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 (4—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 (8—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 —80°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'Universitá 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 Sanità 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 “ApplicazioniCliniche della Ricerca Oncologica―(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, 16—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 37°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,