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
ABSTRACT Most humans in the United States have been
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 hybrid-
ization) 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, in-
cluding 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 tu-
mors. All human DNAs (cell lines, normal tissues, tumors) hy-
bridized 5% with BKV DNA. Hybridization analysis of BKV-
transformed 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 adeno-
carcinomas 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 lym-
phomas (immunosuppressed patients have a high incidence of
Iymphomas), 2 reticulum cel sarcomas (spleen), and 3 skin tu-
mors. We have also analyzed 7 human malignant cell lines
(melanoma, lung, rhabdomyosarcoma, and glioblastomas), in-
cluding 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.
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 new-
born hamsters and transforms (either whole BK virus or trans-
fection with BKV DNA) cultured hamster, rat, and rabbit cells
(8-17). Some transformed hamster cells contain rescuable BKV,
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454
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-
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 ma-
lignant 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 tumor-
igenesis 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 em-
bryo 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 defec-
tive particles. Virus stocks were also prepared from the original
seed stock of the di Mayorca virus.
Human tumor cell lines A375 (melanoma), A204 (rhab-
domyosarcoma), A549 (carcinoma lung), HA188 (carcinoma
lung), and A172 (glioblastoma) were provided by S. Aaronson.
The AlOD cells (melanoma) were received from Naval Bio-
logical 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 pre-
pared by the Hirt procedure (21) and purified by isopycnic
centrifugation in ethidium bromide/CsCl gradients. SV40 DNA
Abbreviations: BKV, BK virus; SV40, simian virus 40; Ca, carcino-
ma.
 Medical Sciences: Wold et al.
4- A
A~~~~~~~~~~~
o 3
11
'X
E2-
n
1 2
16- ~~0
0
'-5
LO
M mol-sec-liter-1. Aliquots were removed and assayed for duplex DNA
X4
E C
a L g0r
12
l
7-
6-
0
E '
12
T-~n5mbe
~~r~tO S 23 (CoIE1
DNA
NA
0 10 20 30 40 50
Fraction number
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 ra-
dioactivity 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 iso-
pycnic 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% su-
crose (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). La-
beled 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 pre-
cipitation with ethanol, sonicated to desired size (300-500 nu-
cleotides 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
Downloaded at Indonesia: PNAS Sponsored on August 23, 2020
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
Proc. Natl. Acad. Sci. USA 75 (1978) 455
;, < 10u
.;r:n /
1.70 -
z
0 0
a
- 1.60
_1.50
e
'D
'a~
40
~~7
60 -g
-
O
- 1.40
-1.30 S.
0.I 0. I I I
-1.20 10-6 10-5
-
10-4 10-3 10-2 lo-'
Q
Cot
FIG. 2. Reassociation of BKV and SV40 [32P]DNA. [32P]DNA
(-1 X 108 cpm/pg, 500 cpm/20 ul, 2.5 X 10-4 Ag/ml) was annealed with
various amounts of homologous DNA to Cot values of 10-6 to 10-1
by batchwise elution from hydroxylapatite. 0, BKV (di Mayorca);
a, BKV (Walker); 0, SV40.
"branch migration" would significantly affect radioactive
hybrid formation. The fraction of duplex DNA was determined
by batch elution from hydroxylapatite (25).
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.
60 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 la-
beled 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 la-
beled 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 ex-
pected 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) hybri-
dized 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 Medical Sciences: Wold et al. Proc. Nati. Acad. Sci. USA 75 (1978)

Table 1. Homology between BKV DNAs and SV40 DNA* Table 2. Hybridization of BKV DNA in the presence of non-
Percent hybridization with unlabeled DNAt human cell DNA, human cultured cell DNA, and
normal human tissue DNA*
Calf % hybridization
[32P]DNA thymus BKV-1 BKV-2 BKV-3 SV40 Source of No. of (uncorrected),t
BKV-1 3.4 88.0 88.2 87.2 15.1 DNA tissues tested mean ± SD
(0) (100) (100) (99.0) (13.9) Non-human Cell DNA
BKV-2 1.2 65.4 66.9 69.8 8.7 Calf thymus 8.3
(0) (97.9) (100) (103) (11.8) Salmon sperm 8.8
BKV-3 4.8 87.3 86.7 90.2 12.9 E. coli 8.8
(0) (98.0) (97.2) (100) (13.9) HEC19 (hamster) 8.8
SV40 5.3 13.4 12.5 13.0 85.4 8617 (rat) 8.4
(0) (11.6) (11.3) (11.9) (100) Human cell DNA
* In vitro labeled [32P]DNA (1 to 2 X 108 cpm/gg, 500 cpm/25-Ml ali- KB cells 13.2
quot) was annealed for 4 hr with unlabeled viral DNA at 1 ,g/ml in HEK cells 13.3
the presence of calf thymus DNA at 6 mg/ml under standard con- HEF cells 14.4
ditions. Raw data are presented; normalized values are given in Human tissue DNA
parentheses. Normal kidney .10 14.8 A 2.2
t BKV-1 refers to DNA from the original seed stock of BKV obtained
Normal spleen 3 13.7 ± 0.9
from G. di Mayorca. BKV-2 refers to DNA from a virus stock pre- Normal lung 13 15.4 ± 1.4
pared from a plaque isolate of the di Mayorca BKV. BKV-3 refers Normal skin 1 13.6
to DNA from a virus stock prepared from a twice plaque-purified
isolate of BKV obtained from D. Walker. Normal ileum 1 14.8
Normal rectum 2 14.2
Analysis of DNA from Normal Human Tissues, Human Normal colon 23 13.8 d 1.8
Tumors, and Human Tumor Cell Lines for BKV DNA Se- * In vitro labeled BKV [32P]DNA (1.5 X 108 cpm/Mg, 500 cpm/25-,ul
quences. Table 2 summarizes the results of our analyses of aliquot) was annealed in the presence of cell DNA at 6 mg/ml to an
normal human tissue DNAs for BKV DNA sequences. After 48 equivalent Cot of 20,000 mol-sec-liter-1. Hybridization values have
hr, the BKV [32P]DNA probe self-annealed (i.e., in the presence not been normalized. In reconstruction experiments performed
of non-human DNA at 6 mg/ml) about 8%. The probe annealed under the same conditions, 0.25 and 1.0 copies per cell ofadded BKV
about 13% in the presence of normal human tissue DNAs. DNA gave 32.2% and 63.9% hybridization, respectively, and BKV
Human cell lines gave the same 5% hybridization above DNA at 4 ,g/ml gave 82.2% hybridization.
t The data are not corrected for self-annealing of the BKV [32P]DNA
background as normal tissue DNAs. We conclude that highly in the absence of human cell DNA during the hybridization.
purified BKV DNA (three different preparations) contains human cell line DNA. We have analyzed DNA from a number
human DNA sequences. This 5% hybridization may represent of malignant human cell lines; as summarized in Table 4, we
human sequences integrated into BKV DNA, or possibly human did not detect BKV sequences in the DNA from any of these
contaminants of BKV DNA.
The results of the analyses of human tumors are given in cell lines.
Table 3. None of the human tumors analyzed contained BKV DISCUSSION
DNA (other than the 5% homology discussed above), i.e., tumor
DNAs hybridized to the same extent as normal tissue and Several lines of evidence ensure that our tumor analyses were
conducted with a representative BKV [32P]DNA probe, able
100 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 bro-
z
mide/CsCl gradients, and sedimentation coefficients in alkaline
(52 S) and neutral (22 S) sucrose gradients. (ii) The three BKV
Ga DNAs were >97% homologous in cross-hybridization reactions
CL
and were 11-14% homologous with SV40 DNA. [BKV and
SV40 have been reported to be 10-20% homologous when hy-
brids are assayed under stringent conditions (27).] (i) The
a)
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 mo-
lecular 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
Hours
four fragments reported previously (28, 29) (data not shown).
(v) Reassociation kinetics of the BKV DNA with BKV-trans-
FIG. 3. Hybridization of in vitro labeled BKV [32P]DNA with formed cell DNA yielded 5-6 viral genome copies per cell. The
0-10 copies per cell of BKV DNA, or BK-HK clone 3 BKV-trans- BKV [32P]DNA gave 11-12% hybridization with cytoplasmic
Downloaded at Indonesia: PNAS Sponsored on August 23, 2020

formed 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 thy- RNA from BK-HK cells, indicating that a minimum of 22-24%
mus DNA containing 0-10 copies of unlabeled BKV DNA. BK-HK of the BKV genome is expressed as mRNA in these cells. This
clone 3 cells are a line of BKV-transformed hamster cells. hybridization probably represents BKV "early" transforming
 Medical Sciences: Wold et al. Proc. Natl. Acad. Sc. USA 75 (1978) 457

Table 3. Analysis of human tumor DNAs -for BKV NA I Table 4. Analysis of human tumor cell lines for BKV-specific
sequences DNA sequences*
Primary site No. of % hybridization % % hybridization
and tumor tumors (uncorrected),t distribution Cell line (uncorrected)*
type* tested mean ± SD of casest
A375 Uncl. (Melanoma) 15.2
Digestive system A375 Cl 3 (Melanoma) 14.7
Ca stomach 5 15.1 I 2.2 3.5 A375 Cl 5 (Melanoma) 18.4
Ca ileum 1 15.7 A375 Cl 10 (Melanoma) 13.7
Adca ileum 1 12.0 0.3 A375 Cl 12 (Melanoma) 11.7
Ca small intestine 1 14.1 AlOlD (Melanoma) 13.2
Ca colon (excluding A549 (Ca lung) 16.2
rectum and caecum) 22 14.4 I 1.9 8.4 A204 (Rhabdomyosarcoma) 13.4
Ca caceum 2 13.6 1.8 A172 (Glioblastoma) 14.3
Adca caecum 2 13.6 HA188 Uncl. (Ca lung) 13.8
Ca rectum 9 14.8 ± 1.8 4.5 HA188 Cl 18 (Ca lung) 12.3
Lung T 98 (Glioblastoma) 12.7
Squamous cell Ca§ 31 15.9 2.1 * Hybridizations were performed under the same conditions as de-
Adca 9 13.4 ± 2.6 13.3 scribed in Table 2. Hybridization values have not been corrected
Oat cell 5 15.1 1.1 for self-annealing of the probe in the absence of human cell
Melanomas 17 15.9 ± 1.8 1.4 DNA.
Prostate 5 15.9+2.3 Q C)

Bladder gene sequences expressed as RNA in BKV-transformed cells,

Ca 3 16.5 + 2.4 in analogy to SV40-transformed cells. Thus, our probes very
Rhabdomyosarcoma 1 13.9 likely contained BKV transforming gene sequences.
Kidney We were unable to detect BKV sequences in DNA from any
Wilms tumor 6 13.4 ± 1.4 human tumors or malignant cell lines. In reconstruction ex-
Hypernephroma 4 13.8 ± 1.9 2.0 periments done under the same conditions as the tumor anal-
Ca 15 13.5 2.1 yses, 0.25 copy per cell and 1 copy per cell of added BKV DNA
Brain gave 34-45% and 50-60% hybridization, respectively, above
Glioma 3 11.2 ± 1.5 the self-annealing background of 13-16%. Thus, we would
Glioblastoma 3 13.0 1.3 1.5 easily have detected 0.05 copies per cell of BKV DNA, or 1 copy
Meningioma 1 14.0 of BKV DNA in tumors containing 5-10% BKV-transformed
Lymphoma cells. All tumors analyzed contained at least 50% transformed
Hodgkin disease cells, as indicated by histological examination. Therefore, we
(lymph node) 3 14.3 ± 1.5 can be virtually certain that these particular tumors and tumor
Hodgkin disease cell lines did not have a BKV etiology (i.e., these tumor types
(lung) 1 16.4 1.1 do not have an obligatory BKV etiology). A "hit and run"
Hodgkin disease mechanism of BKV tumorigenesis cannot be formally excluded,
(spleen) 1 16.1 but this is unlikely from an overwhelming body of data on viral
Lymph node 3 14.2 ± 0.4 tumorigenesis in animal and cell culture systems. We point out
Liver 4 15.4 ± 1.7 that all the BKV isolates examined to date appear to be very
Spleen 1 16.1 2.2
Ileum 1 14.6
similar (13, 16, 29), so that it is likely that our studies are ap-
Colon 1 16.9 plicable to all "strains" of BKV present in the United States
Reticulum cell population.
sarcoma (spleen) 2 13.5 The tumors that we have analyzed represent major categories
Skin of human cancer, and account for about 50% of total cancers
Ca 1 13.9 in the United States (26). For most of the tumor types, we have
Squamous cell Ca 1 14.3 examined quite a large number of samples (Table 3). Therefore,
Neurofibrosarcoma 1 13.5 apparently BKV is not a major cause of human cancer. Because
at least 80% of the United States population has been infected
* About half the tumors tested were obtained through the ( )ffice of by BKV, it is likely that some or most of the patients from which
Program Resources and Logistics of the Virus Cancer I?rogram our tumors were derived had been infected by the virus.
within the National Cancer Institute. The remaining tum4 ors were Therefore, it seems that infection by BKV does not necessarily
obtained from other sources. Our laboratory will supply v
quest (i) the National Cancer Institute tumor designation I mon re- result in the development of any of the common cancers. It
remains possible, however, that BKV could induce specific rare
(ii) our internal designation numbers of the other tumors, ind (iii)
the hybridization data obtained with each individual tuI ior ana- types of cancer, or on occasion induce one of the common
lyzed. Ca, carcinoma; Adca, adenocarcinoma. cancers. A long-range extensive analysis of cancers from all sites,
t The human tumors were analyzed in duplicate under ti ie same including different histological types, is warranted and neces-
conditions and at the same time as the DNA samples desc: ribed in sary to determine whether BKV is ever carcinogenic in
Table 2. The data have not been corrected for self-annealin g of the humans.
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 in the United States diagnosed in 1969-71 by primary site, with both
BKV [32PJDNA probes, as determined by reconstruction experi- sexes and all races combined. The data exclude carcinoma in situ
ments with added BKV DNA, is presented in the no tes for and nonmelanoma skin cancers.
Table 2. § Represents either diagnosed or presumed squamous cell carcinoma
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These incidence data (26) reflect the percent distribution of cases of the lung.
 458 Medical Sciences: Wold et al.
Epidemiological studies have indicated immunosuppressed
renal transplant patients develop cancer at 19 times the inci-
dence 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, par-
ticularly 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 un-
able 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 as-
sayed 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 pas-
sage. 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
Downloaded at Indonesia: PNAS Sponsored on August 23, 2020
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.
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