3.3 Rb/p53

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The first identified tumour suppressor, retinoblastoma (Rb), is a constitutively expressed gatekeeper tumor suppressor, encoded by the Rb1 gene located on chromosome 13. Inactivation of Rb1 is found in at least one-third of human tumors. You may recall from the video in the previous section that one of Rb's functions is to regulate the cell cycle. The Rb protein plays an essential role as a cell-cycle regulator acting at the G1/S cell cycle checkpoint, or more specifically, at the restriction point (R-point). Rb sequesters the transcription factor E2F, preventing entry into S phase (9).



The phosphorylation state of Rb determines its function. Rb is an active repressor of cell proliferation when hypophosphorylated and an inactive repressor when hyperphosphorylated. In response to positive growth signals, Rb is phosphorylated during the end of G1 phase at the R-point, or restriction point, by the cyclin D-Cdk4/6 complex as well as the cyclin E-Cdk2 complex (19). The MAPK signaling pathway controls the phosphorylation of the cyclin D-Cdk4/6 complex. INK4 is a tumor suppressor that inhibits Cdk4 and Cdk6, thus preventing Rb from being phosphorylated. INK4a and ARF, another important tumor suppressor involved in cell cycle arrest and apoptosis (via p53 activation), are expressed from the same locus via alternative splicing (10). INK4a is believed to play a role in limiting cell growth and transitioning the cell into a senescence. This activation can be caused by numerous factors, particularly signals arising from continuous DNA damage (10). After S phase is complete, Rb remains phosphorylated until the end of M phase, where it becomes hypophosphorylated, sequesters E2F, and inhibits G1/S phase progression (4).  

Figure 3.3.1. Dysfunction of tumour suppressor genes leads to Rb hyperphosphorylation during the R-point transition, releasing the transcription factor E2F. Released under the Creative Commons Attribution-ShareAlike 4.0 International license (CC BY-SA 4.0).



Besides phosphorylation, Rb function can be inhibited through several mechanisms including mutation, viral oncoprotein binding, dysregulation of kinases that control Rb, or degradation during apoptosis. The oncogenic viruses SV40 large T antigen, adenovirus E1A protein, and HPV 16 and 18 E7 proteins can bind to Rb, dissociating Rb from E2F so that the virus can manipulate the host cell to replicate viral DNA in the S phase. Amplified Cdk4 and cyclin D genes, or deletion of Cdk inhibitors p15 or p16 can also cause loss of Rb function. If Rb is absent, constitutive expression of cyclins A, E, or D and intact Rb can sometimes rescue osteosarcoma cells (6).


Downstream activities:

1) Blocking transcription

Rb binds to several proteins, one of which is E2F, a transcription factor that activates genes involved in growth control and DNA synthesis. E2F forms a heterodimer with its DP-partner and directly binds DNA to control the transcriptional activity of essential cellular proliferation genes that include cell cycle regulators, nucleotide biosynthesis enzymes, and DNA replication machinery (19). 

The retinoblastoma protein is part of the pocket-protein family that also contains p107 and p130. The members of this family share a conserved pocket domain. When hypophosphorylated, the pocket proteins bind to various members of the E2F transcription factor family (11). Hypophosphorylated Rb predominantly binds to E2F1/2/3, also known as the activating E2Fs, and thus, arrests to cell cycle progression by blocking transcriptional activation of essential genes for proliferation. The Rb:E2F complex is stabilized by interaction of Rb’s pocket domain with the transactivation domain of E2F, and also, by association of its C-terminal domain with the E2F:DP heterodimer (19). When Rb is phosphorylated, Rb is inactivated and allows for dissociation of the Rb:E2F complex which releases E2F and induces entry into S phase for continued cell cycle progression (19).



When overexpressed, E2F1/2/3 can cause quiescent cells to re-enter the cell cycle and override the effect of growth arrest signals. Knocking out any of these transcription factors results in decreased cell proliferation, while a triple knockout mutant will not proliferate at all (11). E2Fs also regulate expression of apoptosis genes when their expression levels exceeds a threshold (11).


E2F4 and E2F5, also known as the repressive E2Fs, interact with either Rb or the other pocket proteins to repress proliferation (11). These two factors, which are essential for cell cycle arrest, are constitutively expressed but are particularly abundant and active in quiescent cells (11).


2) Recruitment of factors involved in repressing transcription

Rb is known to be involved in the recruitment of two factors which result in repression of S phase promoting elements. HDAC1, a histone deacetylase complex, removes an acetyl group from histone H3's lysine 9 (3). The Rb protein then recruits a methylase, SUV39H1, ultimately resulting in inhibition of S phase promoting factors by HP1, which is able to bind the newly methylated histone (3).


Rb and Cancer

Loss of both functional copies of Rb leads to retinoblastoma, a childhood cancer of the eye often diagnosed prior to the age of five years (12). Children develop tumors unilaterally or bilaterally, which may lead to blindness and mental retardation. The disease can either be inherited in an autosomal dominant pattern or sporadic. Often the familial form results in bilateral tumors while the sporadic form results in unilateral tumors (12).


Dr. Alfred Knudson proposed in 1971 that inactivation of the retinoblastoma gene is a two-step process (14). The two-hit hypothesis posits that the “first hit” inactivates the one of the two copies of the Rb1 gene in a retinoblast. A “second hit” is needed to inactivate the remaining functional allele, resulting in the formation of retinoblastoma. To explain the inherited form of the disease, affected patients inherited one defective of copy of Rb1 from a parent and so the “first hit” is already initiated and present in all retinoblast cells. The likelihood for developing a retinoblastoma would occur earlier in heterozygous individuals compared to patients without the mutation. However, there is evidence that additional genetic and epigenetic changes may contribute to the development of this disease (13). Rb1 inactivation is also in osteosarcoma, small cell carcinoma of the lung, and breast/bladder/prostate carcinomas.



p53 is a transcription factor involved in many mechanisms that serve to stabilize the genome and prevent mutation. It is encoded by TP53, a gatekeeper gene located on chromosome 17 in humans. p53 exists in virtually all cells and is especially important in multicellular organisms as its function is disrupted in most cancers. p53 levels are upregulated in response to many stress factors such as lack of nucleotides, UV radiation, ionizing radiation, oncogene signaling, hypoxia, and blockage of transcription.  Further information about p53 including description of its structure can be found here. Two mutated p53 alleles leads to a rare disease called Li-Fraumeni syndrome, which increases a person’s risk of getting malignant tumors by 25 fold. The most common types of cancers affected are breast cancer, brain tumors, acute leukemia, soft tissue sarcomas, bone sarcomas, and adrenal cortical carcinomas.


Downstream activity

1) Cell Cycle Arrest

p53 is expressed at the G1/S checkpoint of the cell cycle. This checkpoint disallows cells from entering the S phase if their genome is damaged. p53 is a transcriptional activator, upregulating gene expression of p21 in damaged cells. The p21 protein forms a bond with the cyclin-CDK1 or cyclin-CDK2 complex, thus preventing S phase from being activated. The cell cycle arrest caused by p53 may be reversible or irreversible, where the cells go into a state of senescence (7).


2) DNA Repair

During cell cycle arrest, p53 may activate genes that are involved in DNA repair. An example is the p53R2 gene that encodes ribonucleotide reductase. Base excision repair proteins such as AP endonuclease and DNA polymerase interact with p53 as well.


3) Blockage of Angiogenesis

Loss of p53 causes changes in gene expression that promote tumor angiogenesis, and is associated with increased levels of vascularization. Thus, p53 loss is involved in the angiogenic switch during tumor formation.


4) Apoptosis

In severely damaged cells, p53 activates Bax, the pro-apoptotic Bcl-2 protein (either directly or via BH3), causing mitochondrial membranes to permeabilize and apoptose.



p53 activity is regulated by the Mdm2 E3-ubiquitin ligase in mice (Hdm2 in humans). Mdm2 interacts with the transactivation domain on the p53, which represses transactivation and causes destabilization of p53 (8). In unstressed cells, Mdm2 binds to p53, prevents p53 from acting on other genes, transports p53 from the nucleus to the cytosol, and attaches ubiquitin to p53. This causes p53 to get degraded by the proteasome via the ubiquitin pathway. Thus, Mdm2 keeps cellular p53 levels low overall. In stressed cells, DNA damage causes a set of kinases to phosphorylate p53. Phosphorylation occurs specifically at one of the Ser15, Thr18 or Ser20 residues on p53 and prevents Mdm2 from degrading it.


Aberrant growth signals can also activate p53 via the ARF protein, encoded by the p16 gene in an alternative reading frame. ARF binds to Mdm2 and sequesters it away from p53 in the nucleolus. p53 is thus undegraded and accumulates in the nucleoplasm. In people with cancer who have normal wild type p53, it is likely that ARF activity has been eliminated.





Figure 3.3.2. Molecular mechanisms of p53 in cells under normal and stressed conditions. Cellular stress prevents growth and cell division--but this can go wrong in cancer. Released under the Creative Commons Attribution-ShareAlike 4.0 International license (CC BY-SA 4.0).


Proteasome inhibitors, such as MG132, can be used to check ubiquitination status of p53. The technique involves the incubation of the samples with proteasome inhibitors, followed by immunoprecipitation using antibodies against p53. The isolated p53 can then be analysed using anti-ubiquitin antibodies on a western blot (15).


p53 and Cancer

When p53 is inactivated, the number of times a cell divides before reaching its terminal proliferation arrest state is increased. p53-null mutant cells bypasses the typical number of divisions that result in senescence, also known as Mortality stage 1. Nevertheless, this mutated cell is not necessarily immortalized and will still arrest proliferation at a greater number of population doublings (18). 



TSGs Work Together

Just now, we have provided examples of two well-known TSG--Rb and p53. But, understanding these genes on their own is not enough. It is important to recognize that a single TSG may not act alone to promote cancer. Cancer genes are highly interconnected. For instance, processes like apoptosis and cell cycle arrest communicate with one another to enable cell death. If the cell undergoes apoptosis, then there must be a way to tell the cell to stop dividing. Recall that Rb controls cell cycle arrest and p53 stimulates apoptosis. Therefore, it comes as no surprise that Rb and p53 pathways are also connected. This connection is mediated by p21 through the p53-Rb pathway.

For a brief review of the E2F sequestering mechanism of Rb and the mechanism of action of p53, watch this video.



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