TT viruses: oncogenic or tumor-suppressive properties?

Torque teno (TT) viruses have been more frequently reported in malignant biopsies when compared to normal control tissue. The possible contribution of TT virus infection to human carcinogenesis or the potential oncolytic functions of these virus infections are being discussed based on available experimental evidence. The data could suggest an involvement of TT virus infections as an indirect carcinogen by modulating T cell immune responses. Significant oncolytic functions, potentially mediated by the inhibition of nuclear factor (NF)-kappaB transcription factor or by apoptin-like gene activities, are emerging to be less likely.

Papillomaviruses--to vaccination and beyond.

High risk human papillomavirus (HPV) types 16 and 18 DNAs were initially identified in 1983-1984. Subsequently the DNA of several other high risk HPV types has been identified. HPV 16 is present in more than 50% of cervical cancer biopsies, and HPV 18 is close to 20%. Some geographic variations exist in the prevalence of HPV high risk types: e.g. HPV 45 is more frequently observed in equatorial Africa, whereas types 58 and 52 have been more often found in East Asia. Molecular as well as epidemiological studies demonstrate that high risk HPV are indeed the causative agents for cervical cancer, they are also involved in other anogenital cancers, and in 25-30% of oropharyngeal carcinomas. Some of the mechanistic aspects are discussed in this review.

Differential sensitivity of human papillomavirus type 16(+) and type 18(+) cervical carcinoma cells to CD95-mediated apoptosis.

When cervical carcinoma cells were monitored for apoptotic signals, HPV18(+) lines were found to be highly sensitive to agonistic CD95 antibodies or recombinant CD95 ligands after co-exposure with CHX (CD95(S)). In contrast, HPV16(+) cervical carcinoma cells and HPV16-immortalized non-malignant human keratinocytes were CD95-resistant (CD95(R)) under equivalent conditions. Somatic cell hybridization between CD95(S) and CD95(R) cervical carcinoma cell lines revealed that CD95 sensitivity was a dominant trait, which could be correlated with abundant c-Myc and low Bcl-X(L) expression. Although CD95(R) cervical carcinoma cells expressed even higher levels of p53 and CD95 receptor at the surface, resistance could be attributed to the inability to form a functional DISC, necessary for successful transmission of the apoptogenic response. These data indicate that resistance to apoptotic stimuli represents an important immunological escape mechanism during virus-induced carcinogenesis.

Inhibitors of histone deacetylase arrest cell cycle and induce apoptosis in cervical carcinoma cells circumventing human papillomavirus oncogene expression.

Histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A arrest human papillomavirus (HPV)-positive carcinoma cells in G1 to S transition of the cell cycle, which is paralleled by an up-regulation of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1 and p27KIP1 as well as the complete loss of cdk2 activity. Although HPV expression was hitherto thought to be required to maintain a proliferative phenotype of these cells, cdk2 suppression is achieved even in the presence of ongoing viral transcription. While CKIs normally cannot exert their cdk2-inhibitory function in the presence of the viral oncoprotein E7, co-immunoprecipitation experiments revealed that E7 binding is prevented. Increase of p27KIP1 correlates with down-regulation of p45SKP2, a component of the ubiquitin-protein ligase SCF(SKP2) controlling the half-life of regulatory proteins during the cell cycle. HDAC inhibition also triggered an E7-dependent degradation of pRb, while the levels of E2F remained unaffected. The presence of free intracellular E2F and the concomitant up-regulation of CKIs during G1 arrest results in a 'conflicting growth situation', which finally renders the cells to undergo apoptosis. These data provide novel molecular insights into how the transforming potential of HPV can be bypassed and open new therapeutical perspectives for the treatment of cervical cancer.

Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference.

The 18th international papillomavirus conference took place in Barcelona, Spain in July 2000. The HPV clinical workshop was jointly organised with the annual meeting of the Spanish Association of Cervical Pathology and Colposcopy. The conference included 615 abstracts describing ongoing research in epidemiology, diagnosis/screening, treatment/prognosis, immunology/human immunodeficiency virus, vaccine development/trials, transformation/progression, replication, transcription/translation, viral protein functions, and viral and host interactions. This leader summarises the highlights presented at the conference (the full text of the abstracts and lectures can be found at www.hpv2000.com). Relevant material in Spanish can be found at www.aepcc.org.

Proliferation-inducing viruses in non-permissive systems as possible causes of human cancers.

Animal viruses, some of which are probably unable to replicate in human cells, could be transmitted to people where they may be linked to tumours currently not attributed to viruses. Several human virus types have oncogenic potential in animals. A potential risk for acquiring such infections by handling and preparation of animal products was analysed against the background of available epidemiological reports. Human tumours should be systematically assessed for proliferation-inducing viruses in non-permissive systems.

Differential transcriptional regulation of the monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic and non-tumorigenic HPV 18 positive cells: the role of the chromatin structure and AP-1 composition.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Genetic complementation to non-tumorigenicity in cervical-carcinoma cells correlates with alterations in AP-1 composition.

The transcription factor AP-1 represents a central key element in the expression of human pathogenic papillomaviruses (HPV). We here propose a novel role for AP-1 as an essential component of an intracellular surveillance mechanism negatively controlling the proliferation of HPV-positive cells under in vivo conditions. The dissection of AP-1 composition in cervical-carcinoma cells revealed an inverse relationship between the Fos-related antigen Fra-1 and the tumorigenic phenotype. Cervical-carcinoma cell lines were either negative or expressed only low amounts of Fra-1 (jointly with c-Fos) within their AP-1 complexes. Somatic-cell hybridization technique was used to fuse different HPV-positive malignant cell lines. This resulted either in tumorigenic hybrids or in cells in which the malignant phenotype of the parental fusion partners was completely suppressed. The monitoring of AP-1 composition in electrophoretic mobility super-shift assays showed that the amount of Fra-1 was substantially increased within the AP-1 complex of non-malignant cells. In contrast, Fra-1 was even diminished in malignant hybrids, while c-Fos remained expressed. This correlation suggests that the concentration of Fra-1 within the AP-1 transcription complex might be an important marker for predicting the in vivo growth properties of HPV-positive cells.

Papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis.

During the past 20 years, several types of human papillomaviruses (HPVs) have been identified that cause specific types of cancers. The etiology of cancer of the cervix has been linked to several types of HPV, with a high preponderance of HPV16. The role of these virus infections has been established 1) by the regular presence of HPV DNA in the respective tumor biopsy specimens, 2) by the demonstration of viral oncogene expression (E6 and E7) in tumor material, 3) by the identification of transforming properties of these genes, 4) by the requirement for E6 and E7 expression for maintaining the malignant phenotype of cervical carcinoma cell lines, 5) by the interaction of viral oncoproteins with growth-regulating host-cell proteins, and 6) by epidemiologic studies pointing to these HPV infections as the major risk factor for cervical cancer development. In addition to cancer of the cervix, a major proportion of anal, perianal, vulvar, and penile cancers appears to be linked to the same HPV infections. In addition, close to 20% of oropharyngeal cancers contain DNA from the same types of HPV. Recent evidence also points to a possible role of other HPV infections in squamous cell carcinomas of the skin. This review covers recent developments in understanding molecular mechanisms of HPV carcinogenesis, mainly discussing functions of viral oncoproteins and the regulation of viral oncogenes by host-cell factors. Modifications in host-cell genes, most likely engaged in the control of HPV gene expression in proliferating cells, emerge as important events in HPV-mediated carcinogenesis.

Papillomaviruses in human cancers.

Papillomaviruses have proved to be the most complex group of human pathogenic viruses. Eighty-five genotypes have been fully characterized; approximately 120 additional isolates represent only partially characterized putative novel genotypes. Specific types, most notably human papillomavirus (HPV) types 16, 18, and a few others, have been shown to cause the majority of cervical cancers and their high-grade precursor lesions. The viral oncogenes E6 and E7 are required for the initiation and maintenance of the malignant phenotype in HPV-positive cancers. Proteins coded by these genes are multifunctional and interfere with important cell cycle regulatory proteins. Expression of viral oncogenes is tightly controlled in nondifferentiated keratinocytes by at least two signaling cascades, one operative at the functional level, the other at the transcriptional level. The latter has been partially characterized. Papillomaviruses are also suspected of playing a role in a subset of oropharyngeal cancers, in squamous cell cancers of the skin, and possibly also in esophageal cancers. Clinical trials are being conducted to test the preventive and therapeutic efficacy of HPV vaccines, directed particularly against HPV 16 and 18. If proven to be effective, their global application should have a measurable effect on the worldwide incidence of cancer.

Conversion of HPV 18 positive non-tumorigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-alpha mediated repression of viral transcription and modification of the AP-1 transcription complex.

AP-1 represents a transcription factor, which plays a pivotal role in initiating and maintaining the expression of human papillomavirus (HPV) oncoproteins E6 and E7 during HPV-linked carcinogenesis of the uterine cervix. AP-1 stands as a synonym for different proteins such as c-Jun, JunB, JunD, c-Fos, FosB as well as the Fos-related antigens Fra-1 and Fra-2, which can either homo- or heterodimerize to build up a functional transcription complex. AP-1 is mainly considered as a positive regulator, which binds to cognate DNA sequences within the viral upstream regulatory region. By using non-tumorigenic HeLa-fibroblast hybrids ('444'), their tumorigenic segregants ('CGL3') as well as HPV 18 positive HeLa cells as a experimental model system, evidence is provided that AP-1 composition differs considerably between these cell lines. In nuclear extracts obtained from non-tumorigenic cells, Jun-family members (in the order c-Jun>JunD>JunB) were mainly heterodimerized with Fra-1, a protein, known to be involved in the abrogation of AP-1 activity under certain experimental conditions. In contrast, Fra-1 concentration is low in extracts from tumorigenic cells. Conversely, c-Fos, the canonical dimerization partner of Jun proteins is expressed in substantial quantity in HeLa- and 'CGL3' cells, but it is completely absent in AP-1 complexes from non-tumorigenic '444' cells. Ectopical expression of c-fos under a heterologous promoter in '444'-cells induces tumorigenicity and a change of the Jun/Fra-1 ratio towards a constellation initially detected in 'CGL3'-and HeLa cells. Furthermore, conversion to tumorigenicity is accompanied with a resistance against TNF-alpha, a cytokine, capable to selectively suppress HPV 18 transcription in formerly non-malignant cells. These data propose a novel role for AP-1 as an essential component of an inter- and intracellular surveillance mechanism negatively controlling HPV transcription in non-tumorigenic cells.

Human adeno-associated virus type 5 is only distantly related to other known primate helper-dependent parvoviruses.

We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.

Immortalization of human cells and their malignant conversion by high risk human papillomavirus genotypes.

Papillomaviruses cause certain common human cancers, most notably carcinoma of the cervix. The viral oncoproteins E6 and E7 are essential components in malignant conversion, although, in spite of being necessary, they are not sufficient for the development of the malignant phenotype. High risk HPV oncogenes fulfill dual functions in genome-harboring cells: their derived oncoproteins stimulate cell growth by pleiotropic effects. At the same time they act as progression factors by inducing mutations in host cell DNA and aneuploidy. The mechanism underlying the process towards malignant conversion, usually covering a long latency period between primary infection and cancer emergence, is presently not fully understood. It emerges, however, that cancer development depends on the interruption of at least two signalling cascades (labeled as CIF I and CIF II) that interfere with the function of viral oncoproteins (CIF I) and with the transcription of viral oncogenes (CIF II). Further modifications of host cell genes most likely mediate the escape from immune surveillance mechanisms of the host and the development of metastases.