Focus: A Multifaceted Battle Against Cancer: Nuns,Warts, Viruses,and Cancer

It has been known for more than 150 years that the risk of carcinoma of the uterine cervix correlates with the number of sexual partners. Laboratory and epidemiological evidence demonstrated that infection with certain human papillomavirus (HPV) types initiates the vast majority of, if not all, cervical cancer, as well as a substantial fraction of other cancers, including other anogenital cancer and oropharyngeal cancer. Pap smear testing resulted in a dramatic reduction in the incidence of cervical cancer in the developed world, and HPV vaccination has the potential to eradicate HPV-associated cancer worldwide and represents a major public health breakthrough. The major current challenge is to ensure that HPV vaccines are widely administered.     

First evidence for the contribution of the genetic variations of BRCA1-interacting protein 1 (BRIP1) to the genetic susceptibility of cervical cancer

BRIP1 (BRCA1-interacting protein 1), a DNA-dependent ATPase and a DNA helicase, is critical for BRCA-associated DNA damage repair functions, and may be involved in the development of cervical cancer. Genetic markers in different regions of the BRIP1 gene have a plausible role in modulating the risk of cervical cancer. In this study, we evaluate the association between the BRIP1 variations and the risk of cervix cancer. We examined the potential association between cervical cancer and eighteen single nucleotide polymorphisms (SNPs, rs2048718, rs16945692, rs4968451, rs6504074, rs4988344, rs8077088, rs10515211, rs9897121, rs9906313, rs2159450, rs4986764, rs11871785, rs4986763, rs11079454, rs7213430, rs34289250, rs4988345 and rs12937080) of the BRIP1 gene using the MassARRAY system. The participants enrolled in this study included 298 patients with cervical cancer and 286 healthy women as the healthy controls from a Chinese Han population. The results showed that rs16945692 (intron 1), rs4968451 (intron 4), rs4986764 (exon 18) and rs7213430 (3′UTR) were significantly associated with cervical cancer (P < 0.05). Furthermore, strong linkage disequilibrium (LD) was observed in three blocks (D′ > 0.9), and significantly more T–A–C–A haplotypes (block 1) (P = 0.001) were found in the patients with cervical cancer. Significantly higher frequencies of C–A–T haplotypes (block 2) (P = 0.018) and A–A haplotypes (block 3) (P = 0.009) were detected in the healthy controls than in the patients with cervical cancer, suggesting that they may show protective effects against cervical cancer. These findings point to a role for the BRIP1 gene polymorphisms in cervical cancer in a Chinese Han population, and may be informative for future genetic or biological studies on cervical cancer.     

头颈部肿瘤基因组中HPV16DNA的同源序列

利用头颈部肿癌临床活检新鲜组织和石蜡包埋组织的两种处理标本,分别采用核酸分子杂交和聚合酶链反应(PCR)两种技术,分析了头颈部肿瘤组织基因组中人乳头瘤病毒16型的同源序列,并研究HPV16的感染同头颈部肿瘤发生发展的关系。结果表明:1.喉鳞状细胞癌DNA中有HPV16 DNA同源序列,检测频率在20.0％以上。2.PCR扩增石蜡包埋肿瘤组织DNA中HPV16的URR(病毒基因上游调控区)中的序列,电泳分析扩增产物在喉乳头状瘤、喉癌、鼻腔内翻性乳头瘤和口腔癌中检测率分别是11.1％、20.0％、42.9％和27.3％。3.研究表明HPV16 DNA阳性率与喉癌原发部位,分化程度和临床分期之间可能有一定的相关性。人乳头瘤病毒可能是头颈部肿瘤的病毒病因之     

Detection and identification of human papillomavirus types isolated from Korean patients with flat warts

Flat warts, also called verruca planna (VP) or juvenile warts, are benign epithelial proliferations of the skin caused by infection with human papillomaviruses (HPV). Several HPV types are known to be associated with flat warts, and particularly HPV type 3 and 10 have been most frequently reported in other countries. In this study, for the detection and typing of human papillomavirus isolated from Korean patients with flat warts, polymerase chain reaction (PCR) and restriction endonuclease digestion were carried out with a set of restriction endonucleases, using the cloned HPV DNA and DNA from clinical specimens. A unique digestion pattern for HPV type 3 and 10, a form of miniature fingerprinting, enabled us to identify HPV type from the amplified fragments. A total of thirty clinical samples, as either frozen tissue or paraffin-embedded tissue, were investigated to verify the type. All the clinical samples except one were con-firmed to be type 3, one of the most frequently observed types in flat warts, and one sample was neither type 3 nor type 10. Further investigation of the unidentified sample by DNA sequencing and sequence alignment with other known HPV types revealed that the sample was a variant of HPV type 94, one of the EV-related HPVs, with the closest evolutionary distance to the HPV type 10 among the known flat wart-associated HPV types.     

Cytokines in the treatment of virus infections

The interferon (IFN) system consists of both the formation of the various IFN proteins, and the diverse cellular responses which these induce: these result from the intracellular changes which follow their binding to a specific cell surface receptor.There is only a single human gamma, omega and beta IFN; in contrast, there are 13 closely related chemical species (subtypes) of human alpha IFN, which are nevertheless chemically and biologically distinct.IFN preparations made from mass cultured human cells or by using recombinant DNA techniques are now readily available for clinical use. IFN have a major role in the defence of the body against virus infections. In acute virus infections, preformed exogenous IFN cannot be given soon enough to be of value. However, IFN- and IFN- have proved of considerable value in some chronic virus infections, particularly chronic virus hepatitis and chronic papillomavirus infections. The doses routinely used are associated with both acute and chronic toxic side effects. Also, some patients form specific neutralising antibodies against the particular IFN preparation injected, which may abrogate all the benefits of the treatment. Nevertheless, IFN are now established as agents for use in routine medical practice.Abbreviations TNF Tumour necrosis factor - IL-6 Interleukin-6 - eIF2 The protein initiation factor     

Model systems to study the life cycle of human papillomaviruses and HPV-associated cancers

The prevalent human papillomaviruses(HPVs) infect either cutaneous or mucosal epithelium. Active Infections lead to epithelial hyperprolifeation and are usually cleared in healthy individuals within a year. Persistent infections in the anogenital tracts by certain high-risk genotypes such as HPV-16, HPV-18 and closely related types, can progress to high grade dysplasias and carcinomas in women and men, including cervical, vulva, penile and anal cancers. A significant fraction of the head and neck cancers are also caused by HPV-16. The viral oncogenes responsible for neoplastic conversion are E6 and E7 that disrupt the pathways controlled by the two major tumor suppressor genes, p53 and members of p RB family. Because HPV cannot be propagated in conventional submerged monolayer cell cultures, organotypic epithelial raft cultures that generate a stratified and differentiated epithelium have been used to study the viral life cycle. This article describes several systems to examine aspects of the viral productive phase, along with the advantages and limitations. Animal model systems of HPV carcinogenesis are also briefly described.     

Overexpression of klotho protein modulates uninephrectomy-induced compensatory renal hypertrophy by suppressing IGF-I signals

The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1–146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA damaged sites.     

Papillomaviruses—to vaccination and beyond

High risk human papillomavirus (HPV) types 16 and 18 DNAs were initially identified in 1983-1984. Subsequently the DNA of several other high risk HPV types has been identified. HPV 16 is present in more than 50% of cervical cancer biopsies, and HPV 18 is close to 20%. Some geographic variations exist in the prevalence of HPV high risk types: e.g. HPV 45 is more frequently observed in equatorial Africa, whereas types 58 and 52 have been more often found in East Asia. Molecular as well as epidemiological studies demonstrate that high risk HPV are indeed the causative agents for cervical cancer, they are also involved in other anogenital cancers, and in 25-30% of oropharyngeal carcinomas. Some of the mechanistic aspects are discussed in this review.     

A potent replicative delta-24 adenoviral vector driven by the promoter of human papillomavirus 16 that is highly selective for associated neoplasms

BACKGROUND: Several human epithelial neoplasms are associated with high-risk strains of human papillomavirus (HPV) such as cervical, anorectal, and other carcinomas. For some tumor types the current therapeutic tools are only palliative. Conditionally replicative adenoviruses (CRAds) are promising antineoplastic agents, which also can trigger confined antitumor effects. METHODS: We constructed a series of CRAds driven by the upstream regulatory promoter region (URR) of an Asian-American variant of HPV-16, which contained different mutations at the E1A region (dl1015 and/or Delta24) and wild-type. All vectors were tested in vitro for viral replication and cytotoxicity. Viral DNA replication and E1A expression were also assessed by quantitative PCR. Finally, we confirmed the antitumoral efficacy of this vector in injected and non-injected xenotransplanted cervical tumors in a murine model for tumor regression and survival studies. RESULTS: A vector denominated Ad-URR/E1ADelta24 displayed a potent cytopathic effect associated with high selectivity for HPV+ cell lines. We found that the oncolytic effect of this CRAd was comparable to Ad-wt or Ad-Delta24, but this efficacy was significantly attenuated in HPV- cell lines, an effect that was contributed by the URR promoter. Ad-URR/E1ADelta24 was very effective to control tumor growth, in both, injected and non-injected tumors generated with two different HPV+ cell lines. CONCLUSIONS: CRAd Ad-URR/E1ADelta24 is a highly selective vector for HPV+ cell lines and tumors that preserves the oncolytic efficacy of Ad-wt and Ad-Delta24. Our preclinical data suggest that this vector may be useful and safe for the treatment of tumors induced by HPV, like cervical cancers.     

光敏生物素标记法制备HPV DNA探针

本文将PBR322与HPV重组质粒DNA转化大肠杆菌HB101株,经氨苄青霉素—四环素平板筛选转化成功的菌落,将其扩增、提取质粒DNA,纯化后用限制性内切酶酶切,琼脂糖凝胶电泳区分不同大小的限制性内切片段,并用电洗脱法回收7.9Kb的HPV DNA片段。在高能卤素灯光照下标记光敏生物素制备HPV DNA探针,并检测其特异性和敏感性。结果表明:此探针具有较高敏感性,可达2.5pg;并且具有较强的特异性。