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
How does p53 cause apoptosis?
Activates PUMA, NOX, Bak/Bim -> Apaf1, cytochrome-c ->caspase9 - caspase 3,6,7
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
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)
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
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.
How to treat patients that develop resistance to RAF inhibitor?
MEK inhibitor
Upstream:
1. Farneysl transferase inhibitor
2. Gerangylgeranyl transferase inhibitor
Which pathway can initially suppress growth then become promoter?
TGF B
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
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.
