Molecular BIology of Cancer Topics             

Retinoblastoma

The retinoblastoma (Rb) gene is 200 kb in length and is located on chromosome 13q14. Its 4.7 kb mRNA is expressed in many tissues. The Rb protein product is a 105 kDa nuclear protein regulated by phosphorylation. Rb protein is phosphorylated in a cell-cycle dependent manner. It is hypophosphorylated during G0 and early G1, hyperphosphorylated during late G1, S and G2, and dephosphorylated during mitosis. Rb is phosphorylated at the restriction point by CDK4 and other cyclin D -dependent kinases. Hypophosphorylated Rb represses the activity of the transcription factor E2F, responsible for expression of genes involved in cell cycle progression and DNA synthesis. Genes with E2F binding sites in their promoter regions include c-myc and N-myc (G1), thymidine kinase, dihydrofolate reductase and DNA polymerase alpha (S), cdc2 and Rb (G2/M).

Rb gene expression in necessary during develoment. Mouse Rb expression is maximal at gestation days 12-14. There is high expression in liver, brain and in differentiating cells. Heterozygotes (Rb +/-) develop normally, this has been observed in both geneticaly altered mice and humans with inherited Rb mutation. Homozygous Rb knockout mice (Rb -/-) survive only to gestation day 12, suggesting that Rb function is important at this stage. They suffer from abnormal neural and hematopoietic development, although their eyes develop normally.

Cancers with Rb loss may be inherited or sporadic. Retinoblatoma and osteosarcoma can be due to either inherited or sporadic loss of Rb. Other cancers that may arrise from sporadic Rb loss only (not inherited) are small cell lung, breast, prostate, and leukemias.

Rb is a tumor suppresor gene, thus there is a loss of Rb protein function in cancer. Loss of function may occur by either deletion of Rb gene, point mutation or loss of heterozygosity in tumor (?). Rb-negative retinoblastoma and osteosarcoma cells regain their ability to produce Rb protein after being transfected with a wild type Rb cDNA. Transfection of Rb cDNA into Rb-negative tumor cells also inhibits thier ability to grow (Huang et. al. 1988; Science 242:1563). Also injection of retinoblastoma cells with reconstitutet Rb gene into mice lead to less tumors than injection of the original retinoblastoma cell line. (Madreperla et al 1991; Cancer Res 51:6381).

<incomplete: discovery of Rb and restriction fragment analysis>

Rb mutations found in tumors are inactivating due to deletion, splice site loss, truncation, frameshift or missense. All of these mutations result in an Rb gene that encodes a nonfuctional protein.

Rb protein can also be inactivated by tumor virus encoded proteins like SV40 large T antigen, human papilloma virus E7 protein and adenovirus E1A. All three of these viral oncoproteins share a common Rb binding site. The viral oncoproteins bind to hypophosphorylated Rb, suggesting that hypophosphorylated Rb is responsible for growth and/or tumor suppression. Viral oncoproteins bind to Rb and release E2F, which may help to drive the infected cells into S phase.

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1- Describe the retinoblastoma gene and mRNA.
The gene is 200 kb in length and is located on chromosome 13q14. The mRNA is 4.7 kb and expressed in many tissues.

2- Describe the Rb protein.
The 105 kDa nuclear protein is regulated by phosphorylation.

3- What controls Rb phosphorylation?
Rb is phosphorylated in a cell-cycle-dependent manner. It is hypophosphorylated during G0 and early G1, hyper phosphorylated during late G1, S and G2, and dephosphorylated during mitosis.

4- Which protein(s) phosphorylate Rb and when?
CDK4 at the restriction point, later other cyclin-dependent kinases.

5- What is the function of Rb?
Hypophosphorylated Rb represses the activity of the transcription factor E2F.

6- What is the function of E2F?
Transcription factor responsible for the expression of genes involved in cell cycle progreassion and DNA synthesis.

7- List 7 genes with E2F response elements and the cell cycle stage that they affect.
c-myc, G1
N-myc, G1
thymidine kinase, S
dihydrofolate reductyase, S
DNA polymerase a, S
cdc2, G2/M
Rb, G2/M

8- Describe the experimental data showing that Rb is necessary for development in mice.
Rb expression is maximal at gestation days 12-14, high in lever, brain and differentiating cells. Heterocygotes (+/-) develop normally by homozygous survive only to gestation day 12 and suffer from normal neural and hematopoietioc development.

9- Which cancer types can be due to inherited vs. sporadic loss of Rb?
Retinoblastoma and osteosarcoma may be due to inherited or sporadic Rb loss. Small cell lung cancer, breat, cancer, prostae cancer and leukemias may arise from sporadic Rb loss only.

10- Which alterations of Rb cause cancer and why?
Rb is a tumor supressor gene, thus a loss of Rb protein function causes cancer. Loss of function may occur by either deletion of Rb gene, pouint mutation or loss of heterozygosity.

11- What are the results of in vitro experiments were wild-type are transfected into cancer cells?
Rb-negative retinoblastoma and osteosarcoma cells regain their ability to produce Rb protein and stop proliferating after being transfected with wild type Rb cDNA.

12- What are the results of in vivo experiments were wild-type was injected into mice?
Injection of retinoblastyoma cells with reconstituted Rb gene into mice lead to less tumors than injection of the original retinoblastoma cell line.

13- Discovery of Rb.
<incomplete>

14- List 5 types of Rb mutations found in tumors that encode nonfunctional protein.
deletion
splice site loss
truncation
franeshift
missense

15- List 3 viral oncoproteins that can inactivate Rb.
SV40 large T antigen
human papilloma virus E7 protein
adenovirus E1A protein

16- What is the mechanism of action of viral oncoproteins that inactivate Rb?
They have a common Rb binding site. They bind to hypophosphorylated Rb and release E2F, which drives the infected cell into S phase.

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