Tumor Suppressor Genes

The story of Retinoblastoma

Retinoblastoma is a cancerous disease
1/20,000 children; 300 per
year
Average age is 18 months
Leukocoria or “white pupil”

Treatment:
enucleation = eye removal
Prognosis is good after
enucleation
over 90% survival with early
detection and treatment

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

Children with bilateral (familial)
Rb have a high risk of developing
non-retinal tumors
Familia
l
Sporad
ic

Germ-line mutations in the Rb
gene lead to predisposition to

In cancer patients with a family history of Retinoblastoma: the inheritance seems to be ? .

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 Knudson’s “Two Hit” Hypothesis for the Generation of RB Alfred Knudson. PNAS 68:820 (1971) .

The Knudson’s “two hit” hypothesis for the generation of RB .

other cancers .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.

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 .

Mutate d matern al Mutate d matern al -/+ Normal patern al Mutated paternal -/- -/- -/The mutated maternal chromosome was .

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 .

Retinoblastoma is inherited as a dominant trait. Cells that would get a second hit will develop Rb or later. but it is recessive at the cellular level People with familial Retinoblastoma carry one mutated copy in ALL their cells. other cancers .

but cancer is not inherited The offspring CANNOT inherit two mutated genes .Predisposition is inherited dominantly.

How can we clone a tumor-recessive gene? How do we test candidate genes? Oncogenes transform cells into cancerous cells But TSGs are recessive .

Rb tumors are associated with a deleted region in chromosome 13 Deletion = loss-of-function probably a recessive mutation in the Rb gene .

Angier book. starting p. 334 .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.

Rb gene expression is absent or altered in Rb tumors WT Other retinoblastoma tumors tumors Friend et al. Nature (86) Lee et al. Science (87) Northern blots (mRNA expression) .

We have correlation What about causation? The RB gene is finally .

Further Work Dr.Bold Predictions. David Abramson. According to Natalie Angier) “I believe that in fifteen years. I call it retino-revert.” . The drug will be an analogue of the natural protein that is missing in retinoblastoma cells … We’ll 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. RB expert at New York Hospital (ca. 1986. we’ll be able to stop retinoblastoma before it begins. or retino-prevent. I know I’m going out on a limb with this one. at the outside. but … Come back to me in 2001 and tell me if I wasn’t right. I’m so sure that I’ve already given the drug a name.

pRb: What does it do? pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in .

hyper-phosphorylated pRb becomes inert and the cell cycle can proceed .The guardian of the cell at earlymid G1 Hypophosphorylated or unphosphorylated pRb inhibits the cell from entering further a Upon new cell cyclephosphorylation at the R point.

.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 Hyperphosphorylation of Rb sequesters Rb.

activation Recruits proteins will “close” the chromatin down .Hypo-phosphorylated Rb binds to E2Fs and: .Inhibits their transcription sites that .

Release of their transcription activation sites .Recruitment of proteins that will “open up” the chromatin .Releasing Rb from the E2Fs leads to: .

the retinoblastoma protein regulates the cell cycleCell 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 w/o 2 copies ofphase Rb: no cell .Rb.

pRb: What does it do? pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in .

Rb activity is tightly regulated by the cell cycle clock Hypo-phosphorylation is catalyzed by cycDCDK4/6 Hyperphosphorylation is catalyzed by cycECDK2 .

pRb is hyper-phosphorylated and inhibited (and released from its role as a guardian). only upon cycE expression .

Rb activity is tightly regulated by the cell cycle clock However. only AFTER Rb is phosphorylated by cycD-CDK4/6 . E-CDK2 can phosphorylate Rb.

cycE can phosphorylate Rb and allow entry to the cell cycle . as a result. enough cycD-CDK4/6 activity).Have I grown enough? Only after we have enough mitogen signaling (and.

mostly involved in the first steps of DNA replication One of the targets: the cycE gene Transcription of cycE starts a positive feedback loop .E2Fs have more than 100 target genes.

As E2Fs are necessary for expression of cycE. think how critical negative regulation by Rb is for cell cycle control E2Fs .

Rb gene alteration is involved many tumors In the majority of tumors you will find mutation involved in the R site .

mutation). loss of CKIs or oncogenic activity of cyclins E and D .Uncontrolled crossing of the R site can be due to loss of Rb function (e.g.

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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. E2Fs.g. p16 and Rb . cycE.What not to focus on Details of the cell cycle (e.