INTRODUCTION

Cancer

- derived from single cell- have characteristics such as uncontrolled

division and invasion of tissues
- Genetic and epigenetic abnormalities due to change in DNA sequence

Neoplasia

- uncontrolled cell division growth and differentiation

- Benign or malignant
- Both need huge amount of nutrients

Benign

- not cancer, only grow locally and cannot spread from their tissue
- localized, clear margins, non-invasive, slow growing, well-differentiated

Malignant

- cancer, invade surrounding tissue, enter blood vessels and metastasize

to different tissues (secondary tumors)
- block blood vessels glands and lungs, rapid growth, invasion without
clear margins, decreased cell adhesion, angiogenesis, uncontrolled cell
division, proteolytic enzymes, aneuploidy (total #of chromosomes
excess 46)

Tumor development stages

- Hyperplasia- division in uncontrolled manner, normal appearance

- Dysplasia- additional genetic changes with abnormal growth, no longer
look normal
- Carcinoma in situ- more abnormality, dedifferentiated cells
- Cancer (malignant tumors)- can invade and metastasize

Statistics

- 200 types of cancer, different causes, symptoms and treatments

- Lifetime risk is 50%, survival rate is also 50%
- Prostate and breast cancer types are leading cancer types for cases by
sex
- Lung and brochus cancer is the leading cancer for death in both sex
Classification of cancer types

By tissue type

- Carcinoma- epithelial tissue, 90% of all cancer, derived from ectoderm

or endoderm (some)
- Sarcoma- connective tissue, 2% of all cancer, derived from mesoderm
- Leukemia and lymphoma- circulatory or lymphatic, 8% of all cancer,
derived from mesoderm

By cell type

- Adenomatous cell- ductal or glandular cell

- Squamous cell- flat cell
- Myeloid- blood cell
- Lymphoid- lymphocytes

Childhood vs adult cancers

- Most adult cancer- carcinoma, increased risk with age

- Most childhood cancer- sarcoma or leukemia, decreased risk with age

Predisposing factors

- Age, sex, heredity

- Infection- 15-20%
- Exposure to DNA damaging compounds
- Precancerous lesions- colon polyps

Cancer is genetic disorder

- It is rarely inherited
- Genetic modifications- somatic (mutation in cancer cell and its offspring
but not in the patient’s healthy cell, and not inherited from parent)
- P53 protein- guardian, error in 50% of all cancer
- Telomerase- protect the end of chromosome, present in 85% of all
cancer allowing infinite division
- Only 5-10%- have hereditary component- BRCA1 and BRCA2 in breast
cancer,
- Somatic changes are also required to develop cancer

Mutation types

- Substitution (change), deletion, insertion (addition), inversion (mirror),

duplication
What causes cancer?

- Arise from mutation in normal cell (oncogenes and tumor suppressor

genes)
- Several mutations
- Cancerous cells don’t self-destruct, continue to divide rapidly

Mutagens and carcinogens

- Mutagens are factors causing mutation

- Carcinogens are agents causing cancer

Carcinogens

- Ionizing radiation- X rays, UV

- Chemicals- tar in cigarettes, alcohol
- Viral infection- human papilloma virus (HPV) for cervical cancer (5%),
hepatitis (HBV/HCV) for hepatocellular cancer
- Hereditary predisposition- more susceptible to get cancer
- Lifestyle factors- diet (overweight and obesity, unhealthy diets and high
salt)

Cancer genes

- 1% of all human genes indicated in cancer- 90% are somatic mutations,

20% germline, 10% both
- Tumor suppressor genes- suppress cell growth, p53, turned off in
cancer
- Oncogenes- induce cell proliferation and growth, turn on in cancer

Genes altered in cancer

- Gatekeeper genes- regulate growth and differentiation, TSG and

oncogenes
- Caretaker genes- maintain genetic integrity, to avoid microsatellite
instability (mismatch repair deficiency) and chromosomal instability
(gain or loss of chromosomal parts)
- Landscaper genes- control microenvironment in which cell grow
TSG

- Brake pedal to control growth and division

- Inhibit growth factor receptor binding, down signaling event,
transcription factor

- P53
o In normal cell, during DNA damage, TP53 activated and binds to
DNA and transcriptional regulation of upregulated genes- p21
(CDK inhibitor for G1 arrest), DNA repair genes and apoptosis
genes
o In cells with TP53 mutations- during DNA damage, no activation
or upregulation of TP53 dependent genes, no cell cycle arrest and
DNA repair, additional mutations occur
- Inactivation of TSG by promoter hypermethylation- silencing

Oncogenes

- Promote cell growth and division in controlled manner

- One allele alteration is enough- proto-oncogene becomes oncogenes

 - Point mutation, gene amplification, chromosomal rearrangement, viral
insertion

Genetic vs epigenetic events

- Epigenetics- inherited changes in gene expression without change in

DNA sequences
- Genome instability due to mutations (genetic)- DNA methylation,
Histone modification, noncoding RNA (epigenetics)
- Promoter hypermethylation- silencing
- miRNAs- tumor suppressor miRNAs and oncogenic miRNAs

Monoclonal and polyclonal of tumors

HALLMARKS

1.Self-sufficiency in growth signal

- Normal cells- require hormones and growth factors to grow and divide
- Cancer cells- no requirements of external signal
- Autocrine signals- produce the signal themselves- signal pathways
activated
- RTK and PI3K pathways- CCND1 transcription and translation
2.Insensitivity to anti-growth signals

- TSG turned off- cannot control abnormal division- p53

- Resistant to growth-preventing signals from neighbors
- Off-switches destroyed- excessive growth
- Negative feedback control- JAK-STAT signaling pathway

- Contact inhibition- stop dividing when fill up the space or touch other
cells, cancer cells continue to divide and piling on top of each other

3.Evading programmed cell death

- Apoptosis- cell suicide/ self-destruct when damaged or infected

- Cancer- bypass apoptosis, become abnormal and don’t die
- Alter the mechanism detecting damage
- Defect in downstream signaling of apoptosis
- Defect in protein in apoptosis
4.Limitless replicative potential

- Cancer cells- indefinite growth and division

- Immortal cells- damaged chromosomes
- Normal cell- after certain # of division- unable to divide (senescence)
and die (crisis) ( 10^-7 immortalized)
- Hayflick limit (60-70 doubling)- Telomere/ Telomeric DNA shortens with
every cell division- activates senescence
- Cancer
o activate enzyme to increase the length of telomere (telomerase)
o disable p53 and Rb TSGs

5.Inducing angiogenesis
 - New blood vessels are formed
- Expanding tumor- To get enough O2 to survive
- Angiogenic switch- disable factors inhibiting blood vessels/ activating
factors promoting blood vessels

6.Activating invasion and metastasis

- Invasion- breaking away from their site or organ to invade surrounding

tissue
- Metastasis- spread of cancer to distant parts
- 1. Local invasion into surrounding tissue
- 2. Invade blood vessels
- 3. Survival in circulatory system
- 4. Exit circulatory system
- 5. Start division in the new tissue

EMERGING HALLMARKS AND ENABLING CHARACTERISTICS

1.Deregulating cellular energetics

- Adjustment of energy metabolism

- Normal cells- use glucose for glycolysis under anaerobic condition but
mostly use oxidative phosphorylation under aerobic conditions
- Cancer cells- reprogram glucose metabolism for glycolysis under every
condition
- Glycolysis- less efficient
 - Cancer cells- upregulate GLUT1 glucose transporters to increase
glucose uptake
- By activated oncogenes (RAS, MYC) and mutant TSGs (p53)

- WHY?
o Diversion of glycolytic intermediates generates nucleosides and
aa – required for macromolecules biosynthesis used in
proliferation
o Oxygenation fluctuates due to instability and disorganization
o One population- Hypoxic (oxygen deficient) one uses glycolysis
and secrete lactate
o Second population- oxygenated one imports lactate and use it in
the oxidative phosphorylation

2.Avoiding immune destruction

- Avoid immune surveillance (recognition and elimination by immune

cells)
- To avoid immune detection OR limit immunologic killing
- Experiment: tumor arising in immunodeficient can be transplanted only
into immunodeficient mice
- Tumor arising in immunocompetent mice can be transplanted into both
- Highly immunogenic cancers eliminated in immunocompetent mice
- Weakly immunogenic cancer can survive in both (immunocompetent
and immunodeficient mice)

3.Genome instability and mutation

 - Cancer cells- increase mutation rate bc genomic maintenance
machinery breaks down- increased sensitivity to mutagenic
- Also, bc monitoring system for genomic integrity damaged- no
senescence and apoptosis

4.Tumor-promoting inflammation

- Immune cells- eradicate tumor (initially), enhance development and

progression (later)
- Supply- GF, survival factors, pro-angiogenic factors, matrix- modifying
enzymes (for angiogenesis, invasion, metastasis), activation of EMT
- Release mutagenic chemicals- ROS- speed up progression

SUMMARY
New Dimensions

APOPTOSIS
Necrosis

- Accidental cell death

- Due to- anoxia, extreme temperature, toxin, physical trauma

Apoptosis

- Programmed cell death

- Cell suicide/murder by immune
cells
- Due to- lack of survival signal or
extensive DNA damage detection
- Caspases- main mediators
- Cysteine in its active site- attack
and cleave target protein after
aspartic acid residue
Initiator caspases

- Normally- inactive, soluble monomers in the

cytosol
- Apoptotic signal- large protein structure with
multiple initiator caspases – dimer formation-
activation
- After binding of adaptor protein (FADD
example), initiator caspase dimerizes and
cleaved within specific location

Effector caspases

- The activated initiator caspase can activate effector (executioner)

caspase by cleaving the dimer in the protease domain and active site
goes under conformational change.
- 1 initiator caspase can activate many- proteolytic cascade

SUMMARY
DNA fragmentation during apoptosis

- Activation of executioner caspases cleaves

iCAD (CAD inhibitor) which frees nuclease
(CAD, caspase activated DNase)
- Activated CAD cuts the chromosomal DNA
between nucleosomes
- Formation of ladder pattern upon gel
electrophoresis
- Detected by TUNEL assay- TdT enzyme
(terminal deoxynucleotidyl transferase) adds
labeled deoxynucleotide (dUTP) to 3’ -OH ends
of DNA fragments
- The presence of large number of DNA
fragment- bright fluorescent dots in apoptotic cells

Extrinsic pathway

- Extracellular signal binding to cell-surface death receptors

(transmembrane)
- Both death receptors and ligands- homotrimers
- Death receptors
o extracellular ligand-binding domain
o transmembrane domain
 o intracellular death domain
- Death domain- required for the activation of apoptotic program
- Death receptors belong to tumor necrosis factor (TNF) receptor family
- Death ligands- belong to TNF family- structurally related
- TNF receptor family- includes TNF receptor and Fas death receptor
- FAS- FAS ligand interaction
o Fas ligand binds to Fas receptor on the surface
o Death domain on the cytosolic tail of Fas binds to intracellular
adaptor proteins (FADD)
o These proteins bind to initiator caspases – formation of death-
inducing signaling complex (DISC)- primarily caspase 8
o Cleavage of caspase 8
o Activation of executioner caspases
o Apoptosis

- Inhibitory proteins – prevent inappropriate activation of extrinsic

pathway
o for example FLIP- resembles initiator caspase but lack of caspase
activity (no cysteine in the active site)
o FLIP dimerize with caspase 8 in DISC
o Caspase 8 appears to be active- but not cleaved so apoptotic
signal blocked

Intrinsic pathway
- Tightly regulate bc cell can kill itself
- Intracellular regulator- Bcl2 family
o Control the release of cytochrome c and other mitochondrial
proteins into the cytosol
o Some are pro-apoptotic and enhance the release
o Others are anti-apoptotic and blocks the release
o Three classes of Bcl2 protein
o BH3 domain- shared by all, direct interaction, heterodimers
formation

- Activation of intrinsic pathway

o Apoptotic stimuli- activate pro-apoptotic Bcl2- form oligomers in
mitochondrial outer membrane
o Release of cytochrome c and other proteins from mitochondria
 o Pro-apoptotic Bcl2 family- Bax and Bak- at least one of them is
required
 Mutant mouse with lack of both- resistant to all pro-
apoptotic signals
o Anti-apoptotic Bcl2 family-Bcl2 and BclXL- located on the
cytosolic surface of outer mitochondrial membrane
 Prevent release of proteins
 At least 1 is required for the survival
 Inhibited for apoptosis- by BH3-only proteins (largest
subclass of bcl2 proteins)
 Apoptotic stimulus activates BH3 only proteins

- Drug strategies targeting Bcl-2 family

- Extracellular factors inhibiting apoptosis
o Increased production of anti-apoptotic
Bcl2
o by survival factor stimulation
 o Inactivation of pro-apoptotic BH3-only proteins
o survival factors
activate protein
kinase Akt
o Akt phosphorylates
and inactivates pro-
apoptotic BH3-only
protein Bad
o When not
phosphorylated, Bad
binds and inhibits
Bcl2 and promote
apoptosis
o Once
phosphorylated, Bad disassociates and frees
Bcl2 to suppress apoptosis

Phagocytosis for the removal of apoptotic cells

- Apoptotic cell and its fragments don’t break open and release their
content and remain contact
- No trace is left and no inflammatory response happens

Insufficient apoptosis

- Causes cancer
- Excessive production of Bcl2 in B cell lymphoma- inhibits apoptosis

ANGIOGENESIS
- Fundamental process regulated by fine balance
- Deregulated in many diseases
 - Formation of new blood vessels out of existing capillaries
- Tumor angiogenesis
o Penetrate
cancerous
growth
o Supply of
oxygen and
nutrients
o Removal of
waste
o Induced by
releasing molecules from cancer cells
o Released molecules triggers neighbor cells to
activate certain genes to make proteins that
enhance new blood vessel growth

Vascular endothelial cells

- Form the wall of blood vessels

- Rarely divide, once every 3 years
- Angiogenesis can only stimulate them to divide

Angiogenesis and cancer

- Before 1960, scientist believed that blood supply can reach the
cancerous cells because pre-existing blood vessels are dilated
- Later, they realized that angiogenesis (formation of new blood vessels)
is necessary for cancerous cells to keep grow and spread

Regulatory molecules

- Normally, inhibitors- predominate (high in concentration) and block

growth
- For the formation of new blood vessels, activators increase in number
and inhibitors decrease
- So, concentration is important
Activators

- Released by tumors, also certain type of normal cells

- Signal for angiogenesis
- Vascular endothelial growth factor (VEGF)
- Basic fibroblast growth factor (bFGF)

- VEGF (Vascular endothelial growth factor)

o Glycoproteins
o Include A-B-C-D-E forms and placenta growth factor (PLGF)
o 6 subtypes
o Loss of 1 allele- embryonic lethality- cardiac complications
o 3 receptors- VEGFR1 (Flt1)- VEGFR2 (Flk1)- VEGFR3 (Flt4), all
tyrosine kinases,
- FGF (Fibroblast growth factor)
o Receptor- High affinity tyrosine kinase receptors
o Cell migration, proliferation, differentiation
- PDGF (Platelet-derived growth factor)
o Recruitment of pericytes (smooth muscle cells) to stabilize new
capillaries
- Signaling Cascade
o Cancer cells release angiogenesis activator molecules (VEGF,
bFGF)
o These activators bind to neighbor endothelial cells’ receptor
molecules and start signaling cascade
o Genes are activated, angiogenesis proteins are produced
 o Also, activated endothelial cells produce MMP (matrix
metalloproteinases, degradative enzymes)
o MMPs are released and break down ECM (extracellular matrix,
support material filling the space btw cells including protein and
polysaccharide)
o So, endothelial can migrate and begin to divide
o They organize into hollow tubes and evolve into mature network
of blood vessels

Inhibitors

- For angiogenesis to start, the concentration of activators must

overcome the concentration of inhibitors
- Inhibitors found in nature- green tea, soy products, fungi, mushrooms,
red wine
- Inhibitor treatment- decrease the chance of metastasis

- Cancer cells remain dormant for years, WHY?

o No angiogenesis
o Primary tumors can
release angiostatin into bloodstream

METASTASIS
Spreading of cancer cells from a place where they first formed to another
part of the body

- How?
o Primary tumor formation
o Local invasion
o Breaking away from primary tumor (intravasation)
o Traveling through blood or lymph system and survival
o Adhesion to blood vessel wall
o Extravasation
o Forming a new tumor in other organs
- Stage IV
- Requirements
o Survival or anti-apoptosis factors (Bcl-2, growth factors,
telomerase)
 o Adhesion or homing factors (integrins, FAK, cadherin)
o Invasion factors (MMP)
o Angiogenic factors (VEGF, FGF)
o Avoiding anoikis (anchorage dependent cell death bc ECM
adherence is lost)
o Resisting shear force
o Escaping immune surveillance
- Hypoxia induces angiogenesis and promotes invasion
- EMT transition is important (for both invasion and metastasis)

CANCER GENOMICS, TRANSCRIPTOMICS AND

EPIGENOMICS
GENOMICS

Next generation technologies

- Type of input
o DNA
o RNA
o Chromatin
- The proportion of genome targeted
o Whole genome (determining complete genome of an organism at
a single time)
 Mutation in promoter/intron/exon/translocation/copy
number variation
 High data storage is required
 Hard computational analysis
o Whole exome (nucleotide changes in only protein coding genes)
 SNV (missense/nonsense/silent mutation)
 Indels (small insertion/deletion)
 Less data storage
  Easier to analyze
o Subset of genes (targeted sequencing)
 Coding sequences of cancer-related genes
 Less time consuming
 Minimal data storage
 Easiest data analysis
- Type of variation studied
o Structural changes
o Point mutation
o Gene expression
o DNA methylation

TRANSCRIPTOMICS

Experimental Approaches

- Array-based
o DNA microarray (measuring expression level of whole
transcriptome)
 For diagnosis and prognosis, pathways and biological
process
 Expression profile
o miRNA analysis
- NGS-based
o Whole-transcriptome sequencing (protein-coding7 lnc/ alternative
transcripts)
 Mutations (SNV, Indels, Fusion)
o mRNA-seq
o miRNA-seq

EPIGENOMICS

- Histone modifications (acetylation/methylation)

- ChIP-seq (DNA+protein- fragmentation- immunoprecipitation
(antibody)- releasing DNA- sequencing)

EPIGENETICS
- The study of biological mechanisms that switch genes on and off
- DNA seq not altered
- Important epigenetic processes in cancer
o DNA methylation (promoter hypermethylation of TSG)
 o Histone modification
o Nucleosome remodeling
o RNA-mediated targeting (miRNA, lncRNA)

Chromatin

- Macromolecular complex of DNA and

histone proteins
- Scaffold for packaging entire genome
- Basic functional unit- nucleosome
- Nucleosome
o 147 bp DNA
o Wrapped around histone octamer (8)
o Histone H2A/H2B/H3/H4
o Two each of these histones
- Chromatin- two major regions
o Heterochromatin
 Condensed
 Late to replicate
 Inactive genes / repetitive regions
 Hypermethylation
 Hypoacetylation of H3/4
 Methylation
o Euchromatin
 Open
 Active genes
 Gene rich
 Hypomethylation
 Hyperacetylation of H3/4
 Methylation

DNA methylation in cancer

DNMT

- DNA methyltransferases (DNMTs) methylate CpG in higher eukaryotes

- DNMT1
o Maintenance
o Recognizes hemi-methylated DNA during replication
o Methylates newly synthesized CpG after replication
- DNMT3a and DNMT3b
o Transcriptional activation / repression
 o De novo methyltransferases during embryogenesis

Methylation of TSG through CpG Islands

- Hypermethylation of promoter by DNMTs-

repression
- Affects the expression of protein coding
and non-coding RNAs
- DNMTs are mutated in cancer cells
 Mutation in DNMT3a- disrupts catalytic
activity

- Therapeutic approach- altering methylation

o Hypomethylating agents

Histone modification in cancer

Histone acetylation

- Lysine acetylation in N-terminal tails of histone- neutralizes positive

charge
- Histone acetyltransferases (HAT/KAT)
- Type A- act on nucleosomal histone
- Type B- act on cytoplasmic free histones
- Transcriptional activators
- Mutation in HAT- alter catalytic activity

Histone deacetylation

- Reversal of acetylation
- Help to compact the chromatin packing
- Histone deacetylases (HDAC)
- Transcriptional repressors

Histone methylation

- Ly and Arginine residues

- Histone tails or sometimes core
- Mono-methylation- activation
- Tri-methylation- repression
- Lysine methyltransferases- mono-di-tri

Histone demethylation

- Demethylase
- Targeted by drugs
- Activation or repression- depends on histone

Mutation in histone genes

- Effects chromatin structure and transcription

- H3.3 and H3.1 common

Chromatin remodeling complexes

1. SWI/SNF/ISWI/NuRD/ CHD
2. Affects composition (canonical or variant) and positioning
3. Mutation
a. May disrupt histone-DNA contacts
b. May cause sliding of nucleosome
c. May result in more accessible chromatin complex to TF
Epigenetic therapies

- Small molecule inhibitors against chromatin regulators (DNMT, HDAC…)

DNA DAMAGE AND REPAIR

Common types of damage

- DNA adduct formation- carcinogens mutate p53 gene by base

alkylation
- Mismatches- mistake in DNA synthesis
- Interstrand- crosslinks
- Double strand DNA breaks

Mutation types

- Spontaneous mutations- error in natural processes

o Tautomerism- repositioning of H atom in a base- incorrect base
pairing
o Depurination- loss of purine base (A or G)
o Deamination- keto group instead of amine group
 C->U or A->HX (hypoxanthine) detected by repair
mechanism
 5MeC->T not detected bc T is normal DNA base
o Slipped strand mispairing- denaturation of new strand,
renaturation occurs different spot- insertion/deletion
- Induced mutations- due to agents
o physically
o chemically
 base analogs (brdU)- 5-bromouracil is analogous to T
 nitrous acid
 hydroxylamine- adding OH to amino group of cytosine and
pairs with A (transition of C-G to T-A
 oxidation- by ROS
 radiation- pyrimidine dimer formation by UV- C and T are
most vulnerable

DNA REPAIR SYSTEM

Pyrimidine dimer

- causes local conformational change

- lesions can be recognized by repair enzymes
- repaired by photoreactivation
 - if absorption wavelength >300nm due to lesions- photoreactivation
enzymes are activated

Base excision repair

- DNA glycosylase recognizes damaged base and cleaves btw base and
deoxyribose
- AP endonuclease cleaves phosphodiester backbone
- DNA poly I initiates repair from free 3’ OH end (5’-3’ endonuclease)
- Nick is sealed by DNA ligase

Nucleotide excision repair

- UvrA recognizes bulky lesion

- Two endonucleases (UvrB and UvrC) bind DNA at bulky lesion site
- One cleaves the 5’ side and other cleaves 3’ side
- DNA segment is removed by helicase (UvrD)
- Gap is filled by DNA polymerase I
- Nick is sealed by DNA ligase

Mismatch repair (prokaryotes)

- MutS/L complex binds mismatch- moves

simultaneously in both directions until
encounters MutH at hemi-methylated
sequence
- MutK links S to H
- MutH recognizes parental strand
- Which strand is parental?- CH3 marks on
parental strands
- DNA helicase II and SSB and many other
endonucleases remove DNA segment
- Gap is filled by DNA poly III
- Nick is sealed by DNA ligase

Mismatch repair (eukaryotes)

- MSH2:MSH6 binds to mismatch

- MLH1 endonuclease binds and recruits helicase and exonuclease
- Gap is filled by Pol gama and sealed by DNA ligase
No template for repair

DS break repair

- End-joining
o Non-homologous- removes several bases at break site so it is
mutagenic
o Mediated by KU80-KU70 heterodimer
- Recombination
- dsDNA activates ATM kinase
- ATM kinase activates exonucleases
- Exonucleases create ss 3’ ends
- Depends on BRCA1-2 and Rad51
DNA REPLICATION
DNA Structure

- Consists of double strand which are

antiparallel
- The function is information storage which can
be passed onto descendant cells

- Hydrogen bonds btw nucleotides

DNA REPLICATION MODELS

DNA ELONGATION
Enzymes involved in replication

- DNA polymerase- joins adjacent nucleotides each other

o Eukaryotic DNA poly- more than one, alpha-beta-deta vs,
different function such as replication, proofreading, repair, and
located in nucleus of mitochondria
o Self-correcting enzyme, correct nucleotide has greater affinity for
moving polymerase
o High fidelity- 1 mistake in 10^9 nucleotides
o
- Primase- provides RNA primer to start polymerization
- Ligase-seals nick by joining adjacent DNA strands together
- Helicase- unwinds DNA and melts it
- Ss binding protein- SSBP- keeps DNA single stranded
- Gyrase- topoisomerase- relieves tension by relaxing the supercoiled
DNA
- Telomerase- finishes off the ends of strands

Initiation of replication

- Human ORC required for initiation

- Gyrase relaxes supercoiled DNA
- Initiator proteins and helicase bind to DNA at replication origin and
untwist it
- Ss binding proteins bind to ss DNA and keep strands from rejoining
- Clamp protein forms a ring around DNA helix, it helps DNA poly binding
to ssDNA, and it releases DNA poly when runs into dsDNA or 5’ end of
Okazaki fragment
- DNA primase binds to helicase and synthesize RNA primer
- DNA poly III add nucleotides 5’ to 3’ on both strands beginning at RNA
primer, requires 3’ end
- RNA primer removed and replaced with DNA poly I
- Gap is sealed by DNA ligase by phosphodiester bond

Telomere

- Eukaryotes- tandem repeated seq at the end of chromosome

- Thousands of repeats of six nucleotide seq- TTAGGG
- Protect chromosomes
- Without- chromosome fusion/ massive genomic instability
- Telomere specific proteins- bind repeated sequence for protection-
TRF1/2
- Telomerase- bind to terminal telomere repeat and catalyze addition of
new repeats
o Reverse transcriptase
o Requires 3’end as a primer
o 5’ to 3’ direction
o Synthesizes one repeat and reposition itself
o RNA + protein
 o Human telomerase- reverse transcriptase- hTERT
o Active cells- germ/ in vitro immortalized/ majority of cancer/ some
stem cells
- Absence/mutation of telomerase- chromosome shortening and cell
division limitation
- RNA primer near the end of lagging strand- cannot be replaced by DNA
since DNA poly must add to primer sequence

CELL CYCLE AND CANCER

Growth and division of single cell into daughter cells and duplication

- G1
- S- DNA replication
- G2
- M- mitosis/cytokinesis
-

- Interphase
- S and G2

Cell cycle control system

 - Without cyclin-> Cdk is inactive

- Concentration of cyclins oscillates

- Concentration of Cdk do not change

Three stages of Cdk activation

- Inactive- Without cyclin bound- active site is blocked by T-loop protein

- Partly active- Binding of cyclin causes T-loop to shift from active site
- Fully active- Phosphorylation of Cdk2 by CAK at threonine residue in T-
loop and cause the conformation change in T-loop, so active site
becomes free

-Inhibition of Cdk-cyclin complex - Kinase Wee1

phosphorylates two closely spaced site above
active site
- Reactivation of complex- Removal of phosphates by phosphatase
Cdc25 activates Cdk-cyclin complex

Cdk- Cyclin complexes

1. G1/S cyclins
a. Activate Ckd in late G1
b. Trigger progression through Start- commitment to cell cycle entry
2. S-cyclins
a. Bind soon after progression through Start
b. Stimulate chromosome duplication
c. Concentration remains elevated until mitosis
d. Control of some early mitotic event
3. M-cyclins
a. Activate Cdks at entry into mitosis at G2/M transitions
b. Concentration level falls in mid-mitosis

Cell cycle phases

Human cell cycle- 90h

Interphase

a. Cell growth
b. Nutrient accumulation
c. DNA duplication
1. G1 phase
a. mRNA/rRNA/tRNA formed
b. New cell organelles formed
c. Cyclin D concentration increases- mitogenic signal sensor
d. Cyclin E- required for G1 to S transition
e. Growth factors bind to receptor on the surface
Mitogen stimulation of cell cycle entry

- Mitogen binds to cell surface receptors-

initiate intracellular signaling
- Activates GTPase Ras
- It activates MAP kinase cascade
- Immediate early genes are activated
- MYC (transcription factor) expression
induced
- MYC induces the expression of delayed
response genes such as Cyclin D
- Cyclin D activates G1-Cdk
- Cyclin D-Cdk4 triggers the
phosphorylation of Rb protein to
inactivate it
- Inactivated Rb frees gene regulatory protein E2F
- Activated E2F
induces the
expression of
G1/S genes
(Cyclin E and
Cyclin A for
example)
- G1/S-Cdk and S-
Cdk further
activate Rb
protein
phosphorylation
and creates
positive loop
- E’F also
stimulates
transcription of
own genes, forming another positive loop
 2. S phase
a. Cyclin E and cyclin A regulates this phase
b. DNA synthesis proteins and enzymes are phosphorylated and
activated
c. DNA duplicated
d. Chromosomes replicated

Control of chromosome duplication

- DNA helicases are loaded at

replication origin
- Pre-replicative complex (preRC)
formed
- S-Cdk activation- causes
helicase activation, also
prevent new preRC formation
- Helicases unwind DNA
- Two replication forks move out
from origin until entire
chromosome is duplicated
- Duplicated chromosomes are
segregated in M
-
Control of DNA replication
initiation

- Replication origin- bound

by ORC
- In early G1- Cdc6 associate
with ORC- for DNA helicase
(Mcm) binding
- Helicase associate with
Cdt1
- Two copies of helicase
loaded in ORC in inactive
form- prereplicative
complex (preRC)
- At S phase- DNA helicases
are activated
- S-Cdk- stimulates initiator
protein assembly on
helicase
- DDK phosphorylates
helicase
- DNA poly and other
replication proteins are
recruited to origin
- Replication begins
- S-Cdk inactivates preRC
such as ORC, Cdt1 and
Cdc6 – no formation again
until the end of mitosis
Cohesin

- Hold sister chromatids together- at the end of S phase

- Each replicated chromosome has a pair of identical sister chromatids
- Smc proteins – two subunit- Smc1 and Smc3
- Two additional subunits- Scc1 and Scc3
- They form ring structure- encircle sister chromatids

G2 Phase

- Has- double the number of chromosomes

- All cellular components are duplicated
- Cyclin A / Cdk1 and Cyclin A / Cdk2 - active
- Cyclin B/ Cdk1- forms but activated in M phase
Mitosis Cyclin / Cdk complex (MPF)

- Mitosis Cyclins + Cdk = mitosis promoting factor (MPF)

- Induce
o Assembly of mitotic spindle
o Each sister chromatid attached to opposite side of spindle
o Chromosome condensation
o Breakdown of nuclear envelope
o Rearrangements of actin cytoskeleton
o Rearrangement of golgi

The activation of M-Cdk

- M-cyclin level rise

- Cdk1 associate with M-cyclin

- Inhibition- M-Cdk complex phosphorylated two times by

o CAK ( Cdk-activating kinase)
o Wee1 kinase
- Inhibited M-Cdk – activated at the end of G2- by Cdc25 phosphatase

- Positive feedback- inhibition of Wee1 and stimulation of Cdc25

M phase

- Cell divides
- Short duration- 45-60 min
- Karyokinesis (mitosis) and cytokinesis (cell division)
- Karyokinesis-> prophase- prometaphase- metaphase- anaphase-
telophase
Prophase

- Chromosomes and their sister chromatids condensed

- Mitotic spindles assemble btw two centrosomes

Condensation of
chromosomes

- Condensin (five subunits) – form ring structure

- Smc2 and Smc4 held together by three subunits
(CAP-G/H/D2)
- Encircle loop of DNA

Prometaphase

- Starts with breakdown of nuclear envelope

- Chromosomes attach to spindle microtubules via kinetochore
- Chromosomes in active motion

Metaphase

- Chromosomes are aligned at equator of spindle

 - Plus ends of microtubules attach sister chromatids through kinetochore

Metaphase Anaphase Transition by APC/C

- APC/C activated by Cdc20

- Activated APC/C- Triggers transition by tagging specific proteins for
degradation- ubiquitination
- Targets- Securin, S cyclins, M cyclins
- Securin degradation- releases separase
- Separase- induce cohesin (Scc1) cleavage (complex that binds sister
chromatids together)

Control of
proteolysis (ubiquitination) by APC/C
 - With the help of E1 and
E2
- Forms polyubiquitin
chains on target
- Polyubiquitinated target-
recognized and
degraded in proteasome

Anaphase

- Chromosome segregation- Sister chromatids separated and pulled

toward spindle pole
- Microtubules get shorter, spindle pole also move apart

Telophase

- Two sets of sister daughter chromosomes

arrive spindle pole and decondensed
- New nuclear envelope reassembles
- Two nuclei formation completed
- Division of cytoplasm by contractile ring

Cytokinesis
 - Cytoplasm divided into two by contractile ring
of actin and myosin
- Cell is pinched in two daughter cells

Regulation of contractile ring

- By GTPase RhoA
o activated by RhoGEF
o inactivated by RhoGAP
- Activated RhoA- concentrated in contractile ring
- Binds formins- for actin filament assembly
- Activates Rock, Rho-activated protein kinase
- Rock- stimulates myosin II filament formation
and activity
- Myosin II filament- promotes contraction of ring

Cell cycle arrest in G1

- By DNA damage detection

- Protein kinases recruited to DNA
damage site – initiate signaling
pathway of cell cycle arrest
o ATM or ATR- first kinases
o Chk1 and Chk2- second kinases
- P53 phosphorylated by these kinases
- Mdm2 normally binds to p53 for
ubiquitination (if no damage)
 - Phosphorylated p53- no binding to Mdm2
- P53 accumulation- stimulates transcription of genes- CKI protein p21
- P21 inactivates G1/S-Cdk and S-Cdk- arrest in G1
- DNA damage may cause
 Mdm2 phosphorylation
 Decrease in Mdm2 production
 Increase in p53 level

Excessive stimulation of mitogenic pathways

- Causes cell cycle arrest or apoptosis

- Due to high level of myc
- Causes Arf activation
- Arf inhibits Mdm2 -> increases p53 level

Overwiev of cell cycle control

Inhibition due to

- Environment
- Cell / DNA
damage

G1 restriction point

- At the end of G1 before S

- Taking the decision- division or delaying division or entering resting
state (G0)
- Controlled by CKI- p16 (Cdk inhibitor p16)

G2-M checkpoint
 - Control MPF
- Any damage- activation of MPF by phosphatase does not occur
- Arrest – till repair of DNA
- Prevents transfer of defected DNA into daughter cells

DNA damage checkpoint at G2-M

- DNA damage- ATM/ ATR signaling

pathway activation
- Phosphorylation and activation of
Chk1/2
- Sequestration of Cdc25 – by
phosphorylation
- Inhibition of Cdc25- causes
Cyclin B / Cdk1 inhibition
- G2 arrest
- Activated ATM / ATR- activates
p53 signaling
- Causes Cyclin B inhibiton
- P53- p21 activation (Cdk
inhibitor)

Metaphase checkpoint

- Regulated by APC/C
- Also called- spindle/mitotic checkpoint
- Due to unattached kinetochore

Cdk Inhibitors

- CKIs
- INK4 – block passage through G1- inhibits Cdk4/6
- CIP/KIP family- p21/27/57- inhibits Cyclin A /Cdk2