2124
Expression of the Simian Virus 40 Large Tumor
Antigen (Tag) and Formation of Tag-p53 and
Tag-pRb Complexes in Human Brain Tumors
Hai-Ning Zhen, M.D.1
Xiang Zhang, M.D., Ph.D.1
Xing-Yao Bu, Post, M.D., Ph.D.1
Zhi-Wen Zhang, M.D., Ph.D.1
Wen-Jin Huang, M.D., Ph.D.2
Ping Zhang, M.D.2
Jing-Wen Liang, M.D.1
Xi-Ling Wang, M.D.1
1
Department of Neurosurgery, Xijing Hospital, The
Fourth Military Medical University, Xi’an, The Peo-
ple’s Republic of China.
2
Institute of Neuroscience, The Fourth Military
Medical University, Xi’an, The People’s Republic of
China.
Supported in part by the Chinese Association for
Cancer Research, Beijing, China.
The authors are indebted to Prof. C. J. Wang for
the HFSV40 cells expressing SV40 Tag.
Address for reprints: Xiang Zhang, M.D., Ph.D.,
Department of Neurosurgery, Xijing Hospital, The
Fourth Military Medical University, Xi’an, 710032,
People’s Republic of China.
Received August 28, 1998; revision received April
28, 1999; accepted July 2, 1999.
© 1999 American Cancer Society
BACKGROUND. The presence of simian virus 40 (SV40) in human brain tumors
remains a controversial issue. Even if SV40 does exist in brain tumors, the ques-
tions of whether it is associated with brain tumorigenesis and by what mechanisms
are unknown.
METHODS. SV40 large tumor antigen (Tag) was investigated by immunoprecipita-
tion, silver staining, and Western blot analysis in 65 brain tumor cases and 8 cases
of normal brain tissue. Tag-p53 and Tag-pRb complexes were screened by immu-
noprecipitation and Western blot analysis in 18 and 15 Tag positive tumor tissues,
respectively.
RESULTS. Tag was found in all 8 cases of ependymoma and 2 cases of choroid
plexus papilloma, 90% of pituitary adenoma cases (9 of 10), 73% of astrocytoma
cases (11 of 15), 70% of meningioma cases (7 of 10), 50% of glioblastoma multi-
forme cases (4 of 8), and 33% of medulloblastoma cases (2 of 6). Five oligoden-
droglioma cases, 1 pineocytoma case, and 8 cases of normal brain tissue were
negative for Tag. The Tag-p53 complex was detected in all 18 Tag positive tumors
tested and the Tag-pRb complex was detected in all 15 Tag positive tumors tested.
CONCLUSIONS. SV40 Tag not only is expressed in brain tumors; it also can form
specific complexes with tumor suppressors p53 and pRb. SV40 is correlated with
brain tumorigenesis. The inactivation of p53 and pRb due to the formation of
Tag-p53 and Tag-pRb complexes possibly is a significant mechanism in the etio-
pathogenesis of brain tumors. Cancer 1999;86:2124 –32.
© 1999 American Cancer Society.
KEYWORDS: simian virus 40, large tumor antigen, brain tumor, p53, pRb.
B rain tumors account for only 2% of all cancers; however, patients
with these tumors have a very poor prognosis. Moreover, brain
neoplasms are the most frequent solid tumors in children. The inci-
dence of brain tumors has increased about 30% in the past 20 years;
therefore, the level of public interest and anxiety is high.1,2 It is
generally accepted that human brain tumors, like other cancers,
represent a genetic disease of somatic cells that is due to several
alterations, such as gene mutations, deletions, translocations, or am-
plifications. The causes of these genetic aberrations are not under-
stood completely; therefore the etiology and risk factors for these
tumors remain to be determined. Simian virus 40 (SV40), one type of
polyomavirus, is able to transform cells from different species, includ-
ing normal human cells, into cells with a neoplastic phenotype.3 SV40
is highly oncogenic in hamsters and transgenic mice, in which it
induces specific types of tumors.4 SV40 oncogenicity and transform-
ing ability are strictly dependent on the expression of the early region
 SV40 Tag, p53, and pRb in Human Brain Tumors/Zhen et al.
gene product, large tumor antigen (Tag), which has
multiple biologic and biochemical properties. SV40
Tag forms complexes with the tumor suppressors p53,
pRb, p107, p130, p300, and p400, inactivating their
functions;5–11 alters the integrity and stability of the
host cell genome, inducing numerical and structural
chromosomal aberrations;12–14 transactivates many
cellular promoters, inducing abnormal gene expres-
sion;15,16 has ATPase and helicase activities; and binds
to cellular DNA, stimulating its replication.9 In addi-
tion, SV40 small t antigen (tag) enhances the ability of
SV40 Tag to transform normal cells in vitro and to
induce tumors in vivo.9 Recently, SV40 sequences
were detected by using polymerase chain reaction
(PCR) followed by Southern blot analysis, and SV40
Tag was detected by immunohistochemistry in some
types of tumors, including brain tumors,17–21 me-
sothelioma,22,23 osteosarcoma,24 and papillary thyroid
carcinoma.25 Even SV40 virion was isolated success-
fully from one choroid plexus tumor.18 All of these
findings indicate that SV40 probably plays a role in
human tumorigenesis. However, there also have been
some conflicting reports.26 –28 Therefore, the presence
of SV40 in brain tumors remains a topic of heavy
debate and investigation. Even if SV40 is present in
brain tumors, until now, there has been no additional
data to show that SV40 is associated with tumorigen-
esis.11,29 –32
In the current study, SV40 Tag was investigated by
immunoprecipitation, silver staining, and Western
blot analysis in brain tumors, and Tag-p53 and Tag-
pRb complexes also were investigated by immunopre-
cipitation and Western blot analysis. We found that
Tag is expressed in brain tumors of most histologic
types and that Tag can form specific complexes with
p53 and pRb. A possible correlation between SV40 and
brain tumorigenesis also is discussed.
MATERIALS AND METHODS
Primary Tumors and Normal Tissues
Sixty-five fresh brain tumor specimens were obtained
by surgical removal. Eight normal brain tissue samples
were obtained from autopsy specimens from road ac-
cident victims. All specimens were provided by the
Department of Neurosurgery of Xijing Hospital, the
Fourth Military Medical University, Xi’an, China. All
neoplastic and normal samples were collected ran-
domly without any selection for specific categories of
patients or normal controls. The histologic types of the
65 tumors, according to the central nervous system
tumor classification criteria established by the World
Health Organization in 1990,33 were as follows: 15
astrocytoma cases, 10 meningioma cases, 10 pituitary
adenoma cases, 8 glioblastoma multiforme cases, 8
2125
ependymoma cases, 6 medulloblastoma cases, 5 oli-
godendroglioma cases, 2 choroid plexus papilloma
cases, and 1 pineocytoma case. The age of the neo-
plastic patients was between 13 months and 72 years
(mean age, 38 years). Thirty-five patients were men,
and 30 patients were women (Table 1). Tumoral and
normal samples were collected, snap frozen in liquid
nitrogen, then stored at 270 °C until the time of anal-
ysis. Human foreskin fibroblasts containing the SV40
Tag (HFSV40 cells34) were a kind gift from Prof. C. J.
Wang and were used as a positive control in immu-
noprecipitation, silver staining, and Western blot ex-
periments. These cells were cultured at 37 °C in RPMI
1640 supplemented with 10% fetal bovine serum (FBS)
in a 5% CO2 humidified atmosphere.
Antibodies
The mouse anti-SV40 Tag monoclonal antibody (MAb)
Pab101, the mouse antihuman p53 MAb DO-1, and
the rabbit antihuman pRb polyclonal antibody (PAb)
C-15 that were used in immunoprecipitation and
Western blot analysis all were purchased from Santa
Cruz Biotechnology Co. (Santa Cruz, CA). The nonspe-
cific mouse hepatocarcinoma ascites fluid ZP9 that
was used as a negative control and to preclear cell
lysates in immunoprecipitation was provided by the
Institute of Digestive Disease of Xijing Hospital, the
Fourth Military Medical University, Xi’an, China. The
secondary antibodies used in Western blot analysis
were goat antimouse and goat antirabbit biotin-con-
jugated immunoglobulin G (IgG), which were pur-
chased from Sigma Chemical Co. (St. Louis, MO).
Immunoprecipitation
For the immunoprecipitation experiments, 0.5 g of
tissue specimen or 107 cells were placed in a 10-mL
centrifuge tube, and 2 mL of ice-cold lysis buffer (50
mM Tris Cl, pH 8.0; 150 mM NaCl; 0.1% sodium do-
decyl sulfate [SDS]; 0.5% sodium deoxycholate; 1%
Nonidet P-40; and 20 mL/mL protease-inhibitor cock-
tail solution [Boehringer Mannheim, Mannheim, Ger-
many]) was added. Tissue was homogenized at 4 °C
and then incubated at 4 °C for 30 minutes. Cell debris
was pelleted by centrifugation at 310,000 g for 15
minutes at 4 °C. One milliliter of supernatant was
transferred to a 1.5-mL centrifuge tube, 10 mL of ZP9
and 20 mL of 50% protein A-Sepharose beads (Phar-
macia Biotech, Uppsala, Sweden) were added, and the
tissues were incubated overnight at 4 °C with rotation,
then centrifuged at 33000 g for 5 minutes at 4 °C.
Supernatant was transferred, and 5 mL (0.5 mg) of
Pab101, DO-1, or C-15 antibody and 10 mL of 50%
protein A-Sepharose beads were added. ZP9 was used
as a substitute for Pab101, DO-1, or C-15 as a negative
2126 CANCER November 15, 1999 / Volume 86 / Number 10

TABLE 1
Brain Tumors and Normal Brain Tissues Studied

Sample Age (yrs) Gender Histologic type SV40 Tag expression Tag-p53 complex Tag-pRb complex

1 14 M E 1 1 n.d.
2 28 F E 1 n.d. 1
3 49 M E 1 n.d. n.d.
4 33 M E 1 n.d. n.d.
5 68 F E 1 1 n.d.
6 51 F E 1 n.d. n.d.
7 36 F E 1 n.d. 1
8 48 M E 1 n.d. n.d.
9 27 M CPP 1 1 1
10 15 M CPP 1 1 1
11 21 F PA 1 1 n.d.
12 35 M PA 2 n.d. n.d.
13 42 F PA 1 n.d. 1
14 27 M PA 1 1 n.d.
15 48 F PA 1 n.d. n.d.
16 52 F PA 1 n.d. n.d.
17 25 M PA 1 1 n.d.
18 37 M PA 1 n.d. 1
19 36 F PA 1 n.d. n.d.
20 34 F PA 1 n.d. n.d.
21 47 M A 2 n.d. n.d.
22 38 F A 1 1 n.d.
23 25 F A 1 n.d. n.d.
24 68 F A 1 n.d. n.d.
25 45 M A 1 1 n.d.
26 28 M A 1 1 n.d.
27 41 F A 2 n.d. n.d.
28 53 M A 1 n.d. 1
29 42 M A 1 n.d. n.d.
30 35 M A 1 n.d. 1
31 29 F A 1 n.d. n.d.
32 47 F AA 1 1 n.d.
33 32 M AA 2 n.d. n.d.
34 65 F AA 1 n.d. 1
35 30 M AA 2 n.d. n.d.
36 51 M MEN 1 1 n.d.
37 45 F MEN 1 n.d. 1
38 68 M MEN 2 n.d. n.d.
39 24 F MEN 1 1 n.d.
40 72 M MEN 1 n.d. n.d.
41 37 M MEN 1 n.d. 1
42 35 F MEN 2 n.d. n.d.
43 60 F MEN 2 n.d. n.d.
44 47 M MEN 1 1 n.d.
45 43 F MEN 1 n.d. n.d.
46 39 F GBM 2 n.d. n.d.
47 46 M GBM 1 n.d. 1
48 33 F GBM 1 1 n.d.
49 28 M GBM 2 n.d. n.d.
50 57 M GBM 2 n.d. n.d.
51 44 F GBM 1 1 n.d.
52 37 M GBM 1 n.d. 1
53 26 M GBM 2 n.d. n.d.
54 14 F MED 2 n.d. n.d.
55 8 M MED 1 1 1
56 22 M MED 2 n.d. n.d.
57 18 F MED 2 n.d. n.d.
(continued)
 SV40 Tag, p53, and pRb in Human Brain Tumors/Zhen et al. 2127

TABLE 1 (continued)

Sample Age (yrs) Gender Histologic type SV40 Tag expression Tag-p53 complex Tag-pRb complex

58 25 F MED 1 1 1
59 13 months M MED 2 n.d. n.d.
60 38 M ODG 2 n.d. n.d.
61 44 F ODG 2 n.d. n.d.
62 40 M ODG 2 n.d. n.d.
63 52 F ODG 2 n.d. n.d.
64 27 M ODG 2 n.d. n.d.
65 35 M P 2 n.d. n.d.
66 25 M NB 2 n.d. n.d.
67 63 M NB 2 n.d. n.d.
68 36 M NB 2 n.d. n.d.
69 15 M NB 2 n.d. n.d.
70 27 M NB 2 n.d. n.d.
71 35 M NB 2 n.d. n.d.
72 32 F NB 2 n.d. n.d.
73 17 M NB 2 n.d. n.d.

SV40 Tag: simian virus 40 large tumor antigen; 1: positive; 2: negative; n.d.: not determined; M: male, F: female; E: ependymoma; CPP: choroid plexus papilloma; PA: pituitary adenoma; A: astrocytoma; AA: anaplastic
astrocytoma; MEN: meningioma; GBM: glioblastoma multiforme; MED: medulloblastoma; ODG: oligodendroglioma; P: pineocytoma; NB: normal brain tissue.

control. Then, the tissues were incubated overnight solution (3% sodium carbonate, 0.02% formaldehyde)
with rotation at 4 °C. Immunoprecipitates were pel- was added, and they were incubated for 5–10 minutes.
leted by centrifugation at 33000 g for 5 minutes at 4 When the desired protein band intensities were
°C, then washed twice with RIPA buffer (50 mM Tris achieved, the reactions were stopped by incubating
Cl, pH 7.5; 150 mM NaCl; 0.1% SDS; 0.5% sodium the gels with 1% acetic acid for 10 minutes, then the
deoxycholate; 1% Nonidet P-40) and once with TN gels were washed three times with tridistilled water for
buffer (10 mM Tris Cl, pH 7.5; 0.1% Nonidet P-40) for 3 minutes each, dried, and stored.
10 minutes both times at 4°C. Immunoprecipitates
were resuspended in 30 mL of loading buffer (50 mM Western Blot Analysis
Tris Cl, pH 6.8; 100 mM dithiothreitol; 2% SDS; 0.1% After 10% SDS PAGE, immunoprecipitated proteins
bromphenol blue; 10% glycerol). Immunoprecipitated were transferred onto nitrocellulose (NC) membranes
proteins were dissociated from the protein A-Sepha- (Millipore, Bedford, MA) by using a Mini-protein II
rose beads by incubation for 5 minutes at 100 °C. electrophoresis unit (Bio-Rad, Hercules, CA). NC
Immunoprecipitated protein supernatant was col- membranes were treated with blocking buffer (PBS,
lected after centrifugation at 33000 g for 5 minutes at pH 7.4; 1% bovine serum albumin [BSA]) for 1 hour at
4 °C and stored at 220 °C. room temperature then probed with primary antibody
in blotting buffer (PBS, pH 7.4; 1% BSA; 0.05% Tween
Silver Staining 20) overnight at 4 °C. Primary antibodies were used at
Equal amounts of protein supernatant (5–20 mL) were the following concentrations: Pab101, 1:5000; DO-1,
subjected to 10% SDS polyacrylamide gel electro- 1:1000; C-15, 1:1000. NC membranes were rinsed three
phoresis (SDS PAGE), and 10-kilodalton (kDa) protein times with rinse buffer (PBS, pH 7.4; 0.05% Tween 20)
ladder markers (Life Technologies, Grand Island, NY) for 5 minutes each then probed with antimouse or
were included for comparison. After electrophoresis, antirabbit secondary antibody (1:2000) in blotting
the gels were treated with fixing solution (50% meth- buffer for 1 hour at room temperature. Then, the
anol, 12% acetic acid) for 3 hours at room temperature membranes were rinsed three times with rinse buffer;
(the following procedures all were performed at room probed with avidin-biotin peroxidase complex (Sigma;
temperature). The gels were washed twice with 10% 1:10,000) in blotting buffer for 1 hour at room temper-
ethanol for 5 minutes each and three times with tri- ature; rinsed three more times with rinse buffer and
distilled water for 3 minutes each, freshly prepared once with PBS, pH 7.4; then the antibody binding was
0.1% silver nitrate solution was added, and the gels visualized using freshly prepared GDN color develop-
were incubated for 30 minutes. The gels were washed ing solution (0.1 M acetate buffer, pH 6.0; 1.25% nick-
with tridistilled water for 30 seconds, color developing el-ammonium sulfate; 0.04% ammonium chloride;
2128 CANCER November 15, 1999 / Volume 86 / Number 10

FIGURE 1. Representative analysis of immunoprecipitation and silver staining of lysates from 65 human brain tumors and 8 normal brain tissues. Lysates were
immunoprecipitated with the mouse antisimian virus 40 (anti-SV40) large tumor antigen (Tag) monoclonal antibody (MAb) Pab101. Immunoprecipitates were
visualized by silver staining. Human foreskin fibroblasts containing the SV40 Tag (HFSV40) were used as a positive control. Pab101 was substituted with the
nonspecific mouse ascites fluid ZP9 in immunoprecipitation as a negative control. The protein bands located between 90 kilodaltons (kDa) and 100 kDa imply the
presence of SV40 Tag (94 kDa). (A) Lane1: Astrocytoma (the same sample is shown in B, lane 3; negative control). Lane 2: HFSV40 cells (positive control). Lane
3: Ependymoma. Lane 4: Choroid plexus papilloma. Lane 5: Meningioma. (B):Lane 1: Normal brain tissue. Lane 2: Pituitary adenoma. Lane 3: Astrocytoma. Lane
4: Glioblastoma multiforme. Lane 5: Medulloblastoma.

FIGURE 2. Representative analysis of immunoprecipitation and Western blot analysis of lysates from 65 human brain tumors and 8 normal brain tissues. Lysates
were immunoprecipitated with the mouse anti-SV40 Tag MAb Pab101. Pab101 also was used in Western blot analysis as the primary antibody. HFSV40 cells were
used as a positive control. Pab101 was substituted by the nonspecific mouse ascites fluid ZP9 in immunoprecipitation as a negative control. SV40 Tag (94 kDa)
and murine immunoglobulin G (IgG) heavy chain ('53 kDa) are indicated. (A) Lane 1: Normal brain tissue. Lane 2: Astrocytoma. Lane 3: Glioblastoma multiforme.
Lane 4: Pituitary adenoma. Lane 5: Meningioma, (B) Lane 1: HFSV40 cells (positive control). Lane 2: Ependymoma (the same sample is shown in lane 3; negative
control). Lane 3: Ependymoma. Lane 4: Choroid plexus papilloma. Lane 5: Medulloblastoma.

0.05% 3,39-diaminobenzidine; 0.2% b-D-glucose;
0.001% glucose oxidase [Sigma]) for '20 minutes at
room temperature.35
RESULTS
Expression of SV40 Tag in Brain Tumors
Because there were no previous reports of the success-
ful detection of SV40 Tag using only Western blot
analysis, we speculated that, even if Tag is expressed
in brain tumors, its quantity is very low. Therefore, in
this study, SV40 Tag first was “concentrated” by im-
munoprecipitation using the mouse anti-SV40 Tag
MAb Pab101. After 10% SDS PAGE, the immunopre-
cipitated proteins were visualized by silver staining,
which is ten times more sensitive than Western blot
analysis for detecting proteins. Obvious protein bands
were seen located between 90 kDa and 100 kDa on
most gel lanes (Fig. 1), suggesting the possible pres-
ence of SV40 Tag (94 kDa) in brain tumors and, sig-
nificantly, also hinting at the possibility of detecting
SV40 Tag with the further use of Western blot analysis,
although it is not as sensitive as silver staining. Indeed,
SV40 Tag was found in most brain tumors by Western
blot analysis using Pab101 as the primary antibody
(Fig. 2). The results of Western blot analysis were in
complete accordance with the results of silver staining
analysis. SV40 Tag was detected in all 8 ependymoma
cases, in 2 choroid plexus papilloma cases, in 90% of
pituitary adenoma cases (9 of 10 tumors), in 73% of
astrocytoma cases (11 of 15 tumors), in 70% of menin-
gioma cases (7 of 10 tumors), in 50% of glioblastoma
multiforme cases (4 of 8 tumors), and in 33% of me-

TABLE 2
Expression of Simian Virus 40 Large Tumor Antigen and Formation
of Large Tumor Antigen-p53 and Large Tumor Antigen-pRb
Complexes in Human Brain Tumorsa
Tissue type SV40 Tag (%)
Brain tumors
Ependymoma 8 of 8 (100)
Choroid plexus papilloma 2 of 2 (100)
Pituitary adenoma 9 of 10 (90)
Astrocytoma 11 of 15 (73)
Meningioma 7 of 10 (70)
Glioblastoma multiforme 4 of 8 (50)
Medulloblastoma 2 of 6 (33)
Oligodendroglioma 0 of 5 (0)
Pineocytoma 0 of 1 (0)
Total 43 of 65 (66)
Normal brain tissues 0 of 8 (0)
SV40: simian virus 40; Tag: large tumor antigen; n.d.: not done.
a
Values represent the number of positive samples in the total samples analyzed. Percentages (in
parentheses) indicate the positive rate.
dulloblastoma cases (2 of 6 tumors). The 8 normal
brain tissue samples all were negative for SV40 Tag
along with 5 oligodendroglioma cases and 1 pineocy-
toma case (Tables 1, 2). When Pab101 was replaced by
the nonspecific mouse ascites fluid ZP9 in immuno-
precipitation, there was no SV40 Tag found on silver
staining and Western blot analyses (Figs. 1, 2).
Detection of Tag-p53 Specific Complex in Brain Tumors
Detection of SV40 Tag by Western blot analysis after
immunoprecipitation led us to wonder whether Tag
can form a specific complex with p53 in brain tumors
as it does in culture cells in vitro. For this, 18 Tag
positive brain tumor samples were selected, including
4 astrocytoma cases, 3 pituitary adenoma cases, 3
meningioma cases, 2 ependymoma cases, 2 choroid
plexus papilloma cases, 2 glioblastoma multiforme
cases, and 2 medulloblastoma cases. In Western blot
analysis of anti-Tag and anti-p53 immunoprecipitates
generated from all 18 specimens, Tag and p53 were
both present when using a cocktail of mouse anti-
SV40 Tag MAb Pab101 and mouse antihuman p53
MAb DO-1 as the primary antibodies (Figs. 3, 4). How-
ever, there was neither Tag nor p53 on Western blot
when Pab101 or DO-1 was substituted by nonspecific
mouse ascites fluid ZP9 in immunoprecipitation (Fig.
3A, lane 3; Fig. 3B, lane 5; Fig. 4, lane 8). These findings
indicated that SV40 Tag still is capable of forming a
specific complex with p53 in brain tumors (Tables 1,
2). However, it should be pointed out that, because
murine IgG heavy chain, which has a molecular
weight of '53 kDa, also can be reacted with the goat
SV40 Tag, p53, and pRb in Human Brain Tumors/Zhen et al. 2129
antimouse secondary antibody and can be visualized
on Western blot analysis, the p53 protein bands in
anti-Tag immunoprecipitates were superimposed
with murine IgG heavy chain. Regardless of the obser-
Tag-p53 Tag-pRb vations that the p53 positive, 53-kDa protein bands
complex complex were wide and heavily colored, whereas the IgG heavy
chain bands were narrow and lightly colored (Figs. 3,
4), the presence of p53 still is implied. Furthermore,
2 of 2 2 of 2
2 of 2 2 of 2 the presence of SV40 Tag on Western blots in anti-p53
3 of 3 2 of 2 immunoprecipitates (Fig. 4) provided further evidence
4 of 4 3 of 3 of the formation of Tag-p53 specific complexes.
3 of 3 2 of 2
2 of 2 2 of 2
Detection of Tag-pRb Specific Complex in Brain Tumors
2 of 2 2 of 2
n.d. n.d. To screen for the presence of Tag-pRb specific com-
n.d. n.d. plex, 15 Tag positive brain tumor specimens were
18 of 18 15 of 15 chosen, including 3 astrocytoma cases, 2 ependy-
n.d. n.d. moma cases, 2 choroid plexus papilloma cases, 2 gli-
oblastoma multiforme cases, 2 medulloblastoma
cases, 2 meningioma cases, and 2 pituitary adenoma
cases. In anti-SV40 Tag immunoprecipitates generated
from all 15 specimens, pRb (110 kDa) was present on
Western blot analysis by using rabbit antihuman pRb
PAb C-15 as the primary antibody (Fig. 5). Further-
more, SV40 Tag was seen in anti-pRb immunoprecipi-
tates on Western blot analysis by using the mouse
anti-SV40 Tag MAb Pab101 as the primary antibody
(Fig. 6A), and pRb was seen on Western blot analysis
by using the C-15 as the primary antibody (Fig. 6B).
However, neither SV40 Tag nor pRb was seen on West-
ern blot analysis when Pab101 or C-15 was replaced by
the nonspecific mouse ascites fluid ZP9 in immuno-
precipitation. These findings indicate that SV40 Tag
also can form a specific complex with pRb in brain
tumors.
DISCUSSION
In the current study, we found that SV40 Tag was
expressed in most histologic types of brain tumors,
including ependymoma (100%), choroid plexus papil-
loma (100%), pituitary adenoma (90%), astrocytoma
(73%), meningioma (70%), glioblastoma multiforme
(50%), and medulloblastoma (33%). However, normal
brain tissue cases all were negative for SV40 Tag. To
the best of our knowledge, this study serves as the first
report of the detection of SV40 Tag in brain tumors by
using immunoprecipitation, silver staining, and West-
ern blot analysis. The expression of SV40 Tag not only
proved indirectly the presence of SV40 but also eluci-
dated that the SV40 early region gene is transcribed
and translated functionally. Martini et al.21 found the
SV40 early region gene by using PCR followed by
Southern blot analysis in 83% of choroid plexus pap-
illoma cases, 73% of ependymoma cases, 47% astro-
cytoma cases, 33% of glioblastoma multiforme cases,
2130 CANCER November 15, 1999 / Volume 86 / Number 10

FIGURE 3. Representative analysis of immunoprecipitation and Western blot of lysates from 18 SV40 Tag positive human brain tumors. Lysates were
immunoprecipitated with the mouse anti-SV40 Tag MAb Pab101. Pab101 was substituted by the nonspecific mouse ascites fluid ZP9 in immunoprecipitation as
a negative control. The cocktail of Pab101 and mouse antihuman p53 MAb (DO-1) was used in Western blot analysis as the primary antibody. SV40 Tag (94 kDa),
p53, and murine IgG heavy chain ('53 kDa) are indicated. (A) Lane 1: Astrocytoma. Lane 2: Ependymoma. Lane 3: Astrocytoma (the same sample is shown in
lane 1; negative control). Lane 4: Choroid plexus papilloma. Lane 5: Glioblastoma multiforme. (B) Lane 1: Meningioma. Lane 2: Pituitary adenoma. Lane 3:
Medulloblastoma. Lane 4: Astrocytoma. Lane 5: Meningioma (the same sample is shown in lane 1; negative control).

FIGURE 5. Representative analysis of immunoprecipitation and Western blot

FIGURE 4. Representative analysis of immunoprecipitation and Western blot
analysis of lysates from 15 SV40 Tag positive human brain tumors. Lysates
analysis of lysates from 18 SV40 Tag positive human brain tumors. Lysates
were immunoprecipitated with the mouse anti-SV40 Tag MAb Pab101. Pab101
were immunoprecipitated with the mouse antihuman p53 MAb DO-1. DO-1
was substituted by the nonspecific mouse ascites fluid ZP9 in immunoprecipi-
was substituted with the nonspecific mouse ascites fluid ZP9 in immunopre-
tation as a negative control. The rabbit antihuman pRb polyclonal antibody
cipitation as a negative control. The cocktail of DO-1 and Pab101 (mouse
(PAb) C-15 was used in Western blot analysis as the primary antibody. pRb
anti-SV40 Tag MAb) was used in Western blot analysis as the primary antibody.
(110 kDa) and murine IgG heavy chain ('53 kDa) are indicated. Lane 1:
SV40 Tag (94 kDa), p53, and murine IgG heavy chain ('53 kDa) are indicated.
Meningioma (the same sample is shown in lane 5; negative control). Lane 2:
Lane 1: Meningioma. Lane 2: Pituitary adenoma. Lane 3: Ependymoma. Lane
Astrocytoma. Lane 3: Glioblastoma multiforme. Lane 4: Medulloblastoma. Lane
4: Choroid plexus papilloma. Lane 5: Astrocytoma. Lane 6: Glioblastoma
5: Meningioma. Lane 6: Pituitary adenoma. Lane 7: Ependymoma. Lane 8:
multiforme. Lane 7: Medulloblastoma. Lane 8: Pituitary adenoma (the same
Choroid plexus papilloma.
sample is shown in lane 2; negative control).

Carbone et al.36 and Deluca et al.37 also found the

and 14% meningioma cases. The positive rate of SV40
Tag in this study was higher than that found by Mar-
tini et al. for the SV40 early region gene. The reasons
for this discrepancy may lie in the differing infection
levels of SV40 due to geographic area and race. In
terms of the high specificity of the reaction of the MAb
Pab101 with SV40 Tag, we believe that our results were
not interfered with by the JC virus Tag or the BK virus
Tag, despite the fact that those two viruses are ubiq-
uitous in the human population.4
We also discovered that SV40 Tag is able to form
specific complexes with p53 and pRb in brain tumors,
a finding that was not reported previously. Recently,
SV40 Tag-p53 as well as SV40 Tag-pRb complexes in
human mesothelioma, respectively; thus, our findings
are supported indirectly by their results. It has been
demonstrated that SV40 Tag specifically can bind
wild-type p53 (wt-p53) and hypophosphorylated
forms of pRb in vitro experiments, and, by seques-
tering and inactivating their activities in cell growth
restraint, Tag can induce cell transformation in cul-
ture cells and tumorigenesis in experimental ani-
mals.5– 8 However, because the mouse antihuman p53
MAb DO-1 used in the current study reacts to both
wt-p53 and mutant p53, whether SV40 Tag binds spe-
cifically wt-p53 but not to mutant p53 in brain tumors
 SV40 Tag, p53, and pRb in Human Brain Tumors/Zhen et al. 2131

FIGURE 6. Representative analysis of immunoprecipitation and Western blot analysis of lysates from 15 SV40 Tag positive human brain tumors. Lysates were
immunoprecipitated with the rabbit antihuman pRb PAb C-15. C-15 was substituted by the nonspecific mouse ascites fluid ZP9 in immunoprecipitation as a negative
control. The mouse anti-SV40 Tag MAb Pab101 (A) or C-15 (B) was used in Western blot analysis as the primary antibody. SV40 Tag (94 kDa) (A) or pRb (110 kDa)
(B) and rabbit IgG heavy chain ('53 kDa) are indicated. Lane 1: Meningioma. Lane 2: Pituitary adenoma. Lane 3: Ependymoma. Lane 4: Choroid plexus papilloma.
Lane 5: Astrocytoma. Lane 6: Glioblastoma multiforme. Lane 7: Meningioma (the same sample is shown in lane 1; negative control). Lane 8: Medulloblastoma.

remains to be determined. Similarly, because the rab-
bit antihuman pRb PAb C-15 reacts to either phos-
phorylated pRb or nonphosphorylated pRb, it could
not be determined whether SV40 Tag forms a specific
complex with nonphosphorylated pRb, and not with
phosphorylated pRb, in this study. Therefore, the bio-
chemical activities of p53 and pRb in complex forma-
tion remain unknown. Nonetheless, in the current
study, because SV40 Tag was shown to have similar
functions of binding p53 and pRb in brain tumors, it
was possible that the other specific properties pos-
sessed by SV40 Tag in vitro also may be identical in
vivo. Thus, the results suggested that the complex
formation of Tag-p53 and Tag-pRb may be another
inactivation pattern of the p53 and Rb genes at the
protein level in addition to the mutations and dele-
tions of the p53 and Rb genes in brain tumors.38 – 42
Consequently, these results implied that SV40 Tag
possibly plays a certain role in the etiopathology of
brain tumors by binding and inactivating p53 and
pRb. The quantitative correlations of SV40 Tag, Tag-
p53, and Tag-pRb complexes were not investigated in
this study; nevertheless, we assume that their quanti-
ties are very low in brain tumors in terms of the
findings. Therefore, it seems reasonable to question
the importance in brain tumorigenesis of SV40 Tag
expression and Tag-p53 and Tag-pRb complex forma-
tion. The current findings do not provide more an-
swers. Along with p53 and pRb, other proteins that
comigrated with SV40 Tag were seen on silver staining
(Fig. 1), suggesting the possibility that SV40 Tag may
bind other host cell proteins, such as p107, p130, p300,
and p400, in brain tumors as it does in vitro9,11 or that
it may bind other unknown host cell proteins.
It is known that SV40 does not infect humans
naturally, and human cells are only semipermissive
for SV40 infection. However, SV40 was introduced into
the human population about 40 years ago by contam-
inated polio vaccines and adenovaccines.4,11,33 Mar-
tini et al.21 found the SV40 early region gene in 23% of
peripheral blood cells samples and in 45% of sperm
fluids from healthy individuals, despite the finding by
Shah et al.43of no SV40 footprints in human urine.
These finding indicate the possible manner of the
transmission of SV40 from person to person in the
normal human population.
In summary, according to the current results,
SV40 Tag not only is expressed in human brain tumors
but also can form specific complexes with p53 and
pRb. SV40 is associated with brain tumorigenesis, and
it would be worthwhile to research further its mode of
infection, course of persistence, mechanisms of onco-
genesis, and appropriate diagnostic and therapeutic
approaches.
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