TSLC1 gene silencing via promoter hypermethylation is a frequent event in the progression from high-risk HPV-containing, high-grade CIN lesions to invasive cervical cancer.

Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

The beta and gamma genera of papillomaviruses consist of epidermodysplasia verruciformis-related human papillomaviruses (HPVs) and phylogenetically related cutaneous HPVs. Here, we have developed a consensus primer PCR assay and reverse line blot typing system coupled thereto (referred to as beta and gamma cutaneous HPV PCR [BGC-PCR]) for detection and typing of 24 beta and gamma HPVs (HPV types 4, 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 48, 49, 50, 60, and 65). Because the HPV-specific PCR products are only 72 bp in size, the system is suitable for formalin-fixed, paraffin-embedded specimens and other samples in which the DNA is of suboptimal quality. This system was able to detect and type as little as 100 ag to 1 fg HPV DNA per reaction (depending on the HPV type) in a background of 100 ng human DNA without any cross-reactivity between the tested types. Beta and gamma HPVs were detected in DNA extracted from plucked eyebrow hairs of 31 of 34 renal transplant recipients. In addition, formalin-fixed, paraffinembedded specimens from nonmelanoma skin tumors of renal transplant recipients (n ‫؍‬ 25) and immunocompetent individuals (n ‫؍‬ 15) scored BGC-PCR positive in 21 and 6 cases, respectively, with HPV type 5 (HPV5) and HPV8 being the predominant types. The data indicate that this method can be a valuable, user-friendly tool for the detection and typing of cutaneous HPV in clinical specimens and may have implications for future monitoring of vaccines or alternative treatment modalities for diseases caused by these cutaneous HPVs.Human papillomaviruses (HPVs) are small DNA viruses that infect cells of epithelial origin and can be grouped into either mucosal or cutaneous HPV types based on tissue tropism. The mucosal types include those HPVs that infect mucosa of the genital and respiratory tracts. They are divided into low-risk and high-risk types according to their ability to induce benign lesions and cancer, respectively. Low-risk types such as HPV type 6 (HPV6) and HPV11 can cause genital warts (condylomata acuminata). High-risk types such as HPV16 and HPV18 are causally involved in carcinogenesis of the uterine cervix and other mucosa of anogenital and oropharyngeal sites (13).The cutaneous HPV types are phylogenetically distant from the mucosal types, and they infect the external skin. A subset of cutaneous HPV types is known to cause benign cutaneous warts. There is also evidence for an association of cutaneous HPV types with skin cancer. This was demonstrated first in patients with epidermodysplasia verruciformis (EV), a rare hereditary disease characterized by disseminated, persistent, flat warts and macular lesions that arise during childhood (reviewed in reference 14). In the macular lesions, the following phylogenetically related HPV types have been detected: HPV types 5,8,9,12,14,15,17, and 19 to 25. Therefore, these HPV types are classified as EV HPVs. EV patients have a high risk of developing cutaneous squamous cell carcinoma (SCC) later in life. HPV DNA, particularly...

To identify cellular genes that may be involved in human papillomavirus (HPV)-mediated immortalization mRNA differential display analysis was performed on preimmortal and subsequent immortal stages of four human keratinocyte cell lines transformed by HPV type 16 or 18 DNA. This yielded a cDNA fragment encoding the transcription factor GATA-3 that was strongly reduced in intensity in all immortal stages of the four cell lines. A marked reduction in both GATA-3 mRNA and protein expression in HPV-immortalized cell lines was confirmed by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry and was also shown to be apparent in cervical carcinoma cell lines. Immunohistochemical analysis of cervical tissue specimens showed a clear nuclear staining for GATA-3 in normal cervical squamous epithelium (n ‫؍‬ 14) and all cervical intraepithelial neoplasia (CIN) I (n ‫؍‬ 6) and CIN II lesions (n ‫؍‬ 2). In contrast, 11% (1 of 9) of CIN III lesions and 67% (8 of 12) of cervical squamous cell carcinomas revealed a complete absence of GATA-3 immunostaining. Hence, complete down-regulation of GATA-3 expression represents a rather late event during cervical carcinogenesis. Whether GATA-3 down-regulation is etiologically involved in HPV-mediated immortalization and cervical carcinogenesis remains to be examined. Infection with high-risk human papillomavirus (HPV) types is the most significant risk factor for the development of cervical cancer and HPV DNA can be detected in almost all cervical squamous cell carcinomas. 1 HPV functions are, however, not sufficient for the development of cervical cancer and additive oncogenic events involving host cell genes are required, consistent with a multistep process of carcinogenesis. To gain better insight in HPV-mediated carcinogenesis in vitro model systems have been proven very valuable. High-risk HPV types, in particular HPV 16 and HPV 18, can induce immortalization of primary human epithelial cells in vitro. [2][3][4] Studies involving somatic cell fusions and microcell-mediated chromosome transfers have indicated that particularly recessive gene alterations are crucial for immortalization of HPV-containing epithelial cells. 5,6 HPV-mediated immortalization is associated with an arrest of telomeric shortening and activation of the telomere lengthening enzyme telomerase. 4,7 Moreover, activation of telomerase, by up-regulation of its catalytic subunit human telomerase reverse transcriptase gene, seemed to be necessary and sufficient for immortalization of HPV-transfected keratinocytes. 6,8,9 In addition, expression of the viral oncogene HPV 16 E6 from a heterologous promoter has been shown to activate telomerase in primary keratinocytes. 10 However, expression of HPV 16 and HPV 18 E6 from their native promoter in the context of the fulllength virus genome did not result in telomerase activation, 4 indicating that additive events are required.Recent studies have shown that yet unknown genes residing at chromosomes 3, 4, and 6 can regulate telomer...