Preferential sites for the integration and disruption of human papillomavirus 16 in cervical lesions.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HPV) is necessary to cause cervical cancer, and integrating viral DNA into the host genome may contribute to the process of carcinogenesis. The underlying mechanisms are still unclear. OBJECTIVE: In this study, we aimed to investigate the distribution of HPV 16 integration in the host genome and disrupted sites in the viral genome. STUDY DESIGN: The physical status of HPV 16 genomes in 46 cervical precancerous and cancerous lesions was determined via ligation-mediated chain reaction (DIPS) using 15 previously published primer sets and 12 newly designed primer sets. RESULTS: A total of 60 viral-cellular junctions were identified in 31 of 46 specimens, and over 80% of the integration sites in the human genome were located in regions of repetitive elements. The proportion of LSIL-, HSIL-, and SCC-containing integration sites near cancer-relevant genes was 10%, 18.8%, and 33.3%, respectively. The frequency of viral gene disruption was significantly higher (P < 0.05) in the L2 gene than in other regions of the viral genome. CONCLUSION: There are sites of preferential HPV 16 integration. The integration sites tend to be located in repetitive regions of the host genome, and some sites are found near cancer-relevant genes. In addition, the HPV 16 genome is more likely to be disrupted in the L2 gene locus.

Determination of integrated HPV58 sequences in cervical lesions.

Integration of high-risk HPV in host genome is an important event in cervical cancer development. This study was aimed to analyze the integration of HPV58, a high-risk type that is prevalent in cervical lesions from southeastern Asia. Detection of integrated papillomavirus sequences by ligation-mediated polymerase chain reaction followed by DNA sequencing revealed 8 integrations in 5 samples, and virus integration was found present in 2 samples with early lesion. Sequence analysis of the viral-cellular junctions showed that E1 disruption in virus genome was an early and common event during HPV58 infection. In 6 integrations, DNA fragments of HPV58 genome integrated into the repetitive element sequences of host genome.

[Viral load, genomic integration frequency of human papillomavirus 16 in cervical cancer and precancerous lesions].

OBJECTIVE: To investigate the association between viral load, genomic integration frequency of HPV 16 and cervical carcinogenesis and assess the probability that HPV 16 integration frequency may be utilized as a marker for cervical cancer. METHODS: Forty cases of HPV16 single infection were selected from 337 cervical scrape samples by PCR (polymerase chain reaction) amplification and DNA sequencing. The copy numbers of E2, E6 and ß-actin were quantified to evaluate the viral load and integration status by real-time PCR. RESULTS: (1) The median number of adjusted viral load of normal group, LSIL (low-grade squamous intraepithelial lesion) group, HSIL (high-grade squamous intraepithelial lesion) group and squamous cervical cancer group were 0.11, 18.55, 44.63 and 7.6 copies per cell respectively. The viral load was higher in LSIL-HSIL group than that in normal group while lower in squamous cervical cancer group than that in HSIL group. (2) The median number of E2/E6 was 0.93 in normal group, 0.84 in precancerous group (LSIL-HSIL) and 0.64 in squamous cervical cancer group respectively. It showed a descending trend with the progression of cervical disease. (3) With the disease development, the proportion of episomal form decreased (normal group 4/5, LSIL group 4/6, HSIL group 10/16, cervical squamous cancer group 5/13) whereas that of integrated form (mixed and totally integrated) increased (normal group 1/5, LSIL group 2/6, HSIL group 6/16, cervical squamous cancer group 8/13). Both totally integrated cases were cervical squamous cancer. CONCLUSION: (1) HPV 16 viral load may not be an ideal marker to predict cervical carcinogenesis. (2) Due to a positive correlation between HPV16 genomic integration frequency and cervical neoplastic progression, HPV 16 integration frequency may be a potential marker of early diagnosis for cervical lesion progression.

[PCR-based sequencing for detection and genotyping of human papillomavirus in 325 cervical cell specimens from Beijing area].

To evaluate PCR-sequencing for clinical detection of human papillomavirus (HPV) genotypes in cervical cell specimens, we applied PCR-sequencing to HPV detection and genotyping by general primer PGMY09/11, which targets the HPV most conserved L1 gene. Samples with multiple infections were subjected to HPV type-specific PCR. Among the 325 cervical samples, 228 were HPV positive, of which 66 showed multiple infections. In all, 27 different HPV genotypes were identified, with HPV 16 being the most prevalent, followed by HPV 58 and 52. The prevalence of high-risk HPV infection increased with the severity of cervical lesions (P < 0.05), whereas the proportion of multiple infections declined significantly from LSIL to SCC (P < 0.05). Both rates of overall and high-risk HPV infection were the highest in 21-30 age groups. There was substantial agreement between the HC2 and PCR-sequencing assay for detection of high-risk HPV (kappa = 0.675). PCR-sequencing was effective in HPV detection and genotyping, and it could be potentially applied to large scale HPV screening.

Mutagenicity of Chinese traditional medicine Semen Armeniacae amarum by two modified Ames tests.

BACKGROUND: Semen armeniacae amarum (SAA) is a Chinese traditional medicine and has long been used to control acute lower respiratory tract infection and asthma, as a result of its expectorant and antiasthmatic activities. However, its mutagenicity in vitro and in vivo has not yet been reported. The Ames test for mutagenicity is used worldwide. The histidine contained in biological samples can induce histidine-deficient cells to replicate, which results in more his+ colonies than in negative control cells, therefore false-positive results may be obtained. So, it becomes a prerequisite to exclude the effects of any residual histidine from samples when they are assayed for their mutagenicity. Chinese traditional herbs, such as SAA, are histidine-containing biological sample, need modified Ames tests to assay their in vitro mutagenicity. METHODS: The mutagenicity of SAA was evaluated by the standard and two modified Ames tests. The first modification used the plate incorporation test same as standard Ames teat, but with new negative control systems, in which different amounts of histidine corresponding to different concentrations of SAA was incorporated. When the number of his+ revertants in SAA experiments was compared with that in new negative control, the effect of histidine contained in SAA could be eliminated. The second modification used a liquid suspension test similar to the standard Ames test, except with histidine-rich instead of histidine-limited medium. The aim of this change was to conceal the effect of histidine contained in SAA on the final counting of his+ revertants, and therefore to exclude false-positive results of SAA in the Ames test. Furthermore, the effect of SAA on chromosomal aberration in mammalian bone marrow cells was tested. RESULTS: The standard Ames test showed a positive result for mutagenicity of SAA. In contrast, a negative response was obtained with the modified plate incorporation and modified suspension Ames tests. Moreover, no apparent chromosomal aberrations were observed in mammalian bone marrow cells treated with SAA. CONCLUSION: The standard Ames test was not suitable for evaluating the mutagenicity of SAA, because false-positive result could be resulted by the histidine content in SAA. However, the two modified Ames tests were suitable, because the experimental results proved that the effect of histidine in SAA and therefore the false-positive result were effectively excluded in these two modified Ames tests. This conclusion needs more experimental data to support in the future. Moreover, the experimental results illustrated that SAA had no mutagenicity in vitro and in vivo. This was in agreement with the clinical safety of SAA long-term used in China.