Dual role of tumor suppressor p53 in regulation of DNA replication and oncogene e6-promoter activity of epidermodysplasia verruciformis-associated human papillomavirus type 8

AbstractHuman papillomavirus 8 (HPV8) is a representative of Epidermodysplasia verruciformis (EV)-associated viruses. Transient assays in the human skin keratinocyte cell line RTS3b have shown that its replication depends in trans on expression of the viral proteins E1 and E2, similarly to other HPVs. Using deletion mutants and cloned subfragments of the noncoding region (NCR) of HPV8 we identified a 65-bp sequence in the 3′ part of the NCR to be necessary and sufficient to support replication in cis. The origin of replication (ori) of HPV8 is composed of the sequence motifs “CCAAC” (nt 57–73) and M29 (nt 84–112), which are highly conserved among the majority of EV HPVs. Analysis of M29 revealed an unconventional binding site of the E2 protein and an overlapping DNA recognition site of the tumor suppressor protein p53. Both these factors competitively bind to M29. In transient replication assays p53 acted as a potent inhibitor of ori activity, most probably in a DNA-binding-dependent fashion. The minimal ori sequences are also functionally critical for the E6 oncogene promoter P175. In contrast to its effect on replication, p53 stimulated promoter activity depending on its interaction with M29. Our observations suggest that p53 is involved in controlling the balance between DNA replication and gene expression of HPV8

Studi Ketoksikan Dinoflagelata Spesies Prorocentrum Minimum (Dinophyceae) Schiller (Pavillard)

This study was carried out to determine the toxicity of the dinoflagellate Prorocentrum minimum species that formed massive blooms in Lido Beach, Johor Bahru in July 2002. Clone cultures were established in ES-DK medium at 26o C under a 14:10 hour light dark cycle. Species toxicity was determined by intra peritoneal (i.p) injection of culture extract into mice. Cultured cell extracts were toxic to mice. The major symptoms were muscular paralysis and diarrhea. However no mouse mortality was observed even after 13 hours. Extracts of cultured cells were also hemolytic on rabbit red blood cells. Keywords: Dinoflagellate, HAB, Prorocentrum minimum, Red Tide dan Toxicity Kajian dilakukan untuk menentukan ketoksikan dinoflagelata spesies Prorocentrum minimum yang menyebabkan kejadian pasang merah besar-besaran di perairan Pantai Lido, Johor Bahru pada Juli tahun 2002. Kultur klon telah dibuat dalam medium ES-DK pada suhu 26o C dan siklus pencahayaan 14:10 jam terang gelap. Ketoksikan spesies diuji dengan penyuntikan ekstrak kultur secara intra peritoneal (i

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis

Background: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (β-PV) play an important role in accelerating the early stages of skin tumorigenesis. Methods: We investigated the effects of UV induced DNA damage in murine models of β-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. Results: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the

A humanized mouse model of HPV-associated pathology driven by E7 expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.This work was supported by Comunidad Autonoma de Madrid (Oncocycle S2006/BIO-0232), by Ministerio de Ciencia e Innovacion (ISCIII-RETIC RD06/0020 and SAF2008-00121), and by Fundación Sandra Ibarra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript