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.

Viruses in human cancers.

Viruses may contribute to the development of human tumors by different mechanisms: indirectly by inducing immunosuppression or by modifying the host cell genome without persistence of viral DNA; directly by inducing oncoproteins or by altering the expression of host cell proteins at the site of viral DNA integration. Human cancers associated with papillomavirus, hepatitis B virus, Epstein-Barr virus, and human T cell leukemia-lymphoma virus infections are responsible for approximately 15 percent of the worldwide cancer incidence. Cancer of the cervix and hepatocellular carcinoma account for about 80 percent of virus-linked cancers. Because experimental and epidemiologic data imply a causative role for viruses, particularly in cervical and liver cancer, viruses must be thought of as the second most important risk factor for cancer development in humans, exceeded only by tobacco consumption.

Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells.

Cultured cells derived from male patients with Burkitt's lymphoma and harboring herpes-type virus particles were lethally irradiated. These irradiated cells induced normal peripheral leukocytes of female infants to grow within 2 to 4 weeks after mixed cultivation. Cells of a line free of this agent failed to stimulate growth. If either type of cell was cultured separately, it did not survive under the experimental conditions. Herpes-type viral antigen and C-group chromosomal marker previously described in cultured Burkitt cells were found in all of the female cell cultures that were obtained.

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.

Seroepidemiologic studies of bovine papillomavirus infections.

Bovine and human sera were analyzed for the presence of antibodies against bovine papillomavirus types 1 and 2 (BPV 1 and 2) and human papillomavirus type 1 (HPV 1) in a solid-phase radioimmunoassay. Human sera did not react with BPV antigens, and bovine sera showed no evidence of antibodies against HPV 1. In contrast, 19% of all bovine sera tested reacted with BPV 1 and 2 antigens, and 35% of human sera revealed antibodies against HPV 1. No serologic evidence was obtained for heterologous infections of persons exposed preferentially to cattle (farmers, butchers, and patients with Q-fever).

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.

Biochemical approaches to detection of Epstein-Barr virus in human tumors.

The application of biochemical studies for the detection of Epstein-Barr virus (EBV)-DNA in human tumor cells is discussed. These studies resulted in the consistent demonstration of viral nucleic acid in African Burkitt's lymphoma biopsies and in epithelial tumor cells of nasopharyngeal carcinomas. The viral DNA resides within those cells regularly in multiple copies per cell. Besides these tumors our group detected significant concentrations of EBV-DNA in a German lymphoma patient revealing histological characteristics of Burkitt's lymphoma. Moreover, virus DNA was also found in a patient suffering from immunoblastic lymphadenopathy. More than 50 additional B-cell lymphomas and more than 40 biopsies from patients with Hodgkin's disease did not contain detectable amounts of EBV-DNA when tested by nucleic acid hybridization. A tentative scheme of EBV-induced pathogenesis is discussed.

Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers.

Infections with specific types of human papillomaviruses (HPV) have emerged as necessary but not sufficient factors for the development, at least, of the majority of cervical, vulvar, penile, and perianal cancers. Evidence has accumulated for their causal role in the induction of anogenital premalignant lesions. Genetic events underlying the mechanism of anogenital carcinogenesis have become increasingly understood. A host cell-mediated intracellular control down-regulating specific HPV genes (E6, E7) in replicating normal cells appears to be interrupted in cancer cells, probably due to structural modifications of the respective host cell genes acquired in the course of HPV DNA persistence. Since genital HPV infections are ubiquitous, cofactors which modify controlling host cell genes are likely to determine the different geographic rates of cervical cancer incidence.

Enhancement of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA amplification in a Simian virus 40-transformed Chinese hamster cell line by 3-aminobenzamide.

A Simian virus 40-transformed Chinese hamster cell line (CO 60) amplifies integrated viral DNA sequences as a response to treatment with a variety of carcinogens. To study a possible involvement of poly(ADP-ribose) synthesis, DNA amplification was induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), an alkylating carcinogen that strongly stimulates poly(ADP-ribose) synthesis. In the presence of 3-aminobenzamide (3AB) (2 mM), a competitive inhibitor of poly(ADP-ribose) polymerase, MNNG-induced amplification was increased two to six times the level induced by MNNG alone. Concomitantly, 3AB reduced cellular poly(ADP-ribose) levels and increased MNNG-induced cytotoxicity, as expected. The effect of 3AB on MNNG-induced amplification depended both on the concentration of 3AB and the duration of its presence after MNNG treatment. By contrast, 3-aminobenzoic acid, a noninhibitory structural analogue of 3AB, had no influence on amplification induced by MNNG. These data strongly suggest an involvement of poly(ADP-ribose) in the process of DNA amplification, as it is shown that inhibition of carcinogen-stimulated poly(ADP-ribose) synthesis by 3AB is correlated with an enhancement of inducible DNA amplification in this cell line.

Effect of indomethacin on Epstein-Barr virus early antigen induction.

The lymphoid cell line, Raji, was derived from a Burkitt's lymphoma and is readily inducible for Epstein-Barr virus (EBV) early antigen synthesis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Treatment of Raji and other EBV genome-positive cells with indomethacin caused a marked inhibition of early antigen induction by 12-O-tetradecanoylphorbol-13-acetate and other chemical inducers. However, this effect did not appear to be due to inhibition of prostaglandin synthesis since the concentration of indomethacin required to inhibit EBV-early antigen induction was 50- to 100-fold higher than that normally required for the inhibition of prostaglandin synthesis. In addition, no prostaglandin synthesis was detected in 12-O-tetradecanoylphorbol-13-acetate-treated Raji cells. EBV-early antigen induction by superinfection was resistant to inhibition by indomethacin and indicates that induction by chemical inducers and by super-infection follows different pathways. Indomethacin at the concentrations required to inhibit EBV-early antigen induction also was cytostatic, which indicates that the cell cycle phase may be an important factor in viral induction.

Growth-regulating functions of human papillomavirus early gene products in cervical cancer cells acting dominant over enhanced epidermal growth factor receptor expression.

Squamous cell carcinomas of the human anogenital tract are usually associated with infection of specific types of human papillomaviruses (HPV 16, 18, 31, 33, 35). The intracellular concentration of human papillomavirus early gene products E6 and E7 has been directly linked to the proliferative capacity of cervical cancer cells. Since the expression rate of epidermal growth factor receptor correlates to growth properties in squamous carcinoma cell lines, it has been presumed that human papillomavirus early genes influence cell growth via enhanced epidermal growth factor receptor expression. This hypothesis implies that growth regulation by epidermal growth factor receptor overexpression dominates over a growth-regulatory influence of human papillomavirus early gene products in squamous carcinoma cells. To test this hypothesis epidermal growth factor receptor expression was analyzed in various clones of the C4-1 cervical cancer cell line which, upon dexamethasone treatment, express either increased or decreased levels of human papillomavirus 18 early gene products. In C4-1 clones expressing reduced levels of viral E6/E7 gene products upon glucocorticoid treatment expression of epidermal growth factor receptor was the same as in those clones displaying increased levels of papillomavirus proteins under identical culture conditions. The growth rate of the cells correlated with the level of viral gene products rather than with the expression of epidermal growth factor receptor. These findings suggest that unregulated overexpression of epidermal growth factor receptor is not the dominant mechanism of growth control in papillomavirus-positive carcinoma cells. Other, yet unknown pathways associated with papillomavirus early genes are essentially involved in growth control mechanisms of human cervical cancer cells.

Potentiation of carcinogen-induced methotrexate resistance and dihydrofolate reductase gene amplification by inhibitors of poly(adenosine diphosphate-ribose) polymerase.

Poly(ADP-ribosyl)ation of nuclear proteins is an immediate response of most eukaryotic cells to DNA strand breaks, as induced by carcinogen treatment. DNA amplification, on the other hand, can be induced in cell culture systems by chemical or physical carcinogens, too, reaching peak levels a few days after induction treatment. We have previously shown that 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation, potentiates carcinogen-induced simian virus 40 DNA amplification in hamster cells which served as a short-term model system (Bürkle et al., Cancer Res., 47: 3632-3636, 1987). Here we report that those results can be extended to the development of methotrexate (MTX) resistance associated with dihydrofolate reductase (DHFR) gene amplification in a different hamster cell line. (a) Treatment with the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 3 days before selection with 350 nM MTX induced the MTX resistance frequency by 17- to 100-fold, as expected. Addition of 3-aminobenzamide (0.1 to 1 mM) before MNNG treatment further potentiated the frequency of MTX resistance by up to 5-fold in a dose-dependent manner, parallel to a potentiation of cytotoxicity. MTX resistance frequency was potentiated not only relative to the decrease in cell survival but also in absolute terms. The same potentiation occurred after cotreatment with benzamide (1 mM), another poly(ADP-ribosyl)ation inhibitor, under conditions which precluded direct drug interactions. Benzoic acid, a noninhibitory analogue, had no effect on the MNNG-induced MTX resistance frequency. (b) Neither 3-aminobenzamide, nor benzamide, nor benzoic acid at 1 mM, respectively, had any effect on the spontaneous frequency of MTX resistance. (c) Individual MTX-resistant colonies were expanded to determine their DHFR gene copy number. The relative frequency of DHFR gene amplification was similar (14% versus 22%) whether clones were derived from cultures induced with MNNG alone or MNNG in the presence of 1 mM 3-aminobenzamide. We conclude that poly(ADP-ribosyl)ation should act as a negative regulatory factor in the induction of DNA amplification, since inhibition of poly(ADP-ribose) polymerase potentiates both MNNG-induced simian virus 40 DNA amplification, as shown previously, and MNNG-induced MTX resistance associated with DHFR gene amplification, as shown in this paper.

Suppression of the malignant phenotype in somatic cell hybrids between Burkitt's lymphoma cells and Epstein-Barr virus-immortalized lymphoblastoid cells despite deregulated c-myc expression.

To approach the question whether the absence of specific cellular gene functions may be involved in Burkitt's lymphoma pathogenesis, somatic cell hybrids were established between a malignant Epstein-Barr virus (EBV) positive Burkitt's lymphoma cell line (BL 60) and a nonmalignant EBV-immortalized lymphoblastoid cell line (IARC 277) derived from the same individual. The hybrids revealed a near tetraploid karyotype including one copy of the 8q+ chromosome resulting from the Burkitt's lymphoma-specific translocation t(8;22) in addition to three apparently normal copies of chromosome 8. Although the hybrid cells exhibited the deregulated c-myc expression pattern of the parental Burkitt's lymphoma cell line with highly abundant transcripts originating from the 8q+ chromosome, their growth characteristics in tissue culture as well as in nude mice were identical to that of the parental nonmalignant lymphoblastoid cell line. These data indicate that, at least in the system described here, the malignant phenotype of Burkitt's lymphoma cells can be suppressed by introduction of an additional set of apparently normal chromosomes from the same individual and that EBV infection and c-myc deregulation may not be sufficient for maintenance of the malignant phenotype.

DNA amplification of adeno-associated virus as a response to cellular genotoxic stress.

We studied DNA amplification of helper virus-dependent parvoviruses [adeno-associated virus (AAV)] following genotoxic treatment of a number of mammalian cell lines from different species including primary, immortalized, and tumorigenic cells. All cell lines, either infected with AAV or transfected with parvoviral DNA, readily amplified AAV DNA in the absence of helper virus following treatment of cells with a wide variety of genotoxic agents like chemical carcinogens, UV, heat shock, and metabolic inhibitors of DNA replication or protein synthesis. In addition, we show that in the SV40-transformed Chinese hamster cell lines CO60 and CO631 carcinogen-induced AAV DNA amplification may result in a complete AAV replication cycle giving rise to infectious AAV progeny. Our results demonstrate that AAV DNA amplification induced by genotoxic agents is completely independent of the presence of viral helper functions. Because its induction is not restricted to a specific cell type or to a malignant phenotype, AAV DNA amplification may represent a marker for cellular genotoxic stress response.

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.

Integration of human papillomavirus type 6a DNA in a tonsillar carcinoma: chromosomal localization and nucleotide sequence of the genomic target region.

Human papillomavirus type 6a (HPV 6a) DNA was detected in a tonsillar carcinoma both as integrated and episomal molecules, and one viral-cellular junction was molecularly cloned (Bercovich et al., J. Gen Virol., 72: 2569-2572, 1991). The cellular sequence was used as a probe for the isolation of a cosmid from a normal human genomic DNA library. A 2.7-kilobase subclone including the integration site was sequenced. It was shown to contain sequences with similarities to the E2 and L2 regions of human papillomaviruses, a 5' truncated long interspersed repeated DNA element type 1 retrotransposon, and a fragment of an O-repeat element. The chromosomal localization of the integration site was determined to be at region 24 of the long arm of chromosome 10 (10q24). This is the region where the fragile site is located in which HPV 18 DNA is integrated in the cell line FEP18-5. In addition it contains the site of breakpoints affecting protooncogenes Hox11 and Lyt10. Other genes related to cell division and DNA repair have also been mapped to this chromosomal band. Analysis of genomic DNA of cell lines and patients using 10q24-derived probes is presented. The integration of human papillomavirus type 6 DNA into chromosome 10q24 may have disrupted a cellular gene critical for normal cell growth, which further analysis should help to identify.

An amplification unit in human melanoma cells showing partial homology with sequences of human papillomavirus type 9 and with nuclear antigen 1 of the Epstein-Barr virus.

By partial homology with the DNA of human papillomavirus type 9 a cellular amplification unit was detected which is amplified in melanoma cells but not in Epstein-Barr virus-transformed B cells of two melanoma patients. A 2.4-kilobase EcoRI fragment of this amplification unit was cloned and designated mel/HPV9. At the chromosomal level we detected mel/HPV9 in homogeneously staining regions or in abnormally banded regions containing different marker chromosomes of both melanoma cell lines. DNA sequence analysis of a part of mel/HPV 9 revealed homology with the third internal repeat array of Epstein-Barr virus nuclear antigen 1.

Association of the human type C retrovirus with a subset of adult T-cell cancers.

To determine whether the human T-cell lymphoma-leukemia virus (HTLV) is associated with particular cancers, patient sera were surveyed for HTLV-specific antibodies. An association was seen with aggressive cancers of mature T-cells, specifically Japanese adult T-cell leukemia (ATL) and T-cell lymphosarcoma cell leukemia (TLCL), a similar cancer of Caribbean blacks. Ninety to 100% of these patients possessed HTLV-specific antibody. Forty-seven and 20% of relatives of ATL and TLCL patients, respectively, and 12 and 4% of healthy donors from ATL and TLCL endemic areas were also antibody positive. Visceral organ involvement, hypercalcemia, and skin manifestation, features of ATL and TLCL, were often seen in other antibody-positive patients. Childhood cancers, most cutaneous T-cell and all non-T-cell leukemias and lymphomas, myeloid leukemias, Hodgkin's disease, and solid tumors were not associated with HTLV. Healthy United States donors and European patients with non-malignant diseases were antibody negative. HTLV is thus associated with a subtype of adult T-cell leukemia-lymphoma, clustered in viral endemic areas, with apparent racial and geographic predilection.

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.