The integration of HPV-18 DNA in cervical carcinoma.

AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV-18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV-18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV-18 E2 region is more consistently deleted.     

The integration of HPV-18 DNA in cervical carcinoma.

AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV-18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV-18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV-18 E2 region is more consistently deleted.     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region.

Homologous, subgenomic fragments of the viral LCR and E6/E7 transforming genes of HPV-18 and HPV-16 were amplified from several primary cervical, penile, and vulvar tumors and cloned into a pUC-18-derived vector. When assayed by a quantitative transformation assay using primary human keratinocytes, the subgenomic regions of HPV-16 and HPV-18 exhibited transforming activities similar to that of the full-length, prototype HPV genomes. More importantly, the HPV-18 LCR-E6-E7 region was approximately 10- to 50-fold more active than that of HPV-16. These studies demonstrate (1) that the transforming activity differences previously observed between prototype HPV-16 and HPV-18 map to the LCR-E6-E7 region, and (2) that individual and independent isolates of HPV-16 and HPV-18 exhibit consistent differences in transforming potential, even when isolated from different anatomic sites.     

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.     

Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16-associated invasive cervical cancer.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.     

Detection of HPV-16 in cell lines and cervical lavage specimens by a polymerase chain reaction-enzyme immunoassay assay.

A gene amplification method that combines the polymerase chain reaction with detection of amplified DNA in a solution hybridization/enzyme immunoassay (PCR-EIA) was developed for HPV-16 DNA. Samples were amplified with primers for the E7-E1 region of HPV-16. Amplified DNA products were identified and quantitated by hybridization in solution with a biotinylated RNA probe. Labeled DNA/RNA hybrids were measured semiquantitatively in an enzyme immunoassay using solid phase anti-biotin antibody and liquid phase B-d-galactosidase labeled monoclonal antibody against DNA-RNA hybrids. Enzyme bound to the solid phase was quantitated with a fluorogenic substrate. The assay was linear over 2 log10 dilutions of SiHa cells and the detection limit was three copies of HPV-16 genome. The sensitivity of PCR-EIA for detection of PCR amplified products compared favorably with slot and Southern blots using a 32P-labeled RNA probe. The assay was used to assess HPV-16 infection of uterine cervix in women attending a clinic for sexually transmitted diseases. Twenty-one of the 81 specimens (25.9%), obtained by cervicovaginal lavage, were positive for HPV-16 by PCR-EIA. The assay provides a convenient means to objectively measure HPV DNA amplified with PCR.     

宫颈癌组织中HPV16,18E6蛋白表达的观察

目的观察ＨＰＶ１６、１８早期蛋白Ｅ６在人宫颈鳞状细胞癌中的表达并评价Ｅ６单抗的应用价值。方法采用ＳＰ免疫组化染色法,检测４０例宫颈鳞状细胞癌,３０例慢性宫颈炎及３０例正常宫颈组织中ＨＰＶ１６、１８Ｅ６蛋白的表达。结果癌组中Ｅ６的阳性率为６７．５％（２７／４０）,慢性宫颈炎中为３．３％（１／３０）,正常宫颈组织中均为阴性,良、恶组ＨＰＶ１６、１８Ｅ６的阳性率差异有显著意义（Ｐ＜０．０１）。结论ＨＰＶ１６、１８感染与本地区宫颈癌病因学密切相关,Ｅ６单抗可作为预测ＨＰＶ１６、１８感染及宫颈癌早期诊断的标记之一。     

人乳头瘤病毒16型E＿6E＿7基因反义质粒对人宫颈癌细胞恶性的逆转作用

构建可为地塞米松诱导表达的人乳头瘤病毒１６型（ＨＰＶ－１６）Ｅ＿６Ｅ＿７基因反义质粒（ｐ１６ａｓＥ＿６Ｅ＿７Ｎｅｏ），利用磷酸钙沉淀法将其分别转染到ＨＰＶ－１６阳性的人宫颈癌细胞株Ｃａｓｋｉ和ＨＰＶ阴性的人宫颈癌细胞株Ｃ－３３Ａ中。地塞米松诱导反义质粒表达后，ＣａｓＫｉ细胞失去其恶性表型，而Ｃ－３３Ａ细胞的生长特性及恶性行为未发生变化。说明反义质粒能够改变Ｃａｓｋｉ细胞的恶性表型，且这种改变是通过特异性抑制Ｅ＿６Ｅ＿７基因表达实现的。     

Physical status of HPV-16 in esophageal squamous cell carcinoma.

BACKGROUND: Infection with high-risk human papillomavirus (HPV) has been implicated as one of the risk factors of esophageal squamous cell carcinoma (ESCC). Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of the HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. OBJECTIVES AND STUDY DESIGN: To investigate the viral integration status of HPV-16 infection in ESCC, 35 HPV-positive ESCC specimens collected from Chinese patients were subject to real-time quantitative PCR for determination of physical status of HPV-16 by analyzing the ratios of E2 to E6 genes. RESULTS: Our results showed that only 8.6% (3/35) of the HPV-16 positive specimens harbored exclusively the episomal form (i.e. E2/E6 ratio > or = 1), whereas the remaining 91.4% contained either only the integrated form (5.7%, with E2/E6 ratio = 0) or a mixture of episomal and integrated forms of viral molecules (85.7%, with E2/E6 ratios > 0 but < 1). Amongst the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/integrated E6 ratios of less than 1, indicating a predominance of integrated form of viral genes in these lesions. CONCLUSION: Our finding of frequent integration of viral DNA in the host genome suggests that integration HPV-16 is common in ESCC from Chinese patients and implies that HPV infection may play a role in the pathogenesis of ESCC.     

Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines.

A series of human carcinoma cell lines was examined for human papillomavirus (HPV) DNA sequences with the use of HPV-6, HPV-11, HPV-16, and HPV-18 DNA probes. Six of eight cell lines derived from human cervical carcinomas were shown to contain integrated HPV DNA sequences. In five of these six lines, HPV-specific polyadenylated RNA species could also be identified. The expression of HPV sequences was detected in three lines with a HPV-18 DNA probe and in two lines with a HPV-16 DNA probe. Of the two lines which contained HPV-16 specific RNA, one contained HPV DNA sequences which hybridized only to an HPV-16 probe, and the other contained HPV DNA sequences which hybridized to both HPV-16 and HPV-18 DNA probes. Six cell lines established from human squamous-cell carcinomas of the bladder, pharynx, lung, esophagus, and vulva were negative for HPV-6, HPV-11, HPV-16, and HPV-18 DNA sequences under stringent hybridization conditions.     

Correlation of laryngeal squamous cell carcinoma and infections with either HHV-8 or HPV-16/18

Each year more than 159,000 new cases of laryngeal cancer are diagnosed globally, and more than 9000 patients die due to this malignancy. Viral infections are a known risk factor for this malignancy. Thus, this study aimed to evaluate the role of HPV-16/18 and HHV-8 infection in patients with laryngeal cancer.     

原发性子宫颈小细胞癌18例临床病理观察

目的探讨原发性子宫颈小细胞癌(small cell carcinoma of the cervix,SCCC)的临床病理学特征、免疫表型及预后。方法采用免疫组化SP法和原位杂交法对18例SCCC进行临床病理学观察,并结合文献复习。结果 18例SCCC患者年龄30~69岁,平均40岁。SCCC患者临床表现均为阴道不规则出血。妇科检查:子宫颈肿块或子宫颈糜烂。组织学特征:SCCC肿瘤细胞小而一致,可呈圆形似淋巴细胞,但较淋巴细胞大,或呈雀麦形,或部分呈短梭形,细胞质少,境界不清,核染色质呈细颗粒状,深染,核仁不明显,核分裂象多见。免疫表型:肿瘤细胞通常表达CKpan,并不同程度地表达Syn、NSE、Cg A、CD56和p16。原位杂交:检测4例肿瘤细胞HPV 16/18阳性。超微结构:肿瘤细胞内可见神经内分泌颗粒。随访10例SCCC,6例患者2年内死亡。结论 SCCC属神经内分泌癌,是子宫颈少见的高度恶性肿瘤,预后差,其发生可能与HPV 16/18感染有关。CKpan、Syn、Cg A、NSE和CD56是SCCC的免疫标志物,有利于辅助诊断。     

Colon carcinoma cell lines stimulate monocytes and lamina propria mononuclear cells to produce IL-10

Cytokines released from tumour cells may have function as signals to neighbouring immune and inflammatory cells. Several studies have shown that the immunoregulatory cytokines IL-10 and transforming growth factor-beta 1 (TGF-β1) as well as prostaglandin-E₂ (PGE₂) play an important role in tumour-induced immunosuppression. The aim of the study was to investigate the effect of colon carcinoma cell lines on IL-10 production in peripheral monocytes (PBMC) and lamina propria mononuclear cells (LPMC). We examined four colon carcinoma cell lines (HT-29, Caco-2, Colo-320 and HCT-116) and determined their production of TGF-β1, IL-10 and PGE₂. Peripheral monocytes were isolated by density gradient centrifugation and LPMC were isolated from surgical specimens using a collagenase digestion method. Monocytes and LPMC were cultured with colon carcinoma cell conditioned medium or in co-culture with colon carcinoma cells. Supernatants were then determined for the production of IL-10 by ELISA assays. All colon carcinoma cell lines stimulated peripheral monocytes as well as LPMC to produce markedly increased levels of IL-10. Colon cancer cells secreted negligible levels of IL-10, but high amounts of TGF-β1 and PGE₂. Neutralization of TGF-β1 by administration of anti-TGF-β as well as neutralization of PGE₂ with anti-PGE₂ antisera reduced the IL-10 production of monocytes markedly, indicating that tumour cell-derived TGF-β1 and PGE₂ are major factors for IL-10 stimulation. In vitro stimulation of monocytes with TGF-β1 and PGE₂ could confirm that TGF-β1 as well as PGF₂ at picogram concentrations were able to prime monocytes for enhanced IL-10 production. Our results demonstrate that colon carcinoma cell lines enhance the ability of monocytes and intestinal macrophages to produce IL-10. The stimulation of monocyte IL-10 by colon cancer cell-derived TGF-β1 and PGE₂ may act as a tumour-protecting mechanism by impairing the activation of anti-tumour cytokines.     

Karyotypic analysis of two related cervical carcinoma cell lines that contain human papillomavirus type 18 DNA and express divergent differentiation

The cell lines C-4I and C-4II were established in culture from a nonkeratinizing squamous carcinoma of the uterine cervix. Both lines contain human papillomavirus (HPV) type 18 DNA (Brandt et al., Cold Spring Harbor Laboratories, 5:179, 1987) and both are hypodiploid with similar, but not identical, karyotypes. Each line expresses multiple characteristics of ectocervical epithelial differentiation, but the characteristics differ between the lines. In the present study, G banding of the lines showed that cells of both lines have two normal chromosomes 1-5, 8-10, 13, 16, and 17, one normal chromosome 12 and 14, and no normal chromosomes 15 and 18. The lines share three abnormal chromosomes, der(8)t(8q;12q), der(18)t(18q;?), and i(5p). There are specific differences between the lines. C-4I has two normal chromosomes 6, while C-4II has one; C-4II has two chromosomes 11 and der(18)t(18q;?), while C-4I lacks both chromosomes 11 and has one der(18)t(18q;?). Each line has unique markers that include del(11)(p11), del(22)(q12), and del(21)(q21) in C-4I and i(15q), der(X)t(Xq;9p), der(6)t(6p;14q), and del(4)(q21) in C-4II. The results show that these phenotypically distinct lines are derived from the same clone and that the 8q arm (the site of HPV 18 integration) is present in three copies in both lines. They also define several chromosome rearrangements that are compatible with the expression of specific differentiation markers.     

逆转录病毒介导IL-18基因在大鼠胶质瘤细胞C6中的表达

目的 :建立表达IL 18基因的大鼠胶质瘤细胞C6 /IL 18,并探讨外源性IL 18基因对C6细胞生长的影响。方法 :应用逆转录病毒载体 ,将IL 18基因导入C6细胞。经G4 18筛选后 ,获得表达IL 18分子的细胞克隆C6 /IL 18。用RT PCR法检测目的基因mRNA的表达 ;用流式细胞和免疫细胞化学染色法检测目的蛋白的表达。用ELISA法检测C6 /IL 18细胞培养上清诱导脾细胞分泌IFN γ的能力 ,以确定IL 18的生物学活性。用MTT比色法检测细胞体外增殖的状况 ,用流式细胞技术检测细胞的增殖活性 ,并建立大鼠胶质瘤模型 ,观察C6 /IL 18细胞体内致瘤性的改变。结果 :外源IL 18基因于mRNA水平和蛋白水平可获得稳定表达 ,并可诱生大鼠脾细胞分泌IFN γ。同时 ,该细胞系的体外增殖率及体内致瘤性 ,均较亲代C6胶质瘤细胞明显下降。结论 :外源性IL 18基因能部分地抑制C6细胞的体外增殖率和体内致瘤性。建立了可进一步用于相关肿瘤基因免疫和基因治疗研究的大鼠胶质瘤细胞系C6 /IL 18。     

CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.     

肼苯哒嗪对宫颈癌细胞系APC基因异常甲基化的影响

目的检测宫颈癌细胞系HeLa、CaSki和SiHa中结肠腺瘤性息肉病基因(APC)启动子区CpG岛甲基化状态;探索肼苯哒嗪(Hyd)能否作为去甲基化药物恢复APC表达。方法应用甲基化特异性聚合酶链反应(MSP)检测细胞系中APC启动子区CpG岛甲基化情况;Hyd处理后,RT-PCR检测APCmRNA表达。结果APC启动子区CpG岛在HeLa细胞中被甲基化,在CaSki细胞中半甲基化,在SiHa细胞中无甲基化;一定浓度Hyd处理HeLa、CaSki细胞后,检测到APCmRNA表达。结论HeLa、CaSki细胞系APC表达沉默与APC启动子区甲基化有关;Hyd能作为去甲基化药物诱导APC表达。     

卵巢癌细胞IL-6、IL-8及其受体表达的研究

目的分析比较五种常见的上皮性卵巢癌细胞系IL-6、IL-8及其受体表达的差异。方法IL-6、IL-8的表达分别采用RT-PCR和ELISA法进行检测,IL-6受体（IL-6Rα和gp130）及IL-8受体（IL-8RA和IL-8RB）的表达采用免疫印迹技术进行测定。结果①五种上皮性卵巢癌细胞均组成性表达IL-6和IL-8。IL-6和IL-8在CAOV-3细胞中的表达水平均最高,而在HO-8910PM细胞中的表达水平均最低,IL-6在SKOV-3、HO-8910、OVCAR-3细胞中的表达水平依次降低,IL-8在OVCAR-3、SKOV-3、HO-8910细胞中的表达水平依次降低。②五种上皮性卵巢癌细胞均表达IL-6Rα、gp130及IL-8RA;除CAOV-3细胞外,其它细胞均表达IL-8RB。结论本研究旨在筛选表达IL-6和IL-8及其相应受体的细胞株,为研究IL-6、IL-8与卵巢癌发生、发展关系奠定基础,同时也为今后卵巢癌的免疫治疗提供一个新的思路。