Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics

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Oncogenes and Cancer

• Genes cell phenotypes
• Transforming viruses
• Retroviral oncogenes

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Cancer Cell Phenotypes

• Immortalization
• indefinite growth (no other phenotype changes)
• Transformation
• independence of factors normally required for
  growth
• Metastasis
• cells are mobile invade normal tissue

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Terminology

• Primary cells
• immediate descendants of cells taken from an
  organism
• mimic in vivo phenotype
• generally survive for only a short period of time
  in culture

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Terminology

• Crisis
• primary cell cultures enter into senescence
• majority of the cells die
• Established cell line
• cells that have passed through crisis
• non-tumorigenic
• immortal, and other phenotypic changes

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Primary cell culture
Primary Cell Culture
Crisis
Established Cell Line
CELLS
TIME
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Fig 30.4 Cancer Cell Properties
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Cell Properties

• Anchorage dependence
• Serum (growth factor) dependence
• Density dependent inhibition
• Cytoskeletal organization

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Cell Properties

• Anchorage dependence
• cells need a solid or firm surface to stick to
• Serum (growth factor) dependence
• growth factors in serum are essential for growth
  of cells in culture

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Cell Properties

• Density-dependent inhibition
• cells grow only to a certain density, then stop
• may involve cell - cell contacts
• Cytoskeletal organization
• cells are flat extended
• elongated network of actin filaments
• Cells also grow as a monolayer

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in vitro vs. in vivo
Established cell lines provide only an
approximation of in vivo control -changes in
the chromosomal complement -not true diploids/
aneuploids
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Cell Properties

• Transformed cells show altered phenotypes
• no anchorage dependence
• no dependence on serum (growth factors)
• no density-dependent inhibition of growth
• altered cytoskeleton

• Form tumors

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Fig 30.6 EM of normal and transformed cells
NORMAL
TRANSFORMED
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Genetic Basis

• What genetic events convert normal cells to
  transformed cells?
• multiple events
• 6-7 events over 20-40 years
• propensity to cancer may be inherited
• Single genetic change
• exposure to carcinogens increases risk

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Genetic Basis

• Two classes of genes which can cause
  transformation when mutated
• Oncogenes
• Tumor suppressor genes

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Oncogenes

• Initially identified as genes carried by viruses
  which transform cells
• viral oncogenes have cellular counterparts
• proto-oncogenes
• about 100 oncogenes have been identified
• many different functions
• transmembrane proteins, TFs, etc.

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Oncogenes

• Oncogene mutations
• Gain of Function mutation
• mutational change of protein
• constitutive activation
• overexpression
• failure to shut off expression

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Tumor suppressor genes

• Loss of function mutation
• detected by deletion or inactivating mutation
• common in hereditary cancers
• patients lack both alleles
• about 10 tumor suppressor genes known
• genes normally constrain cell cycle

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Transformation of Cells

• may occur
• spontaneously
• through exposure to carcinogens
• by viral infection
• transforming virus or tumor virus

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Fig 30.4 Transforming viruses may carry
oncogenes
HPV human papillomaviruses gt60 known warts,
cervical cancers Adeno Adenoviruses gt 80
known mouse cells transformed (not
human) Herpes Epstein-Barr assoc. with variety
of diseases (lymphoma)
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Fig 30.9 Permissive vs. nonpermissivecells
Two cellular responses to viral infection -
infection - transformation
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Permissive vs. Non-Permissive

• Permissive cells
• Productive infection cycle
• Release of new viral progeny
• Cell death
• Non permissive cells
• Abortive infection cycle
• Transformed cells
• Phenotypic changes

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Fig 30.10 Transformation by virusesadenovirus
or polyomavirus
Product of early gene required for transformation
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Fig 30.11 Genetic transfer by
retroviruseshorizontal or vertical transfer of
genetic info
Both RNA and DNA forms of viral genome
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Transforming Retroviruses

• Two general groups
• Non-defective viruses
• Acute transforming viruses

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Transforming Retroviruses

• Acute transforming viruses
• have a viral oncogene
• derived from a cellular proto-oncogene
• replication defective (needs helper virus)

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Fig 30.12 Acute transforming retrovirus
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Fig 30.14 Oncogenes of transforming retroviruses
Each transforming retrovirus caries an oncogene
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Difference in v-onc and c-onc

• v-onc viral oncogene
• c-onc cellular proto-oncogene
• More than 30 c-onc genes identified
• Same oncogene present in several viruses

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Expression of the v-onc sequence is capable of
transformation

• RSV, temp sensitive mutations of v-src
• Transformed phenotype reverted or regained
• v-src gene needed for both initiation and
  maintenance of transformation

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2 Models for Oncogenicity

• Quantitative model
• Qualitative model

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Difference in v-onc and c-onc

• Quantitative model
• v-onc and c-onc are indistinguishable
• v-onc is oncogenic
• more highly expressed
• expressed in inappropriate cell types
• expression cannot be switched off

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Difference in v-onc and c-onc

• Qualitative model
• c-onc lacks oncogenic properties
• v-onc is mutated version
• Gain of function

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v-onc vs. c-onc genes
Gene Changes Identity v-onc region
missing mos 11 / 369 97 None H-ras 3 /
189 98 None K-ras 7 / 189
96 None sis 18 / 220 92 None myc 2 /
417 99 None src 16 / 514 97 C-term
19 aa fms 20 / 930 98 C-term 50 aa erbB 99
/ 600 83 N-term half C-term erbA 22 / 396
95 N-term 12 aa myb 11 / 372 97 N C
termini (268 aa)
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Level of Expression also Important
Oncogenicity when overexpressed
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Fig 30.15 Transfection assay
Tumor
Extract DNA
Transfect
Detection of oncogenes from transformed cell
lines (not normal cells)
Integration
colony of transformed cells
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Transforming Activity

• DNA Isolated only from tumorigenic cells
• Transfection with DNA
• Appearance of foci
• Indicator for transforming ability of DNA
• Genomic DNA
• Specific genes

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Isolated Transforming Genes

• Closely related sequences in DNA of normal cells
• May have counterparts in the oncogenes carried
  by known transforming viruses

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ras transforming gene

• multiple genes
• H-ras, N-ras, K-ras
• collectively known as p21ras
• H-ras and K-ras have v-ras counterparts
• very similar

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ras oncogenes

• Single base mutations can covert proto-oncogene
  into oncogene
• most commonly at aa12 or aa 61
• Glycine at 12 (Gly G)
• any other aa except Pro makes oncogenic
• Glutamine at 61 (Gln Q)
• alteration makes oncogenic some less effective

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ras proto-oncogene

• monomeric guanine nucleotide-binding protein
• active with GTP bound
• inactive with GDP bound
• Two proteins influence ras activity
• GAP - stimulate ras to hydrolyze GTP
• GEF - stimulates replacement of GDP with GTP
• See Fig 30.16

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signal transduction pathway
EGF
EGFR
Sos
GAP
Grb
Pi
MAPKK
fos
jun
Transcription
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signal transduction pathway
Cdc25
Ira 1, 2
Pi
GDP
S. cerevisiae
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Fig 30.16 Ras Activity
(GEF)
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Fig 30.16 Ras Activity
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Fig 30.16 Ras Activity
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Activation of protooncogenes

• Insertion of retroviruses
• Translocation
• Amplification

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Oncogene Amplification

• Established cell lines
• Susceptible to karyotypic changes
• Presence of known oncogenes in the amplified
  regions
• In Tumors
• Amplification of particular oncogenes

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Activation by Retroviral Insertion

• Insertion of a non-defective virus
• First noticed in bursal lymphomas
• T-cell lymphomas
• Ability of LTRs to cause expression of cellular
  genes

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Fig 30.17 Insertion of ALV at c-myc locus
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Fig 30.17 Insertion of ALV at c-myc locus
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Fig 30.17 Insertion of ALV at c-myc locus
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Coding sequence of c-myc does not change

• Oncogenicity
• caused by loss of normal control
• increased expression of the gene
• Other oncogenes activated by the same mechanism
  c-erb, c-myb, c-mos, c-raf

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Activation by Translocation

• Translocation to a new chromosomal location
• Reciprocal translocation
• Illegitimate recombination between two
  chromosomes
• Known examples
• Oncogenes brought by translocation to the
  proximity of Ig or TCR locuses

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Fig 30.18Reciprocal chromosomal translocation
B cells
T cells
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c-myc activation by translocation

• In B cells
• c-myc is translocated to Ig locus (H)
• c-myc brought to actively expressed region
• 2- to 10-fold increase in expression
• structure of c-myc may be changed
• In T cells
• c-myc is translocated to TCR locus

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c-myc and tumorigenic phenotypes

• Continued high expression of c-myc is oncogenic
• Expression of c-myc must be switched off to allow
  immature lymphocytes to differentiate into T and
  B-cells

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c-myc and tumorigenic phenotypes

• c-myc linked to Ig enhancer
• Transgenic mice develop lymphomas
• Present in both mature and immature B-cells
• Over-expression of c-myc is tumorigenic
  throughout the B-cell lineage

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c-myc and tumorigenic phenotypes

• c-myc linked to LTR ( mmtv)
• Transgenic mice develop variety of cancers
• Over-expression of c-myc transforms the cell into
  corresponding tumor

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c-myc activation

• Activation of c-myc via
• retroviral insertion
• chromosomal translocation
• gene amplification
• Activation by increased/altered expression
• NOT by altered amino acid sequence

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Philadelphia Translocation

• Creates a hybrid oncogene
• Philadelphia chromosome
• present in patients with CML
• Translocation of 5000 kb region from chr 9
  carrying c-abl gene to the bcr gene of chr 22

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Figure 30.19
Chr22
Chr9
bcr-abl fusion
Philadelphia Chromosomes
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Why is Bcr-Abl fusion oncogenic?

• Bcr-Abl protein activates Ras pathway via Grb2
  and Shc
• c-abl codes for a tyrosine kinase activity
• In v-abl changes in N-terminal region activate
  kinase activity and transforming capacity
• Lack of N-terminal region may cause inappropriate
  activation of kinase activity in bcr-abl fusion

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Oncogenes

• Components of signal transduction cascades
• Growth factor receptor kinases
• Cytoplasmic kinases
• Regulators of gene expression

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Signal Transduction Cascade
Growth Factor
Growth factor receptor
membrane
Adaptor (Ras)
Kinase Cascade (serine threonine kinases)
Transcription Factors
nucleus
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Fig 30.20 Different Types of Oncogenes
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Fig 30.20 Different Types of Oncogenes
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Fig 30.20 Different Types of Oncogenes
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Common feature

• Trigger changes in cell phenotypes
• initiating or responding to changes related to
  cell growth
• direct changes in gene expression

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Oncogenes

• Components of signal transduction cascades
• Growth factor receptor kinases
• Cytoplasmic kinases
• Regulators of gene expression

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Transmembrane receptors Initiate signal
trasduction pathways
Tyrosine Kinases Targets Tyrosine residues of
target proteins
Cytoplasmic enzymes Provide catalytic functions
for receptors that lack kinase activity
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Transmembrane receptors

• Extra-, Intra-cellular regions
• Kinase activity
• Activated upon dimerization
• Autophosphorylation reaction

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Ligand binding induces receptor dimerization
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Fig 30.21 Activation of GF receptor
Ligand binding induces dimerization Dimers
are active
Mutant is constitutively active - not repressed
Spontaneously forms dimers Constitutively
active
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v-erb

• Truncated version of c-erb/EGF receptor
• lacks N- and C-terminal domains
• N-terminal deletion causes spontaneous
  dimerization
• C-terminal deletion removes inhibitory domain
• Contain activating mutations in the catalytic
  domain
• Oncogenicity Deletions/ mutations that
  constitutively activate the receptor

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v-erb

• erb2 closely related to EGF receptor
• mutations in transmembrane region
• Dimer-formation increased
• c-fms CSF-I receptor
• mutations in extracellular domain
• Dimer-formation increased, constitutively active
• point mutations in C-terminal domain
• Inhibitory effect of c-terminal domain abolished

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Oncogenes

• Components of signal transduction cascades
• Growth factor receptor kinases
• Cytoplasmic tyrosine kinases
• Regulators of gene expression

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src as prototype cytoplasmic tyrosine kinase

• first kinase oncoproteins characterized
• first tyrosine kinase
• multiple viral oncogenes in this group
• v-src, v-yes, v-fgr, v-fps/fes, v-abl, v-ros
• N-terminus is modified
• amino acid removed, end myristoylated

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Fig 30.22 Src protein domains
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Comparison of c-src and v-src
c-Src
v-Src
Property
Phosphorylate target tyrosine residues

20X
Attached to the membrane via myristylation


Autophosphorylated at Tyr-416
-

Phosphorylated at Tyr-527

-
C-terminal sequence replaced including Tyr-527
-

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Src Phosphorylation see Fig 30.23
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Control of src activity by phosphorylation

• Mutation at Tyr527
• activates the transforming potential of c-Src
• c-Src-Phe527
• becomes phosphorylated at Tyr-416
• kinase activity increased 10X,
• transforms target cells
• Mutation at Tyr416
• eliminates ability of c-Src to transform
• c-SrcPhe 416, Phe 527
• Kinase activity reduced

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Control of src Activity by Phosphorylation

• Tyr416 activates oncogenicity of Src
• Tyr527 represses oncogenicity of c-Src

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Fig 30.24 Receptor tyrosine kinase (src)
activation
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Control of src Activation

• Tyr 527-SH2 interaction required for inactivation
  of c-Src
• Tyr 527-SH2 binding critical for oncogenicity
• inactivated by polyoma middle T antigen
• inactivated by SH2-SH3 mutations
• SH2-SH3 domains required for proper activation
  of c-Src

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Oncogenes

• Components of signal transduction cascades
• Growth factor receptor kinases
• Cytoplasmic kinases
• Regulators of gene expression

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Oncogenes

• Regulators of gene expression
• Act directly at the level of expression
• Oncogenes
• rel, jun, fos erbA, myc, myb

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v-Oncogenes

• Quantitative or qualitative changes
• changes in the level of expression
• modifications, defective TFs, dominant negatives
• alter expression of target genes
• acquire specificity for new targets (less likely)

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v-Rel

• Transforming function of avian reticuloendothelios
  is virus
• B-cell lymphomas in chicken
• c-Rel, family of NF-kB
• Lacks 100 a.a. from C-terminal
• Small point mutations in remaining sequence

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Fig 30.26 Function of oncogenic factors

• v-Rel, blocks NF-kB pleiotropic functions
• v-Rel-NFkB dimers are inactive

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AP1

• Mediates transcription induced by phorbol esters
• Activates promoters with AP1- binding sites
• Dimer subunits coded by c-jun and c-fos
• bZip TFs basic region leucine zippers
• v-jun and v-fos
• truncated versions of c-jun and c-fos
• point mutations

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Fig 30.26 Function of oncogenic factors
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v-erbA

• c-erbA
• Thyroid hormone receptor, family of steroid
  receptors
• Located in the nucleus
• binds to response elements in promoters with or
  without ligand
• Binds to triiodothyronine with high affinity
• Hormone binding needed to activate txn of
  previously bound receptor

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v-erbA

• Truncated form of c-erbA
• point mutations
• T3 affinity abolished
• v-erbA independent of hormone binding, cannot
  stimulate transcription

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Fig 30.26 Function of oncogenic factors
v-erbA, dominant negative, blocks functioning of
its normal cellular counterpart
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c-jun, c-rel, c-fos, c-myc

• Immediate early genes
• may be involved in cascades that initiate cycling
• Target genes may be involved in growth initiation

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Transformation by Adenovirus E1A oncogene

• E1A
• Three transcripts, alternative splicing
• 13S, 12S, closely related
• Posess ability to immortalize cells
• Cooperate with other oncogenes for transformation
• Different activates/ represses host gene
  expression
• Different domains required

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Fig 30.27 Adenovirus E1A region is spliced to
form three transcripts
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Adenovirus E1A Oncogene

• Activates RNA polymerase II and III loci (domain
  3)
• Regulates host gene expression via protein
  interactions (domains 1, 2)
• Targets CBP/p300, TBP, RB, p 27

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Tumor Suppressor Genes

• Loss of both alleles is tumorigenc
• Best known
• Rb
• p53

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Retinoblastoma (Rb)

• Childhood disease
• Tumor of the retina
• Inherited or sporadic (somatic mutation)
• Associated with deletions of band q14 of
  chromosome 13
• Loss of function of Rb mutation/deletions that
  abolish gene expression

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Fig 30.28 Retinoblastoma
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Rb protein

• Nuclear phosphoprotein
• influences cell cycle
• Phosphorylated by cyclin/cdk at end of G1
• Dephosphorylated during mitosis
• Binds to specific targets
• phosphorylation releases target proteins

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Target proteins of Rb

• E2F group of transcription factors
• activate genes essential for S phase
• Rb binding inhibits target activation
• Rb represses gene expression via E2F
• Rb prevents cell from entering S phase

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Fig 30.29 Rb and the cell cycle
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Tumor Suppressor Rb

• Overexpression of Rb impedes cell growth
• Rb- osteosarcoma cell lines
• transfected with Rb
• growth is impeded
• Effect of Rb reverted by D cyclins
• form cdk-cyclins that phosphorylate Rb
• Other Rb type proteins
• p107, p130

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Fig 30.30 Tumor suppressors and cell cycle
control


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Other cell cycle control proteins

• Also tumor suppressor proteins
• Small inhibitory proteins
• p16, p21, p27
• inhibit activity of cyclins in the quiescent
  state ( G0)
• inactivating mutations in tumors

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Tumor Suppressor p53

• Nuclear phosphoprotein
• Associates with T antigen in SV40 transformed
  cells
• In early experiments, found to immortalize cells
• these were mutant versions of p53!

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Fig 30.32 p53 Activity
Dominant Negative Mutants
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p53

• Wild type p53 restrains growth
• p53 mutations accumulate in many types of cancer
• Deletion of both alleles or point mutations
  produce the same phenotype
• Unrestrained cell growth
• p53 is involved in general/common control of cell
  proliferation

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p53 - tumor suppressor

• mutant p53 cells have a tendency to amplify DNA
• p53- mice are viable, develop variety of tumors
  at early stages
• wt p53 inhibits transformation of cells in culture

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p53 mutations

• Li Fraumeni syndrome
• cancer predisposition syndrome
• autosomal dominant
• heterozygotes for p53
• p53 mutations
• Dominant negatives

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Tumor suppressor p53

• Normal cells have low levels of p53
• p53 activated by irradation/DNA damage
• functions in growth arrest and/or apoptosis?
• depends on the stage of cell cycle!
• see Fig 30.33

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Fig 30.33 DNA damage activates p53
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Activated p53 Function

• In early G1, p53 triggers a checkpoint that
  blocks further progression through the cell cycle
• If the cell is committed to division, p53
  triggers apoptosis

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Fig 30.34 p53functional domains
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p53 functional domains

• DNA- binding protein
• 10 bp interrupted palindromic motif
• activates transcription (generally)
• N-terminal region - transactivator domain

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p53 functional domains

• Binds to damaged DNA via C-terminal region
• Tetramer/oligomerization via C-terminal region
• PXXP motif, SH-3 binding domain
• putative signaling domain

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p53 regulator of transcription

• N-terminal region
• N-region interacts directly with TBP
• p300/CBP binding required
• E1B 55Kd blocks p53 function
• Interacts directly with N-region
• SV40 T antigen blocks p53 function
• Interacts with DNA- binding region

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p53 Function

• C-terminal domain of p53 binds to damaged DNA
• lt 40nt ssDNA regions, mismatches
• p53 is activated and binds to target genes to
  activate transcription

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Model for p53 Activation

• Damaged DNA binding may cause changes in p53
  properties
• p53 dissociates from damaged site and binds to
  its targets

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p53 mutations

• Increase half-life time
• from 20 minutes to hours
• Change conformation
• detected by specific Ab
• Change cellular location
• from nucleus to cytoplasm
• Prevent DNA binding
• Prevent SV40 T antigen binding

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Fig 30.35 Pathways activated by p53
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Fig 30.35 Pathways activated by p53

• p21 pathway
• p21 cell cycle inhibitor
• prevents cells from proceeding through G1
• GADD45 pathway
• GADD45, repair protein,
• responds to irradiation damage,
• involved in other pathways

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Fig 30.35 Pathways activated by p53

• Apoptotic pathway???
• May be achieved through activation of different
  set of targets
• produce/activate proteins which act on
  mitochondrion to trigger apoptosis
• produce/activate cell surface receptors that
  trigger apoptosis

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p53 Regulation

• p53
• short half life
• response to DNA damage stabilizes the protein
  activates p53 transcription activity
• p53 induces Mdm2
• Mdm2 inhibits p53

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Fig 30.36 Inactivation of p53
129
Fig 30.37 p53 interactions
130
Control of p53

• Negative feedback loop
• p53 Mdm2
• Mdm2
• E3 ubiquitin ligase, triggers p53 degradation
• Acts directly with N-terminal region
• inhibits txn activity of p53

Activation of p53 increases Mdm2 Mdm2 limits p53
activity
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p53 (and Rb) are regulated by multiple pathways

• INK4A, two transcripts
• p16INK4A acts upstream of Rb
• p19ARF acts upstream of p53
• Locus deletions lead to loss of p53 and Rb
• common in human cancers

132
p53 (and Rb) are regulated by multiple pathways

• p16INK4A
• Inhibits cdk 4/6 kinase
• Rb is not phosphorylated, cell cycle is inhibited
• p19ARF
• antagonizes Mdm2
• p53 is stabilized, activity of p53 is increased

133
Control of p53

• p53 responds to enviromental signals that affect
  cell growth
• Most common form of modification
• serine phosphorylation
• lysine acetylation
• Different pathways lead to modifications of
  different residues

134
Fig 30.38 p53 modifications
135
Targets for various pathways located in the
terminal regions

• May affect
• Protein stability
• Oligmerization
• DNA-binding
• Protein- binding