Reduced expression of intercellular adhesion molecule-1 in ovarian adenocarcinomas.

Ovarian adenocarcinomas develop as the result of multiple genetic and epigenetic changes in the precursor ovarian surface epithelial (OSE) cells which result in a malignant phenotype. We investigated changes in gene expression in ovarian adenocarcinoma using a cDNA array containing 588 known human genes. We found that intercellular adhesion molecule-1 (ICAM-1) was expressed at lower levels in the ovarian tumour cell lines OAW42, PEO1 and JAM than in the immortalised human ovarian surface epithelial cell line HOSE 17.1. Further investigation revealed ICAM-1 was expressed in the surface epithelium of normal ovaries and both mRNA and protein expression levels were reduced in the majority of ovarian adenocarcinoma cell lines and primary tumours. ICAM-1 expression was increased in 8/8 cell lines treated with the de novo methyltransferase inhibitor 5-aza-2'-deoxycytidine, indicating that methylation of CpG islands may play a role in the down-regulation of its expression in primary tumours. There was a significant association between patients whose tumours expressed ICAM-1 and survival (P = 0.03), suggesting that expression levels of ICAM-1 may have clinical relevance.     

上皮性卵巢肿瘤中3p14、3p25杂合性丢失

目的经对原发性上皮性卵巢癌的3号染色体短臂(3p14和3p25)杂合性丢失(LOH)分析研究,以探讨3pLOH与卵巢癌的发生发展之间的相关性.方法采用PCR法分别对50例原发性上皮性良恶性卵巢肿瘤(40例癌组织及10例非癌组织)的3p14及3P25处两个特定的位点D3S1228和D3S1038作杂合性丢失(LOH)检测.结果40例原发性上皮性卵巢癌中在3p处总LOH率为80%,其中3p14处发生LOH为24例(60.0%),3p25处为16例(40.0%),两处共同发生LOH的有8例(20.0%).FIGO卵巢癌的Ⅲa及Ⅲb期患者3pLOH率分别为93.8%及91.6%明显高于Ⅰ-Ⅱ期患者50.0%的3p丢失率(P＜0.05).10例良性卵巢肿瘤患者分别在3p14和3p25各处仅1例出现杂合性丢失,但未见有共丢失现象.40例不同病理类型的卵巢癌中以浆液性癌3p14和3p25LOH率为最高,但因病例数少而无统计学意义(P＞0.05).结论3p处可能存在一或多个与卵巢癌发生发展有关的侯选抑癌基因并与卵巢癌的恶性程度相关,有望作为遗传标志而应用于临床.     

沉默Id1基因表达对卵巢癌细胞生长影响的实验研究

目的:通过构建人卵巢癌SKOV3 Id1特异性siRNA慢病毒载体及其裸鼠移植瘤模型,观察沉默分化抑制因子（inhibitor of differentiation-1,Id1）表达对卵巢癌SKOV3生长的影响。方法:根据Id1基因序列设计并合成的siRNA片段转染SKOV3细胞,将转染细胞分为四组:cell+lv-shRNA-Id1（实验组）、cell+lv-shRNA-NC(质粒对照组)、cell+lv-control(空病毒对照组)、cell(空白对照组),筛选出稳定转染的SKOV3细胞克隆,48小时后检测细胞生长活性。将40只裸鼠随机分为四组,分别接种上述转染成功的细胞,30天后称取瘤体重量,计算抑瘤率;然后通过 Western blotting 法检测移植瘤中Id1蛋白的表达。结果:在体外试验中,实验组的细胞生长活性显著低于空白对照组,具有统计学意义（P＜0.05）,三对照组间无明显差异（P＞0.05）;体内实验显示,实验组平均瘤重均低于其他三对照组,差异具有统计学意义（P＜0.05）,三对照组间瘤重差异无统计学意义（P＞0.05）;实验组、质粒对照组、空病毒组抑瘤率分别为45.98%、4.84%、4.50%;Western blotting 结果显示,实验组中Id1表达较对照组明显下调（P＜0.05）。结论:沉默Id1基因能抑制卵巢癌细胞的生长,有望成为卵巢癌治疗新靶点。     

Loss of heterozygosity at 7p in Wilms' tumour development

Chromosome 7p alterations have been implicated in the development of Wilms' tumour (WT) by previous studies of tumour cytogenetics, and by our analysis of a constitutional translocation (t(1;7)(q42;p15)) in a child with WT and radial aplasia. We therefore used polymorphic microsatellite markers on 7p for a loss of heterozygosity (LOH) study, and found LOH in seven out of 77 informative WTs (9%). The common region of LOH was 7p15-7p22, which contains the region disrupted by the t(1;7) breakpoint. Four WTs with 7p LOH had other genetic changes; a germline WT1 mutation with 11p LOH, LOH at 11p, LOH at 16q, and loss of imprinting of IGF2. Analysis of three tumour-associated lesions from 7p LOH cases revealed a cystic nephroma-like area also having 7p LOH. However, a nephrogenic rest and a contralateral WT from the two other cases showed no 7p LOH. No particular clinical phenotype was associated with the WTs which showed 7p LOH. The frequency and pattern of 7p LOH demonstrated in our studies indicate the presence of a tumour suppressor gene at 7p involved in the development of Wilms' tumour.     

Inflammation-associated gene expression is altered between normal human ovarian surface epithelial cells and cell lines derived from ovarian adenocarcinomas

Ovulation is believed to contribute to the development of ovarian cancers that derive from the ovarian surface epithelium (OSE). The process of ovulation is synonymous with inflammation and inflammatory cytokines such as interleukin-1alpha (IL-1alpha) have recently been shown to induce both inflammatory and anti-inflammatory responses in human OSE (HOSE) cells. In this study we directly compared levels of IL-1alpha-induced gene expression by analysing the levels of 11beta-hydroxysteroid dehydrogenase (11betaHSD) types 1 (11betaHSD-1) and 2 (11betaHSD-2), cyclooxygenase-2 (COX-2), IL-1 receptor (IL-1R) and glucocorticoid receptor alpha (GRalpha) mRNA between normal HOSE cells and cell lines derived from poorly differentiated (SKOV-3, BG-1, PEO-4) and well-differentiated (PEO-14) ovarian adenocarcinoma. In HOSE cell cultures, and to a lesser extent PEO-14 cells, the basal mRNA levels of COX-2 and 11betaHSD-1 were relatively high and further shown to be induced in response to IL-1alpha (for HOSE cells; >20-fold, P<0.05 and PEO-14 cells; >3fold, P<0.05). However, whereas HOSE cells expressed a low level of 11betaHSD-2 mRNA that was only mildly responsive to IL-1alpha (1.3-fold, P<0.001), all cell lines exhibited a higher basal level of 11betaHSD-2 mRNA that was in some cases further stimulated in PEO-4 cells (five-fold; P<0.05) or suppressed in SKOV-3 cells (two-fold; P<0.01) in response to IL-1alpha. All cells tested expressed IL-1R and, with the exception of BG-1, GRalpha. These results indicate that cell lines derived from ovarian cancers have lost the ability to respond normally to inflammatory cytokines such as IL-1alpha. The finding that normal OSE cells, in contrast to cell lines derived from patients with ovarian adenocarcinoma, abundantly express 11betaHSD-1 mRNA but are essentially devoid of 11betaHSD-2 mRNA supports the concept that the pattern of 11betaHSD isoform gene expression is a defining feature of neoplastic cellular transformation, which might have particular relevance to the ovary.     

High resolution chromosome 3p, 8p, 9q and 22q allelotyping analysis in the pathogenesis of gallbladder carcinoma

Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p (n=26), 8p (n=14), 9q (n=29) and 22q (n=12) regions. We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis.     

Knockdown of stat3 expression by RNAi inhibits in vitro growth of human ovarian cancer

Background

The aim of the study was to investigate the suppressive effects of pSilencer2.1-U6-siRNA-stat3 recombinant plasmids on the growth of ovarian cancer in vitro.

Material and methods.

Three pairs of DNA template (stat3-1, stat3-2, stat3-3) specific for different target sites on stat3 mRNA were synthesized to reconstruct pSilencer2.1-U6-siRNA-stat3s, which were transfected into SKOV3 cells. The expressions of STAT3, BcL-2, cyclin D1 and C-myc in these cells were detected by Western blot and Northern blot. The cell cycle and the growth were determined by flow cytometry (FCM) and MTT assay, respectively. Cell apoptosis was determined by TUNEL staining.

Results

Of the three siRNAs, only siRNA targeting stat3-3 markedly suppressed the protein expression of stat3 in SKOV3 cells; MTT assay and FCM showed that transfection of stat3-3 siRNA could significantly suppress the growth of SKOV3 cells and arrest the cell cycle in vitro. TUNEL staining also showed massive apoptosis in SKOV3 cells transfected with stat3-3 siRNA.

Conclusions

pSilencer2.1-U6-siRNA-stat3-3 can significantly inhibit the STAT3 expression in human ovarian cancer cells resulting in the inhibition of the cancer growth and the increase of apoptosis of cancer cells.     

正常卵巢上皮细胞和卵巢上皮癌细胞基因表达差异的研究

目的：探讨卵巢上皮癌细胞相关基因的差异表达。方法：采用舍384条肿瘤相关基因的cDNA阵谱检测了卵巢上皮癌细胞株SKOV-3及正常卵巢上皮细胞的基因谱，分析卵巢上皮癌细胞相关基因的差异表达。结果：在384条候选基因中，与卵巢癌相关的差异表达基因33条，其中22条表达上调，11条表达下调。结论：cDNA阵谱技术是筛查卵巢癌相关基因的有效方法。     

Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions.     

Determination of p53 genotypes in oral cancer patients from India

The p53 tumour suppressor gene is inactivated in various types of human cancers, and has been implicated as an early event in several cancers. A p53 Pro/Arg polymorphism at exon 4 codon 72, has been suggested to be involved in susceptibility to cancers as well. Hence, in the current study, we investigated p53 exon 4 codon 72 polymorphism using Proline or Arginine specific primers from the peripheral blood cells (PBC) representing constitutional DNA from 72 oral cancer patients. PBC from 153 normal healthy individuals were used to determine the frequency of the p53 genotypes, Pro/Pro, Arg/Arg and Pro/Arg, in the Indian population. The frequency of distribution of genotypes in the normal healthy individuals was, Pro/Pro - 0.20 (31/153), Arg/Arg -- 0.14 (22/153) and Pro/Arg -- 0.65 (100/153); and in the oral cancer patients was, Pro/Pro -- 0.19 (14/72), Arg/Arg -- 0.08 (6/72) and Pro/Arg -- 0.72 (52/72). Thus, we observed an equidistribution of the genotypes in normal control and oral cancer patients (chi(2)= 1.77, df = 2, 0.3 相似文献   

实时荧光定量PCR法检测卵巢良、恶性肿瘤中Id1 mRNA的表达

目的：检测Id1 mRNA在人卵巢良、恶性肿瘤组织中的表达及其意义。方法：用SYBR GreenI嵌合荧光法进行实时逆转录-聚合酶链反应（RT-PCR）,检测Id1 mRNA在人良性卵巢囊肿和卵巢癌组织中的相对定量,分析Id1 mRNA的含量变化与卵巢良、恶性病变的关系。结果：Id1 mRNA在卵巢癌组织中的含量明显高于良性卵巢囊肿组织（P〈0.01）。卵巢癌组织分化低和恶性度高的组织Id1mRNA的表达量明显高于分化高或恶性程度低的组织。高Id1mRNA表达和患者预后呈显著相关（P〈0.001）。结论：卵巢癌组织中Id1 mR-NA表达显著高于良性囊肿组织,而且与癌组织恶性度和预后密切相关。Id1 mRNA实时定量检测有助于从基因转录水平对卵巢癌进行早期辅助诊断并初步判断预后。     

Identification of differentially expressed genes according to chemosensitivity in advanced ovarian serous adenocarcinomas: expression of GRIA2 predicts better survival

Background:

The purpose of this study was to identify genes that are differentially expressed in chemosensitive serous papillary ovarian carcinomas relative to those expressed in chemoresistant tumours.

Methods:

To identify novel candidate biomarkers, differences in gene expression were analysed in 26 stage IIIC/IV serous ovarian adenocarcinomas (12 chemosensitive tumours and 14 chemoresistant tumours). We subsequently investigated the immunohistochemical expression of GRIA2 in 48 independent sets of advanced ovarian serous carcinomas.

Results:

Microarray analysis revealed a total of 57 genes that were differentially expressed in chemoresistant and chemosensitive tumours. Of the 57 genes, 39 genes were upregulated and 18 genes were downregulated in chemosensitive tumours. Five differentially expressed genes (CD36, LIFR, CHL1, GRIA2, and FCGBP) were validated by quantitative real-time PCR. The expression of GRIA2 was validated at the protein level by immunohistochemistry, and patients with GRIA2 expression showed a longer progression-free and overall survival (P=0.051 and P=0.031 respectively).

Conclusions:

We found 57 differentially expressed genes to distinguish between chemosensitive and chemoresistant tumours. We also demonstrated that the expression of GRIA2 among the differentially expressed genes provides better prognosis of patients with advanced serous papillary ovarian adenocarcinoma.     

Identification of two contiguous minimally deleted regions on chromosome 1p36.31-p36.32 in oligodendroglial tumours

Loss of the short arm of chromosome 1 is a hallmark of oligodendroglial tumours (OTs). Deletion mapping studies in OTs have revealed multiple commonly deleted regions on chromosome 1p, suggesting that there are more than one tumour suppressor gene. To map critical deletion regions on 1p, a series of 25 OTs were examined for loss of heterozygosity (LOH) on 19 polymorphic markers across the 1p arm using microsatellite analysis. Our study revealed that 60% of tumours had LOH of all informative markers on 1p and identified one tumour showing LOH at telomeric markers only. Since this deletion region lies in one of the critical deletion intervals defined previously, we then screened another series of 27 OTs specifically at 1p36.3 for LOH using nine polymorphic markers. A total of 12% (six out of 52) of tumours were found to carry interstitial deletions. The allelic status and the deletion breakpoints in these tumours with interstitial deletion were further verified by fluorescent in situ hybridisation. The small overlapping intervals facilitated the delineation of two contiguous minimally deleted regions of 0.76 Mb, defined by D1S468 and D1S2845, and of 0.41 Mb, bound by D1S2893 and D1S1608, on 1p36.31-36.32. Based on current reference human genome sequence these deletion regions have been sequenced almost to entirety and contain eight annotated genes. TP73, DFFB and SHREW1 are the only known genes located in these deletion regions, while the others are uncharacterised novel genes. In conclusion, our study has narrowed down the critical tumour suppressor loci on 1p36.3, in which two minimally deleted regions are mapped, and markedly reduced the number of candidate genes to be screened for their involvement in OT development.     

Single-nucleotide polymorphism-mass array reveals commonly deleted regions at 3p22 and 3p14.2 associate with poor clinical outcome in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common solid tumors in the world with poor prognosis. Deletion of chromosome 3p is one of the most frequent chromosomal alterations in ESCC, suggesting the existence of one or more tumor suppressor genes (TSGs) at this region. In the present study, a recently developed high-throughput and high-resolution technology, single-nucleotide polymorphism (SNP)-mass array, was applied to investigate loss of heterozygosity on 3p in 100 primary ESCC cases with 386 SNP markers. Four commonly deleted regions (CDRs) at 3p26.3, 3p22, 3p21.3 and 3p14.2 were identified. Absent and down-regulated expression of several candidate TSGs, including CHL1, PCAF, RBMS3, PLCD1 and CACNA2D3, were detected in primary ESCC tumors and ESCC cell lines. Moreover, deletions of CDRs 2 and 4 were correlated with advanced tumor stage and deletion of CDR2 was associated with tumor metastasis in ESCC. Our findings provided evidence that minimal deleted regions at 3p26.3, 3p22, 3p21.3 and 3p14.2 containing potential TSGs may contribute to the pathogenesis of esophageal cancer.