Chemo Flash Cards - SS/AB
1
Q
Drugs metabolized by non-enzymatic hydrolysis?
A
BCNU
Mustargen
Melphalan
2
Q
Drugs metabolized by ubiquitous enzymes?
A
Cytarabine
Gemcitabine
6 mercaptopurine?
3
Q
Which chemo undergo HEPATIC metabolic? Via cyp450 vs conjugation?
A
CYP450: bisulfan, chloarmbucil, cytoxan, ifosfamide, paclitaxel, vinca alkaloids
Conjugation: etopisde
4
Q
Which drugs are excreted really?
A
bleomycin
carbo, cisplatin
etoposide
hydroxyurea
methotrexate
topotecan?
5
Q
Drugs eliminated by biliary excretion?
A
doxo
vinca alkaloids
6
Q
How do lymphs die from RT?
A
apoptosis (interphase death)
7
Q
Why is cytoxan platelet sparing?
A
megakaryocytes have increased ALDH (break down of phosphoramide mustard)
8
Q
L-MOPP with T-cell LSA (Brodsky 2009)?
A
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
9
Q
Respond of dogs to single agent doxo?
A
B: 100% (86% CR, 13% PR)
T: 50%
10
Q
Hodgkins LSA in cats?
A
- 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+
11
Q
MST dogs with splenic MZL?
A
~380d (12 mo) if symptomatic
> 1,100d (36 mo, 3 yr) if asymptomatic
12
Q
DLBCL subtype
A
- centroblastic (multiple central nucleolus)
- immunoblastic (single nucleolus)
13
Q
What is the agreement of IHC with Flow? PARR?
A
Flow: 94%
PARR: 69%
14
Q
T-zone LSA flow?
A
CD45-
CD3, CD21, CD25 +
High MHC class II
variable CD4/8
15
Q
Most common Tcell LSA immunophenotype?
A
CD4+
16
Q
Most common CLL immunophenotpye?
A
Tcell CD8+
<30k/uL cells prognosis 1,098d (36 mo, 3 yr) vs >30k (131d)
17
Q
Thymoma Flow?
A
> /= % CD4+/CD8+
18
Q
Large granular LSA dog
A
- few cytoplasmic granules
- 100% CD8+, most CD3+
- most common TCR alpha, beta 60%
- 8% CD3-
- 92% + for alphadb2 integrin found in spleen/BM
19
Q
Which cells express CD4?
A
- neutrophil: CD4/18
- activated dendritic cell
- T help lymph
20
Q
Two means of T-regs decreased immune response?
A
- Direct interaction
- alpha interferon cytokines; TGFb, IL10
21
Q
What are the Treg markers?
A
- subset of CD4+ (10-15%)
- FOXP3 + TF
- CD25+ IL2 receptor
22
Q
What is the role of NK?
A
kill cells that down regulate MHC class I missing “self recognition”
23
Q
Which immune cells posses MCH I? MCH II?
A
MHC I: all cells
MHC II: dendritic, macrophages, B lymphs
24
Q
Which immune cell is principle to mediate tumor immunity?
A
CD8
25
How do CTLs kill targets via apoptosis?
1. cytotoxic granules
- granzymes - some proteases
- perforin
2, FasL expression -> cas8
26
Which portion of Ab is responsible for Ag binding?
Primarily hypervariable region of Vh and VL CDRs
27
Palladia targets
PDGFR
EGFR
KIT
28
Cat carbo dose?
240 mg/m2 q 3 week
OR
Dose = AUC x 2.6 GFR x BW (kg)
29
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
30
What is terminal half life?
how long it takes a drug to decay in the bosy
31
Weird rxn in people with DTIC?
- Flu syndrome
- photosensitivity
- eosinophilia
- depresses Ab responses
32
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
33
Procarbazine metabolism?
- extensive hepatic microsomal metabolism
- monoamine oxidase inhibitor
- MANY DRUG INTERACTIONS d/t this
34
Cytotoxic mechanism of procarbazine?
methylation at O6 guanine --> increased in AGT, MGMT deficient cells
35
What is the MOA of Mesna?
- 2-mercaptoethane sulfonate
- dimerizes (inactive in plasma) in urine --> hydrolysis --> conjugates with alkylators
disulfide detoxifier
36
Why is cytoxan particularly applying for metronomic chemotherapy?
selective inhibition of Tregs
37
What effect do alkylating agents have on immunity?
suppress humoral and cellular immunity
especially cytoxan (B+T)
38
Why does cytoxan spare BM cells?
high levels of ALDH --> detox
* aldophosphamide --> inactive carboxycyclophosphamide
39
Predominant metabolites of cytoxan?
-nornitorgen mustard
-phosphoramide mustard
40
Alkylating agent metabolism?
1. Spontaneous hydrolysis by H2O
2. Enzymatic (important for PK of phosphor amide mustard and nistrosureas)
41
What is a major difference in MOA and efficacy of oxaliplatin?
1. DNA polymerase alpha cannot pass over adduct
2. decreased thymidylate synthase
42
Which other organs may be affected by platinums?
1. Neuro: peripheral neuropathy
2. ototoxicity
3. Hypersensitivity IgE mediated in 10-15%
43
What can ameliorate platinum induced neuropathies?
amifostine & B6 (pyridoxine)
44
How do platinum agents affect the immune system?
JUN/Jnk increase after cisplatin may cause increased FaS
45
Pulmonary reactions with vinca?
1. acute bronchospasm
2. interstitial infiltrates w/in 1 hour
most common with mitomycin
46
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
47
What happens to platinum agents inside the cell?
1. exported from cell by active transport proteins
2. become reactive by displacement of Cl- (cis) or carboxylic groups --> covalent binding with sulthyonyl groups
3. React with nucleophiles (e- on protein, RNA, DNA)
Affinity for RNA>>>>DNA
48
How do platinum drugs kill cells?
primarily via DNA adduct formation (must be CIS)
49
Cell with what mutation are most sensitive to platinums?
BRCA mutants
(decreased dsDNA break repair)
50
Mechanisms of resistance (increase drug target)
DHFR (dihydrofolate reductase) - methotrexate
RNR (ribonucleotide reductase) - hydroxyurea, gemcitabine
TS (thymidylate synthase) - 5FU
51
Mechanisms of resistance (decrease drug target)
Topo I: topotecan, irinotecan
Topo II: etoposide, anthracylines
52
MRP1 substrates?
- many PGP substrates
- methotrexate
- conjugated metabolites
53
What is the ABCB1-1delta mutation?
4 base pair deletion = frameshift = several premature stops
54
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
55
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
56
How are taxanes different from other microtubule inhibitors?
Vinca alkaloids prevent mitotic spindle formation while taxanes prevent function
57
Taxane resistance?
1. MDR
2. delta binary sites, tubulin dynamics
3. signaling activation (PI3k) --> increase threshold for apoptosis
4. increase MAP--> MAP4 increase Paclitaxel sensitivity
58
What drugs can reverse MDR phenotype?
- Ca2+ channel blockers
- tamoxifen
- alpha arrhythmic
- cremophor/polysorbate
59
How is 5-azacytidne activated?
conversion to monophosphate by uridine-cytidine kinase
60
How does 5-aza enter cells?
facilitated nucleoside transport shared with uridine and cytidine
61
5-aza MOA?
- does not require deoxycitidine activation
1. inhibits methylation
2. competed with CTP for incorporation into RNA
3. incorporates into DNA
62
How does 5-aza structure differ form cytidine?
- S1 Nk of heterocyclic ring unstable --> spontaneous decomposition
63
What is the first strategy for correction of a miscopied nucleotide?
polymerase 8 proof reading and 3'-5' exonuclease activity
64
When is mismatch repair used?
- used during DNA repair
- singe base mistmatch
- MSH/MLH proteins
- MMR deficiency = microsatellite instability
65
How are inter strand crosslinks repaired?
1. problem when replication fork is stalled
2. Fanconi protein recruited with ATR
3. Incision to enable bypass --> broken strand = HR (homologous repair), adduct = NER (nucleotide excision repair)
66
How is ssDNA/ss breaks repaired?
PARP activation
67
How does homologous recombination occur?
1. ds break
2. mRNA complex recruitment (endonuclease) and ATM
3. RPA recruited --> DNA resection (BRCA1 dept)
4. Rad protein loading DNA (BRCA2 controlled)
5. dsDNA invasion
68
Non-homologous end joining (NHEJ) - how does it occur?
1. Break recruits Ku70/80
2. Ku80 interacts with DNA-PKcS --> autophosphorylates
3. DNA polymerase recruitment and strand ligate
Targets of PKcs:
- KU70/80
-XRCC4
- DNApKcs
+/- ATM
*Pic include HR repair
69
G2 DNA damage checkpoint
ATM (chk2), ATR (chk1) --> CDC25C --> Cyclin B/CDK1 (wee1 inhibitory) --> Stop B2
70
What protein is needed for replication checkpoint?
ATR
1. Fork stabilization
2. Origin firing
3. S-m checkpoint
71
G1 DNA damage checkpoint
72
When in the cell cycle are ATM and ATR active?
- ATM: all checkpoints
- ATR: ONLY when sDNA template available (S + G2)
73
What are the major proteins for ssDNA repair?
RPA, RAD1, RAD9, TOPBP1 --> ATR, ATRIIP
also for base alkylation adducts --> MUTs, MUTc (MMR proteins)
74
Via what mechanism does p53 halt the cell cycle?
up regulation of p21
75
Beclin-1
- autophagy promoting protein
- autophagy activated when cells suffer nutrient starvation and digest own intracellular organelles
76
BID function?
- activated (cleaved) by caspase 8 in cytosol --> mitochondrial channel opening
*see figure 8-15 in T&H
77
What are the death receptor ligands?
TNF proteins (TNFalpha, TRIAL, FasL)
78
What are executioner caspases?
3,6,7
79
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
80
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
81
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
82
What is TSP1?
-thrombospondin-1
- blocks blood vessel development
83
What are the majority of p53 mutations?
-AA substitutions in DNA binding domain
84
ARF function?
- AKA p14
- growth inhibitory - depends on functional p53
- ARF binds MDM2 --> sequesters in nucleus --> p53 increases d/t decreased levels of destroyer
85
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
86
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
87
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
88
3 pathways to p53 activation?
1. ds breaks --> ATM --> p53 phosphorylation
2. DNA damaging agents: ATR -->chk2--> p53 phosphorylation
3. deregulation of pRb-E2f
89
What is the role of ATM?
damage --> atm kinase (chk2) --> air kinase --> p53 phosphorylation = protection from destruction
90
UV radiation
- causes pyrimidine dimers
- CC-TT transition characteristic of misrepair or lack of repair
91
What happens in the cell after DNA damage has occurred?
1. DNA repair: most likely
2. 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
92
What bases have amine groups?
-NH2
Purines & cytosine
93
Which base is most commonly incorporated with error prone repair?
adenine
94
Which is the most prevalent oxidized base?
8-oxo-deoxyguanosine = marker of overall oxidative DNA damage
95
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
96
What is the first step in metabolic activation of chemical carcinogens?
oxidative metabolism often mediated by CYP enzyme (change in carbon, nitrogen, sulfur)
97
3 most extensively studied carcinogens requiring bio activation?
1. PAHS
2. aromatic amines
3. nitrosamines
98
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
99
When is relative risk equal to odds ratio?
If disease prevalence is LOW (<10^210)?
100
Information bias
due to errors in obtaining bias (aka misclassification)
101
What is selection bias?
systematic differences between those in study vs those eligible
102
3 criteria for confounder
1. risk factor of disease of interest
2. must be associated with EXPOSURE under the study but not a result of it
3. Should NOT be intermediate factor on the causal pathway between exposure and disease
103
Tumor progression
benign lesions acquire ability to grow, invade tissues, and establish metastasis
104
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
105
What is tumor initiation?
- creation of DNA damage that must be corrected or mutation will become integrated
- irreversible!!
- may die via apoptosis
106
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
107
Fluctuation of cyclins during cell cycle
108
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)
109
What are CDKs?
- serine/threonine kinases
- associated with cyclins to activate CKD catalytic activity
110
What is the neurotoxic metabolite of ifosfamide?
- d/t dechloroethylifosfamide
- Chloroacetyl aldehyde
111
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
112
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
113
How does leptin contribute to tumorigenesis?
leptin (from adipocytes) --> proliferation of BC cells (breast cancer I think) via increased aromatase
114
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
115
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
116
Notch Pathway
Delta/jagged --> NOTCH = surface receptor --> 2x cleavage
1) ectodomain
2) transmemebrane
--> nucleus
117
What is the REL gene?
NFKB subunit amplified in 1/4 DLBCL in people
118
WNT signaling pathway
WNT binds frizzled --> disheveled activated --> blocks GKS3B--> B-catenin accumulation in cytoplasm and nucleus
119
Normal B-catenin cytosolic complex?
B-cateninc + APC + axin } GSK3b executioner
120
NFkb pathway
121
What does B catenin associate with in the nucleus?
TCF/LEF transcription factor
122
Important targets of RAS --> RAF --> MEK--> ERK pathway?
- promoters of Fos + Jun
- Fos + Jun associate --> API --> transcription factor hyper activated in cancer
123
MAPk pathway
RAS --> RAF (MAPKKK) --> MEK (MAPKK) --> ERK (MAPK) --> targets
124
PI3k pathway
PI3k --> PIP3 --> AKT/PKb -->
- Bad inhibition of apoptosis
-mTOR stim
- GSK3b stem of cell proliferation
125
Dual specificity kinase?
- can phosphorylate serine/threonine residues and tyrosine
ex. MEK
126
GRB2 + SHC bridging proteins
127
GRB2 protein structure?
2 SH3 groups --> SOS
1 SH2 --> ligand receptor
128
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
129
Phosphatidyl pathways
PI --> kinases --> PIP2 -->
1) PI3k --> PIP3 --> attracts proteins with PH domain
2) phospholipase C --> DAG --> PKC + IP3 --> Ca2+ reduces
130
Most important PH domain protein?
- AKT
- PI3k --> IP3 attracts AKT/PKI3 via pH domain
- PD1,2 activated kinases
131
Anti-apoptotic substrates of AKT/PKB?
Bad (pro)
Cas 9 (pro)
IKB kinase (alpha)
FOXO TF (pro)
MDM2 (alpha)
132
Proliferative substrates of AKT/PKB?
- GSK3b (alpha)
- FOXO4 (alpha)
- p21 (alpha)
133
Growth substrates of AKT/PKB?
TSC2 (alpha)
134
Ral A/B
- 1/3 major effector pathways of RAS
- Ral A/B gene 58% identity with RAS
135
SOS function?
To induce RAS to shed GDP
136
Ral pathway
RAS --> Ral-GEF -->
RalA
RalB
--> RCLBp --> Cdc42 --> proliferation
137
Examples of JAK/STAt receptor
EPO, TPO
138
JAK/STAT pathway
139
Integrin signaling
- alpha and beta heterodimers
- link cytoskeleton to ECM + signaling
- signaling via SOS--> RAS--> ERK
140
Wnt- B- catenin
- signals via frizzled receptors - GSK3b
- GSK3b normally phosphorylates B catenin --> ubiquinated
141
B-catenin states
1. bound cytoplasmic domain of cell-cell adhesion receptors
2. soluble cytosol
3. transcription factor
142
What are promoters of the cyclin D genes?
D1 - API, TCF/Lef, NFkb, TFs
D2 - myc, increased camp
D3 - Stat 3/5, E2A
143
What is the exception to fluctuating cyclin levels?
Cyclin D
- controlled by extracellular signals (tyrosine kinase receptors)
- others controlled by rapid degradation via ubiquitination
144
What is CD25?
part of IL2 receptor
145
T-zone LSA flow?
Cd3+, Cd5+
CD45-
High CD21
High MHC II
146
PARR sensitivity?
neoplastic 1/100 cells
147
Factors that may affect PARR?
- DNA quality (e.g. formula increases fragmentation)
- Taq inhibitors
- Primer choice
148
Terminal deoxynucleotidyl transferase (TDT) enzyme
Adds nucleotides between V+D, and J.
149
Where does Ig rearrangement occur?
Bone Marrow
* acute leukemia may not have yet, may be -
150
Ig formation
V 80
D 6
J 6
151
T cell markers
Surface: CD3 (TCR), CD5, CD4, CD8
Cytoplasmic: CD3c
152
Where do T cells acquire markers?
CD3 --> BM
CD 4/8 --> thymus
153
B cell markers for flow and IHC?-
Flow: CD21, CD22, CD20
IHC: CD79a (BCR), Pax5 (TF), CD20c, Mum1, IRF4
154
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
155
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
156
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
157
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)
158
Feline MI cut offs: cutaneous & intestinal MCT?
Cutaneous: 5 (same as dog)
Intestine: >2
159
Feline MCT IHC markers?
+ vimentin
alpha 1 alpha trypsin
KIT
160
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
161
What % of dogs with MCT have BM involvement?
- 2.8% on FNA
- if visceral, 37% Buffy coat + and 56% BM+
162
MCT IHC markers?
Vimentin +
Typtase + (skin only)
+/- Chymase (GI and skin)
CD117/KIT +
IL8
163
SQ MCT MI prognosis?
>4
- also if infiltrative +/- multinucleate cells
164
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
165
Cutaneous MCT MI prognosis?
MI > 5 --> 3 mo MST
MI <5 --> 80 mo MST
166
Which molecules control extent of R6 phosphorylation?
Cyclin d + e
167
What is the difference in cyclin D control vs others?
cyclin D = extracellular signals
others = gradual accumulation and rapid destruction
168
What type of kinases are the CDKs?
serine 1 threonine
169
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
170
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
171
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
172
Number 1 target of Mac for cell cycle progression?
cyclin D2 --> R of Rb
* myc also increased expression of e2f1/2/3
173
Cul1
- protein increased by myc
- plays central role in p27 degradation (CDKI) via ubiquitination
174
myc function
- transcription factor
- creates homo-heterodimers
- dimers associated with regulatory sequences termed "e boxes" of promoters "CACGTG"
- phosphorylation of myc regulates activity
175
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
176
What is the most important target gene of ear for cell cycle progression?
-cyclin e
- responsible for complete hyperphosphorylation of pRB
177
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
178
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
179
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
180
How can PI3K activation increase proliferation?
- TGFb can stimulate
- stimulation of RTK
- AKT/PKB --> p21 in nucleus
181
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
182
Cyclin-CDK inhibition
- CDKIs
- INK4 proteins --> CDK 416 (p16, p18, p19)
- p21, p 27, pS7 --> inhibit all others
183
Extrinsic apoptosis pathway
FAS/FADD interaction --> DED-containined procaspase 8 (8 = ex) --> DISC formation --> (+ procasp 8) --> active casp 8 --> Cas 3,6,7, BID
184
What is the function of survivin?
inhibits of apoptosis protein
185
What is the primary means of DNA repair during G1?
non-homologous end joining
186
What triggers DNA replication?
increased cyclin A2-CDK2 at G1/S transition
*cyclin E may also contribute
187
What activates ADP ribosylation factor (ARF)?
excessive activity of E2F
188
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
189
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
190
Which protein cleaves notch?
ADAM
191
WNT pathway ON
WNT --> FX + LPR 5/6 (coreceptor) --> DSH recruitment --> GSK3B/APC/AXN-->Bcatenin to nucleus --> increase myc, cyclin D, mmp7
192
WNT pathway OFF
LRP5/6 + Fz --X DSH --> GSK3B/axin+APC --X bcatenin --> ubiquination
193
Types of transcription factors (4)
1. Homeodomain - 60AA, DNA binding domain called a homeobox
2. Zn finger - 20-30AA with 2 cysteine on histidine residues with zn ion
3. Leucine zipper - helices region of lutein every 7th AA which protrude from some side of helix
4. Helix-loop-helix - similar to leucine but have loop regions separate alpha helexis
194
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)
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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)
196
What 3 distinct MAPk pathways have been characterized in mammalian cells?
1) ERK 1/2
2) C-JUN
3) p38
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Which protein serves as SH2 linker in the PI3k pathways?
p85 subunit of PI3k
198
What do SH3 motifs bind?
proline of target proteins
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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
200
What is the function of SMURF 1 + 2?
regulation of SMAD --> ubiquitination
201
TGFb signaling
binding --> association of type I + 2 (type 2 phosphorylates I) --> SMAD --> SMURF 1/2
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PI3k
signal (phosphorylates)--> 3' hydroxyl of PIs--> activation --> PI3k (p110 and p85 subunit) --> PIP2 -->PIP3 --> PH domain proteins -->PKB/AKT
203
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)
204
polyaromatic hydrocarbons
- in tobacco
- methylated CpGs target of attack
205
What is the most commonly methylated sequence/base?
- CpG islands - cytosine bases
- only when 5' to guansine
206
Deamination of 5-methyl cytosine
= thymine --> normal DNA base may not be repaired
207
Deamination
Removal of amine groups from guanine, adenine, cytosine --> xanthine, hypoxanthine, uracil (respectively) --> causes TRANSITION mutation
208
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
209
Proof reading
- 3'-5' exonuclease activity of DNA polymerase
210
E1A
- deregulation of pRB
- effective at induing apoptosis
- binds effectively sequesters pRb
211
How is p53 targets fine tuned?
C terminus covalent modification
(acetylation, glycosylation, phos, ribosylation, simulation) --> targets protein to specific intracellular site
212
MYC
- induced apoptosis but also has mitogenic functions
- when p53 decreases mitogenic signal more prominent
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How do cells protect telomeres?
1) Telomerase (hTERT)
2) ALT (telomerase independent) exchange of sequences between telomeres = copy choice mechanism
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What is the T-loop?
- G rich strand of telomere in hundreds of nucleotides longer - long 3'
- helps protect DNA
215
Telomeric sequence
5' TTAAGGG 3'
216
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
217
When does p21 affect the cell cycle?
can halt cell cycle progression via blocking all CDKs involved in G1, S, G2, M
218
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
