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.

Intragenomic rearrangement in TT viruses: a possible role in the pathogenesis of disease.

A role for the ubiquitous Torque teno (TT) viruses in the pathogenesis of disease has not been resolved. In vivo and in vitro intragenomic rearrangement of TT virus genomes has been demonstrated. Replication in cell culture of a subviral molecule (411 bp) occurs through oligomerisation of RNA transcripts. Although the functions of the respective TT viral genes, as well as the newly formed genes in the rearranged subviral molecules, are largely unknown, certain similarities to genes of plant viruses of the family Geminiviridae will be described. A degree of similarity to certain cellular genes poses the question as to a role of molecular mimicry in the pathogenesis of autoimmune disease and diabetes.

[Coincidental squamous cell cancers of the esophagus, head, and neck: risk and screening]. / "Field cancerization" im oberen Aerodigestivtrakt: Uberwachungsempfehlungen für Risikopersonen.

The term "field cancerization" was coined by Slaughter in1953 when describing multifocal synchronous and metachronous carcinogenesis in the upper aerodigestive system. Patients suffering from head and neck cancer (HNC) have or develop a second esophageal squamous cell cancer (ESCC) or bronchial cancer (BC) in 5-14% of cases. When a second esophageal cancer occurs in a patient with HNC, the prognosis is generally determined by the ESCC, and, unfortunately, it is poor. Screening and surveillance by Lugol chromoesophagoscopy enable early detection and curative treatment of second esophageal neoplasias. Surveillance appears to result in a survival benefit for HNC patients. Vice versa, patients with ESCC or BC have a risk of about 10% for developing HNC. Periodic pharyngolaryngoscopy is recommended for curatively treated ESCC or BC patients. Patients with field cancerization should be surveilled by a multidisciplinary approach.

TAp63alpha indirectly regulates a cutaneous HPV promoter through complex formation with Jun family members.

The p63alpha isoforms of the p53 family have been demonstrated to play a crucial role in the development and differentiation of the skin. We show that expression of the TAp63alpha isoform leads to an upregulation of the cutaneous papillomavirus HPV 20 promoter, which is increased at least three-fold when c-Jun is co-expressed, in contrast to a minimal increase in activity in the presence of c-Jun alone. Co-expression of TAp63alpha with JunB or JunD, respectively, and in combination, leads to a reduction in the viral promoter activation measured by the expression of TAp63alpha alone. JunB and JunD also inhibits the additive effect exerted on the TAp63alpha activation by c-Jun. Co-immunoprecipitation assays demonstrate a complex formation of c-Jun, JunB and JunD with TAp63alpha through the SAM domain mediating protein-protein interactions, which is characteristic for p63alpha. Co-expression of p53 mutant R248W not only downregulates the differential modulation of the viral promoter by TAp63alpha alone and in the presence of the Jun family members, but leads to a reduction in the protein levels of the overexpressed c-Jun, JunB, JunD, as well as TAp63alpha. This model system provides insight into yet unknown pathways through which TAp63alpha and Jun may cooperate in the pathogenesis of HPV associated cutaneous lesions.

Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients.

BACKGROUND: Nonmelanoma carcinomas of the skin represent the most frequent cancers among the Caucasian population worldwide. They occur with high frequency in renal allograft recipient patients after prolonged immunosuppression. PURPOSE: We analyzed tumors obtained from both immunosuppressed and nonimmunosuppressed patients for human papillomavirus (HPV) DNA. METHODS: Twenty-nine specimens of nonmelanoma carcinomas of the skin were obtained from 19 renal allograft recipient patients; these included 20 specimens of squamous cell carcinoma (SCC) from 11 patients, five specimens of basal cell carcinoma (BCC) from four patients, and four specimens of carcinoma in situ (CIS) from four patients. Forty-one specimens of nonmelanoma carcinomas of the skin were obtained from 32 nonimmunosuppressed patients; these included 26 SCC specimens from 19 patients, 11 BCC specimens from nine patients, and four keratoacanthoma (benign epithelial tumor) specimens from four patients. A polymerase chain reaction method involving use of degenerate oligonucleotide primers, in which the conserved region of the open reading frame of the HPV L1 (major capsid protein) gene is amplified, was used to amplify total cellular DNA purified from individual tumors. The DNA of each specimen was subjected to 16 different amplification reactions; different primer combinations were used in order to increase the sensitivity and specificity of HPV detection. Resulting products were probed with a radioactively labeled, degenerate oligonucleotide. HPV-specific DNA was either sequenced directly after elution from the gel or amplified with semi-nested, degenerate primers, after which the products were cloned and sequenced. Sequences were compared with all known papillomavirus sequences. RESULTS: Thirteen (65%) of the 20 SCC specimens and three of the five BCC specimens from immunosuppressed (renal allograft recipient) patients contained identifiable HPV-related sequences, among them 13 putative novel HPV genomes. In addition, all other malignant tumor specimens from this patient group revealed faint signals upon amplification and hybridization; the origin of these signals has not been identified in the present study. In nonimmunosuppressed patients, eight (31%) of 26 SCC specimens and four (36%) of 11 BCC specimens contained sequences of HPV types. Two putative novel HPV sequences could be identified in this group. Faint signals of yet undetermined origin were observed in eight of the SCC specimens and in two of the BCC specimens. Two of four keratoacanthoma specimens contained sequences of known HPV type. (Keratoacanthoma is a nonmalignant lesion for which the natural history has not been defined.) The spectrum of HPV types in both groups of patients differed substantially. CONCLUSIONS: These data point to the frequent presence of HPV sequences in SCCs and BCCs of the skin. The etiologic relationship of these infections to the respective malignant tumors remains to be evaluated. IMPLICATIONS: The presence of HPV DNA in a large percentage of specimens of nonmelanoma carcinomas of the skin from immunosuppressed patients, as well as from nonimmmunosuppressed patients, renders a papillomavirus infection as a possible factor in the etiology of this disease.

Isolation of a new type of human papillomavirus (HPV52b) with a transforming activity from cervical cancer tissue.

Substantial evidence has implicated human papillomaviruses (HPVs) as etiological agents of human cervical cancer. We detected and cloned an unidentified HPV genome in cellular DNA extracted from a surgical specimen of a Japanese patient with cervical cancer. Hybridization studies suggested that this HPV was the most homologous to HPV33 among more than 51 types of HPV ever identified. A recently identified new HPV, isolated by W. Lancaster and designated HPV52, is also most homologous to HPV33 (W. Lancaster et al., unpublished data). Our HPV was found to be homologous but not identical to HPV52 and hence was designated HPV52b. By introducing it into NIH3T3 cells, we found that HPV52b DNA had growth-stimulating activity as HPV16 DNA. This newly identified HPV52b DNA was present as episomes in cervical cancer cells of two out of two specimens so far examined and the integrated copies were not detected. HPV52b was present in three out of 15 (20%) specimens in Japan. In addition, the presence of three additional unidentified HPV sequences homologous to HPV52b was detected in three other specimens. The results suggest that this group of HPV may be prevalent and involved in carcinogenesis of cervical cancer in Japan.

Specific types of human papillomavirus found in benign proliferations and carcinomas of the skin in immunosuppressed patients.

A total of 118 biopsies from skin lesions of 46 renal allograft patients was analyzed for human papillomavirus (HPV) DNA by polymerase chain reaction with degenerate primers and also partially by subsequent sequencing of the amplified fragment. Sixty-two % of the benign proliferations (31 of 50) contained DNA of known HPV types as well as HPV sequences related to a number of epidermodysplasia verruciformis-associated HPV types. HPV DNA sequences were found in 14 (56%) of 25 biopsies from squamous cell and basal cell carcinomas. One squamous cell carcinoma contained HPV 41 DNA. A novel 640-base pair fragment sharing homology with HPV 29 (82.7%) was found in 15% (3 of 20) of squamous cell carcinomas, in 9.4% (3 of 32) of dysplastic warts and in 8.5% (4 of 47) common warts. The remaining positive carcinoma biopsies contained HPV-related DNA in such a low copy number that additional analysis is required. The identification of new HPV types in skin cancers of immunosuppressed patients (other than epidermodysplasia verruciformis patients) further expands the spectrum of HPV-linked human malignancies and permits new approaches to study the pathogenesis of skin cancers.

Human papillomavirus E7 proteins stimulate proliferation independently of their ability to associate with retinoblastoma protein.

Studies on human papillomavirus type 16 have demonstrated that the product of the early gene, E7, plays a key role in the immortalization and malignant transformation of the host cell. Several of the biological activities of HPV16 E7 are mediated by inactivation of the members of the pocket protein family, pRb, p107 and p130. In this study, we have characterized the in vitro properties of five E7 proteins from benign and malignant HPV types (10, 32, 48, 54, 77). We show that these E7 proteins associate with pRb and p107 with different efficiencies. All E7s increased the proliferative rate of immortalized rodent fibroblasts cultured in 10% calf serum containing medium. This property is completely independent of their ability to associate with the pocket proteins. Furthermore, all E7s, except HPV10 E7, stimulate G1/S progression and activated the cyclin E and cyclin A promoter in the absence of growth factors. This activity also does not correlate with the E7-efficiency of binding the pocket proteins. Together these data provide evidence that different E7s alter the regulation of the cell cycle by diverse mechanism(s). Finally, this comparative analysis of the different E7 proteins demonstrates that the oncogenicity of a HPV type is not determined by the ability of E7 to associate with the pocket proteins.

Human papillomaviruses are commonly found in normal skin of immunocompetent hosts.

We have previously demonstrated, by the combined application of two degenerate polymerase chain reaction primer sets, the presence of human papillomavirus (HPV) DNA in 91% of cutaneous squamous cell cancers from renal allograft recipients, with multiple types being present in one-third of these tumors. Five HPV types--HPV 20, HPV 23, HPV 38, DL40, and DL267--accounted for 73% of positive results. These HPV types are all related to the epidermodysplasia verruciformis group, and HPV 38 was originally isolated from a melanoma. The aims of this study were to determine: (i) whether HPV DNA could readily be demonstrated in skin tumors, as well as in perilesional skin, of immunocompetent patients using two polymerase chain reaction primer sets; (ii) the prevalence of infections in normal skin; and (iii) the prevalence of HPV 38 or HPV 38 related viruses in melanoma. The HPV types detected in lesions from renal allograft recipient were present not only in the perilesional skin and tumors of immunocompetent patients, but also in 35% of normal skin biopsies. HPV DNA was present in 13% of the melanoma samples, but none harbored HPV 38 DNA. We identified four putatively new HPV types. Infections with different types of human papillomavirus are widespread and often occur in clinically normal skin. In vitro studies are required to determine the specific molecular mechanisms by which these HPV types may be involved in the etiology of nonmelanoma skin cancer.

Butcher's wart virus (HPV 7) infections in non-butchers.

The analysis of a total of 654 benign and malignant lesions of the skin, genitalia, lungs and bronchi, intestine, kidneys, bladder, mammae, and of the head and neck region, resulted in the identification of human papilloma virus 7 (HPV 7) infections in 3 individuals. One of these was an ordinary "butcher's wart," whereas the other 2 patients have never been involved in meat-handling or farming. One of the latter revealed extensive verrucosis of hands, feet, axilla, neck, and face, persisting for about 27 years, with new lesions arising in the neck region. Particularly the new lesions showed filiform morphology. The second patient has, for a period of more than 2 years, been showing filiform papillomas in the face which recurred after surgical removal. These 2 patients appear to represent the first cases of HPV 7 infections in non-butchers or non-meathandlers.

Transforming activities of human papillomavirus type 59 E5, E6 and E7 open reading frames in mouse C127 cells.

The DNA sequence from a human papillomavirus type 59 (HPV 59) has been recently determined. The HPV 59 genome consists of 7896 nucleotides (nt). A comparative analysis of this sequence with the sequences of other HPVs revealed the closest homology to HPV 18 (71%). To test the transforming activities of HPV 59 DNA and its gene products, several plasmids expressing HPV 59 open reading frames (ORF) were constructed. The E5, E6, and E7 ORFs of HPV 59 were inserted into pRc/CMV vector containing a promoter of cytomegalovirus to test the transforming activities of these ORFs. When these DNAs were transfected into mouse C127 cells, all three ORFs were independently able to transform C127 cells in the presence of G418, although the full length HPV 59 DNAs failed to induce the focus-formation. The E7 ORF showed the strongest transforming activity and the E5 ORF exhibited the weakest transforming activity. Cell lines transformed by E5, E6, and E7 ORFs were established and they grew anchorage-independently. The presence of HPV 59 ORF DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in HPV 59 ORFs-transformed cell lines.

Nucleotide sequence of human papillomavirus (HPV) type 41: an unusual HPV type without a typical E2 binding site consensus sequence.

The complete nucleotide sequence of human papillomavirus type 41 (HPV-41) has been determined. HPV-41 was originally isolated from a facial wart, but its DNA has subsequently been detected in some skin carcinomas and premalignant keratoses (Grimmel et al., Int. J. Cancer, 1988, 41, 5-9; de Villiers, Grimmel and Neumann, unpublished results). The analysis of the cloned HPV-41 nucleic acid reveals that its genome organisation is characteristic as for other papillomavirus types. Yet, the analysis indicates at the same time that this virus is most distantly related to all other types of human-pathogenic papillomaviruses sequenced thus far and appears to identify HPV-41 as the first member of a new subgroup of HPV. The overall nucleotide homology to other sequenced HPV types is below 50%. The closest other HPV type is represented by HPV-18, sharing 49% identical nucleotides. The typical E2 binding sequence ACCN6GGT, found in all papillomaviruses analyzed to date, does not occur in the URR of the HPV-41 genome. Modified E2 binding sequences, as described for BPV 1 (Li et al., Genes Dev. 1989, 3, 510-526), are located in the domain proximal to the E6 ORF. These are ACCN6GTT, AACN6GGT and the two perfect palindromic sequences AACGAATTCGTT.

A comparative sequence analysis of two human papillomavirus (HPV) types 2a and 57.

HPV 2a is commonly associated with verrucae vulgares, whereas HPV 57 was detected in mucosal lesions of the maxillary sinus and the genital tract, as well as in cutaneous lesions. The complete DNA sequences of HPV 2a and HPV 57 were determined. The HPV 2a genome consists of 7860 base pairs and the HPV 57 genome contains 7861 base pairs. On the nucleotide level an 83% homology between the two sequences could be ascertained. Compared to other HPVs they have a high G/C-content (HPV 2a: 48.8%, HPV 57: 50.1%). The genomic organization of both viruses complies with that of other sequenced HPVs. Significant sequence divergence between the HPV 2a and HPV 57 genomes was found in the long control region (LCR), as well as in the early-late-region (ELR). The latter varies in size between the cutaneous (72 to 103 nucleotides) and the mucosal HPVs (252 to 584 nucleotides). According to the sizes of the ELRs of HPV 2a (377 nucleotides) and HPV 57 (478 nucleotides), as well as DNA sequence comparisons, these two viruses could be grouped with the so-called mucosal HPVs. In a search for possible tissue-specific elements, a common amino acid motif, thr-thr/asp-pro-ala-ile/valile/leu was found in the L2 of all mucosal HPVs, as well as in HPV 2a and 57. The L2 of the cutaneous types contain the motif val-ser/thr-arg-thr-gln-tyr.

Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59.

Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149-7493), central (nt 7493-7742), and proximal (nt 7742-7748). In particular, the 250 bp (nt 7493-7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence.

Characterization of a putative new HPV genomic sequence from a cervical lesion using L1 consensus primers and restriction fragment length polymorphism.

Various methods have been proposed for HPV detection and typing. Prevalence and distribution among types have varied depending upon the methods used and the populations studied. We have applied the polymerase chain reaction (PCR) followed by a Restriction Fragment Length Polymorphism (RFLP) analysis using the MY09/MY11 primers for detection of HPV in cervicovaginal lavages obtained from 323 patients who were referred to our Clinical Department either for genital complaints or an abnormal PAP smear. We assessed (i) the prevalence of HPV and (ii) the reliability of RFLP-typing. For the latter, 35 PCR-HPV products were sequenced. HPV-DNA was detected in 40/197 (20.3%) patients with normal cytology 86/111 (77.5%) with LSIL and 11/15 (73.3%) with HSIL. HPV-16 was the most common type detected in normal cervical cytology samples (10/40, 25%), whereas HPV 16 and 18 were detected in 36/97 (37.1%) of the LSIL and HSIL patients, evidencing the presence of these high-risk HPV types not only in malignant conditions. Results obtained after partial nucleotide sequencing confirmed the results obtained by RFLP analysis. In this study, a putative new HPV fragment (GA6053) was identified. Its closest homology to other known HPV types is 73.8% to HPV-62, 73.0% to HPV-61 and 67.7% to HPV-18. The use of degenerate primers, in conjunction with RFLP, proved to be a reliable method for HPV detection and typing.

Multifocal squamous neoplasia of the female genital tract: significance of human papillomavirus infection of the vagina after hysterectomy.

Six hundred sixteen women with a history of hysterectomy were examined for the presence of human papillomavirus deoxyribonucleic acid (DNA) in vaginal smears using filter in situ hybridization. One hundred twenty patients had had hysterectomy for cervical intraepithelial neoplasia and invasive neoplasia, 54 for noncervical anogenital cancer, and 442 for benign uterine disorders. Human papillomavirus DNA was detected in 18% of all vaginal smears. A history of cervical neoplasia was associated with a significantly higher human papillomavirus infection rate, compared with patients with benign disease (33 versus 14%). Human papillomaviruses 16 and 18 were the most common types detected in patients with a history of cervical carcinoma, whereas the majority of patients with benign uterine disease were infected with human papillomavirus 6/11. Colposcopy identified lesions in more than half of the patients; these appeared in more than 90% as "condylomatous vaginitis." In 5% of the human papillomavirus-positive patients, human papillomavirus 16-positive vaginal intraepithelial neoplasia could be diagnosed. Increased risk of vaginal intraepithelial neoplasia after hysterectomy is associated with vaginal human papillomavirus infection. Virologic results explain the higher risk for vaginal neoplasia in patients with a history of cervical neoplasia.

Colposcopy is superior to cytology for the detection of early genital human papillomavirus infection.

Of 2232 women with no cytologic evidence of intraepithelial neoplasia, 250 (11.2%) were positive for human papillomavirus deoxyribonucleic acid (DNA) by filter in situ hybridization. In 150 of those human papillomavirus-positive patients, an adequate colposcopic examination of the cervix was possible; human papillomavirus infection was diagnosed in 104 women (70%). Cervical cytology showed evidence of human papillomavirus infection in only 23 patients (15%). The following colposcopic features were most common: acetowhite epithelium (29%), punctuation (18%), acetowhite spikes (17%), and mosaicism (9%). Colposcopy was essentially normal in 27%. In 64 hysterectomized patients, vaginal colposcopy showed evidence of human papillomavirus infection in 38 women (59%). Vaginal cytology showed signs of human papillomavirus infection in only 9% (N = 6). Acetowhite spikes were seen in 52%, acetowhite epithelium in 5%, punctuation in 3%, and normal findings in 40%. Histologic examination of 25 biopsy specimens (cervical, N = 15; vaginal, N = 10) showed mainly a lack of glycogenation, acanthosis, and elongation of rete pegs. Koilocytosis and dyskeratosis were seen only in a few cases as rare foci, hence the negative cytology. We conclude that colposcopy is far more sensitive than cytology for the detection of cervical and vaginal human papillomavirus infection.

Failure to detect human papillomavirus sequences at the 3p21 rearrangement site in pleomorphic adenomas.

Eighteen salivary gland pleomorphic adenomas, including nine with a t(3;8)(p21;q12) as the sole chromosome abnormality, were tested for the presence of human papillomavirus (HPV) DNA and for rearrangements of the cellular flanking regions of a HPV 16 integration site mapping to 3p21, as previously described in a cervical carcinoma. In none of the tumors could evidence for either HPV DNA or related sequences, or a rearranged allele of the integration site, be found.

Abnormal function of CD4+ helper/inducer T lymphocytes in a patient with widespread human papillomavirus type 3-related infection.

Human papillomavirus-induced infections may be associated with cellular immunodeficiency. However, very little is known about the dysfunctional interactions among T lymphocytes, B lymphocytes, and antigen-presenting cells. A 30-year-old heterosexual man with a 10-year history of persistent multiple refractory flat wart lesions containing human papillomavirus type 3-related DNA sequence was studied. The patient had a severe depletion of CD4+ T lymphocytes and a compensatory increase in the number of CD8+ T lymphocytes. Impaired T-lymphocyte response to various stimuli was found. Depletion of the increased number of CD8+ T lymphocytes, which suppressed immunoglobulin production in vitro, did not restore the impaired T-lymphocyte response. Immobilized anti-CD3 beads that stimulate the T lymphocyte antigen complex in the absence of antigen-presenting cells indicated a T-lymphocyte defect, rather than a decreased antigen-presenting cell function. Thus, the pronounced cellular immunodeficiency was due to abnormal function of the CD4+ helper/inducer T lymphocytes.