The story of Retinoblastoma

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Tumor Suppressor Genes
The story of Retinoblastoma
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Retinoblastoma is a cancerous disease
1/20,000 children 300 per year Average age is 18
months Treatment enucleation eye
removal Prognosis is good after enucleation over
90 survival with early detection and treatment
Leukocoria or white pupil
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Rb is either sporadic or familial
- Sporadic cancer in 55-65 of all cases -
Sporadic cancers are unilateral
Hereditary childhood cancer - bilateral tumors
in 75 of cases - unilateral tumors in 25 of
cases
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Children with bilateral (familial) Rb have a high
risk of developing non-retinal tumors
Familial
Sporadic
Germ-line mutations in the Rb gene lead to
predisposition to cancer
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In cancer patients with a family history of
Retinoblastoma the inheritance seems to be ?
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Rb tumors are associated with a deleted region in
chromosome 13
Deletion loss-of-function probably a
recessive mutation in the Rb gene
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The Knudsons Two Hit Hypothesis for the
Generation of RB
Alfred Knudson, PNAS 68820 (1971)
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The Knudsons two hit hypothesis for the
generation of RB
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Retinoblastoma is inherited as a dominant trait,
but it is recessive at the cellular level
People with familial Retinoblastoma carry one
mutated copy in ALL their cells. Cells that would
get a second hit will develop Rb or later, other
cancers
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Loss of heterozygosity (LOH)
Loss of heterozygosity (LOH) in a cell represents
the loss of normal function of one allele of a
gene in which the other allele was already
inactivated
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Normal paternal
Mutated maternal
-/-
-/
-/-
Mutated paternal
Mutated maternal
The mutated maternal chromosome was duplicated
-/-
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The presence of one mutated copy increases the
chances of a second mutated copy
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Rb is just one example
Inheritance of brca1(lf) mutation results in
predisposition for breast cancer
Rb A Tumor Suppressor Gene
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Retinoblastoma is inherited as a dominant trait,
but it is recessive at the cellular level
People with familial Retinoblastoma carry one
mutated copy in ALL their cells. Cells that would
get a second hit will develop Rb or later, other
cancers
17
Predisposition is inherited dominantly, but
cancer is not inherited
The offspring CANNOT inherit two mutated genes
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How can we clone a tumor-recessive gene?
How do we test candidate genes?
Oncogenes transform cells into cancerous cells
But TSGs are recessive
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Rb tumors are associated with a deleted region in
chromosome 13
Deletion loss-of-function probably a
recessive mutation in the Rb gene
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Testing a candidate gene
Use a fragment of the candidate gene as a probe
for Southern Blot analysis
Search for absence of the gene in tumors (hoping
both mutated copies are deletions)
More on this- Angier book, starting p. 334
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Rb gene expression is absent or altered in
retinoblastoma tumors
Rb tumors
WT
Other tumors
Friend et al. Nature (86)
Lee et al. Science (87)
Northern blots (mRNA expression)
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We have correlation
What about causation?
The RB gene is finally cloned
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Bold Predictions, Further Work
Dr. David Abramson, RB expert at New York
Hospital (ca. 1986, According to Natalie
Angier) I believe that in fifteen years, at the
outside, well be able to stop retinoblastoma
before it begins. Im so sure that Ive already
given the drug a name. I call it retino-revert,
or retino-prevent. The drug will be an analogue
of the natural protein that is missing in
retinoblastoma cells Well be able to diagnose
a child prenatally and start giving this
retino-revert to the mother to prevent
retinoblastomas from growing as the fetus is
developing. I know Im going out on a limb with
this one, but Come back to me in 2001 and tell
me if I wasnt right.
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pRb What does it do?
pRb is a nuclear protein that undergoes
phosphorylation and dephospharylation in concert
with the cell cycle
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The guardian of the cell at early-mid G1
Hypo-phosphorylated or un-phosphorylated pRb
inhibits the cell from entering a new cell cycle
Upon further phosphorylation at the R point,
hyper-phosphorylated pRb becomes inert and the
cell cycle can proceed
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Hypo-phosphorylated Rb inhibits activity of the
E2F family of transcription factors
E2Fs are needed for transcription of genes that
are essential for the cell to enter the cell cycle
Hyper-phosphorylation of Rb sequesters Rb, and
releases E2Fs
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Hypo-phosphorylated Rb binds to E2Fs and
- Inhibits their transcription activation sites
- Recruits proteins that will close the
chromatin down
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Releasing Rb from the E2Fs leads to
- Release of their transcription activation sites
- Recruitment of proteins that will open up the
chromatin
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Rb, the retinoblastoma protein regulates the cell
cycle
Cell cycle OFF Rb binds to E2F no
transcription, no entry into S phase
Cell cycle ON Rb does not bind to E2F
transcription and entry into S phase
w/o 2 copies of Rb no cell cycle arrest
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pRb What does it do?
pRb is a nuclear protein that undergoes
phosphorylation and dephospharylation in concert
with the cell cycle
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Rb activity is tightly regulated by the cell
cycle clock
Hypo-phosphorylation is catalyzed by cycD-CDK4/6
Hyper-phosphorylation is catalyzed by cycE-CDK2
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pRb is hyper-phosphorylated and inhibited (and
released from its role as a guardian), only upon
cycE expression
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Rb activity is tightly regulated by the cell
cycle clock
However, E-CDK2 can phosphorylate Rb, only AFTER
Rb is phosphorylated by cycD-CDK4/6
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Have I grown enough?
Only after we have enough mitogen signaling (and,
as a result, enough cycD-CDK4/6 activity), cycE
can phosphorylate Rb and allow entry to the cell
cycle
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E2Fs have more than 100 target genes, mostly
involved in the first steps of DNA replication
One of the targets the cycE gene
Transcription of cycE starts a positive feedback
loop
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As E2Fs are necessary for expression of cycE,
think how critical negative regulation by Rb is
for cell cycle control
E2Fs
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Rb gene alteration is involved many tumors
In the majority of tumors you will find mutation
involved in the R site
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Uncontrolled crossing of the R site can be due to
loss of Rb function (e.g. mutation), loss of CKIs
or oncogenic activity of cyclins E and D
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What not to focus on
Details of the cell cycle (e.g. what happens in
prometaphase)
Molecular details of ubiqu. pre-replicative
complex, etc.
What to focus on
Her-2
Cell cycle control
Regulation of CDKs
Mitogens and the cell cycle
Rb genetics
The restriction point cycD, cycE, E2Fs, p16 and
Rb (read the textbook chapter 8)