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.

Decoding Retinoblastoma: Differential Gene Expression

Background: Retinoblastoma, the most common intraocular pediatric cancer, presents complexities in its genetic landscape that necessitate a deeper understanding for improved therapeutic interventions. This study leverages computational tools to dissect the differential gene expression profiles in retinoblastoma. Methods: Employing an in silico approach, we analyzed gene expression data from public repositories by applying rigorous statistical models, including limma and de seq 2, for identifying differentially expressed genes DEGs. Our findings were validated through cross-referencing with independent datasets and existing literature. We further employed functional annotation and pathway analysis to elucidate the biological significance of these DEGs. Results: Our computational analysis confirmed the dysregulation of key retinoblastoma-associated genes. In comparison to normal retinal tissue, RB1 exhibited a 2.5-fold increase in expression (adjusted p Conclusions: Our analysis reinforces the critical genetic alterations known in retinoblastoma and unveils new avenues for research into the disease’s molecular basis. The discovery of chemoresistance markers and immune-related genes opens potential pathways for personalized treatment strategies. The study’s outcomes emphasize the power of in silico analyses in unraveling complex cancer genomics.

Effect of VEGF-targeted antisense gene therapy on retinoblastoma cell line SO-RB50 in vitro and in vivo

AIM: To evaluate the possibility of generation 4 polyamidoamine (G4PAMAM) dendrimers acting as the delivery system of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides (VEGFASODN), and to investigate the anti-tumor effect of G4PAMAM/VEGFASODN complex on the cultured cells and the mouse tumor xenograft model. METHODS: The transfection efficiency was assessed by Row cytometry (FCM). Thiazolyl tetrazolium (MU) assay was performed to determine the relative growth rate (RGR) of the cells after transfection. Then a mouse tumor xenograft model of human retinoblastoma was established. Different interventions were given to the mice by intratumoral injection and the tumor growth was monitored. The expression of VEGF mRNA was detected by reverse transcription PCR (RT-PCR), the expression of VEGF protein was determined by western blot analysis, and the microvessel density (MVD) was measured by immunohistochemistry (IHC) staining. RESULTS: G4PAMAM/VEGFASODN exhibited a high transfection rate in vitro, and the transfection rates of different doses of G4PAMAM/VEGFASODN groups increased with higher doses. This effect was accompanied by a dose-depended reduction in cell viability. The tumor growth in the tumor-bearing athymic mice was significantly inhibited in the G4PAMAM/VEGFASODN group. The expressions of VEGF mRNA and protein were obviously inhibited in the G4PAMAM/VEGFASODN group (p<0.05), and the MVD of the G4PAMAM/VEGFASODN group was lower than that of the other groups(p<0.05). CONCLUSION: VEGFASODN can be delivered into the cultured and transplanted retinoblastoma cells efficiently by G4PAMAM, suppress the expressions of VEGF mRNA and protein, and reduce the MVD of tumor tissues. The G4PAMAM/VEGFASODN complex has antitumor properties vitro and in vivo.

Clinical and genetic characteristics of retinoblastoma patients in a single center with four novel RB1 variants

AIM:To assess the clinical and genetic characteristics of children diagnosed with retinoblastoma(RB)at Gazi University Faculty of Medicine’s Department of Pediatric Oncology.METHODS:All cases diagnosed with RB and received treatment and follow-up in the Ophthalmology and Pediatric Oncology Department,October 2016 to May 2021 were evaluated retrospectively.The RB1 gene was analyzed by next-generation sequencing(NGS)technique in DNAs obtained from peripheral blood samples of the patients.RESULTS:This study included 53 cases with 67 RBaffected eyes during the study period.The mean age was 24.6(median:18.5,range:3–151)mo.There were 15(22.3%)Group D eyes and 39(58.2%)Group E eyes.The RB1 gene was sequenced by the NGS method in 19 patients.Heterozygous RB1:NM_000321.3:c.54_76del(p.Glu19AlafsTer4)variant was detected in a 15-month-old female with bilateral RB.Heterozygous RB1:NM_000321.3:c.1814+3A>T variant was detected in a 5.5-month-old male with bilateral RB.The intronic RB1:NM_000321.3:c.1332+4A>G variant was detected in patient 14,a 13-month-old male with unilateral RB.The RB1:NM_000321.3:c.575_576del(p.Lys192SerfsTer10)variant was found in an 18-month-old female with an allele frequency of 37%.These variants have not been reported in the literature and mutation databases.CONCLUSION:Four novel variants are described and one of them is found in two different patients.This data is crucial for assessing prognosis.It serves as a guide for estimating the long-term risk of secondary malignancy as well as the short-term risk of developing additional malignancies in the same eye and the other eye.

Detection of Tumor Suppressor Gene and Oncogene in SO-Rb_(50) Human Retinoblastoma Cell Line

Retinoblastoma (Rb) is the most common malignant'cancer of eye.So-Rb_(50) is the first Rb cell line established in China in 1988.It has passed to the 387th passage now.We collected cells of the 327th passage of SO-Rb_(50),purified its genomic DNA and detected it with Rb and c-myc cDNA probes respectively(normal human white blood cells DNA was the control).We found the Rb gene was deleted while c-myc gene was amplified three times.This provides a basis for further study of the regulation of tumor development and tumor reversal with this cell line in vitro.Eye Science 1993;9:34-37.

Genetic Predisposition to Retinoblastoma (Rb)

Determination of 8 cytogenetic indicators in 14 cases of Rb,their 21 parents and 14 normal controls revealed various degrees ofchromosome instability and nondisjunction in the patients and their parents,indicating the presence of genetic neoplastic predisposition to neoplasm inRb patients.Eye Science 1993;9:149-152.

Tumor suppressor gene p16 and Rb expression in gastric cardia precancerouslesions from subjects at a high incidence area in northern China

AIM:To further understand the molecular basis for gastric cardia carcinogenesis and to provide etiological clues. METHODS: Endoscopic mucosa biopsy and histopathological examinations were made on 37 subjects from a high incidence area for both esophageal and gastric cardia carcinomas in northern China. All the biopsy samples were fixed in 850 ml. (-1)L alcohol and embedded in paraffin. Each block contained one piece of tissue and was serially section at 5 microm. Immunohistochemistry (ABC) was carried out on these gastric cardia samples to determine the alterations of p16 and Rb. RESULTS: Based on the histopathlogical examination there were 11 cases of chronic superficial gastritis, 12 cases of chronic atrophic gastritis and 14 cases of dysplasia. The immunostaining demonstrated different levels of unclear immunostaining of p16 and Rb in normal gastric cardia tissue and the tissues with different severity of lesions. With the lesions progressing, the positive immunostaining rates for p16 protein had a decreasing tendency. In contrast, the positive immunostaining rate for Rb protein had an increasing tendency. There was a significant negative relationship between the two parameters. Changes of p16 was CSG 11(100%), CAG 7(58%), DYS 4(29%) and changes of Rb was CSG 2(18%), CAG 8(67%) and DYS 12(86%), (P【0.05). CONCLUSION: The alterations of p16 and Rb protein may play a role in the early stages of gastric cardia carcinogenesis.

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

IM To study the alterations of tumor suppressor gene p16 and Rb in the carcinogenesis of the stomach. METHODS Different mucosal biopsies were endoscopically obtained, all samples were immediately fixed with 10% buffered formalin, embedded with paraffin and sectioned serielly. Alterations of p16 and Rb protein in 12 cases of superficial gastritis, 15 atrophic gastritis, 20 atypical hyperplasia and 40 cancerous tissues were detected by the immunohistochemical method (ABC). RESULTS Different degrees of nuclear immunostaining of p16 and Rb occurred on gastric epithelium in different stages of lesions. With the lesions progressing, the positive immunostaining rate of p16 protein had a decreasing tendency (833%→733%→300%→275%), and on the other hand, that of Rb protein had an increasing tendency (250%→467%→600%→675%). A negative correlationship was found between these two parameters in the gastric cancer. Of 40 cases of gastric cancer, a negative relationship was observed in 20 cases. In comparison with both positive (9 cases) and both negative tissues (11 cases), there was a significant difference (500%,225%,275%) (P<005).CONCLUSION Abnormal expression of p16 and Rb plays an important role in gastric carcinogenesis.

Immunohistochemical analysis of p53,cyclinD1,RB1,c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

AIM： TO study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS： Paraffin-embedded tissue samples of 25 patients （18 males and 7 females） with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method （avidin-biotin-peroxidase） for detection of p53, cyclinD1, RB1, c-los and N-ras proteins. RESULTS： Six （24%）, 5 （20%）, 12 （48%） and 2 samples （8%） were positive for p53, cyclinDl, C-los and N-ras expression, respectively. Twenty-two （88%） samples had alterations in the （31 cell-cycle checkpoint protein expression （RBI or cyclinD1）. P53 positive samples showed a higher （9 times） risk of being positive for RBI protein than p53 negative samples. Loss of expression of RBI in association with p53 over-expression was observed in 4 （66.7%） of 6 samples. Loss of expression of RBI was seen in all cyclinD1 positive, 20 （90.9%） N-ras negative, and ii （50%） C-fos positive samples, respectively. CyclinD1 positive samples showed a higher （2.85 and 4.75 times） risk of being positive for c-los and N-ras expression than cyclinD1 negative samples. CONCLUSION： The expression of p53, RB1 and c-los genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.

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.

THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenocarcinoma Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method Infection effects were detected by biochemical staining of β gal and immunohistochemical analysis of Rb protein The Rb cDNA of infected cells were determined by PCR The cell growth rate and cell cycle were observed by cell counting and flow cytometry Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein The transfection of wild type Rb gene could suppress GLC 82 cell proliferation and decrease the cellular DNA synthesis Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer

DETECTION OF LOSS OF HETEROZYGOSITY IN THE RB GENE IN THE PATIENTS WITH GASTRIC CARCINOMA WITH PCR-VNTR

Objective: Detection of loss of heterozygosity (LOH) of RB gene in patients with gastric carcinoma with PCR-VNTR. Methods: We investigated 37 patients with gastric carcinoma. DNA was prepared by standard methods from tumor tissues and normal gastric mucosa (20 cases). The amplification of the VNTR region was performed with PCR. PCR products were run in parallel lanes on 2% agarose gels and 6%PAGE. Results: All the normal gastric mucosa was found to have two different alleles for the VNTR region. Of the 37 cases, LOH was detected in the tumor tissues of 4 patients (10.8%). There was no significantly difference between the high, middle and low, non-differentiation tumor for the LOH of VNTR (P>0.05). Conclusion: PCR-VNTR was a simple and rapid technique for the detection of LOH of RB gene in patients with gastric carcinoma, and it might be useful in the earlier diagnosis of gastric carcinoma.

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.

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.

Analysis of Rb gene Xba Ⅰ polymorphism in Shaanxi aged atherosclerosis population

Objective:To investigate variable number tandem repeat (VNTR) polymorphism of the 17th intron of Rb gene in Shaanxi aged population and the relationship between the polymorphism of Rb gene and atherosclerosis(AS) genetic suscepti- bility. Methods: VNTR polymorphism of the 17th intron of Rb gene were examined in 100 Shaanxi aged AS patients and 100 Shaanxi aged control individuals by PCR-Rb-Xba Ⅰ-RFLP. Results::Two alleles were found both in AS group and control group, which were separately 945 bp(S1) and 630bp + 315bp(S2). S1S2 genotype was the most frequent one in the two populations. Significant difference in allele frequency was not found between AS group and control group, and allele frequency was no significant difference between Chinese and Caucasian. Conclusion: Xba Ⅰ enzyme site of Rb gene could have been certainly stable in AS population, and it was inferred that the polymorphism locus was not liable to cause mutation, which might not implicated in the formation of AS.

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...

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.

Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

SUPPRESSION OF MALIGNANT PHENOTYPE OF A TRANSFORMED MOUSE MAMMARY EPITHELIAL CELL LINE (11A1)BY TUMOR SUPPRESSOR GENE RB

A malignant transformed mammary epithelial cell line (11A1) was transfected with liposome encapsulated eukaryotic expression plasmid pCMV-neo-RB, yielding 4 constant clones which have obvious pheno-typic reversion changes, and named 11A1-R1-R4 respectively. Further experiments showed that the 11A1-R1 behaved like normal epithelial cells in both morphological and biological characteristics, with decreased clonogenicity in solid argar medium as well as decreased tumorigenicity. Northern blot hybridization showed increased expression of RB gene and decreased expression of c-myc gene in 11A1-R1, 11A1-R2 cells compared to 11A1 cells. This was an ideal phenotypic reversion model for epithelial transformed cell line and demonstrated that the RB gene can reexpress and suppress malignant phenotype in RB inactive cells.

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。