VIROLOGY

90, 187-196

Evaluation

(1978)

of Normal and Neoplastic

Human Tissue for BK Virus

MARK A. ISRAEL,* MALCOLM A. MARTIN,* KENNETH PETER M. HOWLEY,$ STUART A. AARONSON,§ DIANE GEORGE KHOURYB

K. TAKEMOTO,? SOLOMON,1 AND

’ Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, iLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, SLaboratory of Pathology, National Cancer Institute, §Laboratory of RNA Tumor Viruses, National Cancer Institute, and BLaboratory of DNA Tumor Viruses, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Accepted June 22, 1978 Despite an extensive search using sensitive DNA hybridization techniques, we were unable to demonstrate the presence of BK Virus DNA sequences in human neoplastic tissues. Evaluation of numerous human tumor cell lines for BK Virus T-antigen and sera from patients with known malignancies for anti-BK Virus T-antibody was also negative. INTRODUCTION

BK Virus (BKV) is a human papovavirus originally isolated from the urine of a renal allograft recipient on immunosuppressive therapy (Gardner et al., 1971). Seroepidemiologic studies indicate that this virus is ubiquitous even among diverse populations of the world (Brown et al., 1975), yet to date, it has been isolated only from immunologically compromised individuals. Although seroconversion indicates that exposure to BKV occurs in early childhood (Gardner, 1973; Shah et al., 1973; Mantyjarvi et al., 1973; Portolani et al., 1974), the nature of the primary infection is not known nor has BKV been implicated as the etiologic agent of any human disease. Morphologically, BKV is related to the simian virus 40 (SV40) and polyoma virus group of papovaviruses. Like SV40 and polyoma, BKV can be productively propagated in the laboratory in certain cell cultures derived from its natural host. Also like SV40 and polyoma, the oncogenic potential of BKV has been established outside of its natural host as evidenced by transformation of cells in culture (Major and DiMayorca, 1973; Portolani et al., 1975; Takemoto and Martin, 1976; Tanaka et al., 1976; Sten et al., 1976; van der Noorda,, 1976; Mason and Takemoto, 1977; Seehafer et al., 1977) and tumor induction in newborn hamsters (Shah et al., 1975; N&e et al., 1975; Costa

et al., 1976; Dougherty, 1976; IJchida et al., 1976; Corallini et al., 1977). Recently, Fiori and diMayorca (1976) reported finding DNA sequences homologous to BKV DNA in DNA preparations from five of twelve human tumor tissues and three of four human tumor cell lines. No BKV sequences were detected in DNA prepared from 12 normal human tissues. Fiori and DiMayorca calculated that from 0.4 to 1.6 BKV genome equivalents per diploid cell were present in seven of the eight tissues found to contain BKV DNA sequences, and 11.0 BKV genome equivalents per diploid cell were present in the eighth positive tissue. In this study, we report the results of our efforts to evaluate the role of the human papovavirus BKV in human neoplasia. Utilizing nucleic acid hybridization studies to survey a broad range of tissues, we were unable to detect BKV DNA sequences in any of the human tumors or human tumor cell lines examined. None of the human tumor cell lines contained BKV T-antigen as monitored by indirect immunofluorescence and no antibodies specific for BKV T-antigen were found in patients with documented malignancy. MATERIALS

AND

METHODS

Cells and Virus Primary human embryonic kidney (HEK) cells (Microbiological Associates,

187 0042~6822/78/0902-0187$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Walkersville, Md.) were propagated in Eagle’s medium containing 2X vitamins and amino acids supplemented with 10% fetal calf serum. Human tumor cell lines were propagated in Dulbecco’s modified Eagle’s Minimal Essential Medium (MEM). The MM isolate of BKV (BKV-MM) was used in our experiments (Howley et al., 1975). Human Tissues and Human Tumor Cell Lines Normal human tissues as well as human tumors were obtained at autopsy from the Laboratory of Pathology of the National Cancer Institute. Human tumor cell lines were isolated as previously described (Giard et al., 1973). Preparation of BKV-MM DNA Unlabeled and 32P-labeled BKV(MM) virions were purified as previously described (Howley et al., 1975) from primary HEK cells infected at a multiplicity of infection (m.o.i.) of 0.1 PFU/cell with plaquepurified BKV(MM) and maintained in either Eagle’s MEM or phosphate-free Eagle’s MEM containing 60 pCi/ml of carrierfree [32P]orthophosphate (New England Nuclear). Unlabeled and 32P-radiolabeled BKV(MM) DNA was prepared from purified virions by incubation in 1% Sarkosyl at 50’ for 1 hr followed by sedimentation through a neutral sucrose gradient (5-30s w/v). BKV(MM) DNA was labeled in vitro essentially as described by Maniatis et al. (1975), except that the reaction mixture contained 1 X lo-’ pg/ml of DNase (Worthington) .

ET AL. Restriction Endonuclease Cleavage of DNA 32P-BKV-MM DNA was cleaved with R.Eco RI and R. Hind III as previously described (Howley et al., 1975). Cellular DNA was similarly cleaved with R&o RI at a DNA concentration of 400 pg/ml. R. Bum HI cleavage of cellular DNA (400 pg/ml) was performed at 37” for 2 hr in a reaction mixture containing 20 n&f Tris-HCl, pH 7.5, 7 miJ4 MgC12, and 2 mM /?-mercaptoethanol. In each case, the enzymatic reactions were monitored for complete cleavage of cellular DNA by evaluating the conversion of “‘P-labeled SV40 DNA I to linear [32P]SV40 DNA III in aliquots of the individual restriction enzyme reaction mixtures. Gel Electrophoresis 32P-Labeled viral DNA and R.Eco RI cleaved [32P]BKV-MM DNA were electrophoresed in a 1.4%agarose slab gel (17 x 12 X 0.3 cm) for 16 hr at 65 V as previously described (Howley et al., 1975). R. Hind III cleaved [32P]BKV DNA was electrophoresed in a composite 3% polyacrylamide, 0.5% agarose slab gel (17 X 12 X 0.3 cm) for 10 hr at 50 V as previously described (Howley et al., 1977).

DNA-DNA Reassociation a. Evaluation of reassociation kinetics. [32P]BKV(MM) DNA and unlabeled cellular DNAs were mechanically fragmented at 50,000 lbs/in2 in a Ribi cell fractionator as previously described (Gelb et al., 1971), mixed in a molar ratio of 1.0, heat denatured, and allowed to reassociate in reaction mixtures containing 0.6 M sodium phosphate buffer (pH 6.8) at 68”. Portions of Preparation of Cellular DNA the reaction mixtures were removed at varHigh molecular weight DNA from tissue ious times and assayed for the fraction of radiolabeled DNA remaining singleculture cells was prepared as previously described (Gelb et al., 1971). DNA from stranded (fss) by hydroxylapatite chromahuman tissues and hamster embryos was tography (Gelb et al., 1971). b. Evaluation of DNA “blots”. Restricsimilarly prepared from cell nuclei (Gelb et al., 1971). DNA from E. coli was prepared tion enzyme-cleaved, cellular DNA (20 essentially as described by Marmur (1961). ~g/sample) was electrophoresed in 1.0% Salmon sperm DNA (Sigma) was depro- agarose slab gels at 15 V for 20 hr, denateinized with phenol and precipitated with tured in situ (Botchan et al., 1976) and transferred to a sheet of nitrocellulose 2 volumes of ethanol before use.

EVALUATION

OF BKV

(BA85, Schleicher and &hull) as described by Southern (1975). The nitrocellulose sheet was then incubated in 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and 4X SSC for at least 4 hr at 60” (Denhardt, 1966). Hybridizations were carried out at 60” for 16 hr in reaction mixtures (2 ml) which contained 1.0 M NaCl, 2 mM TES, pH 8.0, 0.5% sodium dodecyl sulfate, 1 n-G!4EDTA, 2 pg/ml yeast RNA, 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and 0.2 ,ug of denatured in vitro 32P-labeled BKV(MM) DNA (specific activity s6 X lo7 cpm/pg). The nitrocellulose sheets were then extensively washed with 4X SSC at 60”, dried, and subjected to autoradiography for 2-4 weeks on Kodak XR-2 film using a Cronex lightening-plus intensifying screen. FA Tests The indirect procedure for the fluorescent antibody (FA) test was used as previously described (Takemoto and Martin, 1976).

IN HUMAN

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NEOPLASIA

1975) was used in our experiments because after plaque purification, it can be passaged at a low m.o.i. without generation of detectable defective forms. We have previously shown that the prototype BKV and BKV(MM) DNAs are indistinguishable by hybridization techniques (Howley et al., 1975). The homogeneity of the purified radiolabeled BKV(MM) DNA probe was evaluated by electrophoresis in 1.4% agarose (Fig. 1, lane A) and its mobility compared to that observed with [32P]SV40 DNA (lane B). The faster migrating supercoiled and slower migrating nicked circular forms (lane A) run as single, discrete bands; smaller molecules, migrating more rapidly

ABC

D

RESULTS

Characterization of f2P]BKV-MM DNA Two types of nucleic acid hybridization procedures, reassociation kinetics (Gelb et al., 1971) and the Southern DNA transfer technique (Southern, 1975; Ketner and Kelly, 1976; Botchan et al., 1976), were employed to assay for the presence of BKV DNA sequences in normal and malignant cells. Both techniques require the use of homogeneous, radiolabeled viral DNA probes free from contaminating host cell DNA sequences. It is known that propagation of papovaviruses at a high m.o.i. or by serial undiluted passage leads to rearrangements of the viral DNA and to the insertion of host cell sequences into the viral genome. In our experience, defective viral genomes arise quite early during the growth of BKV even when the virus is plaque-purified and grown in primary HEK cells at very low m.o.i. Thus, it was particularly important that the DNA probes used in these experiments be carefully characterized. The MM isolate of BKV (BKV-MM) (Howley et al.,

FIG. 1. Electropherograms of “‘P-labeled BKV(MM) DNA. (A) “P-labeled BKV(MM) DNA. (B) “‘P-labeled SV40 DNA. (C) R. Eco RI digestion of “‘Plabeled BKV(MM) DNA. (D) R.Hin&II digestion of 32P-labeled BKV(MM) DNA. Samples A, B, C were electrophoresed in 1.4% agarose slab gels as described in Materials and Methods. Sample D was electrophoresed in a composite 3% acrylamide, 0.5% agarose slab gel as described in Materials and Methods.

190

ISRAEL ET AL.

than the supercoiled and nicked circular forms of BKV DNA and representing defective viral genomes possibly containing cellular sequences, were not detected. The labeled viral DNA was further characterized by digestion with restriction endonucleases. All of the viral DNA was sensitive to R.&o RI and converted to full length linear molecules (lane C). Cleavage with R.Hind III and electrophoresis in a 3% acrylamide, 0.5% agarose gel revealed three equal molar fragments (lane D) (Howley et cd., 1975). This radiolabeled BKV(MM) DNA probe was used to detect viral DNA sequences in a BKV-transformed hamster cell line (BK-HK-6) (Howley and Martin, 1977). The 32P-labeled viral DNA, reannealing in the presence of fragmented BKHK-6 DNA, reassociated with tl/z (time for the reassociation of 50% of the radiolabeled probe) corresponding to the presence of 3.0 viral DNA equivalents per diploid mass of cellular DNA. This value is in good agreement with previously published results (3.1 copies per diploid mass of DNA) (Howley and Martin, 1977). Analysis of Cellular DNA for Viral DNA Sequences a. Reassociation kinetics. Reassociation kinetics have been successfully employed to quantitate the number of copies of integrated viral DNA in a variety of transformed cell lines (Gelb et al., 1971). We used the same radiolabeled BKV-MM DNA described above to search for viral DNA sequences in cellular DNA from a variety of tissues. When 32P-labeled BKV(MM) DNA was allowed to reassociate in the presence of fragmented DNA prepared from E. coli, salmon sperm, two independent groups of hamster embryos, or primary HEK cells, the tl12 ranged from 25.5 to 28.5 hr. In these studies, such values correspond to the absence of additional BKV(MM) DNA sequences (Fig. 2, panel A) in the added cellular DNA. The failure of HEK cellular DNA to accelerate the reassociation of 32Plabeled viral DNA further indicates that no contamination of the radiolabeled probe by human DNA sequences exists as detected

by this assay. The molar ratio of cellular DNA to viral probe DNA in these experiments and in experiments to be described below was held constant at 1.0. In reconstruction experiments (data not shown), we were able to confidently detect as little as 0.1 copy of BKV DNA per diploid mass of cellular DNA (or approximately 1 copy per diploid genome if only 10%of the cells from which the DNA was prepared contained one viral genome). DNA preparations from twelve normal organs from eight patients were examined by reassociation kinetic analysis for the presence of viral DNA sequences, and the results of these experiments are shown in panels B, C, and D of Fig. 2. The solid lines are the theoretical curves indicating the pattern of reassociation of the probe in the presence of 0, 0.1, 0.25, or 1.0 copies of the viral genome per diploid cellular genome. Six of the twelve DNAs examined did not accelerate the reassociation of the radiolabeled viral DNA (Fig. 2, panel D); three of the preparations caused a small but definite acceleration, corresponding to approximately 0.25 viral DNA equivalents per diploid cellular genome (Fig. 2, panel B), and the remaining three cellular DNA preparations accelerated the viral probe to an extent indicative of the presence of between 0.1 to 0.25 viral DNA equivalents per diploid cellular genome (Fig. 2, panel C). It is of interest to note that brain DNA, which contained less than 0.1 viral DNA equivalents per diploid cell genome (Fig. 2, panel D), was isolated from the same patient (221) whose spleen DNA contained approximately 0.25 viral DNA equivalents. The same 32P-labeled viral DNA probe was also used to monitor cellular DNA prepared from human tumors and human tumor cell lines for the presence of BKV(MM) DNA sequences. No DNA preparation from any human tumor cell line (Fig. 3, panel A) we examined accelerated reassociation of the viral DNA probe to an extent equivalent to the presence of more than 0.1 copy of the viral genome per cell. Similarly, DNA preparations from a number of human tumors did not significantly affect the reassociation of the labeled BKV(MM) DNA probe, indicating the ab-

EVALUATION

OF BKV

IN HUMAN

NEOPLASIA

191

FIG. 2. Reassociation kinetics of [32P]BVK(MM) DNA in the presence of various cellular DNAs. [“‘PIBKV(MM) DNA and unlabeled cellular DNAs were mixed, heat denatured, and allowed to reassociate in reaction mixtures containing 0.6 M sodium phosphate buffer (pH 6.8) at 68’ as described in Materials and Methods. Portions of the reaction mixtures were removed at various times and assayed for the fraction of radiolabeled DNA remaining single-stranded (fss) by hydroxylapatite chromatography. The lines presented represent the least squares best fitting straight line determined by at least six independently determined experimental points. Curve fitting computations were performed on a DEC system-10 computer using the MLAB computer program. Panel A, reassociation of [32P]BKV(MM) DNA in the presence of unlabeled DNA from hamster embryos (--and - -), human embryonic kidney cells (- - - -), E. coli (- - -), and salmon sperm (-----). Panel B, C, D, reassociation of [3ZP]BKV(MM) DNA in presence of DNA from normal human organs. Solid lines represent the hybridization pattern expected when 0, 0.1,0.25, or 1.0 copy of viral DNA is present in the unlabeled cellular DNA. Dashed lines represent the experimentally determined patterns of reassociation for various human DNA preparations. These are described from top to bottom in each panel. Panel B, brain (patient 641), spleen (patient 221), brain (patient 666). Panel C, kidney (patient 290), kidney (patient 180), spleen (patient 180). Panel D, kidney (patient 182), spleen (patient 182), brain (patient 175), kidney (patient 162), brain (patient 221), kidney (patient 175).

sence of detectable viral DNA sequences (Fig. 3, panel B) in such cellular DNA preparations. Cell lines A-549 and A-375 shown in Fig. 3, panel A, have previously been reported by Fiori and DiMayorca (1976) to each contain 0.4 copy of BKV DNA per diploid cell. b. ‘Blotting” analysis. The Southern “blotting” procedure (Southern, 1975) was also employed to detect BKV(MM) DNA

sequences in human tissues. This technique is particularly valuable for distinguishing between integrated and free copies of viral DNA and for providing information about the pattern of virus DNA integration into the host genome (Ketner and Kelly, 1976; Botchan et al., 1976). For these experiments, we used BKV(MM) DNA, purified from virions and characterized by gel electrophoresis to be free of altered genomes,

192

ISRAEL ET AL.

‘/fss

Vfss

HOURS FIG. 3. Reassociation kinetics of [3ZP]BKV(MM) DNA in the presence of cellular DNA from human tumor cell lines (panel A) and human tumors (panel B). These experiments were carried out and analyzed exactly as described in Fig. 2. Solid lines represent the hybridization pattern expected when 0, 0.1,0.25, or 1.0 copy of viral DNA is present in the unlabeled cellular DNA. The DNA preparations from human cell lines (Panel A) and tumors (Panel B) are listed from top to bottom in each panel. Panel A, A-375 (melanoma), A-1186 (rhabdomyosarcoma), A-1117 (carcinoma, intestine), A-673 (rhabdomyosarcoma), A-1146 (carcinoma, lung), A-1632 (fibrosarcoma), A-1306 (melanoma), A-363 (epidermoid carcinoma), A-549 (carcinoma, lung), A-1207 (glioblastoma), A-704 (carcinoma, kidney), A-1115 (adenocarcinoma, undetermined primary, metastatic to brain), A1336 (ovarian cell tumor), A-1235 (astrocytoma), A-1186 (carcinoma, lung), A-204 (rhabdomyosarcoma). Panel B: 72-232-l (reticuloendothelioma), FS136C (melanoma), 74-71 (carcinoma, omentum), 76-198 (carcinoma, rectum), 74-166 (melanoma). Other tumors from which DNA was examined for BKV(MM) homologous sequences and were negative included a carcinoma of the prostrate, a renal cell carcinoma, an astrocytoma, and a Wilm’s tumor.

EVALUATION

OF BKV

which had been highly radiolabeled (specific activity Z 6 X lo7 cpm/pg) in vitro. DNAs prepared from two human tumors, three human tumor cell lines, and five normal human tissues reported in Figs. 2 and 3 were digested with either R.EcoRI or R.BamHI and analyzed as described in Materials and Methods. Figure 4 shows a series of autoradiograms which are typical of the results we obtained. Several sharp bands were observed when R.EcoRI cleaved DNA

O-ABCDEFG

FIG. 4. Autoradiogram of nitrocellulose filter hybridization of cellular DNA following restriction endonuclease digestion and transfer from agarose gels. Restriction endonuclease cleaved cellular DNA was electrophoresed in an agarose slab gel, denatured in situ, transferred to a sheet of nitrocellulose paper, and reacted with denatured 32P-labeled BKV(MM) DNA (specific activity 16 x lo7 cpm/pg) as described in Materials and Methods. The nitrocellulose sheets were then extensively washed with 4~ SSC at W, dried, and subjected to autoradiography for 2-4 weeks on Kodak XR-2 fiim using a Cronex lightening-plus intensifying screen. The DNA preparations shown here are REcoRI-cleaved BK-HK-6 DNA (A), R.EcoRIcleaved BKV(MM) DNA mixed with R.EcoRIcleaved salmon sperm DNA (B), R.BamHI-cleaved HEK DNA (C), R.BamHI-cleaved human spleen DNA (patient 221) (D), R.BamHI-cleaved DNA from tumor line A-549 (E), R.BamHI-cleaved DNA from tumor line A-204 (F), and R.BamHI-cleaved DNA from a carcinoma of the rectum (76-198) (G).

IN HUMAN

NEOPLASIA

1%

from the BKV hamster-transformed line BK-HK-6 (3.1 copies of viral DNA per cell) was examined (Hawley and Martin, 1977) (Fig. 4, panel A). A single band was detected at the position to which the full length linear viral genomes migrate (Fig. 4, panel B) in a reconstruction experiment in which the equivalent of 1 copy of R.EcoRI cleaved BKV(MM) DNA per diploid cellular genome was mixed with R.EcoRI-cleaved salmon sperm DNA. In each of the human DNA preparations digested with R.EcoRI, we observed a similar series of very faint bands; a different series of faint bands was also seen when these cellular DNA preparations were cleaved with R.BamHI (Fig. 4, panel C-G). Panel C depicts the hybridization pattern obtained with BamHI-digested DNA from human embryonic kidney cells. A similar pattern was also observed with BamHI-digested cellular DNA prepared from normal human organs (e.g., Fig. 4, panel D), human tumors (e.g., Fig. 4, panel G) or human tumor cell lines (e.g., Fig. 4, panel E, F). Reconstruction experiments, done in parallel with these “blotting analyses” and employing the same in uitro-labeled BKV(MM) DNA probe, indicated that as little as 0.25 copy of viral genome could be reliably detected if it were located in a single gel band (data not shown); no bands of this intensity were observed in any of the human DNA preparations examined. One of the three cellular DNA preparations isolated from normal human tissues which accelerated the reassociation of the BKV(MM) DNA probe (approximately 0.25 copy of viral DNA per diploid cellular genome) (Fig. 2, panel B) was indistinguishable (Fig. 4, panel D) by the “blotting” procedure from the other human DNA preparations. Analysis of Human Tumor Cell Lines and Sera of Cancer Patients for Evidence of BKV T-Antigen

Intranuclear antigens are readily detected in cell lines derived from tumors induced by inoculation of papovaviruses into animals or in cultured cells transformed by these agents. We, therefore, examined each of the established cell lines from human tumors cited in Fig. 3 by the

194

ISRAEL

indirect fluorescent antibody technique for the presence of BKV T-antigen. None of the tumor cell lines contained BKV T-antigen. We subsequently evaluated four other kidney tumor cell lines, six additional brain tumor cell lines, and 29 other tumor cell lines derived from a wide variety of cancerous tissues. All 55 tumor cell lines examined were negative for BKV T-antigen (Table 1). Evaluation of these human tumor cell lines for JC virus T-antigen by indirect immunofluorescence was also negative (data not shown). We have also sought serologic evidence for the presence of anti-BKV T-antibody in patients with a variety of malignancies. Sera samples from 67 different cancer patients were examined by the indirect fluorescent antibody technique using BKVtransformed hamster cells and all were negative for anti-BKV T-antibody (Table 2). Further examination of these sera for the presence of anti-JC virus T-antibody was also negative. TABLE

1

HUMAN TUMOR CELL LINES NEGATIVE FOR TANTIGEN No. tested

Tumor

5

Kidney Brain: Astrocytoma Glioblastoma Medulloblastoma Mesenchymal sarcoma Melanoma Rhabdomyosarcoma Fibrosarcoma Meningioma Lung Others Total

TABLE

4 2 1 1 5 7 3 8 6 13 55

2

SERUM FROM SELECTED CANCER PATIENTS NEGATIVE FOR T-ANTIBODY Type of tumor Renal cell carcinoma Medulloblastoma Glioblastoma Bladder carcinoma Esophageal carcinoma Total

No. tested 8 10 a 20 21 67

ET AL. DISCUSSION

Despite an extensive search using very sensitive techniques we were unable to demonstrate an association between BKV and the human neoplasms which we investigated. In all, 55 human tumor cell lines were negative for BKV T-antigen by indirect immunofluorescence, serum samples from 67 cancer patients did not contain detectable levels of antibody specific for BKV T-antigen, and 5 tumors as well as 16 tumor cell lines were negative for sequences homologous to BKV DNA. Inasmuch as BKV can replicate in human embryonic kidney cells and has been isolated from the urine of immunosuppressed patients, an effort was made to examine urologic tumors. Two renal tumor cell lines were negative by nucleic acid hybridization studies and five kidney tumor lines were negative for T-antigen. Twentyeight of the sera which did not contain antibodies to BKV T-antigen were from patients with urinary tract malignancies. Because another papovavirus, JCV, has been associated with progressive multifocal leukoencephalopathy (Narayan et al., 1973) and considering that JCV shares DNA sequences with BKV (Howley et al., 1975), we examined human brain tumors for evidence of an association with BKV. Two different types of brain tumors we examined were negative for BKV DNA sequences, six human brain tumor lines were negative for T-antigen, and the sera of 18 patients with brain tumors were negative for antibodies to T-antigen. These findings are compatible with data from several other laboratories. Two groups (Corallini et al., 1976; Costa et al., 1977) which have examined sera from large numbers of cancer patients for papovavirus Tantibody have also been unable to detect an association between these viruses and human neoplasia. Also, Shah et al. (1978) have examined 40 cell lines derived from human tumors of the urogenital tract and found them negative by immunofluorescence for the presence of BKV, JC virus or SV40 T-antigen. Recently, Wold et al. (1978), using a saturation hybridization assay, tested 166 human tissues and 12 human tumor cell lines for the presence of BKV

EVALUATION

OF BKV

IN HUMAN

DNA sequences. DNA from all of the human tissues examined hybridized to 5% of the BKV DNA probe; no tumor or tumor cell line DNA preparation hybridized further. Our evaluation of the human tumor lines A375 and A549 confirms the finding of Wold et al. that no detectable BKV DNA sequences are present; these lines had previously been reported by Fiori and diMayorca (1976) to each contain 0.4 copy of BKV DNA. On the basis of these findings, it seems unlikely that BKV is commonly associated with human cancer. The DNA sequences homologous to BKV DNA in normal human tissues need to be further evaluated. Wold et al. (1978) also found some homology between their BKV DNA probe and human DNA. The multiple, faint bands seen in “blots” of all human DNA samples most likely reflect the presence of minute amounts of human DNA present in the radiolabeled BKV(MM) probe at levels too low to be detected by reassociation kinetics. Indeed, the pattern observed (viz. the series of faint yet discrete bands) is reminiscent of that seen when mammalian cell DNA is digested with restriction enzymes, electrophoresed, and stained with ethidium bromide (Botthan, 1974). Even though such an explanation would account for our data as well as that of Wold et al. (1978), the possibility that we may each be detecting small amounts of viral DNA sequences has not been excluded. Experiments currently in progress hopefully will provide not only further clarification of this issue, but also a better understanding of the biology of human papovaviruses. ACKNOWLEDGMENTS We thank George Hutchinson and Gary Knott of the Division of Computer Research and Technology, NIH, for their introduction to the Decsystem-10 Computer. We also thank Susan Hostler for her secretarial assistance. REFERENCES BOTCHAN, M. R. (1974). Bovine satellite I DNA consists of repetitive units 1,400 base pairs in length, Nature 251, 228. BOTCHAN, M., TOPP, W., and SAMBROOK, J. (1976). The arrangement of simian virus 40 sequences in DNA of transformed cells. Cell 9, 269-287.

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BROWN, P., TSAI, T., and GAJDUSEK, D. C. (1975). Seroepidemiology of human papovaviruses. Amer. J. Epidemiol. 102,331-340. CORALLINI, A., BARBANTI-BRODANO, G., BORTOLONI, W., NENCI, I., CASSAI, E., TAMPIERI, M., PORTALONI, M., and BORGATTI, M. (1977). High incidence of ependymomas induced by BK virus, a human papovavirus. J. Nat. Cancer Inst. 59, 1561-1563. CORALLINI, A., BARBANTI-BRODANO, G., PORTOLANI, M., BALBONI, P. G., GROSSI, M. P., POSSATI, L.. HONORATI, C., LAPLACA, M., MAZZONI, A., CAPUTO, A., VERONESI, U., BREFICE, S., and CARDINALI, G. (1976). Antibodies to BK Virus Structural and Tumor Antigens in Human Sera from normal persons and from patient& with various diseases, including neoplasia. Infect. Immun. 13, 1684-1691. COSTA, J., YEE, C., TRALKA, T. S., and RABSON, A. S. (1976). Hamster ependymomas produced by intracerebral inoculation of a human papovavirus (MMV). J. Nat. Cancer Inst. 56,863-864. COSTA, J., YEE, C., and RABSON, A. S. (1977). Absence of papovavirus T antibodies in patients with malignancies. Lancet 2, 709. DENHARDT, D. (1966). A membrane-fiiter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23,641-646. DOUGHERTY, R. M. (1976). Induction of tumors in Syrian hamsters by a human renal papovavirus, RF strain. J. Nat. Cancer Inst. 57,395-400. FIORI, M., and DIMAYORCA, G. (1976). Occurrence of BK virus DNA in DNA obtained from certain human tumors. Proc. Nat. Acad. Sci. USA 73, 4662-4666. GARDNER, S. D. (1973). Prevalence in England of antibody to human polyomavirus (B.K.) Brit. Med. J. 1, 77-78. GARDNER, S. D., FIELD, A. M., COLEMAN, D. V., and HULME, B. (1971). New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet 1, 1253-1257. GELB, L. D., KOHNE, D. E., and MARTIN, M. A. (1971). Quantitation of Simian Virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes. J. Mol. Biol. 57.129-145. GIARD, D. J., AARONSON, S. A., TODARO, G. J., ARNSTEIN, P., KERSEY, J. H., DOSIK, H., and PARKS, W. P. (1973). In vitro cultivation of human tumors: Establishment of cell lines derived from a series of solid tumors. J. Nat. Cancer Inst. 51, 1417-1424. HOWLEY, P. M., KHOURY, G., BYRNE, J. C., TAKEMOTO, K. K., and MARTIN, M. A. (1975). Physical map of the BK virus genome. J. Viral. 16,959-973. HOWLEY, P. M., and MARTIN, M. A. (1977). Uniform representation of the human papovavirus BK genome in transformed hamster cells. ,I. Viral. 23, 205-208. KETNER, G., and KELLY, T. J. (1976). Integrated Simian Virus 40 sequences in transformed cell DNA: Analysis using restriction endonucleases. Proc. Nat.

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Evaluation of normal and neoplastic human tissue for BK virus.

VIROLOGY 90, 187-196 Evaluation (1978) of Normal and Neoplastic Human Tissue for BK Virus MARK A. ISRAEL,* MALCOLM A. MARTIN,* KENNETH PETER M...
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