Quiz 6 Flashcards
what are the phases of mitosis
prophase prometaphase metaphase anaphase telophase
when are the centrosomes duplicated
during S phase - everything needed for mitosis is synthesized here
when do the centrosomes move to the poles
prophase
what happens during prophase
centrosomes move to poles, nuclear membrane breaks down, chromosomes condense, sister chromatids held together by centromere
what happens during prometaphase
spindle fibers form.
what types of spindle fibers are there
- astral microtubules: position mitotic spindle
- kinetochore microtubules: attach to the chromosomes
- polar microtubules: interdigitate with MTs from opposite pole
what happens during metaphase
alignment of chromosomes in the center of the mitotic spindle. THIS is when we do karyotyping
what happens during anaphase
sister chromatids separate and move to opposite poles, cell elongates
what happens during telophase and cytokinesis
telophase: nuclear membrane reforms, chromosomes recondense, spindle disappears
cytokinesis: actin filaments cause separation of daughter cells
what is the purpose of meiosis 1
reductive division - separation of homologous chromosomes
what is the purpose of meiosis 2
segregation of sister chromatids to be 1n.
what is the diversity generated from meiosis 1 independent assortment
2^23 (2^n)
what else creates genetic diversity (other than independent assortment)
crossing over (THIS is why we don’t just have separation of homologues but rather DNA synthesis and then 2 divisions)
what structure facilitates recombination
synaptonemal complex
describe the synaptonemal complex
highly ordered structure formed during meiosis 1. consists of 2 lateral elements and a central element to facilitate recombination.
what is the term for sites of crossing over
chiasmata (remnants of synaptonemal complex)
where does crossing over happen for the sex chromosomes
at the small region of homology which allows them to pair and cross over. really the main function is to physically keep them together
what is nondisjunction
when homologues fail to separate during meiosis 1 or sister chromatids fail to separate in meiosis 2. get trisomy and monosomy as a result
what percent of gametes have abnormal chromosome numbers if non disjunction occurs in meiosis 1 vs 2
meiosis 1: 100% abnormal
meiosis 2: 50% abnormal, 50% normal
mitotic index
number of cells undergoing mitosis/total number of cells
*done using light microscopy
ki-67
ki-67 is an antigen expressed in cells undergoing active division. can be detected using an antibody tag via immunohistochemistry. ONLY in antigen presenting/proliferating cells, allows us to flag cancerous cells.
flow cytometry
measures fluoresence of a tag that has integrated into DNA - measures the fluorescence PER cell (G2 and M fluoresce twice as much as G1, can’t tell difference between them!)
where do CDKs phosphorylate
ser or thr residue that immediately precedes a proline residue
upregulation vs downregulation of cyclin
upregulation via transcription
downregulation via degradation
CDK at early G1, late G1, S, late G2, M
early G1: CDK 4/6 with Cyclin D Late G1: CDK2 with Cyclin E S: CDK2 with Cyclin A G2: CDK1 with Cyclin A M: CDK1 with Cyclin B
CDK cyclin at early G1
CDK 4/6 with Cyclin D
CDK cyclin at late G1
CDK2 with Cyclin E
CDK cyclin at S
CDK2 with Cyclin A
CDK at G2
CDK1 with Cyclin A
CDK cyclin at M
CDK1 cyclin B
what else is necessary for activation of CDK complex?
Phosphorlylation at T160 – cyclin binding is necessary but NOT sufficient
what phosphorylates CDK subunit
CAK (CDK activating kinase, at T160) and Wee1 at T14 and Y15 (this is inhibitory - juxtaposes where ATP would bind)
what prevents substrate binding in CDK and CDK/cyclin complex
part of the CDK called the T loop is in the substrate binding site and prevents binding. Binding of cyclin causes shift, phosphorylation of t loop on T160 by CAK fully moves it so substrate binding site is completely exposed
why is Wee1 inhibitory
it juxtaposes where ATP would bind and repels ATP via attaching negatively charged phosphates
how are the phosphates put on by Wee1 removed
abruptly
when does CAK/Wee1 phosphorylation occur
simultaneously, waiting for inhibitory phosphates to be removed typically
what removes Wee1 phosphates
Cdc25c phosphatases
feedback loops in CDK
positive feedback of activated CDK/cyclin/P complex on Wee1 – phosphorylates it which makes it less active
positive feedback of CDK/cyclin/P complex on CDC25 phosphatase which makes it MORE active and better at removing inhibitory phosphates
what regulates CDK/cyclin/P activity even if there is no inhibitory phosphates
CIP class (P21, P27, P57, inhibits ALL CDKs)
INK4 class (p15, p16, p18, p19, inhibits only CDK 4/6)
how does CIP class work
binds to Cyclin-CDK complex and induces a conformational change that prevents substrate binding.
how does INK4 class work
binds only to CDK subunit, preventing it from interacting with cyclin
two types of E3 ub ligases
SCF and APC/C
similarities between types of E3 ligases
both have catalytic, scaffold, adaptor, and variable parts
SCF target and function
Cyclin D, E, A. ALWAYS acts as UB ligase, only on things that are already phosphorylated. ALWAYS active and recognizes phosphorylation.
what is SCF important for
cyclins in the G1 to S transition (D, E, A)
APC/C target and function
cyclin B. NOT always active - acts on cyclin B regardless of state it is in. APC itself needs to be activated via phosphorylation. (If it was always on we wouldn’t ever get cyclin B)
what is APC important for
cyclin B, mitotic progression
what phosphorylates APC
cofactor - Cdh1 or Cdc20
3 kinds of signals/feedback in cell cycle
- previous step responsible for activating next step
- negative feedback - when next step is activated, previous inactivated
- positive feedback, aplification of signal
what upregulates cyclin D
downstream in ERK pathway
what does ERK pathway regulate
phosphorylates Cyclin D and RB
what is Rb in natural state
normally bound to E2F, a txn factor. Rb acts as a repressor when bound and E2F gene expression is off
what happens when Rb gets phosphorylated
E2F is released, can now regulate gene expression
what is E2Fs target
cyclin E
what are the two types of E2F targets
- High affinity such as cyclin E
2. low affinity - other enzymes, only binds if these enzymes are present in large amounts (cyclin A)
what is Rb initially phosphorylated by
CDK4 and CDK6
what phosphorylates the rigin
Cyclin A/CDK2 (low affinity E2F target) and Cyclin E/CDK2 (high affinity E2F target). But ONLY if complex is on top and localized. Polymerase is ultimately what gets phosphorylated.
why doesn’t origin fire again
kinase phosphorylates origin proteins at the same time it phosphorylates polymerase. Phosphorylated origin proteins signal to leave and be degraded, and polymerase won’t fire without them despite being in phosphorylated state.
when is pre replication complex formed
G1 phase.
what mediates entry into mitosis (G2/M transition)
cyclin B/CDK1 phosphorylation
what is cyclin B/CDK1 targets
- Lamins: when it becomes phosphorylated, nuclear membrane will break down. Non specific phosphatases will removed it – dynamic process. Phosphorylation only occurs if cyclin B/CDK1 is active
- condensin. Condenses chromosomes. When Phosphorylated it becomes active.
how is cyclin B increased
txn’l regulation caused by loss of a repressor.
what happens at the completion of DNA synthesis
CDC25 becomes phosphorylated, this activates Cyclin B/CDK1.
what regulates exit from mitosis (M to G1)
activation of APC
how is APC activated
becomes associated with a co-factor, either CDC20 or CDH1.
what are APCs substrates
- Securin. Releases it from separase which cleaves Cohesin and causes sister chromatid separation.
- Cyclin B, breaks it down, reversal. Chromosomes decondense, nuclear membrane reforms.
G1 restriction point
if cell does not have GFs, will arrest here. Extracellular signalling and cell cycle control connection. After this restriction point, cell is committed to DNA replication.
what does GF signaling result in
upregulation of txn of cyclin D. this triggers phosphorylation of pRb and release of E2F
what is the DNA damage checkpoint mediated by
p53. Cell wants to stop cycle and repair itself.
where is the DNA damage checkpoint
- prior to entry into S
2. Prior to entry into M
G1 DNA damage checkpoint
upregulation of tumor supressor p53. Kinase cascade results in phophorylation of p53 and negative regulator Mdm2
Mdm2
ub ligase for p53. phosphorylation disrupts binding and p53 levels can rise
what is p53
txn factor. one of its targets is gene encoding p21, CDK inhibitor. p21 binds to CDK complexes and prevents phophorylation of pRb so cell is arrested in G1. p53 can also cause apoptosis
how is entry into mitosis prevented by DNA damage
DNA damage triggers a kinase cascade that inhibits phosphorylation of CDC 25 so there is no activation of CyclinB/CDK1. P21 can also inhibit Cyclin B/CDK1 (p53 plays a role)
what does unreplicated DNA do
prevents entry into mitosis. CDC25 needs to be phosphorylated to be active - phosphorylation doesn’t happen until full 4n content achieved (S phase check point)
spindle checkpint
triggered because of a disruption of the spindle and a loss of attachment of MTs to the kinetochore. causes delay of activation of APC so separase and securin stay attached.
primary cells
finite lifespan, directly removed from organism
immortalized cells
unlimited life span
transformed cells
immortalized cells that have acquired certain properties - if they have the ability to form tumors they are tumorigenic
morphology of cancer cells
spindle shaped - thought to be due to actin depolymerization. EMT – epithelial to mesenchymal transition
what do cancer cells rely on for ATP
glycolysis – this is WEIRD because it is inefficient. Hyptoheses:
- Grow in hypoxic environment (glycolysis is anaerobic)
- Need a lot of AA, fatty acids, etc to grow. This is also whats used in the TCA cycle (which generates more ATP) so wouldn’t work for this to be method of energy production
how does a PET scan work
pt given glucose, look at where a lot of glucose is being used - signals that cell is undergoing rapid glycolysis
contact inhibition
normal cells dont proliferate when in contact with other cells - cancer cells DO
senescence
loss of proliferation without cell death - arrested
how could you experimentally stop senescence and allow cells to grow
by removing p53 and pRb (p53 signals to cell to degrade)
crisis
cells die, lose telomerase activity
how could you get cells to not enter crisis
reactivate telomerase
how can you identify a senescing cell
has fried egg appearance when you do a beta galactosidase assay. Senescent cells have more lysosomes and therefore a lower pH which has an associated beta galactosidase enzyme
what causes initial senescence
activation of p53, which causes p21, CIP, inhibits CDKs
what happens at M2 crisis
there is no telomerase so reaches crisis when real coding portions of gene start to be lost
how do cells maintain telomeres
telomerase OR other way (ALT cells)
RNA tumor virus mechanism
- viral genome contains activated form of cellular gene (viral genome contains oncogene)
- virus integrates into genomic locus that results in aberrant expression of a cellular gene
how does ALV work
inserts into host genome and can lead to aberrant expression of a gene involved in oncogenesis (will insert in many places that has no proliferative advantage)
how can a missense mutation cause cancer
constitutive activation. hyperactive protein made in normal amounts.
- Ras is normally a GTP ase. Bound to GTP and is active. - G12V or G13V- loses GTP ase activity so ALWAYS active.
- Raf - V600E- gets constitutively activated as a kinase
In both of these cases, Ras-Raf-Mek-Erk pathway gets turned on so you get high levels of cyclin D, Rb always phosphorylated, E2F always expressed. Don’t need GF or integrin - growth factor and anchorage independent!
how can we visualize gene amplification on chromosome
- Gene amplified ON chromosome
2. Gene amplified and shuttled out in double minutes.
homogenous staining region
large region stains the same due to gene amplification in aberrant DNA replication
how can gene amplification cause cancer in terms of result
- Gene products normally limited in the cells get amplified. Myc, CyclinD1, CDK4 all examples.
- GF receptors like EGFR or HER2 which require ligand binding to dimerize. If a lot of receptors are made and they are very close to each other, will dimerize independently without GF ligands (GF independent)
- Autocrine loop as in PDGF. GF usually comes from other cells to interact with receptor, PDGF amplification causes SAME cell to express both the growth factor and the receptor (still GF dependent)
gene rearrangements involving translocations have what mechanisms in terms of the chromosomes
- Reciprocal translocation on 2 different chromosomes
2. Inversion on same chromosome
what parts of gene can be altered via translocation
regulatory region (alters expression of a normal protein_ or coding region (causing expression of a novel fusion protein)
what gene rearrangements are common in cancer in terms of cell types
b and t cells
what happens in bcr/abl fusion
Abl is tyrosine kinase, usually has its amino terminus negatively regulated. when it fuses to BCR, loses amino terminus and is always active. This causes the substrate protein to always be phosphorylated and activated, which signals cell proliferation and survival causing CML.
why is fusion protein therapy easier than most cancer therapy
novel protein can be targeted because it will only be found in cancer cells and you don’t run the risk of harming other ells with treatment.
what phenotype dominates - tumor or WT
WT - know this from fusion of cells
what is different about DNA viruses than RNA viruses
DNA viruses contain novel oncogenes.
how does simian virus SV40 work
expresses a large tumor antigen or large T antigen that binds to both cellular p53 and pRb thereby inactivating their function
how do adenoviruses work
express two distinct proteins that inactive p53 and cellular Rb
how does sporadic form of retinal cancer work
two somatic mutations in single retinal cell form the tumor - this would be a rare even
how does hereditary retinoblastoma work
first required mutation is inherited, not THAT uncommon for second mutation to arise which is why it is mostly bilateral
distinction between oncogenes and tumor supressors
oncogene - gain of function, can arise from mutating single allele
tumor supressors - loss of function, need to mutate BOTH alleles
what usually occurs in p53 mutations
missense
why does p53 become more stable with mutation
inability to interact with Mdm2, a Ub ligase for p53.
what does oncogene activation by proteins such as myc, ras, and E1a lead to
upregulation of a protein called ARF. ARF inhibits Mdm2, thereby upregulating p53. This upregulates target genes like p21, a CDK inhibitor, and PUMA/NOXA promoters of cell death.
how is adenamotous polyposis coli protein caused
a truncated protein due to frameshift mutation that doesnt allow APC to bind to beta catenin – beta catenin not being degraded means it can always promote cell proliferation
what is loss of heterozeigosity (LOH)
when there is a missense or frameshift mutation of one allele of a tumor supressor or deletion, the remaining WT allele is deleted.
what is transcriptional silencing of a wild type allele
when one allele is inactivated by mutation, the remaining one can be silenced through epigenetic means.
what does p16 regulate
suppressor of cyclin D-CDK4/6
which phosphorylates Rb
what is HPV E7 function
suppressor of pRb
p16 mutations
missense, deletion, promoter methylation – if p16 is mutated, no longer suppresses Cyclin D-CDK 4/6 and get pRb phosphorylation and E2F expression
cyclin D1 mutations
amplification, chromosomal translocations
CDK 4/6 mutations
amplification and missense mutations that prevent its binding to p16
Rb mutations
deletion, mutation
what does HPV infection lead to
expression of E7 protein
p53 pathway
ARF inhibits MDM2 which inhibits P53
what does p53 cause
arrest or apoptosis
what is the function of HPV E6
targets p53 for degradation
APC pathway
APC and Axin bind beta catenin and degrade it
what does B catenin control
txn of myc and cyclin D
how does the ERK pathway work
GF signaling via cell surface receptors. GF can be overexpressed or receptors can be over expressed and receptor is constitutively active as tyrosine kinase
how does PI3K pathway work
PI3K and downstream Akt can be mutates such that they are always active. PTEN is phosphatase that inhibits PI3K signaling, often deleted in human tumors.
what is PTEN
phosphatase that prevents PI3K signaling and PTEN often deleted in human tumors
what does AKT promote
cell survival
what does INK4A gene encode
two proteins, p16 and ARF. p16 and RF share second and third exon but has their own first exon and promoter. Therefore, any mutation in the second or third exon will affect BOTh proteins!
what will get altered if there is a mutation in exon 2 and 3 of INK4
p16 will be mutated: therefore pRb affected
ARF mutated: therefore p53 affected
what do mutations in Myc and Ras often trigger
upregulation of ARF which inhibits MDM2, which activates p53.
what is ARF a txnl target for
E2F. Inactivation of pRb pathway will upregulate p53.
why do cells lose cell-cell junctions in EMT transition
loss of E cadherin expression and cell polarity, increased migratory behaviour
tumor groth and metastasis steps
- Angiogenesis
- Invasion
- Intravasion
- Metastasis
- Extravasation
- Secondary Growth
angiogenesis
when tumor reaches 1-2 mm, angiogenesis factors produces that induce blood vessel formation. Inhibitors also reduced.
invasion
tumor cells become attached to sub endothelial cellular matrices via cell surface receptors. Followed by protease mediated degradation of matrix. Migration via chemotaxis using degradation products or tumor associated autocrine motility factors also occurs
intravasation
tumor cells invade through vascular endothelial cells and their sub endothelial basement membranes and enter vasculature
metastasis
tumor cells must survive shear of blood flow and attack by immune system, then adhere to endothelial cells of target organ or exposed sub endothelial ECM basement membranes
extravasation
tumor cells extravasate out of the vasculature and into perivascular stroma. reverse of invasion
secondary growth
at these distant sites, there is formation and growth of secondary tumor metastasis
oncogene addiction
tumor cells become dependent upon activated oncogene pathways for their proliferation. targeted chemo uses this. EX: inhibitor of EGF receptor blocks proliferation in normal cells but causes death in cancer cells.
necrosis vs apoptosis
necrosis: cells and organelles swell and rupture, leakage induces inflammatory response
apoptosis: cells shrink and condense, organelles and membrane retain integrity, remnants phagocytosed, NO inflammatory response
DNA ladder
way of measuring size – 80-100 bp is nucleosome, see this when cell is undergoing apoptosis
flow cytometer in apoptosis
cell will be hypodiploid because DNA is getting degraded
TUNEL assay
breaks in DNA that occur can be labeled using enzyme terminal deoxynucleotidyl transferase - biotinylated dUTP and can be detected using a strepavidin that has a fluorescent tag
annexin and phosphatidyl serine flip
phosphatidyl serine normally localize to the cytoplasmic side, during phagocytosis, flips to extracellular side but membrane remains intact. Annexin is dye that binds it
*membrane integrity NEEDS to be maintained, otherwise will interact with internal and assay wont tell you anything!
how is apoptosis activated in T cells
glucocorticoids and antibody binding to the cell surface
caspase
cysteine aspase – cleave at aspartic acid and have cysteine at the active site
how do caspases become active
must be processed – get cleaved and lose their pro domains.
what are the two types of caspases
initiator caspase (8 and 9) and executioner caspase (2, 3, 6, 7) - executioner is what cleaves particular substrates
NOTE: 1 initiator caspase can cleave multiple executioners to make them active
what do executioner caspases cleave
cellular substrates that result in apoptosis
what are the substrates for executioner caspases
Proteins involved in DNA degradation (CAD/ICAD)
Proteins involved in DNA repair ( PARP)
Proteins involved in the cytoskeleton (gelsolin)
CAD
endonuclease that cleaves nuclear DNA. Normally bound to ICAD which inhibits it. ICAD gets cleaved by executioner caspase which frees CAD and makes it active to degrade DNA
how do initiator caspases get activated
two main pathways:
1) Extrinsic/cell surface receptors
2) Intrinsic (mitochondrial and Bcl-2)
how does extrinsic pathway of caspase activation work
- killer lymphocyte with Fas ligand attaches to Fas Death Cell receptor on target cell
- This triggers formation of the disk (FADD adaptor protein with procaspase 8/10)
- Caspase 8 or 10 gets activated, executioner caspase gets activated
- Cells undergo apoptosis
why does the cluster occur with assembly of disk
pro molecule has low level of proteolytic activity. if you cluster enough together, get more proteolysis – this triggers cleavage of caspase 8 - THIS can cleave executioner caspase
what is the hallmark of instrinsic apoptosis
loss of mitochondrial transmembrane potential - diagnostic of the opening of permeability transition pores
what is the intrinsic pathway
- Apoptotic stimulus triggers release of cyt c from mitochondria
- Cyt C activates Apaf1 which hydrolyzes dATP to dADP
- Apoptosome formed by release of dADP in exchange for dATP or ATP
- Apoptosome recruits and activates pro-caspase 9
- Caspase 9 cleaves and activates executioner caspases, leading to apoptosis
what kind of gene is Bcl2
oncogene involved in B cell lymphoma but has nothing to do with proliferation – it is an anti apoptotic gene! (BH 1234)
what kind of protein is BH123
pro apoptotic - Bax and Bak
what kind of protein is BH3 only protein
pro apoptotic - Bad, im, Bid, Puma, Noxa
what are BH 1-2-3-4 referred to as
homology domains
what is the basic function of bax and Bak
they form pores in the mitochondrial membrane
how do bax and bak work
an apoptotic stimulus causes the inactive BH123 protein to aggregate and form pores. Cyt C gets released through the pores, or the formation of pores may just disrupt the entire membrane. Bax and Bak are normally cytosolic and monomeric - death signal leads to their insertion.
what is the function of Bcl-2
binds to bax or bak and inhibits their activity so pores cant be formed and apoptosis doesnt occur
what is the function of Bid
BIH3 only protein that directly activates Bax and Bak (pro - apoptotic).
PUMA and Noxa function
block Bcl-2 inhibition of Bax and Bak – this means that pores can be formed and apoptosis can occur
what does p53 target in terms of txn’l regulation of apoptosis
Bax, Puma, Noxa
