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Retinoblastoma is a cancerous disease
1/20,000 children; 300 per yearAverage age is 18 months
Treatment: enucleation = eye removal
Prognosis is good after enucleationover 90% survival with early detection and treatment
Leukocoria or “white pupil”
- Sporadic cancer in 55-65% of all cases- Sporadic cancers are unilateral
Rb is either sporadic or familial
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
Germ-line mutations in the Rb gene lead to predisposition to cancer
Sporadic
Familial
Rb tumors are associated with a deleted region in chromosome 13
Deletion = loss-of-functionprobably a recessive mutation in the Rb
gene
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
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
Loss of heterozygosity (LOH)
Normal paternal
Mutated maternal
Mutated maternal
-/+
-/-
-/-
-/-
Mutated paternal
The mutated maternal chromosome was duplicated
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, 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
Predisposition is inherited dominantly, but cancer is not inherited
The offspring CANNOT inherit two mutated genes
How can we clone a tumor-recessive gene?
Oncogenes transform cells into cancerous cells
But TSGs are recessive
How do we test candidate genes?
Rb tumors are associated with a deleted region in chromosome 13
Deletion = loss-of-functionprobably a recessive mutation in the Rb
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)
Testing a candidate gene
More on this- Angier book, starting p. 334
Rb gene expression is absent or altered in retinoblastoma tumors
Friend et al. Nature (86) Lee et al. Science (87)
Northern blots (mRNA expression)
Rb tumors WT Other tumors
Dr. David Abramson, RB expert at New York Hospital (ca. 1986, According to Natalie Angier)
“I believe that in fifteen years, at the outside, we’ll be able to stop retinoblastoma before it begins. I’m so sure that I’ve 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 … 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. I know I’m going out on a limb with this one, but … Come back to me in 2001 and tell me if I wasn’t right.”
Bold Predictions, Further Work
pRb: What does it do?
pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in concert with the cell cycle
Hypo-phosphorylated or un-phosphorylated pRb inhibits the cell from entering a new cell cycle
The guardian of the cell at early-mid G1
Upon further phosphorylation at the R point, hyper-phosphorylated pRb becomes inert and the
cell cycle can proceed
Hypo-phosphorylated Rb inhibits activity of the E2F family of transcription factors
Hyper-phosphorylation of Rb sequesters Rb, and releases E2Fs
E2Fs are needed for transcription of genes that are essential for the cell to enter the cell cycle
Hypo-phosphorylated Rb binds to E2Fs and:
- Inhibits their transcription activation sites
- Recruits proteins that will “close” the chromatin down
Releasing Rb from the E2Fs leads to:
- Release of their transcription activation sites
- Recruitment of proteins that will “open up” the chromatin
Rb, the retinoblastoma protein regulates the cell cycle
Cell cycle = OFFRb binds to E2F: no transcription, no entry into S phase
Cell cycle = ONRb does not bind to E2F: transcription and entry into S phase
w/o 2 copies of Rb: no cell cycle arrest
pRb: What does it do?
pRb is a nuclear protein that undergoes phosphorylation and dephospharylation in concert with the cell cycle
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
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, E-CDK2 can phosphorylate Rb, only AFTER Rb is phosphorylated by cycD-CDK4/6
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
Have I grown enough?
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
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 tumorsIn the majority of tumors you will find mutation
involved in the R site
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
What to focus on
Cell cycle controlRegulation of CDKs
Mitogens and the cell cycle
Rb: genetics
The restriction point: cycD, cycE, E2Fs, p16 and Rb(read the textbook – chapter 8)
Details of the cell cycle (e.g. what happens in prometaphase)
What not to focus on
Molecular details of ubiqu. pre-replicative complex, etc.
Her-2