Transcript Transcription factors

BioSci 145A Lecture 15 - Oncogenes and Cancer
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Topics we will cover today
– Important motifs in transcription factors
• helix-turn-helix
– homeobox genes
• helix-loop-helix
– myogenic genes
• bZIP proteins
– Introduction to normal and cancer cells
• Characteristics of cells in culture
• Cancerous changes in cells
• Viruses can harbor transforming genes
• DNA from tumor cells can transform normal
cultured cells
• Oncogenes and cell growth
• tumor suppressor genes
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Last year’s final exam is posted
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Next two lectures are by Dr. LaMorte in BLI
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No lecture on 3/12
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Review session on 3/14
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Final on Tuesday 3/19
BioSci 145A lecture 15
page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Homeobox genes
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helix-turn-helix motif is widely used in transcription
factors
– phage repressors
– 2 helices come to lie at almost right angles to each
other
– the recognition helix fits in the major groove of DNA
while other helices make minor groove contacts
– target sequence discrimination is by only a few
residues in the recognition helix
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Homeobox genes (contd)
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Homeobox is a highly-conserved, 180 bp DNA sequence
– only DNA has a homeobox
homeodomain is the protein product of the homeobox
and.
– proteins encoded by homeobox genes are
homeodomain proteins
very large family of genes
– first discovered in Drosophila homeotic selector
genes, Antennapedia and Ultrabithorax
– low stringency hybridization showed that there were
many such genes in the Drosophila genome
– in a “secret” experiment, Bill McGinnis (Walter
Gehring’s lab) and Andrés Carrasco (Eddy De
Robertis’s lab) decided to test whether such
sequences occurred in vertebrates
• low stringency Southern blot was performed, the
first “zoo blot”
• several genes were identified, Cell papers
published and feelings were hurt
Drosophila (Hom-C) and vertebrate Hox genes control
the identity of body segments during development. Loss
of function mutations cause changes in segment identity
– incredibly, corresponding vertebrate genes can
completely rescue fly mutations
– Transcription factors are developmental switches
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Homeobox genes (contd)
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this residue is very important for determining specificity
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Homeobox genes (contd)
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We will talk more about homeobox genes in the last two
lectures
– for now, it is sufficient to note that homeobox genes
are critical for normal development
– more than 400 different types already known
– homeodomain proteins can act as transcriptional
activators or repressors
• many people spent years trying to demonstrate
activation of reporter genes by homeodomain
proteins with little success
• it later turned out that the ones that were being
tested were repressors
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mutations in homeobox genes cause developmental
defects in humans
– mutations in emx2 homeobox gene (related to
Drosophila empty spiracles) causes schizencephaly
• cortical malformation that manifests
developmental delay, blindness, seizures, and
other neurological disabilities
– mutations in MSX-2 lead to Boston-type
craniosynostosis
• cranial bones fuse inappropriately
BioSci 145A lecture 15
page 5
©copyright
Bruce Blumberg 2000. All rights reserved
Helix-loop-helix proteins
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HLH proteins are a large group of dimeric proteins
– signature motifs are two stretches of amphipathic helices flanking a central loop (linker) of variable
size
• protein protein interaction is mediated via
hydrophobic interactions
• regions of strong sequence conservation within
the helices among related proteins
– not all have the ability to bind DNA, these are
typically negative regulators
– those that can bind DNA tend to have a basic region
adjacent to the HLH motif
• these are called bHLH proteins
dimerization regulates function
– two basic regions are required for DNA binding
– two groups of bHLH proteins exist
• Class A are ubiquitously expressed (eg E12/E47)
• Class B are tissue-specific (MyoD, myogenin)
– a common strategy among tissue-specific proteins is
to heterodimerize with ubiquitous partners
– homodimers are not very stable and do not bind
DNA with high affinity
– heterodimers between bHLH and HLH proteins are
typically nonfunctional, an important regulatory
mechanism
BioSci 145A lecture 15
page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Helix-loop-helix proteins (contd)
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Helix-loop-helix proteins (contd)
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bHLH proteins and muscle development
– MyoD was the first discovered. Identified in an
expression screen as a single protein that could
transform cultured fibroblasts (3T3) into muscle
(myotubes)
– MyoD acts first to kick ID off of E12 and/or E47 and
initiates the muscle program.
• Later bHLH genes such as myogenin and myf5
are also important
– this family of genes illustrates the general principle
that combinatorial associations of transcription
factors can yield complexes with different functions
• DNA binding
• transcriptional regulation
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Leucine zipper (b-ZIP) proteins
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Leucine zipper is a protein:protein interaction domain
characterized by coiled-coil -helical structure
– coiled-coil is a common structural motif in proteins
(e.g. myosin)
– coiled-coil is formed from two helices wound around
each other and typified by large hydrophobic amino
acids (leu, ile) repeated every 7 residues
– the helices are usually amphipathic
– leucine zipper flanked by a basic region is common
in transcription factors, so-called b-ZIP motif.
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Leucine zipper (b-ZIP) proteins (contd)
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bZIP is a common motif in viral transcriptional activators
and some enhancer binding proteins
– eg C/EBP (CAAT box enhancer binding protein)
like bHLH proteins, bZIP proteins are regulated by
heterodimerization.
– dimers have distinct functions
– not all proteins can homodimerize
• eg c-jun can homodimerize to bind DNA
• c-fos can not
• c-jun and c-fos can heterodimerize to produce
the transcription factor AP-1
– the jun/fos heterodimer binds DNA ~10x better than
the jun homodimer although both prefer the same
DNA target sequence
target sequences for all dimeric proteins have two
half-sites.
– dyad symmetry
• AGGTCACACTGACCT
• AGGTCAAAGGAGGTCA
– this is a signature feature and should always cause
you to suspect a dimeric transcription factor
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Introduction to normal and cancer cells
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Most cells in the organism have a finite lifetime
– majority of differentiated cells are postmitotic
• stem cells can divide nearly endlessly
• other cell types that typically divide
– skin
– lining of gut
– hematopoeitic stem cells
– hair follicles
• liver cells can dedifferentiate, re-enter the cell
cycle
– cell growth and division are tightly controlled
• most cells that can divide are only capable of a
finite number of cell divisions
– so-called Hayflick limit
• cancer cells are a notable exception
Cancer cells have lost their ability to regulate their own
growth or to respond to normal growth regulatory cues or
to sense their proper location in the organism
– each of these characteristics of cancer cells
contributes to disease progression
– a variety of genetic events are responsible
– different genetic events can be associated with
characteristics of the developing tumor.
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Introduction to normal and cancer cells (contd)
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BioSci 145A lecture 15
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©copyright
Three types of changes occur
as a cell becomes
tumorigenic
– immortalization - cells
retain the ability to
divide endlessly
• not necessarily
detrimental to
organism
• telomerase
– transformation - cells
stop responding to
normal growth controls
• do not need growth
factors and/or
• do not respond to
growth inhibitors
• transformed cells
typically form
tumors in situ
– metastasis - cells gain
the ability to move from
their normal location and
invade other tissues
• very dangerous
feature of cancer
cells
• aberrant regulation
of extracellular
matrix proteases
Bruce Blumberg 2000. All rights reserved
Cells in culture
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growth characteristics of normal and tumor cells differ
– normal cells do not grow well, in vitro, typical
cancer cells grow very well
– primary cells are the immediate descendents of cells
taken directly from a tissue.
• such cells divide a small number of times and
then stop growing - senescence
• subsequently, most cells will die.
– Lewin calls this the crisis stage
– if the cells are kept and fed for a long time,
a small number may begin to grow
– cell lines are cells that successfully pass through
crisis and gain the ability to divide indefinitely
• many, if not most, overexpress telomerase
Fundamental rule - Cells (even primary cells) change
their phenotype almost immediately when they are placed
in culture
– degree of difference depends on the similarity of
their microenvironment to their usual environment
• extracellular matrix
• type and density of surrounding cells
– change usually comes after several cell divisions.
• primary cells that stop dividing will maintain
more of their phenotype than those that divide
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
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Characteristics of cells in culture - most cells grow as a
monolayer for the following reasons:
– anchorage dependence - cells require a substrate to
grow on
• solid or semi solid medium
– serum dependence - cells require substances in serum
to grow
• commonly called growth factors but in reality
there are two different types
– mitotic factors - required for cells to grow
and divide
» typically peptide growth factors, e.g.
FGF, EGF, PDGF, NGF, etc
– survival factors - not strictly required for
cell division, but required for cells to
survive in culture
» typically lipids or other small
molecules, e.g. retinol, 14-hydroxy
retroretinol
– density-dependent inhibition (contact inhibition) cells only grow until confluence
• surface is completely covered
• at this time cells go into G0 and exit the cell
cycle
– cytoskeletal organization - cells are flat and extended
on the surface
BioSci 145A lecture 15
page 14
©copyright
Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
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illustrates
– morphological differences
• flat vs rounded up
– contact inhibition
• transformed cells pile up on plates and cluster in
3D
– nuclear morphology
• note strong staining in transformed cells, a
higher resolution picture would show
multinucleate cells and mitotic figures
BioSci 145A lecture 15
page 15
©copyright
Bruce Blumberg 2000. All rights reserved
Cells in culture (contd)
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How does one judge the “normalcy” of cultured cells?
– much can be surmised from morphology
– what is the chromosomal constitution of the cells?
• chromosomal duplications, deletions and
translocations are common in culture
• cells that have changed from normal, diploid
state are aneuploid
– are the cells anchorage dependent?
• most normal cells (except blood cells) are
anchorage dependent
• many transformed cells can grow in soft agar
– are the cells serum dependent?
• many abnormal cells are serum independent
• but many “normal” cell lines can be adapted to
low or serum-free conditions
– do the cells express normal protein complement?
– do the cells form tumors if injected into animals?
• if not, they are not “transformed”
cells originating from tumors are typically transformed
– reduced serum dependence
– reduced anchorage independence
– reduced contact inhibition - cells grow in foci
– will cause tumors if injected into animals
• typically use nude mice (lack significant part of
immune system). somewhat cheating
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells
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benign vs malignant tumors
– Tumor = swelling caused by new tissue growth
– benign tumors contain cells that look and function
like normal cells
• express normal complement of proteins
• typically remain localized to appropriate tissues
– often surrounded by a fibrous capsule of
connective tissues
• can become problematic if:
– their size interferes with normal function of
the tissue (e.g. brain tumor)
– they secrete excessive amounts of
biologically active substances such as
hormones (e.g. pituitary tumor)
– malignant tumors look qualitatively different from
normal tissues of origin
• close enough to determine tissue of origin but
not identical to normal tissue
• express only a subset of normal proteins
• many grow and divide more rapidly than normal
• can remain encapsulated in situ for a time (e.g.
carcinoma in situ)
• later become invasive and metastatic (definition
of malignant)
– many tumors produce growth factors that increase
the local blood supply
• Inhibiting angiogenesis is promising treatment
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells (contd)
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Induction of tumors
– discovery of oncogenes led to the model that genetic
changes could cause cancer
– tumor incidence increases with age -> a series of
events is required to cause a tumor
• believed that 6-7 discrete genetic events are
required to get a cancer
– agents that increase frequency of cell transformation
are called carcinogens
• can be classified according to properties
• tumor initiators cause tumors
– typically cause DNA damage (e.g.
benzapyrene-diol-epoxide)
• tumor promoters aid in the growth of
transformed cells, typically by inhibiting growth
control (e.g. phorbol esters)
BioSci 145A lecture 15
page 18
©copyright
Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells (contd)
– two classes of genes are targets of mutations that
cause transformation
• oncogenes encode proteins that can transform
cells or cause cancer in animals
– most are dominant gain of function
mutations - three basic types
– point mutations that cause constitutively
active protein products
– gene amplification that leads to
overexpression
– translocations that result in inappropriate
expression (Dr. La Morte)
• tumor suppressor genes are recessive, loss-offunction mutations that inactivate cellular genes
that regulate growth or cell cycle
– five classes of tumor suppressor genes
– intracellular proteins that regulate or inhibit
progression through the cell cycle
– receptors for secreted hormones that should
inhibit cell proliferation (e.g. TGF-beta)
– checkpoint control proteins that arrest the
cell cycle if DNA is damaged or
chromosomes are abnormal
– proteins that promote apoptosis
(programmed cell death)
– enzymes that participate in DNA repair
BioSci 145A lecture 15
page 19
©copyright
Bruce Blumberg 2000. All rights reserved
Viruses can harbor transforming genes
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Peyton Rous (1911)
– took chicken fibrosarcomas, ground them up, filtered
out all cells, cellular debris and bacteria
– injected this filtrate into other chickens ->
fibrosarcomas
• Rous sarcoma virus remains one of the most
virulent tumor viruses ever discovered
– received the Nobel Prize in 1966 (55 years later, at age
86) when it was finally discovered that a virus was the
cause of the cancer
– http://www.nobel.se/medicine/laureates/1966/rousbio.html
– RSV contains an oncogene v-src that was demonstrated
to be required for cancer induction
• RSV is a retrovirus with only 4 genes so this was
relatively easy to demonstrate
Bishop and Varmus (1977)
– showed that normal cells from chickens and other
species contained a cellular homolog of v-src.
– This c-src (cellular src) was the first proto-oncogene
– fundamental discovery that revolutionized the field
(and got them a Nobel prize in 1989) was that cancer
may be induced by the action of normal, or nearly
normal cellular genes that were incorporated into
transducing viruses
– turns out that c-src is a protein tyrosine kinase that is
constitutively active when mutated
BioSci 145A lecture 15
page 20
©copyright
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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BioSci 145A lecture 15
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©copyright
many acutely
transforming
retroviruses exist
– affect a variety of
species
– impact many
cellular signaling
pathways
fundamental
mechanism is
transduction of
cellular gene and later
mutation due to
inaccurate viral
reverse transcriptases
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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oncogenes may be involved in many types of cancers
– same c-onc (cellular oncogene) may be represented
as v-onc (viral oncogenes) in a variety of cancers
• sis in both simian and feline sarcoma viruses
– viruses may contain related v-onc genes
• Harvey and Kirsten sarcoma viruses contain vras genes derived from two different members of
the c-ras family
evidence exists directly linking oncogenes from acutely
transforming retroviruses with cancer
– first obtained from RSV using temperature sensitive
mutations in v-src that allowed the phenotype to be
reverted and regained
identification of dominant oncogenes from acutely
transforming retroviruses led to the model that single
gene changes could cause cancers
– major opponent to this idea was Peter Duesberg who
later became somewhat infamous for his criticism of
the involvement of HIV in AIDS
• in this case, Duesberg was correct
– although the data linking acutely transforming
retroviruses with cancer are strong, this mechanism
is considered to be a relatively minor cause of cancer
in humans
BioSci 145A lecture 15
page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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most acutely transforming retroviruses require normal
retroviruses to get packaged into infective particles
growth-promoting genes transduced by retroviruses
confer a selective advantage because they increase the
proliferation of infected tissues
– retroviruses cannot replicate unless cell is
proliferating
BioSci 145A lecture 15
page 23
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viruses can be transferred
laterally from one organism
to another, carrying the
cancer potential along
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viruses can also be
transferred to offspring
©copyright
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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Not all viruses are acutely carcinogenic
– slow-acting retroviruses
• cause cancers by integrating near cellular
protooncogenes and activating them
inappropriately
• act slowly because integration into cellular
protooncogenes is a rare event and other
mutations may be required
– various DNA viruses
• oncogenic potential resides in a single function
or group of related functions that are activated
early in viral lytic cycle
• many oncogenes act by inactivating tumor
suppressor genes
– polyoma T antigens
– papilloma virus E6,7 antigens and cervical
cancer
– adenovirus E1A,B
BioSci 145A lecture 15
page 24
©copyright
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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Models for differences in properties between c-onc and
v-onc
– quantitative model
• viral genes are functionally indistinguishable
from normal cellular genes
• oncogenesis comes from
– overexpression
– expression in inappropriate cell types
– failure to turn expression off
– qualitative model
• c-onc genes are not intrinsically oncogenic
• mutations can convert proto-oncogenes into
oncogenes
– that acquire new properties
– or lose old properties
– as usual, both models are correct
• mos, sis and myc genes can confer oncogenesis
without significant mutation
• ras and src are changed by point mutations into
dominant transforming oncogenes
BioSci 145A lecture 15
page 25
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor cell DNA can transform cultured cells
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BioSci 145A lecture 15
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©copyright
DNA from any of a
variety of tumors can be
transfected into cultured
cells (typically NIH 3T3
cells)
– a small number take
up DNA and form
foci of transformed
cells
– DNA is extracted
from these foci and
re-transfected into
fresh cells to enrich
for the specific
human sequence
– genomic library is
prepared and human
clones selected by
hybridizing with
repetitive DNA
(Alu)
– oncogene
responsible is
isolated and
characterized
Bruce Blumberg 2000. All rights reserved
Tumor cell DNA can transform cultured cells (contd)
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Using such methods, a variety of human oncongenes were
identified
– two important properties were identified in oncogenes
isolated in this way
• many have closely related sequences in the DNA
of normal cells
– this argues that the transformation was
caused by mutation of a normal cellular gene
(proto-oncogene)
– could be a point mutation or reorganization
of genomic DNA
• many have counterparts in the oncogenes carried
by acutely transforming retroviruses
– e.g.mutations were found in human bladder
cancer DNA that corresponded those in the
Ha-ras gene from harvey sarcoma virus.
– oncogenes found in this manner frequently do not
cause tumors when introduced into normal cells
• NIH-3T3 cells already have a mutation in a tumor
suppressor gene that, in combination with the
introduced oncogene, could lead to transformation
It is important to note that DNA with transforming activity
can only be isolated from tumorigenic cells
– not present in normal DNA
– in general, this is not such a great way to identify
oncogenes
BioSci 145A lecture 15
page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth
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Seven classes of proteins control cell growth
– Collectively, these genes comprise the known set of
genes involved in tumor formation
BioSci 145A lecture 15
page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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BioSci 145A lecture 15
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©copyright
Dominant
transforming
oncogenes are
frequently created
from proteins
involved in
regulating cell
growth
– Growth factors
– Growth factor
receptors
– Intracellular
transducers of
above
– Transcription
factors that
mediate the
terminal effects
of extracellular
signaling
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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Growth factors - proteins secreted by one cell that act on
another cell (eg sis, wnt, int)
– oncoprotein growth factors can only transform cells
that harbor the specific receptor
Growth factor receptors - transmembrane proteins that
are activated by binding to extracellular ligand (protein)
– very frequently protein tyrosine kinases
– oncogenicity usually results from constitutive
(ligand-independent) activation
Intracellular transducers - several classes
– protein tyrosine kinases, e.g. src
– G-protein signal transduction pathways - primary
effectors of activated growth factors (e.g. ras)
– protein serine/threonine kinases (e.g. mos, raf)
Transcription factors - these regulate gene expression
directly
– myc - HLH protein
– fos, jun - b-ZIP proteins
– erbA - nuclear receptor
common feature among these is that each type of protein
can trigger general changes in cell phenotypes by:
– initiating changes that lead to cell growth
– respond to signals that cause cell growth
– altering gene expression directly
BioSci 145A lecture 15
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©copyright
Bruce Blumberg 2000. All rights reserved