Loss of Drop1 expression already at early tumor stages in a wide range of human carcinomas.

In a study on gene deregulation in ovarian carcinoma we found a mRNA coding for a 350 kDa protein, Drop1, to be downregulated 20- to 180-fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5' region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two-hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors.

Characterization of two novel cutaneous human papillomaviruses, HPV93 and HPV96.

Two novel human papillomaviruses (HPVs), HPV93 and HPV96, with genomes of 7450 and 7438 bp, respectively, are described. The L1 open reading frame of HPV93 showed highest identity to HPV24 (79%) and that of HPV96 had highest identity to HPV92 (71%). Real-time PCR for HPV92, 93 and 96 on stripped biopsies from tumours and healthy skin from 269 immunocompetent patients found HPV DNA in 2.6% of tumours and in 0.4% of healthy skin samples. Double infections were observed in two tumours. HPV92 was detected in four, HPV93 in two and HPV96 in three tumours. The range of viral loads spanned from one copy per 45 cells to one copy per 10,000 cells. The E7 proteins of HPV92, 93 and 96 were found to bind the retinoblastoma protein (pRb). These results suggest a possible role for these HPV types in skin carcinogenesis that deserves further study.

Isolation of multiple TT virus genotypes from spleen biopsy tissue from a Hodgkin's disease patient: genome reorganization and diversity in the hypervariable region.

We report the isolation of 24 novel genotypes of TT viruses from a surgically removed spleen of a patient with Hodgkin's disease. The sequence analysis of our 24 isolates revealed the remarkable heterogeneity of TT virus isolates not only from the same patient but also from the same biopsy material. These isolates belong to four phylogenetic groups of TT viruses. Nucleotide sequence analyses revealed five distinct genotypes (tth3, tth4, tth5, tth6, and tth7). The limited variation in sequence identity of the other isolates defines the latter as variants of four of these genotypes. A group of 6 isolates (the tth7 group) revealed a reorganization of open reading frame 1 (ORF1) leading to one larger and a varying number of smaller ORFs. The nucleotide difference of the full-length genomes was less than 1%. A variation of 69 to 97% in amino acids of a second group of 8 isolates (the tth3 group) was restricted to the hypervariable region of ORF1, indicating the existence of a quasi-species. These isolates differed by less than 2% in the remainder of their nucleotide sequences. An alignment of these isolates with 79 previously reported TT virus genotypes permits the proposal of TT virus genera and species within the family Anelloviridae in analogy to a previous proposal for the papillomaviruses (family Papillomaviridae).

Human papillomavirus type 16 E6 and E7 genotypes in head-and-neck carcinomas.

Human papillomavirus type 16 (HPV16) is associated with squamous cell carcinomas of the head and neck (HNSCC) particularly from the Waldeyer's tonsillar ring. A causal role of HPV16 in carcinogenesis is linked to the activity of the viral oncoproteins E6 and E7 which inactivate the cellular tumor suppressors p53 and pRB, respectively. Lack of E6 expression in HPV16-positive HNSCC has been reported, in some cases caused by disruption of the E6 gene. We have examined the status of the HPV16 E6-E7 gene region in tumor and metastasis samples of 24 HNSCC patients employing genomic PCR. No cases with a disrupted E6-E7 region could be identified. Sequence analysis of the E6-E7 segments revealed three different HPV16 E6-E7 genotypes: the HPV16 prototype (6 of 21 cases), the E6 variant T350G (8 of 21 cases), and the E6-E7 variant A131G/C712A (7 of 21 cases). The E6 variants T350G and A131G have been associated with increased oncogenic potential in cervical cancer patients depending on host genetic factors. Their high prevalence in the HNSCC samples studied indicates that they may be important also in HNSCC development.

Nucleotide sequence and phylogenetic classification of candidate human papilloma virus type 92.

From a basal cell carcinoma (BCC) the complete genome of candidate human papillomavirus (HPV) type 92 was characterized. Phylogenetically, the candidate HPV 92 was relatively distantly related to other cutaneous HPV types within the B1 group. By quantitative real time PCR, 94 viral copies were present per cell in the BCC and another BCC contained 1 viral copy per cell. Lower copy numbers were found in two solar keratoses (1 copy per 33 cells and 1 copy per 60 cells) and two squamous cell carcinomas (1 copy per 436 cells and 1 copy per 1143 cells). The high viral load of HPV 92 in two BCCs differs from the low amount of HPV DNA reported from nonmelanoma skin cancers.

The evolutionarily conserved single-copy gene for murine Tpr encodes one prevalent isoform in somatic cells and lacks paralogs in higher eukaryotes.

Vertebrate Tpr and its probable homologs in insects and yeast are heptad repeat-dominated nuclear proteins of M(r) 195,000 to M(r) 267,000 the functions of which are still largely unknown. Whereas two homologs exist in Saccharomyces cerevisiae, it has remained uncertain whether metazoans possess different paralogs or isoforms of Tpr that might explain controversial reports on the subcellular localization of this protein. To address these possibilities, we first determined the sequence and structure of the murine tpr gene, revealing a TATA box-less gene of approximately 57 kb and 52 exons. Southern hybridization of genomic DNA and radiation hybrid mapping showed that murine tpr exists as a single-copy gene on chromosome 1; RNA blotting analyses and EST (expressed sequence tag) database mining revealed that its expression results in only one major mRNA in embryonic and most adult tissues. Accordingly, novel antibodies against the N- and C-terminus of Tpr identified the full-length protein as the major translation product in different somatic cell types; reinvestigation of Tpr localization by confocal microscopy corroborated a predominant localization at the nuclear pore complexes in these cells. Antibody specificity and reliability of Tpr localization was demonstrated by post-transcriptional tpr gene silencing using siRNAs that eliminated the Tpr signal at the nuclear periphery but did not affect intranuclear staining of Tpr-unrelated proteins. Finally, we defined several sequence and structural features that characterize Tpr polypeptides in different species and used these as a guideline to search whole-genome sequence databases for putative paralogs of Tpr in higher eukaryotes. This approach resulted in identification of the Tpr orthologs in Arabidopsis thaliana and Caenorhabditis elegans, but also in the realization that no further paralogs of Tpr exist in several metazoan model organisms or in humans. In summary, these results reveal Tpr to be a unique protein localized at the nuclear periphery of the somatic cell in mammals.