Proc. Nati. Acad. Sci. USA Vol. 75, No. 1, pp. 454-458, January 1978 Medical Sciences
Analysis of human tumors and human malignant cell lines for BK virus-specific DNA sequences (molecular hybridization/BK virus-transformed hamster cells/human cancer)
WILLIAM S. M. WOLD, JESSE K. MACKEY, KARL H. BRACKMANN, NOBUYUKI TAKEMORI, PATRICIA RIGDEN, AND MAURICE GREEN Institute for Molecular Virology, St. Louis University School of Medicine, 3681 Park Avenue, St. Louis, Missouri 63110
Communicated by Robert M. Chanock, October 11, 1977
express T antigen, and are tumorigenic in hamsters (12, 14, 15). BKV is related to but distinct from simian virus 40 (SV40) and JC virus, another human papovavirus (18). BKV and SV40 T antigens crossreact strongly (9, 12, 14, 15, 19, 20) (the SV40 T antigen is believed to be a protein encoded by the SV40 early region and to play a role in SV40-induced cell transforma-
Most humans in the United States have been ABSTRACT infected with BK virus (BKV), a human papovavirus. Because BKV has oncogenic properties, we have investigated whether it may be a cause of human cancer. Basic principles of tumor virology imply that BKV-induced tumors should contain BKV DNA sequences. Therefore, we assayed (by molecular hybridization) DNA from human tumors and malignant cell lines for BKV DNA, using BKV [32P]DNA as probe. The BKV [32PJDNA was labeled in vitro (nick translation) to specific activities of 1 to 2 X 108 cpm/gg. The BKV DNA used to prepare our probes had the properties expected of authentic BKV genomes, including density of superhelical DNA, sedimentation velocity in alkaline and neutral sucrose gradients, production of one fragment by endonuclease EcoRI cleavage and four fragments by endonuclease Hin II + III cleavage and reassociation properties. From these studies we conclude that our BKV probes hybridized well, and represented bona fide BKV DNA. Using three different BKV [32PJDNA probes, i.e., from three distinct plaque isolates, we have analyzed DNA from BKV-transformed cells, normal human tissues, and a large number of human tumors. All human DNAs (cell lines, normal tissues, tumors) hybridized 5% with BKV DNA. Hybridization analysis of BKVtransformed hamster cell DNA indicated 5-6 copies of at least 88% of the BKV genome per cell. No BKV DNA sequences were detected (above the normal 5% hybridization to all human DNAs) in the following normal human tissues: 10 kidney (BKV is usually isolated from urine), 3 spleen, 13 lung, 23 colon, 2 rectum, 1 ileum, and 1 skin. No BKV-specific DNA was found in 166 tumors, including 5 carcinomas (Ca) of stomach, 3 Ca small intestine, 26 Ca colon, 9 Ca rectum, 31 Ca lung, 9 adenocarcinomas and 5 oat cell carcinomas of lung, 17 melanomas, 5 Ca prostate, 4 Ca bladder, 6 Wilms tumors, 4 hypernephromas, 15,Ca kidney, 7 brain tumors, 5 Hodgkin lymphomas, 10 lymphomas (immunosuppressed patients have a high incidence of Iymphomas), 2 reticulum cel sarcomas (spleen), and 3 skin tumors. We have also analyzed 7 human malignant cell lines (melanoma, lung, rhabdomyosarcoma, and glioblastomas), including several clones of a lung melanoma line; no BKV DNA sequences were detected. Because our probes could detect one copy of BKV DNA if only 10% of the cells were tumor cells, our results are very strong evidence that the tumors we analyzed did not have a BKV etiology. The tumors we tested represent about 50% of all cancers in the United States; there is no evidence that BKV is involved in the etiology of these types of tumors.
tion). Because BKV is widespread in the human population and has oncogenic properties, it is important to determine whether it is a cause of human cancer. To test this, we assayed for BKV DNA sequences in DNA from 166 human tumors and 7 malignant cell lines, using in vitro 32P-labeled BKV DNA (1 to 2 X 108 cpm/,gg) as probe in saturation molecular hybridization reactions. On the basis of studies of transformation and tumorigenesis in animals with papovaviruses and other DNA tumor viruses, tumors induced by BKV would be expected to contain BKV DNA (probably integrated). In this report we describe the preparation and characterization of BKV DNA, and show that our in vitro labeled BKV [32P]DNA is a representative probe for BKV DNA sequences. With this probe, we did W detect BKV sequences in DNAs from any human tumors or malignant
cell lines tested. MATERIALS AND METHODS Virus and Cells. BK seed virus was provided by D. Walker and G. di Mayorca, and was grown on secondary human embryo kidney (HEK) cells. The Walker and di Mayorca viruses were plaque-purified twice and once, respectively. Virus stocks were prepared, using a low multiplicity of infection (0.01 plaque-forming unit per cell) to avoid accumulation of defective particles. Virus stocks were also prepared from the original seed stock of the di Mayorca virus. Human tumor cell lines A375 (melanoma), A204 (rhabdomyosarcoma), A549 (carcinoma lung), HA188 (carcinoma lung), and A172 (glioblastoma) were provided by S. Aaronson. The AlOD cells (melanoma) were received from Naval Biological Research Laboratories. The T98 (glioblastoma) and BK-HK (BKV-transformed hamster kidney cells) cell lines were furnished by H. Pinkerton and K. Takemoto, respectively. Human normal and tumor tissues were obtained from J. Gruber and I. Sekely (Office of Program Resources and Logistics, National Cancer Institute), the late E. Harrison (Mayo Clinic), M. Gardner (University of Southern California), and from H. Pinkerton and K. Smith (St. Louis University). Preparation and Labeling of Viral DNA and Viral DNA Restriction Endonuclease Fragments. BKV DNA was prepared by the Hirt procedure (21) and purified by isopycnic centrifugation in ethidium bromide/CsCl gradients. SV40 DNA
BK virus (BKV) is a human papovavirus that has been isolated from a number of immunoincompetent patients (1-5). Seroepidemiological studies indicate that over 80% of the population of the United States and Great Britain have been infected with BKV (6,7). BKV is weakly tumorigenic in newborn hamsters and transforms (either whole BK virus or transfection with BKV DNA) cultured hamster, rat, and rabbit cells (8-17). Some transformed hamster cells contain rescuable BKV, 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 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: BKV, BK virus; SV40, simian virus 40; Ca, carcinoma. 454
Medical Sciences: Wold et al. 4- A
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 1. Characterization of BKV DNA used to prepare [32P]DNA probes. (A) Density equilibrium centrifugation of BKV DNA in ethidium bromide/CsCl gradients. The Hirt supernatant fraction of BKV-infected HEK cells was centrifuged at 40,000 rpm for 60 hr at 200 in a Beckman Ti 50 rotor. Gradients were fractionated and radioactivity in each fraction was determined by liquid scintillation counting. 0, 3H cpm; A, density. (B) Rate zonal centrifugation of BKV DNA in alkaline sucrose gradients. BKV DNA purified by isopycnic centrifugation was centrifuged at 40,000 rpm for 3 hr at 150 in 5% sucrose (0.2 M NaOH/0.8 M NaCl/0.002 M EDTA) to 20% sucrose (0.8 M NaOH/0.2 M NaCl/0.002 M EDTA) in an SW 41 rotor, using intact adenovirus 5 (Ad5) DNA as a marker. (C) Rate zonal centrifugation of BKV DNA in neutral sucrose gradients. BKV DNA purified by isopycnic centrifugation was centrifuged at 40,000 rpm for 24 hr at 40 in 10-30% sucrose (1 M NaCl/0.02 M Tris-HCl, pH 8.0/0.05 M EDTA) gradient in an SW 41 rotor, using ColEl DNA and Ad5 DNA as markers.
was the gift of L. Gelb and ColEl plasmid DNA was provided by D. Grandgenett. Endonuclease EcoRI was purified from Escherichia coli RY13 (22) and endonuclease Hin 11 + III was purchased from New England Biolabs. BKV and SV40 DNA were labeled by the micro-nick translation reaction (23). Labeled DNA was fractionated on alkaline sucrose gradients, and fractions of 300-500 nucleotides were isolated for use as probe in hybridization reactions. Isolation of Tissue and Cell DNA. Tissue was homogenized, treated with Pronase and sodium dodecyl sulfate, and extracted with chloroform/phenol. Nucleic acid was recovered by precipitation with ethanol, sonicated to desired size (300-500 nucleotides in length), treated with alkali to hydrolyze RNA, and purified by gel filtration using Sephadex G-50 (24). Hybridization Conditions. Hybridizations were in 0.72 M NaCI/10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.7)/1 mM EDTA/0.05% sodium dodecyl sulfate (25). Tissue and cell DNA concentrations were 6 mg/ml. This large excess of cell DNA over probe DNA ensures that a small fraction of the BKV genome in a tumor would be sufficient to drive the hybridization reaction. The tissue, cell, and probe DNAs were 300-500 nucleotides in length, eliminating the possibility that
RESULTS Preparation and Characterization of BKV DNA. BKV DNA was prepared from three sources of virus: stocks generated from (i) the di Mayorca seed virus, (ii) a plaque isolate of the di Mayorca virus, and (iii) a plaque isolate of the Walker virus. DNA was purified by isopycnic centrifugation. As shown in Fig. IA, two peaks of DNA were observed in ethidium bromide/ CsCl gradients: one peak representing BKV DNA had a density of 1.593 g/cm3; the second peak, with density 1.541 g/cm3, consisted of contaminating cell DNA. The purified BKV DNA sedimented at 52 S in alkaline sucrose gradients (Fig. 1B) and at 22 S in neutral sucrose gradients (Fig. IC), as expected of superhelical DNA. Therefore, our DNA preparations consisted mainly of superhelical complete BKV DNA molecules. Preparation and Characterization of In Vitro 32P-Labeled BKV DNA. BKV DNA preparations described above were labeled in vitro (23) with 32p to specific activities of 1 to 2 X 108 cpm/,ug. DNA labeled by this procedure is uniformly labeled and hybridizes with the same kinetics as in vivo labeled DNA (23). Fig. 2 illustrates the reassociation kinetics of in vitro labeled BKV DNA (from di Mayorca and Walker) and SV40 DNA. All three DNAs displayed similar reassociation kinetics, with a Cotl/2 (product of DNA concentration and incubation time) of about 3 x 10-4 mol of nucleotide-sec-liter-1, as expected of DNAs of about 3 X 106 daltons. Table 1 presents cross-hybridization data, using SV40 DNA and three different preparations of BKV DNA. All three preparations of BKV [32P]DNA hybridized to the same extent with all preparations of BKV DNA, and hybridized 12-14% with SV40 DNA. SV40 [`2P]DNA hybridized completely with SV40 DNA, and 11-12% with the three BKV DNAs. Thus, the three preparations of BKV DNA are indistinguishable by hybridization, and were 11-14% homologous with SV40 DNA. We conclude that the BKV probes used in our human tumor analyses (see below) hybridized well and were representative of BKV DNA. Sensitivity of BKV [32P]DNA Probes and Analysis of BKV-Transformed Hamster Cells. As shown in Fig. 3, under the same conditions used for analyses of tumor DNAs, the BKV [32P]DNA probe could readily detect 0.1 copies per cell of BKV DNA, as 20% hybridization above background after 48 hr. DNA from BKV-transformed cells (BH-HK) gave 60% hybridization after 4 hr. These cells contain at least 88% of the BKV genome (Fig. 3) and about six copies per cell of BKV DNA, as indicated by reassociation kinetic analysis (not shown). Cytoplasmic RNA from BK-HK cells gave 11-12% hybridization (not shown).
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Medical Sciences: Wold et al. Table 1. Homology between BKV DNAs and SV40 DNA* Percent hybridization with unlabeled DNAt
Calf
[32P]DNA
SV40 15.1 BKV-1 3.4 88.0 88.2 87.2 (13.9) (0) (100) (100) (99.0) BKV-2 8.7 1.2 65.4 66.9 69.8 (11.8) (0) (97.9) (100) (103) 12.9 BKV-3 4.8 87.3 86.7 90.2 (13.9) (0) (98.0) (97.2) (100) 5.3 13.4 12.5 13.0 85.4 SV40 (0) (11.6) (11.3) (11.9) (100) * In vitro labeled [32P]DNA (1 to 2 X 108 cpm/gg, 500 cpm/25-Ml aliquot) was annealed for 4 hr with unlabeled viral DNA at 1 ,g/ml in the presence of calf thymus DNA at 6 mg/ml under standard conditions. Raw data are presented; normalized values are given in parentheses. t BKV-1 refers to DNA from the original seed stock of BKV obtained from G. di Mayorca. BKV-2 refers to DNA from a virus stock prepared from a plaque isolate of the di Mayorca BKV. BKV-3 refers to DNA from a virus stock prepared from a twice plaque-purified isolate of BKV obtained from D. Walker.
thymus
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Analysis of DNA from Normal Human Tissues, Human Tumors, and Human Tumor Cell Lines for BKV DNA Sequences. Table 2 summarizes the results of our analyses of normal human tissue DNAs for BKV DNA sequences. After 48 hr, the BKV [32P]DNA probe self-annealed (i.e., in the presence of non-human DNA at 6 mg/ml) about 8%. The probe annealed about 13% in the presence of normal human tissue DNAs. Human cell lines gave the same 5% hybridization above background as normal tissue DNAs. We conclude that highly purified BKV DNA (three different preparations) contains human DNA sequences. This 5% hybridization may represent human sequences integrated into BKV DNA, or possibly human contaminants of BKV DNA. The results of the analyses of human tumors are given in Table 3. None of the human tumors analyzed contained BKV DNA (other than the 5% homology discussed above), i.e., tumor DNAs hybridized to the same extent as normal tissue and 100
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FIG. 3. Hybridization of in vitro labeled BKV [32P]DNA with 0-10 copies per cell of BKV DNA, or BK-HK clone 3 BKV-transformed cell DNA. BKV [32P]DNA (1.7 X 108 cpm/,g, 500 cpm/50 ,ul) was annealed with 6 mg/ml of BK-HK DNA, or 6 mg/ml of calf thymus DNA containing 0-10 copies of unlabeled BKV DNA. BK-HK clone 3 cells are a line of BKV-transformed hamster cells.
Table 2. Hybridization of BKV DNA in the presence of nonhuman cell DNA, human cultured cell DNA, and normal human tissue DNA* % hybridization (uncorrected),t Source of No. of mean ± SD DNA tissues tested Non-human Cell DNA Calf thymus 8.3 8.8 Salmon sperm 8.8 E. coli 8.8 HEC19 (hamster) 8617 (rat) 8.4 Human cell DNA KB cells 13.2 HEK cells 13.3 14.4 HEF cells Human tissue DNA 14.8 A 2.2 Normal kidney .10 13.7 ± 0.9 Normal spleen 3 15.4 ± 1.4 Normal lung 13 Normal skin 13.6 1 14.8 Normal ileum 1 14.2 Normal rectum 2 13.8 d 1.8 Normal colon 23 * In vitro labeled BKV [32P]DNA (1.5 X 108 cpm/Mg, 500 cpm/25-,ul aliquot) was annealed in the presence of cell DNA at 6 mg/ml to an equivalent Cot of 20,000 mol-sec-liter-1. Hybridization values have not been normalized. In reconstruction experiments performed under the same conditions, 0.25 and 1.0 copies per cell ofadded BKV DNA gave 32.2% and 63.9% hybridization, respectively, and BKV DNA at 4 ,g/ml gave 82.2% hybridization. t The data are not corrected for self-annealing of the BKV [32P]DNA in the absence of human cell DNA during the hybridization. human cell line DNA. We have analyzed DNA from a number of malignant human cell lines; as summarized in Table 4, we did not detect BKV sequences in the DNA from any of these
cell lines. DISCUSSION Several lines of evidence ensure that our tumor analyses were conducted with a representative BKV [32P]DNA probe, able to detect BKV transforming DNA sequences integrated in the DNA of transformed cells. BKV DNAs from three different virus preparations were indistinguishable. (i) The properties of the three BKV DNAs were those expected of superhelical BKV DNA in terms of density (1.593 g/cm3) in ethidium bromide/CsCl gradients, and sedimentation coefficients in alkaline (52 S) and neutral (22 S) sucrose gradients. (ii) The three BKV DNAs were >97% homologous in cross-hybridization reactions and were 11-14% homologous with SV40 DNA. [BKV and SV40 have been reported to be 10-20% homologous when hybrids are assayed under stringent conditions (27).] (i) The reassociation kinetics of the three BKV DNAs and of SV40 DNA were very similar, and as expected of DNA genomes with complexity of about 3 X 106 daltons. (iv) Digestion of the three BKV DNAs with EcoRI produced linear molecules with molecular weight 3.2 to 3.4 X 106, (28, 29), and digestion of the three BKV DNAs by Hin 11 + III appeared to yield the same four fragments reported previously (28, 29) (data not shown). (v) Reassociation kinetics of the BKV DNA with BKV-transformed cell DNA yielded 5-6 viral genome copies per cell. The BKV [32P]DNA gave 11-12% hybridization with cytoplasmic RNA from BK-HK cells, indicating that a minimum of 22-24% of the BKV genome is expressed as mRNA in these cells. This hybridization probably represents BKV "early" transforming
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Proc. Natl. Acad. Sc. USA 75 (1978)
Table 3. Analysis of human tumor DNAs -for BKV NA sequences Primary site No. of % hybridization % and tumor tumors (uncorrected),t distribution type* tested mean ± SD of casest Digestive system Ca stomach 5 15.1 I 2.2 3.5 1 Ca ileum 15.7 Adca ileum 1 12.0 0.3 Ca small intestine 1 14.1 Ca colon (excluding rectum and caecum) 22 14.4 I 1.9 8.4 Ca caceum 2 13.6 1.8 Adca caecum 2 13.6 Ca rectum 9 14.8 ± 1.8 4.5 Lung 31 15.9 2.1 Squamous cell Ca§ Adca 9 13.4 ± 2.6 13.3 Oat cell 5 15.1 1.1 Melanomas 17 15.9 ± 1.8 1.4 Q C) Prostate 5 15.9+2.3 Bladder Ca 3 16.5 + 2.4 1 13.9 Rhabdomyosarcoma Kidney 6 Wilms tumor 13.4 ± 1.4 4 Hypernephroma 2.0 13.8 ± 1.9 Ca 15 13.5 2.1 Brain 3 11.2 ± 1.5 Glioma 1.5 3 Glioblastoma 13.0 1.3 1 14.0 Meningioma Lymphoma Hodgkin disease 3 14.3 ± 1.5 (lymph node) Hodgkin disease 1 16.4 (lung) 1.1 Hodgkin disease 1 (spleen) 16.1 3 14.2 ± 0.4 Lymph node 4 Liver 15.4 ± 1.7 2.2 1 16.1 Spleen Ileum 1 14.6 Colon 1 16.9 Reticulum cell 2 sarcoma (spleen) 13.5 Skin Ca 1 13.9 1 14.3 Squamous cell Ca Neurofibrosarcoma 1 13.5 * About half the tumors tested were obtained through the ( )ffice of Program Resources and Logistics of the Virus Cancer I?rogram within the National Cancer Institute. The remaining tum4 ors were obtained from other sources. Our laboratory will supply v request (i) the National Cancer Institute tumor designation I (ii) our internal designation numbers of the other tumors, ind (iii) the hybridization data obtained with each individual tuI ior analyzed. Ca, carcinoma; Adca, adenocarcinoma. t The human tumors were analyzed in duplicate under ti ie same conditions and at the same time as the DNA samples desc: ribed in Table 2. The data have not been corrected for self-annealin g of the probe (8-9%) in the absence of human cell DNA. The data in1ImPON1'M jlaues, 2 and 3 were combined from three separate experiments, us3ing two different preparations of BKV [32P]DNA. The sensitivityy of the BKV [32PJDNA probes, as determined by reconstruction experiments with added BKV DNA, is presented in the no tes for Table 2. These incidence data (26) reflect the percent distribution of cases
mon
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Table 4. Analysis of human tumor cell lines for BKV-specific DNA sequences* % hybridization (uncorrected)* A375 Uncl. 15.2 A375 Cl 3 14.7 A375 Cl 5 18.4 A375 Cl 10 13.7 A375 Cl 12 11.7 AlOlD 13.2 A549 16.2 A204 13.4 (Rhabdomyosarcoma) A172 (Glioblastoma) 14.3 HA188 Uncl. (Ca lung) 13.8 HA188 Cl 18 (Ca lung) 12.3 T 98 (Glioblastoma) 12.7 * Hybridizations were performed under the same conditions as described in Table 2. Hybridization values have not been corrected for self-annealing of the probe in the absence of human cell DNA. Cell line (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Ca lung)
gene sequences expressed as RNA in BKV-transformed cells, in analogy to SV40-transformed cells. Thus, our probes very likely contained BKV transforming gene sequences. We were unable to detect BKV sequences in DNA from any human tumors or malignant cell lines. In reconstruction experiments done under the same conditions as the tumor analyses, 0.25 copy per cell and 1 copy per cell of added BKV DNA gave 34-45% and 50-60% hybridization, respectively, above the self-annealing background of 13-16%. Thus, we would easily have detected 0.05 copies per cell of BKV DNA, or 1 copy of BKV DNA in tumors containing 5-10% BKV-transformed cells. All tumors analyzed contained at least 50% transformed cells, as indicated by histological examination. Therefore, we can be virtually certain that these particular tumors and tumor cell lines did not have a BKV etiology (i.e., these tumor types do not have an obligatory BKV etiology). A "hit and run" mechanism of BKV tumorigenesis cannot be formally excluded, but this is unlikely from an overwhelming body of data on viral tumorigenesis in animal and cell culture systems. We point out that all the BKV isolates examined to date appear to be very similar (13, 16, 29), so that it is likely that our studies are applicable to all "strains" of BKV present in the United States
population. The tumors that we have analyzed represent major categories of human cancer, and account for about 50% of total cancers in the United States (26). For most of the tumor types, we have examined quite a large number of samples (Table 3). Therefore, apparently BKV is not a major cause of human cancer. Because at least 80% of the United States population has been infected by BKV, it is likely that some or most of the patients from which
our tumors were derived had been infected by the virus. Therefore, it seems that infection by BKV does not necessarily result in the development of any of the common cancers. It remains possible, however, that BKV could induce specific rare types of cancer, or on occasion induce one of the common cancers. A long-range extensive analysis of cancers from all sites, including different histological types, is warranted and necessary to determine whether BKV is ever carcinogenic in humans. in the United States diagnosed in 1969-71 by primary site, with both sexes and all races combined. The data exclude carcinoma in situ and nonmelanoma skin cancers. § Represents either diagnosed or presumed squamous cell carcinoma of the lung.
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Medical Sciences: Wold et al.
Epidemiological studies have indicated immunosuppressed renal transplant patients develop cancer at 19 times the incidence in the normal population, and often get very rare types of cancer (30). For example, reticulum cell sarcomas occurred at 150 times the normal incidence. Other cancers with increased incidence were cancers of skin, hepatobilary tract and bladder, adenocarcinomas of the lung, leukemia, melanomas, and soft tissue sarcomas. Because BKV apparently replicates readily in immunosuppressed patients, these types of cancer could be particularly suspect of having a BKV etiology. Our study, which examined a limited number of tumors in these categories, suggests that there is not an obligatory link between these cancers and BKV; additional tumors must be analyzed, particularly from immunosuppressed patients, to exclude BKV as an agent of these types of cancer. Fiori and di Mayorca (31) recently reported that BKV DNA was present (0.4-11.0 copies per cell) in 5/12 human tumors and 3/4 malignant human cell lines examined. The cell lines were negative for expression of BKV T antigen. We were unable to detect BKV DNA in the same three cell lines (A375, passage 114; A549, passage 95; and AlOlD, passage 60) or in one of the tumors (FT 750038, rhabdomyosarcoma of bladder) reported to contain BKV sequences by those authors. Our analyses were conducted using viral probes prepared from the same stock of BKV used by Fiori and di Mayorca. We received the A375 line on Oct. 9, 1974, at passage 78; the clones we assayed varied between passage 80 and 110. The* uncloned preparation of A375 was received as a frozen pellet, and therefore these cells were assayed at less than passage 78. We received the A549 cells on Mar. 30, 1973, at passage 38; these cells had been passaged less than 70-80 times when assayed for BKV sequences. We received the AlOlD cells on Mar. 30, 1973, at passage 20; when assayed for BKV DNA, the AlOlD cells had been passaged less than 60 times. Therefore, the cells tested in our experiments had been passaged fewer times than those in the experiments of Fiori and di Mayorca (31), ruling out the possibility that our cells had lost the BKV genome during passage. Our assay procedure (saturation hybridization with highly radioactive DNA) is more sensitive than the procedure used by Fiori and di Mayorca (reassociation kinetics). In addition, our hybridizations were assayed using hydroxylapatite, a less stringent and therefore more sensitive method than the S-1 nuclease procedure used by those authors. The malignant cell lines were negative when assayed after both 20-hr and 48-hr hybridization, excluding the possibility that DNA-DNA hybrids were somehow degraded after 48-hr hybridization. We have no explanation for the apparent discrepancy between our results and those reported by Fiori and di Mayorca. We thank K. Takemoto for the BKV-transformed cells, S. Aaronson, H. Pinkerton, and Naval Biological Research Laboratories for stocks of the malignant human tumor cell lines, and D. Walker and G. di Mayorca for seed stocks of BKV. We thank L. Gelb for the SV40 DNA, D. Grandgenett for the ColE1 DNA, and J. Gruber, I. Sekely, M. Gardner, H. Pinkerton, K. Smith, and the late E. Harrison for human tumor tissues. We thank H. Thornton for cell culture assistance and L. Young, E. Bentley, C. Self, A. Pearson, and C. Boudreau for technical assistance. This work was supported by Contract N01 CP 43359 from the Virus Cancer Program within the National Cancer Institute. W.S.M.W. was partially supported by a fellowship from the Medical
Proc. Natl. Acad. Sci. USA 75 ('1978) Research Council of Canada. M.G. is the recipient of a Research Career Award (KO 6 Al 04739) from the National Institutes of Health. 1. Gardner, S. D., Field, A. M., Coleman, D. V. & Hulme, B. (1971) Lancet i, 1253-1257. 2. Coleman, D. V., Gardner, S. D. & Field, A. M. (1973) Br. Med. J. 3, 371-375. 3. Dougherty, R. M. & di Stefano, H. S. (1974) Proc. Soc. Exp. Biol. Med. 146, 481-487. 4. Takemoto, K. K., Rabson, A. S., Mullarkey, M. F., Blaese, R. M., Garon, C. F. & Nelson, D. (1974) J. Natl. Cancer Inst. 53, 1205-1207. 5. Gardner, S. D. (1975) Abstracts of the 3rd International Congress of Virology, Madrid, p. 185. 6. Gardner, S. D. (1973) Br. Med. J. 1, 77-78. 7. Shah, K. V., Daniel, R. W. & Warszawaski, R. M. (1973) J. Infect. Dis. 128, 784-787. 8. Shah, K. V., Daniel, R. W. & Strandberg, J. D. (1975) J. Natl. Cancer Inst. 54, 945-949. 9. Nase, L. M., Kirkkirnen, M. & Mantyjarvi, R. A. (1975) Acta Pathol. Microbiol. Scand. 83,347-352. 10. Van der Noordaa, J. (1976) J. Gen. Virol. 30, 371-373. 11. Major, E. 0. & di Mayorca, G. (1973). Proc. Natl. Acad. Sci. USA 70,3210-3212. 12. Portolani, M., Barbanti-Brodano, G. & LaPlaca, M. (1975) J. Virol. 15, 420-422. 13. Wright, P. J. & di Mayorca, G. (1975) J. Virol. 15, 825-835. 14. Takemoto, K. K. & Martin, M. A. (1976) J. Virol. 17,247-253. 15. Seehafer, J., Salmi, A. & Colter, J. S. (1977) Virology 77,356366. 16. Miao, R. & Dougherty, R. (1977) J. Gen. Virol. 35, 67-75. 17. Mason, D. H. & Takemoto, K. K. (1977) Int. J. Cancer 19, 391-395. 18. Osborn, J. E., Robertson, S. M., Padgett, B. L., Walker, D. L. & Weisblum, B. (1976) J. Virol. 19,675-684. 19. Takemoto, K. K. & Mullarkey, M. F. (1973) J. Virol. 12,625631. 20. Shah, K. V., Ozer, H. L., Ghazey, H. N. & Kelly, T. J. (1977) J. Virol. 21, 179-186. 21. Hirt, B. (1967). J. Mol. Biol. 26,365-369. 22. Mulder, C. & Delius, H. (1972). Proc. Natl. Acad. Sci. USA 69, 3215-3219. 23. Mackey, J. K., Brackmann, K. H., Green, M. R. & Green, M. (1977) Biochemistry 16, 4478-4483. 24. Green, M. R., Mackey, J. K. & Green, M. (1977) J. Virol. 22, 238-242. 25. Green, M. R., Chinnadurai, G., Mackey, J. K. & Green, M. (1976) Cell 7, 419-428. 26. Culter, S. & Young, J., eds. (1975) Third National Cancer Survey: Incidence Data (National Cancer Institute, Bethesda, MD), pp. 18-19. 27. Khoury, G., Howley, P. M., Garon, C., Mullarkey, M. F., Takemoto, K. K. & Martin, M. A. (1975) Proc. Natl. Acad. Sci. USA 72,2563-2567. 28. Howley, P. M., Mullarkey, M. F., Takemoto, K. K. & Martin, M. A. (1975) J. Virol. 15, 173-181. 29. Howley, P. M., Khoury, G., Byrne, J. C., Takemoto, K. K. & Martin, M. A. (1975) J. Virol. 16,959-973. 30. Hoover, R. (1977) in Cold Spring Harbor Conferences on Cell Proliferation, eds. Hiatt, H. H., Watson, J. D. & Winsten, J. A. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 4A, pp. 369-379. 31. Fiori, M. & di Mayorca, G. (1976). Proc. Natl. Acad. Sci. USA 73,4662-4666.
Analysis of human tumors and human malignant cell lines for BK virus-specific DNA sequences (molecular hybridization/BK virus-transformed hamster cells/human cancer)
WILLIAM S. M. WOLD, JESSE K. MACKEY, KARL H. BRACKMANN, NOBUYUKI TAKEMORI, PATRICIA RIGDEN, AND MAURICE GREEN Institute for Molecular Virology, St. Louis University School of Medicine, 3681 Park Avenue, St. Louis, Missouri 63110
Communicated by Robert M. Chanock, October 11, 1977
express T antigen, and are tumorigenic in hamsters (12, 14, 15). BKV is related to but distinct from simian virus 40 (SV40) and JC virus, another human papovavirus (18). BKV and SV40 T antigens crossreact strongly (9, 12, 14, 15, 19, 20) (the SV40 T antigen is believed to be a protein encoded by the SV40 early region and to play a role in SV40-induced cell transforma-
Most humans in the United States have been ABSTRACT infected with BK virus (BKV), a human papovavirus. Because BKV has oncogenic properties, we have investigated whether it may be a cause of human cancer. Basic principles of tumor virology imply that BKV-induced tumors should contain BKV DNA sequences. Therefore, we assayed (by molecular hybridization) DNA from human tumors and malignant cell lines for BKV DNA, using BKV [32P]DNA as probe. The BKV [32PJDNA was labeled in vitro (nick translation) to specific activities of 1 to 2 X 108 cpm/gg. The BKV DNA used to prepare our probes had the properties expected of authentic BKV genomes, including density of superhelical DNA, sedimentation velocity in alkaline and neutral sucrose gradients, production of one fragment by endonuclease EcoRI cleavage and four fragments by endonuclease Hin II + III cleavage and reassociation properties. From these studies we conclude that our BKV probes hybridized well, and represented bona fide BKV DNA. Using three different BKV [32PJDNA probes, i.e., from three distinct plaque isolates, we have analyzed DNA from BKV-transformed cells, normal human tissues, and a large number of human tumors. All human DNAs (cell lines, normal tissues, tumors) hybridized 5% with BKV DNA. Hybridization analysis of BKVtransformed hamster cell DNA indicated 5-6 copies of at least 88% of the BKV genome per cell. No BKV DNA sequences were detected (above the normal 5% hybridization to all human DNAs) in the following normal human tissues: 10 kidney (BKV is usually isolated from urine), 3 spleen, 13 lung, 23 colon, 2 rectum, 1 ileum, and 1 skin. No BKV-specific DNA was found in 166 tumors, including 5 carcinomas (Ca) of stomach, 3 Ca small intestine, 26 Ca colon, 9 Ca rectum, 31 Ca lung, 9 adenocarcinomas and 5 oat cell carcinomas of lung, 17 melanomas, 5 Ca prostate, 4 Ca bladder, 6 Wilms tumors, 4 hypernephromas, 15,Ca kidney, 7 brain tumors, 5 Hodgkin lymphomas, 10 lymphomas (immunosuppressed patients have a high incidence of Iymphomas), 2 reticulum cel sarcomas (spleen), and 3 skin tumors. We have also analyzed 7 human malignant cell lines (melanoma, lung, rhabdomyosarcoma, and glioblastomas), including several clones of a lung melanoma line; no BKV DNA sequences were detected. Because our probes could detect one copy of BKV DNA if only 10% of the cells were tumor cells, our results are very strong evidence that the tumors we analyzed did not have a BKV etiology. The tumors we tested represent about 50% of all cancers in the United States; there is no evidence that BKV is involved in the etiology of these types of tumors.
tion). Because BKV is widespread in the human population and has oncogenic properties, it is important to determine whether it is a cause of human cancer. To test this, we assayed for BKV DNA sequences in DNA from 166 human tumors and 7 malignant cell lines, using in vitro 32P-labeled BKV DNA (1 to 2 X 108 cpm/,gg) as probe in saturation molecular hybridization reactions. On the basis of studies of transformation and tumorigenesis in animals with papovaviruses and other DNA tumor viruses, tumors induced by BKV would be expected to contain BKV DNA (probably integrated). In this report we describe the preparation and characterization of BKV DNA, and show that our in vitro labeled BKV [32P]DNA is a representative probe for BKV DNA sequences. With this probe, we did W detect BKV sequences in DNAs from any human tumors or malignant
cell lines tested. MATERIALS AND METHODS Virus and Cells. BK seed virus was provided by D. Walker and G. di Mayorca, and was grown on secondary human embryo kidney (HEK) cells. The Walker and di Mayorca viruses were plaque-purified twice and once, respectively. Virus stocks were prepared, using a low multiplicity of infection (0.01 plaque-forming unit per cell) to avoid accumulation of defective particles. Virus stocks were also prepared from the original seed stock of the di Mayorca virus. Human tumor cell lines A375 (melanoma), A204 (rhabdomyosarcoma), A549 (carcinoma lung), HA188 (carcinoma lung), and A172 (glioblastoma) were provided by S. Aaronson. The AlOD cells (melanoma) were received from Naval Biological Research Laboratories. The T98 (glioblastoma) and BK-HK (BKV-transformed hamster kidney cells) cell lines were furnished by H. Pinkerton and K. Takemoto, respectively. Human normal and tumor tissues were obtained from J. Gruber and I. Sekely (Office of Program Resources and Logistics, National Cancer Institute), the late E. Harrison (Mayo Clinic), M. Gardner (University of Southern California), and from H. Pinkerton and K. Smith (St. Louis University). Preparation and Labeling of Viral DNA and Viral DNA Restriction Endonuclease Fragments. BKV DNA was prepared by the Hirt procedure (21) and purified by isopycnic centrifugation in ethidium bromide/CsCl gradients. SV40 DNA
BK virus (BKV) is a human papovavirus that has been isolated from a number of immunoincompetent patients (1-5). Seroepidemiological studies indicate that over 80% of the population of the United States and Great Britain have been infected with BKV (6,7). BKV is weakly tumorigenic in newborn hamsters and transforms (either whole BK virus or transfection with BKV DNA) cultured hamster, rat, and rabbit cells (8-17). Some transformed hamster cells contain rescuable BKV, 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 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: BKV, BK virus; SV40, simian virus 40; Ca, carcinoma. 454
Medical Sciences: Wold et al. 4- A
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 1. Characterization of BKV DNA used to prepare [32P]DNA probes. (A) Density equilibrium centrifugation of BKV DNA in ethidium bromide/CsCl gradients. The Hirt supernatant fraction of BKV-infected HEK cells was centrifuged at 40,000 rpm for 60 hr at 200 in a Beckman Ti 50 rotor. Gradients were fractionated and radioactivity in each fraction was determined by liquid scintillation counting. 0, 3H cpm; A, density. (B) Rate zonal centrifugation of BKV DNA in alkaline sucrose gradients. BKV DNA purified by isopycnic centrifugation was centrifuged at 40,000 rpm for 3 hr at 150 in 5% sucrose (0.2 M NaOH/0.8 M NaCl/0.002 M EDTA) to 20% sucrose (0.8 M NaOH/0.2 M NaCl/0.002 M EDTA) in an SW 41 rotor, using intact adenovirus 5 (Ad5) DNA as a marker. (C) Rate zonal centrifugation of BKV DNA in neutral sucrose gradients. BKV DNA purified by isopycnic centrifugation was centrifuged at 40,000 rpm for 24 hr at 40 in 10-30% sucrose (1 M NaCl/0.02 M Tris-HCl, pH 8.0/0.05 M EDTA) gradient in an SW 41 rotor, using ColEl DNA and Ad5 DNA as markers.
was the gift of L. Gelb and ColEl plasmid DNA was provided by D. Grandgenett. Endonuclease EcoRI was purified from Escherichia coli RY13 (22) and endonuclease Hin 11 + III was purchased from New England Biolabs. BKV and SV40 DNA were labeled by the micro-nick translation reaction (23). Labeled DNA was fractionated on alkaline sucrose gradients, and fractions of 300-500 nucleotides were isolated for use as probe in hybridization reactions. Isolation of Tissue and Cell DNA. Tissue was homogenized, treated with Pronase and sodium dodecyl sulfate, and extracted with chloroform/phenol. Nucleic acid was recovered by precipitation with ethanol, sonicated to desired size (300-500 nucleotides in length), treated with alkali to hydrolyze RNA, and purified by gel filtration using Sephadex G-50 (24). Hybridization Conditions. Hybridizations were in 0.72 M NaCI/10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.7)/1 mM EDTA/0.05% sodium dodecyl sulfate (25). Tissue and cell DNA concentrations were 6 mg/ml. This large excess of cell DNA over probe DNA ensures that a small fraction of the BKV genome in a tumor would be sufficient to drive the hybridization reaction. The tissue, cell, and probe DNAs were 300-500 nucleotides in length, eliminating the possibility that
RESULTS Preparation and Characterization of BKV DNA. BKV DNA was prepared from three sources of virus: stocks generated from (i) the di Mayorca seed virus, (ii) a plaque isolate of the di Mayorca virus, and (iii) a plaque isolate of the Walker virus. DNA was purified by isopycnic centrifugation. As shown in Fig. IA, two peaks of DNA were observed in ethidium bromide/ CsCl gradients: one peak representing BKV DNA had a density of 1.593 g/cm3; the second peak, with density 1.541 g/cm3, consisted of contaminating cell DNA. The purified BKV DNA sedimented at 52 S in alkaline sucrose gradients (Fig. 1B) and at 22 S in neutral sucrose gradients (Fig. IC), as expected of superhelical DNA. Therefore, our DNA preparations consisted mainly of superhelical complete BKV DNA molecules. Preparation and Characterization of In Vitro 32P-Labeled BKV DNA. BKV DNA preparations described above were labeled in vitro (23) with 32p to specific activities of 1 to 2 X 108 cpm/,ug. DNA labeled by this procedure is uniformly labeled and hybridizes with the same kinetics as in vivo labeled DNA (23). Fig. 2 illustrates the reassociation kinetics of in vitro labeled BKV DNA (from di Mayorca and Walker) and SV40 DNA. All three DNAs displayed similar reassociation kinetics, with a Cotl/2 (product of DNA concentration and incubation time) of about 3 x 10-4 mol of nucleotide-sec-liter-1, as expected of DNAs of about 3 X 106 daltons. Table 1 presents cross-hybridization data, using SV40 DNA and three different preparations of BKV DNA. All three preparations of BKV [32P]DNA hybridized to the same extent with all preparations of BKV DNA, and hybridized 12-14% with SV40 DNA. SV40 [`2P]DNA hybridized completely with SV40 DNA, and 11-12% with the three BKV DNAs. Thus, the three preparations of BKV DNA are indistinguishable by hybridization, and were 11-14% homologous with SV40 DNA. We conclude that the BKV probes used in our human tumor analyses (see below) hybridized well and were representative of BKV DNA. Sensitivity of BKV [32P]DNA Probes and Analysis of BKV-Transformed Hamster Cells. As shown in Fig. 3, under the same conditions used for analyses of tumor DNAs, the BKV [32P]DNA probe could readily detect 0.1 copies per cell of BKV DNA, as 20% hybridization above background after 48 hr. DNA from BKV-transformed cells (BH-HK) gave 60% hybridization after 4 hr. These cells contain at least 88% of the BKV genome (Fig. 3) and about six copies per cell of BKV DNA, as indicated by reassociation kinetic analysis (not shown). Cytoplasmic RNA from BK-HK cells gave 11-12% hybridization (not shown).
456
Proc. Nati. Acad. Sci. USA 75 (1978)
Medical Sciences: Wold et al. Table 1. Homology between BKV DNAs and SV40 DNA* Percent hybridization with unlabeled DNAt
Calf
[32P]DNA
SV40 15.1 BKV-1 3.4 88.0 88.2 87.2 (13.9) (0) (100) (100) (99.0) BKV-2 8.7 1.2 65.4 66.9 69.8 (11.8) (0) (97.9) (100) (103) 12.9 BKV-3 4.8 87.3 86.7 90.2 (13.9) (0) (98.0) (97.2) (100) 5.3 13.4 12.5 13.0 85.4 SV40 (0) (11.6) (11.3) (11.9) (100) * In vitro labeled [32P]DNA (1 to 2 X 108 cpm/gg, 500 cpm/25-Ml aliquot) was annealed for 4 hr with unlabeled viral DNA at 1 ,g/ml in the presence of calf thymus DNA at 6 mg/ml under standard conditions. Raw data are presented; normalized values are given in parentheses. t BKV-1 refers to DNA from the original seed stock of BKV obtained from G. di Mayorca. BKV-2 refers to DNA from a virus stock prepared from a plaque isolate of the di Mayorca BKV. BKV-3 refers to DNA from a virus stock prepared from a twice plaque-purified isolate of BKV obtained from D. Walker.
thymus
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Analysis of DNA from Normal Human Tissues, Human Tumors, and Human Tumor Cell Lines for BKV DNA Sequences. Table 2 summarizes the results of our analyses of normal human tissue DNAs for BKV DNA sequences. After 48 hr, the BKV [32P]DNA probe self-annealed (i.e., in the presence of non-human DNA at 6 mg/ml) about 8%. The probe annealed about 13% in the presence of normal human tissue DNAs. Human cell lines gave the same 5% hybridization above background as normal tissue DNAs. We conclude that highly purified BKV DNA (three different preparations) contains human DNA sequences. This 5% hybridization may represent human sequences integrated into BKV DNA, or possibly human contaminants of BKV DNA. The results of the analyses of human tumors are given in Table 3. None of the human tumors analyzed contained BKV DNA (other than the 5% homology discussed above), i.e., tumor DNAs hybridized to the same extent as normal tissue and 100
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FIG. 3. Hybridization of in vitro labeled BKV [32P]DNA with 0-10 copies per cell of BKV DNA, or BK-HK clone 3 BKV-transformed cell DNA. BKV [32P]DNA (1.7 X 108 cpm/,g, 500 cpm/50 ,ul) was annealed with 6 mg/ml of BK-HK DNA, or 6 mg/ml of calf thymus DNA containing 0-10 copies of unlabeled BKV DNA. BK-HK clone 3 cells are a line of BKV-transformed hamster cells.
Table 2. Hybridization of BKV DNA in the presence of nonhuman cell DNA, human cultured cell DNA, and normal human tissue DNA* % hybridization (uncorrected),t Source of No. of mean ± SD DNA tissues tested Non-human Cell DNA Calf thymus 8.3 8.8 Salmon sperm 8.8 E. coli 8.8 HEC19 (hamster) 8617 (rat) 8.4 Human cell DNA KB cells 13.2 HEK cells 13.3 14.4 HEF cells Human tissue DNA 14.8 A 2.2 Normal kidney .10 13.7 ± 0.9 Normal spleen 3 15.4 ± 1.4 Normal lung 13 Normal skin 13.6 1 14.8 Normal ileum 1 14.2 Normal rectum 2 13.8 d 1.8 Normal colon 23 * In vitro labeled BKV [32P]DNA (1.5 X 108 cpm/Mg, 500 cpm/25-,ul aliquot) was annealed in the presence of cell DNA at 6 mg/ml to an equivalent Cot of 20,000 mol-sec-liter-1. Hybridization values have not been normalized. In reconstruction experiments performed under the same conditions, 0.25 and 1.0 copies per cell ofadded BKV DNA gave 32.2% and 63.9% hybridization, respectively, and BKV DNA at 4 ,g/ml gave 82.2% hybridization. t The data are not corrected for self-annealing of the BKV [32P]DNA in the absence of human cell DNA during the hybridization. human cell line DNA. We have analyzed DNA from a number of malignant human cell lines; as summarized in Table 4, we did not detect BKV sequences in the DNA from any of these
cell lines. DISCUSSION Several lines of evidence ensure that our tumor analyses were conducted with a representative BKV [32P]DNA probe, able to detect BKV transforming DNA sequences integrated in the DNA of transformed cells. BKV DNAs from three different virus preparations were indistinguishable. (i) The properties of the three BKV DNAs were those expected of superhelical BKV DNA in terms of density (1.593 g/cm3) in ethidium bromide/CsCl gradients, and sedimentation coefficients in alkaline (52 S) and neutral (22 S) sucrose gradients. (ii) The three BKV DNAs were >97% homologous in cross-hybridization reactions and were 11-14% homologous with SV40 DNA. [BKV and SV40 have been reported to be 10-20% homologous when hybrids are assayed under stringent conditions (27).] (i) The reassociation kinetics of the three BKV DNAs and of SV40 DNA were very similar, and as expected of DNA genomes with complexity of about 3 X 106 daltons. (iv) Digestion of the three BKV DNAs with EcoRI produced linear molecules with molecular weight 3.2 to 3.4 X 106, (28, 29), and digestion of the three BKV DNAs by Hin 11 + III appeared to yield the same four fragments reported previously (28, 29) (data not shown). (v) Reassociation kinetics of the BKV DNA with BKV-transformed cell DNA yielded 5-6 viral genome copies per cell. The BKV [32P]DNA gave 11-12% hybridization with cytoplasmic RNA from BK-HK cells, indicating that a minimum of 22-24% of the BKV genome is expressed as mRNA in these cells. This hybridization probably represents BKV "early" transforming
Medical Sciences: Wold et al.
Proc. Natl. Acad. Sc. USA 75 (1978)
Table 3. Analysis of human tumor DNAs -for BKV NA sequences Primary site No. of % hybridization % and tumor tumors (uncorrected),t distribution type* tested mean ± SD of casest Digestive system Ca stomach 5 15.1 I 2.2 3.5 1 Ca ileum 15.7 Adca ileum 1 12.0 0.3 Ca small intestine 1 14.1 Ca colon (excluding rectum and caecum) 22 14.4 I 1.9 8.4 Ca caceum 2 13.6 1.8 Adca caecum 2 13.6 Ca rectum 9 14.8 ± 1.8 4.5 Lung 31 15.9 2.1 Squamous cell Ca§ Adca 9 13.4 ± 2.6 13.3 Oat cell 5 15.1 1.1 Melanomas 17 15.9 ± 1.8 1.4 Q C) Prostate 5 15.9+2.3 Bladder Ca 3 16.5 + 2.4 1 13.9 Rhabdomyosarcoma Kidney 6 Wilms tumor 13.4 ± 1.4 4 Hypernephroma 2.0 13.8 ± 1.9 Ca 15 13.5 2.1 Brain 3 11.2 ± 1.5 Glioma 1.5 3 Glioblastoma 13.0 1.3 1 14.0 Meningioma Lymphoma Hodgkin disease 3 14.3 ± 1.5 (lymph node) Hodgkin disease 1 16.4 (lung) 1.1 Hodgkin disease 1 (spleen) 16.1 3 14.2 ± 0.4 Lymph node 4 Liver 15.4 ± 1.7 2.2 1 16.1 Spleen Ileum 1 14.6 Colon 1 16.9 Reticulum cell 2 sarcoma (spleen) 13.5 Skin Ca 1 13.9 1 14.3 Squamous cell Ca Neurofibrosarcoma 1 13.5 * About half the tumors tested were obtained through the ( )ffice of Program Resources and Logistics of the Virus Cancer I?rogram within the National Cancer Institute. The remaining tum4 ors were obtained from other sources. Our laboratory will supply v request (i) the National Cancer Institute tumor designation I (ii) our internal designation numbers of the other tumors, ind (iii) the hybridization data obtained with each individual tuI ior analyzed. Ca, carcinoma; Adca, adenocarcinoma. t The human tumors were analyzed in duplicate under ti ie same conditions and at the same time as the DNA samples desc: ribed in Table 2. The data have not been corrected for self-annealin g of the probe (8-9%) in the absence of human cell DNA. The data in1ImPON1'M jlaues, 2 and 3 were combined from three separate experiments, us3ing two different preparations of BKV [32P]DNA. The sensitivityy of the BKV [32PJDNA probes, as determined by reconstruction experiments with added BKV DNA, is presented in the no tes for Table 2. These incidence data (26) reflect the percent distribution of cases
mon
I
457
Table 4. Analysis of human tumor cell lines for BKV-specific DNA sequences* % hybridization (uncorrected)* A375 Uncl. 15.2 A375 Cl 3 14.7 A375 Cl 5 18.4 A375 Cl 10 13.7 A375 Cl 12 11.7 AlOlD 13.2 A549 16.2 A204 13.4 (Rhabdomyosarcoma) A172 (Glioblastoma) 14.3 HA188 Uncl. (Ca lung) 13.8 HA188 Cl 18 (Ca lung) 12.3 T 98 (Glioblastoma) 12.7 * Hybridizations were performed under the same conditions as described in Table 2. Hybridization values have not been corrected for self-annealing of the probe in the absence of human cell DNA. Cell line (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Melanoma) (Ca lung)
gene sequences expressed as RNA in BKV-transformed cells, in analogy to SV40-transformed cells. Thus, our probes very likely contained BKV transforming gene sequences. We were unable to detect BKV sequences in DNA from any human tumors or malignant cell lines. In reconstruction experiments done under the same conditions as the tumor analyses, 0.25 copy per cell and 1 copy per cell of added BKV DNA gave 34-45% and 50-60% hybridization, respectively, above the self-annealing background of 13-16%. Thus, we would easily have detected 0.05 copies per cell of BKV DNA, or 1 copy of BKV DNA in tumors containing 5-10% BKV-transformed cells. All tumors analyzed contained at least 50% transformed cells, as indicated by histological examination. Therefore, we can be virtually certain that these particular tumors and tumor cell lines did not have a BKV etiology (i.e., these tumor types do not have an obligatory BKV etiology). A "hit and run" mechanism of BKV tumorigenesis cannot be formally excluded, but this is unlikely from an overwhelming body of data on viral tumorigenesis in animal and cell culture systems. We point out that all the BKV isolates examined to date appear to be very similar (13, 16, 29), so that it is likely that our studies are applicable to all "strains" of BKV present in the United States
population. The tumors that we have analyzed represent major categories of human cancer, and account for about 50% of total cancers in the United States (26). For most of the tumor types, we have examined quite a large number of samples (Table 3). Therefore, apparently BKV is not a major cause of human cancer. Because at least 80% of the United States population has been infected by BKV, it is likely that some or most of the patients from which
our tumors were derived had been infected by the virus. Therefore, it seems that infection by BKV does not necessarily result in the development of any of the common cancers. It remains possible, however, that BKV could induce specific rare types of cancer, or on occasion induce one of the common cancers. A long-range extensive analysis of cancers from all sites, including different histological types, is warranted and necessary to determine whether BKV is ever carcinogenic in humans. in the United States diagnosed in 1969-71 by primary site, with both sexes and all races combined. The data exclude carcinoma in situ and nonmelanoma skin cancers. § Represents either diagnosed or presumed squamous cell carcinoma of the lung.
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Epidemiological studies have indicated immunosuppressed renal transplant patients develop cancer at 19 times the incidence in the normal population, and often get very rare types of cancer (30). For example, reticulum cell sarcomas occurred at 150 times the normal incidence. Other cancers with increased incidence were cancers of skin, hepatobilary tract and bladder, adenocarcinomas of the lung, leukemia, melanomas, and soft tissue sarcomas. Because BKV apparently replicates readily in immunosuppressed patients, these types of cancer could be particularly suspect of having a BKV etiology. Our study, which examined a limited number of tumors in these categories, suggests that there is not an obligatory link between these cancers and BKV; additional tumors must be analyzed, particularly from immunosuppressed patients, to exclude BKV as an agent of these types of cancer. Fiori and di Mayorca (31) recently reported that BKV DNA was present (0.4-11.0 copies per cell) in 5/12 human tumors and 3/4 malignant human cell lines examined. The cell lines were negative for expression of BKV T antigen. We were unable to detect BKV DNA in the same three cell lines (A375, passage 114; A549, passage 95; and AlOlD, passage 60) or in one of the tumors (FT 750038, rhabdomyosarcoma of bladder) reported to contain BKV sequences by those authors. Our analyses were conducted using viral probes prepared from the same stock of BKV used by Fiori and di Mayorca. We received the A375 line on Oct. 9, 1974, at passage 78; the clones we assayed varied between passage 80 and 110. The* uncloned preparation of A375 was received as a frozen pellet, and therefore these cells were assayed at less than passage 78. We received the A549 cells on Mar. 30, 1973, at passage 38; these cells had been passaged less than 70-80 times when assayed for BKV sequences. We received the AlOlD cells on Mar. 30, 1973, at passage 20; when assayed for BKV DNA, the AlOlD cells had been passaged less than 60 times. Therefore, the cells tested in our experiments had been passaged fewer times than those in the experiments of Fiori and di Mayorca (31), ruling out the possibility that our cells had lost the BKV genome during passage. Our assay procedure (saturation hybridization with highly radioactive DNA) is more sensitive than the procedure used by Fiori and di Mayorca (reassociation kinetics). In addition, our hybridizations were assayed using hydroxylapatite, a less stringent and therefore more sensitive method than the S-1 nuclease procedure used by those authors. The malignant cell lines were negative when assayed after both 20-hr and 48-hr hybridization, excluding the possibility that DNA-DNA hybrids were somehow degraded after 48-hr hybridization. We have no explanation for the apparent discrepancy between our results and those reported by Fiori and di Mayorca. We thank K. Takemoto for the BKV-transformed cells, S. Aaronson, H. Pinkerton, and Naval Biological Research Laboratories for stocks of the malignant human tumor cell lines, and D. Walker and G. di Mayorca for seed stocks of BKV. We thank L. Gelb for the SV40 DNA, D. Grandgenett for the ColE1 DNA, and J. Gruber, I. Sekely, M. Gardner, H. Pinkerton, K. Smith, and the late E. Harrison for human tumor tissues. We thank H. Thornton for cell culture assistance and L. Young, E. Bentley, C. Self, A. Pearson, and C. Boudreau for technical assistance. This work was supported by Contract N01 CP 43359 from the Virus Cancer Program within the National Cancer Institute. W.S.M.W. was partially supported by a fellowship from the Medical
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