Chemo Flash Cards - SS/AB
Drugs metabolized by non-enzymatic hydrolysis?
BCNU
Mustargen
Melphalan
Drugs metabolized by ubiquitous enzymes?
Cytarabine
Gemcitabine
6 mercaptopurine?
Which chemo undergo HEPATIC metabolic? Via cyp450 vs conjugation?
CYP450: bisulfan, chloarmbucil, cytoxan, ifosfamide, paclitaxel, vinca alkaloids
Conjugation: etopisde
Which drugs are excreted really?
bleomycin
carbo, cisplatin
etoposide
hydroxyurea
methotrexate
topotecan?
Drugs eliminated by biliary excretion?
doxo
vinca alkaloids
How do lymphs die from RT?
apoptosis (interphase death)
Why is cytoxan platelet sparing?
megakaryocytes have increased ALDH (break down of phosphoramide mustard)
L-MOPP with T-cell LSA (Brodsky 2009)?
CR 78%
CR 1 (achieved and maintained through 28d cycle) 56%
CR 2 (lost during cycle but subsequent CR at next MOPP) 22%
PFS 189d
Respond of dogs to single agent doxo?
B: 100% (86% CR, 13% PR)
T: 50%
Hodgkins LSA in cats?
- Head, neck (can be other regions e.g. inguinal)
- cats >6, all FIV/FeLV-
Histo: mostly non-neoplastic T cells with REED-STERNGERG cells scattered throughout
- tumor cells are B-CELL (CD79a+, BLA36+)
- RS cells are CD79-, BLA36, CO3, Mac38+
MST dogs with splenic MZL?
~380d (12 mo) if symptomatic
> 1,100d (36 mo, 3 yr) if asymptomatic
DLBCL subtype
- centroblastic (multiple central nucleolus)
- immunoblastic (single nucleolus)
What is the agreement of IHC with Flow? PARR?
Flow: 94%
PARR: 69%
T-zone LSA flow?
CD45-
CD3, CD21, CD25 +
High MHC class II
variable CD4/8
Most common Tcell LSA immunophenotype?
CD4+
Most common CLL immunophenotpye?
Tcell CD8+
<30k/uL cells prognosis 1,098d (36 mo, 3 yr) vs >30k (131d)
Thymoma Flow?
> /= % CD4+/CD8+
Large granular LSA dog
- few cytoplasmic granules
- 100% CD8+, most CD3+
- most common TCR alpha, beta 60%
- 8% CD3-
- 92% + for alphadb2 integrin found in spleen/BM
Which cells express CD4?
- neutrophil: CD4/18
- activated dendritic cell
- T help lymph
Two means of T-regs decreased immune response?
- Direct interaction
- alpha interferon cytokines; TGFb, IL10
What are the Treg markers?
- subset of CD4+ (10-15%)
- FOXP3 + TF
- CD25+ IL2 receptor
What is the role of NK?
kill cells that down regulate MHC class I missing “self recognition”
Which immune cells posses MCH I? MCH II?
MHC I: all cells
MHC II: dendritic, macrophages, B lymphs
Which immune cell is principle to mediate tumor immunity?
CD8
How do CTLs kill targets via apoptosis?
- cytotoxic granules
- granzymes - some proteases
- perforin
2, FasL expression -> cas8
Which portion of Ab is responsible for Ag binding?
Primarily hypervariable region of Vh and VL CDRs
Palladia targets
PDGFR
EGFR
KIT
Cat carbo dose?
240 mg/m2 q 3 week
OR
Dose = AUC x 2.6 GFR x BW (kg)
Doxo MOA?
- inhibition of RNA and DNA polymerase
- Topo II inhibition
- DNA alkylation
- ROS generation
- Perturb Ca2+ homeostasis
- Change plasma membrane
- inhibition of thioredoxin reductase
What is terminal half life?
how long it takes a drug to decay in the bosy
Weird rxn in people with DTIC?
- Flu syndrome
- photosensitivity
- eosinophilia
- depresses Ab responses
anti tumor activity of DTIC?
- Methylation of O6 guanine
- originally developed as a purine alpha metabolite but not cell cycle dependent nor AIC portion needed for activity
Procarbazine metabolism?
- extensive hepatic microsomal metabolism
- monoamine oxidase inhibitor
- MANY DRUG INTERACTIONS d/t this
Cytotoxic mechanism of procarbazine?
methylation at O6 guanine –> increased in AGT, MGMT deficient cells
What is the MOA of Mesna?
- 2-mercaptoethane sulfonate
- dimerizes (inactive in plasma) in urine –> hydrolysis –> conjugates with alkylators
disulfide detoxifier
Why is cytoxan particularly applying for metronomic chemotherapy?
selective inhibition of Tregs
What effect do alkylating agents have on immunity?
suppress humoral and cellular immunity
especially cytoxan (B+T)
Why does cytoxan spare BM cells?
high levels of ALDH –> detox
- aldophosphamide –> inactive carboxycyclophosphamide
Predominant metabolites of cytoxan?
-nornitorgen mustard
-phosphoramide mustard
Alkylating agent metabolism?
- Spontaneous hydrolysis by H2O
- Enzymatic (important for PK of phosphor amide mustard and nistrosureas)
What is a major difference in MOA and efficacy of oxaliplatin?
- DNA polymerase alpha cannot pass over adduct
- decreased thymidylate synthase
Which other organs may be affected by platinums?
- Neuro: peripheral neuropathy
- ototoxicity
- Hypersensitivity IgE mediated in 10-15%
What can ameliorate platinum induced neuropathies?
amifostine & B6 (pyridoxine)
How do platinum agents affect the immune system?
JUN/Jnk increase after cisplatin may cause increased FaS
Pulmonary reactions with vinca?
- acute bronchospasm
- interstitial infiltrates w/in 1 hour
most common with mitomycin
How do platinum drugs enter the cell?
- Cisplatin: diffusion at pH 7.4, Cl- replaced by -OH
also transmembrane transporters: CTR1, ATP7A, ATP7B, Cu transport
What happens to platinum agents inside the cell?
- exported from cell by active transport proteins
- become reactive by displacement of Cl- (cis) or carboxylic groups –> covalent binding with sulthyonyl groups
- React with nucleophiles (e- on protein, RNA, DNA)
Affinity for RNA»»DNA
How do platinum drugs kill cells?
primarily via DNA adduct formation (must be CIS)
Cell with what mutation are most sensitive to platinums?
BRCA mutants
(decreased dsDNA break repair)
Mechanisms of resistance (increase drug target)
DHFR (dihydrofolate reductase) - methotrexate
RNR (ribonucleotide reductase) - hydroxyurea, gemcitabine
TS (thymidylate synthase) - 5FU
Mechanisms of resistance (decrease drug target)
Topo I: topotecan, irinotecan
Topo II: etoposide, anthracylines
MRP1 substrates?
- many PGP substrates
- methotrexate
- conjugated metabolites
What is the ABCB1-1delta mutation?
4 base pair deletion = frameshift = several premature stops
What happens when MDR1 mutants are treated with substrate drugs?
- severe GI upset
- 3x higher exposure
- 20-25% decrease with mut/norm, 50% decrease in mut/mut
- Doxo decrease to 10.7 mg/m2 in mut/mut
Taxane MOA
-disruption of microtubule function
- Microtubules are essential to cell division
- taxanes stabilize GDP-bound tubulin in the microtubule inhibiting the process of cell division
- depolymerization is prevented
- B tubulin
- Cannon dissolve but can assemble
How are taxanes different from other microtubule inhibitors?
Vinca alkaloids prevent mitotic spindle formation while taxanes prevent function
Taxane resistance?
- MDR
- delta binary sites, tubulin dynamics
- signaling activation (PI3k) –> increase threshold for apoptosis
- increase MAP–> MAP4 increase Paclitaxel sensitivity
What drugs can reverse MDR phenotype?
- Ca2+ channel blockers
- tamoxifen
- alpha arrhythmic
- cremophor/polysorbate
How is 5-azacytidne activated?
conversion to monophosphate by uridine-cytidine kinase
How does 5-aza enter cells?
facilitated nucleoside transport shared with uridine and cytidine
5-aza MOA?
- does not require deoxycitidine activation
1. inhibits methylation
2. competed with CTP for incorporation into RNA
3. incorporates into DNA
How does 5-aza structure differ form cytidine?
- S1 Nk of heterocyclic ring unstable –> spontaneous decomposition
What is the first strategy for correction of a miscopied nucleotide?
polymerase 8 proof reading and 3’-5’ exonuclease activity
When is mismatch repair used?
- used during DNA repair
- singe base mistmatch
- MSH/MLH proteins
- MMR deficiency = microsatellite instability
How are inter strand crosslinks repaired?
- problem when replication fork is stalled
- Fanconi protein recruited with ATR
- Incision to enable bypass –> broken strand = HR (homologous repair), adduct = NER (nucleotide excision repair)
How is ssDNA/ss breaks repaired?
PARP activation
How does homologous recombination occur?
- ds break
- mRNA complex recruitment (endonuclease) and ATM
- RPA recruited –> DNA resection (BRCA1 dept)
- Rad protein loading DNA (BRCA2 controlled)
- dsDNA invasion
Non-homologous end joining (NHEJ) - how does it occur?
- Break recruits Ku70/80
- Ku80 interacts with DNA-PKcS –> autophosphorylates
- DNA polymerase recruitment and strand ligate
Targets of PKcs:
- KU70/80
-XRCC4
- DNApKcs
+/- ATM
*Pic include HR repair
G2 DNA damage checkpoint
ATM (chk2), ATR (chk1) –> CDC25C –> Cyclin B/CDK1 (wee1 inhibitory) –> Stop B2
What protein is needed for replication checkpoint?
ATR
- Fork stabilization
- Origin firing
- S-m checkpoint
G1 DNA damage checkpoint
When in the cell cycle are ATM and ATR active?
- ATM: all checkpoints
- ATR: ONLY when sDNA template available (S + G2)
What are the major proteins for ssDNA repair?
RPA, RAD1, RAD9, TOPBP1 –> ATR, ATRIIP
also for base alkylation adducts –> MUTs, MUTc (MMR proteins)
Via what mechanism does p53 halt the cell cycle?
up regulation of p21
Beclin-1
- autophagy promoting protein
- autophagy activated when cells suffer nutrient starvation and digest own intracellular organelles
BID function?
- activated (cleaved) by caspase 8 in cytosol –> mitochondrial channel opening
*see figure 8-15 in T&H
What are the death receptor ligands?
TNF proteins (TNFalpha, TRIAL, FasL)
What are executioner caspases?
3,6,7
What is the role of smac/diablo?
protein released rom the mitochondria with cytochrome C –> inactivated proteins (alpha-apoptotic IAPS[inhbitiors of apoptosis]) –> increase apoptosis
- IAPs normally block caspase reaction:
1. bind directly –> block proteolysis
2. mark caspases for ubiquitination
What are the components of the apoptosome?
- heptameric protein complex of the intrinsic apoptotic pathway
pro-caspase 9 + apaf1 + cytochrome C (activated caspase 9) –> pro-caspases 3,6,7 –> caspases 3,6,7 –> cleavage of death substrate
How does the AKT/PKB kinase act to inhibit apoptosis?
phosphorylation of BAD by AKT/PKB –> decreased ability to keep mitochondrial channel open –> decreased apoptosis
What is TSP1?
-thrombospondin-1
- blocks blood vessel development
What are the majority of p53 mutations?
-AA substitutions in DNA binding domain
ARF function?
- AKA p14
- growth inhibitory - depends on functional p53
- ARF binds MDM2 –> sequesters in nucleus –> p53 increases d/t decreased levels of destroyer
PI3k pathway connection to p53?
anti-apoptotic pathways
PI3K–> AKT/PKB kinase–> MDM2 phosphorylation –> MDM2 translocation to nucleous
RAS–> RAF–> MAPk –> ETS –> API (Fas+Jun) –> increased MDM2
How is p53 protected from ubiquitination?
- phosphorylation (n-terminus) blocks MDM2s ability to bind and ubiquitinate
- ATM, chk1/2
*ATM kinases can also phosphorylate mdm2 –> functional inactivation
Role of MDM2?
*function in nucleus
- ubiquitination of p52 as it is synthesized
- mdm2 expression is induced by p53
- mdm2 bind p53 and blocks transcription activity –> directs ubiquitin attachments and export from nucleus –> polyubiquinated in cytoplasm
3 pathways to p53 activation?
- ds breaks –> ATM –> p53 phosphorylation
- DNA damaging agents: ATR –>chk2–> p53 phosphorylation
- deregulation of pRb-E2f
What is the role of ATM?
damage –> atm kinase (chk2) –> air kinase –> p53 phosphorylation = protection from destruction
UV radiation
- causes pyrimidine dimers
- CC-TT transition characteristic of misrepair or lack of repair
What happens in the cell after DNA damage has occurred?
- DNA repair: most likely
- No DNA repair: mutation
- error prone replication = base substitution
- frame-shift mutations occur when an adduct is bound to a nucleoside base (usually base deletion)
- DNA breaks from incomplete excision repair or alkylation and cleavage of backbone
What bases have amine groups?
-NH2
Purines & cytosine
Which base is most commonly incorporated with error prone repair?
adenine
Which is the most prevalent oxidized base?
8-oxo-deoxyguanosine = marker of overall oxidative DNA damage
How do genotoxic chemicals damage DNA?
- either add carcinogen adduct or oxidative damage
- strong electrophiles –> bind wide array of weak bases –> must bind strong nucleophiles
- may also be oxidized by hydrocylanine of nucleotide bases
What is the first step in metabolic activation of chemical carcinogens?
oxidative metabolism often mediated by CYP enzyme (change in carbon, nitrogen, sulfur)
3 most extensively studied carcinogens requiring bio activation?
- PAHS
- aromatic amines
- nitrosamines
What properties do all genotoxic carcinogens share?
- electrophilic or capable of being converted to electrophiles –> interact with nucleophilic groups on DNA/protein
- most require enzymatic bio activation typically by drug-metabolizing enzymes
When is relative risk equal to odds ratio?
If disease prevalence is LOW (<10^210)?
Information bias
due to errors in obtaining bias (aka misclassification)
What is selection bias?
systematic differences between those in study vs those eligible
3 criteria for confounder
- risk factor of disease of interest
- must be associated with EXPOSURE under the study but not a result of it
- Should NOT be intermediate factor on the causal pathway between exposure and disease
Tumor progression
benign lesions acquire ability to grow, invade tissues, and establish metastasis
Tumor promotion
- clonal expansion of initiated cell d/t changes in altering gene expression
- agents ten to be NON-genotoxic and cause division w/o death/differentiation
What is tumor initiation?
- creation of DNA damage that must be corrected or mutation will become integrated
- irreversible!!
- may die via apoptosis
Ionizing radiation mechanisms of genetic instability?
- mutation of genes involves in control of DNA synthesis/repair
- chromosome instability
- aberrant production of oxygen radicals–> DNA damage
Fluctuation of cyclins during cell cycle
What CDK/cyclin is responsible for each phase of the cell cycle?
- G1 = D, CDK 4,6
- S = cyclin E, CDK2
- G2 = cyclin A, CDK2
- m = cyclin B, CDK 1 (CDC2)
What are CDKs?
- serine/threonine kinases
- associated with cyclins to activate CKD catalytic activity
What is the neurotoxic metabolite of ifosfamide?
- d/t dechloroethylifosfamide
- Chloroacetyl aldehyde
What is the most important methyltransferase? What drugs target this?
-DNMT1
- 5-azacytidine
- 5-cita-deocycitidine
- incorporate into DNA and inhibit DNMT while allowing replication to proceed
What are the most prominent cancer stem cell subpopulation in BCL and TCL?
BCL = CD34, CD90, CD117, OCT 3/4
TCL = OCT 3/4
Tx with CHOP enriched for CSC (increase CSC markers, increase ALDH)
Hartley et al 2018
How does leptin contribute to tumorigenesis?
leptin (from adipocytes) –> proliferation of BC cells (breast cancer I think) via increased aromatase
TGFb signaling pathway
*receptor is a heterodimer
TGFb binds type II receptor –> dimerization with type I and phosphorylation of type I –> SMAD 2/3 recruitment –> phosphorylation –> associated of SMAD4 + 2/3 –> nucleus
Patched Pathway
Hedgehog –> patch receptor –> release of SMO = activation –> cytoplasmic GLI stabilization
- ON: normally cleaved into 2 fragments; 1 nuclear repressor of transcription
- SMO protect GLI from cleavage –> trans ACTIVATION
Notch Pathway
Delta/jagged –> NOTCH = surface receptor –> 2x cleavage
1) ectodomain
2) transmemebrane
–> nucleus
What is the REL gene?
NFKB subunit amplified in 1/4 DLBCL in people
WNT signaling pathway
WNT binds frizzled –> disheveled activated –> blocks GKS3B–> B-catenin accumulation in cytoplasm and nucleus
Normal B-catenin cytosolic complex?
B-cateninc + APC + axin } GSK3b executioner
NFkb pathway
What does B catenin associate with in the nucleus?
TCF/LEF transcription factor
Important targets of RAS –> RAF –> MEK–> ERK pathway?
- promoters of Fos + Jun
- Fos + Jun associate –> API –> transcription factor hyper activated in cancer
MAPk pathway
RAS –> RAF (MAPKKK) –> MEK (MAPKK) –> ERK (MAPK) –> targets
PI3k pathway
PI3k –> PIP3 –> AKT/PKb –>
- Bad inhibition of apoptosis
-mTOR stim - GSK3b stem of cell proliferation
Dual specificity kinase?
- can phosphorylate serine/threonine residues and tyrosine
ex. MEK
GRB2 + SHC bridging proteins
GRB2 protein structure?
2 SH3 groups –> SOS
1 SH2 –> ligand receptor
What is the difference in the SRC domains?
SH1 = catalytic domain
SH2 = enables association with partner protein displaying specific phosphotyrosine + AA
- may or may not have catalytic activity
SH3 = binds purine rich sequence domaines in partner proteins –> ligands of SHC domain
Phosphatidyl pathways
PI –> kinases –> PIP2 –>
1) PI3k –> PIP3 –> attracts proteins with PH domain
2) phospholipase C –> DAG –> PKC + IP3 –> Ca2+ reduces
Most important PH domain protein?
- AKT
- PI3k –> IP3 attracts AKT/PKI3 via pH domain
- PD1,2 activated kinases
Anti-apoptotic substrates of AKT/PKB?
Bad (pro)
Cas 9 (pro)
IKB kinase (alpha)
FOXO TF (pro)
MDM2 (alpha)
Proliferative substrates of AKT/PKB?
- GSK3b (alpha)
- FOXO4 (alpha)
- p21 (alpha)
Growth substrates of AKT/PKB?
TSC2 (alpha)
Ral A/B
- 1/3 major effector pathways of RAS
- Ral A/B gene 58% identity with RAS
SOS function?
To induce RAS to shed GDP
Ral pathway
RAS –> Ral-GEF –>
RalA
RalB
–> RCLBp –> Cdc42 –> proliferation
Examples of JAK/STAt receptor
EPO, TPO
JAK/STAT pathway
Integrin signaling
- alpha and beta heterodimers
- link cytoskeleton to ECM + signaling
- signaling via SOS–> RAS–> ERK
Wnt- B- catenin
- signals via frizzled receptors - GSK3b
- GSK3b normally phosphorylates B catenin –> ubiquinated
B-catenin states
- bound cytoplasmic domain of cell-cell adhesion receptors
- soluble cytosol
- transcription factor
What are promoters of the cyclin D genes?
D1 - API, TCF/Lef, NFkb, TFs
D2 - myc, increased camp
D3 - Stat 3/5, E2A
What is the exception to fluctuating cyclin levels?
Cyclin D
- controlled by extracellular signals (tyrosine kinase receptors)
- others controlled by rapid degradation via ubiquitination
What is CD25?
part of IL2 receptor
T-zone LSA flow?
Cd3+, Cd5+
CD45-
High CD21
High MHC II
PARR sensitivity?
neoplastic 1/100 cells
Factors that may affect PARR?
- DNA quality (e.g. formula increases fragmentation)
- Taq inhibitors
- Primer choice
Terminal deoxynucleotidyl transferase (TDT) enzyme
Adds nucleotides between V+D, and J.
Where does Ig rearrangement occur?
Bone Marrow
- acute leukemia may not have yet, may be -
Ig formation
V 80
D 6
J 6
T cell markers
Surface: CD3 (TCR), CD5, CD4, CD8
Cytoplasmic: CD3c
Where do T cells acquire markers?
CD3 –> BM
CD 4/8 –> thymus
B cell markers for flow and IHC?-
Flow: CD21, CD22, CD20
IHC: CD79a (BCR), Pax5 (TF), CD20c, Mum1, IRF4
BRCA in canine mammary tumors?
- no association between polymorphisms and development
- several SNPs identified
- CMT have decreased nuclear and increased cytoplasmic BRCA but this is likely not significant
1) What is AgNOR?
2) Where is it located?
3)When are levels highest?
- Crucial for formation of nucleolus
1) chromosome segments involved in ribosome biogenesis composed of agrophylic proteins that localize with DNA loops of nucleolus, decondensation of DNA loops during high transcription –> segregation of associated proteins
2) nucleus, nucleolus
3) interphase –> give an indication of duplication role
1) what is ki67?
2) where is it located?
3) when are levels highest?
4) half life?
1) proliferation associated protein
2) nucleus
3) peak in M, low in G1/S
4) <1hr –> rare detection if cells aren’t cycling
*Ab - MIb1
1) what is PCNA?
2) where is it normally located?
3) when is expression highest?
1) cofactor of DNA polymerase delta –> increased DNA replication
- also plays a role in RAD6 dependent DNA repair and inhibitory apoptosis via negative regulation of abl stability
2) nucleus
3) high in G1/S but also M because of half life (8-20 hours)
Feline MI cut offs: cutaneous & intestinal MCT?
Cutaneous: 5 (same as dog)
Intestine: >2
Feline MCT IHC markers?
+ vimentin
alpha 1 alpha trypsin
KIT
What’s the difference between metric and proportional approach for MCT margins?
- metric: 1 cm for low and 2 cm intermediate
- proportional: lateral proportional to maximum MCT dimension
What % of dogs with MCT have BM involvement?
- 2.8% on FNA
- if visceral, 37% Buffy coat + and 56% BM+
MCT IHC markers?
Vimentin +
Typtase + (skin only)
+/- Chymase (GI and skin)
CD117/KIT +
IL8
SQ MCT MI prognosis?
> 4
- also if infiltrative +/- multinucleate cells
Kueppel grading criteria
1) MI >/= 7
2) 3 bizarre nuclei /10 hpf
3) 3 multinucleate cells/10 hpf
4) karyomegaly
- if any of these = high grade
Cutaneous MCT MI prognosis?
MI > 5 –> 3 mo MST
MI <5 –> 80 mo MST
Which molecules control extent of R6 phosphorylation?
Cyclin d + e
What is the difference in cyclin D control vs others?
cyclin D = extracellular signals
others = gradual accumulation and rapid destruction
What type of kinases are the CDKs?
serine 1 threonine
Most critical CDKI in cancer? How is it controlled?
- p27 = cyclone E , CDk2
cell enter G0 –> increased p27
enter G1 –> decreased p27, fall during late G1 by cyclin E/CDk
*p27 decreased by SKP2 protein - acts with cul1 to recognize p27 and ubiquitance –> tagged for destruction = decrease skp2 at G0 = increased p27
How does TGFb guarantee cells do not proliferate?
- must ensure p21 is not inhibited
- in normal cells –> TGFb keeps Mac away from promoters by decreasing Mac expression via smad3 promoter sequence
TGFb basics
- uses serine/threonine kinases
- targets mad 2/3 –> smad4 association –> heterodimer moves to nucleus –> TF
- most important targets: CDKs, p21, each has CAGAC seq in promoter
Number 1 target of Mac for cell cycle progression?
cyclin D2 –> R of Rb
- myc also increased expression of e2f1/2/3
Cul1
- protein increased by myc
- plays central role in p27 degradation (CDKI) via ubiquitination
myc function
- transcription factor
- creates homo-heterodimers
- dimers associated with regulatory sequences termed “e boxes” of promoters “CACGTG”
- phosphorylation of myc regulates activity
How do mitogenic signals reduce CC progression?
mitogen signals –> RAS (–> Jun/fas –> cyclin D1 transcription) –> PI3k –> AKT/PKb–> GSK3b –> increase b cat
- cyclin D is not deposited on EaF
- cyclins D+e inactivat E2f –> S phase entrance
What is the most important target gene of ear for cell cycle progression?
-cyclin e
- responsible for complete hyperphosphorylation of pRB
How does Rb inhibit E2F?
- E2F sits on promoters
- when hypO phosphorylated Rb bound, transactivation domain is blocked, which is needed for transcription
- pRb recruits other proteins that decrease transcription
- E2F1/2/3 –> binds Rb
- E2f 4 + 5 –> gene repression via p107/130 to attract repressors
- E2F6–> doesn’t interact with proteins –> acts exclusively as a repressor
What controls Rb phosphorylation?
- D type cyclins with CDK 4/6 (controlled by extracellular signals)
- cyclin e +CDK2 increased at R and drives pRB phosphorylation to completion
Importance of pRB phosphorylation status?
- unphosphated in G0
- weak in G1
- hyper on ser/thre residues to advance through R
- remains hyper throughout cell cycle
- once cells exit mitosis, phosphorylation stripped of by PP1
How can PI3K activation increase proliferation?
- TGFb can stimulate
- stimulation of RTK
- AKT/PKB –> p21 in nucleus
Role of TGFb in cell cycle progression?
- increase levels of p15 –> blocks cyclin D/CDK4/6 complex and inhibits those already formed
- without active complex cannot proceed through G1-R
Cyclin-CDK inhibition
- CDKIs
- INK4 proteins –> CDK 416 (p16, p18, p19)
- p21, p 27, pS7 –> inhibit all others
Extrinsic apoptosis pathway
FAS/FADD interaction –> DED-containined procaspase 8 (8 = ex) –> DISC formation –> (+ procasp 8) –> active casp 8 –> Cas 3,6,7, BID
What is the function of survivin?
inhibits of apoptosis protein
What is the primary means of DNA repair during G1?
non-homologous end joining
What triggers DNA replication?
increased cyclin A2-CDK2 at G1/S transition
*cyclin E may also contribute
What activates ADP ribosylation factor (ARF)?
excessive activity of E2F
2 roles of cyclin d CDK4/6 at G1/S?
1) non-catalytic binding to p27 and prevention of p27 inhibition of cyclin E-CDK2
2) cyclin D-CDK 4/6 phosphorylation 2 pocket proteins:
- Rb
- p107
- p130
All release from E2f
How do cells ensure each origin of replication fires only once per cycle?
- pre-RC complexes in early G1
- replication only occurs when DNA polymerase is recruited in S phase
- temporal separate = safe
Which protein cleaves notch?
ADAM
WNT pathway ON
WNT –> FX + LPR 5/6 (coreceptor) –> DSH recruitment –> GSK3B/APC/AXN–>Bcatenin to nucleus –> increase myc, cyclin D, mmp7
WNT pathway OFF
LRP5/6 + Fz –X DSH –> GSK3B/axin+APC –X bcatenin –> ubiquination
Types of transcription factors (4)
- Homeodomain - 60AA, DNA binding domain called a homeobox
- Zn finger - 20-30AA with 2 cysteine on histidine residues with zn ion
- Leucine zipper - helices region of lutein every 7th AA which protrude from some side of helix
- Helix-loop-helix - similar to leucine but have loop regions separate alpha helexis
What is mTOR and what are its targets?
-serine/threonine kinase that complexes to form TORC1 (proliferation, inhibits autophagy) and TORC2 (activation of AKT, change cytoskeletal dynamics)
What happens following PIP3 degeneration?
- recruitment of PH-domain containing serine/threonine kinases PDK/AKT
- full AKT activation requires phosphorylation at second site by TORC2
- regulator of cell survival (phosphorylation of FOXO)
- regulator for cell proliferation (mTOR)
What 3 distinct MAPk pathways have been characterized in mammalian cells?
1) ERK 1/2
2) C-JUN
3) p38
Which protein serves as SH2 linker in the PI3k pathways?
p85 subunit of PI3k
What do SH3 motifs bind?
proline of target proteins
What is the function of BRCA1/2?
- Tumor suppressor
- BRCA1 = critical to DNA reapir, CC checkpoint and chromatin remodeling, ubiquitination
-BRCA2 = binds rad51 –> ssDNA repair by HR
What is the function of SMURF 1 + 2?
regulation of SMAD –> ubiquitination
TGFb signaling
binding –> association of type I + 2 (type 2 phosphorylates I) –> SMAD –> SMURF 1/2
PI3k
signal (phosphorylates)–> 3’ hydroxyl of PIs–> activation –> PI3k (p110 and p85 subunit) –> PIP2 –>PIP3 –> PH domain proteins –>PKB/AKT
Lead time vs length time bias?
types of selection bias
- lead: appearance of longer survival d/t diagnosis earlier in the course of disease = longer knowledge of disease vs tx response
- length: screened subjects with better prognosis detected by screening ( detected a disproportionate number of slowly growing)
polyaromatic hydrocarbons
- in tobacco
- methylated CpGs target of attack
What is the most commonly methylated sequence/base?
- CpG islands - cytosine bases
- only when 5’ to guansine
Deamination of 5-methyl cytosine
= thymine –> normal DNA base may not be repaired
Deamination
Removal of amine groups from guanine, adenine, cytosine –> xanthine, hypoxanthine, uracil (respectively) –> causes TRANSITION mutation
Why are areas of DNA repeat likely to have more error?
- increased strand slippage = higher/lower copy number of repeat in to daughter strand
- insertions/deletion of repeat may evade polymerase proof reading
Proof reading
- 3’-5’ exonuclease activity of DNA polymerase
E1A
- deregulation of pRB
- effective at induing apoptosis
- binds effectively sequesters pRb
How is p53 targets fine tuned?
C terminus covalent modification
(acetylation, glycosylation, phos, ribosylation, simulation) –> targets protein to specific intracellular site
MYC
- induced apoptosis but also has mitogenic functions
- when p53 decreases mitogenic signal more prominent
How do cells protect telomeres?
1) Telomerase (hTERT)
2) ALT (telomerase independent) exchange of sequences between telomeres = copy choice mechanism
What is the T-loop?
- G rich strand of telomere in hundreds of nucleotides longer - long 3’
- helps protect DNA
Telomeric sequence
5’ TTAAGGG 3’
When does p16 affect the cell cycle?
G1 –> blocks phosphorylation of Rb by cyclin D/CDK 4/6 –> hypophosphorylates of Rb - halt cell cycle progression
When does p21 affect the cell cycle?
can halt cell cycle progression via blocking all CDKs involved in G1, S, G2, M
miz-1
when no myc present miz1 associates with smad 3/4 –> increase p15/p21 expression
if myc associates decreased expression of p15/p21 CKDI –> increase action of CDK 4/6 and 2
= way to confer resistance to TGFb alpha growth signals
