DELETION AND INACTIVATION OF RETINOBLASTOMA SUSCEPTIBILITY GENE IN PRIMARY RETINOBLASTOMA

The status and expression of Rb gene was detected and analyzed in 19 surgical retinoblastoma specimens using Rb cDNA 3. 8 kb and 0. 9 kb fragment as probe and antibodies specific for synthetic Rb peptide or expressive product of Rb gene expression plasmld. DNA from those tumors had the hemlzygous deletion in 3 cases, the homozygous internal deletion In 2 cases and alterated restriction fragment involving In one copy of Rb gene In 1 case. The quantity of Rb protein demonstrated either absence of reduction in all the 16 cases examined In comparison with that in normal adult retina. It suggested that there were structural or/ and functional defects of Rb gene In retinoblastoma cells and provided evidence to support Knudson' s two hit hypothesis.

Epidemiology and Rb 1 gene of retinoblastoma

Retinoblastoma (Rb) is the most common eye cancer in children and it can be inherited. Rb is quite rare and originators from the neural retina with a significant genetic component in etiology, which occurs in approximately 1 in every 20 0000 births. In children with the heritable genetic form of Rb, there is a mutation on chromosome 13, called the retinoblastoma 1 (Rb1) gene. Early diagnosis and intervention is critical to the successful treatment of the Rb. The Rb1 gene is the first cloned tumor suppressor gene. As a negative regulator of the cell cycle, Rb1 gene could maintain a balance between cell growth and development through binding to transcription factors and regulating the expression of genes involved in cell proliferation and differentiation. Thus, it is involved in cell cycle, cell senescence, growth arrest, apoptosis and differentiation. We summarized the recent advances on the epidemiology and Rb1 gene of Rb in this review.

BamHI AND RsaI RESTRICTION FRAGMENT LENGTH POLYMORPHISMS IN RETINOBLASTOMA GENE IN A CHINESE POPULATION

The retinoblastoma (Rb) gene probe p123 M 1.8 andP68 Rs2.0 were used to study the frequencies of the.BamHI and Rsal restriction fragment length polymorphisms (RFLPs) in the Rb gene among the population of Han nationality in Guangdong Province.The result showed that the heterozygotic rate of the Rsal locus was only 55.0%. Linkage study of the BamHI and the Rsal RFLPs demonstrated that 10.s% of the Rsal polymorphic loci were heterozygous for the BamHI RFLP.Thus the combined use of the two RFLPs can give information for about 65.5% of the population.

Preliminary Report of Molecular Detection of Retinoblastoma Gene Mutations

To develop gene diagnosis for retinoblastoma predisposition, it is necessary to disclose the retinoblastoma gene mutations or deletions in detail. Genomic DNA from tumor and peripheral white blood cells in 33 patients with retinoblastoma was detected with 3.8kb probe derived from 3' end of retinoblastoma gene cDNA. The gene abnormalities, including deletion, partial deletion and rearrangement, were found in 18 patients. Further research will be aimed at microdeletions or mutations for those patients wti...

Prognostic significance of retinoblastoma gene mutation in retinoblastoma eye with respect to pathological risk factors

Retinoblastoma (Rb) was reported firstly by Benedict is the commonest pediatric intraocular malignant tumor in children younger than 5 years of age. The study was conducted to detect the RB-1 gene for prognostic evaluation in retinoblastoma and to see the frequency of RB-1 gene in our population. This was a retrospective descriptive analytical study. Five years biopsies (January, 2006 to December 2011) of the retinoblastoma, from the Pathology department, was retrieved to see optic nerve involvement in all the retrieved specimens. The study was taken to see the mutation of RB1 gene by immunohistochemistry and PCR. The study plan was approved from Institutional Review Board (IRB) of the University. All the cases showed positivity of abnormal Rb-1 gene proteins expression by Immunohistochemistry staining. On PCR, 51/52 (98%) tumors expressed gene mutation as compared to 100% expression shown by IHC. Out of these, 28/51 (55%) cases showed ONI and ODI with positivity for mutated RB gene. A positive association was seen among RB gene mutation with ONI and ODI (p = 0.05). There were 33/51 (65%) cases who did not show any EOE but showed PCR positivity for RB gene mutation. While there were 18/51 (35%) cases who showed EOE and positivity of PCR for Rb-1 gene mutation and a positive association was seen with EOE and gene mutation (p = 0.005). The most common sequence of mutation was on 13 with 33 cases for double mutation, 12 cases for single and 6 patients for triple pattern of mutation. Most of the double and triple sequences of mutations were associated with ONI, ODI and EOE. We concluded that mutation of RB-1 gene is responsible in causation of the tumors with a positive association with tumor size and tumor extension (optic nerve, and extraocular extension), and mutation affects patients with all ages, both gender and unilateral and bilateral tumors.

Epidemiological aspect of retinoblastoma in the world: a review of recent advance studies

·AIM: To collect and present updated evidence about epidemiological aspects of retinoblastoma(Rb) in the world.·METHODS: A comprehensive search without the time and language restrictions was conducted in international databases, including MEDLINE, Scopus, Web of Science, and Pub Med. The search keywords were “retinoblastoma” OR “retinal Neuroblastoma” OR “retinal glioma” OR “retinoblastoma eye cancer” OR “retinal glioblastoma”.·RESULTS: The worldwide incidence of Rb is 1 in 16 000-28 000 live births, but was higher in developing compared to developed countries. Several attempts for improving early detection and treatment had increased the Rb survival rate from 5% to 90% in developed countries over the past decade, but its survival was lower in developing countries(about 40% in low-income countries) and the majority of mortalities occurred in developing countries. The etiology of Rb could be viewed as genetics in the heritable form and environmental and lifestyle factors in the sporadic form. Some environmental risk factors such as in vitro fertilization;insect sprays;father’s occupational exposure to oil mists in metal working, and poor living conditions might play a role in the occurrence of the disease. Although ethnicity might affect Rb incidence, sex has no documented effect and the best treatment approaches were now ophthalmic artery chemosurgery and intravitreal chemotherapy.·CONCLUSION: Determining the role of genetics and environmental factors helps to accurately predict the prognosis and identify the mechanism of the disease, which can reduce the risk of tumor development.

Exploring the structural and functional effect of pRB by significant nsSNP in the coding region of RB1 gene causing retinoblastoma

In this study,we identified the most deleterious nsSNP in RB1 gene through structural and functional properties of its protein (pRB) and investigated its binding affinity with E2F-2.Out of 956 SNPs,we investigated 12 nsSNPs in coding region in which three of them (SNPids rs3092895,rs3092903 and rs3092905) are commonly found to be damaged by I-Mutant 2.0,SIFT and PolyPhen programs.With this effort,we modeled the mutant pRB proteins based on these deleterious nsSNPs.From a comparison of total energy,stabilizing residues and RMSD of these three mutant proteins with native pRB protein,we identified that the major mutation is from Glutamic acid to Glycine at the residue position of 746 of pRB.Further,we compared the binding efficiency of both native and mutant pRB (E746G) with E2F-2.We found that mutant pRB has less binding affinity with E2F-2 as compared to native type.This is due to sixteen hydrogen bonding and two salt bridges that exist between native type and E2F-2,whereas mutant type makes only thirteen hydrogen bonds and one salt bridge with E2F-2.Based on our investigation,we propose that the SNP with an id rs3092905 could be the most deleterious nsSNP in RB1 gene causing retinoblastoma.

Biological Function of Retinoblastoma Gene (Rb Gene).

The human wild-type Rb gene cDNA has been cis- or trans-inserted into the retrovirus vector DOL, resulting in a sense-expression vector DOLRS and an antisense-expression

THE BIOLOGICAL FUNCTION OF RETINOBLASTOMA GENE

The human wild-type Rb gene cDNA has been cis or trans inserted into the retrovirus vector poL,resulting in a sense-expression vector DOLRB and an antisense-expression vector DOLR-BAS of Rb gene.By eletroporation transfection techniques,the vector DOLRB has been introduced into the human breast carcinoma cell ilne MDAMB468 and human hepatocellular carcinoma cell line SMMC7721 both of which have an inactivated Rb gene and the vector DOLRBAS,into normal human embryonic lung fibroblasts HEL cells.With the expression of Rb protein, the growth rate of the MDAMB468 cells is decreased by about 50%,their colony formation ability in soft agar is repressed completely, and their tumorigenicity in nude mice is repressed partially.Meanwhile,the cell population of Gl phase of Rb￣+MDAMB468 cells is iiicreased markedly.About 75%of transfected SMMC7721 cells have been killed by Rb gene product.For HEL cells,with the transient expression of antisense Rb gene,the Rb protein synthesis is reduced and the growth rate of those cells increased,but no colonies of HEL cells are formed in soft agar.

Analysis of Rb gene in primary acute lymphoid leukemia

The structure of the Rb gene in 32 cases of acute lymphoid leukemia (ALL) were studied by Southern blotting using 32P-labeled Rb cDNA 3. 8 kb probe- Structural abnormalities of Rb gene were found in 8 cases of ALL, an incidence of 25%. Two novel fragments (3. 1 kb, 2- 3 kb)were observed in 5 of 8 cases. We used five pairs of Rb gene primers of exons 18, 19, 21, 22, 27 and amplified Rb gene from 6 cases of ALL with abnormal Rb gene- Only one case was free from products of exons 18 and 2l. The results seemed to indicate that abnormalities of Rb gene might be closely associated with initiation and/or promotion of ALL.

Rb基因诱导视网膜母细胞瘤移植瘤细胞凋亡的实验研究

目的观察外源性Ｒｂ基因对视网膜母细胞瘤（ＲＢ）细胞凋亡的影响。方法建立裸鼠眼玻璃体腔ＲＢ移植瘤模型及构建Ｒｂ基因的逆转录病毒表达载体Ｐｂａｂｅ－Ｒｂ，用脂质体Ｄｏｓｐｅｒ介导法将Ｒｂ基因导入裸鼠ＲＢ移植瘤，采用流式细胞术（ＦＣＭ），光镜、电镜及ＴＵＮＥＬ细胞凋亡原位末端标记法进行ＲＢ细胞凋亡的检测。结果Ｒｂ基因在ＲＢ移植瘤内表达至少持续７天。ＦＣＭ检测发现在Ｒｂ基因转染后第４天，各实验组治疗眼ＲＢ中均可检测到凋亡细胞峰，凋亡细胞百分率高于对照眼；第２０天，各实验组治疗眼细胞凋亡百分率与对照眼比无差异（Ｐ＞０．０５）。透射电镜下可见到典型的早期凋亡细胞及晚期凋亡小体形成。ＴＵＮＥＬ方法可在荧光显微镜下见到发黄绿色荧光的凋亡细胞，光镜下可见到紫红色的被标记的凋亡细胞。结论Ｒｂ基因可诱导ＲＢ移植瘤细胞凋亡，这可能是Ｒｂ基因体内抗癌的又一作用机制。

视网膜母细胞瘤细胞系HXO-Rb_(44)抑癌基因的表达

目的检测视网膜母细胞瘤（ＲＢ）细胞系ＨＸＯＲｂ４４中抑癌基因Ｒｂ的表达状况，为进一步研究ＲＢ的发病机制，利用抑癌基因Ｒｂ进行ＲＢ的基因治疗提供实验依据。方法用ＳＰ免疫组织化学（ＩＨＣ）及流式细胞术（ＦＣＭ）间接免疫荧光法对Ｒｂ基因的表达进行定性、形态学定位及定量检测。结果ＩＨＣ法检测显示：正常视网膜组织中Ｒｂ蛋白呈阳性反应，位于细胞核呈棕黄色；ＨＸＯＲｂ４４中Ｒｂ蛋白表达阴性。ＦＣＭ检测显示：正常人淋巴细胞Ｒｂ蛋白表达量是ＨＸＯＲｂ４４细胞的３．７１倍，ＨＸＯＲｂ４４中Ｒｂ蛋白表达量极低。结论ＲＢ细胞系ＨＸＯＲｂ４４中存在野生型Ｒｂ基因的功能失活，Ｒｂ基因的突变失活与ＲＢ的发生密切相关。

MCM2和Rb蛋白在胃癌中的表达及临床意义

目的:探讨微小染色体维持蛋白2(MCM2)和视网膜母细胞瘤易感基因(Rb)在胃癌组织中的表达及其意义。方法:采用免疫组化法检测MCM2和Rb蛋白在20例正常胃黏膜、30例非典型增生、80例胃癌组织中的表达,并以标记指数(labellingindex,LI)量化其表达。结果:MCM2和Rb蛋白在正常胃黏膜、非典型增生和胃癌组织中的表达之间均有显著性差异(P<0.05);两者的LI在胃癌组织中表达与临床分期、浆膜浸润、术后复发、肝转移及淋巴结转移密切相关(P<0.05),与肿瘤大小及组织学类型无关(P>0.05)。胃癌组织中MCM2和Rb的LI表达呈显著负相关(r=-0.321,P=0.004)。结论:MCM2表达升高和Rb蛋白表达下降与胃癌的发生及发展密切相关,检测MCM2和Rb有助于判断胃癌恶性程度及预后。

急性淋巴细胞白血病Rb基因的分析

应用限制性内切酶片段长度多态性分析了３２例原发急性淋巴细胞白血病Ｒｂ基因的等位基因变化，采用３２Ｐ－标记的ＲｂｃＤＮＡ３．８ｋｂ探针，结果在８例患者中发现Ｒｂ基因结构异常（２５％）．其中４例出现新的３．１ｋｂ和２．３ｋｂ片段，选用Ｒｂ基因外显子１８，１９，２１，２２，２７五对引物，对已发现Ｒｂ基因异常的６例患者进行了分析，发现１例在外显子１８和２１无扩增产物．结果提示：Ｒｂ基因异常与急性淋巴细胞白血病的疾病加剧的关系较疾病的始发关系更为密切。

Rb和MCM2蛋白在前列腺癌中的表达及临床意义

目的:探讨视网膜母细胞瘤易感基因(Rb)和微染色体维持蛋白2(MCM2)在前列腺癌发病机制中的作用及其临床意义。方法:应用免疫组化EliVisionTM plus二步法,检测49例前列腺癌(PCa)组织,20例良性前列腺增生(BPH)及10例正常前列腺组织(NP)中pRb蛋白与M CM 2的表达。结果:pRb在PCa,BPH与NP组织中的阳性表达率分别为44.90%,80%,90%,PCa中pRb的水平明显低于BPH(P<0.05)及NP(P<0.05),且与PCa病理分级(P<0.05)和临床分期(P<0.05)呈负相关。M CM 2在PCa,BPH与NP组织中的阳性表达率分别为67.35%,25%和10%,PCa中M CM 2的水平明显高于BPH(P<0.05)及NP(P<0.05),且与PCa病理分级(P<0.05)和临床分期(P<0.05)呈正相关。M CM 2表达与pRb表达呈负相关(rs=-0.596,P<0.01)。结论:Rb基因参与了前列腺癌的发生、发展过程,检测Rb基因和M CM 2有助于判定前列腺癌恶性程度及预后。

抑癌基因p16及Rb在肺癌细胞中表达的实验研究

目的研究中药组方对小鼠肺癌细胞的抑制作用及其分子机制。方法将小鼠Lewis肺癌细胞复苏后,于小鼠右腋窝皮下传代后构建小鼠Lewis肺癌模型30只,随机分为3组,各10只,雌雄各半。接种后第2d开始灌胃,中药治疗组予中药稀释液(相当于生药量60g/kg),替加氟组予替加氟稀释液(相当于替加氟药量78mg/kg),0.9%氯化钠注射液组予0.9%氯化钠注射液,各组均为0.4mL,每日1次灌注。观察中药组方、替加氟和0.9%氯化钠注射液对各组瘤体大小的影响,并用免疫组化方法检测不同组别间抑癌基因p16和Rb蛋白的表达情况,比较各实验组的变化。结果治疗3周后,中药治疗组和替加氟组小鼠皮下肿瘤平均质量均比0.9%氯化钠注射液组小(P<0.01);中药治疗组、替加氟组抑瘤率均>40%,2组比较差异无统计学意义(P>0.05),说明中药组方具有一定的抑制肿瘤生长的作用。0.9%氯化钠注射液组p16的标记指数(LI)、Rb-LI显著低于中药治疗组和替加氟组(P<0.01,P<0.01)。镜下可见,p16、Rb蛋白阳性细胞在各组中表达不一,0.9%氯化钠注射液组阳性细胞呈棕黄色,细胞形态较完整,数量较少,呈散在分布;中药治疗组和替加氟组阳性细胞呈棕黄色,深浅不一,细胞形态相对不完整。结论中药组方对小鼠Lewis肺癌的细胞生长具有抑制作用,其机制可能与中药激活抑癌基因,影响细胞周期的发生和阻断细胞脱氧核糖核酸(DNA)合成有关。

乳腺癌中RB基因缺失及与乳腺癌发病危险因素关系的研究

应用PCR扩增方法检测 6 9例女性乳腺癌患者肿瘤组织RB基因外显子 14～ 16和外显子 2 1的存在状态 ,同时分析有关乳腺癌发病危险因子对这种存在状态的可能影响。结果显示 :外显子 14～ 16的缺失率为 2 0 3% (14/6 9) ,外显子 2 1的缺失率为 2 1 7% (15 /6 9) ,总的缺失率为 37 78%。有家族肿瘤史的患者其相应外显子缺失率显著高于无家族肿瘤史的患者 ,而发病年龄、绝经状态、生育史以及ER或PgR状态等因素均与RB相应外显子的缺失无显著性相关。PolychotomousLogistic回归分析显示肿瘤家族史明显增加存在RB基因相应外显子缺失或突变的概率。结果提示RB基因在女性乳腺癌发生过程中有一定的作用 。

视网膜母细胞瘤细胞系SO-Rb_(50)瘤细胞Rb基因突变动态变化研究

目的:研究SO-Rb50细胞系瘤细胞Rb基因突变的动态变化。方法:1.用 Southern blot杂交法分析 SO-Rb50细胞系第327代瘤细胞DNA。2.用PCR-SSCP法对SO-Rb50细胞系第415代及第713代瘤细胞的Rb基因27个外显子和1个启动子逐个筛查。3.将SO-Rb50细胞系的第775代克隆出3个细胞株:MC2、MC3及MC4。用PCR-SSCP-HA法对MC2、MC3及MC4第11代细胞和MC3第138代细胞的Rb基因的27个外显子逐个筛查。结果:SO-Rb50细胞系第327代细胞DNA缺失3.5Kb、2.9Kb及1.0Kb的三条带,证明SO-Rb50细胞系瘤细胞Rb基因缺失。第415代瘤细胞Rb基因第23外显子少一条单链;第713代第25外显子发生新的突变。SO-Rb50细胞系第775代的3个克隆株MC2、MC3及MC4的第11代细胞,只有MC4第24外显子突变;而MC3第138代第24外显子突变,提示MC3经多次传代后第24外显子发生新突变。结论:SO-Rb50细胞系在长期培养传代过程中,瘤细胞的Rb基因突变有动态变化。眼科学报2001;17:111～113。

Rb1基因第16内含子内21个碱基缺失1例

目地研究双眼视网膜母细胞瘤患者Rb1基因杂合性突变的分子生物学特性。方法应用PCR—SSCP直接测序技术检测双眼视网膜母细胞瘤患者白细胞DNA中Rb1基因杂合性突变。结果50例证实有Rb1基因杂合性突变的病例中有1例发生于第16内含子中可以用3种定位方法解释、具有相同序列的21个碱基缺失。结论这种极为少见的Rb1基因突变方式可能是由于破坏了正常拼接位点的结构而激活了“隐蔽拼接位点”，导致异常的Rb1基因mRNA产生或由此影响整个拼接过程。

口腔鳞状细胞癌Rb基因的检测

目的研究Ｒｂ基因异常与口腔鳞状细胞癌发生及临床病理指标的关系。方法应用地高辛高效引物标记Ｒｂ３．６ＫｂＤＮＡ探针，结合抗地高辛抗体碱性磷酸酶显色法，对口腔鳞癌病人中Ｒｂ基因改变进行了研究。结果在７／２８例口腔鳞癌中发现Ｒｂ基因结构异常，其中６例出现５．４、４．１及２．３Ｋｂ片段缺失，１例出现６．２Ｋｂ新片段；Ｒｂ缺失与临床病理指标无显著相关性。结论口腔鳞癌中存在Ｒｂ基因缺失、重排。