Relationship between the Expression of RASSF1A Protein and Promoter Hypermethylation of RASSF1A Gene in Bladder Tumor

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma （BTCC）. The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR （MSP）. The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF 1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.     

Expression of Twist Gene in Primary Liver Cancer

In order to investigate the possibility of overexpression of Twist in primary liver cancer （PLC）, the Twist expression was detected by using immunohistochemical analysis and RT-PCR assay in 45 patients with PLC. Control tissues were obtained from 9 patients with liver hemangioma. It was found that in 36 （80.0%） out of 45 PLC patients, cancerous regions showed positive cytoplasm and nucleus staining for Twist with a diffuse pattern. In noncancerous adjacent areas and control liver tissues, the expression of Twist was 57.8% and 22.2% respectively. The results of RT-PCR assay revealed that the expression of Twist was stronger in the cancerous tissues than that in the noncancerous adjacent tissues. It was suggested that the expression of Twist was up-regulated in PLC, which play an important role in the progression of PLC.     

Expression of HLA-DR antigen in hepatocellular carcinoma and its up-regulation by interferon

Objective: To observe the expression of human leukocytic antigen DR (HLA-DR) in primary hepatocellular carcinoma (HCC) and its up-regulation by interferon (IFN). Methods: The expression of HLA-DR in 46 specimens of human HCC tissues, 4 human HCC cell lines (SMMC-7721, HCC-9204, BEL-7402 and HHCC) and a human hepatocyte cell line QZG was respectively detected by immunohistochemical ABC staining and flow cytometry. The expression of HLA-DR in the 5 cell lines was detected by ELISA before and after the cells were treated with IFN-γ or IFN-α. Results:Eighteen out of 46 HCC tissues (39.1%) expressed HLA-DR, whereas all the normal liver tissues immediately adjacent to HCC tissues were HLA-DR-negative. No obvious HLA-DR-positive staining was found in all the 5 cell lines. The expression of HLA-DR was up-regulated in all the 5 cell lines after IFN-γ or IFN-α treatment. The up-regulation of HI A-DR in QZG cells was less obvious than that in HCC cell lines. The effect of IFN-γ was more significant than that of IFN-α. Conclusion: HCC tissues can express HLA-DR to some extent, but HCC cell lines do not express detectable HLA-DR. IFN can up-regulate HLA-DR expression in HCC cells.     

Expression of cancer-testis antigen CT10 gene mRNA in hepatocellular carcinoma and prediction of HLA - A2 restricted CTL epitopes of CT10

Objective To investigate the expression of cancer-testis antigen CT10 gene mRNA in hepatocellular carcinoma and predict the HLA-A2-restricted CTL epitopes of CT10. Methods The expression of CT10 mRNA was detected by using RT - PCR in HCC tissues and the corresponding adjacent non-HCC tissues from 45 HCC patients, among those 3 samples selected randomly from CT10 PCR positive products were sequenced. HLA - A2-restricted CTL epitopes of CT10 was predicted by peptide supermotif prediction combined with quantitative motif. Results CT10 mRNA was detectable in 19/45 (42. 2%) of HCC samples, while the corresponding adjacent non-HCC tissue were all negative in expression of CT10 mRNA. In addition, The DNA sequence confirmed that the RT- PCR products were truly CT10 cDNA. No June 2003 Vol12 No2 relationship was found between the expression of CT10 and demographic and clinical features such as age, sex, tumor size, degree of tumor differentiation, serum α-fetoprotein level and infection of hepatitis B virus or h     

Expression of p28GANK and its correlation with RB in human hepatocellular carcinoma

Background：Aberrance of retinoblastoma protein （RB） signal pathway is known to play an important role in the carcinogenesis of human hepatocellular carcinoma （HCC）. p28GANK, originally purified from human 26S proteasome as a nonATPase subunit, was recently found in HCC and shown to interact with RB. The aim of this study was to investigate the expression profile of p28GANK and its correlation with RB in HCC. Methods：The expression of p28GANK was evaluated in 55 surgically resected HCCs by immunohistochemistry （IHC）, and the associations were explored between p28GANK level and clinicopathologic features as well as tumor suppressor RB. Western blotting was performed to determine p28GANK expression level in 12 HCCs. Immunofluorescence stainings of p28GANK and RB in U2-OS cells were examined by confocal microscopy. Results：Positive p28GANK cytoplasmic staining was recognized in 55 HCCs. Nuclear positive occurrence of p28（GANK） in HCCs was more frequent than paracancerous hepatic tissues （P〈0. 05）. The overexpression probability of p28GANK was inversely associated with Edmonson＇s grade：overexpression occurred in nine out of 11 （81.8%）, 22 out of 35 （62.9%） and two out of nine （22.2%） in Ⅰ -Ⅱ,Ⅲ and Ⅳ graded cases, respectively （P=0. 004）. Total cellular expression of p28GANK had curvilinear correlation with the nuclear expression of RB （r= 0. 475, P = 0.019）, while the nuclear expression of p28GANK had not. Western blot analysis showed that up-regulation of p28GANK expression was found in nine out of 12 HCCs compared with paracancerous liver tissues. Exogenously expressed p28GANK colocalized with RB in cytoplasm of U2-OS cells. Conclusion： These results confirm the role of p28GANK as a highly expressed oncoprotein in HCC by in situ examination. Its overexpression correlates with the differentiation status of HCC. The whole cellular p28GANK activation, not nuclear portion only, influences the alteration of RB. Underlying nuclear translocation of p28GANK may contribute to the counteraction against RB through a feed back loop. These data provide new evidence for p28GANK to be used as a promising drug target of a therapeutic agent against HCC.     

Expression of Survivin, p53 and its relationship with apoptosis, proliferation in hepatocellular carcinoma（HCC）

Objective:To investigate the expression of Survivin p53 and its relationship with apoptosis、proliferation in hepatocellular carcinoma(HCC). Methods:The expression of Survivin, p53 and the proliferation of tumor cells marked by proliferation cell nuclear antigen (PCNA) in 42 cases of HCC were assessed by immunohistochemical method. TUNEL was used to detect apoptosis. Results:Survivin protein was expressed in 30 of 42 cases of HCC(71.4%) and in 4 of 34 cases of adjacent cirrhosis tissues(11.8%). Expression of Survivin protein was negative in 10 cases of normal tissues. Survivin protein positive expression rate in HCC was significantly higher than adjacent cirrhosis tissues and normal tissues(P < 0.001). The increased Survivin protein expression in cancer was significantly associated with histological grade(P = 0.003), p53 protein(P = 0.013), survival time(P < 0.05) and the ratio of proliferative index to apoptotic index(P < 0.01), but was not significantly correlated with age, sex, clinical stage, tumor size, metastasis, AFP, HBs-Ag(P > 0.05).Conclusion:There is a marked increased expression of Survivin in HCC, which may play an important role in breaking the balance of proliferation and apoptosis of HCC cells. The correlation between Survivin and p53 expression in HCC indicates that cooperation between Survivin and p53 plays a certain role in occurrence and /or development of HCC.     

Study on MXR7 methylation in hepatocellular carcinoma

Objective：To obtain information at the molecular level on the possible mechanism of MXR7 gene overexpressed in hepatocellular carcinoma （HCC） and also to provide a clue for further study. Methods：Genomic DNA was isolated from 20 samples of hepatoma and paired non-HCC liver tissues, 2 cases of blood tumor and two types of cells （HepG2, MCF-7） and digested with two kinds of endonucleases （EcoR I and Eag I which is methylation sensitive endonuclease）. And the condition of MXR7 gene methylation was examined and analyzed by Southern blot. Results： MXR7 was unmethylated neither in tested tumorous liver samples nor in paired non HCC liver tissues. In addition, the same result was found in 2 blood tumor samples and HepG2. Only two paired samples had different methylation outcome, one was unmethylated and the other was partly methylated. Conclusion： MXR7 is unmethylated in HHC, suggesting methylation of MXR7 may have no relation with its expression and regulation.     

Expression of the MAGE-1 gene in human hepatocellular carcinomas

Objective To further investigate the expression of MAGE-1 gene in hepatocellular carcinom a (HCC). Methods The tumors and adjacent liver tissue from 45 HCC patients and liver tissue from 28 non-HCC patients (16 with liver cirrhosis and 12 with normal liver) were cha racterized by RT-PCR. A 421 bp PCR product from a cDNA fragment spanning exon s 1, 2 and 3 was sequenced. The HLA type was assayed by standard ELISA in 43 HC C patients.Results Thirty-two of 45 tumor tissues from HCC patients expressed MAGE-1 mRNA (71.1% ). In contrast, MAGE-1 mRNA was not detected in adjacent tissues. Three were found to have point mutations at 3 identical sites resulting in the substitutio n of two amino acid residues. The most frequent HLA types in 43 HCC patients we re: HLA-A2, 53.5%; A11, 25.6%; A24, 20.9%; A33, 20.9%; HLA-B13, 28.3% and B35, 23.2%. Expression of HLA-A33 (20.9%) was higher in HCC patients than t hat predicted in the normal Chinese population (8.8%). There was no discer nable correlation between MAGE-1 expression and α-FP level, tumor size and he patitis B or C virus infection. The identification of peptides which are restri cted by haploptypes other than A1 should increase the opportunity for peptide ba sed immunotherapy.Conclusions This study shows that MAGE-1 mRNA is highly expressed in HCC tumor tissue in Ch inese patients. Previously unreported point mutations in the MAGE-1 gene are d escribed and may also provide additional opportunities for immunotherapy.     

Methylation of RAR-β2, RASSF1A, and CDKN2A Genes Induced by Nickel Subsulfide and Nickel-carcinogenesis in Rats

Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide (Ni 3 S 2 ) at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5’ region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of t...     

Relationship between alterations of p16INK4a and p14ARF genes of CDKN2A locus and gastric carcinogenesis

Objective To investigate the relationship between alterations of p16INK4a and p14ARF genes and gastric carcinogenesis. Methods The tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied. The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16INK4a and p14ARF genes were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR. Results ① The homozygous deletion rate of p16INK4a and p14ARF was 35.4% （17/48）, and no homozygous deletion was examined in any gastric tissue neighboring the tumor. ② There was no point mutation of p16INK4a and p14ARF in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor. ③ Methylation of the CpG islands of p16INK4a and p14ARF was detected in 47.9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference (P&lt;0.01). ④ The loss rate of p16INK4a mRNA was 47.9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1α and 2 had a higher loss rate (100%, 6/6) of p16INK4a mRNA than those of the methylation of the other exons (11.8%, 2/17, P&lt;0.01); the loss rate of p14ARF mRNA was 45.8%(22/48) in gastric cancer, and patients with the combined methylation of exons 1β and 2 had a higher loss rate (100%, 3/3) of p14ARF mRNA than those of the methylation of the other exons (15%, 3/20, P&lt;0.05). ⑤ The combined loss of p16INK4a and p14ARF mRNAs was examined in 1 (5.6%) of 18 patients of well and moderately-differentiated carcinomas, and 11 (36.7%) of 30 patients of poorly and not-differentiated carcinomas with a significant difference (P&lt;0.05). Conclusion p16INK4a and p14ARF genes are frequently inactivated by homozygous deletion and methylation of the 5’CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer.     

Expressions of Nucleoside Diphosphate Kinase （nm23） in Tumor Tissues are Related with Metastasis and Length of Survival of Patients with Hepatocellular Carcinoma

Objective To evaluate the relationship of expressions of nucleoside diphosphate kinase （nm23） and proliferating cell nuclear antigen （PCNA）, as well as apoptosis, with the prognosis of HCC patients by analyzing their pathological and clinical data. Methods The expressions of nm23 and PCNA were analyzed by immunohistochemistry and the apoptotic phenomena were detected by TUNEL technique in the liver samples from 43 HCC tissues, 39 para-neoplastic tissues, and 10 normal tissues. The mean apoptosis index （AI） and proliferative index （PI） in individual sample were calculated. Results As shown by the detection, 32.6% of carcinomas had negative nm23 signal in tumor tissues, whereas all para-neoplastic and normal tissues had positive nm23. The AI in nm23 positive HCC was significantly higher than that in nm23 negative one, with statistical difference （P0.05）. Furthermore, the expressions of nm23, and the values of AI and PI were contrastively analyzed with some main pathological and clinical data of HCC. It revealed that HCC with extrahepatic metastasis showed remarkable correlation with the negative nm23 （P=0.013） and higher PI values of HCC （P=0.015）. The disease-free survival in HCC patients with negative nm23 expression was significantly poorer than that in patients with positive nm23 expression. Conclusion These data suggest that expressions of nm23 protein in tumor tissues are correlated with occurrences of metastasis and length of survival of the HCC patients, which may be an indicator for their prognosis.     

Expression and Prognostic Significance of Livin in the Progression of Bladder Cancer

It has been suggested that progression of bladder transitional cell cancer (BTCC) may be regulated at the molecular level by a typical pattern of expression of genes involved in apoptosis. Re-cently Livin, belonging to the inhibitors of apoptosis (IAP) family, has been found to be expressed in most solid tumors, where its expression is suggested to have clinical significance. In order to explore the significance of Livin expression in the development of BTCC, immunohistochemistry and RT-QPCR were used to detect the expression of Livin mRNA in tumor tissues and adjacent normal tissues of 30 cases of BTCC. The results showed that the positive rate of Livin expression in adjacent normal tissues and tumor tissues was 0 and 60% (18/30) respectively. The -△△CT value of Livin in BTCC tissues was 8.0454 (7.4264-8.6644) times of that in adjacent normal tissues. The expression of Livin mRNA had no correlation with tumor pathological grades and clinical stages. It was sug-gested that there was weak expression of Livin mRNA in adjacent normal tissues, but strong in tumor tissues.     

Up-regulated manganese superoxide dismutase expression increases apoptosis resistance in human esophageal squamous cell carcinomas

Background Esophageal cancer is one of the most common malignancies in the world. In order to identify the proteins associated with esophageal squamous cell carcinomas （ESCC）, we analyzed the protein profiles of ESCC cases with tumor and matched adjacent normal tissues. Methods Two-dimensional electrophoresis （2-DE） was carried out to analyze the protein profiles. Dysregulated protein spots were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight （MALDI-TOF） and verified by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry （LC-ESI-IT MS）. RT-PCR and immunohistochemistry on tissue microarray were performed to confirm the gene dysregulation in esophageal cancerous tissues. RNA interference （RNAi） was used to knock down the gene expression in ESCC cell lines. Apoptosis assay with annexin V-FITC/PI staining was conducted and cells were analyzed by flow cytometry. Results 2-DE showed that two protein spots with approximate molecular weights and different pl were elevated in 12 out of 18 ESCCs as compared to the corresponding normal tissues. Both the two spots were identified as MnSOD by MALDI-TOF and were verified by LC-ESI-IT MS. MnSOD overexpression was detected in 14 tumors out of 24 cases by RT-PCR and 52 tumors out of 116 cases by immunohistochemistry comparing to normal epithelia, siRNA-mediated silencing of MnSOD in KYSE450 and KYSE150 cell lines revealed that MnSOD protected ESCC cells from apoptosis induced by ultraviolet （UV） and doxorubicin （DOX）. Conclusions These findings suggest that there existed two isoforms of MnSOD protein in normal and tumor esophageal tissues. MnSOD was overexpressed in ESCC and its up-regulation in esophageal cancer cells was associated with apoptosis resistance.     

Expression of Vascular Endothelial Growth Factor and Cyclooxygenase-2 in Laryngeal Squamous Cell Carcinoma and Its significance

n order to study the expressions of vascular endothelial growth factor （VEGF） and cyclooxygenase-2 （COX-2） in human laryngeal squamous cell carcinoma （LSCC） and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues （both P〈0. 01）, and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ⅲ＋ Ⅳtissues of LSCC as compared with the stage Ⅰ ＋ Ⅱ tissues of LSCC （P 〈0.01）. There was a high positive correlation between VEGF and COX-2 expression in LSCC （r= 0. 756,P〈0.01）. These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.     

Treatment of hemangioma by transfection of antisense VEGF gene

The effect of transfection of antisense vascular endothelial growth factor （VEGF） gene on the growth of hemangioma was studied. A total of 49 cases of capillary hemangiomas of the skin were collected. Immunohistochemical method was used to detect the expression of PCNA in hemangioma tissues. According to the finding, 49 cases of hemangiomas fell into proliferating phase （27 cases） and involuting phase （22 cases） respectively. Another 5 cases of normal skin tissues adjacent to the tumor tissues served as control. Immunohistochemical staining was performed to detect the expression of VEGF in the tumor tissues and the normal tissues. The average absorbance （A） values and the average positive area rate of VEGF were measured by image analysis system （HPIAS-2000）. Endothelial cells from the tumor tissues in proliferating phase were cultured. Eukaryotic expression vector was constructed by sub-cloning, and transfected into human hemangioma endothelial cells by using cation liposome as vector. The expression of VEGF mRNA and protein was detected by RT-PCR and indirect immunofluorescence assay （IFA）, respectively, and the biological characteristics of the transfected endothelial cells were examined by MTT assay and flow cytometry （FCM） after transfection. Immunohistochemical results showed that the expression of VEGF in proliferating endothelial cells was remarkably higher than those in involuting endothelial cells and normal endothelial cells （P〈0.01）, but there was no significant difference in the expression of VEGF between involuting endothelial cells and normal ones （P〉0.01）. Electrophoresis and sequencing indicated that the eukaryotic expression vector containing antisense VEGF gene, i.e. pcDNA3.1-VEGF, was success- fully constructed. After VEGF antisense RNA recombinant was transfected into hemangioma endothelial cells, RT-PCR revealed that the expression of VEGF mRNA in pcDNA-VEGF （V） group and blank group was obviously higher than that in pcDNA-VEGF （A） group, and that the expression of endogenous VEGF mRNA in pcDNA-VEGF （A） group was significantly inhibited. Immunohistochemical result demonstrated that, compared with blank group, there was statistically significant difference between pcDNA-VEGF （A） and pcDNA-VEGF （V） groups （P〈0.01）, but there was no significant difference between pcDNA-VEGF （V） group and blank group （P〉0.05）. The activity of endothelial cell proliferation was reduced significantly after transfection, and obvious apoptosis occurred in hemangioma endothelial cells after transfection of antisense VEGF. It was suggested that VEGF plays an important role in the pathological change of hemangiomas by promoting endothelial cell proliferation and angiogenesis. Antisense VEGF gene transfection could effectively inhibit the growth of hemanioma endothelial cells.