DEFINITION
The retinoblastoma protein (abbreviated pRb or Rb) is a tumor suppression protein of 110 kDa that is dysfunctional in many types of cancer. One highly studied function of pRb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. It is also a recruiter of several chromatin remodelling enzymes such as methylases and acetylases. ( W ).
pRB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorilation during the G1/S CHECKPOINT. pRB plays an essential role in the G1 arrest induced by a variety of growth inhibitory signals. For example pRb prevents the cell from replicating damaged DNA by preventing its progression along the cell cycle through G1 into S.
CHEMICAL STRUCTURE
The gene of the retinoblastoma (RB1) encodes a nuclear phosphotrotein of 928 aa (pRb) and 110 kDa. RB1 is a gene of 178,14 Kb localized on the 13th cromosome in q arm. It's aminoacids percentage is:
pRb contains different functional domains, and the most important of them is known as pocket domain. The pocket domain is constituted by 2 subdomain called A and B, separated by a spaced sequence. The two subdomain A and B, interacting each other, constitute the pocket, from whose integrity depends the tumour suppression role of pRb. Mutations that alter the structure of the pocket domain have been found in patients affected by hereditary retinoblastoma.
Some viral oncoproteines, but also many cellular proteins that interact with pRb, contain a preserved motive LxCxE with which they tie the pocket domain. The site which bonds LxCxE is located on the subdomain B. The subdomain A is necessary for the dominion B to assume an active conformation.
The motive LxCxE is surely the more characterized, but it's certainly not the only one. The transcriptional factors E2Fs don't contain LxCxE motive and they are tied to pRb in a separate site.
CELLULAR FUNCTIONS
The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Its fundamental role in Cycle Cell is shown in G1/S Checkpoint. pathway
PATHOLOGICAL INACTIVATION OF pRB
Dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F residues 409–426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. Within the E2F construct there are nine residues that are conserved across E2F’s from all animal species. Amino acid substitutions at five of these positions have been shown to lead to loss of binding to pRb but retention of E2F’s transactivation potential. The following description focuses on the structural role of these five residues:
- Tyr(411)-E2F appears to play an important role in peptide binding because its phenolic ring occupies a hydrophobic pocket created by Ile(536)-pRb, Ile(532)-pRb, Ile(547)-pRb;
- Phe(413)-E2F, whereas its hydroxyl group makes a hydrogen bond to the invariant Glu(554)-pRb.
- toward the C-terminal part of the E2F peptide, Leu(424)-E2F and Phe(425)-E2F make several hydrophobic interactions, two of which involve conserved residues.
- Leu(424)-E2F makes contacts with the aliphatic portion of the side chain of Lys (530)-pRb and also packs against Leu (415)-E2F and Phe(425)-E2F.
- In addition, Phe(425)-E2F itself packs against Phe(482)-pRb. Unlike the residues of E2F just discussed, the side-chains of Glu(419)-E2F and Asp(423)-E2F do not point into the groove formed between the A and B domains of pRb, but instead point away from it.
Permanent inactivation of pRB is involved in the pathogenesis of many tumours. The reason is that RB controls DNA before it will be duplicated and for this reason prevents mutations.
There are many way thought which pRb can be permanently inactivated:
- loss of heterozygosity
- mutations or deletions of the RB1 gene
- pRB hyperphosphorylation
The alterations of pRB pathway are involved in many kind of tumours in different ways:
- Gastrointestinal Tract Tumours: Many cell cycle regulators modulate pRB function through its phosphorylation status. G1 cyclins (cyclin D, E)/cyclin-dependent kinases (cdk2, 4) inactivates pRB through its phosphorylation, while p21 (WAF1) and p16 inhibit cdks. In several kinds of cancer, Rb gene alteration or functional inactivation of pRB has been reported. In esophageal cancer, loss of heterozygosity of Rb gene and cyclin D gene amplification were frequently detected. But in gastric and colorectal cancer, Rb gene loss or deletion has been shown to be rare. In this study the expression of pRB, G1 cyclins, cdks and cdk-inhibitors in adenoma-carcinoma sequence of colorectum has been investigated. And it is compared the phosphorylation status of pRB in colorectal normal mucosa and cancer tissue. In adenoma only cyclin D and E were overexpressed but not cdks. In cancer pRB and cdk2 were overexpressed with high frequency, and cdk4 overexpression was detected in advanced cancer. p16 overexpression was detected in almost all cancers, but in contrast p21 overexpression was rare event. Comparative study showed that pRB-positive cancer cells also expressed both cdc2/cdk2 and cyclin E. Densitometric analysis revealed that in advanced cancer pRB was hyperphosphorylated compared with normal mucosa. These results indicate that overexpression of cyclin D/cdk4 and cyclin E/cdk2 would phosphorylate pRB, and insufficient expression of p21 may accelerate pRB inactivation. RB gene expression in gastrointestinal tract, 1996
- Breast Cancer: inactivation of the retinoblastoma protein (pRB) by mutations or abnormal phosphorylation is a mechanism by which tumour cells can subdue normal growth control. Among molecules involved in control of pRB phosphorylation, cyclin D1 and E have been found to be deregulated and overexpressed in various types of cancers. In order to study the cell cycle regulatory mechanisms in breast cancer, the study has analysed the protein expression of cyclin D1 and E in 114 tumour specimens from patients with primary breast cancer using Western blotting. Twenty-five out of 34 tumours with overexpression of cyclin E showed uniform low cyclin D1 expression, and by immunohistochemical analysis of pRB we present evidence for the existence of pRB defects in approximately 40% of these tumours in contrast to no pRB defects in the other group of tumours. This result was supported by a high protein expression of the cyclin-dependent kinase inhibitor p16 in 44% of the tumours with high cyclin E and low D1 expression, and all immunohistochemical pRB defect tumours showed a high p16 protein level. Additionally, an abnormal low pRB phosphorylation in relation to a high proliferative activity and loss of heterozygosity of the retinoblastoma susceptibility gene locus were found in all but one tumour with immunohistochemical defect pRB. Interestingly, tumours with high cyclin E and low D1 expression were generally oestrogen receptor negative suggesting a role for cell cycle regulators in the mechanisms leading to oestrogen independent tumour growth. Furthermore, the prognosis differed markedly for the patients in the various groups of tumours, indicating that the heterogeneous nature of breast cancer pathogenesis and the clinical course in part could be explained by different and distinctive sets of cell cycle defects.
Taken from Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein, 1997
- Sporadic Pituitary Adenomas: Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution this study simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.
Taken from Aberrant expression of G(1)/S regulators is a frequent event in sporadic pituitary adenomas, 2001
- Bladder Cancer: Constitutive hyperphosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function.
Taken from Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer, 2004
- Uveal Melanoma: Uveal melanoma is the most common primary eye cancer, yet its molecular pathogenesis is poorly understood. The immunohistochemical expression of proteins in the Rb and p53 tumor suppressor pathways have been investigated, in one study, in 33 uveal melanomas from enucleated eyes. Strong nuclear staining for Rb was present in most tumors. However, a few cases displayed weak nuclear staining and strong cytoplasmic staining ( possibly indicating Rb mutation ), and this aberrant staining correlated strongly with failed radiotherapy or thermotherapy before enucleation. Staining for cyclin D1 was positive in most tumors and was associated with advanced age and larger tumor size, which are both poor prognostic factors. Generally, immunostaining for p53 was weak (suggesting a lack of p53 mutations), although p53 positivity correlated strongly with staining for phosphorylated Rb, supporting the notion that inappropriate phosphorylation of Rb can induce p53. Strong immunostaining for MDM2, which can functionally block p53 activity, was observed in most tumors and correlated significantly with female sex. Strong cytoplasmic staining was observed for Bcl2, which can inhibit both p53-dependent and -independent apoptosis. The study concludes that Rb and p53 are mutated infrequently in uveal melanoma, but their respective pathways may be functionally inactivated.
Taken from Deregulation of the Rb and p53 Pathways in Uveal Melanoma, 2005
- Viral Oncoprotein : The interaction between viral oncoproteins such as Simian virus 40 TAg, adenovirus E1A, and human papilloma virus E7, and the retinoblastoma protein (pRB) occurs through a well characterized peptide sequence, LXCXE, on the viral protein and a well conserved groove in the pocket domain of pRB. Cellular proteins, such as histone deacetylases, also use this mechanism to interact with the retinoblastoma protein to repress transcription at cell cycle regulated genes. For these reasons this region of the pRB pocket domain is thought to play a critical role in growth suppression. RESULTS: In this recent study ( published 5 days ago ), they identify and characterize a tumor derived allele of the retinoblastoma gene (RB1) that possesses a discrete defect in its ability to interact with LXCXE motif containing proteins that compromises proliferative control. To assess the frequency of similar mutations in the RB1 gene in human cancer, they screened blood and tumour samples for similar alleles. They screened almost 700 samples and did not detect additional mutations, indicating that this class of mutation is rare. A cancer derived mutation in the Retinoblastoma gene with a distinct defect for LXCXE dependent interactions, 2010
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Pietro Garofalo
