HPV16 E2 variants correlated with radiotherapy treatment and biological significance in cervical cell carcinoma.

Specific genetic mutations in human papillomavirus type 16 (HPV16) DNA are considered important in cervical lesion progression. This study analyzes to what extent radiotherapy treatment contributes to viral DNA mutation in cervical cell carcinomas, and the biological significance of these mutations. Serial tumor tissue, including 44 cervical cancer samples, collected before and after radiotherapy, and 52 biopsies with benign cervices, were tested and analyzed for the presence of HPV16, and for the integrity of the E2 gene. Analysis was performed with polymerase chain reaction (PCR), and a bidirectional sequencing assay was performed to find HPV16 E2 gene variants. HPV16 E2 accounted for 81.8% and 37.5% among tumor and benign cervices respectively (p = 0.02). The incremental number of DNA mutations was associated with radiotherapy treatment. Most E2 gene mutations involved regions encoding the amino-terminal and carboxy-terminal regions of E2 in the tumor irradiated samples. Amino acid changes T135 K, A143T, N203D and P208A in the amino-terminal region were the most common mutations across the irradiated samples. Rather, the mutations in the carboxy-terminal region (T3694A and T3805G) were synonymous changes. Specific nucleotide deletions were detected in the hinge domain, at positions 3455A > -, 3466 T > -, and 3501A > -. The mutation degree is influenced by the irradiation modalities, interestingly E2 sequence mutation being found widely after radiotherapy treatment with a total fractioned dose of 50 Gy (p = 0.004). E2 mutation has predictive and biological significance in cervical cancer patients receiving curative radiation therapy. Possibly, E2 mutation could influence viral genome intactness and could serve as an intrinsic marker for cervical cancer.

STAT1 and STAT6 Act as Antagonistic Regulators of PPARγ in Diabetic Patients with and without Cardiovascular Diseases.

BACKGROUND: The processes that mediate an inflammatory environment and increase atherosclerosis in diabetes are not well understood. Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the nuclear hormone receptor superfamily of ligand-activated transcription factors which play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM) and atherosclerosis. PPARγ promotes changes in lipid metabolism, especially in fatty acid (FA) trafficking, and the activity of PPARγ could be modulated by diabetes phenotype patients. Fatty acid translocase CD36 is one of the advanced PPARγ targets to arbitrate this action. In the current study, we investigated the potential role of signal transducer and activator of transcription STAT1 and STAT6 signaling linked to PPARγ and its implication in the modulation of lipid metabolism. METHODS: Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify target genes in Peripheral Blood Mononuclear Cells (PBMCs) isolated from two diabetic groups: diabetic patients with cardiovascular diseases (D.CVD) and without cardiovascular diseases (D). RESULTS: We demonstrated that PPARγ and CD36 mRNA expressions were downregulated along D.CVD compared to D (p = 0.002; p = 0.04; respectively). Decreased CD36 was accompanied by elevated levels of plasma triglyceride (TG) concentrations, 0.83 ± 0.29 vs. 2.46 ± 0.22), respectively. Furthermore, STAT1 was significantly more expressed in D.CVD (p = 0.01). On the other hand, we demonstrated that STAT6 induces a significant level of PPARγ mRNA expression in D patients (p = 0.01). CONCLUSIONS: Our results suggest that the expression and activity of PPARγ mediates CD36 in PBMCs and varies with respect to STAT6 and STAT1 trafficking in diabetic patients with and without cardiovascular diseases.

Molecular detection of human papillomavirus and viral DNA load after radiotherapy for cervical carcinomas.

AIMS AND BACKGROUND: Infection with high-risk types of human papillomavirus (HPV) is a necessary cause for cervical carcinoma. Radiation therapy together with surgery is the most effective treatment. The purpose of this study was to investigate the molecular basis of the response to radiotherapy in cervical cancer cells. METHODS AND STUDY DESIGN: Tumor cells were obtained from biopsies of 44 cervical cancers, collected before and after radiotherapy. The presence of HPV was analyzed by polymerase chain reaction (PCR) using primers specific for the L1 region. RESULTS: The prevalence of HPV was 70.4%, with HPV16 being the most common (54.5%) and HPV18 the second (15.9%). Our analyses show that ionizing radiation does not influence HPV detection, as the percentage of HPV-positive biopsies was similar in patients before and after radiotherapy (HPV16 60% vs. 51.7% and HPV18 20% vs. 13.7%, respectively). However, the detection of HPV did vary by tumor stage, with the highest proportion observed in late-stage tumors (HPV16 and HPV18 in 80% and 60% of stage III tumors, respectively). We also found that HPV viral load is influenced by radiotherapy and tumor stage, with the highest viral loads in late-stage tumors (stage III) after 1 day since radiotherapy (p<0.05). According to Kaplan-Meier curves, higher HPV viral load was associated with significantly shortened progression-free survival (p = 0.04). CONCLUSIONS: Our data provide prospective evidence that ionizing radiation can affect the HPV viral load and this might offer the best strategies for assessment of therapeutic efficacy.

Sequence variation in the E2-binding domain of HPV16 and biological function evaluation in Tunisian cervical cancers.

HPV16 E2 variants have different effects on the transcriptional activity of the LCR. In this study, we examined the nucleotide and amino acid sequence variation within the HPV16 E2 gene and to correlate with disease progression. E2 gene disruption was detected by PCR amplification of the entire E2 gene using a single set of primers. Nucleotide variations were analyzed by bidirectional sequencing. mRNA expression patterns of E6 and E7 gene transcripts were evaluated by a reverse transcriptase-PCR method (RT-PCR). The detection of intact E2 genes was significantly higher among controls than cases (81.8% versus 37.5%, resp., P < 0.05). Among the E subgroup, variation at position 3684 C>A results in the amino acid substitution T310K and was more common among the E2 undisrupted cases (7/9; 77.7%), compared to controls (2/9; 22.2%). In addition, specific sequence variations identified in the E2 ORF at positions 3684 C>A were associated with increased viral oncogenes E6-E7 production. Besides HPV16 E2 disruption, the 3684 C>A variation within undisrupted E2 genes could be involved in an alternative mechanism for deregulating the expression of the HPV16 E6 and E7 oncogenes and appears to be a major factor contributing to the development of cervical cancer in Tunisian women.

Situation of HPV16 E2 gene status during radiotherapy treatment of cervical carcinoma.

BACKGROUND: Human papillomavirus (HPV) integration within the E2 gene has been proposed as a critical event in cervical carcinogenesis. This study concerned whether HPV16 status and E2 gene intactness are predictive of radiation response in patients with cervical cancer. MATERIALS AND METHODS: Biopsies of 44 patients with cervical cancer were collected before or after radiotherapy. The presence of HPV16 was assessed by polymerase chain reaction (PCR) using specific primers for the L1 region. E2 disruption was detected by amplifying the entire E2 gene. RESULTS: HPV16 DNA was found in 54.5% of the clinical samples. Overall, 62.5% of the HPV16 positive tumors had integrated viral genome and 37.5% had episomal genome. There was a tendency of increase of HPV16 E2 negative tumors compared with HPV16 L1 ones in advanced stages (75% versus 20% in stage III respectively). Detection of E2 gene appeared influenced by the radiotherapy treatment, as the percentage of samples containing an intact HPV16 E2 was more frequent in pretreated patients compared to radiotherapy treated patients (66.6% versus 20%). The radiation therapy caused an eight-fold [OR= 8; CI=1.22-52.25; p=0.03] increase in the risk of HPV16 genome disruption. The integration status is influenced by the irradiation modalities, interestingly E2 disruption being found widely after radiotherapy treatment (75%) with a total fractioned dose of 50 Gy. CONCLUSIONS: This study reveals that the status of the viral DNA may be used as a marker to optimize the radiation treatment.

HPV-16 E2 physical status and molecular evolution in vivo in cervical carcinomas.

A key event in the development of cervical carcinoma is the deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly due to HPV integration into host DNA. Here we explored whether HPV-16 E2 gene integrity is a biomarker of progressive disease with oncogenes expression. HPV-16 genome disruption was assessed by amplification of the entire E2 gene, while mRNA expression patterns of the E1, E2, E6, and E7 genes were evaluated by reverse transcription PCR (RT-PCR). As expected, E2 disruption was significantly higher among patients with cervical cancers than subjects with benign lesions (p=0.02). The status of the E2 gene correlated with tumorogenesis, and seemed also to correlate with the stage of the carcinomas, since integrated HPV-16 DNA was frequently detected in patients with advanced cancer stages (75% of stage III vs 60% stages I and II). In bivariate analysis, the lesions’ grade was most significantly associated with HPV-16 DNA disruption (p<0.05). In cervical carcinoma the deletion pattern involved more frequently the E2 gene rather than the E1 gene (62.5% vs 45.8%). The prevalence of the E6/E7 HPV-16 transcripts in cervical carcinoma specimens and in benign cervical lesions were detected with frequencies of, respectively, 91.6% and 45.4%. The mRNA levels of the HPV-16 E6/E7 genes were expressed at approximately the same levels in each physical state. We consistently observed that E6/E7 were absent or weakly detectable in the presence of E2. However, in the absence of E2 the levels of E6/E7 markedly increased (p<0.05). This study underscores the significance of investigating alternative mechanisms of E2 expression and oncogenes E6/E7 transcripts in vivo as biomarkers for disease severity in cervical carcinomas.

Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients.

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

Analysis of antibody response to HPV 16 and HPV 18 antigens in Tunisian patients.

OBJECTIVE: The aim of this present study was to use Luminex technology to detect antibodies against the late antigen L1 as well as those directed against the early antigens E6 and E7. BACKGROUND DATA: Human papillomavirus (HPV) serology is complex because infection and disease lead to distinct type-specific antibody responses. MATERIALS AND METHODS: Viral antigens were expressed with pGEX vectors in Escherichia coli and then used in Luminex as coating antigens for antibody detection in 205 human sera samples: 71 cervical cancer cases, 64 cases of cervical inflammation, and 70 controls. RESULTS: The data showed that 90.14% of sera among the cervical cancer patients had seropositivity toward at least one of the HPV 16 or HPV 18 antigens. Moreover, the percentages of positivity toward E6 and E7 HPV 16 antigens were 44% and 61%, respectively, versus only 21% for the L1 antigen. Among cervical cancer patients, the data showed different distributions in women of different ages. In addition, the intensity of the antibody response was also different for the six antigens analyzed. CONCLUSIONS: Antibody detection depends on the type of antigen, and is well correlated with international scientific findings. The differences in antibody response between patients with inflammation and patients with cervical cancer were significant.