利用FISH分析HPV16在Hela细胞中的整合位点

 目的 探讨HPV16病毒感染导致子宫颈癌发生的染色体畸变。方法 利用荧光原位杂交（FISH）技术检测HPV16病毒感染导致的Hela细胞中染色体核型的变化。结果 Hela细胞系中存在9号染色体的三体畸变，但3号染色体表现为正常的二倍体核型；Hela细胞中没有发现HPV16的整合位点，但当外源性的HPV16感染Hela细胞后，HPV16病毒DNA整合在3号染色体上；9号和3号染色体变异在子宫颈癌的发生发展中起着重要的作用，而HPV16的感染主要引起3号染色体的变异。结论 HPV16可以引起Hela细胞的3号染色体的畸变，从而导致子宫颈癌的发生。     

Common fragile sites are preferential targets for HPV16 integrations in cervical tumors

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at both the cytogenetic and molecular levels. To further explore this relationship at the molecular level, we used RS-PCR to rapidly isolate cellular sequences flanking the sites of HPV16 integration in 26 primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on aphidicolin-stimulated metaphases. Our data demonstrate that 11/23 HPV16 integrations in cervical tumors occurred within CFSs (P&<0.001). In addition, we show that deletions and complex rearrangements frequently occur in the cellular sequences targeted by the integrations and that integrations cluster in FRA13C (13q22), FRA3B (3p14.2), and FRA17B (17q23). Finally, our data suggest that cellular genes, such as Notch 1, are disrupted by the HPV16 integrations, which may contribute to the malignant phenotype.     

HPV16 E6-mediated stabilization of ErbB2 in neoplastic transformation of human cervical keratinocytes

Whether ErbB2 receptor tyrosine kinase contributes to cervical cancer is controversial. We have examined the effects of E6 and E7 genes of human papillomaviruses type 16 (HPV-16) on ErbB2 expression in primary human cervical keratinocytes (HCK) immortalized with hTERT (HCK1T). In E6-positive cells (HCK1T-E6 and HCK1T-E6E7), ErbB2 expression levels increased with the cell density. HCK1T-E6E7 showed impaired contact inhibition and anchorage-independent growth in soft agar which were abrogated with introduction of ErbB2-specific short hairpin RNA (shRNA) or an ErbB2 specific inhibitor AG825. Furthermore, increased ErbB2 expression was also observed in HPV16 positive cervical cancer cell lines and this was diminished by introduction of HPV16E6- or E6AP-shRNA. At post-confluence cell densities, ErbB2 protein was stabilized in the presence of E6 whereas increased ErbB2 expression was not obvious with E6 mutants incapable of degrading p53. Furthermore, introduction of p53-shRNA to HCK1T resulted in increased ErbB2 protein stability, indicating possible ErbB2 regulation through p53. Finally, we showed that tumor formation of ErbB2-shRNA introduced SiHa cells were almost abolished. Taken together, these data indicate an important role of ErbB2 regulation by HPV16 E6 in oncogenic transformation of human cervical keratinocytes.     

子宫颈癌患者人乳头瘤病毒16、18检测

 目的　探讨人乳头瘤病毒（HPV）16、18型在子宫颈癌的发生、发展中的意义。方法　应用实时荧光定量聚合酶链反应技术对子宫颈原位癌13例，子宫颈癌Ⅰ期32例，子宫颈上皮内瘤样病变（CIN）Ⅰ~Ⅱ级12例，对照组54例（慢性子宫颈炎组37例、非研究疾病组17例）进行HPV16、HPV18型荧光基因定量检测，计算出HPV DNA的拷贝数。结果　研究组、对照组HPV16、HPV18的感染率差异有统计学意义（P＜0.05），在子宫颈癌发生的不同阶段，HPV16、HPV18的感染定量差异亦有统计学意义。子宫颈癌HPV含量与肿瘤直径大小、浸润间质深度、淋巴结阳性个数无相关性（r = 0.168， r = 0.280， r = 0.333，P＞0.05）；局部肿瘤的直径大小与浸润间质深度呈正相关（r = 0.473，P＜0.05）。结论　致癌性HPV的持续、高浓度存在是子宫颈癌发生、发展的主要因素之一。     

HPV16 integration probably contributes to cervical oncogenesis through interrupting tumor suppressor genes and inducing chromosome instability

Background

The integration of human papilloma virus (HPV) into host genome is one of the critical steps that lead to the progression of precancerous lesion into cancer. However, the mechanisms and consequences of such integration events are poorly understood. This study aims to explore those questions by studying high risk HPV16 integration in women with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (SCC).

Methods

Specifically, HPV integration status of 13 HPV16-infected patients were investigated by ligation-mediated PCR (DIPS-PCR) followed by DNA sequencing.

Results

In total, 8 HPV16 integration sites were identified inside or around genes associated with cancer development. In particular, the well-studied tumor suppressor genes SCAI was found to be integrated by HPV16, which would likely disrupt its expression and therefore facilitate the migration of tumor. On top of that, we observed several cases of chromosome translocation events coincide with HPV integration, which suggests the existence of chromosome instability. Additionally, short overlapping sequences were observed between viral derived and host derived fragments in viral-cellular junctions, indicating that integration was mediated by micro homology-mediated DNA repair pathway.

Conclusions

Overall, our study suggests a model in which HPV16 might contribute to oncogenesis not only by disrupting tumor suppressor genes, but also by inducing chromosome instability.

     

Correlation between load of HPV 16 DNA in cervical cancer and HPV 16 DNA in lymph nodes

OBJECTIVE To determine the association between viral load of human papillomavirus 16 （HPV16） DNA in the primary focus of cervical carcinoma and HPV16 DNA in pelvic lymph nodes. METHODS The HPV16 DNA load was measured by fluorescent quantitation polymerase chain reaction （FQ-PCR） in 17 primary foci. HPV16 DNA was detected by polymerase chain reaction （PCR） using HPV16 type-specific primers in 296 pelvic lymph nodes which were from 17 cases of cervical cancer. RESULTS The viral load of HPV16 DNA showed statistically significant differences between tumors with a diameter of 〈 4 cm and ≥ 4 cm （P 〈 0.05）. Seven of 17 cervical cancer cases had HPV16 DNA positive lymph nodes, designated as the positive group, while the remaining 10 without positive lymph nodes was designated the negative group. The average load of HPV16 DNA showed no significant difference between the 2 groups （P 〉 0.05）. The load of HPV16 in the primary lesion was not associated with that in the lymph nodes. There were 38 HPV16 DNA positive nodes in the total 296 nodes. The rate of positivity of HPV16 DNA in lymph nodes showed statistically significant differences in consideration of maximum tumor diameter, tumor differentiation, histologic type, depth of myometial infiltration and the metastatic status of the nodes, respectively （P 〈 0.05）. CONCLUSION Viral load of HPV16 in the primary cancer focus correlated with the quantity of tumor cells in the primary focus but not with the existence of HPV DNA positive lymph nodes. Detection of HPV DNA may help to find the early metastases that cannot be evaluated histopathologically, but the prognostic value of HPV positive lymph nodes needs further examination.     

Roles of the PDZ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes

The high‐risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ‐domain‐containing proteins through its carboxy‐terminal motif. This PDZ‐binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6‐transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRAS^G12V) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild‐type E6. Interestingly, we found decreased expression of PAR3 in E6‐expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ‐domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRAS^G12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild‐type E6. These data highlight the importance of the carboxy‐terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.     

HPV typing and HPV16 E6-sequence variations in synchronous lesions of cervical squamous-cell carcinoma from Swedish patients.

We microdissected 15 specimens of invasive cervical cancer co-existing with some of its precursors. Out of 15 cases, 10 carried HPV16, 2 HPV31, 1 HPV18 and 2 were HPV-negative. We found 3 HPV16 E6 variants among the 10 cases; one was A --> G in nt 131 (one case) and a second was A --> G in nt 276. The third, T;--> G in nt 350, was common, and was found in 5 of the 10 patients infected by HPV16. The type of HPV and the E6 variant were identical in all lesions within the same patient. Viral DNA present in normal epithelium was identical in type and E6 variant to HPV in the same patient's lesions. Multiple samples from invasive cancers with HPV were consistently positive. The data suggest that the originally infecting HPV, including its variant type in the E6 gene, persists unaltered in the whole series of CIN that precedes invasive cancer. Our data are compatible with an essential role of HPV manifested by persistence of the viral genome during the entire natural life history of cervical cancer. We did not confirm previous data on the specific association of invasive cancer with an HPV E6 variant (G at nt 350 rather than T). The discrepancy may depend on the relatively few cases investigated or selection of a special sub-set with progression from CIN to invasive cancer already manifest.     

HPV16 genetic variation and the development of cervical cancer worldwide

Background:

Factors that favour a small proportion of HPV16 infections to progress to cancer are still poorly understood, but several studies have implicated a role of HPV16 genetic variation.

Methods:

To evaluate the association between HPV16 genetic variants and cervical cancer risk, we designed a multicentre case–control study based on HPV16-positive cervical samples (1121 cervical cancer cases and 400 controls) from the International Agency for Research on Cancer biobank. By sequencing the E6 gene, HPV16 isolates were classified into variant lineages and the European (EUR)-lineage isolates were subclassified by the common polymorphism T350G.

Results:

Incidence of variant lineages differed between cases and controls in Europe/Central Asia (P=0.006, driven by an underrepresentation of African lineages in cases), and South/Central America (P=0.056, driven by an overrepresentation of Asian American/North American lineages in cases). EUR-350G isolates were significantly underrepresented in cervical cancer in East Asia (odds ratio (OR)=0.02 vs EUR-350T; 95% confidence interval (CI)=0.00–0.37) and Europe/Central Asia (OR=0.42; 95% CI=0.27–0.64), whereas the opposite was true in South/Central America (OR=4.69; 95% CI=2.07–10.66).

Conclusion:

We observed that the distribution of HPV16 variants worldwide, and their relative risks for cervical cancer appear to be population-dependent.     

Characterization of the biological activity of gamma-glutamyl-Se-methylselenocysteine: a novel, naturally occurring anticancer agent from garlic

Gamma-glutamyl-Se-methylselenocysteine (GGMSC) has recently been identified as the major Se compound in natural garlic and selenized garlic. Our working hypothesis is that GGMSC serves primarily as a carrier of Se-methylselenocysteine (MSC), which has been demonstrated in past research to be a potent cancer chemopreventive agent in animal carcinogenesis bioassays. The present study was designed to examine the in vivo responses to GGMSC or MSC using a variety of biochemical and biological end points, including (a) urinary Se excretion as a function of bolus dose; (b) tissue Se accumulation profile; (c) anticancer efficacy; and (d) gene expression changes as determined by cDNA array analysis. Our results showed that like MSC, GGMSC was well absorbed p.o., with urinary excretion as the major route for eliminating excess Se. When fed chronically, the profile of Se accumulation in various tissues was very comparable after treatment with either GGMSC or MSC. In rats that had been challenged with a carcinogen, supplementation with either GGMSC or MSC resulted in a lower prevalence of premalignant lesions in the mammary gland, and fewer mammary carcinomas when these early lesions were allowed to progress. More importantly, we found that a short term GGMSC/MSC treatment schedule of 4 weeks immediately after carcinogen dosing was sufficient to provide significant cancer protection, even in the absence of a sustained exposure past the initial 4-week period. With the use of the Clontech Atlas Rat cDNA Array, we further discovered that the gene expression changes induced in mammary epithelial cells of rats that were given either GGMSC or MSC showed a high degree of concordance. On the basis of the collective biology, biochemistry, and molecular biology data, we conclude that GGMSC is an effective anticancer agent with a mechanism of action very similar to that of MSC.     

HPV 16 infection and progression of cervical intra-epithelial neoplasia: Analysis of HLA polymorphism and HPV 16 E6 sequence variants

High-risk human papillomavirus (HPV) infection plays an important role in cervical intra-epithelial neoplasia (CIN), but HPV infection alone is not sufficient for progression to cervical cancer. Several lines of evidence suggest that cellular immune surveillance is important in the control of HPV infection and the development of CIN. The presentation to T cells of target viral peptides in the context of HLA molecules is influenced by the genetic polymorphisms of both HPV and HLA and thereby influences the host immune response and clinical outcome of HPV infection. HLA class I and II polymorphism in susceptibility for HPV 16 infection, development and progression of CIN was analyzed in a group of 118 patients participating in a prospective study of women with initial abnormal cytology. Patients were stratified according to HPV status and course of the disease. HLA-B*44 frequency was increased in the small group of patients with a lesion that showed clinical progression during follow-up [OR = 9.0 (4.6–17.5),p= 0.007]. HLA-DRB1*07 frequency was increased among HPV 16-positive patients compared with patients who were negative for all HPV types [OR = 5.9 (3.0–11.3), p= 0.02]. Our results are consistent with the immunogenetic factors associated with disease progression being different from those associated with susceptibility to HPV 16 infection. Sequencing of the HPV 16 E6 and E7 open reading frames of a subset of these patients (n = 40) showed the frequency of HPV 16 variants to be similar to other studies. However, there was no significant correlation between variant incidence and disease progression or viral persistence and no significant correlation with any HLA allele. It appears that multiple HLA types can influence HPV 16-associated cervical dysplasia but the role of HPV 16 variants in disease progression and susceptibility in relation to HLA polymorphism remains unclear. Int. J. Cancer 78:166–171, 1998.© 1998 Wiley-Liss, Inc.     

Increased expression of Dyrk1a in HPV16 immortalized keratinocytes enable evasion of apoptosis

Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually.