cell cycle
o Tagged Cdc25 with Myc12 epitopes (short peptides) – didn’t have antibodies to Cdc25 but did have antibodies to Cdc25 epitope
o Also used fluorescent dye DAPI which binds to dsDNA and shows where nucleus is
o Findings:
- Wild type cells: Cdc25 is found in nucleus in late G2 and mitosis. No longer in cytoplasm in late Mitosis and early G2.

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26
Q

Does the cdc25 distribution in WT cells and in ∆chk1 cells change when exposed to ionizing radiation?

A

o In WT cells, there is no accumulation of Cdc25 in nucleus if exposed to ionizing radiation (gets exported out).

o In ∆chk1 mutant yeast, Cdc25 remains localized in cell nucleus even in response to ionizing radiation (Cdc25 isn’t exported out, cells don’t stay in G2 arrest)

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27
Q

Is rad24 (14-3-3s) required for export of Cdc25 in response to radiation?

A

o Yes

o Cdc25 accumulates in the nucleus in ∆ rad24 cells in the presence and absence of ionizing radiation

28
Q

What is causing the movement of Cdc25 out of the nucleus?

Can’t be chk1 (a protein kinase) and can’t be rad24 (small acidic protein)

A

o CRM1 nuclear export factor is a possible candidate – so tested CRM1-809 cells (cold-sensitive mutant)
o In crm1-809 cells (crm1 inactive), Cdc25 remains highly localized in cell nucleus in presence and absence of ionizing radiation
o Means that CRM1 is exporting cdc25 out of nucleus, because no export happens when crm1 is inactivated

29
Q

What is the tTA experimental system?

A
  • Inducible system In order to facilitate control of overexpression of Cdc25A.
  • Artificial inducible tetracyline off system.
  • Bacterial operator (tetO) is recognized by the tetracyline repressor (tetR).
  • To make the tetR functional in mammalian cells, researchers fused the tetR with a mammalian transcription factor transactivation domain (tTA, tet repressor with transactivation domain).
  • In the absence of tetracyline, tTA binds to the tetO sequence (tetracycline response element TRE), and is a potent activator of transcription.
  • If tetracyclin is added to the medium, it binds to the tet repressor domain in tTA, which prevents tTA from binding to the TRE.
  • TRE (regulatory region) placed upstream of gene of interest that you want over expressed.
30
Q

What is the effect of gama-irradiation on cdc25A levels?

A
  • Cdc25A protein disappears within 30mins
    o Cdk2-pTyr15 levels increase within an hour
    o Cdk2/CyclinE ability to phosphorylate histone H1 drops
    o NA synthesis is inhibited
31
Q

What regulates the rapid destruction of cdc25A in response to gama-irradiation?

A

o G-irradiation activates ATM which activates Chk2 which phosphorylates S123 on Cdc25A
o Ubiquitin ligases recognises pS123 on Cdc25A and ubiquitinates it -> degradation by proteasome.
o Absence of Cdc25A -> T14 & Y15 remain phosphorylated on Cdk2 -> S-phase delay

32
Q

What type of gene are ATM and Chk2?

A
  • Tumour suppressors
  • If ATM has double mutation it will be silenced -> not activated by DNA damage -> Chk2 not activated -> Cdc25A not phosphorylated -> Cdk2 not dephosphorylated -> RDS (Radioresistant DNA synthesis)
  • RDS is very dangerous because mutated/damaged DNA (from radiation) is replicated which leads to more mutations
33
Q

DNA Damage control at the G1/S Checkpoint

A
  • p53 Independant pathway
    o DNA Damage detected ATM -> Phosphorylation of Chk2-S123 -> Cdc25A-P -> Ubiquitinisation by ubiquitin ligases -> degradation by proteasome.
  • p53 Dependant pathway
34
Q

p53 Dependant pathway for DNA Damage control at the G1/S checkpoint

A
  • Both copies of gene most commonly mutated in many forms of cancers
  • In normal mammalian cells: P53 associated with MDM2 (ubiquitin ligase; targets p53 for degradation) and MDM4 (Actively inhibits transcription of p53)
  • In presence of DNA damage, ATM phosphorylates p53, MDM2, MDM4 (And chk2 which can also phosphorylate p53)
  • P-p53 doesn’t associate with MDM2/4 (no longer ubiquitinated and degraded) and can transcribe itself and downstream targets that are active in DNA repair pathway and stopping cell cycle (cdc inhibitors (p21) and 14-3-3 proteins)
  • P-MDM2/4 opens them up to ubiquitination and they are targeted for degradation
35
Q

How does cancer arise?

A
  • When there is a failure of genes that regulate growth and cell proliferation, or if there is a failure in genes that encode and regulate DNA damage repair pathways.
  • Cancers arise in cells that get replaced rapidly in the adult life (that’s why you rarely see cancer of the heart etc.)
36
Q

What is the cancer phenotype?

A
  • Self-sufficiency in growth signals (not dependant on growth factors to leave G0)
  • Insensitivity to antigrowth signals
  • Evasion of apoptosis
  • Limitless replicative potential
  • Tissue invasion and metastasis
  • Sustained angiogenesis[= growth of blood vessels] (For O2 supply)
  • Unstable genotype & uncontrolled cell growth
37
Q

Benign vs malignant tumour

A

Benign:

  • First stages of cancer - cell proliferation but still contained within basal membrane
  • Suffix - Oma

Malignant tumour:

  • Has ability to break through membrane and invade other organs
  • Suffix - sarcoma or carcinoma
38
Q

What are polyps?

A
  • Small pre-cancerous growths along the colon wall. (Often have inactivating mutation in both of APC alleles from chromosome 5)
    o APC is tumour suppressor gene, so if born with one mutation then a single second (somatic) mutation will form polyps
  • If polyps not removed, they will develop into a malignant cancer within 12 years.
39
Q

What is the multi-hit model?

A
  • Cancers arise from clonal selection.
  • First mutation that affects cell cycle gives slight growth advantage.
  • Progeny of this cell acquires second mutation which gives further growth advantage.
  • Progeny of THIS cell might form small benign tumour.
  • Theirs mutation in one of these cells might affect DNA damage repair pathway -> further mutations accumulating.
  • Fourth mutation in one of its progeny will lead to malignant population of cells that have mutations in four genes, allowing the progeny to escape the blood and establish daughter cells at other sites.
40
Q

Multi-hit model in colorectal cancers

A
  • Requires successive somatic mutations in 5-10 genes
  • Polyps form (inactivating mutation on both APC alleles)
    o APC suppresses expression of Myc1, transcription factor that is important for transition through G1 checkpoint.
    o Inactivation of APC leads to inappropriate expression of Myc1 -> cells progress through START without control.
  • Then if K-ras proto-oncogene is activated (only needs 1 mutation to super activate) turns signal transduction on constitutively
    o Polyps will carry on growing despite no growth factors
  • Third mutation = deletion of DCC (tumor-supressor)
  • Loss of p53 = 4th mutation often found in crc -> now malignant melanoma develops
    o Cells start to proliferate, get through basal lamina into blood vessel and transport to secondary sites in body.
41
Q

What is an oncogene?

A
  • Cancerous cell that causes unnecessary cell proliferation
  • Viral oncogenes:
    o Encoded by viruses which stimulate the cell cycle; can be viral-specific genes, or they can be mutant forms of cellular genes (proto-oncogenes)
    o Bind to hypophosphorylated form of Rb which frees E2F which activates cyclinE -> premature entry in START
  • Cellular oncogenes:
    o Gain-of-function mutations of cellular genes that lead to uncontrolled cell growth
  • Examples:
    o Cyclin D,E
    o SV40 large T antigen from a simian virus
42
Q

What is an example of a DNA Tumour Virus?

A

Papilomavirus

43
Q

Action of human papilomavirus

A
  • Infects human warts & carcinomas of the human cervix (6% of human cancers)
  • Papillomavirus infects epithelial cells, which are retained in basal layer of cells as extrachromosomal plasmids -> stimulates cells to divide (warts)
  • Papillomavirus induces cancer if it infects stem cells in epithelium
  • E6 protein: Binds to p53
    o P53 not available to halt cell cycle in DNA damage
  • E7 protein: Binds to Rb
    o Cells never arrested in G0 because Rb is bound to E7
  • Cells replicate in an uncontrolled way once infected by papillomavirus
44
Q

Ionizing Radiation as a cancer treatment

A
  • Normal cells will stop dividing & repair DNA in response to IR
  • Tumor cells: defects in cell-cycle checkpoints, continue to multiply following irradiation, die due to catastrophic DNA damage when they divide with defective chromosomes

NB: if the cell doesn’t die, it could just introduce new mutations into the DNA

45
Q

Chemotherapy

A
  • Administered via the blood, so can target all cancer cells in all areas of the body.
  • Drugs take advantage of increase replication and division rate of cancer cells.
    o Target either transcription or DNA replication or mitotic events
  • Many of these treatments are toxic to normal cells as well, and treatment must be balanced between killing cancer cells but not too many normal cells.
46
Q

Mechanisms of action of different Chemo drugs

A 
1.	Promote microtubule polymerisation and stabilisation
	o	Paclitaxel
	o	Docetaxel
2.	Act as a pyrimidine nucleoside antimetabolite
	o	Mimic nucleotides, but they stop DNA replication
	o	Gemcitabine
3.	Inhibit thymidylate synthase
	o	Inhibits production of thymidine
	o	S-FU
4.	Cross-link DNA
	o	Cis-platinum
	o	Carboplatin
5.	Intercalate into DNA and inhibit topoisomerase action during DNA replication
	o	Doxorubicin
6.	Bind to minor groove of DNA
	o	Trabectidin
47
Q

Cancer Hormonal Treatments

A

Prevent cancer cell growth by preventing cells from receiving mitotic signals:
o Anti-oestrogen (tamoxifen treatment of breast cancer) or
o Anti-androgen treatment (prostrate cancer)

  • Specific inhibitors:
    o Drugs that target specific proteins that are found to be more prevalent in cancer cells (designed to specifically bind to oncoproteins)
    o E.g. Herceptin- a monoclonal antibody that binds to the external domain of Her2 receptors- over-expressed in breast cancer cells
  • Vaccines:
    o Vaccines usually contain proteins found or produced by cancer cells. Aim is to use immune system to recognize cancer cells
    o E.g. vaccine against E6 or E7 from papillomavirus
  • Anti-angiogenesis factors:
    o Block blood supply to tumours
48
Q

What is cancer diagnosis based on?

A
  • Used to be based on tumours primary location
    o Would look at sections to try and identify the type of cell that went rogue
  • Now based on genetic features of Cancer DNA
    o Gives better diagnosis and guides treatments
  • Combine DNA, RNA analysis with tissue biopsy
49
Q

Radiation and wee1 inhibitors combination treatments for Cancer

A
  • Cancer cells that are defective in p53 can’t mount a DNA damage response -> susceptible to radiation induced DNA damage -> mitotic catastrophe
  • Found that patients defective in p53 (G1/S checkpoint) have an upregulation of wee1 at mid S-phase checkpoint & G2/M point -> delay allowing cells a chance to repair DNA
  • Wee1 active at G1/S checkpoint (phosphorylates cdk2) & G2/M checkpoint
  • If wee1 is inhibited this will prevent the cell stopping cycle at G2/M checkpoint, so DNA cannot be repaired
50
Q

How was Mitotic cyclin discovered?

A

Early development in Sea Urchin Embryos
- Tim Hunt was investigating proteins that were newly synthesized at early stages of embryonic development is sea urchins

  • Suspension of 20 000 eggs were fertilized
  • After 6 min 35S-methionine was added (labels all newly synthesized proteins)
  • Samples taken at 10 min intervals, and analyzed on SDS-PAGE gels & autoradiography
  • At same time, sample fixed in 1% glutaraldehyde (kills cells and fixes them exactly where they are) for microscope examination (measured cleavage index)
  • Findings:
    o Cyclin would increase then suddenly disappear, then increase, then suddenly disappear throughout cell cycle.
51
Q

How did they isolate cdc mutants?

A
  1. Grow up a culture of haploid S. pombe yeast.
  2. Mutagenize with a chemical mutagen
  3. Plate the culture of mutagenized yeast out on rich media agar plates.
  4. Incubate plates at 25degC
  5. Once colonies have grown, replica plate each master plate onto two, fresh rich media plates.
  6. Place one plate in a 25oC incubator, and the other in a 36degC incubator
  7. After several days, compare the plates, and identify yeast colonies that grow at the
    permissive temperature of 25degC but not at the restrictive (or non-permissive temperature) of 36degC. These are temperature-sensitive mutants.
  8. Streak the temperature-sensitive mutants out from the 25degC plate (to make a stock). Pick a single colony and inoculate into yeast broth and incubate at 25degC.
  9. Shift the yeast culture to 36degC, and after five hours examine the morphology of the temperature-sensitive yeast mutant under a microscope to identify cell division cycle (cdc) mutants.
  10. Mutants that are defective in genes that regulate the cell cycle will either not divide at the restrictive temperature (and grow into very long S. pombe cells) or may divide prematurely (in which case you would see S. pombe cells that are much shorter). These long or tiny mutant S. pombe cells are likely to have temperature-sensitive mutations in cell division cycle genes.
52
Q

How would you clone by complementation?

A
  1. Incubate a colony of cdc25ts mutants at a permissive temperature.
  2. Transform those cells with a plasmid (human cDNA if looking for human homologue) library and incubate them at a non-permissive temperature (36 degC).
  3. After incubation observe which cells formed colonies. Only cells that were transformed with insert DNA that was complimentary to cdc25 would be able to form colonies at a non-permissive temperature.
  4. Then identify the nucleotide sequence of the insert DNA, and use that to determine the amino acid sequence it codes for.
  5. Compare that aa sequence to know protein banks/human sequences and find a match.
53
Q

What is the cdc2 homologue in budding yeast?

A

CDC28

54
Q

What is the mechanism of co-immunoprecipitation?

A
  1. Incubate whole cell extract with antibody to protein X
    a. Antibody will bind to target protein (X)
  2. Incubate extract with Protein-A-Sepharose, which binds antibody
    a. Creates Protein-antibody-sepharose complex
  3. Collect Sepharose beads by centrifugation
    a. Because Sepharose beads are large, they will pellet in centrifugation… with protein X, antibody, and any other protein that physically interacts with protein X
  4. Discard supernatant
  5. Wash pellet several times
  6. Dissociate proteins from Protein-A-Sepharose
  7. Separate proteins by SDS Page gel
55
Q

What experimental procedure was used to determine if cdc2 protein was present in partially purified Xenopus MPF fraction?

A

o Raised anti-cdc2 antibodies
o Did a Western blot
o Loaded E. coli protein extract in lane 1
o Lane 2: Xenopus MPF partially purified by chromotography
o Lane 3: total protein from human fibroblast
o Found 34kDa protein (cdc2) in Xenopus MPF and the human fibroblast
o Confirms that anti-cdc2 recognize very similar protein in human cells and Xenopus MPF

56
Q

How was cyclin identified as a part of MPF?

A

Co-immunoprecipitation of MPF run on SDS page with antibody shows 2 proteins (cdc2 and cyclin).

57
Q

How was the role of Cyclin in MPF activity investigated?

A
  • G2 arrested frog eggs are electrically activated to start the cell cycle
  • Centrifugation separates contents of eggs into 3 layers:
    o Lipid droplets at the top,
    o Yolk particles at the bottom
    o Cytoplasm in the middle
  • Withdraw middle cytoplasmic layer, & see whether it can drive a cell cycle in a test tube
  • Added sperm nuclei to the above Xenopus oocyte cytoplasm extract
  • DNA morphology: Chromosomal condensation (mitosis) & de-condensation (interphase) will show if cell cycle is happening
    o Followed with DAPI = DNA-binding fluorescent dye
  • Microscopy:
    o In interphase - nuclear envelope present which would diffuse the DNA staining
    o As cell enters mitosis: nuclear envelope breakdown, chromosome condenses -> sharp fluorescent signal

Findings:
o DNA fluctuated from being in a condensed and uncondensed form.
- Add 35S-methionine to cell extract
- Take samples out at regular intervals and SDS Page Gel to look for new proteins being synthesised

58
Q

Is cyclinB degradation required for exit from the cell cycle?

A

The following 3 experiments were set up, and each time the ability of the frog oocyte extract to run sperm nuclei through the cell cycle was measured.

1) All RNA in the frog extract was destroyed by adding RNase.
2) Following RNAse treatment, a RNAase inhibitor (RNAasein) was added to the frog oocyte extract, as well as only cyclin B mRNA.
3) Following RNAse treatment, a RNAase inhibitor (RNAasein) was added to the frog oocyte extract, as well as a mutant form of cyclin B, which lacked the N-terminal domain.

Findings:
1) RNAase treatment destroyed the in vitro cell cycle
2) The addition of only cyclinB mRNA was sufficient to restore it.
o This result proved that the accumulation of Cyclin B during interphase is a prerequisite for MPF activity.
3) When the researchers added the mutant form of cyclin B, lacking the N-terminal domain, they found that the cell cycle started, but that it was unable to move beyond entry to mitosis.
o An analysis of the newly synthesized protein showed that the 46kDA protein did not disappear when the mutant form of Cyclin B mRNA was added to the RNAase depleted frog extracts.

This result showed that destruction of Cyclin B was required for the exit from mitosis.

59
Q

What assay was performed to determine which amino acid was phosphorylated on cdc2?

A
  • cdc25ts mutants grown at 25degC, shifted to 36degC for 4.5 hrs
    o Results in all cells being blocked in late G2
  • Released from arrest by shifting cells back to 25degC
  • Samples taken at different points, cells lysed and Cdc2 immunoprecipitated
  • Immunoppt run on SDS-PAGE gel, transferred to nylon membrane
  • Incubate membrane with anti-phosphotyrosine (A) or anti-cdc2 antibodies (B)
  • Results:
    o Lots of cdc2 constant throughout hour – antibody is also very specific because if peptide block added, no cdc2 is pulled out
    o Tyrosine only phosphorylated at time 0 (when cells blocked)
    o Proves that in late G2 cdc2 is phosphorylated on Tyrosine residues, and then dephosphorylated when it enters the cell cycle again
60
Q

Assay for dephosphorylation of cdc2

A
  • Cdc25 seemed to be a likely candidate for dephosphorylation of Cdc2 because mutation in cdc25 at nonpermissive temp blocks cell division and overexpression of cdc25 accelerates cell division
  • Purified cdc25 added to inactive MPF (purified from G2 arrested starfish oocytes)
  • Histone H1 kinase assay used to test whether this was sufficient to convert inactive MPF to active MPF
  • Found that recombinant cdc25 could dramatically activate MPF
  • Assay of immunodepleted bacterial lysate (not expressing cdc25) did not activate MPF
  • Then checked phosphotyrosine status of cdc2 before and after addition of cdc25
    o Western Blot using anti-cdc2 antibodies and anti-phosphotyrosine antibodies
    o Showed phosphorylation of tyrosine absent once cdc25 added.
  • Further proof in addition of vandate (known inhibitor of phosphates)
    o Addition of vandate reversed dephosphrylation of cdc2 by recombinant cdc25.

PROOF that cdc25 encodes phosphotyrosyl phosphatase.

61
Q

Cell Cycle in S. cerevisiae

A
  • CDC28 (CDK) active at G1/S transition (START) and G2/S transition
    o Associates with different cyclins at different stages in cell cycle
  • Leaves mitosis, heads through G1 towards START -> CLN3 (G1 cyclin) associates with CDK
  • In S phase it associeates with Clb5,6
  • Then with Clb3,4 (late S-phase, early M phase cyclins)
  • Then finally as it enters Mitosis it associates with Clb1,2 (Late M phase cyclins)
62
Q

Mammalian cyclins

A

cdk4-cyclinD: G0/G1

cdk2-cyclinE: G1/S

cdk2-CyclinA: S-phase

cdk1-CyclinA: Late G2
cdk1-CyclinB: Entry to M phase

63
Q

How were CDK inhibitors investigated?

A
  • Found that Cdk4/CyclinD phosphorylated Rb protein
  • Set up assay to measure ability of cdk4/cyclinD to phosphorylate Rb (32P-y-ATP in medium) in the presence of increasing amounts of recombinant p16.
  • Found p16 directly inhibits cdk4/cyclinD
64
Q

What happens to Rad24 localisation in a crm1 mutant?

A

o Would expect that it is stuck in nucleus IF it associates with crm1
o WT cells: Rad24 predominantly in cytoplasm
o In crm1-809cs mutant: Rad24 in nucleus & cytoplasm
o Conclusion: there is a link between Rad24 localization and Crm1

65
Q

What happens to Cdc25 localisation in Rad24-NES mutant?

A

o Rad24-nes mutant can’t associate with crm1
o crm1 associates with proteins containing leucine-rich nuclear export signal (NES)
o Cdc25 cannot be exported in Rad24-nes mutant

66
Q

What are the DNA Damage Checkpoints?

A
  1. G2/M: Entrance to M blocked if DNA replication not complete
  2. M: Anaphase blocked if chromatids not properly assembled on mitotic spindle
  3. G1/S: Entrance into S blocked if genome is damaged
  4. S: DNA replication is halted if genome is damaged
67
Q

Is cancer a result of genetics or the environment?

A

Oncogenesis is an interplay between genes and the environment and is the result of the accumulation of somatic mutations throughout life.

MCB2023S (9 decks)

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