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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. 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. 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). 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.

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.

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.

Glioblastoma multiforme (GBM) is the most severe form of cancer among gliomas. Indeed, GBM is a World Health Organization grade IV tumor of the CNS, with a mean survival time of 0.5 years.1 Its incidence is ∼1/100 000, which represents 30% of all CNS malignancies.2 According to the recent Eurocare 3 report,3 the mean age of patients affected by glioblastoma is 66 years. The extremely grim prognosis of GBM patients probably explains why such a rare tumor accounts for 2% of all cancer-related deaths in both Europe and the United States.4 GBM is characterized by high genotypic and phenotypic heterogeneities, but it is invariably and homogeneously resistant to conventional chemo/radiotherapy.1 The vast majority (95%) of GBM is represented by primary de novo glioblastomas.5 GBM is considered, like other tumors, a genetic disease of the somatic cell due to an impressive series of alterations of the cellular genome, such as point mutations, gene amplifications, translocations, and deletions, as well as epigenetic modifications, including methylation of specific genes. A limited percentage (5%) of GBM is linked to genetic predispositions, which are mainly associated with rare hereditary syndromes, such as Li–Fraumeni syndrome, Turcot syndrome, Peutz–Jegher syndrome, and neurofibromatosis 1 and 2.2 Exposure to chemical and physical carcinogenic agents, the genetic predisposition, and/or infections by oncogenic viruses, including simian virus 40 (SV40), have been repeatedly advocated in the literature.6,7 SV40 is a nonenveloped small DNA virus of ∼5.2 kb in size. SV40 was recognized in the 1960s as a contaminant of both inactivated (Salk) and live attenuated (Sabin) antipoliomyelitis vaccines. As a consequence, SV40 was inadvertently administered to human populations worldwide mainly by antipoliomyelitis vaccines, produced in SV40 naturally infected monkey kidney cells. Soon after its isolation, SV40 was characterized as a transforming and tumorigenic viral agent.6,7 Cell transformation and tumorigenesis by SV40 is induced by 2 oncoproteins, the large T antigen (Tag) and the small t antigen (tag), which target key cellular proteins, such as the tumor suppressor p53 and retinoblastoma family proteins, inactivating their functions.6,8 This small DNA tumor virus was found to be associated with some human cancers, including GBM. SV40 footprints were detected at high prevalence in GBM specimens, but SV40 sequences have also been revealed, although at lower prevalence, in blood specimens from healthy donors.9–11 In this context it is worth recalling that other investigations did not find SV40 footprints in human brain tumor specimens12,13 or higher SV40 antibodies in serum samples from brain tumor patients than in normal individuals.14 It has been reported that SV40-negative data on human tumor samples obtained by some groups were affected by technical flaws that invalidate the conclusions.15–17 A considerable debate, because of these contrasting reports, has developed in the scientific community.6,7 SV40 antibody detection had been performed in most cases using serologic methods with SV40 virus-like particles, but due to the high protein homology among the 3 main polyomaviruses—SV40, BKV, and JCV—final results were always influenced by some cross-reactivity.18–24 Altogether these reports prompted us to use an indirect ELISA with synthetic peptides employed as specific mimotopes, mimicking SV40 viral capsid antigens. Indeed, recent investigations have reported on the development of a specific and sensitive serologic test for SV40. The test is based on an indirect ELISA employing synthetic peptides as mimotopes/antigens of SV40 viral capsid proteins (VPs), without cross-reactivity with other closely related human polyomaviruses, such as BKV and JCV.25–28 This immunologic assay was used to detect specific serum antibodies against SV40 VPs in normal individuals of different ages and oncologic patients affected by malignant pleural mesothelioma and breast cancer (BC).25–28 In this investigation, serum samples from GBM patients were comparatively analyzed for the presence and titer of antibodies against SV40 infection, with serum samples from healthy individuals and from other reliable controls. No other slow virus infections were investigated.

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico-pathological features. This report describes the analysis of 63 "microsatellite instability-high" (MSI-H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFbetaRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFbetaRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico-pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI-H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico-pathological traits.

To study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS.

Loss of constitutive heterozygosity at 11q23 has been detected in various human solid tumors. Here, we described the analysis of a series of normal and tumor pairs from 110 breast carcinomas for the presence of loss of heterozygosity at 11q23 loci. The overall frequency of LOH was 48%, confirming the importance of deletions at 11q23 in breast tumorigenesis. Previously, we have identified two independent regions of LOH at 11q23, the LOH region 1 at 11q23.1 and the LOH region 2 at 11q23.3. The most telomeric region was recently refined between loci D11S1345 and D11S1316, a region of about 1 Mb. However, the LOH region 1, most centromeric, was still not finely refined: the boundaries were defined by loci D11S2000 and D11S897, separated by about 8 Mb. Here, we refined its boundaries between loci D11S1347 and D11S927, a region of about 2 Mb. We have mapped 11 expressed sequence tags (ESTs) within this region and excluded another 20. This study represents a further step toward the identification of the putative tumor suppressor gene found within the LOH region 1 at 11q23.1.

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi’s sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi’s sarcoma in AIDS patients.

We have analyzed by PCR skin lesions from classic, endemic and AIDS-related Kaposi's sarcoma (KS), as well as from KS-derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full-length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57-67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11-20% of KS specimens and was detected with a low prevalence (0-21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20-25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 18 and benign HPV types 6 and 11 were detected in KS specimens with a similar prevalence of 11-83%, suggesting that the presence of HPV-transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS-HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV-transforming functions may participate in the development of KS.

Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression.

A BK virus (BKV) episomal vector (pRPneoCMV) was constructed for expression of cDNAs under control of the cytomegalovirus (CMV) immediate-early promoter. Transfection of pRPneoCMV for expression of the chloramphenicol acetyltransferase (CAT) gene in several human cell lines showed that the CMV promoter is more efficient than the HIV-1 and RSV LTRs in directing gene expression from episomal vectors. In 293 human cells pRPneoCMV/CAT is twenty times more active in CAT expression than the well known pSV2CAT vector in COS7 cells. Stable expression of the gene of the herpes simplex virus type 1 and type 2 glycoprotein G, cloned into pRPneoCMV, was obtained in 293 cells. This vector will allow direct cloning of newly synthesized cDNAs whose expression can be monitored in human cells.

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.

BK virus (BKV), JC virus (JCV) and Simian Virus 40 (SV40) are polyomaviruses, highly homologous at the DNA and protein levels. While the human polyomaviruses BKV and JCV are ubiquitous in humans, SV40 is a simian virus which was introduced in the human population, between 1955 and 1963, by contaminated poliovaccines produced in SV40-infected monkey cells. Alternatively, SV40 or an SV40-like virus may have entered the human population before anti-poliovirus vaccination. Epidemiological evidence suggests that SV40 is now contagiously transmitted in the human population by horizontal infection, independently from the earlier contaminated poliovaccines. All three polyomaviruses transform rodent and human cells and are oncogenic in rodents. JCV induces tumors also in experimentally inoculated monkeys. Transformation and oncogenicity induced by BKV, JCV and SV40 are due to the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag), encoded in the early region of the viral genome. Both proteins display several functions. The large Tag acts mainly by blocking the functions of p53 and pRB family tumor suppressor proteins and by inducing in host cells chromosomal aberrations and instability. The principal effect of small tag is to bind the catalytic and regulatory subunits of the protein phosphatase PP2A, thereby constitutively activating the β-catenin pathway which drives cells into proliferation. All three polyomaviruses are associated with specific human tumor types which correspond to the tumors induced by experimental inoculation of the three viruses in rodents and to the neoplasms arising in mice transgenic for the polyomavirus early region gene directed by the native viral early promoter-enhancer. Human tumors associated with BKV. JCV and SV40 contain viral DNA, generally episomic, express viral RNA and are positive for large Tag by immunohistochemistry. The low copy number of viral genomes in human tumors suggests that polyomaviruses may transform human cells by a “hit and run” mechanism. An autocrine-paracrine effect, involving secretion of growth factors by cells expressing polyomavirus Tag, may be responsible for recruiting to proliferation Tag-negative cells in polyomavirus-associated human tumors.

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.

The human immunodeficiency virus type 1 (HIV-1) Tat protein stimulates cell proliferation, inhibits apoptosis, displays angiogenic functions and is believed to be involved in the pathogenesis of Kaposi's sarcoma (KS) and other tumours arising in AIDS patients. Tat-transgenic (TT) mice, which constitutively express Tat in all tissues and organs, may therefore be predisposed to tumorigenesis. To test this hypothesis, we treated TT mice with urethane, a general carcinogen inducing tumours of various organs. The results indicate that, after injection of urethane, the incidence of lung tumours and lymphomas is not significantly different in the TT and control (CC) mice, whereas liver preneoplastic lesions and tumours show a significantly greater incidence in TT than in CC mice. This remarkable carcinogenic effect of urethane for the liver may be due to a tat-induced predisposition, manifested as a liver cell dysplasia (LCD), spontaneously affecting most of the TT mice. LCD may exert a promoting effect by stimulating proliferation of cell clones initiated by the mutagenic effect of urethane. In addition, LCD, which is associated with aneuploidy and chromosome instability, may enhance the progression to malignancy of the preneoplastic lesions induced by urethane. Interestingly, a significantly greater incidence of vascular ectasias and haemangiomas was detected in the liver of urethane-treated TT mice, most likely due to the marked angiogenic properties of Tat. This study suggests a role for Tat in the promotion and progression of tumours initiated by exogenous and endogenous carcinogens in HIV-1-infected patients, thereby contributing to the tumorigenesis in the course of AIDS.

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.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

The human immunodeficiency virus-1 Tat protein is suspected to be involved in the neoplastic pathology arising in AIDS patients. tat-transgenic (TT) mice, which constitutively express Tat in the liver, develop liver cell dysplasia (LCD) that may represent a preneoplastic lesion. To test if TT mice are predisposed to liver carcinogenesis, we treated them with diethylnitrosamine, a hepatotropic carcinogen. Diethylnitrosamine-treated TT mice developed both preneoplastic and neoplastic lesions in the liver. They showed an enhancement of LCD and developed basophilic liver cell nodules (BLCN), hepatocellular adenomas (HA), and hepatocellular carcinomas (HC). Both preneoplastic (LCD and BLCN) and neoplastic (HA and HC) lesions were significantly more frequent in TT than in control mice: 29.7% versus 12.7% for LCD, 57.9% versus 23.3% for BLCN, 40.6% versus 10.0% for HA, and 50.0% versus 12.7% for HC. These results indicate that Tat expression in the liver predisposes to both initiation of hepatocarcinogenesis and to malignant progression of liver tumors. This study supports a role for Tat in enhancing the effect of endogenous and exogenous carcinogens in human immunodeficiency virus-1-infected patients, thereby contributing to tumorigenesis in the course of AIDS.

The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the beta isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both alpha (PPP2R1A) and beta isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci D11S601 and D11S679 resulted in the identification of six transcriptional units. Three genes, hNAP2 , CDKN1C , and KVLQT1 , are well characterized, whereas three genes are novel. The three additional genes were designated BWR1A , BWR1B , and BWR1C . Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress, BWR1C cDNA was described as IPL [Qian, N., Franck, D., O’Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997) Hum. Mol. Genet. 6, 2021–2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith–Wiedemann syndrome samples that resulted in the identification of genetic alterations in the BWR1A gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that BWR1A may play a role in tumorigenesis.

T-antigen (TAg) coding sequences specific for BK virus (BKV) were detected, by PCR amplification followed by Southern-blot hybridization, in a high percentage of human tumors and tumor-cell lines, as well as in normal tissues, by analysis of 189 specimens. Specifically, the BKV early region was detected in 85% of brain tumors and in all normal brain tissues, in 78% of osteosarcomas, in 38% of Ewing's tumors, in 40% of normal bone specimens and in 71% of normal peripheral blood cell samples. Wilms' tumor tissues used as a control were all negative for BKV sequences. RT-PCR analysis indicated that TAg coding sequences were expressed in specimens carrying BKV early region, ranging from 64% of the osteosarcomas to 100% of glioblastomas, Ewing's tumors, peripheral blood cells and normal bone. Moreover, DNA sequencing performed in 12 different positive samples revealed that the amplified PCR products are identical to the early-region sequence of wild-type BKV. The role of BKV TAg and its possible mechanism of action in human tumorigenesis are discussed.

The human polyomaviruses BK (BKV) and JC (JCV) are ubiquitous in human populations and have a worldwide distribution.(1) They are oncogenic in rodents and monkeys and transform cells in vitro to a neoplastic phenotype. For all these reasons, BKV and JCV have been considered possible candidates in the etiology of human tumors. Association of BKV, but not of JCV, with human tumors has been described, although a formal proof for an etiological role of BKV in human oncogenesis is still lacking. SV40, which is not a ubiquitous human virus, has also been sporadically detected in human tumors. In this chapter, we consider their general characteristics, the natural history of infection, experimental transformation and oncogenicity by BKV and JCV, as well as the evidence linking BKV, JCV, and SV40 to human neoplasia.

BK virus (BKV) is a human papovavirus that readily transforms rodent cells, but not human cells, to a neoplastic phenotype, suggesting that tumor-suppressor functions expressed in human cells control BKV oncogenicity. Transfer of a normal human chromosome 11 to BKV-transformed mouse cells suppresses the malignant phenotype. In this report we map the regions of chromosome involved in tumor suppression. Transfer of chromosome 11 to the BKV-transformed hamster cell line HKBK produces monochromosomic hybrids retaining only portions of the transferred human chromosome. We have compared the tumorigenicity of the hybrids with the molecular mapping of chromosome 11 retained regions. This analysis indicated that 3 regions of human chromosome 11, 11p15.5, 11p13 and 11q13, cooperate in tumor suppression. However, 11q13 seems the most important, since all the HKBK/H11-induced tumors analysed had lost this region, whereas 11p15.5 and 11p13 were sometimes retained. The chromosomal regions identified in this study are deleted in several types of human tumors, suggesting that the BKV transformation system specifically detects tumor-suppressor genes on chromosome 11 that are involved in human oncogenesis. This model may be of use in isolating and cloning such genes. The results of this report raise the possibility that BKV may have a synergistic tumorigenic effect in human cells where tumor-suppressor genes controlling its oncogenk potential are inactivated. © 1994 Wiley-Liss, Inc.

A BK virus (BKV) expression vector, specific for human cells, was engineered to express antisense human immunodeficiency virus type 1 (HIV-1) tat cDNA (tat-AS) or a tat mutant in cysteine 22 (tat22). Cysteine residues in the cysteine-rich domain of tat are necessary for tat transactivation of the HIV-1 long terminal repeat (LTR). Both the AS tat and the tat mutant significantly inhibited transactivation by tat when assayed in cells cotransfected with an expression vector where the reporter gene for chloramphenicol acetyl transferase was driven by the HIV-1 LTR. Infection of Jurkat cell clones stably expressing tat22 (Jurkat/tat22) or tat-AS (Jurkat/tat-AS) with HIV-1 did not show differences in virus titer in comparison to HIV-1-infected control cells. However, in two Jurkat/tat22 cell clones, entrance of HIV-1 into latency was accelerated significantly and reactivation of HIV-1 from latency induced by tumor necrosis factor-alpha (TNF-alpha) or tat was blocked. These results suggest that, in a combined and integrated approach to the treatment of acquired immunodeficiency syndrome (AIDS), anti-tat genetic therapy could be successfully applied to maintain virus in latency, thereby extending the duration of the asymptomatic phase preceding full-blown AIDS.

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi's sarcoma and in cell lines from brain tumours. Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable elements in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.

Genomic instability has been reported at microsatellite tracts in few coding sequences. We have shown that the Bloom syndrome BLM gene may be a target of microsatelliteinstability (MSI) in a short poly-adenine repeat located in its coding region. To further characterize the involvement of BLM in tumorigenesis, we have investigated mutations in nine genes containing coding microsatellites in microsatellite mutator phenotype (MMP) positive and negative gastric carcinomas (GCs). We analyzed 50 gastric carcinomas (GCs) for mutations in the BLM poly(A) tract aswell as in the coding microsatellites of the TGFβ1-RII, IGFIIR, hMSH3, hMSH6, BAX, WRN, RECQL and CBL genes. BLM mutations were found in 27% of MMP+ GCs (4/15 cases) but not in any of the MMP negative GCs (0/35 cases). The frequency of mutations in the other eight coding regions microsatellite was the following: TGFβ1-RII (60 %), BAX (27%), hMSH6 (20%),hMSH3 (13%), CBL (13%), IGFIIR (7%), RECQL (0%) and WRN (0%). Mutations in BLM appear to be more frequently associated with frameshifts in BAX and in hMSH6 and/or hMSH3. Tumors with BLM alterations present a higher frequency of unstable mono- and trinucleotide repeats located in coding regions as compared with mutator phenotype tumors without BLM frameshifts. BLM frameshifts are frequent alterations in GCs specifically associated with MMP+tumors. We suggest that BLM loss of function by MSI may increase the genetic instability of a pre-existent unstable genotype in gastric tumors.

BK virus (BKV)-induced tumors in hamsters were investigated for the presence of viral DNA by the blot-transfer hybridization technique. Several viral genomes per cell were found in tumor tissues and in their derived cell lines and clones. Most of the detected viral genomes were integrated into the cellular DNA, but some tumors also contained free viral DNA sequences. Integration patterns were different from each other, and many different integration sites were available on the cellular or on the viral DNA or on both. Typical features of integration patterns were found in ependymomas, which were the most frequent (72%) among BKV-induced tumors. Readily detectable viral DNA sequences were only found in neoplastic tissues, but traces of BKV DNA were also present in the apparently normal portion of the brain of an animal that had developed an ependymoma and in the brain (but not in the liver) of another animal 15 days after virus inoculation. A cell line and a single-cell clone derived from a tumor had hybridization patterns considerably simpler that the pattern of the original tumor, lacking several integrated viral genomes and all free viral sequences.

Analysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing theneo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E 1 A and E 1 B which may directly or indirectly activate BKV origin of replication.

Hamster kidney cells were transformed, with comparable efficiency, by circular or linear molecules of complete BK virus (BKV) genome and by agarose gel-purified fragments of BKV DNA obtained by single or double digestions with various restriction endonucleases. Only fragments containing the complete early region of BKV DNA displayed transforming activity. Analysis by blot hybridization of the arrangement of viral DNA sequences in a cloned cell line transformed by a 3.8-kilobase fragment, obtained after sequential digestion of BKV DNA with HhaI and BamHI, showed the presence of seven viral integrations into the cellular DNA. Apparently all of the integrated viral molecules contained the entire early region of BKV DNA. Large T antigen, small t antigen, and the 56,000-dalton nonviral Tau antigen were detected in transformed cells by immunoprecipitation. The pattern of integration of viral sequences in transformed cells was constant over many generations. Likewise, large T antigen was always detected in transformed cells at various passage levels. These results may suggest that all of the sequences of the early region coding for large T antigen are required for transformation by BKV. Alternatively, subgenomic segments of the BKV DNA early region may be unable to transform because the appropriate polyadenylation site, necessary to obtain a complete functional transcriptional unit, is removed by the restriction enzyme cleavage.

Tandem integrations of complete viral genomes, which are not usually found in hamster cells transformed by BK virus (BKV) or circular BKV DNA can be established when hamster cells are transfected with linear BKV DNA with cohesive ends. Treatment of linear BKV DNA with S1 nuclease or use of linear BKV DNA with staggered but non-complementary ends prevents tandem integration. These results support a model in which formation of tandems of free viral DNA is a preliminary step for tandem integration.

BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the central nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.

Human fibroblasts, transfected with a recombinant DNA containing the neo gene and BK Virus (BKV) early region, which expresses BKV large T antigen (TAg), show cytogenetic alterations characterized by dicentric chromosomes and other structural aberrations such as deletions, duplications, translocations, and ring chromosomes. Such alterations were absent or significantly less frequent in human fibroblasts transfected with a plasmid expressing only the neo gene. The chromosome damage in BKV-transfected cells was evident before the appearance of the morphologically transformed phenotype and therefore seems to be a primary effect of TAg expression in human cells. The specific pattern of chromosome aberrations suggests the prevalence of an indirect clastogenic effect, determined by the inhibition of p53 regulatory functions on genome stability by BKV TAg. Due to the widespread distribution of BKV in the human population and to the latent state of BKV DNA in many human organs, the clastogenic activity of BKV TAg may potentially participate in an oncogenic process involving BKV latently infected cells.

The Tat protein of the human immunodeficiency virus type 1 promotes survival and growth and inhibits apoptosis of different cell types. These effects of Tat are attributed to the induction of bcl-2 gene expression. In this study we show that the blocking of both intracellular and extracellular Tat correlates with a decrease of bcl-2 transcripts, leading in vitro to a lower growth rate and attenuation of the transformed phenotype and in vivo to a reduced angiogenic and oncogenic activity of Tat-expressing cells. These results support the notion that bcl-2 is an effector of Tat-induced angiogenesis and oncogenesis and indicate that the blocking of Tat functions by immunoprophylactic, pharmacological, and gene therapy approaches may help to control oncogenesis during AIDS.

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.

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.

Expression of cloned viral and cellular genes in eukaryotic cells is relevant to production of vaccines (Melnick, 1986; Moss and Flexner, 1987) and gene therapy (Anderson, 1984). To this purpose numerous eukaryotic vectors have been developed. Since the level of expression of exogenous genes is directly related to the degree of gene amplification (Rigby, 1983) episomal eukaryotic vectors have been constructed where a viral origin of replication directs vector amplification in suitable cells (DiMaio, 1987; Milanesi et al., 1984). However, only two eukaryotic vectors (Milanesi et al., 1984; Sugden et al., 1985) are specific for human cells and they allow expression of genes endowed with their own regulatory sequences. We have therefore devised a viral vector (pBK-1) that replicates and persists episomally in human cells; The structure and general characteristics of this vector have been described in detail (Milanesi et al., 1984). It contains DNA sequences from the plasmid pML, a deletion derivative of pBR322, as well as the origin of replication and the early region of BK virus (BKV), a human papovavirus that efficently replicates in human cells.

Tumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with gamma-radiation (60Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of glucose were detected in blood of animals with tumors of pancreatic islets. No antibodies to BKV tumor antigen (TAg) and low levels of hemagglutination-inhibition antibodies to BKV viral coat protein Ag were detected in hamster sera. BKV TAg was found in tumors by complement fixation. Blot hybridization analysis of tumor DNA showed the presence of both free and integrated BKV genomes in tumor cells. BKV DNA inoculated intravenously and subcutaneously in immunosuppressed or immunocompetent hamsters was not oncogenic, whereas it was weakly oncogenic when inoculated intracerebrally.

Summary After exposure of human embryonic fibroblasts to BK virus, virus particles adsorbed to the plasma membrane were engulfed by pinocytosis or captured by vesicles, possibly originating from the endoplasmic reticulum, within 2 h after infection. Most of the virus particles were then transported into lysosomes or into the nucleus, while a small amount of virus was found free in the cytoplasm. Virus particles entered the nucleus between 2 and 12 h after infection, were still detectable in the nucleus at 24 h after infection and became morphologically undiscernible at 30 h after infection, suggesting that a nuclear uncoating mechanism was active between 24 and 30 h after infection. Virus progeny started to appear in the nucleus of infected cells at 4 days after infection, but not until 7 to 8 days after infection did the virus escape into the cytoplasm and cell degeneration became evident. The possible explanations for the long replicative cycle of BK virus are discussed.

Sera from 1,279 patients with various diseases were examined for the presence of antibodies to BK virus (BKV) capsid antigens. The percentage of positive sera was comparable in all the diseases except rheumatoid arthritis and chronic nephropathies, where a slightly higher prevalence was found. Sera from 952 patients with tumors were examined for the presence of antibodies to BKV tumor and capsid antigens in comparison with a matched control group of 501 blood donors. Sera from 11 tumor patients (1.15%) and from 4 normal controls (0.80%) had antibodies to BKV tumor antigen. No higher prevalence of antibodies to BKV capsid antigens was found in any cancer type except in carcinomas of the urinary bladder, where the percentage of positive sera and of sera with high titers was higher than in other groups. BKV infection is discussed in relation to its possible connection with human non-neoplastic diseases as well as with human tumors and to its activation under conditions of immunosuppressive therapy.

Early-passage hamster embryo cells were transformed by recombinant DNA molecules containing BK virus (BKV) early-region gene and either the activated c-Ha-ras oncogene (pBK/c-rasA) or the normal c-Ha-ras proto-oncogene (pBK/c-rasN). The recombinant DNAs had a greater transforming ability and converted hamster cells to a more malignant phenotype than the single genes transfected separately. pBK/c-rasA was significantly more powerful than pBK/c-rasN in conferring to cells all the characteristics of transformation. Transfected DNA sequences were integrated mostly as single insertions into cellular DNA. Specific c-Ha-ras and BKV transcripts as well as c-Ha-ras p21 and BKV T antigen were detected in transformed cells. Although stimulation of c-Ha-ras expression by BKV enhancers cannot be excluded in recombinants, super-transfection and co-transfection experiments in hamster embryo cells and pre-neoplastic cell lines showed that BKV early-region and c-Ha-ras co-operate in transformation by contributing separate and independent functions.

Summary The construction of the first stable human cell lines that express and secrete authentic hepatitis B virus surface antigen (HBsAg), using a BK virus (BKV) episomal plasmid vector, is described. The amount of HBsAg produced by BKV vectors (up to 600 ng/107 cells) was comparable to other eukaryotic vector systems. The level of HBsAg expression remained the same regardless of the orientation of the HBsAg gene, substitution of the HBsAg gene promoter with the mouse metallothionein I gene promoter or the tissue origin of the human cell lines used to establish stable cellular transformants. Northern blot analysis also indicated synthesis of normal HBsAg transcripts. Surprisingly, however, the vectors were maintained at far lower than expected copy number (one to five copies/cell). Reasons for this are discussed.

The early promoters of BKV and SV40 plasmids were transactivated in both BKV and SV40-transformed cells which failed to support replication of these plasmids. This suggests that the T-antigen of either virus can transactivate BKV and SV40 early promoters by either increasing the availability of cellular transcription factors or by directly interacting with specific sequences which comprise the transcriptional control region of the early promoters. We also observed that removal of 8-bp on the early side of T-antigen binding site I of BKV does not alter viral-plasmid replication.

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.

BK virus (BKV)-transformed cells and BKV-induced tumors as well as in vitro derived tumor cell lines were all found to contain BKV DNA sequences when analysed by DNA-DNA reassociation kinetics. In transformed cells the number of viral genome equivalents per diploid cell genome (CG divided by VG) decreased with increasing generations. Likewise, single cell clones had a lower CG divided by VG than parental transformed cells. BKV-induced tumors had a high CG divided by VG. Tumor cells cultivated in vitro and their clones had a lower CG divided by VG than BKV-induced tumors from which they were derived, suggesting a multiclonal origin of tumors. Hamster tumors induced by subcutaneous inoculation of BKV-transformed cells or tumor cell lines had a higher CG divided by VG than cells producing them. Variation in CG divided by VG is discussed in terms of cell selection depending on different in vitro or in vivo conditions of cell growth. In some cases, however, the decrease in CG divided by VG most likely depends on loss on free viral sequences from transformed cells or tumors.