Mtc Exam 3 Cell Cycle & Cancer Biology

Front 1

Interphase

Back 1

all cell cycle phases EXCEPT mitosis (Go, G1, S, G2)

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Go phase
-duration
-event

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-days/mos/years
-metabolically active but NOT cycling
-low protein expression
-receptive to mitogens
*induced by contact inhib, growth fact w/d

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G1 phase
-duration
-event(s)

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- 12-24hrs
-lg incr in cell size
-receptive to extracell growth factors
-chroms prepped for duplic
*variability in fast/slow growing cells

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S phase
-duration
-event(s)

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-8 hrs
-chroms duplicated
*sensitive to DNA damage
*cancer cells spend more time in S-phase --> why radiation Tx works

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G2 phase
-duration
-event(s)

Back 5

-some addt'l cell size increase

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Mitosis
-duration
-events

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-1 hr total
-split into two daughter cells

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Mitosis: prophase

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-Chroms condense
-nuc env breakdown

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Mitosis: prometaphase

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-spindle assembly
-chrom congression (start to align on spindle)

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Mitosis: metaphase

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-chrom align
-chrom attach to spindle

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Mitosis: anaphase

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-chrom sep
-spindle disassembly
*sensitive to alignment defects

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Mitosis: telophase

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-chrom decondense
-nuc env assembly

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Mitosis: cytokinesis

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-divide cytoplasm btwn 2 daughter cells

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Genome instability causes

Back 13

-replic errors or DNA repair defects (S phase)
-chrom segreg errors (M)

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Mitosis promoting factor (MPF)

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Cdk1/CyclinB
-Cdk1 = Ser/Thr kinase
-substrates = LaminA, histones (+Pi = promote mitosis)
-levels of Cdk DON'T change over cell cycle; CYCLIN levels change

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Mitotic index

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-how many cells are in mitosis on the microscope field
-higher % = more likely to be cancerous (spend more time in M b/c cycling faster & make more errors)

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Mitosis Cdk/Cyclin pair

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Cdk1/CyclinB
(Cdk1 = cdc2)

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S phase Cdk/Cyclin pair

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Cdk2/Cyclin A1/A2

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G1-to-S transition Cdk/Cyclin pair

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Cdk2/Cyclin E1,2

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Cyclin D

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ONLY cyclin that responds to extracellular growth factors
(Ras/Raf/MAPK: increase transcription
PI3K pathway: +Pi makes Cdk/CycD more stable complex)
--> helps cell go from Go to G1
CycD/Cdk4,6 inhibs Rb, so E2F active --> S phase genes on

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Rb protein

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-inhibits E2F (so inhib S phase genes)
-bound to HDAC (histone deacetyl)
-CycD/Cdk4,6 inhibs Rb binding to E2F (Cdk4,6 phosphorylates Rb= weakens binding to E2F)

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E2F protein

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-controls expression of S phase genes (directly binds to DNA)
-inhib by Rb

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HDAC (histone deacetylase)

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-removes acetyls from histones
-fewer acetyls = less accessible DNA (more compact)
-bound with Rb to E2F to inhib S phase genes

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Tumor suppressors
-mech of fcn
-examples

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-inhibit cell cycle progression
-Rb, p53, p16, RAF

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Proto-oncogenes
-mech of fcn
-examples

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-stimulate cell cycle progression
-Cyclin D1, Mdm2, c-myc, Ras

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Oncogene

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mutated proto-oncogene
gain of fcn/overexpression --> cancer

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Effect of ↑ Cyclin D1 & ↓ Rb

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↑ cell cycle progression --> cancer
↑ CycD = ↓ inhib of Rb = E2F active
↓ Rb = less inhib of E2F => Sphase genes made

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C-myc

Back 27

transcription factor
promotes cell growth (↑ mass)
-MAPK & PI3K pathways promote c-myc activity --> prot synth genes made --> Go-G1-S

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Effect of ↑ c-myc & ↓ Rb

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Cancer!
c-myc = more prot synth genes made
Rb del means E2F active & Go to G1 trans promoted

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S-phase promoting factor
-mechs of fcn

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CyclinE/Cdk2
-promotes DNA replic via +Pi to helicase
-phosphorylates Rb so ACTIVE (E2F suppr - no more S genes made- not needed)
-blocks new licensing (G1/S)

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Cak

Back 30

Cyclin activating kinase
-Pi on T160 of Cdk's activation loop

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Cdk4,6 inhibitors
-ID
-mech of fcn

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p16(INK4) family:
-p15, p16, p18, p19
-competitive inhibition (bind at same spot as cyclin D)

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how does p16 alteration ==> cancer

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-↓ p16 = ↑ CycD = ↓ inhib of Rb = E2F active--> S phase genes --> cell prolif

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Cdk1, 2 inhibitors
-ID
-mech of fcn

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p27 family:
-p21, p27, p57
-bind to catalytic cleft of Cyc/Cdk = trimeric
-affects CycA,E/Cdk2 & CycB/Cdk1

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How do cells go from Go--> G1?
-mech

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-Go= high levels p27, p16, p21 & low levels CycE/Cdk2
-cell receives signal--> ↑ CycE
-(G1/S) some CycE/Cdk2 Pi's p27
-p27-Pi recog by Spk2--> Cul1 recruited
-Cul1 ubiquinates p27-Pi-->degrad
-(S) CycE/Cdk2 self-Pi's for recog by Cul1, ubiq--> degrad

Front 35

p27 -/- mice: larger or smaller than WT?

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larger: more cells in body b/c p27 doesn't inhib CycE,A/Cdk2 or Cdk1

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Skp2 -/- mice: larger or smaller than WT?

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smaller: Skp2 levels low = higer p27 --> more CyclinE/Cdk2 inhib --> CyclinE/Cdk2 inactive
suppressed cell prolif

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Wee1 protein

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-kinase that Pi's CyclinB/Cdk1 at Y15 site (NOT on activ loop) --> Cyc/Cdk inactive
-when Wee1 inactive, cell goes thru mitosis too quickly = cells too small

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pre-mitosis DNA damage pathway

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detector --> (+) ATR kinase --> (+) Chk 1 kinase --> (-) Cdc25
= CycB/Cdk1 inactive ==> nuc disassembly & chrom condens'n

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Cdc25 protein

Back 39

-phosphatase
-does reverse of Wee1
-removes Pi on CycB/Cdk1 so Cyc/Cdk is active
-inhib by Chk1 kinase

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DNA damage inhibition of Cdk1 activity (2 mechs)

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-inactivation of phosphatase (Cdc25: detector/ATR kinase/Chk1 kinase/Cdc25)
-activation of p53 (--> more p21 so more inhib of Cyc/Cdk)

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p53 protein
-general fcn
-activators
-inhibitors

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-cell cycle prolif inhibitor: activates p21 (which inhibs Cdk1 &2 = complex w/ Cdk & cycB, E/A)
-induced by stressors--> +Pi = active -->cell cycle arrest, DNA repair, senescence, apoptosis
-activ by kinases; Pi weakens binding w/ Mdm2
-Mdm2 inhibits (ubiq ligase)
[ARF inhib Mdm2 = ↑p53]

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Mdm2 protein
-mech of fcn
-activators
-inhibitors

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-ubiquitin ligase; inhibs p53 when bound to it (via ubq'n)
-Mdm2 default to be bound to p53?
-ARF inhibs

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protein degradation regulation mechanisms (4)

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-substrate phosphorylation to induce E3 ubiq ligase binding
-regulate activity of E3 itself
-substrate phosphorylation to block
-regulate expression of E3

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Cohesin protein

Back 44

deposited on chroms as replic occurs
-holds sister chroms together until anaphase
-removed by separase

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anaphase promoting complex (APC)
-mech of fcn
-inhibitors
-activators

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-ubiqs CycB & securin for degrad (which makes separase active)
-inhib when sis chroms NOT attached to spindle [detector-kinase-effector pathway]
-activ'd when detector leaves (b/c finds sis chroms attch'd to spindle)

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Securin protein
-mech of fcn
-inhibs
-activs

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-inhibits separase; when degrad, separase active
-inhib (degrad) when Ubq'd by APC for degrad
-activ when sis chroms NOT attached to spindle

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Separase
-mech of fcn
-inhibs
-activs

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-removes cohesins from btwn sis chroms--> anaphase
-inhib when bound to securin
-activ when securin degrad (Ubq'd by APC when sis chrom attch'd to spindle)

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Taxol, vinblastine fcn

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-cancer Tx
-make difficult for cells to go through mitosis
-highly toxic

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Tumor-causing viruses
-RNA mech
-DNA mech

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-RNA: integrates into genome, carry or activate oncogenes
-DNA: does NOT integrate, carry viral oncogenes; if accidentally integr, viral prot constit expressed

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HPV
-viral type
-general fcn of cancer causing

Back 50

-DNA
-makes host cell move into S phase so can use DNA replic machinery for own protein expression purposes
-integr of fragment w/ E6 & E7 genes is worst-- knock out two tumor suppressor elements (p53 & Rb)

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HPV E6 gene fcn

Back 51

-E6 binds to p53 & induces its deregulation
(E6AP is E3 ubiq ligase)

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HPV E7 gene fcn

Back 52

-binds to Rb & blocks Rb intrxn w/ E2F --> E2F active --> Sphase

Front 53

Examples of mutations --> cancer
-describe mech of fcn
1) ↑Mdm2 ; ↓ Rb
2) ↑CyclinD ; ↓ p53
3) ↓ p16 ; ↓ ARF *

Back 53

1) ↑Mdm2 = ↓ p53; ↓ Rb= ↑E2F activity
2) ↑CyclinD=Go-G1 trans ; ↓ p53 = ↓p21=Cyc/Cdk remains active
3) ↓ p16 = ↑ CycD = ↓ inhib of Rb = E2F active ; ↓ ARF= ↓ inhib Mdm2 = ↓p53
*p16 & ARF encoded by same gene; alt reading frame

Front 54

ARF-Mdm2-p53 pathway: which changes cause cancer?

Back 54

↓ ARF ; ↑ Mdm2 ; ↓ p53 = cancer prone… one hit enough

Front 55

Nutlins

Back 55

molecs that bind Mdm2 & compete for p53 intrxn
-Useful for cancers where Mdm2 is overexpressed (too much degrad of p53)

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Hallmarks of cancer

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- Resisting cell death
- Sustaining prolif signal
- Evading growth suppressors
- Activating invasion & metastasis
- Enabling replicative immortality
- Inducing angiogenesis

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Cancer contributors: abnormal ↑ in cell #

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=more cell divisions/less apoptosis
Eg: loss of Cdk control: prob w/ upstream signaling or downstream core regulator problems

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Cancer contributors: Genetic instability

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cell pop becomes increasingly heterogeneous
○ Loss of checkpoint control
○ Loss of DNA repair efficiency
○ Loss of chrom segreg control

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Cancer contributors: Changes in cell adhesion & migration

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b/c of changes in integrin signaling, etc

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Cancer contributors: - Induction of angiogenesis (new blood vessels)

Back 60

○ Stim of VEGF receptor in tumors (vasc endothel growth fac)

Front 61

Cancer contributors: mutation in growth factor signaling pathways
3 overall ways to have errors

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1) Growth factor over pdc'n
2) RTK overexpression (receptor TyrKin)
3) Ras activation (or player downstream of this)

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Receptor Tyrosine Kinase (RTK)
2 major signaling pathways
-ultimate effect on ___

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1) Ras-Raf-MAPKK-MAPK
2) PI3K
**both work to ↑ CyclinD & C-myc (promote cell prolif)

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Cancer contrib: Growth factor-independent signaling- 3 ways

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-Mutation of Ras causes it to be stuck in GTP (active) form
-Receptor over-production; sheer mass action
-One of players in pathway (after receptor) is mutated so constit active (eg: Raf)

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Philadelphia chromosome
-mutation
-effect

Back 64

-Reciprocal translocation btwn chroms 9 & 22 in chronic myelogenous leukemia = new oncogene (BCR-ABL)
○ ABL: non-receptor TyrKin like Src?. Mut moved one of domains --> constit active kinase
○ BCR: signaling protein; when fuse BCR to ABL, disrupts auto-inhib domain of ABL --> constit active
OVERALL: Activates Ras & PI3K pathways b/c can put Pi on Tyr when not supposed to

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Gleevec
-how fcn

Back 65

-small-molec inhib. of TyrKin specific to BCR-ABL (Philadel) protein that not usually in normal cells (so few side effects as Tx b/c only targeted cancerous cells)

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Mutations/deletions that cause cancer: Rb
-what mutation affects

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-no Rb = E2F active --> transcription of S-phase genes

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Mutations/deletions that cause cancer: p16INK4
-what mutation affects

Back 67

-Cdk 4 & 6 inhib;
-p16 family competes for same binding spot as CyclinD

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Mutations/deletions that cause cancer: p27 family
-what mutation affects

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-Inhib Cdk 1 & 2
-trimeric: binds btwn Cdk & Cyclins (B, E/A)

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Mutations/deletions that cause cancer: DNA damage chkpts

Back 69

-BRCA1
-ATM
-Chk1/Chk2
-p14ARF
-p53