Mutations in the retinoblastoma-related gene RB2/p130 in primary nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an endemic cancer in southern China and northern Africa, and its pathogenesis is not yet well defined at the molecular level. Although the involvement of p53 and of the retinoblastoma gene (RB/p105) in NPC has been well studied, there is paucity of mutational data regarding the retinoblastoma-related gene RB2/p130 in primary tumors and particularly in NPC. We have shown previously that RB2/p130 could be rearranged in a nasopharyngeal cell line. In the present study, we screened by single-strand conformation polymorphism and sequence analysis the retinoblastoma-related gene RB2/p130 for mutations within exons 19-22. Mutations in the RB2/p130 gene were detected in 3 of 10 primary human NPCs from Northern Africa (30%). These findings, along with previous data showing that genetic replacement of RB2/p130 restores a normal growth pathway in the nasopharyngeal cell line Hone-1, strengthen the hypothesis that genetic changes of RB2/p130 may be involved in the development and/or progression of nasopharyngeal cancer and suggest that RB2/p130 could be considered a tumor suppressor gene and may be a candidate for novel gene therapeutic approaches for NPC.     

Mutations in the retinoblastoma-related gene RB2/p130 in adult T-cell leukaemia/lymphoma

The retinoblastoma (Rb) family consists of the tumor suppressor Rb/p105 and related proteins p107 and Rb2/p130. Although the involvement of the RB / p105 gene in Adult T-cell leukaemia/lymphoma (ATL) has been studied, no mutational data is reported regarding the RB2 / p130 gene in ATL. We screened for mutations of the RB2 / p130 gene. Mutation was detected in 1 of 41 primary ATL sample. This is the first report describing mutation of the RB2 / p130 gene in ATL, suggesting that RB2/p130 may be involved in the development of ATL, and may behave as a tumor suppressor gene in T lymphocytes.     

The RB2/p130 gene: the latest weapon in the war against lung cancer?

Lung cancer is the second cause of death after cardiovascular diseases and is the major cause of cancer deaths in the Western world. Large scale screening trials conducted 15-20 years ago using chest X-rays and sputum cytology were able to detect stage I cancers but failed to impact on survival. This is because of the early metastatic potential of small primary tumors. It is important then to detect lung cancer at an earlier stage, studying and identifying genetic lesions that could indicate a new target(s) for gene therapy. The retinoblastoma-related gene pRb2/p130, a new tumor suppressor gene cloned in 1993, is emerging as one of the candidate markers and targets for gene therapeutic approach. Effective genetic therapy requires both a genetic material to be used therapeutically and a means to deliver it. A scope for this review is to examine some of the gene delivery systems mostly used, discussing their weaknesses and strengths, and to discuss the role of pRb2/p130 in lung cancer.     

Retinoblastoma-related p107 and pRb2/p130 proteins in malignant lymphomas: distinct mechanisms of cell growth control.

pRb/p105, p107, and pRb2/p130 compose the retinoblastoma (RB) family of proteins and regulate cellular growth and differentiation. Because recent functional studies have indicated that the expression of the RB-related proteins p107 and pRb2/p130 are tightly cell cycle regulated, we were interested in investigating their expression along with cellular kinetic characteristics and proliferative features of non-Hodgkin's lymphomas (NHLs). p107 and pRb2/ p130 expression was determined immunohistochemically in biopsy specimens from 83 untreated patients with NHLs of various histiotypes. The expression of these two RB-related proteins was correlated with the mitotic index, apoptotic index, and percentages of Ki-67(+), cyclin A(+), p34(+), and cyclin B(+) cells. The overall survival rate was evaluated according to the Kaplan-Meier method and the log-rank test. We found a positive correlation between the percentages of cells positive for p107 and proliferative features such as mitotic index and percentage of Ki-67(+) and cyclin A(+) cells, whereas such correlation could not be demonstrated for the percentages of pRb2/p130 positive cells. Low immunohistochemical levels of pRb2/p130 detected in untreated patients with NHLs of various histiotypes inversely correlated with a large fraction of cells expressing high levels of p107 and proliferation-associated proteins. Such a pattern of protein expression is normally observed in continuously cycling cells. Interestingly, such cases showed the highest survival percentage (82.5%) after the observation period of 10 years. Thus, down-regulation of the RB-related pRb2/p130 protein could be one of the reasons why these cases display such a high rate of proliferation and why they respond so well to therapy.     

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.     

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.     

RB2/p130 gene-enhanced expression down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in vivo

Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.     

Genetic and epigenetic alterations of RB2/p130 tumor suppressor gene in human sporadic retinoblastoma: implications for pathogenesis and therapeutic approach

Human retinoblastoma occurs in two forms (familial and sporadic) both due to biallelic mutation of the RB1/p105 gene even if its loss is insufficient for malignancy. We have recently reported that loss of expression of the retinoblastoma-related protein pRb2/p130 correlates with low apoptotic index, suggesting that RB2/p130 gene could be involved in retinoblastoma. Mutational analysis of RB2/p130 in primary tumors showed a tight correlation between Exon 1 mutations and pRb2/p130 expression level in sporadic retinoblastoma. These mutations are located within a CpG-enriched region prone to de novo methylation. Analysis of RB2/p130 methylation status revealed that epigenetic events, most probably consequent to the Exon 1 mutations, determined the observed phenotype. Treatment of Weri-Rb1 cell line by 5-Aza-dC induced an increase in expression level of pRb2/p130, E2F1, p73 and p53. Overall, our results highlight a crucial role of epigenetic events in sporadic retinoblastoma, which opens a perspective for new therapeutic approaches.     

Rb2／p130基因在骨肉瘤中的表达及临床意义

目的：研究骨肉瘤中Rb2／p130基因表达情况及其在骨肉瘤发生发展中的作用。方法：用免疫组织化学S—P法检测Rb2／p130基因在62例骨肉瘤、39例骨软骨瘤及51例非肿瘤患者正常骨组织中的蛋白表达情况,采用SPSS11．0统计学软件包对实验结果进行统计学分析。结果：骨肉瘤组中pRb2／p130阳性率为27．42％,骨软骨瘤组阳性率为76．92％,正常骨组织阳性率为82．35％,骨肉瘤组与骨软骨瘤组及正常骨组织组中Rb2／p130基因表达差异均有统计学意义（P〈0．01）。结论：Rb2／p130基因在骨肉瘤中以低表达方式存在,它与骨肉瘤的发生、发展及预后关系密切,并且有可能因其低表达而促使骨肉瘤的发生、发展,其机理有待进一步研究。     

Rb2/p130基因在骨肉瘤中的表达及临床意义

目的:研究骨肉瘤中Rb2/p130基因表达情况及其在骨肉瘤发生发展中的作用.方法:用免疫组织化学S-P法检测Rb2/p130基因在62例骨肉瘤、39例骨软骨瘤及51例非肿瘤患者正常骨组织中的蛋白表达情况,采用SPSS 11.0统计学软件包对实验结果进行统计学分析.结果:骨肉瘤组中pRb2/p130阳性率为27.42%,骨软骨瘤组阳性率为76.92%,正常骨组织阳性率为82.35%,骨肉瘤组与骨软骨瘤组及正常骨组织组中Rb2/p130基因表达差异均有统计学意义(P<0.01).结论:Rb2/p130基因在骨肉瘤中以低表达方式存在,它与骨肉瘤的发生、发展及预后关系密切,并且有可能因其低表达而促使骨肉瘤的发生、发展,其机理有待进一步研究.     

Rb2／p130与肺癌

ｐＲｂ蛋白家族 (ｒｅｔｉｎｏｂｌａｓｔｏｍａｐｒｏｔｅｉｎｆａｍｉｌｙ)又称口袋蛋白家族 (ｐｏｃｋｅｔｐｒｏｔｅｉｎｆａｍｉｌｙ) ,由三个成员组成 :视网膜母细胞瘤易感基因 (Ｒｂ)的产物Ｒｂ/ｐ10 5、ｐ10 7和Ｒｂ2 /ｐ13 0。人类Ｒｂ2 /ｐ13 0基因由 3 85 3个核苷酸构成 ,包含一个编码 113 9个氨基酸的可读片断 ,分子量 13 0× 10 3ｕ ,有 2 2个外显子 ,位于 16ｑ12 .2 [1 ] 。国外资料显示Ｒｂ2 /ｐ13 0是一个典型的抑癌基因 ,在人体内的各种正常组织中具有广泛的表达 ,与多种肿瘤的发生、发展密切相关。本文将对Ｒｂ2 /ｐ13 0…     

EGFR gene amplification - rearrangement in human glioblastomas

Immunostaining using an affinity-purified rabbit polyclonal antibody against the extracellular domain of the epidermal-growth-factor receptor (EGFR) showed over-expression occurring in a fraction of tumor cells in 17 out of 18 human glioblastomas and in a majority of cells in 7 of the 18. Southern-blotting technique using a full-length EGFR cDNA probe showed a variable degree of amplification in 10 of the 17 glioblastomas, which was associated with EGFR over-expression in each case. In 2 of the glioblastomas with EGFR gene amplification, a rearrangement of the gene affecting the extracellular domain of the receptor was identified and DNA sequence analyses revealed an identical deletion-rearrangement of 801 base pairs between exons 2 to 7, resulting in an in-frame fusion of exons I and 8. © 1995 Wiley-Liss, Inc.     

Regulation of cellular senescence by Rb2/p130

Growth regulatory functions of Rb2/p130, which aim at a sustained arrest such as in quiescent or differentiated cells, qualify the protein also to act as a central regulator of growth arrest in cellular senescence. In this respect, Rb2/p130 functions are connected to signaling pathways induced by p53, which is a master regulator in cellular senescence. Here, we summarize the pathways, which specify pRb2/p130 to control this arrest program and distinguish its functions from those of pRb/p105.     

The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas

Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.