Effect of radiofrequency therapy on HPV16-E7 lentivirus infection in the reproductive tract of mice and its effect on immune function of splenic lymphocytes

Objective To investigate the effect of radiofrequency therapy (RFT) on HPV16-E7 lentivirus infection in the reproductive tract of mice and reveal its effect on immune function of splenic lymphocytes.Materials and Methods The mouse reproductive tract model was established by infection with HPV16-E7 lentivirus. Fluorescence microscope was used to evaluate successful injection. The expression of HPV16-E7 protein was detected by Western blotting test. The levels of CD4+ and CD8+ were determined by flow cytometry, and the ratio was calculated. The proliferation of splenic lymphocytes was detected by MTT assay. Expression of Interleukin (IL)-2 and interferon-γ (IFN-γ) messenger RNA (mRNA) in lymphocyte was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).Results Fluorescence microscope determined the successful injection of HPV16-E7 lentivirus. Compared with model group, RFT treatment decreased HPV16-E7 protein, and increased CD4+/CD8+ ratio and the proliferation activity of splenic lymphocytes. Besides, RFT treatment increased the mRNA expression levels of IL-2 and IFN-γ compared to the model group. In particular, the proliferation activity of spleen lymphocytes and the expression levels of IL-2 mRNA and IFN-γ mRNA in RFT were higher at 12 days than at 6 days after treatment.Conclusion RFT could eliminate HPV16-E7 lentivirus infection in the reproductive tract of mice, and the mechanism was related to the immune system (AU)

Human myoma tissue-based extracellular matrix models for testing the effects of irradiation on the HPV positive cells.

BACKGROUND: This study was designed to investigate the invasion of human papillomavirus (HPV) positive human cervical carcinoma cell lines in human leiomyoma-based extracellular matrices in vitro, and to test the suitability of the model for studying the irradiation effects on the cancer cell invasion. METHODS: HPV positive cervical carcinoma cell lines SiHa and CaSki, and HPV negative squamous cell carcinoma cell line HSC-3 were used. CaSki cells contain around 600 copies of HPV 16 virus in the genome, whereas SiHa have only 1-2 copies per cell. Cells were analyzed using two different human tumor derived extracellular matrix methods (3D myoma disc model, and Myogel Transwell invasion assay). Cultures were irradiated with 4 Gy. Myoma invasion area and the depth of invasion were measured with ImageJ 1.51j8 software. Statistical analyses were performed with SPSS Statistics (IBM SPSS® Statistics 25). RESULTS: All cells invaded through Myogel coated Transwell membranes and within myoma discs. In myoma discs, a difference in the invasion depth (p = 0.0001) but not in invasion area (p = 0.310) between the HPV positive cell lines was seen, since SiHa (less HPV) invaded slightly better than CaSki (more HPV). HSC-3 cells (HPV negative) invaded deepest (p = 0.048) than either of the HPV positive cell line cells. No difference was detected in the invasion area (p = 0.892) between HPV positive and HPV negative cells. The ionized radiation significantly reduced the invasion depth of HSC-3 (p = 0.008), SiHa (p = 0.0001) and CaSki (p = 0.005). No significant effect on the invasion area was detected in any of the cell lines. However, a significant difference was observed between SiHa and CaSki in the reduction of the invasion depth after radiation (p = 0.013) as the reduction was greater with SiHa than CaSki. CONCLUSIONS: Both solid and gelatinous human leiomyoma-based extracellular matrix models were suitable platforms to study the invasion of HPV positive cervical carcinoma cells in vitro. SiHa cells with less HPV copy number cells invaded slightly better and were slightly more sensitive to irradiation than CaSki cells with high HPV copy number. However, there was no drastic differences between the invasion properties of these carcinoma cells.

A Phase 1 Trial Assessing the Safety and Tolerability of a Therapeutic DNA Vaccination Against HPV16 and HPV18 E6/E7 Oncogenes After Chemoradiation for Cervical Cancer.

PURPOSE: This study assessed the safety and tolerability of therapeutic immunization against the human papillomavirus (HPV) viral oncoproteins E6 and E7 in patients with cervical cancer after chemoradiation. METHODS AND MATERIALS: MEDI0457 (INO-3112) is a DNA-based vaccine targeting E6 and E7 of HPV-16/18 that is coinjected with an IL-12 plasmid followed by electroporation with the CELLECTRA 5P device. At 2 to 4 weeks after chemoradiation, patients with newly diagnosed stage IB1-IVA (cohort 1) or persistent/recurrent (cohort 2) cervical cancers were treated with 4 immunizations of MEDI0457 every 4 weeks. The primary endpoints were incidence of adverse events and injection site reactions. Immune responses against HPV antigens were measured by ELISpot for interferon-γ (IFNγ), enzyme-linked immunosorbent assay for antibody responses and multiplexed immunofluorescence for immune cells in cervical biopsy specimens. RESULTS: Ten patients (cohort 1, n = 7; cohort 2, n = 3) with HPV16 (n = 7) or HPV18 (n = 3) cervical cancers received MEDI0457 after chemoradiation. Treatment-related adverse events were all grade 1, primarily related to the injection site. Eight of 10 patients had detectable cellular or humoral immune responses against HPV antigens after chemoradiation and vaccination: 6 of 10 patients generated anti-HPV antibody responses and 6 of 10 patients generated IFNγ-producing T cell responses. At the completion of chemoradiation and vaccination, cervical biopsy specimens had detectable CD8+ T cells and decreased PD-1+CD8+, PD-L1+CD8+, and PD-L1+CD68+ subpopulations. All patients cleared detectable HPV DNA in cervical biopsies by completion of chemoradiation and vaccination. CONCLUSIONS: Adjuvant MEDI0457 is safe and well tolerated after chemoradiation for locally advanced or recurrent cervical cancers, supporting further investigation into combining tumor-specific vaccines with radiation therapy.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.

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.

HSV-1 Infection Modulates the Radioresponse of a HPV16-positive Head and Neck Cancer Cell Line.

BACKGROUND: The combined effects of Human papillomavirus (HPV) and Herpes simplex type 1 (HSV-1) infections and their effects on cancer cell radioresistance are unexplored. MATERIALS AND METHODS: An HPV16-positive hypopharyngeal carcinoma cell line (UD-SCC-2) was infected with wt-HSV-1 at low multiplicity of infection (MOI) and irradiated with 2 Gy at 24 h postinfection. Viability assays and quantitative reverse-transcriptase PCR for HPV16 E6, E7, nuclear factor kappa B1, B-cell CLL/lymphoma 2 (BCL2), and caspases 3, 8 and 9 at 24, and 72 h, as well as immunocytochemistry for BCL2, caspase 3, cyclin E, mouse double minute 2 homolog (MDM2), HSV-1 and Ki-67 were performed at 144 h postirradiation. RESULTS: At 144 h, cell viability was significantly lowered by irradiation only in uninfected cells. Infection combined with irradiation resulted in increased expression of E6, E7, BCL2 and NF-κB1 at 144 h. Simultaneously, E6 and E7 were down-regulated in non-irradiated infected cells. Irradiation and infection with 0.00001 MOI separately up-regulated caspase 3 but infection with 0.0001 MOI halved its expression in irradiated cells. CONCLUSION: HSV-1 infection modulates radioresistance of HPV16-positive hypopharyngeal carcinoma cells.

UVB irradiation down-regulates HPV-16 RNA expression: implications for malignant progression of transformed cells.

A new cell line obtained from normal human epithelial keratinocytes transfected with the whole HPV-16 genome has been extensively characterised. This cell line, named HK-168, has a basal/para-basal keratinocyte phenotype, requires the use of serum-free chemically defined media and maintains the ability to differentiate toward pluri-stratified epithelia. Although immortalised it is not capable of anchorage independent growth and is not tumorigenic. HK-168 has a distinctive kariotype, with a complete, transcriptionally active HPV-16 genome integrated at an almost 1:1 ratio into the host haploid genome thus providing a convenient experimental model for viral transformed pre-neoplastic cell phenotype. The oxidative stress, induced by mild UVB irradiation, caused in HK-168 a general suppression of viral transcription, accompanied by a moderate growth arrest, an appropriated response of cellular antioxidant enzymes, the activation of cell repair mechanisms and a mild induction of apoptosis. This response is similar to the one observed in the highly resistant diploid keratinocytes. Conversely, transformed cells devoid of HPV sequences (HaCaT), appeared extremely susceptible to apoptosis. We propose that reported suppression of viral oncogenes, restoring the cell control on growth and repair mechanisms, allows the damage repair, ultimately resulting in a surviving response.