Final Exam Flashcards
How many types of cancer have been classified
Over 110
What is cancer?
characterized by unregulated cell growth
Due to mutations in DNA
Normal cells lose powerful genetic circuits regulating cell death and cell division
Invasion and spread of cells from the primary site to other sites in the body
It’s a clonally evolving disease
Imhotep
An Egyptian physician in 2625 BC described breast cancer in detail and described it as a distinct disease
Herodus
Greek historian who recorded the story of Atossa, the queen of Persia, who had a bleeding lump in her breast
A Greek slave named Democedes persuaded her to allow him to excise her tumor
The increase in life span has resulted in
an accumulation of mutations in our genome, and as a result an increase in cancer incidence in older people
Risk of Breast cancer
1 in 400 for a 30 year old woman
1 in 9 for a 70 year old woman
How do different types of cancer manifest (characteristics)
cancers of different origins have distinct features
Factors that cause cancer in each tissue is different
Differences in molecular mechanisms involved in different cancers
Treatments are different
Carcinomas
85% of cancers which occur in epithelial cells
Basal cell carcinoma, Ductal carcinoma, squamous cell carcinoma
Sacrcomas
Cancers that occur in the mesoderm
Adenocarcinomas
Cancers originating in the glandular tissue
Normal Cell characteristics
Grow in monolayers Exhibit contact inhibition Cannot grow in low serum media Flat and extended morphology Grow attached to the substrate
Cancer cell characteristics
Grow in piles of cells called foci Do not exhibit contact inhibition Can grow in low serum media Round morphology Exhibit anchorage independence
Neoplasia
New growth, not reversible
Dysplasia
Disordered growth, which is reversible, but often results in neoplasia
Tumor
abnormal growth of cells, can be benign or maignant
Cancer
Malignant neoplasm or tumors that invade nearby tissue
Benign Neoplasms
Small in size
Slow growing
Well-defined borders
Well differentiated
Malignant Neoplasms
Large, Rapid growth Poorly demarcated Poorly differentiated Increased nuclear to cytoplasmic ratios Nuclear hyperchromasia and prominent nucleoli High Mitotic activity
Benign tumor
No evidence of cancer
Do not metastasize
Some can be life-threatening
Malignant tumor
Not encapsulated
Invades and metastasizes to other tissues
Benign and malignant epithelial tissues
Benign-Adenoma, Papilloma
Malignant- Adenocarcinoma, Papillary carcinoma
Mesenchyme Benign and malignant tissues
Benign-Fibroma, Lipoma
Malignant- Sarcoma
Melanocyte benign and malignant tissues
Benign- Nevus
Malignant- Melanoma
Lymphocyte benign and malignant tissues
Benign- N/A
Malignant- Lymphoma
Breast cancer sites of metastasis
Lungs, Liver, bones
Colon cancer sites of metastasis
Liver, Peritoneum, lungs
Kindey cancer sites of metastasis
Lungs, liver, Bones
Lung cancer sites of metastasis
Adrenal gland, liver, lungs
Melanoma cancer sites of metastasis
Lungs, Skin/muscle, liver
Why are malignant tumors life-threatening?
Physical obstruction
Invading other organs and compromise their function
Compete for nutrients and oxygen and produce waste products
In normal tissue, overall cell number in an individual is contributed by
Cell proliferation (cell growth and division) Apoptosis (Programmed cell death) Cell Differentiation (Inactive phase of cell growth)
How can marijuana affect symptoms of cancer?
small studies showed marijuana effectiveness in treating nausea and vomiting
neuropathic pain
less of a need for pain medicine
THC and cannabinoids can slow growth and cause death in certain types of cancer cells
slow growth and reduce spread of some forms of cancer in animal studies
marijuana has not been proven to help control or cure the disease
Why would marijuana be harmful?
Benzo(a)pyrene Benz(a)anthracene Phenols vinyl chlorides Nitrosamides Reactive Oxygen species Possibly posing greater danger to the lungs
Overall goal of cancer therapies
cytostatic and cytotoxic
The best drug is the one that can be used in the lowest dose with minimal side-effects
Therapeutic Index
• Value between minimum effective dose and
maximum tolerated dose
• The larger the value the better the drug
• Many drugs are given at maximum tolerated dose
(MTD)
Function of Chemotherapy
Target DNA, RNA, and Protein to disrupt the cell cycle
Main goal of chemotherapy
Cause DNA Damage and trigger Apoptosis
Side effects of Chemotherapy
Alopecia( loss of hair)
Ulcers
Anemia
Types of clinical trials
Observational
Interventional
Interventional clinical trials
the research subjects are assigned by the investigator to a treatment or other intervention, and their outcomes are measured
Observational studies
those in which individuals are observed and their outcomes are measured by the investigators
Phase I Clinical Trials
researchers test an experimental drug or treatment in a small group of people (20-80) for the first time To evaluate safety, determine a safe dosage range, and identify side effects.
Phase II Clinical trials
larger group of people (100-300) to see if it is effective and to further evaluate its safety
Phase III clinical trials
even larger groups of people (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the experimental drug or treatment to be used safely
Phase IV Clinical Trials
post marketing studies delineate additional information including the drug’s risks, benefits, and optimal use
A Gene
a specific stretch of DNA that programs the amino acid sequence of a polypeptide.
Parts of a Gene
▪ Promoter region
▪ Terminator region
▪ Start codon
▪ Stop codon
Operon
- An operon is a collection of genes all under the control of the same promoter.
- Genes in an operon tend to have related functions
Bases of DNA and RNA
▪ Adenine (A) ▪ Guanine (G) ▪ Thymine (T) ▪ Cytosine (C) Uracil (U) * RNA ONLY
Nucleotide Base Pairing
The functional groups hanging off the base determine which bases pair up: ▪ A only pairs with T. ▪ G can only pair with C
Pure As Gold
CUT Purines to get Pyrimidines
CUT The PIE
Purines
Adenine and Guanine
Pyrimidines
Cytosine
Thymine
Uracil
___ is the genetic material for all organisms
DNA
What is coded in DNA
Genetic information
____ Directs the production of proteins needed for the structure and function of cells
DNA
Intermediate genetic molecule
RNA
Changes to DNA sequence may have
severe consequences for the cell and its progeny
Mutations can occur in our DNA due to
environmental agents and endogenous
processes during metabolism
Cells are equipped with defense mechanisms
against mutations, such as
the detection and repair of DNA damage
Where does transcription occur in eukaryotic cells?
Nucleus
Where does Translation occur in eukaryotic cells
Cytoplasm
Does mRNA processing occur in eukaryotic cells?
Yes
True or false: Eukaryotic cells do not have introns and exons
FALSE
Eukaryotic cells have introns and exons
Features of MRNA in eukaryotic cells
5’ Cap and Poly A Tail
Can one mRNA code for multiple proteins in eukaryotic cells?
No
This only applies in prokaryotic cells (within operons) and this means eukaryotic cells are monocistronic
True or false: Eukaryotic cells have polyribosomes
False
Polyribosomes are only present in prokaryotic cells
True or False: Eukaryotic cells do not have a formyl methionine
True
Formyl methionine is only present in prokaryotic cells
Where does transcription occur in prokaryotic cells?
Cytoplasm
Where does translation occur in prokaryotic cells?
Cytoplasm
True or False: mRNA is processed (with introns and exons) in prokaryotic cells?
False
mRNA processing only occurs in eukaryotic cells
True or false: mRNA in prokaryotic cells do not have a 5’ cap and poly-A Tail
True
In Prokaryotic cells, can one mRNA code for more than one protein?
Yes
This means they are polycistronic
This occurs in operons
Are Eukaryotic cells polycistronic or monocistronic?
Monocistronic:
Only one mRNA can code for each protein
True or False:
Formyl Methionine is present in Prokaryotic cells
True
True or false: Polyribosomes are absent in prokaryotic cells
False
Polyribosomes are present in prokaryotic cells
Promoter region is involved in
regulating the expression of genes
True or False:
A promoter cannot control when and where a gene is expressed?
False
Promoter controls when and where a gene is
expressed
The promoter region interacts with
with proteins that affect the activity of RNA polymerase
Tata Box sequence
TATAAAA
Tata Box
An important regulatory element for most genes
is able to define the direction of transcription and also indicates the DNA strand to be read
transcription factors can bind to the TATA box and recruit RNA polymerase
Where is the tata box located?
near the start site of transcription
Binding of Tata Box-binding protein (TBP) is important for what proces?
initiation of transcription
Response Element (RE)
a short sequence of DNA within a promoter that
is recognized by a specific protein and contributes to the regulation of the gene
Response elements can either be _____ or ______
enhancer elements (EE – enhance transcription) or Inhibitor elements (IE – inhibit transcription)
What are the types of point mutations?
Silent
Missense
Nonsense
Silent Mutation
Does not affect amino acid sequence
Missense Mutations
Amino acid changed; polypeptide altered
Nonsense Mutation
Codon becomes stop codon; polypeptide is incomplete
Nonsense codons or STOP codons
UAA, UAG, UGA – do not
code for amino acids
Frameshift Mutations
Deletions or insertions that result in a shift in the
reading frame, Often result in complete loss of gene function
insertion Mutation
addition of 1 base causes a frame shift
Deletion Mutation
removal of 1 base causes a frame shift
Transition Mutation
- substitution of one purine with another purine
▪ substitution of one pyrimidine with another pyrimidine
Transversion Mutation
substitution of one purine to another pyrimidine T to A/G C to G/A A to T/C G to C/T
Translocation
exchange of part of one chromosome with part of another chromosome
Burkitt’s Lymphoma Translocation
8: 14
Acute Myeloblastic Leukemia (AML) Translocation
8:21
Gene Amplification
a number of natural and artificial processes by which the number of copies of a gene is increased “without a proportional increase in other genes
Elephants have amplified p53 genes and often do not get cancer
Chromothripsis
when fragments of single shattered chromosome are pieced together
3 possible reasons for chromothripsis
▪ Ionizing radiation that leads to chromosome breaks
▪ Telomere dysfunction which may lead to end-end chromosome fusions
▪ Aborted apoptosis such that cells which have initiated DNA fragmentation survive
Biological effects of radiation on living cells results in 3 outcomes:
- Injured or damaged cells repair themselves, no residual damage
- Cells die, being replaced through normal biological processes
- Cells incorrectly repair themselves, resulting in a biophysical change
Ionizing Radiation Types
Includes (α) alpha particles, (β) beta particles and gamma (γ) rays
Function of Ionizing radiation
Convert electrically neutral molecules into ions
▪ Cause radiolysis generating intermediates called reactive oxygen species (ROS)
▪ ROS may react with DNA or with other biomolecules and cause damage
Free Radicals
potent carcinogens because they can
cause oxidation of DNA by oxidizing DNA bases
8-oxoguanine
an oxidized guanine nitrogen base
▪ DNA polymerase mispairs 8-oxyguanine with adenine during replication of leading to G ->T transversion
DNA polymerase mispairs 8-oxyguanine with
adenine during replication of leading to G ->T transversion
UVA Damage mechanism
via free radicle-mediated damage
UVA Damage Process
Water is fragmented generating electron-seeking ROS that cause DNA damage and G → T transversion
cyclobutane pyrimidine dimers causes
a bend in DNA helix and DNA polymerase cannot read the DNA template
cyclobutane pyrimidine dimers causes
a bend in DNA helix and DNA polymerase cannot read the DNA template
TT dimers are often restored but
TC and CC
dimers result in TC→TT and CC → TT
UV damaged skin is eliminated by apoptosis familiar to us as
peeling of the skin after sunburn
Mutations in P53 is important in
initiation of squamous cell and basal cell carcinomas it provides important growth advantage
Mutations in what gene are found in 66% of malignant melanoma
BRAF Gene
Major Mutations identified in skin cancers
(T → A) in gene
First demonstration that chemicals can be used for inducing cancer
Polycyclic aromatic hydrocarbons (PAH’s)
What happens in Polycyclic aromatic hydrocarbons (PAH’s)
Additional rings and/or methyl groups in the bay region
convert inactive phenanthrene into active carcinogen
DMBA (7,12-dimethyl benz(a)anthracene)
one of the most potent carcinogens
Benzo(a)pyrene (BP)
well known carcinogen in cigarette smoke
CYP1A1
metabolizes BP in BP diol epoxides
• Cause G → T transversions
Aromatic Amines in cancer
Heterocyclic amines (HCA’s)
• About 20 HCA’s have been identified
• Are carcinogens produced by cooking meat formed from heated amino acids and proteins
Asbestos
a group of fibrous silicate minerals that was
used extensively in building materials.
• its association with several diseases of the lung,
including lung cancer and mesothelioma
Erionite
a fibrous zeolite mineral formed from volcanic
rock
_______ is an important line of defense against mutations caused by
radiation and endogenous mechanisms
DNA Repair
Mutations not fixed by DNA Repair Mechanisms
can contribute to carcinogenesis
5 types of DNA Repair Systems
- One-step repair
- Nucleotide excision repair (NER)
- Base excision repair
- Mismatch repair
- Recombinational repair
One-Step Repair
Direct reversal of DNA damage
•N-methylnitrosourea
(Nitrosamines and
Nitrosamides)
damages guanine by adding alkyl group
Repaired with One-Step Repair
DNA alkyltransferase enzyme
directly removes alkyl group from O6 atom of guanine and adds to itself and gets inactivated
One-Step Repair mechanism
Nucleotide-Excision Repair (NER)
Specific for helix distorting lesions such as pyrimidine dimers caused by UVB
• Cuts out 24-32 bases of one strand with the help of
exonucleases and DNA polymerase fills the gap
Disease associated with Nucelotide Excision Repair (NER) mutation
Xeroderma pigmentosum (XP)
Two subpathways of NER
Global Genome NER
Transcription-Coupled-Repair
Global Genome NER
surveys genome for helix distortion
Transcription Coulpled Repair
surveys damage that interferes
with transcription
(NER Subpathway)
Mismatch Repair
Corrects errors that have escaped editing by polymerase and also repairs insertion and deletion mutations (frameshift)
Recognition of mismatch is carried out by what proteins?
MutS homolog 2 (hMSH2/3)
Mutations in hMSH2/3 or hMLH1 or hPMS1/2 causes
HNPCC
Hereditary non-polyposis colorectal cancer (HNPCC)
most common cancer syndrome in humans
Steps of Mismatch Repair
MutL homolog 1/ homolog 1, mismatch repair system
(hMLH1/hPMS1) and hMLH1/hPMS2 are recruited
• Newly synthesized strand with mutation is identified
• Endonucleases and exonucleases remove bases around and including mismatch
• DNA polymerase synthesizes new strand
HNPCC Characteristics
- HNPCC – Lynch syndrome
- It is a autosomal dominant (AD) mutation disease
- if you inherit the abnormal gene from only one parent, you can get the disease.
- AD mutation in mismatch repair leading to microsatellite instability.
- 80% progress to cancer.
- Cancer of proximal colon
- Increases your risk of other cancers
Recombination Repair Types
Homologous Recombination
Non-Homologous End Joining
Homologous recombination depends on
The presence of Sister Chromatids
Non-Homolgous-End-Joining
Does not depend of presence of sister chromatids and can lead to frame shift mutation and chromosomal translocation
A double-Strand break activates
ataxia telangiectasis mutated (ATM) kinase
The RAD50/MRE11/NBS1 complex uses its 5’ → 3’
exonuclease activity to
create single-stranded 3’ ends
Breast cancer type1/2 (BRCA1/2) aids in
the nuclear transport of RAD51
RAD52 facilitates
RAD51 binding to these exposed ends to form a nucleoprotein filament
RAD51 can
exchange a homologous sequence from a single strand within a double-strand molecule with a single-stranded sequence
Resolvace restores the junctions formed as a result of homologous recombination, called
Holliday Junctions
Alkylating drugs and platinum based drugs
- They have similar mode of action
* They form DNA adducts by covalent bonds via an alkyl group
Chlorambucil
is a member of nitrogen mustard family of
drugs
Alkylating drugs and platinum based drugs Target
N7 position of guanine forming intra-strand and inter-strand cross-linking preventing the separation of DNA strands and interfere with replication
Cyclophosphamide
requires metabolic activation within the body
-Oxidases in liver produce an aldehyde form that
decomposes to yield an active form called phosphoramide mustard
Cisplatin and Carboplatin
are platinum-based drugs that form covalent bonds via platinum atom
Cisplatin and Carboplatin method
The molecule binds to N7 position of guanine and adenine in its DNA target
• The GG, AG and GXG adducts comprise over 90% of the total resulting in apoptosis
Pros and Cons of Cisplatin and Carboplatin
Works well with ovarian cancer but has irreversible kidney damage hence carboplatin
Antimetabolites
are structurally similar to nitrogen bases of DNA and inhibit role and nucleic acid synthesis
Examples of Metabolites
Fluorodeoxyuridylate (F-dUMP) and Methotrexate
F-dUMP competes with
dUMP for the catalytic site of thymidylate synthase , the enzyme that produces thymidylate (dTMP), inactivating the enzyme through covalent modification
Methotrexate is a competitive inhibitor of
dihydrofolate reductase (DHFR) required in thymidylate synthase reaction
Doxorubicin
a fungal anthracycline antibiotic that inhibits topoisomerase II enzyme
Topoisomerase II Enzyme
releases torsional stress during DNA replication, by trapping single-strand and double strand DNA intermediates
Uses and Side Effects of Doxorubicin
- Cardiac damage is its most sever side effect
* Used for treating solid tumors (breast or lung)
Mechanisms of drug resistance
Increase the efflux of drugs
• A family of ATP-dependent transporters that are involved in the movement of nutrients and other molecules across membrane
The multiple-drug resistance gene (MDR1) codes for
P-glycoprotein (P-gp) which can bind to drugs such as doxorubicin, vinblastin and taxol and release the drug extracellularly
Transcription Factors
proteins that bind to gene promoters and regulate transcription
Transcription factors contain
a set of independent protein modules or domains, each having a specific role important for the function
of transcription factors.
Transcription Factor Domains
▪ DNA-binding domains
▪ Transcriptional activation domains
▪ Dimerization domains
▪ Ligand-binding domains
Transcriptional activation domains
function by binding to other components of the transcriptional apparatus in order to induce transcription by RNA polymerase
Dimerization domains
Some transcription factors work in pairs (“dimer”) and require a dimerization domain which facilitates protein-protein interactions between the two molecules
Ligand-binding domains
Some transcription factors only function upon binding of a ligand and therefore require a ligand-binding domain.
The activity of a transcription factor can be
regulated by several means
▪ Synthesis /localization in particular cell types only
▪ Covalent modification such as phosphorylation
▪ Interaction with partner proteins
▪ Ligand binding
AP-1 is itself activated in response to
specific signals such as growth factors, ROS, and radiation
AP-1 binds
either to the 12-0-tetradecanoylphorbol13-acetate (TPA) response element or the cAMP response element in the promoter region of their target genes
That interaction controls the processes of growth, differentiation, and death, and plays a role in carcinogenesis
The AP-1 transcription factor is actually composed of
two components and can be produced by dimers of proteins from the Jun (Jun, Jun B and Jun D) and Fos families (Fos, Fos B, FRAl, and FRA2)
Steroid hormones are lipid-soluble signaling
molecules that exert their effects by regulating
the transcription of sets of genes via specific receptors.
Can result in self-sufficiency growth signals
DNA is wrapped around proteins called
Histones
Histones are basic proteins with
A positive charge
which allows them to combine with negative charged DNA
Function of Histones
package and protect DNA
The simplest or primary level of organization of chromatin is
the wrapping of DNA around a protein “spool” and is referred to as the “beads on a string” array.
What does beads on a string mean?
The beads represent the nucleosome, which contains 147 base pairs (bp) of DNA wrapped 1.7 times around a core of histone proteins
The histone core is an octomer of histones containing
two copies of histones H2A, H2B, H3, and H4.
Each histone contains domains for
▪ histone-histone
▪ histone-DNA interactions
▪ NH2 -terminal lysine-rich
▪ COOH-terminal “tail” domains
Epigenetics refers to
heritable changes that is encoded by modifications of the genome and chromatin components.
Can Epigenetic changes cause a change in nucleotide sequence?
NO
They do not cause a change in the nucleotide
sequence of the DNA and therefore are not
mutations.
Epigenetic changes are influenced by
how DNA gets wraparound histones making genes readable or unreadable
Epigenetics instructs cells on
how to differentiate and develop
Two types of epigenetic mechanisms
▪ Histone modifications
▪ DNA methylation
Covalent posttranslational modifications (PTM) to histone proteins
can alter gene expression by altering chromatin structure
Histone proteins are subject to
diverse post-translational modifications
▪ Acetylation (Add Acetyl Group)
▪ Methylation (Add Methyl Group)
▪ Phosphorylation (Add Phosphorus Group)
▪ Ubiquination (Tag for degredation) via proteasome
Acetylation plays an important role in the following
▪ Transcription ▪ DNA replication and repair ▪ Cell cycle progression ▪ Differentiation ▪ Gene silencing
HAT
Histone Acetyltransferase
HDAC
Histone Deacetylases
DNA methyltransferases (DNMTs)
mediate the covalent addition of a methyl group
DNA methylation
the addition of a methyl group to position 5 of cytosine.
Methylation or demethylation can
turn gene on or off
Causing repression or activation of genes
Histone Phosphorylation
a transient histone modification induced by
extracellular signals such as DNA damage
Histone Phosphorylation is associated with various biological processes
▪ DNA damage response
▪ DNA repair
▪ Apoptosis
▪ Chromatin compaction
The 3’ end of the parental chromosomal DNA is not
replicated and thus chromosomes
progressively erode during each round of replication
When the chromosomes reach a threshold length
cells enter a stable and irreversible state of growth
arrest called cellular senescence
If cells bypass this stage because of mutation and
telomeres become critically short, chromosomal
instability results and apoptosis is induced
Cellular scenscence
Telomeres are composed of
several thousand repeats of the sequence TTAGGG bound by a set of associated proteins called the shelterin complex, which functions to control telomere length and protect the
chromosomal ends.
Telomeres shorten
by 100-200 bases with each round of DNA replication owing to the limits of DNA polymerases during DNA
Telomerase, a ribonucleoprotein containing human
telomerase reverse transcriptase activity (hTERT) and a human telomerase RNA (hTR)
maintain telomere length in certain cell types, such as stem cells
The hTERT contains
11 complementary base pairs to the
TTAGGG repeats and acts as a template for the reverse transcriptase to add new repeats to telomeric DNA on the 3’ ends of chromosomes.
Several oncogenes have been demonstrated
to regulate the expression of
Telomerase
the transcription factor c-myc ( an oncogene) increases the expression of
the hTERT gene via specific response elements in
the promoter region
miRNA (Micro RNA)
• ~50-70 nucleotides long • Endogenous RNA • They are part of our genome • Coded by our own genes • Post-transcriptional regulation of genes • Conserved • Production and processing involves nucleus and cytoplasm • Full complementarity is not needed
siRNA
- Long double stranded RNA
- Exogenous RNA
- Not part of our genes
- Viral origin or from transposons
- Post-transcriptional regulation of genes
- Not conserved
- Processing happens in cytoplasm
- Requires full complementarity
MicroRNAs (miRNAs)
small, non-proteincoding RNAs (18-25 nucleotides in length) that regulate the expression of mRNAs
Each miRNA may be able to
repress hundreds of gene targets post-transcriptionally
miRNAs (micro RNAs) are products of
dsRNAs encoded in genes of our genome
MiRNA do not require full complementarity to
bind with target mRNA, e.g. one type of miRNA may
regulate many genes, as well as one gene can be
regulated by several miRNAs
siRNAs (short interfering RNAs)
products of double-stranded RNAs, which can have viral origin
Silence genes by the same mechanism as miRNA (Micro RNA)
MicroRNA (miRNA) Processing
▪ After they are transcribed by RNA polymerase II from intergenic regions or from regions that code for introns, the primary transcript is processed by ribonucleases Drosha and DGCR8 in the nucleus.
▪ This processing produces pre-miRNAs, hairpin-shaped intermediates of 70-100 nucleotides.
▪ Exportin-5 transports pre-miRNAs into the
cytosplasm where they are further processed by
ribonuclease Dicer into a double-stranded miRNA.
▪ The strands separate and a mature single-stranded molecule joins a RNA-induced silencing complex (RISC).
MicroRNA (miRNA) Repression
▪ The miRNA hybridizes perfectly to the 3’
untranslated region (UTR) of their target mRNA.
The formation of this complex in the RISC leads to mRNA cleavage and subsequent degradation.
▪ Alternatively, miRNAs may bind to imperfect
complementary sites in the 3’ UTR of their target
mRNAs. The formation of this complex in the
RISC blocks translation.
Net Result of miRNA
a decrease in the amount of the protein encoded by the gene from which the mRNA has been transcribed
Inhibitors of histone deacetylases
Several classes of drugs that bind to the catalytic site of HDACs and block the binding to their substrates (acetylated lysines of histone proteins) are used
▪ Short-chain fatty acids such as sodium n-butyrate;
hydroxamic acids such as SAHA; cyclic peptides such as romidepsin (formerly FK-228); and benzamide derivatives such as entinostat.
sodium n-hydroxamic acids SAHA (vorinostat; Zolinza™)
have been approved by the FDA for use in the clinic for Cutaneous T cell lymphoma
Cell growth is regulated by many external and internal signals and involves the following steps
- Extracellular growth factors must bind to specific receptors
- Signals must be transduced across the membrane and into cell
- Signal must be conducted through cytoplasm
- Signal must reach nucleus
- Genes involved cell replication are transcribed
- Proteins involved in cells replication are translated
- Translated proteins must interact with other proteins resulting in cell division
Four types of proteins involved in transduction
of growth factor signals
▪ Growth factors
▪ Growth factor receptors
▪ Intracellular signal transducers
▪ Nuclear transcription factors (regulate gene expression)
Kinases
are enzymes that catalyze the transfer of gamma phosphate group from ATP/GTP to hydroxyl groups on a specific amino acid in a target group
Serine/threonine kinases
phosphorylate serine and threonine residues in target proteins
Tyrosine kinases
phosphorylate tyrosine residues in target proteins
Phosphatase
is an enzyme that removes phosphate group from a specific amino acid in a target group
The addition / removal of the phosphate group
may
▪ Serve as a recognition site for new protein-protein interactions ▪ May cause conformational change ▪ Result in the activation ▪ Inactivation of an enzyme activity
EGF binds to domains i and iii and then
Causes conformational change in receptor and results in domain iii binding to EGF
▪ This exposes domain ii and helps it to form a dimer with another EGF bound to EGFR
Autophosphorylation
Dimerization enables the kinase domains of one receptor to phosphorylate the other receptor and vice versa
▪ The change in receptor conformation permits access to ATP and substrate to the catalytic kinase domain
Autophosphorylation is crucial for
recruitment of cytoplasmic proteins
At this step signals from outside the cell have been
transduced to inside the cell
Mechanisms of Termination include
• Additional phosphorylation and conformational change
• Dephosphorylation of tyrosine residues by tyrosine
phosphatase
• Binding of negative regulators (ERRF1 -ERBB receptor
feedback inhibitor 1 or RALT)
• Receptor endocytosis and degradation
Autophosphorylation exposes
domains in intracellular fragment of EGFR which act as docking sites for recruitment of other specific intracellular proteins
Grb2 (Growth factor receptor-bound protein 2)
an intracellular protein that contains one SH2 and two SH3 domains
Grb2 binds to EGFR with its SH2 domain and two SH3 domains
interact with exchange protein son of sevenless (SOS)
SOS facilitates the activation of
the pivotal intracellular transducer RAS
RAS Activation
They are “star players” in regulating cell growth
▪ They are responsible for integrating growth factor signals from membrane to nucleus
RAS family has 3 members
N-,H- and K-RAS. These are most common oncogenes
Ras and GTP
RAS are are GTP-binding proteins and are activated when bound to GTP and inactivated when bound to GDP
SOS catalyzes
the exchange of GDP→ GTP and GTPase activating protein (GAPs) catalyzes the hydrolysis of GTP → GDP
Steps for RAS Activation
▪ RAS-GTP binds to and contributes to the activation of serine/threonine kinase RAF
▪ Activated RAF (rapidly accelerated fibrosarcoma ) phosphorylates MEK
▪ Activated MEK phosphorylates another family of serine/threonine kinase mitogen-activated protein kinase (MAPK Mitogen-Activated Protein Kinase or
ERK -extracellular signal–regulated kinases
▪ MAPK affects the activity of transcription factors via phosphorylation
▪ RAF and MEK are cytoplasmic links between RAS and MAPK
group of proteins that bind to DNA and regulate
the expression of genes involved in growth, differentiation and death
Transcription Factors
Examples of Transcription Factors
AP-1 family (Jun and Fos) and Myc family (Myc, Max,
Mad and Mxi) of transcription factors which control genes, involved in growth, differentiation and death
AP-1 gene transcription factor is target of
MAP-Kinase
AP-1 gene products activate
cyclin D genes which are critical regulators of cell cycle
Knowledge of EGFR signaling and other pathways have led to development of
many therapeutics targeting individual components
Herceptin (trastuzumab)
a monoclonal antibody that binds the extracellular domains of ErB2 receptor with high affinity
Herceptin functions through
a combination of mechanisms including enhanced receptor degradation, inhibition of angiogenesis, cell proliferation and recruitment of immune cells, resulting in antibody-dependent cellular cytotoxicity
Erbitux
another drug has 5 mechanisms of action
Cell cycle arrest of cancer cell and inhibits proliferation,
prevents metastasis, inhibits angiogenesis, antibodydependent cellular cytotoxicity, and inhibits DNA repair mechanisms
Iressa and Tarceva (erlotinib)
small molecule kinase inhibitors that are directed against tyrosine kinase activity of EGFR family members
The Reagent ISis5132 (Phase 1 clinical trials) uses
sense oligonucleotide against Raf mRNA resulting in
formation of RNA hybrid
The RNA hybrids are most likely targeted for degradation or block translation and result in reduction of Raf protein
Nexavar (Sorafenib)
approved for renal cancer is a serine/threonine kinase inhibitor
Normal cellular genes that can be converted to
oncogenes are referred to as
proto-oncogenes
Proto-oncogenes are
Not “bad genes”
These are normal genes that make essential contributions to the regulation of cell proliferation and survival
RAS proto-oncogene normal gene produces
normal protein that controls proliferation
Mutations in RAS proto-oncogene can
transform it into RAS oncogene which can cause cancers due to uncontrolled proliferation
Oncogenes
are genes whose presence can contribute to uncontrolled cell proliferation and cancer
What type of inheritance pattern do Oncogenes display
Dominant mutations
How many oncogenes have been discovered?
More than 100 oncogenes have been identified and
proteins they produce fall into different categories
What function do oncogenes usually have?
They are usually growth factor receptors, growth
factors, enzymes that catalyze phosphorylation, and
transcription factors
Most proteins produced by oncogenes are
components of signaling pathways that
promote cell proliferation survival
Mechanism of oncogenes
producing abnormal amounts they interfere
with the normal signaling mechanisms and foster
excessive proliferation and survival of cancer cells
Point mutation are mutations in
A single nucelotide
Single mutation in RAS proto-oncogene results in
RASoncogene that produces mutated Ras protein in which single amino acid is converted from glycine to valine
RAS oncogene point mutations have been observed in
many types of cancers such as bladder, lung, colon and pancreas
What can convert proto-oncogenes to oncogenes
Point Mutations
Gene Amplification
Chromosomal Translocation
Gene amplification
creating multiple duplicate copies of the same gene
Results in abnormal amounts of protein
chromosomes have a distinct abnormal appearance when stained
What is the most common oncogene
MYC Gene
Neuroblastoma with extensively amplified MYC gene
are more likely to invade and metastasize and lower survival rates
Gene Amplification in breast cancers
also seen in ERBBs (HER2) gene in 25% of breast cancers
Chromosomal translocation
Process in which a piece of one chromosome is broken off and moved to another chromosome
Philadelphia chromosome
abnormal version of chromosome 22 in 90% of all cases of chronic myelogenous leukemia
Translocation of ABL gene (tyrosine kinase) in chromosome 9 to BCR gene (Serine/threonine kinase) on chromosome 22 and results in
abnormal BCR-ABL fusion gene and fusion protein
Autophagy
Self-eating
-Involves degradation of proteins and lipids in cells
through autophagosomes
-Also involves degradation of organelles (mitophagy,
reticulophagy, nucleophagy, lipophagy, xenophagy)
a mechanism used for recycling nutrient subunits
in cells and waste clearence
▪ Helps in maintaining cellular homeostasis inside a
cell
▪ Also helps in cell death
▪ Plays a critical role in health and disease in our body
Autophagy
Process of autophagy
▪ Starts with ER and formation of preautophagosome
▪ Matures into autophagosomes and fuses with lysosomes in cells
Activation signals for autophagy are triggered by
starvation
cellular stress
infection
Proteins involved in autophagy
LC3, Beclin-1, and ULK
Autophagy is inhibited by
▪ Cell growth and proliferation
▪ Excess cellular energy
▪ mTOR and PI3K complex
Necrosis
a passive form of cell death that happens in injured tissue or organs
Is ATP needed for necrosis?
No, it is passive (requires no ATP)
Characterisitics of Necrosis
▪ It involves group of cells ▪ Involves extrinsic stimuli ▪ Increase in cell size ▪ Cell membrane damage is involved ▪ Involves local inflammatory responses ▪ No cellular markers -Passive
Apoptosis
▪ Active cell death that happens in normal cells and diseased organs
Characterisitics of Apoptosis (#1)
Active cell death in normal cells and diseased organs ▪ Need ATP for this process ▪ Involves a single cell ▪ Programmed cell death ▪ Decrease in cell size ▪ Cell membrane is intact ▪ Does not trigger local inflammatory responses or absent ▪ Involves annexin V
True or false: Apoptosis causes the cell to explode and causes inflammation
FALSE:
It is organized, neat, and tidy, leaving behind little
evidence of the preexisting cell
A special group of proteases called ________ play
main role in apoptosis.
Caspases
Characteristics of Apoptosis (#2)
▪ Cell shrinking ▪ Cytochrome C release ▪ Inversion of phosphatidyl serine from inside cell to outside ▪ Membrane blobbing and budding ▪ Chromatin condensation ▪ Precise fragmentation
Caspases
They are cysteine-rich aspartate proteases
(caspases)
▪ They are proteases that recognize and cleave
at aspartate residues.
How many mammalian caspases have been identified?
13
How are caspases synthesized?
Synthesized as inactive enzymes (procaspase)
which when cleaved at aspartate residues results in activation of enzymes (caspase).
caspases participate in a cascade of activation, activating
downstream caspases and amplifying the signals
Cells may be induced to undergo apoptosis by
Extracellular signals
Internal or chemical signals
Extracellular Signals in apoptosis
• Death factors can be soluble – TNF tumor necrosis
factor.
• Membrane bound – Fas ligand bound to neighbouring cells or certain immune cells.
Internal signals in apoptosis
DNA damage or oxidative damage (ionizing radiation- reactive oxygen species)
Cell stress signals/DNA damage active ATM/ ATR kinases which in turn activate
Chk1/Casein Kinase II
CHk1/Casein Kinase II disrupt
p53-MDM2 complex, which activates P53 protein
P53 activates BAX protein, which in turn
Activates and binds to BID protein
The conformational change in BAX causes
BAX to insert into the outer mitochondrial membrane and oligomerize (6-8 molecules)
In apoptosis, the mitochondria releases
Cytochrome C and Procaspase 9 into the cytoplasm
In apoptosis, Cytochrome C is released, what happens
Procaspase 9 is released with cytochrome C and binds with APAF-1 to form the apoptosome
Caspase aggregation leads to
the activation of procaspase 9 which in turn triggers caspase cascade activating caspase 3
Caspase 3 cleaves
target proteins and causes apoptosis of cell
Anti-Apoptotic Proteins
BCL-2
BCL-X
BCL-W
BOO
Pro-Apoptotic Proteins
BAX BAD BID BAK NOXA PUMA
Death signals, TNF (tumor necrosis factor) and Fas activate
their death receptors TNF receptor and Fas receptor
Binding of death receptors (like TNF and Fas) cause
changes in shape and oligomerization of receptors
Adaptor proteins ___________ and _________ recognize the activated receptors and lead to the aggregation of ___________.
TRADD (TNF receptor associated death domain)
FADD (Fas associated death domain)
Procaspase 8
Procaspase 8 aggregation leads to
the activation of caspase 8
Caspase 8 initiates
a caspase cascade, proteolysis and apoptosis
Caspase cascade activates
caspase 3 which causes proteolysis of target proteins
Both the intrinsic and extrinsic pathways of apoptosis activate _______ at the end
Caspase 3
Caspase 3 is
an important mediator of apoptosis as it moves the cell into execution phase of apoptosis
Caspase 3 causes
proteolysis of different target proteins
Target proteins of Caspase 3
Nuclear Lamins- allowing for nuclear membrane shrinkage
Cytoskeletal proteins- such as actin filaments affecting cell structure
Activation of DNAse- resulting in cleavage of DNA
Regulation of the Intrinsic Pathway of Apoptosis
Inhibitors of apoptosis proteins (IAP) regulates apoptosis by binding to and inhibiting the activity of caspase-3 and caspase-7
Smac (Second mitochondria-derived activator) / DIABLO
is released from mitochondria eliminates the inhibition by IAP’s
True or false:
The intrinsic and extrinsic pathways of apoptosis are completely separate and do not interact with each other
False:
There is a cross-talk between extrinsic and intrinsic pathways
Caspase 8
a key regulator of extrinsic pathway also cleaves
and activates Bid in intrinsic pathway
Where is another point in which the intrinsic and extrinsic pathways of apoptosis coincide or converge?
at the activation of caspase 3 which causes proteolysis
Mutations in p53 genes
provide the cancer cells with a survival advantage by disrupting apoptosis is common in lymphomas
In addition mutations in upstream regulators p53 such as
ATM and chk2 are also common
Chromosomal translocation of anti-apoptotic protein Bcl2 is observed by
Many B-Cell Lymphomas
What is the consequence of the chromosomal translocation of BLC-2
over-expression of Bcl-2 protein leads to insufficient
apoptotic turnover and accumulation of B-cell
Mutations in BAX and BID genes that code for anti-apoptotic proteins are
mutated in 50% of colon tumors
A successful chrmotherapy will be one that
triggers apoptosis
Many chemotherapeutic agents
trigger DNA mutations or DNA damage thus activating intrinsic apoptotic pathway
many tumors have
defective apoptotic pathways
and are inherently resistant to chemotherapy
Mutations in p53 which is common in cancers inherently contributes to
drug resistance
Upregulation of anti-apoptotic members of Bcl-2 family
and the downregulation of the pro-apoptotic members
increase the resistance to chemotherapy
Totipotent (AKA Omnipotent) Stem Cells
▪ These cells are produced from the fusion of an egg and sperm cell.
▪ Cells produced by the first few divisions of the fertilized egg are also totipotent.
▪ Such cells can construct a complete, viable, organism.
▪ These cells can differentiate into embryonic (inner cell mass) and extraembryonic cell types (placenta
and other supporting tissue).
Pluripotent stem cells
▪ Are the descendants of totipotent cells.
▪ Can differentiate into nearly all cells.
▪ Cells derived from any of the three germ layers.
▪ Can become any of the 200 different cell types in the
body (under right conditions)
Multipotent stem cells
can differentiate into a number of cells, but only those of a closely related family of cells
Hematopoietic stem cells
Hematopoietic stem cells
Oligopotent stem cells
can differentiate into only a few cells, such as lymphoid or myeloid stem cells.
▪ Lymphoid stem cells and myeloid stem cells
Unipotent stem cells
can produce only one cell type, their own.
▪ Epidermal stem cells in our skin give rise to epithelial skin cells
What are stem cells?
▪ Cells of variable potency that can self renew. ▪ Normally found in our body. ▪ Help in organ maintenance. ▪ Help in organ repair. ▪ Somewhat committed
Characteristics of stem cells
▪ They have ability to migrate to other tissues.
▪ High level of telomerase activity.
▪ Stem cells must maintain a balance between self-renewal and differentiation
When not dividing, stem cells are in what type of phase?
Quiescent/ G0 Phase
Obligator Asymmetric Replication
When a stem cells divides it becomes another stem
cell and a progenitor cell
Progenitor cell in stem cells
will divide rapidly, and their progeny divide and differentiate into a specific cell type
True or False:
Differentiated cells remain in the cell cycle
FALSE: Differentiated cells withdraw from the cell cycle
A block in cell differentiation results in
a higher net number of cells and therefore is a mechanism for tumor formation
When do normal stem cells multiply?
only when you need to
Stem cells are kept
Quiescent by the environment
On stem cell activation by environmental input
the niche cells induce them to divide into a stem cell and progenitor cell
▪ They do this because of their signaling
IPS Cells
Pluripotent stem cells that have been programmed to differentiate into a different type of cell than their original type
What are cancer stem cells?
rare cells within a tumor that have the ability to self-renew
What type of cells do cancer stem cells generate
Can give rise to phenotypically diverse cancer cells
Cancer stem cells have surface proteins called markers
which are characteristic of the stem cell normally
present in the tissue
Breast Cancer stem cells express what cell surface markers?
CD44+ and CD24 (low)
Colon cancer stem cells over express what surface antigen?
CD 133
WNT pathway
plays a role in stem (cancer) cell self-renewal
Characteristics of the wnt pathway
It's a stimulatory factor 19 wnt genes discovered in mammals Involved in embryonic development Development of the heart involves a destruction complex Involves Beta Catenin
Describe the WNT signaling pathway when WNT is absent
WNT is not present to bind to Frizzled to create a complex with LRP
The degredation complex is present
Beta Catenin is phosphorylated and tagged with ubiquitin
Sent to proteasome
Beta Catenin Degredation
Acts as a transcriptional repressor
Groucho prevents Tcf/Lef from transcribing genes
Describe WNT pathway when WNT is present
WNT is present and binds to Frizzled/LRP complex
LRP is phosphorylated and binds to axin
Degredation complex is disassembled
Beta Catenin is activated
Activated Beta Cateinin can bind to TCF/LEF and forms a complex with Pygopus and BCL9
Allows target genes to progress (ex. c-myc , cyclin-D (Cell cycle))
FAP
Familial Adenomatous poly posiscoli
FAP Characteristics
▪ 85% of colon cancers
▪ Caused by chromosomal instability
▪ There is a mutation in tumor suppressor gene (APC) on chromosome 5q
What protein is important in signaling with FAP (disease)
Beta-Catenin
Describe the process for which a normal colon can develop carcinoma in FAP (Disease)
Normal colon –→ loss of APC gene -→ colon at risk –→K-RAS mutation –→ adenoma ——-→ loss of p53 —–→ carcinoma
True or false:
FAP develops into cancer 100% of the time and involves the entire colon (Pancolonic)
True
Hedgehof signaling pathway
plays a role in stem (cancer) cell self-renewal
Hedgehog proteins are involved in
graded signaling and organ/tissue specific gene induction
Hedgehog proteins are involved in
▪ Embryo development ▪ Tissue self-renewal ▪ Tissue repair ▪ Carcinogenesis ▪ Role in digit formation in mammals ▪ Formation of neural tube, skin, and gut
Hedgehog signaling
important roles in embryonic development, tissue self-renewal, and carcinogenesis
Hedgehog pathway is essential for
pattern formation in many tissues, including the neural tube, skin, and gut
Describe the hedgehog pathway when HH is absent
No HH present
Patched cannot bind to smoothened
SUFU, PKA, and Gli bind together
inhibits target genes from exiting the nucleus
Gli
a Zinc-finger transcription factor
Describe the hedgehog pathway when HH is Present
HH is present Binds to patched Patched and smoothened associate Sufu, PKA, and Gli Dissociate Cyclin Ds, BCL2, VEGF, and SNAIL (effector proteins) Promote target genes exiting the nucleus
The process of stem cell differentiation is dependent on
the expression of a specific subset of genes that defines a particular type of cell
The polycomb group (PcG) of proteins
represses the transcription of specific sets of genes by epigenetic modifications
What proteins are called the guardians of stemness?
polycomb group (PcG) of proteins
What are the Polycomb Group of proteins (PCG)?
HOX FOX SOX PAX POU
Metastasis
the spread of tumor cells from a primary tumor that is not clonal
A primary tumor is composed of
subpopulations of genetically identical cells, called subclones, that differ by mutations obtained through an ongoing evolutionary process
How many metastasized cells survive transport?
Only 1 in 10,000 metastasizing cells survives transport
Steps of Metastasis
Invasion Intravasation Transport Extravasation Metastatic Colonization Angiogenesis
Integrins (Proteins)
are a family of more than 24 heterodimersmade up of a range of α and β subunits that mediate cell-ECM interactions and intracellular signal transduction
Integrin function
cluster in the membrane and affect the cytoskeleton through interaction with actin-binding proteins and specific kinases, such as focal adhesion kinase (FAK)
Intracellular signals mediated at the cytoplasmic
domain of integrins induce
a conformational change in the extracellular domain and thus regulate the affinity of the integrins for their ECM ligands
Integrins also have a role in anoikis
apoptosis triggered in response to lack of ECM ligand
binding and loss of cell adhesion
lntegrins without suitable ECM ligands
recruit caspase-8 to the membrane and trigger apoptosis.
The role of Integrins in motility
is obvious in melanoma cells in which their invasive front edge shows a strong pattern of expression of integrin ανβ3 that is absent in preneoplastic melanomas
Protease
2 common types
- serine proteases
- Matrix metalloproteinases (MMPs)
Some proteases are synthesized by tumors or they
can induce surrounding stromal cells to produce
MMPs (extracellular matrix metalloprotease inducer
EMMPRIN
MMPs play an important role in
Metastasis, including angiogenesis
EMT is characterized by
▪ Loss in cell polarity
▪ Downregulation of epithelial markers such as E cadherin,
▪ Upregulation of mesenchymal proteins such as N-cadherin
▪ Secretion of MMPs.
Extravasation
Tumor cell must attach to the endothelial side of the
blood vessel
▪ Binding of tumor to E-selectin induces tyrosine phosphorylation in endothelial cells and also modifies endothelial cell shape.
▪ stress-activated protein kinase-2 (SAPK2/p38) similar to MAPK is induced in cancer cells is necessary for transendothelial migration
Pre-Metastatic Niche
tumor-type specific factors released from the primary
tumor facilitate changes to the microenviromnent of a
distant and future colonization site before tumor cells
arrive
Exosomes
small vesicles (30-100 nm) that carry protein and nucleic acids
Exosomes are an important means of
intercellular communication between cancer cells and non cancer cells in their microenvironment and further away.
They can carry and transfer DNA, RNA, and protein to
cells to which they can
fuse, and this is referred to as horizontal transfer
Exomes are packaged in
multivesicular bodies that fuse with the cell membrane and are released into the circulation
Pancreatic exosomes
promote the establishment of a pre-metastatic niche for liver metastasis in, a stepwise fashion
Steps of Pancreatic exosomes to set-up the prematastatic niche
▪ First, pancreatic exosomes selectively fuse with Kupffercells in the liver.
▪ Kupffer cells upregulate genes involved in fibrosis,
including TGF-β, in response to macrophage migration
inhibitory factor (MMIF) enriched in the exosomes.
▪ TGF-β then induces the expression of fibronectin in
another liver cell type, stellate cells.
Fibronectin deposits function
recruit bone marrow-derived cells, an important component of the pre-metastatic niche
The altered microenvironment of pancreatic exomes
supports the survival and growth of pancreatic tumor cells
Metastatic Colonization
the establishment of a progressively growing tumor at a distant site
Metastasis suppressor genes
regulate/controls the growth of metastatic cells at secondary sites
What happens in loss of function in normal cells?
increases the metastatic propensity of a cancer cell
NM 23
functions as a nucleoside diphosphate kinase
and a histidine kinase that possesses metastasis inhibitory effects
MKK4 (mitogen-activated protein kinase kinase 4)
a metastasis suppressor gene whose protein product
affects metastatic colonization by inducing apoptosis
miR-335 and miR-126
metastasis suppressor microRNAs
Metalloproteinase inhibitors
▪ These molecules appear to function in several steps of metastasis, including invasion and metastatic colonization
▪ Targeting membrane-bound MMPs by selective therapeutic antibodies
G0 Phase
▪ Is outside the cell division / cell cycle
▪ The cell is in non-diving state
▪ It is dependent on the growth signals for division
G1 Restriction point in the cell cycle
- Part of G1 phase
- Is a control point in cell division
- If a cell crosses this point, it becomes irreversibly committed to progress through cell cycle without the need for growth factors
Cyclins
proteins that have a critical role in cell cycle
Different cyclins for different stages of the cell cycle
True or False:
There is a cyclical change in cyclin concentration during cell division
True
Cyclins coordinate and regulate
the passage of cell through different phases of cell cycle
Cyclins act as
regulatory subunits of cyclindependent kinase (cdks)
Upon binding of cyclin to cdk partner, what happens?
cyclin undergoes a conformational change in the catalytic domain, exposing an active site
Concentration of cyclins during cell division is dependent on
▪ Transcription of cyclin genes
▪ Regulated protein degradation
True or false:
CDK concentration fluctuates throughout the cell cycle, depending on which phase is active at the time.
False:
There is no change in cdk concentration during
cell division
CDKs along with cyclins coordinate and regulate what?
the passage of cell through different phases of cell cycle
CDK proteins are regulated by
binding of cyclins
Cell cycle checkpoints are important in
maintaining the integrity of the genome
Cell cycle checkpoints are connected to
different pathways which sense and induce apoptosis in response to DNA damage
G1 Checkpoint of the cell cycle
arrest of cell cycle in response to DNA damage
G2 Checkpoint of the cell cycle
arrest of cell cycle in response to DNA damage and/or unreplicated DNA to ensure proper completion of S phase
M Checkpoint of the cell cycle
arrest of chromosomal segregation in response to misalignment on the mitotic spindle
Disruption of a cell cycle checkpoint leads to
mutation and carcinogenesis
In cyclin-CDK complexes, p16 binds with cdk 4/6 and interferes with the binding of
cyclin D to cdk4/6
In cyclin-CDK complexes, p21 inhibitor binds to
both cyclin E and cdk 2blocking ATP –binding site, thus disabling kinase activity.
Inhibitors of association with CDK inhibitors are regulated by
ubiquitin-mediated degradation
When CDK is bound to P16 or P21
CDK is inactive (P16 and P21 are inhibitors)
A key substrate of the cyclin D-cdk 4/6 complex is
the
RB Protein
Rb protein serves as
a molecular link for the G1-S phase transition
Rb binds to transcription factor E2F
which is crucial for the expression of genes needed for S phase
Rb protein comprises the A domain and B domain
joined by
a linker region
Histone deacetylase (HDAC) binds to
domain B and E2F binds to domain A
The G2 checkpoint
blocks entry into M phase in cells that have incurred DNA damage in previous phase or have not correctly completed S phase
At the G2 Checkpoint, DNA damage activates
either of two kinases, ATM or ATR
Activated ATM and ATR kinases, in response to DNA Damage
phosphorylate and activate chk1 and chk2 kinases
One Target of ATM/ATR Kinases
cdc25 tyrosine phosphatase that regulate cdk activity by removing inhibitory phosphatase
Activation of G2 checkpoint results in
the inhibition of cdc25 by chk1
There is also a decatenation during G2 checkpoint
involving topoisomerase II which
helps in detangling daughter chromatids after DNA synthesis
If CDC is active/not inhibited
CDC dephosphorylates CDK Protein
CDK Activated
Progress into M Phase of the cell cycle (Mitosis)
If CDC is inactivated by CHK1
No dephosphorylation of CDK
No Progression of cells into M Phase
Prophase (Mitosis)
▪ Characterized by appearance of the chromosomes
▪ Nuclear membrane starts to break down.
▪ Separation of duplicated centrosomes.
▪ Assembly of mitotic checkpoint at the centromeres.
Metaphase (Mitosis)
▪ Chromosomes align on a central plate.
▪ Assembly of microtubules to form mitotic spindles.
▪ Microtubules capture of both centromere regions of a
chromatid pair results in checkpoint silencing.
Anaphase (Mitosis)
▪ Characterized by spindle pulling apart.
▪ Separating sister chromatid pairs
Telophase (Mitosis)
▪ Chromosomes reach their poles.
▪ Re-forming of nuclear membrane.
▪ Chromosome condensation.
▪ Cytokinesis
Mitotic Checkpoint in cell cycle is also known as
The spindle assembly checkpoint
Function of the Mitotic Checkpoint
▪ Ensures chromosomal segregation during mitosis and production of two genetically identical nuclei.
▪ Prevents mis-segregation of chromosomes.
If any of the sister chromatids are not attached to
microtubules at their centromeres during mitosis
they recruit checkpoint proteins that act as inhibitors of anaphase-promoting complex such as securin
When securin is attached to sister chromatids without microtubules
they inhibit enzyme separase
Separase
cleaves the link (cohesin) between sister chromatids and helps their separation during anaphase
Genes encoding cell cycle regulators are
frequently mutated in tumors
Mutations in cell cycle regulators can result in
aberrant regulation of cell cycle, uncontrolled proliferation and carcinogenesis
Some melanomas have been observed to contain
mutations in
CDK4 where p16 inhibitors bind
. This prevents inhibition of cyclin dependent kinases and helps in progression of cell from G1 to S phase
Chromosomal translocations cause over-expression
of ______ in some leukemias
CKD6
DNA amplification of cyclin D and E by gene
amplification occurs in
15% of breast cancers and 20% squamous cell carcinoma
p16 inhibitor deletions have been observed in
Pancreatic Cancers
Abnormal chromosome numbers or aneuploidy
caused by defects in centrosomes, mitotic spindle, or cytokinesis is often observed in many solid tumors.
Over expression of Aurora kinase A gene has
been commonly reported in
94% of invasive ductal breast adenocarcinomas
Flavopiridol acts as
competitive inhibitor of all cdks
Flavopiridol Mechanisms
▪ Its induces cell cycle arrest at G1/S and G2/M phase.
▪ It also regulates gene expression of cyclin D1 and D3
Paclitaxel/taxol
a mitotic inhibitor (Prevents Mitosis)
The Yew tree
contains a mixture of poisons, paclitaxel and taxine B. Taxine B is what causes death in those that eat the leaves/seeds of the plant due to cardiac arrest. Taxol contains the purified paclitaxel
Paclitaxel’s mechanism of action
(at the doses seen with poisoning) is to prevent microtubules from depolymerizing. If the microtubules attached to chromosomes cannot shrink, chromosomes cannot be separated. The cell is ”frozen“ in mitosis. This event causes the failure of the affected cell to pass certain cell cycle check points, and the cell undergoes programmed cell death (Apoptosis)
Tumor suppressor genes
are genes whose loss or inactivation can lead to cancer
Tumor suppressor genes are also involved in
DNA repair mechanisms
Tumor Suppressor Genes are also called
“anti-oncogenes”
The function of tumor suppressor genes is
to inhibit cell proliferation and/or promote cell death
Tumor Suppressor Genes mutation type
loss-of-functionmutations
RB1 Chromosome Location
13q14
RB1 Gene Function
Transcriptional regular of the cell cycle
RB1 Human tumors associated with sporadic mutation
Retinoblastomas , Osteosarcoma
RB1 Associated cancer syndrome
Familial Retinoblastoma
P53 Chromosome Location
17q11
P53 Gene Function
Transcriptional Regulator/ Growth arrest/ apoptosis
P53 Human tumors associated with sporadic mutation
Sarcomas, breast, brain tumors
P53 Associated cancer Syndrome
Li-Fraumeni
BRCA 1 and 2 Human Tumors
Breast/Ovarian Tumors
VHL Gene Function
Regulates proteolysis
VHL HUman Tumors
Hemangiomas, Renal, Pheochromocytoma
VHL Associated Cancer syndrome
Von Hippel Lindau
APC Chromosome Location
5q21
APC Gene Function
Binds and Regulates Beta Catenin Activity
APC Human tumors
Colon Cancer
PTEN Associated Cancer Syndrome
Cowden Syndrome
BZS
Ldd
Describe P13K Activation by RAS
EGF Binds to EGFR Binds to PI3K PTEN binds to PIP 2 and PIP 3 Activates AKT MTOR Leads to Proliferation or Apoptosis This pathway inhibits Apoptosis
Tumor suppressor genes are often recessive in nature because
one intact allele is sufficient to inhibit growth
In tumor suppressor genes, One of the alleles can be mutated due to
any known mechanisms of mutagenesis
The mutated allele in tumor suppressor gene can be transmitted to daughter cells during cell division through any of the following mechanisms of
“Loss of Heterozygosity”
Knudson’s 2-Hit Hypothesis
- Explains the mechanism of tumor suppressor genes
- States that both alleles must be mutated to trigger carcinogenesis
- Explains certain individuals have increased risk of cancer
- They inherit mutated tumor suppressor allele
Tumor suppressor genes arise through a
phenomenon known as
“Loss of Heterozygosity”
Methods for losing heterozygosity
▪ Mitotic nondisjunction
▪ Mitotic recombination
▪ Gene conversion
Methods for losing heterozygosity
▪ Mitotic nondisjunction
▪ Mitotic recombination
▪ Gene conversion
Retinolastoma
Rare type of childhood cancer occurs 1 in 20,000 ▪ There are two forms of disease ▪ Familial form Sporadic Form
Retinolastoma
Rare type of childhood cancer occurs 1 in 20,000 ▪ There are two forms of disease ▪ Familial form Sporadic Form
Familial Retinoblastoma
Rare type of childhood cancer occurs 1 in
20,000
▪ There are two forms of disease
▪ Familial form
Sporadic Retinoblastoma
▪ 60% of cases
▪ The individual acquires two somatic mutations in two
alleles
▪ They usually affect one eye
Sporadic Retinoblastoma
▪ 60% of cases
▪ The individual acquires two somatic mutations in two
alleles
▪ They usually affect one eye
Retinoblastoma (RB) Tumor suppressor gene
▪ First tumor suppressor gene to be isolated and characterized
▪ RB gene produces a protein called Rb protein
Rb protein controls
cell proliferation in the absence of growth factors
In normal nondividing cells, Rb protein controls cell proliferation
binding to E2F transcription factor
E2F transcription factor when bound to Rb protein
cannot activate transcription genes coding for
enzymes and other proteins required for initiating DNA replication
Hence, No Cell division
E2F transcription factor when bound to Rb protein
cannot activate transcription genes coding for
enzymes and other proteins required for initiating DNA replication
Hence, No Cell division
RB Protein in dividing cells that receives growth factor
signals through growth signaling pathways leads
to
production of cyclin-cdk complexes
Cyclin-cdk complexes catalyze
the phosphorylation of Rb protein which results in
release of E2F transcription factor
E2F transcription factor when unbound to Rb protein
can activate transcription genes coding for enzymes and other proteins required for initiating DNA replication
This results in cell division
What happens to Rb when interacting with The viral proteins adenovirus ElA, papilloma virus E7, and SV40 Large T antigen
Inactivates Rb
The ability of both E1A and E7 to degrade RB, uses
the ubiquitin-proteasome system
The biochemical events involved in RB degradation by E7 involves
E7 binds to a ubiquitin-protein ligase (E7-AP) and forms a dimer that subsequently binds to RB.
After E7 binds to a ubiquitin-Protein ligase and forms a dimer that binds to RB,
RB is then ubiquitinated and tagged for recognition by the proteosome for degradation
After E7 binds to a ubiquitin-Protein ligase and forms a dimer that binds to RB,
RB is then ubiquitinated and tagged for recognition by the proteosome for degradation
What is the most important tumor suppressor gene?
P53
2 homolgs of P53
P63 and P73
What is the msot commonly mutated genes in human cancers?
P53
True or False:
P53 is known as the Guardian of Stemness?
False:
It is known as the guardian of the genome because products of P53 are at the heart of tumor suppressive mechanisms
p53 protein plays a very important role in
Protecting cells from the effets of DNA Damage
P53 Regulates How many genes
over 300 different genes promoter regions
P53 Regulates How many genes
over 300 different genes promoter regions
P53 Chromosome Location
Located on chromosome 17 and contains 11 exons that code of 53kDa protein
P53 is a transcription factor containing 4 distinct domains
▪ Amino-terminal transactivation domain
▪ DNA binding domain which binds to DNA
▪ Oligomerization domain (helps in the formation of tetramer by binding to other monomers)
▪ Carboxy-terminal regulatory domain
As p53 can induce inhibition of cell proliferation or apoptosis in a cell, the factors that determine biological outcome depends on
Absence of Myc
Presence of Myc
In the absence of oncogene transcription factor Myc
p53 interacts with transcription factor MIZ-1 which in turn causes the expression of cyclin-cdk inhibitor p21 protein resulting in cell cycle inhibition
In the presence of oncogene transcription factor Myc
Myccompetes with p53 and binds to MIZ-1 thus preventing the expression of p21 genes
p53 interacts with apoptosis stimulating protein of p53 (ASPP) and
triggers apoptosis in the cell
Li-Franmeni syndrome is predominantly characterized by
a germline mutation of the p53 gene and leads to a
predisposition to a wide range of cancers.
Li Fraumeni Characteristics
• It is an autosomal dominant disease, so an affected
individual has a 50% chance of passing the mutation to
each offspring.
• Patients have a 25-fold increased risk of developing
cancer before they are 50 years old compared with the
general population.
• The young age at which individuals develop cancer and the frequent occurrence of multiple primary tumors in individuals are characteristic features of the syndrome.
• The types of cancer seen within families that carry the
mutation include sarcomas, breast cancer, leukemia, and brain tumors.
P53 interaction with Adenovirus ElB, and papilloma virus E6 and SV40 Large T antigen
inactivate p53
The ability of E6 to degrade p53
uses the ubiquitin-proteasome system
The biochemical events involved in p53
degradation by E6 are as follows
6 binds to a ubiquitin-protein ligase (E6-AP) and forms a dimer that subsequently binds to p53
p53 is then ubiquitinated and tagged for
recognition by the proteasome for degradation
Angiogenesis
the process of forming new blood vessels from pre-existing ones by the growth and migration of endothelial cells in a process called “sprouting.”
In adults angiogenesis is reserved for
Wound Healing
Female Reproductive Cycle
The Neovasculature in cancer is different from normal vessels
▪ Leaky
▪ Tortuous and chaotic flow pattern
▪ Allow cells easy access to the circulation
▪ Loose association with pericytes and extracellular matric (ECM)
▪ Lack normal hierarchy artery-arteriole-capillary-venule-vein
▪ Endothelial cells express more of integrins αvβ3 and αvβ5
▪ Pericytes and ECM show specific angiogenic markers (e.g. NG2 and oncofetal fibronectin, respectively
Anti-Angiogenic Factors
Angiostatin Endostatin Prolactin (16kD) P53 Thrombospondin 1,2
Pro-Angiogenic Factors
Vascular Endothelial Growth Factor (VEGF)
Fibroblast Growth Factor (FGF )
Hepatocyte-Derived Growth Factor (HGF)
Epidermal Growth Factor (EGF)
Pro-Angiogenic Factors
Vascular Endothelial Growth Factor (VEGF)
Fibroblast Growth Factor (FGF )
Hepatocyte-Derived Growth Factor (HGF)
Epidermal Growth Factor (EGF)
The VEGF family currently consists of five family
members
VEGF A, B, C, D, E
VEGF A-E transmit their signal via
3 receptor tyrosine kinases
VEGFR 1, 2, 3
The interaction of VEGF-A with its receptor VEGFR-2 is responsible for
the majority of angiogenic effects.
VEGFR-1 acts as a decoy by regulating or inhibiting the amount of
VEGF-A available to VEGFR-2
VEGFR-3 and its ligand VEGF-C play a role in
development of the lymphatic vascular system
Plasminogen can be cleaved by proteinases, such as
matrix metalloproteinases(MMPs), to release the angiogenic inhibitor, angiostatin
Angiostatin binds to
its endothelial cell surface receptors and stops the VEGF-VEGFR
Endostatin is a fragment of collagen XVIII and can be
proteolytically released by elastase and cathepsin
Endostatin blocks
MAPK activation in endothelial cells and also MMPs
Name the Ten Hallmarks of cancer
Susatained Proliferation Signals Tumor-Supporting Inflammation Evading Apoptosis Deregulating cellular energetics/metabolism Genome instability and Mutation Invasion and Metastasis Inducing Angiogenesis Enabling replicative immortality Avoiding immune destruction Evading growth suppressors
