Sv4oearly Region and Large T Antigen in Human Brain Tumors

Home , Choroid plexus papilloma

ICANCER RESEARCH56. 4820-4825. October 5. 1996j SV4O Early Region and Large T Antigen in Human Brain Tumors, Peripheral Blood Cells, and Sperm Fluids from Healthy Individuals'

Fernanda Martini, Laura Iacchen, Lorena Lazzarin, Paolo Carinci, Aifredo Corallini, Massimo Gerosa, Paolo luzzolino, Giuseppe Barbanti-Brodano, and Mauro Tognon2 Institute of Histology and General Embryology. (F. M., L 1., L L, P. C.. M. TI, Interdepartment Center for Biotechnology (L I., A. C.. C. B-B., M. TI, and institute of Microbiology (A. C.. G. B-B.]. School of Medicine, Unis'ersitv of Ferrara. Via Fossato di Mortara 64/B, 44100 Ferrara, italy; Department of Neurosurgery. University of Verona (M. G.]. and Pathological Anatomy Sers'ice,Geriatric Hospital of Verona(P. 1.], 37100 Verona.Italy

ABSTRACT tected in human brain tumors and in normal brain tissue by Southern blotting and PCR (4â€”7).JCV sequences were found associated with SV4OT antigen (Tag) coding sequences were detected by PCR ampli normal brain tissue (6) but were not detected in brain tumors in two fication followed by Southern blot hybridization in human brain tumors different investigations (4, 5). Recently, SV4O sequences were de and tumor cell lines, as well as in peripheral blood cells and sperm flUids tected by PCR in ependymomas and choroid plexus papillomas of of healthy donors. SV4Oearly region sequences were found in 83% of choroid plexus papillomas, 73% of ependymomas, 47% of astrocytomas, childhood, as well as in other brain tumors, and in pleural mesothe 33% of glioblastoma multiforme cases,14% of meningiomas,50% of liomas (8â€”10).SV4O is an infectious agent for monkeys and does not glioblastoma cell lines, and 33% of astrocytoma cell lines and in 23% of normally infect humans. However, millions of children and adults in peripheral blood cell samples and 45% of sperm fluids from normal the United States, Canada, and Europe were injected between 1955 individuals. None of the 13 normal brain tissues were positive for SV4O and 1963 with polio vaccines contaminated by infectious SV4O (1 1). DNA, nor were seven oligodendrogliomas, two spongioblastomas, one SV4O is capable of transforming to the neoplastic phenotype cells neuroblastoma, one meningioma, or four neuroblastoma cell lines. Ex from different species including human cells (12). It is highly onco pression of SV4Oearly region was found by reverse transcription PCR, genic in hamsters, where it induces specific tumor types, mainly and SV4O-speciflcTag was detected by indirect immunofluorescence in ependymomas, choroid plexus papillomas, osteosarcomas, soft-tissue glioblastoma cell lines. DNA sequence analysis, performed in four positive sarcomas, mesotheliomas, and lymphomas (3). SV4O oncogenicity samples, confirmed that the amplified PCR products belong to the SV4O early region. Sixty-one % of the neoplastic patients positive for SV4O and transforming ability are strictly dependent on the expression of sequences had an age excluding exposure to SV4O-contaminated polio the early region of the viral genome, coding for the Tag, a nuclear vaccines, suggesting a contagious transmission of SV4O.The possible role phosphoprotein of 96,000 molecular weight with multiple biological of SV4O Tag in the etiopathogenesis of human brain tumors and the and biochemical properties. SV4O Tag has ATPase and helicase spread of SV4O by horizontal infection in the human population are activities, binds to viral and cellular DNA, inducing their replication, dhscussed. and forms a complex with the products of p53 and Rb tumor suppres sor genes, inactivating their functions (13). It has been reported that INTRODUCTION expression of SV4O Tag alters the integrity and stability of the host cell genome, inducing numerical and structural chromosomal aberra Human brain tumors account for 2% of all cancers and show a poor tions (14, 15). prognosis (1). Moreover, brain neoplasms are the most frequent solid In this study, SV4O Tag coding sequences were investigated by tumors in children, and their incidence has increased 30% in the last PCR in human brain tumors of different histotypes, as well as in 20 years (1, 2). It is generally accepted that human brain tumors, like normal brain tissue, blood, and sperm fluids from healthy donors. other cancers, represent a genetic disease of somatic cells that is due Moreover, we analyzed the expression of SV4O early region by to several alterations such as gene mutations, deletions, translocations, RT-PCR and by detection of Tag with immunofluorescence. The or amplifications. The causes of these genetic aberrations are not specificity of SV4O DNA detected by PCR was confirmed by se completely understood. Consequently, the etiology and risk factors for quencing the amplified products. these tumors remain to be determined. Infectious agents, such as human polyomaviruses BKV3 and JCV, as well as simian polyoma virus SV4O, have been considered as possible cofactors in the etio MATERIALS AND METHODS pathogenesis of human brain tumors, due to their neurotopism and oncogenic potential in experimental animals (3). BKV and JCV are Primary Tumors, Normal Tissues, and Cell Lines. Fresh biopsies were ubiquitous in the human population (3). BKV sequences were de obtained from primary tumors of 41 patients. Specimens were collected, quickly frozen in liquid nitrogen, and kept at â€”80Â°Cuntilthe time of analysis. Forty-two tumor samples, fixed and embedded in paraffin, were taken from our Received 5/7/96; accepted 8/8/96. histological archives. The histological diagnosis of the 83 tumors was as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with follows: 30 glioblastomas, I 1 ependymomas, 17 astrocytomas, 9 oligodendro 18 U.S.C. Section 1734 solely to indicate this fact. gliomas, 7 meningiomas, 6 choroid plexus papillomas, 2 spongioblastomas, I Supported by grants from the Associazione Italiana per la Ricerca sul Cancro (to and I neuroblastoma. The age of the neoplastic patients was between 3 months M. T. and G. B-B.), from Ministero dell'UniversitÃ¡ e Ricerca Scientifica e Tecnologica. and 79 years. Thirteen normal brain tissue specimens were used. These Consiglio Nazionale delle Ricerche Bilateral Project 95.00945.CTO4, Istituto Superiore di SanitÃ AIDS Project (to M. T.), and Consiglio Nazionale delle Ricerche Progetto Final consisted of 4 fresh biopsies from accidental road victims, 7 formalin-fixed izzato â€œApplicazioniCliniche della Ricerca Oncologicaâ€•(to 0. B. B.). L. I. was supported samples, and 2 fixed and paraffin-embedded specimens from patients not by a fellowship from the Fondazione Cassa di Risparmio di Cento (Ferrara. Italy), and affected by tumors or neurological diseases. Seventy whole blood samples L. L. was supported by a fellowship from the Associazione Italiana per Ia Ricerca sul Cancro. from healthy adult donors were obtained from the Blood Bank of the General 2 To whom requests for reprints should be addressed, at Institute of Histology and Hospital in Ferrara, Italy, whereas 15 B lymphocyte and 15 T lymphocyte General Embryology, School of Medicine, University of Ferrara. via Fossato di Mortars preparations were obtained from the Institute of Medical Genetics, School of 64/B, 44100 Ferrara, Italy. Phone: (39) 532-247-335; Fax: (39) 532-247-461; E-mail: Medicine, University of Ferrara, Ferrara, Italy. Twenty samples of seminal [email protected]. fluid were obtained from the Endocrinology Laboratory of the General Hos 3 The abbreviations used are: BKV, BK virus; iCy, JC virus; Tag, large T antigen; RT-PCR, reverse transcription PCR; MAb, monoclonal antibody; PBC, peripheral blood pital in Modena, Modena, Italy. All neoplastic and normal samples were cell. randomly collected, without any selection for specific categories of patients or 4820

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1996 American Association for Cancer Research. sv4o ANDHUMANBRAINTUMORS normal persons. Cell lines from 18 glioblastoma multiforme cases (A172, quencing, after two amplification rounds of 30 cycles each, the PCR products DBTRGO5MG,GBM, GLI5, GL22, Hu70, HulO4, Hul 12, Hul26, Hul95, were purified by the Gene Clean kit and cloned into the plasmid TA (Invitro Hu197, Hu255, Hu256, Hu257, T98G, U87MG, U138MG, and U373MG), 3 gen, San Diego, CA). To screen for the recombinant clones, bacterial colonies astrocytomas (l32INl, MOG-G-CCM, and MOG-G-UVW), 4 neuroblastomas were hybridized to the SV4O-specific oligoprobe and to a 20-base BKV (L697, SKNMC, SKNBE2C, and LAN5), and 1 meningioma (HM) have been specific oligoprobe (5'-AATC'VI'CATCCCA' FlFF lCA-3') comprising the described (7, 16â€”18)or purchased from the European Collection of Animal 9-base insert missing in the SV4Oearly region and showing a 4-base mismatch Cell Cultures (Salisbury, United Kingdom). Cell monolayers were grown at to the SV4Osequence among the residual nucleotides. Clones hybridizing to 37Â°CinDMEM or RPM! 1640 supplemented with 10% fetal bovine serum in the SV4O probe and not hybridizing to the BKV probe were picked and a 5% CO2 humidified atmosphere. sequenced by standard procedures as described previously (7). DNA and RNA Isolation. Each samplewas cut andmincedwith a single Indirect Immunofluorescence. Cells grown on glass coverslips were an disposable scalpel. Paraffin was removed from paraffin-embedded specimens alyzed by indirect immunofluorescence as described previously (22). Briefly, with xylol, ethanol, and acetone, whereas formalin-fixed specimens were cell monolayers were fixed in acetone and reacted with the anti-SV4OTag extensively rinsed with water. Cultured cells were scraped off the culture specific PablOl MAb (Santa Cruz Biotechnology, Santa Cruz, CA) and then vessel with a rubber policeman and sedimented by centrifugation. PBCs were with FITC-conjugated antimouse immunoglobulins (Sigma Chemical Co., isolated by gradient centrifugation onto Hystopaque-l077 and washed three Saint Louis, MO). Cos-l cells, expressing SV4OTag, and T53 cells from a times with saline. Samples of sperm fluid were stored at â€”80Â°Cuntilthe time BKV/tat transgenic mouse, expressing BKV Tag (23), were used as specific of analysis. All samples were digested with SDS and proteinase K followed by controls in each experiment. Other immunofluorescence tests were performed extraction with a mixture of phenol-chloroform-isoamyl alcohol (25:24:1), with the PablÃ³l4 MAb (24) and with hamster serum to BKV Tag (23), which dialyzed for 24 h with TEN buffer (10 mMTris, pH 7.5, 1 mMEDTA, 1 M react with both SV4Oand BKV Tag. NaCI) and for a further 24 h period with TE buffer, which is identical to TEN buffer but lacks NaCl. Total cytoplasmic RNA was extracted from cell lines RESULTS and tissues according to standard procedures as described (7). PCR, RT-PCR, and DNA Sequence Analysis. Plasmid pACTSV2 (19), Detection of SV4O Tag Coding Sequences in Human Brain containing the entire genome of the SV4O wild-type strain 776 (20, 21), was Tumors. In a preliminary PCR assay, three different recombinant used as a control in PCR amplification experiments and sequence analysis. plasmids carrying SV4O, BKV, or JCV DNA were amplified as a Each DNA sample was first tested for suitability for PCR analysis by ampli control with primers PYV.for and PYV.rev for the polyomavirus early fication of @-globingene sequences. Only positive samples were further region (Fig. 1A). To prove the specificity of the hybridization system, investigated for amplification of SV4O DNA sequences. To confirm the re producibility of PCR assays and to avoid problems related to contamination the three different PCR amplification products were hybridized to the during the PCR procedures, most of the samples were analyzed at least five SV4O oligoprobe. Only the PCR product containing SV4O early times by different operators in laboratories with appropriate PCR facilities. region sequences produced a positive signal (Fig. IB). Since brain Primers for f3-globin gene amplification were KM38 (5'-TGCITCTCCTFA tumors are frequently induced in experimental animals by SV4O, we AACCTGTCTrG-3') and PCO3 (5'-ACACAACTGTGUCACTAGC-3'). 5V40 early region sequences were amplified by using the primers PYV.for (5'-TAG GTGCCAACCFATGGAACAGA-3', from nucleotide 4574 to 4552 in the SV4O genome) and PYV.rev (5'-GAAAGTC'lTfAGGGTC'Vl'CTACC BK JC SV4O R@ 3', from nucleotide 4425 to 4402; Refs. 8 and 10). These primers allow amplification of an amino-tenninal Tag coding sequence, which is conserved in the early region of SV4O, BKV, and JCV, with a size of 172 bp for SV4O and 181 bp for BKV and iCy. The 172-bp sequence is therefore specific for SV4O because it lacks a 9-bp insert between positions 4516 and 4517 that is present in BKV and JCV sequences (8, 10, 20, 21). DNA (0.5 @g)was amplified in a total volume of 50 @lcontaining10 mMTris-HCI, pH 8.3, 50 A mM KCI, 1.5 mM MgCl2, 0.01% gelatin, 150 p@ ofeach dNTP, 50 nt@iof each primer. Taq DNA polymerase (0.75 units; Perkin-Elmer Cetus, Norwalk, CT) was added at 80Â°Cafter DNA denaturation at 95Â°Cfor 5 mm. Cycling parameters were 1 rain at 94Â°C,30s at 56Â°C,16s at 72Â°Cfor35 cycles. Sometimes the DNA extracted from paraffin-embedded tissues was partially degraded; in these cases, to avoid false-negative results, two subsequent PCR reactions, each of 35 cycles, were performed. PCR products were subjected to electrophoresis in 2.5% agarose gels and transferred to nylon membranes. DNA was cross-linked to filters by UV irradiation for 2 mm. All filters were BK JC SV4O R always hybridized to a SV4O-specificinternal oligoprobe (5'-ATGTTGAGA GTCAGCAGTAGCC-3'), which shows a 6-base mismatch with respect to the BKV sequence,at 37Â°CforI h in 5X SSPE (20X SSPE stock solution:3.6 M NaCl, 0.2 MNaH2PO4,0.02 MNa2EDTA),5X Denhardt's solution, 100 @&g/ml salmon sperm DNA, 0.5% SDS. The oligoprobe was previously end-labeled with 32Pby polynucleotide kinase. Hybridization for SV4Owas carried out in B high-stringency conditions (52Â°Cin2X SSC) as described by Bergsagel et aL (8). Autoradiography was performed at â€”80Â°C.overnightfor the control samples of viral DNA and for 3â€”idaysfor the samples of tumor and normal tissues. For RT-PCR, total cytoplasmic RNA (5 @g)wasresuspended in 100 @.dofa buffercontaining40 nmiTris-HCI,pH 7.5, 10 maiNaCl, 6 nmiMgCI2. Contaminant DNA was removed by two repeated treatments with RNase-free DNase (50 units; Boehringer Mannheim, Mannheim Germany) at 37Â°Cfor20 ruin followed by phenol extraction and ethanol precipitation. Reverse tran Fig. 1. A, agarose gel electrophoresis of PCR-amplified early region from control plasmids containing BKV, JCV, and SV4ODNA, stained by ethidium bromide. Lane R, scription was carried out with a kit from Invitrogen (San Diego, CA) as negative control of the PCR reaction without DNA template. The first lane on the left indicated by the supplier. The cDNA obtained was then amplified by PCR and contains the molecular weight markers. B, autoradiogram of the samples shown in A, subjected to Southern blot hybridization as described above. For DNA se hybridized to the oligoprobe specific for SV4Oearly region. Exposure time was overnight. 4821

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1996 American Association for Cancer Research. sv@io@r@oHUMANBRAINTUMORS C 1 2 3 4 5 6 Râ€” 1). These samples were reamplified by PCR for 35 cycles with negative results. Since the SV4O Tag coding sequences were detected in brain tumors but not in normal brain tissue, we addressed the analysis to other normal tissues such as PBCs, B and T lymphocytes, A$@ and sperm fluids. To this purpose, 70 PBC samples, 15 B and 15 T lymphocyte preparations, and 20 sperm fluid samples obtained from w w w. healthy individuals were analyzed. Sixteen of 70 (23%) PBC samples, 5 of 15 (33%) B lymphocyte and 3 of 15 (20%) T lymphocyte preparations, and 9 of 20 (45%) sperm fluid specimens were positive for SV4O Tag coding sequences (Table 1). C 1 2 3 4 R Detection of SV4O Early Region RNA. The expression of SV4O early region was investigated by RT-PCR only in brain tumor derived cell lines because the fresh biopsies of primary tumors were too small, allowing only the extraction of DNA, and the RNA extracted from @ . formalin-fixed and paraffin-embedded tissues was degraded, the B larger RNA bands being of only approximately 70 nucleotides. Seven of 9 (78%) glioblastoma multiforme cell lines and 1 astrocytoma cell line, which were positive for SV4O DNA, showed expression of SV4O Fig. 2. Hybridization of SV4Oearly region amplified DNA to a SV4O-specificoligo early region sequences when analyzed by RT-PCR (Fig. 2B; Table 1). probe. A, Lane C, SV4O early-region amplified from SV4O DNA cloned in plasmid DNA Sequence Analysis of the Amplified Products and Detec pACTSV2 as a control. Lanes 1-6, DNAS from primary brain tumors: Lane 1, glioblas toma multiforme; Lane 2, meningioma; Lane 3, choroid plexus papilloma; Lane 4, tion of Tag by Immunofluorescence. To assess the SV4Ospecificity ependymoma; Lane 5, astrocytoma; Lane 6, meningioma. Lane R, negative control of of the amplified DNA, sequence analysis was performed on the the PCR reaction without DNA template. B, hybridization of PCR-amplified cDNAs to a SV4O-specificoligoprobe. cDNAs were obtained by reverse transcription of total cyto amplified products from four different specimens: two primary brain plasmic RNAs from Cos-l, positive control (C), and four glioblastoma-derived cell lines tumors (one glioblastoma multiforme and one ependymoma) and two (Lanes 1â€”4).Lane R contains the PCR-negative control reaction without DNA. glioblastoma multiforme derived cell lines. Viral DNA sequences detected in the four samples were found to correspond to SV4O searched for SV4O early region sequences in 83 primary brain tumors Tag-coding sequences because they lacked the 9-bp insert between of different histotypes, in 26 brain tumor derived cell lines, and in 13 nucleotides 4516 and 4517, typical of BKV and JCV early region normal brain tissues used as a control. Using PCR, followed by sequences (Refs. 8, 10, 20, and 21; Fig. 3). Two samples (the ependy Southern blotting and hybridization with an internal oligoprobe spe moma and one glioblastoma cell line) had the sequence of wild-type cific for SV4O early region DNA, we detected SV4O Tag coding SV4O DNA, whereas the other two specimens (the primary sequences in 5 of 6 (83%) choroid plexus papillomas, 8 of 11 (73%) glioblastoma and the other glioblastoma-derived cell line) ependymomas, 8 of 17 (47%) astrocytomas, 10 of 30 (33%) glioblas showed point mutations. The primary glioblastoma had two distinct tomas, 1 of 7 (14%) meningiomas, 9 of 18 (50%) glioblastoma Câ€”sTand Aâ€”G transitions at codons 110 (CCAâ€”s'TCA)and 115 multiforme derived cell lines, and 1 of 3 (33%) astrocytoma cell lines (GAG-'+GGG), respectively, whereas the glioblastoma cell line had (Fig. 2A; Table 1). Thirteen normal brain tissues, 9 oligodendroglio only one mutation, which was the same Aâ€”*Gtransition at codon 115 mas, 2 spongioblastomas, 1 neuroblastoma, 1 meningioma, and 4 detected in the primary glioblastoma. These transitions occurred at neuroblastoma cell lines were all negative for SV4O sequences (Table nucleotides 328 (Câ€”*T)and 344 (Aâ€”+G)of the Tag coding sequence

Table1 Presenceof SV4OfluidsSV4O and BKV early region inhuman brain tumors, brain tumorcell lines, normal PBCs, and sperm sequenceTissues DNA, positiveBKV DNA, positiveSV4O RNA, positiveSV4O amplified DNA, analyzedPrimaryand cell linesasamples/samples analyzed (%)bsamples/samples analyzed (%)b.csamples/samples analyzed (%)dof samples braintumorsChoroid (100)Ependymoma8/11plexus papilloma5/6 (83)6/6 (91)1Astrocytoma8/17 (73)10/11 (94)Glioblastoma10/30 (47)16/17 (93)1Spongioblastoma0/22/2 (33)28/30 (100)Oligodendroglioma0/97/9 (77)Meningioma1/7 (57)Neuroblastoma0/11/1 (14)4/7 (100)Brain linesAstrocytoma1/3tumor cell (100)Glioblastoma9/18 (33)3/3 (100)1/1 (78)2Meningioma0/11/1 (50)14/18 (78)7/9 (100)Neuroblastoma0/43/4 (75)Normal tissuesBrain0/1313/13 (100)Peripheral (76)B blood cells16/70 (23)53/70 (86)Tlymphocytes5/15 (33)13/15 (93)Spermlymphocytes3/15 (20)14/15 fluids9/20 (45)18/20(90) aTotalnumberofspecimensis242:45freshbiopsies,44formalin-fixedandparaffin-embeddedsamples,7formalin-fixedsamples,26celllines,and100bloodand20spermfluid samples. b Early region SV4O and BKV DNA was detected by PCR amplification.

C Data concerning BKV DNA, except for sperm fluids and part of cell lines, PBCs, B lymphocytes, T lymphocytes, and brain tumor samples, were obtained from the results reported in the article by Dc Mattei et al. (7). d Early region SV4O RNA was detected by RT-PCR. 4822

ACGT A CGT 4, B, D, F, and H) when they were reacted with a hamster serum to BKV Tag (23) and with Pabl6l4 MAb (24), both of which react with both BKV and SV4O Tag.

DISCUSSION

The presence of SV4O sequences in human tumors is still a matter of investigation. Indeed, SV4O does not naturally infect humans, and human cells are only semipermissive for SV4O infection. However, 9bp Insert SV4O was introduced in the human population about 40 years ago by

BKV JCV contaminated polio vaccines. Some studies reported detection of SV4O DNA or Tag in human tumors by Southern blot hybridization or immunofluorescence, whereas others failed to detect SV4O footprints in human neoplastic tissues (3). Most of these early investigations were conducted by nucleic acid hybridization techniques, which show a limited sensitivity of detection, approximately 0.1 genome equiva lents per cell. More recently, Bergsagel et a!. (8) found SV4O early region sequences by PCR in ependymomas and choroid plexus tumors of children not exposed to SV4O-contaminated vaccines, and Carbone â€˜/1/I et a!. (10) detected SV4O DNA in human pleural mesotheliomas. An infectious SV4O wild-type strain was rescued by transfection of DNA from a human choroid plexus carcinoma into permissive monkey kidney cells (27). In our study, 9% of neoplastic patients positive for Fig. 3. DNA sequence analysis of the PCR-amplified products from control DNA of SV4O sequences were more than 33 years old in 1955, and 52% were SV4Owild-type strain 776, cloned in the vector pACl'SV2 (panel I), and from a primary born after 1965, suggesting that these two groups of patients had not ependymoma (panel 2). The SV4ODNA sequence is shown up to down in a clockwise direction from nucleotide 4450 to nucleotide 4530, corresponding to nucleotides 368 and been vaccinated with SV4O-contaminated polio vaccines. These ob 288 in the Tag coding sequence. The 9-bp sequence absent in SV4ODNA and present in servations therefore imply that SV4O has spread in the human popu BKV and JCV DNA is shown as an insert between SV4O nucleotides 4516 and 4517. The lation by horizontal infection. In our study, SV4O-Tag coding se SV4O DNA sequences in a primary glioblastoma and in a glioblastoma cell line were identical to that of the primary ependymoma, except for the presence of a Câ€”sTandan quences were detected in a high proportion of ependymomas and Aâ€”.Gtransition at nucleotides 4490 and 4474, respectively. choroid plexus papillomas, whereas other brain tumors, astrocytomas, glioblastomas, and meningiomas showed a lower frequency of posi tive samples. Normal brain tissue was negative for SV4O sequences. (corresponding to nucleotides 4490 and 4474 of the SV4O DNA This result is surprising, because SV4O sequences were detected in sequence), resulting in two amino acid substitutions from Pro to Ser PBCs and in B and T lymphocytes. However, the positive hybridiza and from Glu to Gly, respectively. Computer-assisted analysis of the tion signals of the amplified products from blood DNA samples were mutated Tags did not suggest any relevant conformational changes of always faint, indicating that the concentration of SV4O DNA in PBCs the proteins. However, the amino-terminal region of Tag, comprising is very low. Consequently, the SV4O DNA present in normal brain amino acid residues 102â€”115,is required for transformation because tissue due to circulation of PBCs is probably not detectable in our it binds the products of tumor suppressor genes plO5Rb, p107, and conditions of PCR amplification. Lack of SV4O sequences in normal p130, inactivating their functions (13, 25). Tag mutants in this region human brain and lung tissues was previously reported by others (8, loose transforming activity, although they maintain the ability to 10). On the other hand, a specific association of SV4O sequences with immortalize primary mouse and rat embryo fibroblasts (25, 26). It is tumor cells was demonstrated in this study by the presence of SV4O noteworthy that mutations in the Tag sequence, as compared to SV4O DNA in tumor cell lines. Cell lines derived from glioblastoma mul laboratory strains 776 and SV4O-B2, were detected in other strains of tiforme that were positive for SV4O DNA showed expression of SV4O SV4O isolated from humans (27), namely from a case of progressive early region sequences when analyzed by RT-PCR, and SV4O Tag multifocal leukoencephalopathy (28), a meningioma, an astrocytoma was detected by indirect immunofluorescence with a specific MAb. (29),andachoroidplexuscarcinoma(27). DNA sequencing of the amplification products from two primary Indirect immunofluorescence with the PablOl MAb specific for brain tumors and two brain tumor cell lines confirmed that they were SV4O Tag was carried out on seven glioblastoma cell lines and one derived from SV4O. Since we amplified and sequenced the amino astrocytoma cell line positive for the expression of SV4O RNA by terminal region of SV4O Tag and since the epitope recognized by RT-PCR (Table 1). Three of seven glioblastoma cell lines positive for PablOl MAb resides at the carboxy terminus of the protein, between SV4O RNA sequences showed a nuclear fluorescence typical of Tag, amino acid residues 512 and 708 (30), it is likely that the whole SV4O detectable as discrete foci of positive cells in the monolayer (Fig. 4, E Tag coding sequence is present in human brain tumors. Our data and G), whereas the single astrocytoma cell line expressing SV4O therefore indicate that SV4O Tag coding sequences can be detected in RNA was negative in the immunofluorescence test. Positive cells in a variety of human brain tumors, suggesting that SV4O is associated the immunofluorescence assay were estimated to be 1 out of 1,000. with human neoplasms with a higher frequency than previously re Since all brain tumor cell lines positive for SV4O sequences contain ported. It is not clear at present whether the SV4O sequences detected also BKV sequences (Table 1), controls were carried out to assess the in human brain tumors maintain their functional properties. The specificity of the reaction of the PablOl MAb with SV4O Tag. As mutated Tags in the amino-terminal region have probably lost their shown in Fig. 4, A and C, nuclear fluorescence was detected with transforming capacity but may still be able to immortalize cells (25, PablOl MAb on Cos-l cells expressing SV4O Tag, whereas it was 26). They might therefore be involved in the early steps of develop absent in T53 cells transformed by BKV (23). Positive signals were ment of human brain tumors. Rescue of infectious SV4O by transfec obtained in all cell lines (Cos-l, T53; and human tumor cell lines; Fig. tion of SV4O-positive tumor DNA into permissive monkey cells 4823

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Fig. 4. Detection of SV4O and BKV Tag by indirect immunofluores if cence. Nuclear fluorescence was detected with MAb PablOl, specific for SV4O Tag, in Cos-1 cells, expressing SV4O Tag and used as a positive control (A) in the two glioblastoma cell lines, Hul95 (E) and Hul97 (G), whereas in T53 cells (C), transformed by BKV and used as negative D control, fluorescence was absent. Tag-specific nuclear fluorescence was also detected with a hamster polyclonal serum to BKV Tag, which cross reacts with SV4O Tag, in cell lines Cos-l (B), T53 (D), Hul95 (F), and Hu197 (H). Specific fluorescence for SV4O Tag was detected as discrete foci in the monolayer. A. X500; Bâ€”H,X250. ..i@ .@ *

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would allow researchers to assess the biological activity of Tag coded polyomaviruses and SV4O to other tissues of the host. The presence of by the SV4O sequences detected in human brain tumors. Interestingly, SV4Osequences in sperm fluids, as previously demonstrated for BKV SV4O early region sequences were also detected in PBCs, B and T and JCV (33), indicates that sexual transmission may contribute to lymphocytes, and sperm fluids from healthy individuals. This result, spreading infection by polyomaviruses in humans. All specimens in in agreement with the detection of BKV and JCV sequences in PBCs this study, brain tumors, PBCs, B and T lymphocytes, and sperm (7, 31, 32), suggests that PBCs may be vectors for transfer of human fluids positive for SV4O sequences were found in previous investiga 4824

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1996 American Association for Cancer Research. sv@sou@@uHUMANBRAINTUMORS tions to contain BKV DNA (Refs. 7, 9, and 34; Table 1), suggesting coding sequences in normal human tissues and tumors of different histotypes. mt. j. that BKV may act as a helper for SV4O replication. Formation of Cancer, 61: 756â€”760,1995. 8. Bergsagel, D. J., Finegold, M. J., Butel, I. S., Kupsky, W. J., and Garcea, R. L. DNA hybrid viruses between SV4O and BKV coinfecting the same cells sequences similar to those of simian virus SV4Oin ependymomas and choroid plexus cannot be ruled out. It is possible, therefore, that circulation of SV4O tumors of childhood. New EngI. J. Med., 326: 988â€”993,1992. 9. Martini, F., Dc Mattei, M., Iaccheri, L., Lazzarin, L., Barbanti-Brodano, G., Tognon, in the humanpopulationis enhanced by the helper functions of the M., and Gerosa, M. Human brain tumors and simian virus 40. J. Nail. Cancer Inst., ubiquitous BKV infecting the same hosts. Under these conditions, 87: 1331, 1995. SV4O could establish a latent infection in specific human cells and 10. Carbone, M., Pass, H. I., Rizzo, P., Mannetti, M. R., Di Muzio, M., Mew, D. J. Y., Levine, A. S., and Procopio, A. Simian virus 40-like DNA sequences in human display its oncogenic potential. pleural mesothelioma. Oncogene, 9: 1781â€”1790,1994. The incidence of human brain tumors has risen about 30% in the 11. Shah, K. V., and Nathanson, N. Human exposure to SV4O:review and comments. last 20 years (2). The reasons for this effect are not known, although Mn. J. Epidemiol., 103: 1â€”12,1976. 12. Topp, W. C., Lane, D., and Pollack, R. Transformation by SV4O and polyomavirus. it is firmly established that the increase is real and not simply due to in: J. Tooze (ed), DNA Tumor Viruses, Part 2, pp. 205â€”296.Cold Spring Harbor, improvement in diagnostic procedures. 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Fernanda Martini, Laura Iaccheri, Lorena Lazzarin, et al.

Cancer Res 1996;56:4820-4825.

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