Significant association between human osteosarcoma and simian virus 40.

BACKGROUND: Simian virus 40 (SV40) has been considered to be an oncogenic viral agent in the development of osteosarcoma (OS), which to the authors' knowledge continues to be of unknown etiology. METHODS: In the current study, serum samples from patients with OS were investigated with an indirect enzyme-linked immunoadsorbent assay (ELISA) to test for the presence of immunoglobulin G antibodies, which react with SV40 antigens. In ELISA, SV40 antigens were represented by 2 synthetic polypeptides that mimic epitopes of the viral capsid proteins 1 to 3. Additional sera from patients with breast cancer and undifferentiated nasopharyngeal carcinoma as well as healthy subjects were the controls. RESULTS: Immunologic results suggested that antibodies that react with SV40 mimotopes were more prevalent (44%) in serum samples from patients with OS compared with healthy subjects (17%). The difference in prevalence between these cohorts was statistically significant (P<.001). It is interesting to note that in the patients with OS, significance indicated the difference between OS versus breast cancer (44% vs 15%; P<.001) and OS versus undifferentiated nasopharyngeal carcinoma (44% vs 25%; P<.05). CONCLUSIONS: The data from the current study indicate an association between OS and SV40. These data could be transferred to clinical applications for innovative therapies to address SV40-positive OS.

Immunologic evidence of a strong association between non-Hodgkin lymphoma and simian virus 40.

BACKGROUND: Non-Hodgkin lymphoma (NHL), the most common cancer of the lymphatic system, is of unknown etiology. The identification of etiologic factors in the onset of NHL is a key event that could facilitate the prevention and cure of this malignancy. Simian virus 40 (SV40) has been considered an oncogenic agent in the onset/progression of NHL. METHODS: In this study, an indirect enzyme-linked immunosorbent assay with 2 synthetic peptides that mimic SV40 antigens of viral capsid proteins 1 to 3 was employed to detect specific antibodies against SV40. Serum samples were taken from 2 distinct cohorts of NHL-affected patients (NHL1 [n = 89] and NHL2 [n = 61]) along with controls represented by oncologic patients affected by breast cancer (BC; n = 78) and undifferentiated nasopharyngeal carcinoma (UNPC; n = 64) and 3 different cohorts of healthy subjects (HSs; HS1 [n = 130], HS2 [n = 83], and HS3 [n = 87]). RESULTS: Immunologic data indicated that in serum samples from NHL patients, antibodies against SV40 mimotopes were detectable with a prevalence of 40% in NHL1 patients and with a prevalence of 43% in NHL2 patients. In HSs of the same median age as NHL patients, the prevalence was 16% for the HS1 group (57 years) and 14% for the HS2 group (65 years). The difference was statistically significant (P < .0001 and P < .001). Interestingly, the difference between NHL1/NHL2 patients and BC patients (40%/43% vs 15%, P < .001) and between NHL1/NHL2 patients and UNPC patients (40%/43% vs 25%, P < .05) was significant. CONCLUSIONS: Our data indicate a strong association between NHL and SV40 and thus a need for innovative therapeutic approaches for this hematologic malignancy.

High prevalence of serum antibodies reacting with simian virus 40 capsid protein mimotopes in patients affected by malignant pleural mesothelioma.

Human malignant pleural mesothelioma (MPM) is considered a rare tumor, but recent estimations indicate that one-quarter million people will die of this neoplasm in Europe in the next three decades. The mineral asbestos is considered the main causative agent of this neoplasm. MPM is largely unresponsive to conventional chemotherapy/radiotherapy. In addition to asbestos exposure, genetic predisposition to asbestos carcinogenesis and to simian virus (SV)40 infection has also been suggested. SV40 is a DNA tumor virus found in some studies to be associated at high prevalence with MPM. SV40 sequences have also been detected, although at a lower prevalence than in MPM, in blood specimens from healthy donors. However, some studies have failed to reveal SV40 footprints in MPM and its association with this neoplasm. These conflicting results indicate the need for further investigations with new approaches. We report on the presence of antibodies in serum samples from patients affected by MPM that specifically react with two different SV40 mimotopes. The two SV40 peptides used in indirect ELISAs correspond to viral capsid proteins. ELISA with the two SV40 mimotopes gave overlapping results. Our data indicate that in serum samples from MPM-affected patients (n = 97), the prevalence of antibodies against SV40 viral capsid protein antigens is significantly higher (26%, P = 0.043) than in the control group (15%) represented by healthy subjects (n = 168) with the same median age (66 y) and sex. Our results suggest that SV40 is associated with a subset of MPM and circulates in humans.

Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders.

HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.

Serologic investigation of undifferentiated nasopharyngeal carcinoma and simian virus 40 infection.

BACKGROUND: The association between undifferentiated nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) is well established. Nevertheless, available evidence suggests that other cofactors are required for the development of undifferentiated NPC. Several investigations reported simian virus 40 (SV40) footprints in human tumors of different histotypes. METHODS: Serum samples from patients with undifferentiated NPC (n = 64) and healthy subjects (n = 130) were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) with SV40 synthetic peptides to detect antibodies against viral peptide (VP) capsid proteins VP1, 2, and 3. RESULTS: Immunologic data indicate that in sera from patients with undifferentiated NPC, the prevalence of SV40 antibodies was 25%, whereas in controls it was 16%. This difference is not statistically significant (p > .05). CONCLUSION: A similar prevalence of SV40 antibodies was detected in undifferentiated NPC and healthy subjects. Our serologic data suggest no association between undifferentiated NPC and SV40 infection. This investigation may stimulate further studies aimed at determining the possible contribution of other risk factors in the pathogenesis of undifferentiated NPC.

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

Oncogenic transformation by BK virus and association with human tumors.

BK virus (BKV), a human polyomavirus closely related to JC virus and Simian Virus 40, is ubiquitous in human populations worldwide. After primary infection, BKV establishes a lifelong latent infection in many organs. BKV transforms rodent cells to the neoplastic phenotype and is highly oncogenic in rodents. This review considers the oncogenic potential of BKV in humans and its possible involvement in human tumors. BKV sequences and T antigen (Tag) are detected in several types of human neoplasms, although the viral load is generally low, with less than one copy of the viral genome per cell. The possible causative role of BKV in human oncogenesis rests on the ability of BKV Tag to inactivate the functions of tumor suppressor proteins p53 and pRB family as well as on its ability to induce chromosomal aberrations in human cells. A 'hit and run' mechanism and secretion of paracrine growth factors by BKV Tag-positive cells, recruiting into proliferation neighboring and distant cells, are discussed as possible BKV pathogenic elements in human oncogenesis.

Cloning and characterization of the common fragile site FRA6F harboring a replicative senescence gene and frequently deleted in human tumors.

The common fragile site FRA6F, located at 6q21, is an extended region of about 1200 kb, with two hot spots of breakage each spanning about 200 kb. Transcription mapping of the FRA6F region identified 19 known genes, 10 within the FRA6F interval and nine in a proximal or distal position. The nucleotide sequence of FRA6F is rich in repetitive elements (LINE1 and LINE2, Alu, MIR, MER and endogenous retroviral sequences) as well as in matrix attachment regions (MARs), and shows several DNA segments with increased helix flexibility. We found that tight clusters of stem-loop structures were localized exclusively in the two regions with greater frequency of breakage. Chromosomal instability at FRA6F probably depends on a complex interaction of different factors, involving regions of greater DNA flexibility and MARs. We propose an additional mechanism of fragility at FRA6F, based on stem-loop structures which may cause delay or arrest in DNA replication. A senescence gene likely maps within FRA6F, as suggested by detection of deletion and translocation breakpoints involving this fragile site in immortal human-mouse cell hybrids and in SV40-immortalized human fibroblasts containing a human chromosome 6 deleted at q21. Deletion breakpoints within FRA6F are common in several types of human leukemias and solid tumors, suggesting the presence of a tumor suppressor gene in the region. Moreover, a gene associated to hereditary schizophrenia maps within FRA6F. Therefore, FRA6F may represent a landmark for the identification and cloning of genes involved in senescence, leukemia, cancer and schizophrenia.

Loss of methylation at chromosome 11p15.5 is common in human adult tumors.

Chromosome 11p15 deletion is frequent in human tumors, suggesting the presence of at least one tumor suppressor gene within this region. While mutation analyses of local genes revealed only rare mutations, we have previously described a mechanism, gain of imprinting, that leads to loss of expression of genes located on the maternal 11p15 chromosome in human hepatocarcinomas. Loss of expression was often associated with loss of maternal-specific methylation at the KvDMR1 locus. Here, we show that loss of the maternal KvDMR1 methylation is common, ranging from 30 to 50%, to a variety of adult neoplasms, including liver, breast, cervical and gastric carcinomas. We found that other 11p15.5 loci were concomitantly hypomethylated, indicating that loss of KvDMR1 methylation occurred in the context of a common mechanism affecting the methylation of a large 11p15 subchromosomal domain. These epigenetic abnormalities were not detected in any normal somatic tissue. Therefore, it seems possible that, contrary to the repression of promoter activity caused by hypermethylation, loss of gene expression at 11p15.5 may result from the activation, by hypomethylation, of one or more negative regulatory elements.

Tumor and metastasis suppression by the human RNASET2 gene.

The region 6q27 from human chromosome 6 has been reported to contain one or more tumor suppressor genes on the basis of cytogenetic, molecular and functional studies. We have recently carried out a detailed analysis of a candidate gene from 6q27 to evaluate its putative role as a tumor suppressor gene involved in ovarian cancer pathogenesis. The RNASET2 gene was shown to behave as a class II tumor suppressor and abolish the tumorigenic potential of an ovarian cancer-derived cell line. In this study, we have started the cellular and biochemical characterization of RNASET2 and showed that it is a secreted glycoprotein. Moreover, we have extended our previous studies by evaluating the effect of RNASET2 on the metastatic behavior of the highly-invasive ovarian cancer cell line HEY3MET2. From such analysis, RNASET2 was found to significantly decrease the metastatic potential of this cell line in vivo. Moreover, RNASET2-mediated suppression of tumorigenesis and metastasis was not affected by a double point mutation targeted to the putative ribonuclease catalytic sites, suggesting that tumor suppression by RNASET2 is not mediated by its ribonuclease activity. The potential biological implications of this unexpected finding are discussed in relation to the current evolutionary models.

Genetic chaos and antichaos in human cancers.

Cancer is considered the genetic disease with the most complex pathogenetic mechanism. Contrary to normal human tissues, which function in a highly ordered and uniform manner, tumor tissues were considered to develop chaos. Here we propose that analysis of human neoplasia according to the concepts of chaos and antichaos allowed to distinguish two main types of cancers: the hematopoietic cancers, characterized by the orderly antichaos and solid cancers governed by a disordered complex of genetic and molecular events characteristic of a chaotic pattern. Genetic chaos and antichaos have generated rules that can be applied to the diagnosis and therapy of human tumors.

Significant prevalence of antibodies reacting with simian virus 40 mimotopes in sera from patients affected by glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is a rare tumor, which affects 1/100 000 individuals, but it represents 30% of central nervous system malignancies. GBM is a severe tumor responsible for 2% of all cancer-related deaths. Although characterized by genotypic and phenotypic heterogeneities, GBM invariably resists conventional chemo- and radiotherapies. Several chromosome alterations and gene mutations were detected in GBM. Simian virus 40 (SV40), a small DNA tumor virus, has been found in GBM specimens by some studies, while other investigations have not confirmed the association. METHODS: An indirect enzyme-linked immunosorbent assay with 2 synthetic peptides mimicking SV40 antigens of viral capsid proteins 1-3 was employed to detect specific antibodies against SV40 in serum samples from GBM-affected patients, together with controls represented by patients affected by breast cancer and normal subjects of the same median age. RESULTS: Our data indicate that in serum samples from GBM-affected patients (n = 44), the prevalence of antibodies against SV40 viral capsid protein antigens is statistically significantly higher (34%, P = .016 and P = .03) than in the control groups (15%), represented by healthy subjects (n = 101) and patients affected by breast cancer (n = 78), respectively. CONCLUSION: Our data indicate that SV40, or a closely related yet undiscovered human polyomavirus, is associated with a subset of GBM and circulates in humans. Our study can be transferred to the clinical oncology application to discriminate different types of heterogeneous GBM, which in turn may address an innovative therapeutic approach to this fatal cancer.

Serological evidence of an early seroconversion to Simian virus 40 in healthy children and adolescents.

At present Simian virus 40 (SV40) infection in humans appears to be transmitted independently from early contaminated vaccines. In order to test the spread of SV40 infection in children, an immunologic assay employing specific SV40 synthetic peptides corresponding to its viral protein (VP) antigens was employed to estimate the seroprevalence of this polyomavirus in Italian infants and adolescents. Serum samples from 328 children and adolescents, up to 17 years, were investigated. Serum antibodies against SV40 VPs were detected by indirect enzyme-linked immunosorbent assays. The seroprevalence of this polyomavirus was calculated after stratifying the subjects by age. Anti-viral capsid protein 1-2-3 SV40 IgG antibodies were detected in 16% of the study participants. The prevalence of antibodies against SV40 VPs tended to increase with age in children, up to 10 year old (21%). Then, in the cohort of individuals aged 11-17 years, the prevalence decreased (16%). A higher prevalence rate (23%) of SV40 VP antibodies was detected in the cohorts of 1-3 year and 7-10 year old children, than in children and adolescents of the other age groups. This age corresponds to children starting nursery and primary school, respectively, in Italy. IgM antibodies against SV40 VP mimotopes were detected in 6-8 month old children suggesting that SV40 seroconversion can occur early in life. SV40 VP antibodies are present at low prevalence in Italian children (16%), suggesting that SV40 infection, although acquired early in life, probably through different routes, is not widespread. The low SV40 seroprevalence suggests that SV40 is less transmissible than other common polyomaviruses, such as BKV and JCV. Alternatively, our immunologic data could be due to another, as yet undiscovered, human polyomavirus closely related to SV40.

Specific antibodies reacting with simian virus 40 capsid protein mimotopes in serum samples from healthy blood donors.

Simian virus 40 (SV40), a small DNA tumor virus, was inadvertently administered to human populations with the use of contaminated vaccines. SV40 sequences have mainly been detected in healthy individuals and cancer patients using polymerase chain reaction techniques. However, some studies have failed to reveal the presence of SV40 in human specimens. These conflicting results indicate the need for new research to verify whether SV40 is circulating in humans. Mimotopes from SV40 structural peptides were tested to investigate for specific reactions to human sera antibodies. An indirect enzyme-linked immunosorbent assay with synthetic peptides from SV40 viral capsid proteins 1-2-3 (VPs 1-2-3) was set up and employed to test 855 serum samples from healthy blood donors. Data from immunologic assays indicate that serum antibodies against SV40 VP mimotopes are detectable, although with a low titer, in blood donors 18 to 65 years old. The overall prevalence of serum samples that reacted with the 2 SV40 VP peptides was 18%. The strong points for this novel method include the simplicity of its approach and the potential to discriminate between SV40-specific antibody responses and to draw correlations between responses to the 2 independent SV40 peptides. These data suggest that SV40, or a yet undetected closely related polyomavirus, is circulating in human populations, but with lower prevalence than that of the ubiquitous BK and JC human polyomaviruses.

Simian virus 40 infection in humans and association with human diseases: results and hypotheses.

Simian virus 40 (SV40) is a monkey virus that was introduced in the human population by contaminated poliovaccines, produced in SV40-infected monkey cells, between 1955 and 1963. Epidemiological evidence now suggests that SV40 may be contagiously transmitted in humans by horizontal infection, independent of the earlier administration of SV40-contaminated poliovaccines. This evidence includes detection of SV40 DNA sequences in human tissues and of SV40 antibodies in human sera, as well as rescue of infectious SV40 from a human tumor. Detection of SV40 DNA sequences in blood and sperm and of SV40 virions in sewage points to the hematic, sexual, and orofecal routes as means of virus transmission in humans. The site of latent infection in humans is not known, but the presence of SV40 in urine suggests the kidney as a possible site of latency, as it occurs in the natural monkey host. SV40 in humans is associated with inflammatory kidney diseases and with specific tumor types: mesothelioma, lymphoma, brain, and bone. These human tumors correspond to the neoplasms that are induced by SV40 experimental inoculation in rodents and by generation of transgenic mice with the SV40 early region gene directed by its own early promoter-enhancer. The mechanisms of SV40 tumorigenesis in humans are related to the properties of the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag). Tag acts mainly by blocking the functions of p53 and RB tumor suppressor proteins, as well as by inducing chromosomal aberrations in the host cell. These chromosome alterations may hit genes important in oncogenesis and generate genetic instability in tumor cells. The clastogenic activity of Tag, which fixes the chromosome damage in the infected cells, may explain the low viral load in SV40-positive human tumors and the observation that Tag is expressed only in a fraction of tumor cells. "Hit and run" seems the most plausible mechanism to support this situation. The small tag, like large Tag, displays several functions, but its principal role in transformation is to bind the protein phosphatase PP2A. This leads to constitutive activation of the Wnt pathway, resulting in continuous cell proliferation. The possibility that SV40 is implicated as a cofactor in the etiology of some human tumors has stimulated the preparation of a vaccine against the large Tag. Such a vaccine may represent in the future a useful immunoprophylactic and immunotherapeutic intervention against human tumors associated with SV40.

Tumor-host interaction mediates the regression of BK virus-induced vascular tumors in mice: involvement of transforming growth factor-beta1.

Several sexually transmitted viruses have been associated with the development of Kaposi's sarcoma (KS), a highly vascularized multi-focal neoplasm, characterized by the presence of spindle-shaped and endothelial cells, fibroblasts and macrophages. As BK virus (BKV) sequences were found in 100% of primary KS and 75% of KS cell lines, we established an experimental model to test whether BKV may be involved in the pathogenesis of KS. For this purpose, we transformed primary and spontaneously immortalized murine endothelial cells with BKV or with a plasmid containing BKV early region, which encodes BKV T antigen. Murine endothelial cells lost endothelial markers after transformation by BKV and, when inoculated s.c. in nude mice, induced tumors which regressed 7-30 days after onset, whereas spontaneously immortalized murine endothelial MHE cells induced progressing tumors, which brought the animals to death. Histologic examination showed an initial formation of vessels around the tumors, followed by the appearance of a dense population of fibroblasts and mononuclear cells in the peritumoral tissue. Subsequently, tumors appeared to be infiltrated by mononuclear cells and surrounded by a thick fibrous wall with scattered fibroblasts and without vessels. Areas of necrosis developed in the tumor mass and finally the neoplastic tissue completely degenerated. The medium conditioned by BKV-transformed cells induced proliferation and migration of human fibroblasts and NIH3T3 cells. These effects were inhibited by an anti-transforming growth factor-beta1 (TGF-beta1) antibody. Northern blot analysis revealed that BKV-transformed cells express a greater amount of TGF-beta1 RNA than normal murine endothelial cells. Besides, TGF-beta1 was not expressed in progressing tumors induced by spontaneously immortalized endothelial MHE cells, whereas it was highly expressed during the regression of tumors induced by BKV-transformed MHE and primary endothelial cells. Over-expression of TGF-beta1 may be responsible for the mononuclear cell infiltration, inhibition of angiogenesis and formation of the fibrotic wall around tumors, inducing tumor regression through tumor cell necrosis and nutritional starvation. These results prompt us to test whether production of TGF-beta1 is associated with spontaneous KS regression in human patients. In this case, KS regression could be induced or accelerated by any means that enhances TGF-beta1 production at the tumor site.

Reactivation of infectious simian virus 40 from normal human tissues.

In this study, 82 DNA samples of simian virus 40 (SV40)-positive human tumors and normal tissues were transfected into SV40-permissive monkey cells. SV40 wild-type strain 776 was reactivated from two DNA samples, derived from peripheral blood mononuclear cells (PBMCs) of a blood donor and from a vulvar tissue. SV40 reactivation was confirmed by obtaining rescue of SV40 from the DNA of the vulvar tissue in a second transfection experiment. This investigation indicates that infectious SV40 is present in normal human tissues and suggests that (i) PBMCs are probably vectors of SV40 to different tissues of the host and (ii) blood and sexual transmission may be routes of SV40 infection in humans, leading to (iii) virus spread in the human population.

Different simian virus 40 genomic regions and sequences homologous with SV40 large T antigen in DNA of human brain and bone tumors and of leukocytes from blood donors.

BACKGROUND: Many studies found only a small fragment of the large T-antigen coding sequences in human tumors, raising doubts on authenticity of SV40 sequences detected in these samples. METHODS: Five different regions of SV40 DNA were investigated in 106 fresh human tumor biopsies (25 brain, 69 bone, 12 Wilms' tumors), 71 tumor-derived cell cultures (38 from brain and 33 from bone tumors) and normal tissues (5 fresh bone biopsies and 38 buffy coats) by polymerase chain reaction (PCR) techniques and filter hybridization with specific oligoprobes. Expression of SV40 Tag sequences was analyzed in human tumor specimens by RT-PCR. RESULTS: SV40 large T-antigen sequences were detected at high prevalence, in human biopsies of primary brain (37-44%) and bone (21-37%) tumors, in cell cultures derived from brain (30-54%) and bone (53-80%) tumors. SV40 Tag sequences were detected in 29% of buffy coats of blood donors. However, only four brain tumor cell lines showed all the five regions of the SV40 genome investigated. Expression of SV40 Tag sequences was found in 11 of 27 (41%) human tumor samples. DNA sequence analysis indicated that the PCR-amplified products belong to the SV40 wild type. Polymerase chain reaction products of Tag middle portion from 20 of 78 (26%) samples showed a 97% homology with telomeric sequences of human chromosomes 10 and 11. CONCLUSIONS: Authentic SV40 sequences were detected in human samples. The expression of SV40 Tag sequences indicates that SV40 could play a role, as a cofactor, in the onset/progression of specific human cancers. The inability to detect some regions of the virus genome may suggest that those regions are not required for tumor persistence or growth and have been lost or, alternatively, may be the result of assay conditions that were unable to PCR-amplify those regions in the tumors.