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BioSci 145A Lecture 15 - Oncogenes and Cancer
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Topics we will cover today
– 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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Don’t forget that the next two lectures will be held in the
Beckman Laser Institute Library
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No office hours on the following dates (I will be out of
town)
– 3/1
– 3/6
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Office hours for 3/8 will be held on 3/9 from 2-3
BioSci 145A lecture 15
page 1
©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
– generally speaking, different genetic events can be
associated with characteristics of the developing
tumor.
BioSci 145A lecture 15
page 2
©copyright
Bruce Blumberg 2000. All rights reserved
Introduction to normal and cancer cells (contd)
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BioSci 145A lecture 15
page 3
©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
page 4
©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, 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 5
©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 6
©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
page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Cancerous changes in cells
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benign vs malignant tumors
– 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
BioSci 145A lecture 15
page 8
©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 9
©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 10
©copyright
Bruce Blumberg 2000. All rights reserved
Viruses can harbor transforming genes
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•
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Peyton Rous (1911)
– took chicken fibrosarcomas, ground them up, filtered
out all cells, cellular debris and things as small as
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)
when it was finally discovered that a virus was the
cause of the cancer
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) 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 11
©copyright
Bruce Blumberg 2000. All rights reserved
Viral oncogenes (contd)
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•
BioSci 145A lecture 15
page 12
©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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•
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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 13
©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 14
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viruses can be transferred
laterally from one organism
to another, carrying the
cancer potential along
•
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 15
©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 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 16
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor cell DNA can transform cultured cells
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BioSci 145A lecture 15
page 17
©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 18
©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 19
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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BioSci 145A lecture 15
page 20
©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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•
•
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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
page 21
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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One example signaling pathway - MAPK (Bardwell lab)
growth factor
receptor tyrosine kinase
ras
kinase cascase (serine/threonine)
•
•
transcription factors
since the signal passes from one component to the next,
inappropriate activation of one element in the cascade
canl lead to widespread changes in gene expression
– these pathways are not strictly linear but branch and
interact with many other signaling pathways
• can cause wider effects
• may require mutations in parallel pathways to
get oncogenesis
central importance of this pathway is illustrated by the
number of components that can be mutated into
oncogenes
– aberrant activation of mitogenic pathways can
contribute to oncogenicity
BioSci 145A lecture 15
page 22
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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Growth factor receptors are ligand modulated dimers
– EGF receptor (v-erbB) is the prototype member
• EGF binding stimulates dimerization and
activates tyrosine kinase cascade
• one oncogenic variant can dimerize in the
absence of ligand and signals constitutively
• another lacks an internal regulatory domain
resulting in constitutive signaling
– activated kinase domain autophosphorylates and can
then interact with src family proteins
BioSci 145A lecture 15
page 23
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
• transforming activity of
src-family kinases is
related to kinase activity
– autophosphorylation
controls activity
• Y416 -> active
• Y527 -> weak,
normally
suppresses
phosphorylation
of Y416
– some oncoproteins
activate src by
interfering with
phosphorylation of
Y527
BioSci 145A lecture 15
page 24
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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modulation of transcription factor activity is important
for oncogenesis
– can’t cause cancer without altering gene expression!
BioSci 145A lecture 15
page 25
©copyright
Bruce Blumberg 2000. All rights reserved
Oncogenes and cell growth (contd)
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transcription factors and cancer
– several prominent families of oncogenes are
transcription factors - rel, jun, fos, erbA, myc, myb
– actions may be quantitative or qualitative
• effects may be to increase activity of the
oncoprotein
– increased expression could upregulate
target genes and influence growth, e.g. AP1
• alternatively, the mutations could make the
oncoprotein a dominant negative inhibitor of
other cellular transcription factors (e.g. v-erbA)
– many members are “immediate early” genes
• transcription is immediately upregulated without
the requirement for new protein synthesis when
cells are treated with mitogens
– likely to be involved with initiating or
promoting growth
• increased activity would be expected to increase
oncogenesis and it does with some but not others
BioSci 145A lecture 15
page 26
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes
•
•
oncogenesis is not
typically dominant.
A growing number of
“tumor suppressor” genes
have been identified that
confer a genetic
predisposition to cancers
– several types of
genes are involved
• apoptosis
proteins (eg p53)
• cell-cycle
control proteins
(RB)
• DNA-repair
proteins (p53)
– classic examples are
RB (retinoblastoma)
and p53
– loss of tumor
suppressor genes is
implicated in several
infrequent cancers of
childhood
• retinoblastoma
• Wilm’s tumor
BioSci 145A lecture 15
page 27
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
• RB is a nuclear
phosphoprotein that
influences the cell cycle
– unphosphorylated
RB prevents cell
proliferation by
binding to E2F and
blocking G1/S
transition
– phosphorylation of
RB inhibits binding
to E2F and releases
block
– some oncogenes (e.g.
SV40 T-antigen,
E1A) function by
sequestering RB and
removing block to
cell growth
– similar effects by
loss of both alleles in
human disease
BioSci 145A lecture 15
page 28
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
•
•
A variety of other cell-cycle control proteins are tumor
suppressor genes
– p16, p21 and D cyclins
– shown by identification of inactivating mutations in a
variety of human tumors
in quiescent cells
– RB is not phosphorylated
– D cyclin levels are low or absent
– p16, p21 and p27 prevent activity of cdk-cyclin
complexes
• cdc2, cdk2 and cdk4,6 interact with cyclins and
promote cell cycle
• this is blocked by tumor suppressor genes
BioSci 145A lecture 15
page 29
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
•
P53 suppresses cell growth or triggers apoptosis
– more than 50% of human tumors have lost p53
protein or harbor mutations in the gene
– a variety of mutations are possible
• recessive mutations cause loss of p53 function
allowing unrestrained growth (eg. ko mice)
• others are dominant negative p53 mutants that
interfere with normal p53 subunits in cells and
allow unrestrained growth (eg rare cancers)
BioSci 145A lecture 15
page 30
©copyright
Bruce Blumberg 2000. All rights reserved
Tumor suppressor genes (contd)
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•
•
BioSci 145A lecture 15
page 31
p53 has dual functions
– cells normally have
low levels of p53
– DNA damage induces
large increase in p53
levels
– increased p53 leads to
growth arrest until
DNA is repaired if
cells are in G1
– cells in S-phase or later
are triggered to
become apoptotic
p53 is a transcription factor
that typically activates
– one target is p21 ->
cell cycle arrest
– another is GADD45 - a
DNA repair protein
– role in inducing
apoptosis is unknown
at present
apoptosis is an important
pathway in preventing
tumor formation - blocking
it is a common strategy
©copyright
Bruce Blumberg 2000. All rights reserved
Cancer - putting it all together
BioSci 145A lecture 15
page 32
©copyright
Bruce Blumberg 2000. All rights reserved