Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

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cThe E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6-and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level. Human papillomaviruses (HPV) are associated with epithelial cancers of the genital tract, and high-risk HPV of the genus Alphapapillomavirus (alphaPV) have been proven to be a necessary causative factor in the development of cervical cancer (42). HPV types of the genus Betapapillomavirus (betaPV) have been implicated in the development of cutaneous tumors, especially squamous cell carcinomas (SCC) and actinic keratoses (AK) as their precursor lesions (13). The first evidence implicating cutaneous HPV types in the pathogenesis of cutaneous SCC was based on the detection of betaPV types, predominantly HPV5 or HPV8 and rarely HPV type 14, 17, 19, 20, 24, or 47, in SCC of epidermodysplasia verruciformis (EV) patients (11,15,27,29). Also, in non-EV patients, betaPV are frequently detected in cutaneous SCC and AK. Interestingly, the high prevalence and viral load of betaPV types in actinic keratoses (30, 38) and the decrease of viral load during skin carcinogenesis may point to a particular involvement of HPV in the early stages of skin cancer development. UV radiation is recognized as the main risk factor in SCC development (28), and HPV is discussed as a cofactor in concert with UV radiation (7,16,18,21,33,35,37). However, the role of betaPV and their cell-transforming mechanisms are still less clear than those of high-risk alphaPV (4).The ...

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Background: In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough.