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.     

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.     

Retinoblastoma-related gene RB2/p130 exons 19-22 are rarely mutated in glioblastomas

The retinoblastoma gene family RB1, p107 and RB2/p130 cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Previous data demonstrated that RB2/p130 inhibits proliferation of the glioblastoma cell line T98G, which is resistant to the growth suppressive effects of both RB1 and p107, and that RB2/p130 gene overexpresion induces astrocyte differentiation. We screened by single-strand conformation polymorphism and sequence analysis the structure of exons 19 through 22 of the RB2/p130 gene, which encodes the B domain and C terminus, in a series of 42 glioblastomas (32 primary and 10 secondary). Sequence variations were identified in one tumor, suggesting that mutation inactivation of RB2/p130 is a rare event in glioblastoma.     

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.     

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.     

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.     

Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma

RASSF2 can bind directly to K-Ras and function as a negative effector of Ras protein. RASSF2A is the only isoform of RASSF2 that contains CpG islands in its promoter and it has been reported to be inactivated by its promoter methylation in several human cancers. In the present study, we investigated the correlation of RASSF2A expression with its promoter methylation in nasopharyngeal carcinoma (NPC). Expression of RASSF2A was down-regulated in 80% (4/5) of NPC cell lines. Decreased RASSF2A expression was also observed in NPC primary tumors compared with normal nasopharyngeal epithelia. Promoter methylation of RASSF2A could be detected in all the RASSF2A-silenced cell lines (4/5) of the NPC cell lines and 50.9% (27/53) of primary tumors, but not in any of the normal epithelia. RASSF2A-methylated cases showed a significantly lower level of RASSF2A expression than unmethylated cases. Loss of RASSF2A expression can be greatly restored by the methyltransferase inhibitor 5-aza-dC in NPC cell lines. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (p < 0.05). Ectopic expression of RASSF2A in RASSF2A-silenced and -methylated NPC cell line CNE2 shows that RASSF2A could inhibit cell cycle progression, colony formation and cell migration, which provided further evidence that RASSF2A is a candidate tumor suppressor gene. In conclusion, RASSF2A, a candidate tumor suppressor gene (TSG), is frequently inactivated by its promoter methylation and this aberrant methylation correlates with lymph node metastasis in NPC.     

鼻咽癌表达下调基因NASG3′UTR可变剪接分析及其在多种肿瘤中的表达

背景与目的：采用抑制性消减杂交技术已分离获得了人鼻咽组织特异性基因NASG。本研究对人鼻咽组织特异性表达且在鼻咽癌表达下调的NASG基因3′非编码区(untranslated region,UTR)的可变剪接进行分析,并考察NASG基因在多种肿瘤组织中的表达。方法：在NASG基因3′UTR存在可变剪接部位的两端设计引物进行RT—PCR扩增,分离扩增产物并测序。用RT—PCR检测NASG基因在鼻咽癌中的表达,采用了肿瘤表达谱阵列(cancer profiling array)杂交分析NASG基因在多种肿瘤组织的表达状况。结果：NASG基因3′UTR存在3种剪接产物,NASG基因在71％的鼻咽癌活检组织中表达下调,25％的肺癌组织中表达上调．而在其他肿瘤及其配对的正常组织末见明显表达。结论：NASG基因3′UTR存在3种剪接产物,NASG基因的表达异常是鼻咽癌和肺癌发生、发展过程中重要的分子事件。     

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.     

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.     

MiR-663, a microRNA targeting p21(WAF1/CIP1), promotes the proliferation and tumorigenesis of nasopharyngeal carcinoma

MicroRNAs (miRNAs) may function as either oncogenes or tumor suppressors in the malignant progression of different tumor types. MiR-663 was recently reported to be decreased and identified as a tumor suppressor in gastric cancer. We also verified its role in repressing cell proliferation of a gastric cancer cell line. In this study, however, miR-663 was found to be upregulated in nasopharyngeal carcinoma (NPC) cells compared with human immortalized nasopharyngeal epithelium cells, using a miRNA microarray, and this higher expression was confirmed in NPC tissue samples. Indeed, inhibition of miR-663 impaired the proliferation of NPC cells in vitro and the NPC tumor growth of xenografts in nude mice. Mechanistically, miR-663 directly targeted p21(WAF1/CIP1) to promote the cellular G1/S transition, as the inhibitory effects of miR-663 on the G1/S transition could be rescued by p21(WAF1/CIP1) silencing. Our results imply that miR-663 may act as an oncogene in NPC. The newly identified miR-663/p21(WAF1/CIP1) axis clarifies the molecular mechanism of NPC cell proliferation and represents a novel strategy for the diagnosis and treatment of patients with NPC.     

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

Background

Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

Methods

We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

Results

Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

Conclusion

Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.     

p16基因的表达上调对鼻咽癌细胞恶性表型的影响：p16基因在鼻咽癌中的改变

探讨上调Ｐ１６基因的表达对鼻咽癌细胞恶性表型逆转的作用,为Ｐ１６基因在处鼻咽癌发病过程中具有一定的作用提供直接的语气方法,本文采用电穿孔的方法向Ｐ１６基因表达下调的鼻咽癌细胞系ＨＮＥ１中导入全长野生型的Ｐ１６ｃＤＮＡ,通过Ｇ４１８筛选获得可以稳定传代的转当细胞系,对其中４个转染细胞系以ＰＣＲ和Ｎｏｒｈｅｒｎ杂交鉴定转染细胞系中外源Ｐ１６ｃＤＮＡ整合以及Ｐ１６基因表达的状况,并借助比较细胞倍增时间,     

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.     

组织芯片技术检测人鼻咽癌组织及细胞株KIAA1173基因的表达

背景与目的:鼻咽癌(nasopharyngealcarcinoma,NPC)致病的分子机制至今仍不清楚,已有研究表明,染色体3p21～22区域存在与鼻咽癌发生密切相关的抑癌基因。KIAA1173基因是定位于3p22.1的一个新的肿瘤相关基因,其与NPC发病的关系尚未见报道。本研究采用KIAA1173基因特异性原位杂交探针,检测其在NPC组织及细胞株中的表达,探讨KIAA1173基因与NPC发病的关系。方法:克隆KIAA1173基因片段(354bp),并制备cDNA探针;采用组织芯片技术,通过原位杂交检测73例鼻咽部不同组织标本(41例NPC、18例鼻咽非典型增生上皮、14例正常鼻咽粘膜上皮)和6种NPC细胞株(CNE1、CNE2、HNE1、HNE2、6-10B、5-8F)中KIAA1173基因mRNA的表达情况。结果:KIAA1173基因在NPC细胞、非典型增生上皮和正常鼻咽粘膜上皮的阳性率分别为21.9%(9/41)、83.3%(15/18)、92.8%(13/14),而6种NPC细胞株均未见表达;在正常鼻咽粘膜上皮和非典型增生上皮中强阳性率分别是64.3%(9/14)和38.9%(7/18),而NPC中无强阳性,在鼻咽部不同上皮组织中的表达差异有显著性(P<0.001);在38例伴有淋巴细胞浸润的NPC组织中,癌细胞与浸润淋巴细胞之间KIAA1173基因表达有显著性差异(P=0.026),并且呈明显负相关(κ=-0.337,P=0.020)。结论:KIAA1173基因在鼻咽部不同组织中表达不同,正常鼻咽上皮强表达,而NPC细胞中低表达甚至不表达,提示该基因可能参与NPC演变的过程。     

Absence of p53 gene mutations in primary nasopharyngeal carcinomas.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.