principles of cancer biology 22% LC Flashcards
define aneuploidy
have an abnormal number of chromosomes due to gain or loss
cat has 38 chromosomes normally
dog has 78 chromosomes normally
in what phase does genomic instability occur
s phase
what percent of solid tumors are aneuploid
> 90%
unclear if its a cause or effect of cancer
causes of aneuploidy
- mitotic segregation error
- DNA damage repair deficiency
ie Compromised checkpoint functions, defective chromosome segregation, formation fo tetraploid cells, chromothripsis - replication stress prior to mitosis
DNA replication steps
- helicase unwinds the parental double helix
- single strand binding proteins stabilize the unwound parent DNA
- leading strand synthesized continuously 5’ to 3’
- lagging strand synthesize discontinuously. primate synthesizes rna primer which is extended by dna polymerase to form okazaki fragment
- After the RNA primer is
replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing
protein synthesis
DnA transcriptions then translation
over view:
1. DNA in nucleus as a template.
2. mRNA is processed and released into
cytoplasm.
3. mRNA binds to ribosomes.
4. tRNA carries amino acid to mRNA.
5. Anticodon-codon complementary base
pairing occurs.
6. Peptide chain is transferred from
resident tRNA to incoming tRNA.
7. tRNA departs.
8. Protein modification after translation.
DNA transcription 3 steps
- Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can “read” the bases in one of the DNA strands. The enzyme is ready to make a strand of mRNA with a complementary sequence of bases. The promoter is not part of the resulting mRNA
- Elongation is the addition of nucleotides to the mRNA strand.
- Termination is the ending of transcription. As RNA polymerase transcribes the terminator, it detaches from DNA. The mRNA strand is complete after this step.
processing mRNA
pre-mRNA, and it must go through more processing before it leaves the nucleus as mature mRNA
The processing may include the addition of a 5’ cap, splicing, editing, and 3’ polyadenylation (poly-A) tail. These processes modify the mRNA in various ways. Such modifications allow a single gene to be used to make more than one protein.
- 5’ cap protects mRNA in the cytoplasm and helps in the attachment of mRNA with the ribosome for translation.
- Splicing removes introns from the protein-coding sequence of mRNA. Introns are regions that do not code for the protein.
- The remaining mRNA consists only of regions called exons that do code for the protein.
- Editing changes some of the nucleotides in mRNA. One form is smaller than the other because editing adds an earlier stop signal in mRNA.
- Polyadenylation adds a “tail” to the mRNA. The tail consists of a string of As (adenine bases). It signals the end of mRNA. It is also involved in exporting mRNA from the nucleus, and it protects mRNA from enzymes that might break it down.
RNA translation 3 steps
Translation Initiation: The small subunit binds to a site upstream (on the 5’ side) of the start of the mRNA. It proceeds to scan the mRNA in the 5’–>3’ direction until it encounters the START codon (AUG). The large subunit attaches and the initiator tRNA, which carries methionine (Met), binds to the P site on the ribosome.
Translation Elongation: The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. The energy for each bond between amino acids is derived from GTP. Briefly, the ribosomes interact with other RNA molecules to make chains of amino acids called polypeptide chains, due to the peptide bond that forms between individual amino acids. Inside the ribosome, three sites participate in the translation process, the A, P, and E sites.
Translation Termination: Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released with the help of various releasing factors and the ribosome subunits dissociate and leave the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.
DNA-binding domain definition
importance in cancer
an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA
> 80% p53 mutations occur in the region encoding the central DNA binding domain
examples of dna binding sites
- Homeodomain (helix-turn-helix)
Homeobox: 60 amino acid DNA binding domain. 3 helical regions, third contacts DNA - Zinc-finger
Binding zinc ion folds polypeptides and inserts into DNA
SCAN domains - ZSCAN → differentiation, growth, cancer development - Leucine zipper
Basic zipper proteins bind to DNA, include FOS/JUN pair → activated by stress - Helix-loop-helix
Carboxyterminal a helix → formation of homo/heterodimers → contact DNA in aminoterminal helix
10 hallmarks of cancer and the genes related to each one
- sustained cell growth - Ras Myc
- evading growth inhibition - p53, Rb1, pTEN, CDKN2A (p16)
- angiogenesis - VEGFA, VHL
- Invasion and metastasis - N- cadherin, SNAIL, SLug, TWIST, Zeb1/2
- evasion of apoptosis - upregulated Bcl 2, Bcl XL, suppress Bax, Bim, Bak
- replicative immortality - TERT, WNT, notch, Hh pathway
- evasion of the immune sytem - TGF b, T reg, MDSC, TAMs
- reprograming metabolism - warburg effect, GLUT1 transporters, Ras, Myc, p I 53 mutants
- genomic instability - mutations in house keeping genes
- tumor promoting inflammation - promotion of growth factors and proangiogenic factors
K9 BRAF mutation - dog? location? human? frequency?
Somatic mutation in V595E on Chr16
Homologous to human V600E
Present in 87% of invasive TCC
tumor supressor mutation in human and k9 lsa
p53, p16
Which mutation occurs in ~50% of K9 HSA
PTEN -> AKT/mtor disruption
MEN2 mutation casues what
- hyperparathyroid
- thyroid (medullary) cancer
- pheochromocytomas
mulitple endocrine neoplasia
MEN1 mutation causes what
- pituitary adenomas
- parathyroid tumors
- insulinomas
- gastrinomas
- facial angiofibromas
multiple endocrine neoplasia
Human clear cell renal carcinoma (CCRCC) is associated with extremely high levels of VEGF, florid angiogenesis and hypervascularity. This is attributed to the high frequency (70%) of loss of function mutation in what gene?
- Von Hippel-Lindau tumor suppressor gene VHL
- Ubiquitin ligase targeting Hif-1a in oxygen rich environment
- Recessive mutation, loss of heterogeneity common
- Renal clear cell CA due to active HIF
Li Fraumeni syndrome occurs in people with germline mutations of what gene
p53 mutation
autosomal dominant
that increases the risk of developing cancer throughout a person’s life
Which canine cancer has STAT3 and p53 mutations?
OSA
DLBCL in dogs and humans share which common pathway mutations?
NFKB and Pi3K, Jak Stat (N-ras, p53, Rb, p16 CDK, telomerase)
Which mutation can inhibit and then promote cancer?
c-myc, TGF-B
chromosome structure
short arms (p) and long arms (q) - centromere in the middle
What are zinc fingers and leucine zippers?
Transcription factors that can activate or repress histone acetylation
Relationship of β-catenin and GSK-3β
GSK-3β degrades β-cat and prevents nuclear transcription
a part of the destruction complex that degrades b catenin
What does SMAD do?
transcription factor that is activated by TGF b - regulate growth and proliferaiton
cna be tumor supressor or tumor promoting ( smad blocks c-myc)
MOA of HDACi
Increased transcription by preventing histone deacetylation and gene silencing;
Ex. valproic acid, vorinostat
induce apoptosis, dna damage, cell cycle arrest, inhibit angiogenesis, generate ROS
Patient with OSA. Which HDACi potentiates doxorubicin?
Valproic acid
how does valproic acid potentiate doxorubicin
increased apoptosis in a dose dependant manner
hyperacetylation
epigenetics definition
heritable change that impact dna template processes that are not encoded in the primary linear dna sequence
epigenetics is mediated by
attachments of methyl or acetyl groups to DNA associated proteins, histones, and chromatin
what is dna methylation
covalent addition of methyl group at position 5 of cytosine nucleotide
mediated by DNA methytransferase DNMT
where does dna methylation occur
occurs in CpG island - 70% of promotor regions of genes
CpG islands associated with tumor suppressor genes are unmethylated in normal tissues but often become hypermethylated during tumor formation
Methylation in promoters =
Methylation in gene bodies =
hyperMethylation in promoters (CpG) = silencing
Methylation in gene bodies = typifies transcribed genes
gen body = region of the gene past the first exon because methylation of the first exon, like promoter methylation, leads to gene silencing
what is purpose of acetylation
neutralizes positive charge of lysine on histone tails - weakens the interaction with DNA.
increases availability for gene transcription - active promotors and enhancers
what do writers readers and erasers do
Writers: add modifications to histones
Readers: recognize modified histones
Erasers: mediate removal of histone modifications
histone variants
not cell cycle specific
contribute to control of transcription
early signalling of dsDNA break repair
DNA methylation on cancer cells globally and locally
(hyper or hypo?)
Cancer cells globally are hypOmethylated and locally hypERmethylated at promoters
promotor methylation and gene expression in carcinogenesis
Promoter methylation correlates w/ reduced gene expression. Therefore, hypERmethylation results in silencing tumor suppressor genes → carcinogenesis
demethylation at gene promoters during carcinogenesis
CTCFL/BORIS (promoter normally limited to testis) is associated with overexpression in cancer development
Promoter demethylation driving aberrant expression of uPA leads to
→ tumor progression and metastasis
Increased expression of urokinase plasminogen activator (uPA) has been reported in various malignancies including prostate cancer
Variant enhancer loci (VELs)
enhancer variants that are either lost or gained in cancer cells compared to normal tissue
histone deacetylases
HDAC definition
enzymes responsible for removing acetyl groups from histone leading to decreased transcrption and inhibits gene expression
will silence tumor supressor genes
tightening the histone coils
tazemetostat is a
EZH2 inhibitor
enhancer of zeste homolog 2 - methyltransferase enzyme - methylates = gene silencing
inhibitors promote the reexpression of silenced genes
Samples do not express p53 initially. You treat a tumor with decitabine and 5-azacytidine. Western blot provided shows expression of p53. why?
Removing inhibitory methylation of p53’s promoter with decitabine
DNMT inhibitors - can reactivate methylated tumor suppressor genes
decitabine
treats myelodysplastic syndrome
Antimetabolite antineoplastic agent
inhibits DNMT
risk of DNA methyltransferase (DNMT) inhibitors
global hypomethylation leading to genomic instability and demethylation could trigger reactivation of gene promoting more aggressive or metastatic phenotype
expression of uPA in cancer
The uPA/uPAR interaction can promote the expression of oncogenes and cell proliferation, eventually leading to the development of tumors, invasion or metastasis
what drug has been shown to increase uPA
5-azacitidine shown to increase expression of uPA
match the cyclin with the kinase and the cell cycle
G0 : cyclin C + CDK3
G1: cyclin D and E + CDK2,4,&6
S: cyclin A and E + CDK2
G2: cyclin A + CDK2 & CDK1
M: cyclin B + CDK1
match CDK inhibitors to their binding
INK 4 binds to CDK4 and D type cyclins - regulate the g1 phase
CIP/KIP proteins bind to cdk/cyclin 1/B, 2/a, 2/E - regulate at any check point
s phase
DNA synthesis
G2 phase
growth and prep for mitosis
M phase
mitosis - cell division
prophase
anaphase
metaphase
telophase
G0 phase
resting phase
G1 checkpoint “restriction point”
- purpose
looking for:
cell size
nutrients
DNA damage
growth factors
how are double strand breaks repaired in the G1 phase
nonhomologous end joining - NHEJ
how are double strand breaks repaired in the S/ G2 phase
use NHEJ and homologous recombination - use the replicated sister chromatid as a template for repair
Homologous recombination is considered error free
if growth factors or nutrients are withdrawn before restriction point what happens to the cell
cell enters G0
what is the importance of the restriction point in cancer
Restriction point is lost in most cancers allowing proliferation without requirement for extracellular mitogenic signals
inter s phase checkpoint
- purpose
DNA damage
DNA replication
G2M checkpoint
cell size
DNA replication
HR and NHEJ
M phase check point
spindle assembly
extrinsic apoptosis
mediated by Natural Killer (NK) lymphocytes or CD8-positive Cytotoxic T lymphocytes (CTLs) ligand binding on the cell surface receptors (FAS, TNFa, TRAIL) bind to death receptor
activate caspase 8 and (maybe 10)
then cleave and activate executioner caspase 3, 6, 7
caspase 3 and 7 action
caspase 3 controls DNA fragmentation
caspase 7 control cell viability (such as DNA repair, genomic stability, and programmed cell death) by cleaving PARP
intrinsic apoptosis
mitochondrial pathway
cell stress or damage to dna - activates
p53 (Bax, BAK) and then transcription of proapoptotic proteins (Bcl -> BID) . leading to increased mitochondrial permeability and leakage of cytochrome c. this binds to apaf1 forming the 7 spoke apoptosome and cleave procaspase 9 - caspase 9 to activate executionaer caspases 3,6,7
malignant transformation 5 steps
- mutagenic event
- leads to repair, apoptosis or failed repair
- failed repair leads to perpetuation of genomic defect in daughter cells
- progression = clonal expansion of mutated cell. premalignant
- promotion = acquisition of additional genetic mutations to support growth and survival
gate keeper tumor suppressor genes
control cell growth through cell cycle regulation and programed cell death
loss of function leads to cancer
care giver tumor suppressor genes
protect the genome through regulation of dna repair
loss of function leads to cancer
onco genes
code for normal cell machinery involved in cell growth and differentiation
gain of function mutations lead to cancer
- point mutation, gene amplification, chromosomal translocation, viral insertions
APC
- what kind of gene
- what does it do
- what associated cancer
- tumor suppressor
- cell adhesion and signal transduction and cell division - a part of wnt destruction complex
- colorectal cancer
VHL
- what kind of gene
- what does it do
- what associated cancer
- tumor suppressor
- transcriptional elongation regualtion - angiogenesis
- schwannoma, meningioma, etc
PTEN
- what kind of gene
- what does it do
- what associated cancer
- tumor suppressor
- phosphatase that covert PIP3 back to PIP2 stopping PDK1 -mtor- cell survivial
- hamartoma, glioma, etc
RB1
- what kind of gene
- what does it do
- what associated cancer
- tumor suppressor
- cell cycle control - when bound to E2F prevents cell cycle progression when phosphorylated (cancer) allow progression through cell cycle by releasing E2F
- OSA others
TP53
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- cell cycle control ( activates p21 (cip) which blocks CDk2/cyclinE and p27 (KIP) which blocks other cyclins/cdks) , apoptosis via activation of caspase 9
- sarcoma, leukemia, etc
NF1
- what kind of gene
- what does it do
- what associated cancer
- Tumor supressor
- blocks Ras ( similar to PTEN - converts RAS GTP to RAS GDP)
- neurofibroma, sarcoma , etc
CDKN2A
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- cell cycle control ( encode p16 or INK ie blocking CDK4/6 CyclinD whihc then cannot phosphorylate Rb1)
- melanoma, pancreatic cancer
WT1
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor and oncogene
- transcription factor of p53 - activates p21, and degrades b catenin. or blocks apoptosis and promotes cell EMT via blocking e cadherin
- nephroblastoma ( in embyro helps develop kidneys and testes) also on lymphoblastic leukemias
BRCA 1/2
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- DNA repair (HR) , cycle checkpoint control (p21 ie CIP block CDK/Cylins)
- Breast and ovarian cancer
ATM
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- dna repair and apoptosis via AKT and p53
- lymphoma
FANCA
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- dna rapair of intrastrand crosslinks - Fanconi anemia (FA) pathway - complex of proteins that rec and activate NHEJ and HR and NER
- acute myeloid leukemia
MLH1
- what kind of gene
- what does it do
- what associated cancer
- tumro supressor
- dna mismatch repair microsattelite
- lymphoma, sarcoma
NER
- what kind of gene
- what does it do
- what associated cancer
- tumor supressor
- nucleotide excision repair
- skin cancer
ALK
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- receptor tyrosine kinase - constituatively activates jak,pi3k,ras, nfkb, etc
- lymphoma
BCL2
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- anti-apoptotic protein
- lymphoma leukemia
C-MYC
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- transcription of both apoptotic proteins and cell proliferation and cell cycle progression
- leukemia, carcinoma, etc
EGFR
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- cell surface receptor - binds TNFa or EGF to start PI3k or RAS - survival
- SCC
GLI
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- transcription factor - HH path and ras - > repair, metastasis, and theapeutic resistance
- glioblastoma, basal cell carcinoma, panc carcinoma, medulloblastoma
KIT
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- receptor tyrosine kinase
- sarcoma gi stromal tumor mct
JUN
- what kind of gene
- what does it do
- what associated cancer
- oncogene with FOS
- transcription factor self renewal and differentiation
- sarcom
RAS
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- g protein signal transduction
- carcinoma
RET
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- receptor tyrosine kinase protein- binding domains and fusion proteins
- thryoid carcinoma, endocrine neoplasia
SIS
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- encode growth factor PDGF
- glioma, fibrosarcoma
SRC
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- encodes non-receptor tyrosine kinase protein c-srs which phosphorylates tyrosine residues
- sarcoma
TRK
- what kind of gene
- what does it do
- what associated cancer
- oncogene
- receptor tyrosine kinase - Neurotrophic tyrosine receptor kinase
- colon, thyroid cancer
what phase of the cell cycle does homologous recombination occur
G2 S phase
requires homology between broken DNA strand and template - usually newly replicated chromatid
phosphorylation occurs at what phase of the cell cycle
CHK2 at G1-S -
dsDNA break -> ATM phosphorylates CHK 2
CHK 2 phosphorylates CDC25 to prevent activation of CDKS and E2F to inhibit cell cycle progression
chk 2 = serine/threonine kinase
emerging hallmarks of cancer
deregulating cellular energetics and avoiding immune destruction
enabling hallmarks of cancer
genome instability and mutation
tumor promoting inflammation
cellular features of senescence
senescent cells ar elarger and more morphologically diverse
what phosphorylates threonine on CDK
Cdk-activating kinase (CAK)
steps of activation of CDK/cyclin complexes
- cyclin and cdk1 bind - inactive
- CAK phosphorylates threonine 160 on CDK - inactive
- other active cyclin/cdk can phosphorylate CDC25 which in turn activates the inactive cyclin/cdk complex (positive feedback)
- active CDK/cyclin can also activate Wee1 - whcih is an inhibitory CDK kinase that inhibits CAK phosphorylation
which protein are in the INK4 family and what do they inhibit
p15, p16, p18, p19
inhibit CDK4/6 to block Cyclin D interaction
** cannot progress through G1**
Which proteins are in the CIP/KIP family and what do they inhibit?
p21, p27, p 57
inhibit Cyclin E-CDK2 and Cycklin A-CDK2 preventing entry into S phase
inhibit Cyclin A/CDK1 and Cyclin B/CDK1 for progression through G2 and M phase
how are cells in the S or G2 phase stopped from entering mitosis
inactivate all 3 CDC25 and activate Wee1 kinase
prevents removal of the inhibitory phosphate group preventing activation of Cycline B and CDK1
p21 and p27 inhibit Cyclin A/CDK1 and Cyclin B/CDK1 for progression through G2 and M phase
Cell is overexpressing cycD1 causing continuous proliferation. How do you reverse?
Dominant negative CDK2 should halt cell cycle in late G1
During what phase of the cell cycle does Rb protein function and how does it function
in G1 phase Sequential phosphorylation by kinase complexes Cyclin D:Cdk 4/6 and Cyclin E:Cdk 2, causes conformational changes to the Rb structure and release of E2F.
The release of E2F is necessary for the expression of S-phases genes
E2F gene targets
regulation of cell cycle and DNA replication, such as cyclins E and A
how does phosphorylation control Rb
Hypophosphorylated Rb is active and binds to E2F, thereby silencing E2F-dependent activity therefore play a central role in maintaining cells in a resting or quiescent state.
Phosphorylation of Rb promotes its dissociation from E2F, allowing transcriptional activation of E2F targets such as cyclin E. represents the transition from mitogen-dependent to mitogen-independent cell cycle progression (or passage through the restriction point)
P53 causes a cell cycle arrest in what phase when there is DNA damage?
G1
How does p53 cause cycle arrest?
ATM activate CHK which stabilizes/phosp p53 when DNA DSB,
p53 activates CIP/KIP (p21) stops G1/S checkpoint and less G2/M
What does PTEN do?
Inhibits PIP3 so no PI3k, stabilize p27 which inhibits G2/M
micro satellite instability is found in what human cancers
canine cancers
colorectal cancer, gastric cancer, endometrial cancer
mammary gland tumors and OMM, HCC in dogs
what is microsatellite instability (MSI)
short tandem repeats that are prone to mutations. located in inherited genes
when you have mismatch repair deficiency (MSI) host cannot see the errors
what is high MSI - microsatellite instability in relation to cancer development and treatment
tumors that have mismatch repair deficiency - cant recognize errors - have insertions and deletions. leads to carcinogenesis
very responsive to immunotherapy
Hereditary nonpolyposis colorectal cancer is associated with what mutation?
Mismatch repair; Proteins MLH1, MSH2
mismatch repair
steps
proof reading failure
either insertions or deletions
or polymerase creates helical distortions
1. recognition
2. excision
3. synthesis by polymerase ligation
base excision repair
Enzymatic removal of the damaged DNA base by DNA glycosylases
- Cleave glycosidic bonds
- Specific to DNA base lesions
major and minor pathways of base excision repair
major pathway - single nucleotide replacement
Minor pathway is long patch base excision repair which enable repair of 2-13 damaged nucleotides
what germline mutation is responsible for BER deficiency in enhanced colorectal carcinogenesis
MUTYH - glycolase
base excision repair and p53
P53 can stimulate BER by direct interaction with APE1 and DNA polymerase
BER defective in cells with p53 mutations
mechanism of repair of oxidative damage
eg by ionizing radiation
BER
ionizing radiation ds break repaired via hr and nhej
PARP - poly ADP ribose polymerase
is used during what repair
Required at an intermediate step preceding DNA synthesis during BER or when ssDNA region are recognized
what happens when PARP is inhibited
leads to ss gaps that result in dna collapse or repair via other mechanisms
how is PARP inhibition repaired?
Can be repaired by Homologous recombination
When HR is altered as in BRCA1/2 mut, PARP inhibitors can cause cell death via Synthetic lethality
Match the defect with the disease and defective protein:
hereditary nonpolyposis colorectal cancer
mismatch repair
MLH1, MSH2
microsatellite instability
Match the defect with the disease and defective protein:
XP - xeroderma pigmentosum
nucleotide excision repair
XP, CS - point mutations
Match the defect with the disease and defective protein:
ataxia telangiectasia
DNA DSB - non homologous end joining
ATM - chromosome aberration
Match the defect with the disease and defective protein:
renal clear cell carcinoma
VHL Von Hippel-Lindau Disease
Match the defect with the disease and defective protein:
Li fraumeni
p53
DNA DSB response - DNA checkpoint damage
Match the defect with the disease and defective protein:
Familial adenomatous polyposis (FAP)
APC -adenomatous polyposis coli (APC) gene
inherited point mutation
APC is a tumour suppressor gene that controls beta-catenin turnover in the Wnt pathway
Match the defect with the disease and defective protein:
fanconi anemia
dna crosslink repair
FANC - chromosome abberation
Match the defect with the disease and defective protein:
breast cancer
homologous recombination
BRCA1/2
What occurs when giving a PARP inhibitor with platinum agent or RT to patients with BRCA mutations?
Synthetic lethality
BRCA mutations = HR defect, dependent on BER
PARP inhibitor impairs BER → cell death
patients with xeroderma pigmentosum are sensitive to what
UV damage due to nucleotide excision repair failure
Nucleotide excision repair
4 steps
involves 30 genes
- Recognition of damaged dna
- Dual incision of damaged dna strand on each side of the lesion leading to excision of an oligonucleotide of 24-32 residues
- Filling in of the gap by dna polymerase
- Ligation of the nick
Nucleotide excision repair
2 sub pathways
· Transcription coupled repair
· Transcription coupled repair - stalled RNA polymerase induces the recognition of the DNA lesions on the transcribed strands
- efficient
- facilitated by CSA and CSB proteins
Nucleotide excision repair
2 sub pathways
· Global genome repair
· Global genome repair - transcription independent and surveys the entire genome for DNA lesions
- slow
Why are carboplatin and gemcitabine synergistic/what results when given together?
Decreased DNA repair - inhibit NER
BRCA2 and RAD 51 are needed for what repair mechanism?
homologous recombination
In what phase of the cell cycle does Homologous recombination occur?
G2 & S phase Requires homology between broken DNA strand and template - typically newly replicated sister chromatid
In what scenario would a PARP inhibitor be effective for cell death?
If compromised dsDNA repair (ex. BRCA1/2 or RAD51 loss) PARPi blocks ssDNA repair
UV radiation induces pyrimidine dimers that are repaired by which DNA repair process
nucleotide excision repair
what repair mechanism is deficient in rabbit mammary carcinoma
homologous recombination
what repair involves DSB
homologous recombination and non homologous end joining
HPV is a cancer causing virus in humans with many viral proteins. What is the function of the E7 protein?
E7 protein is a transforming protein that binds to Rb
What is a molecular diagnostic that can be utilized to detect chromosome aberrations (such as Raleigh/Philadelphia chromosome)?
Genomic hybridization in situ FISH mitosis
how does homologous recombination occur
- Requires homology between broken DNA strand and the template strand used in repair
- Typically provided by a newly replicated sister chromatid
- Restricted to S and G2 phases
- Error free repair
homologous recombination is mediated by what protieins
RAD proteins, MRE11, and BRCA1
non homologous end joining NHEJ
- fast pathway
heterodimer recognizes the DNA ends and recruits the DNA dependent protein kinase catalytic subunit which autophosphorylates and forms the active DNA-PK complex while downstream a ligase dimer associates with the proteins and joins the DNA ends together
non homologous end joining NHEJ
- slow pathway
end are repaired by ATM kinase and may require chromatin modification for repair then follow fast pathway
- Used when DSB ends are unligatable by fast pathway
major protein complexes in NEHJ pathway
DNA dependent protein kinase (DNA-PK)
MRE11-RAD50-NBS1 (MRN) protein complex
Mutations in genes encoding DNA-PKcs result in what?
sensitivity to ionizing radiation and reduced ability to repair radiation-induced DNA DSBs
Mutations in MRE11-RAD50-NBS1 (MRN) protein complex result in
various human diseases that can lead to increased incidence of lymphoid tumors and death
Fanconi anemia pathway (FAP):
specialized pathway for repair of interstrand crosslinks (ICLs)
People with deficiencies of any of the proteins in this pathway have a predisposition to cancer
Increased protein of FAP found in metastasizing canine OMM
how do interstrand crosslinks cause dna damage
highly toxic lesions that prevent DNA strand separation interfering with transcription and replication
check points are initiated by what protein kinases
ATM, ATR, DNA Pk
act upstream of cell signaling cascades and detect damaged chromosomes and block cell cycle progression
mitotic spindle checkpoints
ensure that microtubules are assembled into a functional mitotic spindle that connects sister kinetochores to opposite centrosomes prior to division
DNA integrity checkpoints
delay progression through the cell cycle in response to DNA damage or replication defects
DNA damage check point process (ie steps)
“Sensor” proteins (ATM, DNA-PK) activated→ phosphorylation of histone proteins → assembly of “mediator” proteins (mediator of DNA checkpoint [MDC1]) around DSB → concentration of “effector” proteins (p53) to facilitate enactment of downstream processes like DNA repair & checkpoints
p53 function in DNA damage check points
P53 stabilization activates cyclin D/E kinase complex inhibitor which leads to hypophosphorylation of Rb protein to effect G1 cell cycle arrest
Second G1 checkpoint pathway activated that targets CDC25A phosphatase that is essential for G1/S transition → inactive CDC25 is incapable of removing inhibitory phosphate group which prevents entry into mitosis
Kinetochore proteins crucial to Spindle assembly checkpoint pathway
MAD2, BUBR1, BUB1, and BUB3
MAD2 + BUBR1 function in spindle checkpoint
regulate mitotic progression by direct interaction and inhibition of the APC machinery
BUB1/3 function in spindle checkpoint
mediate mitotic arrest after disruption of microtubules
what happens to cells that lack BUB1/3
do not undergo mitotic arrest when treated with spindle-disrupting agents such as docetaxel or vinblastine
Chromosomal passenger complex (CPC)
central regulator of the tension checkpoint
If activated by lack of tension will result in removal of improper attachments and activation of spindle assembly checkpoint pathway
How does TRAP assay work?
Detects telomerase activity using primer and amplification
What is the TUNEL assay used to detect?
DNA fragmentation or strand breaks
- tells you if cells are apoptotic
DLBCL in dogs and humans share which common pathway mutations?
NFKB and Pi3K, Jak Stat (N-ras, p53, Rb, p16 CDK, telomerase)
Telomerase effects on cell signaling pathways
Wnt and β-catenin;
Wnt/β-cat activity regulates telomerases by increasing TERT (telomerase reverse transcriptase), involved in apoptotic resistance
. Without β-cat you get shorter telomeres. Cancer and CSCs can upregulate β-cat
promotes cell survival
What interferes with telomerase inhibitors and why?
alternate lengthening of telomeres (Alt) -alternate route for telomerase so will always be resistant
What cells have telomerase activity?
Tumors, germline/embryo, testicles/ovary
Differences in the telomeres of mice, dogs, and men
Mice 40-60 bps, Dogs 20 bps, Men 10-15 bps
Mouse telomeres are 5-10x longer compared to dogs and humans
telomeres
proteins on the ends of chromosomes to protect them from being inappropriately recognized as DNA damage
how do cancer cells control telomeres
Cancer cells most often protect their telomeres through the activation of an enzyme called Telomerase that adds new telomere DNA onto chromosome ends
Those that do not express telomerase maintain cells with ALT mechanisms
Several telomerase chemotherapeutic targets have been proposed but with limited efficacy bc of ALT - will always be resistant
Breakage-fusion-bridge cycle
Loss of telomeres led to fusion or loss of chromosomes and an ensuing cycle of chromosome breakage, chromosome fusion, and anaphase bridges during cell division
BFB cycles are a major source of genome instability
could be cause of heterogeneity in tumors
TIF (Telomere-dysfunction induced foci)
DNA damage foci at uncapped telomeres
Coincides with activation of checkpoint kinases and lead to p53 and p21 dependent apoptosis or cell cycle arrest
TERT (Telomere Reverse Transcriptase)
directs new telomere DNA synthesis
TERT and cell senescence
In normal cells, levels of TERT is undetectable due to transcriptional repression and therefore cells undergo telomere shortening with each cell division
Cellular senescence - eventual loss of replicative potential
what are shelterins
Shelterin “shelters” the chromosome ends and protects telomeric DNA from degradation and end-to-end fusion events
Telomeres are bound by protein complexes called Shelterins
what does stat doe int he cell
Antiapoptotic, proliferation, angiogenesis
How does p53 cause apoptosis?
Activates PUMA, NOX, Bak/Bim -> Apaf1, cytochrome-c ->caspase9 - caspase 3,6,7
What does PTEN do?
Inhibits PIP3 so no PI3k, stabilize p27
How does Rb control the cell cycle?
Hypophos blocks cyclinD/cdk4/6, HyperphosRb, release E2F, cell cycle progression (wont produce cyclin e /a), Halt cells at G1/S checkpoint if hypophos
Which requires energy apoptosis or necrosis?
Apoptosis; Necrosis causes inflammation
What are the 2 apoptosis pathways?
Death receptor (Fas/FADD, TRAIL, TNFa) - extrinsic
mitochondrial - intrinsic
What connects the two apoptotic pathways?
caspase 8
What pathways does VEGF stimulate?
PI3K, BcL2
Which VEGF receptor is associated with lymphatics?
vegfr-3
Where is NFkB activated? What happens?
Activated in cytoplasm after released from IkB (phos->ubiquinated),Travels to nucleus; Produces cytokines
What are 4 ways Myc is activated?
Retrovirus transduction, amplification, translocation, proviral insertion
MYC can be directly genetically activated through chromosomal translocation, genomic amplification, retroviral integration, and mutation, as well as activated through increased gene expression and/or protein stability by the activation of other oncogenes, including RAS, SRC, NOTCH, or the inactivation of tumor suppressor genes such as APC.
What are integrins and what pathways are activated by them?
Attachment/communication with ECM, Tumor suppressor ; Activate FAK/Src->Ras, ERK, Rho
What does Ras do in the cell?
Regulated cycle, survival, cytoskeleton; Works through RTK and Gcoupled
How is Ras usually mutated?
Point mutation
what pathways are activated by Ras
Raf, Ral, PI3K
What pathway does GF activate?
PI3K, Stat, MAPK, HER2/neu
Name important GF
VEGF, IGF1, TGFb, PDGF, kit, Ret, Met, Erb2
Describe Wnt pathway
Wnt->receptor Frizzled-> disheveled ->GSK3b, APC/Axin - DSH blocks GSK3b so B catenin released to promote survival
noncanonical wnt not bound so gsk3b apc complex bound to b catenin
What diseases are the Wnt pathways mutated
FAP Familial adenomatous polyposis and colon cancer - APC point mutation
Name ways LOH occur in tumor suppressor genes
Mitotic recombination, chromosomal nondysjunction, gene conversion
What are gatekeepers and caretakers? Give examples of each
Gate-direct against proliferation, promote tumor death, ex. Rb, p53,PTEN, BRCA1/2, APC, VHL
Care-Gene encodes protein that helps with genomic integrity, sensitive to DNA damage, inherited diseases, ex. ATM, XP, MSH2/MLH1
How can p53 act like an oncogene
Point mutation cause dominate negative effect
How does MDM2 and Arf work?
MDM2 ubiquinate p53 for degredation, p53 makes MDM2; Arf moves MDM2 to nucleolus where it cant bind p53; E2f makes Arf
mechanism of SMAC
o Aka DIABLO, pro-apoptotic
o Inhibits IAP (inhibitor of apoptosis)
o Promotes caspase activation in cytochrome c/Apaf-1/caspase 9 pathway
o Promotes caspase 9 activation by binding to inhibitor of apoptosis proteins and removing their inhibitor activity
o Normal mitochondrial protein – release into cytosol when undergo apoptosis
o Mitochondrial import and cleavage of its signal peptide are required for SMAC
o Overexpression of SMAC increases cells’ sensitivity to apoptotic stimuli
Which activates extrinsic apoptotic pathway?
o DR5 (TRAIL - death receptor 5)
o FAS
o TNFR
o Also: granzyme via caspase 3 and 8 cleavage
How does yunnan baiyao mediate apoptosis in vitro?
o Caspase-mediated apoptosis
o Caspase 3/7
What are features of apoptosis?
Condensed chromatin - cell shrinkage
no inflammatory response
phagocytes
versus necrosis
- inflammation
- swelling of ER and mitochondria
in the intrinsic pathway, what are mediators of pro-apoptosis and anti-apoptosis
anti apoptosis - Bcl2, Bcl XL, Bcl w, Mcl 1, A1
pro apoptosis - Bax Bad Bid Bck Bak Bak
what are effector/initiators of the cascade cascade?
o Effector/executioner: caspase 3, 6, 7
o Initiators:
1. Extrinsic: 8, 10
2. Intrinsic: 9
Which BH3 family member causes necrosis and apoptosis?
Bid via Caspase-8
MOA of thrombospondin-1?
o Binds CD36
o Blocks proliferation
o Activates FasL→ apoptosis
How does Bcl prevent apoptosis?
Prevents release of Cyt-C from mitochondria
Regulators of autophagy?
Beclin-1, p62
Detection of early and late necrosis on flow cytometry
o Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability
o One of the first steps in apoptosis is flipping the cell membrane so that phosphatidylserine (PS) is exposed on the outside. Annexin V binds PS with high specificity, making it a good indicator of early apoptosis
o PI will penetrate the membrane and bind nucleic acids in late apoptosis and necrosis
o Flow gated cells = viable (Annexin V– PI– ), early (Annexin V+PI– ), and late (Annexin V+ PI+ ) apoptosis/ necrosis
Propidium iodide (PI) incorporation into DNA. Treatment with a taxane would affect which peak?
4N (S/G2/mitosis peak)
Mutations in gene Beclin 1 have been observed in some types of cancer. What does Beclin 1 regulate/why is this important?
Beclin 1 regulates autophagy (the ability to remove and degrade these otherwise toxic cellular components is thought to provide a tumor-suppressor function in some instances during early tumorigenesis)
What is the mechanism of action of elsamitrucin?
Elsamitrucin, the most potent topoisomerase II inhibitor available, is unique in that it does not cause neutropenia or cardiotoxicosis (Fiocchi 2011 JVIM)
Topoisomerase 1 inhibitor?
Camptothecin (also irinotecan)
●What are suicide genes
what cancers have they bene used in
Delivery of a gene enzyme directly into tumor followed by delivery of a prodrug that is normally non-toxic but converted to active compound in tumor to kill
Cytokine-enhanced vaccine and IFN-beta plus suicide gene for canine sarcomas - administered IFN-beta and HSVtk-carrying lipoplexes and ganciclovir
Combination of suicide and cytokine gene therapies as surgery adjuvant for canine mammary carcinomas.
What is a gene gun
DNA absorbed onto gold particles and fired into tissues under high pressure
autophagy
lysosome mediated degradation of membrane bound vesicles called autophagosomes
cell response to stress
Beclin 1 is key protein
how does chemotherapy induce immunogenic death
Chemotherapy can induce a type I interferon response through production of DAMPs that are sensed by pattern-recognition receptors (PRRs) on innate and adaptive immune cells
initiates an immune response towards antigens expressed by the dying cells
Ferroptosis
an intracellular iron-dependent form of cell death that is distinct from apoptosis, necrosis, and autophagy
ferroptosis is initialized through GSH depletion or inactivation of GPX4 activity leads to iron-dependent accumulation of lipid peroxidation products ( ROS) leading to cell death
what inhibits ferroptosis
lipophilic antioxidants (e.g., ferrostatin-1 and α-tocopherol) and iron chelators (e.g., deferoxamine) can prevent ferroptotic cell death
Intrinsic (Mitochondrial) Apoptotic Pathway:
Triggered by internal cellular signals such as DNA damage, oxidative stress, or lack of growth factors.
Key regulators include members of the Bcl-2 protein family, which control mitochondrial membrane permeability.
When activated, pro-apoptotic Bcl-2 proteins such as Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP).
MOMP leads to the release of cytochrome c and other pro-apoptotic proteins from the mitochondria into the cytoplasm.
Cytochrome c, along with apoptotic protease activating factor 1 (Apaf-1) and procaspase-9, forms the apoptosome, initiating the caspase cascade.
The caspase cascade leads to the activation of executioner caspases (caspase-3, -6, and -7), which dismantle the cell by cleaving specific substrates.
Extrinsic (Death Receptor) Apoptotic Pathway:
Initiated by external signals such as binding of death ligands (e.g., Fas ligand, TNF-alpha, TRAil (DR5)) to death receptors (e.g., Fas, TNFR1).
Binding of death ligands induces clustering of death receptors, leading to the formation of the death-inducing signaling complex (DISC). The DISC recruits and activates procaspase-8.
Activated caspase-8 can directly cleave and activate executioner caspases (e.g., caspase-3), initiating the caspase cascade.
In some cases, caspase-8 cleaves and activates the pro-apoptotic Bcl-2 family member Bid.
Activated Bid triggers the intrinsic pathway by inducing mitochondrial outer membrane permeabilization.
Endocrine Signaling:
Process: Hormones released into the bloodstream, hormones travel to distant target cells, where they bind to receptors on the cell surface or inside the cell, initiating cellular responses.
Autocrine signaling
occurs when a cell releases signaling molecules that bind to receptors on its own surface or neighboring cells of the same type. In other words, the signaling molecule acts on the same cell or cell type that secreted it.
regulate cell proliferation, differentiation, and other cellular functions.
Paracrine signaling
involves the release of signaling molecules by one cell that act on neighboring cells in the immediate vicinity. The signaling molecules (paracrine factors) are released into the extracellular space and affect nearby target cells.
Cell survival pathways
molecular mechanisms that cells utilize to promote their survival and prevent programmed cell death (apoptosis).
Wnt/β-catenin Pathway
NF-κB Pathway
PI3K-Akt Pathway
MAPK/ERK Pathway
JAK-STAT Pathway
PI3K-Akt Pathway
The pathway is typically initiated by the binding of growth factors (e.g., insulin, insulin-like growth factor 1, epidermal growth factor) to their respective receptor tyrosine kinases (RTKs) on the cell surface.
This leads to receptor dimerization and autophosphorylation of tyrosine residues in the receptor’s cytoplasmic domain
Activated RTKs recruit and activate phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3)
Akt (also known as protein kinase B) is recruited to the plasma membrane by binding to PIP3 through its pleckstrin homology PH domain.
Once localized to the membrane, Akt is phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK1) at threonine 308.
Akt is further activated by phosphorylation at serine 473, primarily by the mammalian target of rapamycin complex 2 (mTORC2).
Akt phosphorylates and inhibits pro-apoptotic proteins such as Bad and caspase-9, while activating anti-apoptotic proteins such as Bcl-2 and Mdm2.
MAPK/ERK Pathway
cell proliferation
Activation of RTKs or G protein-coupled receptors by growth factors (e.g., EGF, FGF, PDGF) leads to receptor dimerization and autophosphorylation of tyrosine residues in the receptor’s cytoplasmic domain -> recruit and activate the small GTPase Ras (Rat sarcoma protein) by facilitating the exchange of GDP for GTP.
Ras-GTP activates a cascade of protein kinases. First, it activates a MAP3K (Mitogen-Activated Protein Kinase Kinase Kinase), such as Raf (Rapidly Accelerated Fibrosarcoma), through direct interaction.
Raf phosphorylates and activates MAP2K (Mitogen-Activated Protein Kinase Kinase or MEK).
Activated MAP2K/MEK phosphorylates and activates MAPK/ERK (Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase) by phosphorylating specific threonine and tyrosine residues within the activation loop
Activated ERK then phosphorylates various cytoplasmic and nuclear targets, including transcription factors (e.g., Elk-1, c-Myc, c-Fos), protein kinases, and other signaling proteins.
Phosphorylation of these targets leads to changes in gene expression, cytoskeletal rearrangements, and modulation of various cellular proliferation
MAPK/ERK pathway regulates what cellular responses:
Cell Proliferation: ERK activation promotes cell cycle progression by upregulating cyclins and cyclin-dependent kinases (CDKs) and downregulating CDK inhibitors.
Cell Differentiation: ERK signaling induces differentiation in various cell types by activating specific transcription factors.
Cell Survival: ERK activation promotes cell survival by phosphorylating and inactivating pro-apoptotic proteins such as Bad.
Cell Migration: ERK signaling regulates cell migration by controlling cytoskeletal dynamics and cell adhesion.
how is AKT activated
PIP3 serves as a second messenger that recruits proteins containing pleckstrin homology (PH) domains, such as Akt, to the plasma membrane.
Akt is phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK1) at threonine 308. and by mtor2 on serine
cell responses of PI3K/ERK
Cell Growth and Protein Synthesis: Akt activates mTORC1 (mammalian target of rapamycin complex 1) by phosphorylating and inhibiting the tuberous sclerosis complex 2 (TSC2), leading to increased protein synthesis and cell growth.
Cell Survival: Akt promotes cell survival by phosphorylating and inactivating pro-apoptotic proteins such as Bad, caspase-9, and Forkhead box O (FoxO) transcription factors.
Glucose Metabolism: Akt stimulates glucose uptake and glycolysis by promoting translocation of glucose transporter proteins (GLUT4) to the cell membrane and activating glycolytic enzymes.
Cell Proliferation and Cell Cycle Progression: Akt promotes cell proliferation by phosphorylating and activating various proteins involved in cell cycle progression, including cyclin-dependent kinases (CDKs) and the mTORC1 substrate p70S6 kinase (p70S6K).
NF-κB Proteins
NF-κB proteins are transcription factors that exist in the cytoplasm as inactive complexes bound to inhibitory proteins called IκBs (inhibitors of κB).
The NF-κB family includes various dimers, most commonly p50/p65 (RelA), p52/RelB, and c-Rel
The NF-κB pathway
activated in response to various extracellular signals, such as pro-inflammatory cytokines (e.g., TNF-α, IL-1β), microbial products (e.g., lipopolysaccharide, LPS), and stress signals.
Activation of the pathway typically involves the phosphorylation and degradation of IκB proteins, allowing NF-κB to translocate into the nucleus and regulate gene expression
2 pathways - canonical and non canonical
The NF-κB canonical pathway
In the canonical NF-κB pathway, activation is initiated by the engagement of cell surface receptors, such as TNF receptor (TNFR) or Toll-like receptors (TLRs), by their ligands.
This triggers the recruitment of adaptor proteins such as TNF receptor-associated factors (TRAFs) and TGF-β-activated kinase 1 (TAK1), leading to the activation of the IκB kinase (IKK) complex.
The IKK complex consists of two catalytic subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ or NEMO).
IKK phosphorylates IκB proteins, marking them for ubiquitination and proteasomal degradation.
Degradation of IκB proteins frees NF-κB dimers, allowing them to translocate into the nucleus.
In the nucleus, NF-κB binds to specific DNA sequences called κB sites and regulates the transcription of target genes.
Transcriptional Regulation by NF-κB
Inflammation: Pro-inflammatory cytokines (e.g., IL-6, IL-8, TNF-α), chemokines, adhesion molecules (e.g., ICAM-1, VCAM-1).
Immune Response: Genes encoding immune receptors, antimicrobial peptides, and immunoregulatory molecules.
Cell Survival: Anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL), cell cycle regulators, and growth factors.
Non-canonical NF-kB pathway
The non-canonical NF-κB pathway is initiated primarily by members of the TNF receptor superfamily, such as lymphotoxin β receptor (LTβR), CD40, B-cell activating factor receptor (BAFF-R), and receptor activator of NF-κB (RANK).
Activation of these receptors leads to the recruitment of TRAF3 (TNF receptor-associated factor 3) and subsequent activation of the NF-κB-inducing kinase (NIK).
TRAF3, in its unbound state, mediates the degradation of NIK through the proteasomal pathway.
However, upon receptor activation, TRAF3 is degraded, leading to the stabilization and accumulation of NIK.
Accumulated NIK then phosphorylates and activates IKKα, also known as IKK1, but not IKKβ.
Activated IKKα phosphorylates p100, a precursor protein that binds to RelB to form an inactive complex in the cytoplasm.
Phosphorylation of p100 targets it for partial proteasomal degradation, resulting in the generation of p52, the active form of NF-κB.
This processing allows the release of RelB/p52 dimers from p100 and their translocation into the nucleus.
In the nucleus, RelB/p52 dimers regulate the expression of specific target genes.
The non-canonical pathway’s target genes often include those involved in lymphoid organogenesis, B-cell maturation, bone metabolism, and immune responses, distinct from those regulated by the canonical NF-κB pathway.
JAK-STAT Pathway
The pathway is initiated by the binding of cytokines or growth factors to their respective cell surface receptors, such as cytokine receptors or receptor tyrosine kinases (RTKs).
Binding of the ligand induces dimerization of the receptor subunits, leading to activation of associated Janus kinases (JAKs) that are constitutively associated with the receptor cytoplasmic domains.
Activated receptors induce conformational changes in the associated JAKs, leading to their transphosphorylation and activation.
Once activated, JAKs phosphorylate tyrosine residues on the cytoplasmic tails of the receptor (receptor-associated tyrosine residues)
Phosphorylated tyrosine residues on the receptor serve as docking sites for signal transducer and activator of transcription (STAT) proteins. they are phosphorylated by activated JAKs on a conserved tyrosine residue near their C-terminus
Phosphorylated STAT proteins undergo a conformational change that promotes their dimerization through reciprocal interactions between phosphorylated tyrosine residues.
Dimerized STATs then dissociate from the receptor complex and translocate to the nucleus as transcriptionally active dimers
STAT dimers bind to specific DNA sequences known as gamma-activated sites (GAS) or interferon-stimulated response elements (ISRE), located within the promoters of target genes.
Binding of STATs to these DNA elements leads to the recruitment of co-activators and chromatin-modifying enzymes, resulting in transcriptional activation or repression of target genes.
Negative regulators, such as protein tyrosine phosphatases (PTPs) and suppressor of cytokine signaling (SOCS) proteins, inhibit JAK activity and dephosphorylate activated receptors or STATs.
Additionally, unphosphorylated STAT proteins are actively exported out of the nucleus by exportins, limiting their transcriptional activity.
JAK STAT cell process
Immune Responses:
JAK-STAT signaling is crucial for the regulation of immune responses, including the development, differentiation, and activation of immune cells such as lymphocytes (T cells, B cells), natural killer (NK) cells, and myeloid cells.
Cytokines such as interleukins (ILs), interferons (IFNs), and colony-stimulating factors (CSFs) activate the JAK-STAT pathway to modulate immune cell proliferation, differentiation, and function.
Inflammation:
Cytokines such as IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) activate the JAK-STAT pathway to induce the expression of inflammatory mediators and adhesion molecules, promoting inflammation.
Dysregulation of JAK-STAT signaling can lead to chronic inflammation and autoimmune diseases.
Cell Growth and Proliferation:
JAK-STAT pathway regulates cell growth, proliferation, and survival by controlling the expression of genes involved in these processes.
Growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) activate the JAK-STAT pathway to promote cell proliferation and survival.
Hematopoiesis:
Cytokines such as erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), and thrombopoietin (TPO) activate the JAK-STAT pathway to stimulate the production of red blood cells, granulocytes, and platelets, respectively.
Development and Differentiation:
JAK-STAT signaling is involved in the development and differentiation of various tissues and organs during embryogenesis and tissue regeneration.
Antiviral Responses:
The JAK-STAT pathway is a critical component of the innate antiviral immune response.
IL-2 activates STAT5 promoting what?
promoting T cell proliferation and differentiation
Activation of what STAT proteins are associated with cell survival
particularly STAT3 and STAT5, is associated with cell cycle progression and inhibition of apoptosis.
leukemia inhibitory factor (LIF) - cytokine that does what
activate STAT3 to promote stem cell pluripotency and self-renewal
Wnt/β-catenin pathway
The pathway is initiated by secreted glycoprotein ligands called Wnts.
Wnts bind to Frizzled (Fzd) family receptors on the cell surface, in conjunction with co-receptors LRP5/6 (low-density lipoprotein receptor-related protein 5/6).
leads to the recruitment and activation of Dishevelled (Dsh)
Activated Dsh inhibits a the destruction complex consisting of Axin, APC (adenomatous polyposis coli), GSK-3β (glycogen synthase kinase-3β), and CK1 (casein kinase 1).
Inhibition of the Axin complex prevents the phosphorylation and subsequent degradation of β-catenin and it accumulates in the cytoplasm.
Stabilized β-catenin then translocates to the nucleus.
In the nucleus, β-catenin associates with transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family.
This complex activates the transcription of target genes by binding to specific DNA sequences known as Wnt response elements (WREs) in the promoters of target genes.
Target genes include those involved in cell proliferation (e.g., c-Myc, cyclin D1), cell fate determination (e.g., Axin2, CD44), and stem cell maintenance (e.g., Lgr5, Sox9).
Dysregulation of the Wnt/β-catenin pathway is associated with what various cancers
colorectal cancer, hepatocellular carcinoma, and breast cancer.
TGF-B pathway
Active TGF-β ligands bind to TGF-β type II receptor (TβRII) on the cell surface.
Upon ligand binding, TβRII phosphorylates and activates TGF-β type I receptor (TβRI or ALK5), a serine/threonine kinase.
Activated TβRI phosphorylates receptor-regulated Smads (R-Smads), primarily Smad2 and Smad3.
Phosphorylated R-Smads form complexes with the common Smad (Co-Smad), Smad4.
The R-Smad/Co-Smad complexes translocate into the nucleus.
In the nucleus, Smad complexes regulate the transcription - can act as transcriptional activators or repressors - cmyc
Target genes include those involved in extracellular matrix production, cell cycle regulation, apoptosis, and differentiation.
what does tgf B pathway regulate
Regulation of Target Gene Expression:
TGF-β regulates the expression of a wide range of target genes, including:
Extracellular matrix proteins (e.g., collagen, fibronectin) and proteases (e.g., MMPs) involved in tissue remodeling.
Cell cycle inhibitors (e.g., p21, p15) and pro-apoptotic factors (e.g., Bim) that inhibit cell proliferation and induce apoptosis.
Transcription factors (e.g., Snail, Slug) involved in epithelial-mesenchymal transition (EMT) and cell differentiation.
Cytokines and growth factors (e.g., TGF-β itself, IL-6) that modulate immune responses and inflammation.
Cross-talk with Other Signaling Pathways:
The TGF-β pathway interacts with other signaling pathways, including the MAPK/ERK, PI3K-Akt, and Wnt/β-catenin pathways, to regulate cellular responses.
negative regulators for TGF - B
Negative regulators include inhibitory Smads (Smad6 and Smad7), which inhibit TGF-β receptor activation, and various phosphatases and ubiquitin ligases that target components of the pathway for degradation.
HER2/neu pathway:
The HER2/neu receptor, also known as ErbB2 or HER2 (human epidermal growth factor receptor 2), is a member of the epidermal growth factor receptor (EGFR) family.
HER2 is a transmembrane receptor tyrosine kinase that lacks a ligand-binding domain. Instead, it is activated through heterodimerization with other EGFR family members, particularly EGFR (ErbB1), HER3 (ErbB3), or HER4 (ErbB4).
Heterodimerization leads to activation of the intracellular kinase domain of HER2, resulting in autophosphorylation of tyrosine residues.
Activation of HER2 leads to the activation of several signaling pathways, including the PI3K-Akt pathway, the MAPK/ERK pathway, and the PLC-γ (phospholipase C-gamma) pathway.
These pathways regulate cellular processes such as cell proliferation, survival, differentiation, and migration.
In normal cells, HER2 signaling is tightly regulated and plays a role in tissue development and homeostasis.
Dysregulation of the HER2 pathway, often due to HER2 gene amplification or overexpression, is associated with various cancers, particularly breast cancer and gastric cancer.
HER2 in cancer
HER2 overexpression is a hallmark of a subset of breast cancers known as HER2-positive breast cancers.
Therapies targeting HER2, such as monoclonal antibodies (e.g., trastuzumab, pertuzumab) and small molecule inhibitors (e.g., lapatinib), have revolutionized the treatment of HER2-positive breast cancer,
what is MDM2/ARF pathway in normal cells
MDM2 (Mouse double minute 2) is an E3 ubiquitin ligase that plays a key role in the negative regulation of p53.
Under normal conditions, MDM2 binds to p53 and promotes its ubiquitination, targeting it for proteasomal degradation.
This interaction forms an auto-regulatory feedback loop, as p53 can induce the transcription of the MDM2 gene, leading to increased MDM2 levels and subsequent inhibition of p53 activity.
ARF (Alternative Reading Frame) is a tumor suppressor protein encoded by the INK4a/ARF locus, also known as CDKN2A.
ARF is activated in response to oncogenic stress, such as hyperproliferative signals or DNA damage.
In normal cells, ARF acts as a nucleolar stress sensor, detecting abnormalities in ribosome biogenesis or other cellular stresses.
Upon activation, ARF binds to MDM2 and inhibits its E3 ubiquitin ligase activity, preventing the ubiquitination and degradation of p53
When ARF binds to MDM2, it disrupts the MDM2-p53 interaction, leading to the stabilization and activation of p53.
Stabilized p53 accumulates in the nucleus and functions as a transcription factor, activating the expression of genes involved in cell cycle arrest, apoptosis, and senescence.
MDM2/ARF pathway in cancer
Dysregulation of the MDM2/ARF pathway is common in cancer and contributes to tumorigenesis.
Loss of ARF function or overexpression of MDM2 can lead to the inactivation of p53 and promote tumor formation.
Mutations in the p53 gene itself are also common in cancer and can disrupt its function independently of the MDM2/ARF pathway.
Small molecule inhibitors of MDM2, such as Nutlin-3a, disrupt the MDM2-p53 interaction, leading to p53 stabilization and activation.
These inhibitors are being investigated as potential cancer therapeutics, particularly in tumors with wild-type p53. Additionally, strategies to restore ARF function or reactivate mutant p53 are also under investigation.
Test for proliferation?
PCNA, Ki67, AgNOR
Which growth factor is not a RTK and what does it do?
TGFβ-immunosupressive/inhibit dendritic cells up regs tregs tams, increase fibrin, expressed in tumors
What pathway/ligand activates osteoclast?
RANKL
PI3K/Akt leads to what changes in the cell?
Antiapoptotic, cell proliferation
What is the Jak receptor? What are some ligand examples?
cytokines;
(ILs), (IFNs), (CSFs)
Inflammation:
IL-6, IL-1β, (TNF-α) a
Cell Growth and Proliferation:
(EGF), (PDGF)
Hematopoiesis:
(EPO), (G-CSF), (TPO)
What does Stat do in the cell?
Antiapoptotic, proliferation, angiogenesis
What does southern, northern and western blot determine?
Southern-dna, Northern-mRNA, Western-proteins
What assay is used to assess carcinogenesis and how does it work?
Cell Transformation Assays (CTAs)
The assay uses histidine-dependent strains of Salmonella typhimurium that are unable to synthesize histidine due to mutations in genes
Bacterial strains with specific mutations (usually his- or rfa-) are exposed to the test chremical or compound in the presence or absence of a metabolic activation system,
If the test compound induces reverse mutations in the bacterial strains, resulting in the reversion of the his- or rfa- mutation, colonies of revertant bacteria will grow on the agar plates.
An increase in the number of revertant colonies compared to the negative control indicates a positive result for mutagenicity.
Positive results suggest that the compound has the potential to cause mutations in DNA, which could lead to carcinogenesis.
Negative results suggest that the compound is not mutagenic under the conditions tested.
dose-response relationship
What is the difference between FISH and comparative genomic hybridization?
FISH is used for detecting specific chromosomal abnormalities and gene mapping - you know the abnormality you are looking for - use of fluorescently labeled DNA probes that bind to specific target sequences on chromosomes
CGH provides a genome-wide view of DNA copy number changes - dont know the abnormality you are searching for one - compares the relative DNA copy number between a test sample and a reference sample - bets combined wiht micro array
What pathways synthesize Hif?
PI3K, mTOR
Why is NFkB important?
Link between inflammation & cancer
What IL connects innate & adaptive immunity
IL12
What are HMGA1/HMGB1?
High mobility group/box that regulate transcription binding, HMGB enables NfKB and both elevated in dogs with LSA compared to normal
What is Herceptin and what is the generic name?
Humanized monoclonal Ab against Her2/neu (Erb2 growth factor); Trastuzumab
How are RTK mutated? Give an ex for each
Overexpression-Her2/neu,
fusion-Bcr-Abl, (CML)
autocrine loops-MET/HGF
What mutation has been found in Polycythemia Vera?
jak2
mTORC1:
sensitive to rapamycin. bound to RAPTOR
Activation of mTORC1 occurs in response to growth factors (e.g., insulin, insulin-like growth factor 1), amino acids (particularly leucine), and cellular energy status (e.g., AMP:ATP ratio).
Key regulators of mTORC1 include:
Rheb (Ras homolog enriched in brain), a small GTPase that activates mTORC1 when in the GTP-bound state.
TSC1 (tuberous sclerosis complex 1) and TSC2, form a complex that inhibits Rheb when activated by upstream signals.
Akt (protein kinase B), phosphorylates and inhibits TSC2, leading to activation of mTORC1.
mTORC2
bound to RICTCHER
Akt is phosphorylated and activated by mTORC2 at a serine residue (Ser473)
mTORC2 regulates various downstream targets, including Akt, SGK1 (serum- and glucocorticoid-regulated kinase 1), and PKC (protein kinase C
The mTOR pathway plays a central role in regulating numerous cellular processes including
Protein synthesis: promoting ribosome biogenesis and translation initiation.
Cell growth and proliferation
mTORC1 inhibits autophagy,
mTORC1 regulates cellular metabolism by modulating nutrient uptake, glycolysis, lipid metabolism, and mitochondrial function.
Both mTORC1 and mTORC2 regulate cell survival pathways through their downstream effectors.
How to treat patients that develop resistance to RAF inhibitor?
MEK inhibitor
Upstream:
1. Farneysl transferase inhibitor
2. Gerangylgeranyl transferase inhibitor
In the PI3K pathway, what does PIK3 phosphorylate?
PIP2 to PIP3
PIP3 attracts AKT to cell membrane where PDK1 phosph threonine and mtor phosphorylates serine
Consequence of Patched dysfunction?
Patched is TSG; without → Gli cleaved to active TF, stimulating proliferation
Increased SHH signaling causes?
o Proliferation
o Invasion
o Metastasis
o Also: embryogenesis, angiogenesis, and activation of CSCs
hedge hog pathway receptors andligands
The Hedgehog family includes three ligands in mammals: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)
primary receptor for Hedgehog ligands is the transmembrane protein Patched (Ptch)
When Hedgehog ligands bind to Ptch, it relieves the inhibition on Smo.
Once activated, Smo initiates downstream signaling by modulating the activity of the Gli (Glioma-associated oncogene) transcription factors - cyclin D, cmyc, Bcl, nanog
in the absence of ligands what does patched do?
In the absence of Hedgehog ligands, Ptch inhibits another transmembrane protein called Smoothened (Smo) - therefore GLI2 and GLI3 are phosphorylated by PKA, CK1 and GSK3β, which create binding sites for the E3 ubiquitin ligase β-TrCP. GLI3 and GLI2 undergo partial proteasome degradation, leading to the formation of repressor forms (GLI3/2R), that translocate into the nucleus where they inhibit the transcription of HH target genes.
When Hedgehog ligands bind to Ptch, it relieves the inhibition on Smo.
non canoniucal pathway
hedge hog canonical pathway
in the absence of Hedgehog ligands, Ptch inhibits Smo, preventing Gli activation.
When Hedgehog ligands bind to Ptch, it relieves the inhibition on Smo, allowing Smo to accumulate and activate Gli proteins.
Activated Gli proteins translocate into the nucleus, where they regulate the transcription of target genes. GliA promotes the expression of Hedgehog target genes, such as Ptch1, Gli1, and Hhip (Hedgehog-interacting protein), while GliR inhibits their expression.
Target Genes: cell proliferation (e.g., Cyclin D1), survival (e.g., Bcl-2), differentiation (e.g., Ptch1, Gli1), and tissue patterning (e.g., Hox genes).
dysregulation of HH leads to what cancers
basal cell carcinoma, medulloblastoma, and pancreatic cancer
notch pathway receptors ligands and complexes
Notch receptors are single-pass transmembrane proteins. In mammals, there are four Notch receptors: Notch1, Notch2, Notch3, and Notch4.
Notch ligands are transmembrane proteins expressed on neighboring cells. In mammals, there are five Notch ligands: Jagged1, Jagged2, Delta-like 1 (Dll1), Delta-like 3 (Dll3), and Delta-like 4 (Dll4).
Intracellular Domain: Upon ligand binding, Notch receptors undergo proteolytic cleavage, releasing the intracellular domain of Notch (NICD), which translocates to the nucleus to regulate gene expression.
NOTCH canonical pathway
Notch ligands (e.g., Jagged or Delta-like) on one cell bind to Notch receptors on neighboring cells.
Ligand binding induces conformational changes in Notch receptors, leading to proteolytic cleavage by γ-secretase.
The NICD is released from the cell membrane and translocates to the nucleus.
In the nucleus, NICD binds to the DNA-binding protein CSL (CBF1/RBPJκ in mammals), converting it from a transcriptional repressor to an activator.
The NICD-CSL complex recruits coactivators, target genes such as Hes (Hairy and enhancer of split) and Hey (Hes-related with YRPW motif), which regulate cell fate decisions.
what does NOTCH regulate
influences the choice between different cell lineages (e.g., neuronal vs. glial cells in the nervous system, secretory vs. absorptive cells in the intestine).
Notch signaling regulates cell proliferation, differentiation, and stem cell maintenance in various tissues and organs.
In certain contexts, Notch signaling can inhibit apoptosis and promote cell survival.
Notch signaling is involved in the development and function of the immune system, including T cell development and activation.
Dysfunction or loss of Patched leads to
the inability to inhibit Smo, resulting in constitutive activation of Smo even in the absence of Hedgehog ligands
cell proliferation, survival, and differentiation
Dysregulated HH Patched function is associated with the development of various cancers, particularly basal cell carcinoma (BCC) and medulloblastoma.
Which pathway can initially suppress growth then become promoter?
TGF B
What are markers of hypoxia that can be used with PET/CT?
18-FDG
How does FDG accumulate?
FDG taken up by hypermetabolic cells via GLUT1
Phosphorylated by hexokinase → traps metabolically active cells
PET takes advantage of what? This allows us to obtain what additional information vs. regular CT
o Metabolism of cancer cells (Warburg effect)
o Proliferation, hypoxia, etc. vs. anatomic localization only (with CT)
molecular tests for chromosome numbers
karyotype
chromosomal microarray
SNP array
Comparative genomic hybridization
microsatelitte array
FISH
karyotype
visual assessment of metaphase chromosomes using microscopy = size shape and number
combines with FISH or pcr it could be a molecular test - should detect balanced chromocomal abnormalities
chromosomal microarray
high resolution method for detecting copy number changes - gains or losses accross the entire genome in a singe assay and is sometimes called molecular karyotypes
can detect aneuploidies as well as smaller deletions and duplications (microdeletions/microduplications) in chromosomes.
CMA can be performed using array comparative genomic hybridization (aCGH) or single nucleotide polymorphism (SNP) arrays.
Lactate Dehydrogenase (LDH) Assay
assay measures LDH activity in cell lysates, reflecting lactate production. It can be used to assess cell viability and glycolytic activity.
Quantitative Polymerase Chain Reaction (qPCR)
qPCR can be used to quantify the copy number of specific chromosomal regions by measuring the relative abundance of target DNA sequences
By comparing the amount of target DNA to reference DNA (e.g., a housekeeping gene), qPCR can detect aneuploidies
Multiplex qPCR assays are available for detecting aneuploidies in specific chromosomes, such as chromosomes 13, 18, 21, X, and Y
SNP
SNPs are variations in a single nucleotide base at specific positions in the DNA sequence, representing the most common type of genetic variation in humans
SNP arrays
SNP arrays consist of thousands to millions of DNA probes, each designed to hybridize to a specific SNP locus.
SNP arrays are used for large-scale genotyping studies to identify genetic variations associated with diseases, traits, and drug responses.
SNP arrays can detect chromosomal gains and losses (CNVs) by analyzing changes in signal intensity across adjacent SNP loci.
SNP arrays are used in family-based linkage studies and case-control association studies to identify genetic variants linked to complex diseases.
SNP arrays are used to study population genetics, ancestry, and evolutionary relationships
variations of pcr
Reverse Transcription PCR (RT-PCR): Used to amplify RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase enzyme.
Quantitative PCR (qPCR): Used to measure the amount of DNA or RNA present in a sample by monitoring the PCR amplification in real-time using fluorescent dyes or probes.
Multiplex PCR: Amplifies multiple target DNA sequences in a single reaction using multiple primer sets.
Nested PCR: Utilizes two sets of primers to increase the specificity and sensitivity of amplification.
pcr basic steps
Denaturation: The PCR reaction starts with heating the reaction mixture to around 94-98°C to denature the double-stranded DNA into single strands, separating the two DNA strands.
Annealing: The reaction temperature is lowered to around 50-65°C to allow the primers to anneal (bind) to their complementary sequences on the single-stranded DNA template.
Extension (Elongation): The temperature is raised to around 72°C, the optimal temperature for DNA polymerase activity. The DNA polymerase synthesizes new DNA strands by extending from the primers, using the template DNA and incorporating dNTPs.
Cycle Repeats: These three steps (denaturation, annealing, extension) are repeated for multiple cycles (usually 20-40 cycles), with each cycle doubling the amount of DNA produced.
PCR Product:
After each cycle, the target DNA sequence is amplified exponentially, resulting in a large amount of DNA product.
The PCR product is typically analyzed by gel electrophoresis, DNA sequencing, or other downstream applications to confirm the presence and size of the amplified DNA fragment.
Microsatellite array
also known as short tandem repeat (STR), arrays are used to analyze microsatellite loci throughout the genome
Microsatellites are repetitive DNA sequences consisting of short (1-6 base pair) tandem repeats (e.g., CA, AGAT).
Microsatellite arrays use fluorescently labeled probes to detect variations in the number of repeats at specific microsatellite loci following PCR amplificaiton
micrsatelitte array uses
Genotyping: Microsatellite arrays are used for genotyping in research and forensic applications.
Linkage Mapping: Microsatellite markers are used in linkage analysis to identify regions of the genome associated with inherited traits or diseases
.
Population Genetics: Microsatellite arrays are used to study genetic diversity and population structure in various species.
Cancer Research: Microsatellite instability (MSI) analysis using microsatellite arrays is used to detect genetic instability in cancer cells, which can be indicative of certain types of cancer or response to treatment.
Apoptosis Assays
Detects apoptotic cells using techniques like TUNEL assay, Annexin V staining, or caspase activity assays
B Cell Development in bone marrow
differentiation of hematopoietic stem cells (HSCs) into common lymphoid progenitors (CLPs) in the bone marrow which express specific transcription factors and surface markers, committing them to the B cell lineage.
Rearrangement occurs in the Ig heavy (IgH) and light (IgL) chain gene loci, leading to the assembly of variable (V), diversity (D), and joining (J) gene segments.
Recombination-activating genes (RAG1 and RAG2) mediate the DNA rearrangement process.
Successful rearrangement of IgH genes results in the production of μ (mu) heavy chains, which combine with surrogate light chains (VpreB and λ5) to form the pre-B cell receptor.
Pre-B cells that express functional pre-B cell receptors undergo positive selection, leading to further differentiation into immature B cells.
Immature B cells undergo negative selection
Immature B cells exit the bone marrow and migrate to secondary lymphoid organs, such as the spleen and lymph nodes.
In secondary lymphoid organs, mature B cells encounter antigens and receive signals from helper T cells, leading to activation, proliferation, and differentiation into antibody-secreting plasma cells or memory B cells.
T cell development in the thymus
Thymic Seeding:
T cell development begins with the migration of bone marrow-derived lymphoid progenitors to the thymus.
Progenitors enter the thymus through the bloodstream and settle in the thymic cortex.
T cell development involves the rearrangement of T cell receptor (TCR) genes, which encode the TCRα and TCRβ chains.
TCR gene rearrangement occurs through V(D)J recombination
Developing thymocytes express both TCRα and TCRβ chains on their surface, forming the pre-TCR complex.
Pre-TCR-expressing cells undergo positive selection in the thymic cortex, where cells with functional receptors that interact with self-major histocompatibility complex (MHC) molecules survive.
Positively selected thymocytes migrate to the thymic medulla, where they undergo negative selection to eliminate self-reactive T cells through apoptosis or regulatory T cell differentiation.
Thymocytes that successfully pass positive and negative selection differentiate into mature CD4+ or CD8+ T cells.
Mature T cells migrate to secondary lymphoid organs, where they circulate in search of antigens presented by antigen-presenting cells (APCs).
Interaction with APCs and recognition of antigen-MHC complexes lead to T cell activation, proliferation, and differentiation into effector or memory T cells.
What is the immunophenotype of the most common peripheral lymphocyte in dogs?
cd4
What is the function of an NK cell?
Innate cytotoxic cell
o CD56+
Cytotoxicity:
1. Perforin/granzyme
2. Fas-FasL
3. Cytokines (MAFs, IFNɣ, GM-CSF, TNF-ɑ)
Which are NK activating receptors?
CD16
KARs (KIRS are inhibitory)
C-type lectins
Target recognition:
1. FcyRII (CD16) - ADCC
2. Absence of MHC I
TLR ligands and their PAMPs
TLR 4 - LPS, RSV-F protein
(Recognition of LPS is aided by CD14 and MD-2)
TLR 2, 6, 1 work together - peptidoglycan, lipoprotein, mycobacterium, LPS of letup, porphyromonas, Glycophosphoinositol on trypanosome Cruzi, yeast zymosan
TL3 - dsDNA
TL5 - bacterial flagellin
TLR9 - unmethylated CpG DNA
innate immunity fast facts
- defects?
- onset, recognition, memory?
- how do neutrophils control pathogens
- macrophages
- major pathways for cell activation and cytokines
- inherited defect are rare
- acquired defects are common
- rapid onset
- recognition of DAMPs and PAMPs
- no memory
- PMN control pathogens by phagocytosis, cytokines, ROS, apoptotic vs necrotic cell death and inflammation
- macrophages are separate lineage - endogenous and self renewing specific to each organ
- TLRs, NLRs, PIG-I pathways
role of monocytes in innate immunity
monocytes respond rapidly, fxn determined by the environment, immune suppression in chronic inflammation
NK cells and the innate immunity
NK cell generate rapid anti-viral and antitumor activity
key early producers of IFNy
dendritic cells and immunity
dendritic cells are the link between the innate and adaptive immunity
antigen processing and presentation to TCells
3 major complement pathways
classical
MB lectin
alternative
What is the difference between innate and adaptive immunity?
Innate: immediate, no memory
1. IgM > IgG
2. Lower affinity, non-Sp
Adaptive: delayed response, memory
1. Increase in Igs (IgG)
2. Higher affinity, more Sp
3. IL-17**, IL-15
What is the major costimulatory signaling on T cells/APCs?
CD40L (T cell) = CD40 (APC)
Which of the following are co-stimulatory molecules?
CD28:B7 family
CD2
4-1BB
OX40
Also*
1. GITR
2. ICOS
3. CD27
4. CD40L
5. CD122
What tumors express PD-L1?
o Melanoma
o OSA
o Mammary
o Prostatic adenocarcinoma
o TCC
o HSA
What is the most potent APC for induction of naive T cells?
o Dendritic cells
o (CD11b drives CD4+)
o Interdigitating DC (CD8+): LN/spleen
o Interstitial DC (CD103): perivascular
**costimulation necessary for activation of naive T cells
What are the subsets of T helper cells and what do they produce?
Th1 - produce IFNy, IL2 and TNF
- stimulate cell mediated immunity
- trigger production of certain IgG isotypes that fix complement
Th-2 - produce IL-4, IL-5, IL-13
- trigger production of high levels of IgG and IgE
- associated with helminth responses and allergy
What is the activity of IL-2 and IL-4?
IL-2 (from Th1)→ proliferation of T, NK and B cells
IL-4 (from Th2)→ more Th2 cells, B cell isotype switch to IgE, inhibition of IFN-ɣ
- IFNy
- IFNy - Key Th1 cytokine produced by activated T cells and NK cells; promotes the differentiation of naïve CD4+ T cells to Th1 phenotype; activates macro- phages, increases MHC Class I/II expression.
- IFN a and b
IFN a and b - Induce apoptosis of tumor cells; enhances CTL effector function, activates NK cells, modulates MHC Class I/II expression, inhibits tumor angiogenesis
what cytokines are immunosuppressive
IL10, TGFb, TNFa
What does IL-12 do?
- Activate NK and CTLs
- Directly causes B cells to produce Abs
- stimulates synthesis of IFN-γ and TNF-α - dec angiogenesis
- stimulates differentiation of naïve CD4+ T cells to T cells with the Th1 phenotype.
What are the functions of IFN-ɑ, -β, and -ɣ?
- Pro-inflammatory, anti-angiogenic = IFN-ɑ, -β, -ɣ
- Activation of macrophages = IFN-ɣ
- Direct tumor effects = IFN-ɑ
- Anti-angiogenic, induces TSGs, downregulates OGs, inhibits tumor cell proliferation
Which cells recognize MHC class I and II?
MHC II = CD4+
MHC I = CD8+
MHC I on all nucleated cells
MHC II on professional APCs
NK cells attack cells that do not express MHC I
what’s a key feature of Th17 response
produce IL 17 which stimulate neutrophil recruitment and inflammation
where are naiive and effector T cells found
native T cells in lymph nodes and spleen
effector t cell sin tissues
how does flow cytometry measure t cells
binding fluorescent antibodies to CD4 or CD8 or CD3 on surface of T cells
passing as fluid stream past lasers whihc excite fluorescent dyes and number of positive events are counted electronically
what is normal cd4 cd8 T cell ratio in the blood
2:1
acquired CD4 deficiency in cats
FIV
how does CD4 deficiency present clinically
low IgG recurrent bacterial infections with intracellular pathogens
how would you manage an immune deficient patient
give plasma transfusion, non specific immune stiumulation recombinant IFN (cats(
how do T cell receptors differ from B cell receptors
TCR recognize peptides presented by MHC - Ag INSIDE of cell
Vs. B cell recognize naive Ag OUTSIDE of cell
B cell receptor is a membrane-bound soluble Ab. B cells recognize Ag in native form. TCR recognize Ab as peptides presented by MHCs
Which of the following is a feature of the B cell receptor?
Undergo Ag-independent maturation
Which portion of the Ig is responsive for cytotoxic effects?
Fc/constant region (determines immune response)
what drives antibody isotope switching
T cell cytokine and B cell CD40
how are antibody responses different than booster vaccination
more rapid response higher titer and higher affinity antibodies
what 2 factors determine whether antigen antibody complexes form
antigen concentration and antibody cross linking efficiency
which cytokines promote IgE
IL 4, 5, 13
how do oyu assess humoral immunity
measure IgA IgM and IgG concentrations
detemrine b cell numbers
measure vaccine titres
most common inherited immune deficiency
hypogammaglobulinemia
most abundant type of antibody ? where is it located?
IgA in the Gi tract and lung
how do anti bodies fix complement
complement binds to Fc molecules on certain isotope of antibodies
clinical signs of Ig deficiency
recurrent bacterial infections esp Gi urinary lung anf skin
how does antibody deposition trigger inflammation
activation of complement C5a release which functions as a chemoattractant for PMN
What are Treg markers?
o Membrane: CD4, CD25
o Intracellular: FoxP3
What is the function of PD-1?
Inhibits activation of effectors cells by binding to PD-L1
Mechanisms of immune evasion
o Downregulation of MHC class 1
o Induction of immune cell tolerance
o Activate PD-L1
o Loss of tumor antigen expression
o T cell exclusion from TME
o Masking of tumor antigens
how do tumors modulate/evade the immune system through Immune Checkpoint Activation
Tumors can upregulate immune checkpoint molecules, such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), to inhibit T cell activation and function.
Interaction between PD-L1 on tumor cells and PD-1 on T cells leads to T cell exhaustion and dysfunction, inhibiting anti-tumor immune responses.
how do tumors modulate/evade the immune system through Recruitment of Immunosuppressive Cells:
Tumors recruit immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), to the tumor microenvironment.
Tregs suppress effector T cell responses and promote immune tolerance, while MDSCs inhibit T cell activation and proliferation.
TAMs exhibit an M2-like phenotype and promote immunosuppression, angiogenesis, and tissue remodeling.
how do tumors modulate/evade the immune system through Production of Immunosuppressive Cytokines:
Tumors secrete immunosuppressive cytokines, such as interleukin-10 (IL-10), transforming growth factor-beta (TGF-β), and vascular endothelial growth factor (VEGF), to suppress anti-tumor immune responses.
IL-10 and TGF-β inhibit effector T cell function and promote Treg differentiation, while VEGF contributes to the formation of an immunosuppressive tumor microenvironment.
how do tumors modulate/evade the immune system through Induction of T Cell Anergy and Exhaustion:
Tumors can induce T cell anergy and exhaustion through chronic antigen exposure and signaling.
Anergic T cells lose their ability to respond to antigen stimulation, while exhausted T cells display reduced cytokine production and cytotoxicity.
how do tumors modulate/evade the immune system through Loss or Downregulation of Tumor Antigens
Tumors may downregulate or lose expression of tumor antigens, making them less recognizable by the immune system.
Loss of tumor antigens reduces T cell recognition and targeting of tumor cells.
how do tumors modulate/evade the immune system through Induction of Metabolic Reprogramming:
Tumors undergo metabolic reprogramming, change from kreb cycle to glycolysis to use glucose to make pyruvate and lactate due to hypoxia.
Tumors can consume nutrients such as glucose and amino acids, limiting the availability of these nutrients for immune cells.
Metabolites produced by tumors, such as lactate and adenosine, inhibit T cell function and promote immunosuppression.
how do tumors modulate the immune system through Extracellular Matrix Remodeling:
Dense ECM structures and increased collagen deposition limit T cell migration and function within the tumor microenvironment.
how do tumors evade the immune system through Induction of Immune Tolerance:
Tumors promote immune tolerance by inducing the expression of immune regulatory molecules, such as indoleamine 2,3-dioxygenase (IDO) and arginase-1 (Arg-1).
These molecules inhibit T cell activation and promote the generation of regulatory T cells.
how do tumors evade the immune system through Escape from NK Cell Recognition
Tumors downregulate the expression of ligands for natural killer (NK) cell activating receptors, such as NKG2D ligands.
Decreased expression of NKG2D ligands prevents NK cell-mediated killing of tumor cells.
how do tumors evade the immune system through Resistance to Apoptosis:
Dysregulation of apoptosis pathways, such as increased expression of anti-apoptotic proteins (e.g., Bcl-2) and decreased expression of pro-apoptotic proteins, contributes to tumor survival.
Extracellular Matrix (ECM) in normal tissue
The ECM is a complex network of proteins, glycoproteins, and polysaccharides that provides structural support to tissues and organs.
Cells interact with the ECM through cell surface receptors, such as integrins, which bind to ECM components like collagen, fibronectin, and laminin.
ECM receptors transmit signals bidirectionally, regulating cell adhesion, migration, proliferation, differentiation, and survival.
Cells within tissues exhibit polarity, with distinct apical and basal surfaces and specialized junctions for cell-cell adhesion.
Stem cell niches provide microenvironments that support self-renewal and differentiation of stem cells.
Role of Cellular adhesion molecules CAMs in a Normal State:
cell - cell: CAMs mediate cell-cell adhesion and form specialized junctions between adjacent cells.
Cadherins, including E-cadherin, N-cadherin, and P-cadherin, are calcium-dependent CAMs that form adherens junctions and maintain tissue integrity.
Adherens junctions provide mechanical strength to tissues, regulate cell polarity, and facilitate cell signaling.
Cell-ECM Adhesion:
CAMs mediate cell-ECM adhesion by binding to ECM components such as collagen, fibronectin, and laminin. Cell adhesion to the ECM activates intracellular signaling pathways, including focal adhesion kinase (FAK), Src, and Rho GTPases, which regulate cytoskeletal dynamics, cell motility, and gene expression.
Integrins are the primary CAMs involved in cell-ECM adhesion and signaling.
cell signaling:
CAMs transmit signals bidirectionally across cell membranes, activating downstream signaling pathways involved in cell growth, differentiation, and survival.
Tissue Morphogenesis and Development:
CAMs regulate cell sorting, tissue patterning, and organogenesis during embryonic development.
- TGF b
- TGF b - immune suppressive, produced by macrophages, DC and T regs, inhibits B cells
- TNFa
- TNFa - Produced by Th1 T cells, Cytotoxic T cells, activated DCs and macrophages; induces NO production by macrophages, induces tumor apoptosis; important pro-inflammatory cytokine
IL-2
- IL-2, 7 - T cell growth, proliferation and survival. induces B cell proliferation. approved by FDA for animals
- IL3
- IL 3 - multi colony stimulating factor- production and differentiation of macrophages, monocytes, granulocyts, and DC
- IL 4
- IL 4, 5, - produced by Th2 CD4 T cells. promote IgG and IgE. inhibits macrophage activation, induces B cell growth and differentiation,
- IL6
- IL 6 - Supports B-cell proliferation and differentiation to plasma cells; proinflammatory, antiapoptotic cytokine that may contribute to tumor development associated with chronic inflammation. Causes upregulation of PD-1 on monocytes that are triggered to produce IL-10
- IL 8
- IL 8 - Chemotactic/activation factor for neutrophils and T cells; induces matrix metalloproteinase-2 activity; plays a role in inflammation and tumor metas- tasis.
- IL 10
- IL 10 - Immunosuppressive cytokine produced by activated DCs, macrophages, and T cells; induces regulatory T cell function; also overexpressed by some tumors and tumor-associated leukocytes
IL 11
- IL 11 - Stimulates proliferation of hematopoietic stem cells; induces megakaryocyte maturation resulting in increased platelet production.
IL 13
- IL 13 - Th2 promoting cytokine, tumor immune surveillance
- IL 15
- IL 15 - T cell growth factor; supports survival of memory CD8+ T cells; promotes NK cell activation and survival and triggers cytotoxic activity.
- IL 17
- IL 17 - Induces proinflammatory response. Role in cancer is currently controversial
- IL 19
- IL 19 - Promotes T-cell differentiation toward the Th2 phenotype.
- IL 21
- IL 21 - Member of IL-2 cytokine family; enhances cytotoxicity and proliferation of CTL and NK cells
- GM-CSF/CSF-1
- GM-CSF - Promotes growth and differentiation of pleuripotent progenitor cells; stimulates growth of cells of the granulocyte, macrophage, and eosinophil lineage.
How to MDSC exert their effects?
myeloid-derived suppressor cells
Suppression of T cells by:
1. Arginase deprivation
2. NOS
3. ROS
Also:
1. Cysteine deprivation
2. Produce TGF-β and IL-10
What is the MOA of mAb on RTKs?
Can bind extracellular site to block or trigger cell signaling cascade → cell lysis via complement or antibody-dependent cell-mediated cytotoxicity
Mechanisms of LOH
o Gene conversion
o Translocation
o Mitotic recombination
Most common LOH?
Mutations in TSGs (ex. p53)
PTCH, BRAF (human melanoma), loss of INK4a (familial cut melanoma)
ways to manipulate myeloid derived suppressor cells
depletion/inhibit proliferation
promote maturation
inhibit recruitment
block interactions
block function
Alterations of CAMs in Cancer Progression:
Loss of E-Cadherin and Adherens Junctions:
- epithelial-mesenchymal transition (EMT) in cancer
- leading to loss of cell-cell adhesion and increased cell motility and invasion.
- associated with increased tumor aggressiveness, invasion, and metastasis.
Upregulation of N-Cadherin (Cadherin Switch):
- N-cadherin-mediated adhesion promotes cell migration, invasion, and metastasis.
Integrin Dysregulation:
- Upregulation of specific integrin subunits promotes cancer cell adhesion to the ECM and facilitates tumor cell dissemination and metastasis.
- Dysregulated integrin signaling promotes tumor angiogenesis, survival in the circulation, and colonization of distant organs.
CAM-Mediated Signaling Pathways:
- Aberrant CAM signaling activates downstream pathways involved in cell proliferation, survival, migration, and invasion.
CAM-mediated signaling promotes cancer cell survival under stress conditions and resistance to apoptosis and cytotoxic therapies.
CAMs as Therapeutic Targets:
CAMs and their associated signaling pathways represent potential targets for cancer therapy.
Cellular migration
- Migration begins with the polarization of the cell, establishing a leading edge (front) and a trailing edge (rear).
- At the leading edge, the cell forms dynamic protrusions called lamellipodia and filopodia. - The leading edge of the migrating cell establishes transient adhesions with the extracellular matrix (ECM) or neighboring cells.
Integrins, a family of cell surface receptors, mediate adhesion to the ECM by binding to specific ECM proteins such as fibronectin, collagen, and laminin. - As the leading edge adheres to the ECM, the cell generates traction forces by contracting its cytoskeleton, pulling itself forward.
- Actin polymerization drives the extension of lamellipodia and filopodia at the leading edge. The Arp2/3 complex nucleates actin polymerization, generating a branched actin network in lamellipodia.
- Myosin motor proteins, particularly myosin II, generate contractile forces that retract the trailing edge of the cell.
- As the leading edge advances, the cell body moves forward by a process known as cytoplasmic streaming.
Cytoplasmic streaming involves the flow of cytoplasmic contents toward the leading edge, driven by actin polymerization and myosin II-mediated contraction. - Integrin-mediated adhesions are dynamic and undergo turnover, allowing the cell to detach from the substrate at the trailing edge and move forward.
Cell-cell adhesions, such as cadherin-mediated adherens junctions, stabilize cell-cell contacts and coordinate collective migration of groups of cells. - Polarity proteins, such as Par complex and Rho GTPases, regulate the distribution of signaling molecules and cytoskeletal components within the cell.
Directional sensing mechanisms, including chemotaxis, haptotaxis, and durotaxis, guide cells toward specific cues in the microenvironment, such as chemical gradients, ECM stiffness, or topographical features. - At the trailing edge, integrin-mediated adhesions disassemble, allowing the cell to detach from the substrate.
Myosin II-mediated contraction generates contractile forces that retract the rear of the cell, completing the migration cycle.
