IGSF4 promoter methylation and expression silencing in human cervical cancer

OBJECTIVES: Functional assays of tumor suppression and loss of heterozygosity point to a tumor suppressor gene (TSG) for cervical cancer (CC) on chromosome 11q23. We evaluated IGSF4, a putative TSG located in the region, for promoter methylation and gene silencing in CC cell lines and cervical tissues. METHODS: IGSF4 expression was detected by both RT-PCR and Northern blot analysis. Methylation maps of the IGSF4 promoter region were generated for 11 CC cell lines based upon bisulfite-genomic sequencing, using seven nested-PCR primer sets covering 97 CpG sites. Methylation fingerprints in primary cervical tissues were evaluated by denaturing high performance liquid chromatography. RESULTS: A 4.4-kb mRNA was seen in cell lines, consistent with the RT-PCR results for both cell lines and primary cervical tissue. IGSF4 was expressed in 6/11 cell lines, 6/8 CC tissues and in all seven normal cervical epithelia. In the cell lines, IGSF4 silencing was associated with promoter hypermethylation. The methylation status in the region covering the -18 to -2 CpG sites correlated most strongly with expression, pointing to the existence of an unmethylated core in the IGSF4 promoter in cell lines expressing IGSF4. This unmethylated core spans approximately 180 bp and is immediately upstream of the ATG site. In primary tissues, methylation was detected in 15/23 (65%) CC specimens but in none of seven normal cervical epithelia. CONCLUSIONS: Our data strongly suggest that IGSF4 is a TSG and that gene silencing by aberrant hypermethylation may contribute to the development of CC.     

Allelic Losses at Chromosome 3p Are Seen in Human Papilloma Virus 16 Associated Transitional Cell Carcinoma of the Cervix

OBJECTIVE: Transitional cell carcinomas (TCCs) of the cervix are rare neoplasms of the female genital tract. Although these tumors display urothelial differentiation, there is controversy regarding their histogenetic relationship to squamous cell carcinomas (SCC) of the cervix versus transitional cell carcinomas of the bladder. METHODS: We performed partial allelotyping of five TCCs of the cervix using 23 polymorphic markers located on chromosomes 3p and 9, which demonstrate frequent and early losses in cervical SCC and urothelial TCC, respectively. Multiplex polymerase chain reaction was used on DNA extracted from archival paraffin-embedded tissue using precise microdissection. Additionally, P53 gene mutation analysis was performed using single-strand confirmation polymorphism (SSCP) and the presence of human papilloma virus (HPV) sequences was analyzed using general and specific (types 16 and 18) primers. RESULTS: General HPV sequences were demonstrated in all cases, but the oncogenic strain HPV 16 was present in only three (60%) of the five tumors; no HPV 18 was detected in any sample. Three of five TCCs, all harboring HPV 16 sequences, demonstrated concurrent allelic losses at several 3p loci (specifically 3p12, 3p14.2 [the FHIT gene locus], 3p21.3, and 3p22-24.2). LOH at a single locus on 9q32-qter was demonstrated in one tumor; no other deletions were seen on chromosome 9. P53 gene mutations in exons 5-8 were absent by SSCP analysis. CONCLUSIONS: The infrequent involvement of chromosome 9 in TCCs of the cervix, along with the concurrent presence of 3p LOH and oncogenic HPV 16 in a subset of tumors, suggests a closer histogenetic relationship of this neoplasm to cervical SCCs rather than urothelial TCCs.     

人端粒酶逆转录酶在子宫颈癌组织中的表达变化及其意义

目的研究端粒酶在宫颈癌及其癌前病变组织中的表达,探讨人端粒酶逆转录酶(hTERT)作为宫颈癌早期诊断标志物的可能性。方法采用RTPCR方法测定端粒酶mRNA在3株宫颈癌细胞、2株正常的人二倍体细胞、38例宫颈癌、16例宫颈上皮内瘤变(CIN)和20例正常宫颈组织中的表达水平;端粒重复序列扩增酶联免疫吸附法测定端粒酶活性;免疫组化链酶菌抗生物素蛋白过氧化物酶(SP)连接法检测3株宫颈癌细胞、2株正常的人二倍体细胞、21例正常宫颈组织、2例CIN和55例宫颈癌切片中hTERT蛋白的表达水平。结果hTERTmRNA在3株宫颈癌细胞中均表达,而在2株正常的人二倍体细胞中无表达,宫颈癌组织中的阳性表达率为81.6%,高于CIN的37.5%和正常宫颈组织的5.0%,差异有统计学意义(P<0.05);hTERTmRNA的阳性表达率与端粒酶活性有相关性(P<0.01);hTERT蛋白在3株宫颈癌细胞中有表达,2株正常的人二倍体细胞中无表达,正常宫颈组织中阳性表达率为4.8%、CIN为28.0%,均低于宫颈癌组织的65.5%,差异有统计学意义(P<0.05)。结论宫颈癌组织和细胞中hTERTmRNA和蛋白阳性表达率均高于正常宫颈组织,其有可能作为宫颈癌早期诊断的标志物。     

Chlamydia trachomatis modulates expression of tumor suppressor gene caveolin-1 and oncogene C-myc in the transformation zone of non-neoplastic cervical tissue

OBJECTIVES: The obligate intracellular bacterium Chlamydia trachomatis is frequently found in association with benign proliferative, pre-neoplastic and malignant changes in cervical epithelium. The present study addresses the possible role of C. trachomatis infection of the uterine cervix in modulating human cancer gene expression. METHODS: RNA was extracted from both C. trachomatis infected and non-infected human fibroblast cultures treated with ITFgamma. The extracted RNA was used for cDNA microarrays carrying 33,000 human genes to detect abnormal gene expression induced by Chlamydia. Forty specimens of cervix dissected from the transformation zone had previously tested negative for HPV and positive for C. trachomatis by standard DNA PCR (20). These samples were subjected to RT-PCR to detect the expression of the abnormal genes induced by Chlamydia infection. RESULTS: The ITFgamma-induced, non-replicative Chlamydia-infected fibroblast cultures showed significant modulation of gene expression. The cultures showed a 2-fold decrease in the expression of the gene coding for the tumor suppressor caveolin-1, and increased expression of the oncogene C-myc, a promoter of cervical carcinogenesis. In tissues from the Chlamydia-infected cervical transformation zone, real-time RT-PCR demonstrated a highly significant average 4.7-fold reduction of caveolin-1 mRNA (P < or = 0.0001) and an average 2.1-fold increase in C-myc (P < 0.05). CONCLUSIONS: Human ITFgamma-treated fibroblasts as well as non-neoplastic cervical tissues responded to C. trachomatis with a strong down-regulation of caveolin-1 mRNA and a light up-regulation of C-myc mRNA. These changes were independent of the HPV high-risk types. This study reveals possible mechanisms by which C. trachomatis infection may contribute to neoplastic changes in the transformation of uterine cervix. These possible mechanisms require further evaluation.     

p53 Protein Expression and Gene Analysis in Clear Cell Adenocarcinoma of the Vagina and Cervix

Objective:p53 is the most commonly mutated gene in human cancers. The objective of this study was to determine if clear cell adenocarcinomas (CCAs) of the vagina and cervix are associated with p53 gene mutations or alterations in p53 tumor-suppressor protein expression.Methods:Paraffin-embedded tissue specimens from 21 women (median age 22 years) with clear cell adenocarcinoma of the vagina or cervix were studied. Fifteen women had a prior history ofin uteroexposure to diethylstilbestrol. p53 protein expression was detected by immunohistochemical (IHC) analysis with monoclonal antibody DO-7 (Dako Corp.) which recognizes both wild-type and mutant p53 proteins. For p53 gene analysis, genomic DNA from malignant tissue was isolated and exons 4–10 were amplified by PCR and subjected to mutation screening by single-stranded conformation polymorphism (SSCP) analysis.Results:p53 protein was detected by IHC in tumors from 14 of 21 cases (67%). The observed p53 staining patterns were heterogeneous in both the proportion and intensity of tumor cells stained but were clearly overexpressed relative to the surrounding benign stroma. Metastatic tumors from 3 women with metastatic disease were also positive for p53 staining. SSCP analysis did not identify p53 mutations in any of the cases and strongly suggests that the tumors contained only wild-type p53 alleles.Conclusions:Recent studies have demonstrated that wild-type p53 may accumulate in response to DNA damage which normally leads to growth arrest or programmed cell death. Our observations are consistent with the hypothesis that p53 overexpression in CCAs of the vagina and cervix is a response to generalized DNA damage, rather than a result of p53 protein half-life prolongation resulting from mutational inactivation of p53. Overexpression of wild-type p53 protein in vaginal and cervical CCA may relate to the more favorable prognosis of this subset of tumors in comparison to other gynecologic tumors containing mutated p53 genes.     

survivin、FHIT及bFGF在宫颈鳞癌组织中的表达及意义

目的:研究凋亡抑制基因survivin、抑癌基因FHIT及碱性成纤维细胞生长因子(bFGF)在正常宫颈组织、宫颈上皮内瘤变组织(CIN)及宫颈鳞癌组织中的表达及其与宫颈鳞癌临床分期、病理分级及淋巴结转移的关系,探讨其在宫颈鳞癌发生、发展中的作用。方法:分析郑州大学第二附属医院病理科2008年1月至2009年11月手术切除、病理检查证实为宫颈鳞癌的组织标本45例,另取CIN45例、正常宫颈组织15例(子宫肌瘤行子宫全切除术的宫颈组织)作为对照,应用免疫组化SP法检测survivin、FHIT及bFGF在3组宫颈组织中的表达情况。结果:sur-vivin在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达呈上升趋势(χ2=11.429,P<0.05),survivin的表达在宫颈鳞癌不同临床分期、病理分级间比较,差异有统计学意义(P<0.05);FHIT在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达呈下降趋势(χ2=24.640,P<0.05),FHIT的表达与宫颈鳞癌病理特征的关系中,仅在淋巴结有无转移方面比较差异有统计学意义(P<0.05);bFGF在正常宫颈组织、CIN及宫颈鳞癌组织中的阳性表达依次呈上升趋势(χ2=17.552,P<0.05),bFGF的表达在宫颈鳞癌不同临床分期、病理分级及淋巴结有无转移方面比较,差异均有统计学意义(P<0.05)。结论:survivin、FHIT、bFGF的表达与宫颈病变的进展有一定的关系,并且可能与宫颈鳞癌的发生、发展及浸润和转移有关。     

子宫颈癌组织中DNA聚合酶β基因突变的研究

目的　研究宫颈癌组织中是否存在DNA聚合酶 β(POLB)基因的突变。 方法　采用逆转录 聚合酶链反应 (RT PCR)技术、聚合酶链 单链构象多态性分析 (PCR SSCP) DNA序列分析法对34例宫颈癌组织进行POLB基因突变的调查。结果　宫颈癌组织中存在POLB基因突变 ,且基因突变率与癌组织的病理分级有关 ,G1、G2 、G3 宫颈癌组织中POLB基因突变率分别为 9例中 1例 ,2 9%(4/ 14 )、73% (8/ 11) ,除G1与G2 比较 ,差异无显著性外 (P >0 0 5 ) ,余两比较 ,差异均有极显著性 (P<0 0 0 5 )。测序结果表明 ,POLB基因的第 6 6 0位核苷酸由A变为G ,其氨基酸的第 182位由精氨酸(Arg)变为甘氨酸 (Gly)。结论　宫颈癌组织中存在POLB基因突变现象 ,可能与宫颈癌的发生、发展相关。     

Inverse correlation between RASSF1A hypermethylation, KRAS and BRAF mutations in cervical adenocarcinoma

OBJECTIVE: Although the incidence of cervical adenocarcinoma is increasing, few genetic and epigenetic changes in its progression have been described. We hypothesized that RASSF1A methylation and KRAS and BRAF mutations may play an important role in cervical adenocarcinoma. METHODS: Archival primary carcinoma tissues (n=258) in uterine cervix consisting cervical adenocarcinomas (n=115) and squamous cell carcinomas (n=143) were evaluated for activating mutations of BRAF and KRAS and promoter hypermethylation of RASSF1A using methylation specific PCR and specific sequence analysis. HPV E7 Type-specific PCR was used for HPV-16 and -18 status. RESULTS: KRAS mutations were found in 16 adenocarcinomas (13.9%), while BRAF mutations were found in 5 (4.3%). RASSF1A methylation was found in 27 adenocarcinomas (23.5%) and inversely correlated with KRAS and/or BRAF mutation (p=0.002) in cervical adenocarcinoma. In cervical squamous cell carcinomas, KRAS mutations were detected only in 1 (0.7%) cases and RASSF1A hypermethylation was detected in 2 (1.4%). The frequency of KRAS mutation and RASSF1A methylation were significantly different between adenocarcinomas (P<0.001) and squamous cell carcinomas (P<0.001). Neither KRAS mutation nor RASSF1A methylation were associated with HPV status. RASSF1A hypermethylation and KRAS mutations and BRAF mutations are inversely correlated and play an important role in the development of adenocarcinomas. CONCLUSIONS: These results are suggesting that these two histological types of cervical cancer arise through different molecular pathways in tumor development. Different genetic/epigenetic alterations may explain the possible different therapeutic responsiveness between adenocarcinoma and squamous cell carcinoma of uterine cervix seen in clinic.     

子宫颈癌及其癌前病变组织端粒酶活性的研究

目的研究宫颈癌及其癌前病变组织中端粒酶激活的意义.方法应用端粒酶重复序列扩增-酶联免疫吸附法(TRAP-ELISA)及电泳-银染法,对36例宫颈浸润癌及16例宫颈上皮内瘤变(CIN)组织进行端粒酶活性测定,以吸光度(A)值判断端粒酶活性.同时测定11例正常宫颈、6例慢性宫颈炎症及8例癌旁组织端粒酶活性作为对照.结果CIN、宫颈癌及对照组端粒酶活性A值分别为0.398±0.293、1.580±0.819和0.050±0.012,3组比较,差异有极显著性(P＜0.01).端粒酶活性高低与肿瘤分化程度呈负相关,与淋巴结转移呈正相关;与组织学类型、分期、体积大小无关.结论端粒酶的激活发生在宫颈癌病变的早期,可能成为宫颈癌及癌前病变早期诊断和鉴别诊断的指标.