The risk of radiotherapy-related secondary cancers in children with constitutional retinoblastoma 1 (RB1) mutations has led to reduced use of external beam radiotherapy (EBRT) for RB. Presently, tumor reduction with chemotherapy with or without focal surgery (chemosurgery) is most commonly undertaken; EBRT is avoided as much as possible and is considered only as the last treatment option prior to enucleation. Nevertheless, approximately 80% of patients are diagnosed at a locally advanced stage, and only 20-25% of early stage RB patients can be cured with a chemosurgery strategy. As a whole, chemotherapy fails in more than two-thirds of eyes with advanced stage disease, requiring EBRT or enucleation. Radiotherapy is still considered necessary for patients with large tumor(s) who are not candidates for chemosurgery but who have visual potential. When radiation therapy is indicated, the lowest possible radiation dose combined with systemic or local chemotherapy and focal surgery may yield the best clinical outcomes in terms of local control and treatment-related toxicity. Proton beam therapy is one EBRT method that can be used for treatment of RB and reduces the radiation dose delivered to the adjacent orbital bone while maintaining an adequate dose to the tumor. To maximize the therapeutic success of treatment of advanced RB, the possibility of integrating radiotherapy at early stages of treatment may need to be discussed by a multidisciplinary team, rather than considering EBRT as only a last treatment option.
Child ; Child, Preschool ; Eye Neoplasms/genetics ; Genes, Retinoblastoma/genetics ; Humans ; Radiotherapy Dosage ; Retinal Neoplasms/*radiotherapy ; Retinoblastoma/genetics/*radiotherapy

OBJECTIVE(1) To investigate the promoter methylation status of gene p16(INK4a) and gene RB in breast carcinoma and the adjacent non-neoplastic hyperplastic epithelial tissue. (2) To study the correlation of p16(INK4a) gene expression at protein level with the abnormal gene methylation, the clinical manifestation and the pathological parameters.

METHODSMethylation status of promoters of p16(INK4a) gene and RB gene was detected by using methylation specific PCR in 46 cases of breast cancer, 22 cases of the adjacent non-neoplastic hyperplastic epithelium tissue and 7 cases of normal breast tissue. In addition, the p16(INK4a) gene protein expression level was also detected using immunohistochemical technique(SP method) in 46 cases of breast cancer and 22 cases of the adjacent hyperplastic epithelial tissue.

RESULTSThe methylation rate of p16(INK4a) gene was 23.9% (11/46) in breast cancer, 18.2% (4/22) in the adjacent non-neoplastic hyperplastic epithelial tissue and 1/7 in normal breast tissue, respectively. The methylation rate of RB gene was relatively low, which was 10.8% (5/46), 9.1% (2/22) and 0(0/7) in the above 3 groups, respectively. Methylation rate of p16(INK4a) gene and RB gene was not significantly different among the breast cancer, the adjacent non-neoplastic hyperplastic tissue and the normal tissues (P > 0.05). However, the methylation status of p16(INK4a) gene was closely correlated with its protein expression level and the negative ER expression result of the breast cancer (P < 0.05), but not correlated with the size of the cancer, differentiation status, lymph node metastasis, and age. The methylation status of RB gene was correlated with lymph node metastasis, but not with the size, the differentiation status, ER expression of the breast cancer and the age of the patients.

CONCLUSIONSThe abnormal methylation of p16(INK4a) gene may not play a significant role in the early stage of breast cancinogenesis, but may play a role of in the progression of the cancer. RB gene methylation may also be a indicator in choice to identify the progression and prognosis of breast cancer.

Adult ; Aged ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Genes, p16 ; Humans ; Lymphatic Metastasis ; Middle Aged ; Receptors, Estrogen ; metabolism ; Retinoblastoma Protein ; genetics ; metabolism

Apoptosis ; Brain Neoplasms ; genetics ; pathology ; Cell Proliferation ; Genes, Retinoblastoma ; genetics ; Genes, erbB-1 ; genetics ; Genes, p53 ; genetics ; Glioma ; genetics ; pathology ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; Mutation ; Nuclear Proteins ; genetics ; Tumor Suppressor Proteins ; genetics

Acute Disease ; Blast Crisis ; genetics ; Genes, Retinoblastoma ; Genes, Wilms Tumor ; Humans ; Leukemia ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Polymerase Chain Reaction ; U937 Cells

OBJECTIVETo detect the loss of heterozygosity (LOH) in esophageal squamous cell carcinoma and adjacent high-grade squamous dysplasia, and to evaluate possible tumor suppressor genes in the development and progression of invasive malignancy.

METHODSLOH was detected in normal esophageal mucosa, high grade squamous dysplasia and esophageal squamous cell carcinoma using microdissection and polymerase chain reaction technology. The changes of LOH at seven microsatellite markers and the relationship between LOH rate and clinicopathologic parameters were analyzed.

RESULTSIn high grade squamous dysplasia, LOH was detected at D13S802 (40%), D13S267 (32%), D13S221 (31%), D9S942 (30%), D17S520 (24%) and D9S171 (33%). However, D17S1798 LOH was not detected. In invasive squamous cell carcinoma, LOH was detected as follows: D13S267 (71%), D13S802 (58%), D17S520 (55%), D13S221 (45%), D9S942 (43%), D9S171 (33%) and D17S1798 (11%). The frequency of LOH in the seven microsatellite markers, the pathologic grade, clinical stage and occurrence of lymph node metastasis did not show any statistically significant correlation (P > 0.05).

CONCLUSIONSThe progression from normal squamous epithelium to high grade squamous dysplasia and subsequently to invasive squamous cell carcinoma of the esophagus was associated with accumulation of genetic errors. Possible tumor suppressor genes related to the development of esophageal squamous cell carcinoma may exist near D13S802 (13q12.12). Possible tumor suppressor genes near D13S267 (13q13.1), D17S1798 (17p13.3) and D17S520 (17p13.1) may be related to the progression of esophageal squamous cell carcinoma.

Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; Chromosome Mapping ; Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 9 ; Esophageal Neoplasms ; genetics ; Female ; Genes, Retinoblastoma ; Genes, Tumor Suppressor ; Genes, p16 ; Genes, p53 ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged ; Precancerous Conditions ; genetics

OBJECTIVETo screen and clone the novel genes related to cellular proliferation of oral squamous cell carcinoma.

METHODSWe selected the Rb gene as the bait protein gene to construct the fusion bait plasmid of yeast two-hybrid. The whole code sequence of Rb gene was acquired by digestion with restricted enzyme EcoRI and BamH1 and reclaimed from its original vector pGBT9-pRb. After being confirmed by electrophoresis, the Rb gene was cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-pRb and the sequence of the recombined plasmid was detected in company. According to the protocol of yeast two hybrid system III, the competent Y187 yeast was prepared, and transformed with recombined plasmid pGBKT7-pRb. Following that, the toxicity and transcriptional activation of this recombined plasmid pGBKT7-pRb in Y187 yeast were tested.

RESULTSThe sequence of the recombined plasmid was correct compared with the sequence provided in Genbank. The protein could be correctly synthesized in vitro, and no self-activating transcriptional activation and toxicity was observed in Y187 yeast.

CONCLUSIONSThe construction of the recombined plasmid was capable to be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombined Rb-protein could be used as the bait protein successfully.

Carcinoma, Squamous Cell ; genetics ; Genes, Retinoblastoma ; genetics ; Genetic Vectors ; Humans ; Mouth Neoplasms ; genetics ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Recombination, Genetic ; Retinoblastoma Protein ; biosynthesis ; genetics ; Two-Hybrid System Techniques ; Yeasts ; genetics

OBJECTIVEBoth tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene.

METHODSAfter identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry.

RESULTSNeither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene.

CONCLUSIONp15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

Apoptosis ; Carcinoma, Hepatocellular ; genetics ; Cell Cycle Proteins ; genetics ; Cell Division ; Cyclin-Dependent Kinase Inhibitor p15 ; Genes, Retinoblastoma ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Liver Neoplasms ; genetics ; pathology ; RNA, Messenger ; analysis ; Tumor Suppressor Proteins ; genetics

OBJECTIVEE2F-1 and Rb are involved in cell cycle regulation. This study was to illustrate the mechanism of transformation from benign papillomatosis to ductal carcinoma in situ (DCIS) of the breast in relation to E2F-1 and Rb expression.

METHODSIn situ hybridization (ISH) was used to determinate the expression of E2F-1 and Rb mRNA of mild papillomatosis (MP, n = 40), severe papillomatosis (SP, n = 40) and DCIS (n = 40). Immunohistochemistry (IHC) was used to examine the expression of E2F-1 and Rb protein.

RESULTSThe positive rate of E2F-1 mRNA expression in MP, SP and DCIS was 17.5%, 45.0% and 80.0%, and that of E2F-1 protein expression was 20.0%, 47.5% and 77.5%, respectively. There were significant differences among the three groups (P < 0.01), and between any two groups (P < 0.01). The positive rate of Rb mRNA expression in MP, SP and DCIS was 90.0%, 50.5% and 20.0%, and that of Rb protein expression was 85.0%, 52.5% and 22.5%, respectively, with statistically significant difference similar with that of E2F-1. With the progression of papillomatosis to DCIS, the expression of E2F-1 mRNA and protein increased, while that of Rb decreased. The protein expression by IHC was positively correlated with the mRNA expression by ISH. However, that of E2F-1 was negatively correlated with Rb.

CONCLUSIONE2F-1 and Rb might provide a valuable basis for screening high risk papillomatosis and new target of gene therapy for pre-cancerous lesions of the breast.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; Cell Cycle Proteins ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; E2F Transcription Factors ; E2F1 Transcription Factor ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Retinoblastoma ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Papilloma ; genetics ; metabolism ; Precancerous Conditions ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Retinoblastoma Protein ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the methylation of p16(INK4a) and RB gene, and the expression of p16(INK4a) in meningiomas.

METHODSMethylation-specific polymerase chain reaction (MSP) was used to detect the methylation of p16(INK4a) and RB in 50 cases of meningiomas, and immunostaining was performed to analyze the protein expression of p16(INK4a) in 25 of those cases.

RESULTSNo methylation was found in the benign meningiomas, whereas methylation of p16(INK4a)or RB occurred in 6(37.5%) cases of grade II tumors and 4(28.6%) cases of grade III tumors, and among these cases, an atypical meningioma showed methylation of both genes. Thirteen cases showed p16(INK4a) positive expression, but none of them was methylated.

CONCLUSIONThe methylation of p16(INK4a) or RB is related with the tumorigenesis and progression of atypical and anaplastic meningiomas, and a probable mechanism is that methylation causes the loss of expression and leads to dysfuncation of the p16(INK4a)/cyclin D1/CDK4/RB pathway.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cyclin D1 ; genetics ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinases ; genetics ; DNA Methylation ; Female ; Genes, Retinoblastoma ; Genes, p16 ; Humans ; Male ; Meningeal Neoplasms ; genetics ; Meningioma ; genetics ; Middle Aged ; Proto-Oncogene Proteins

OBJECTIVESTo explore the effects of p210 bcr/abl fusion gene on expression of beta1 integrin and L-selectin mRNAs in mouse chronic myeloid leukemia (CML) cells.

METHODSComparisons of beta1 integrin and L-selectin mRNA levels among p210 bcr/abl negative, p210 bcr/abl positive, and p210 bcr/abl-Rb-C-Box positive cells were undertaken by quantity RT-PCR.

RESULTSIn p210 bcr/abl positive cells, L-selectin mRNA level was decreased, but beta1 integrin mRNA expression had no change as compared to those in p210 bcr/abl negative cells. When inhibition of bcr-abl tyrosine kinase activity by Rb-C-Box, the L-selectin mRNA expression restored to normal (similar to p210 bcr/abl negative cells).

CONCLUSIONp210 bcr/abl oncoprotein inhibits expression of L-selectin mRNA, but not of beta1 integrin mRNA.

Animals ; Cell Line, Tumor ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Leukemic ; Genes, Retinoblastoma ; genetics ; Integrin beta1 ; genetics ; L-Selectin ; genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Mice ; RNA, Messenger ; genetics ; Transfection