Download Morphological Transformation by Early Region Human

J, gen. Virol. (1988), 69, 467-471.
Printed in Great Britain
467
Key words: B K virus~transformation~human chromosome 11
Morphological Transformation by Early Region Human Polyomavirus BK
DNA of Human Fibroblasts with Deletions in the Short Arm of One
Chromosome 11
By A. D E R O N D E , 1 M. M A N N E N S , 2 R. M. S L A T E R , 2 J. H O O V E R S , 2
C. H E Y T I N G , 2 E. M. B L E E K E R - W A G E M A K E R S , 3 N. J. L E S C H O T , 2
A. V A N S T R I E N , 1 C. J. A. SOL, 1 J. T E R S C H E G G E T 1 AND
J. V A N D E R N O O R D A A 1.
1Department of Virology, 2Institute of Human Genetics and 3Interuniversity Ophthalmology
Institute, University of Amsterdam, Academic Medical Centre, Meibergdreef 15,
1105 A Z Amsterdam, The Netherlands
(Accepted 5 November 1987)
SUMMARY
Human fibroblasts derived from four individuals with various deletions in the short
arm of one chromosome 11 were susceptible to morphological transformation by early
region BK virus DNA, whereas diploid human fibroblasts were not. This difference in
susceptibility to transformation by early region BK virus DNA might be explained by a
putative 'transformation suppressor' locus situated within the deleted region on the
short arm of chromosome 11.
Morphological transformation of human cells by BK virus (BKV) (Purchio & Fareed, 1979;
Takemoto et al., 1979) and by BKV DNA and its subgenomic fragments (Grossi et al., 1982) has
been reported. From these studies it has emerged that the transforming capacity of BKV in
human cells is extremely low and that the properties of the transformed cells are markedly
different from report to report. Recently we described (van der Noordaa et al., 1986) the
persistence of BKV in human foetal pancreas cells after infection in vitro. Although all cells
continuously expressed BKV T antigen they did not exhibit the transformed phenotype.
From our comparative studies on morphological transformation by BKV and simian virus 40
(SV40), which share 75~ homology at the DNA and protein levels, we concluded that the
corresponding early coding regions of BKV and SV40 differed in their ability to transform
diploid human fibroblasts (de Ronde et al., 1987). The early regions of both BKV and SV40 were
able to induce the morphological transformation of rodent cells, whereas the early region of
BKV DNA in contrast to that of SV40 DNA did not induce the transformation of diploid
human fibroblasts derived from embryonic tissue, embryonic lung or foreskin (de Ronde et al.,
1987). The striking difference between rodent cells and human cells in their susceptibility to
transformation by BKV indicates that, besides viral factors, cellular factors determine the
transformation process.
It has been postulated that the loss of tumour suppressor factors plays an important role in the
development of certain human tumours such as retinoblastoma and Wilms' tumour (Comings,
1973; Knudson, 1985; Friend et al., 1986; Bishop, 1987). In Wilms' tumour there is strong
evidence that genetic changes in the short arm of chromosome 11 play a role in tumour
development. Congenital deletions of region 1 l pl 3 predispose to wilms' tumour development
(Riccardi et al., 1980) and loss of heterozygosity for genetic markers on the short arm of
chromosome 11 has been demonstrated for specific cases of Wilms' tumour (Koufos et al., 1984;
Fearon et al., 1984; Orkin et al., 1984). In addition, studies on somatic cell hybrids between
tumourigenic HeLa cells and diploid human fibroblasts have shown that the tumourigenic
0000-8119 © 1988 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 23:41:07
468
Short communication
llp
WAGR l
TG/GC
SH/FH
Fig. 1. Schematic presentation of the short arm of the human chromosome 11. The G-banding pattern
of the arm of the chromosome is shown. The del (11) human fibroblasts used in this study have deletions
as indicated in the figure. The Wilms' tumou~aniridi~genito-urinary abnormalities-mental
retardation (WAGR) locus is assigned to llp13 (Riccardi et al., 1980). The clinical data of the
individuals are given in Table 1.
Table 1. Clinical data o f the individuals*
Subject
SHI"
FH
TG
GC
AN2
?
+
+
+
M.R.
9
+
+
+
G.A.
?
+
WILMS
9
~
+
-
CAT
1
1
ND
1
Chromosome 11 abnormalities
del (11) (pll.llpl5.1)
del (11) (pl 1.1 lp15.1)
del (11) (p12p14.3)
del (11) (p12p14.3)
Other abnormalities
?
Microcephaly
Microcephaly
Microcephaly
* AN2, sporadic aniridia; M.R., mental retardation; G.A., genito-urinary abnormalities; WILMS, Wilms'
tumour; CAT, catalase gene copy number (catalase gene is located at band 1lp13); ND, not determined.
t SH and FH are siblings.
phenotype of the hybrids is suppressed in cells which have retained the diploid number of the
fibroblast-derived chromosome 11 (Klinger & Kaebling, 1986; Saxon et al., 1986). Recently it
was demonstrated that the introduction of a fibroblast-derived chromosome 11 in a Wilms'
tumour cell line suppressed its tumourigenic phenotype (Weissmann et al., 1987). These data
indicate that the short arm of chromosome 11 bears loci with tumour suppressor properties
deletion or mutation of which leads to an increased susceptibility to tumourigenesis. These
observations led us to study the transforming capacity of the early region of BKV D N A in vitro
in human cells with various deletions of the short arm of one chromosome 11.
Transformation studies were carried out on human fibroblast cultures derived from normal
embryonic lung tissue and from three patients (FH, T G and GC) with deletions in the short arm
of chromosome 11, one of which (TG) had developed a Wilms' tumour. Two of these (TG and
GC) had microscopically identical deletions (l lp12p14. 3). Patient F H had a much larger
deletion (1 lpl 1.1 lp15.1) and died at the age of 15 months with no evidence of Wilms' tumour.
In addition, embryonic fibroblasts with the same deletion were available from a sibling of
patient FH (SH) (Fig. 1 and Table 1). In this study of the transformation of human fibroblasts by
the BKV early region we have included the SV40 early region, which is able to transform diploid
human fibroblasts, as a positive control. For comparative reasons both the BKV and SV40 large
T and small t coding regions were placed downstream from the Rous sarcoma virus long
terminal repeat resulting in the plasmids pRSV-BK V and pRSV-SV40 (see Fig. 2). The plasmids
pRSV-BKV and pRSV-SV40 were transfected into the human fibroblasts using the calcium
phosphate method (Graham & Van der Eb, 1973), followed by a 15 ~ glycerol shock 4 h after the
addition of the D N A (Frost & Williams, 1978). The results are presented in Table 2. Six to 8
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 23:41:07
469
Short communication
pBR
RSV
BKV/SV40
Large T
Small t
-I
--t--]
~
Fig. 2. Structure of pRSV-BKV and pRSV-SV40. The early coding regions of BKV and SV40 were
placed downstream from the strong heterologous enhancer-promoter of the Rous sarcoma virus long
terminal repeat. Using standard cloning techniques (Maniatis et al., 1982) the neomycin resistance
coding sequences of pRSV-neo (Gorman et al., 1983)between the unique HindlII and BamHI sites were
replaced by the large T and small t coding regions of BKV and SV40 (nucleotides 5148 to 2246 and 5238
to 2533 respectively; numbering as in Tooze, 1981) resulting in the plasmids pRSV-BKV and pRSVSV40.
Table 2. Dense focus formation by B K V and SV40 early region D N A
Number of foci per 60 mm dish*
A
t
Diploid
r
DNA
pRSV-BKV
pRSV-SV40
pBR322
Del (11) SH
•
(
;
Expt. 1
Expt. 2
Expt. 1
Expt. 2
0/0/0
0/0/0
2/2/2/4
3/5/5/7
23/25/26 21/26/30 11/12/13/16 4/13/14/14
0/0/0
0/0/0
0/0/0/0
0/0/0/0
del (11) FH
4/0/0
16/9/7
0/0/0
del (11) TG
3/2/0
18/12/11
0/0/0
del (11) GC
2/2/1
23/21/18
0/0/0
* Five rtg of plasmid DNA (pBR322 as a negative control plasmid) was transfected onto 60 mm dishes of
semiconfluent human fibroblasts [see legend of Fig. 1 for an explanation of the size of the deletion of the del (11)
cells]. The dishes were followed for the appearance of macroscopically visible foci of morphologically transformed
cells. Foci were scored 6 to 8 weeks after the transfection. Dishes that did not show foci were followed for an
additional 8 week period. Large T expression in the transformed cells was confirmed by indirect
immunofluorescence using an anti-SV40 hamster tumour serum or an SV40 and BKV large T- and small trecognizing monoclonal antibody (PAB1614; Ball et al., 1984).
weeks after transfection, dense foci of morphologically transformed cells were observed in the
human fibroblasts with constitutional deletions of the short arm of chromosome 11 transfected
by p R S V - B K V or pRSV-SV40. N o morphological transformation of diploid human fibroblasts
was induced by pRSV-BKV, but pRSV-SV40 did induce dense foci of morphologically
transformed cells. Despite the variation between different experiments inherent to the
transfection procedure, it can be concluded that the efficiency of transformation of the human
fibroblasts with deletions in the short arm o f one chromosome 11 induced by p R S V - B K V was
lower than the efficiency of transformation induced by pRSV-SV40. The difference between
B K V and SV40 with respect to efficiency of transformation is in agreement with the findings in
rodent cells as shown by Yoshiike & T a k e m o t o (1986) and by ourselves (de Ronde et al., 1987).
F o r further analysis the pRSV-BKV-transformed cells of the various strains of human
fibroblasts with a deletion in the short arm of one chromosome 11 were picked from separate
dishes and expanded. Intranuclear B K V T antigen could be detected in the majority of the
transformed cells by immunofluorescence. Three foci of pRSV-BKV-transformed cells of one
strain (SH) were examined by immunoblotting (Fig. 3). Both the B K V large T and small t
antigens were detected (Fig. 3, lanes 1, 2 and 3). Cytogenetic studies on the transformed
fibroblasts revealed no additional clonal chromosome abnormalities apart from the original 11 p
deletion.
In this report we have described the morphological transformation by B K V of human
fibroblasts derived from four individuals with various deletions in the short arm of one
chromosome I 1. No transformation of apparently normal diploid human fibroblasts could be
obtained under similar conditions. F r o m our results it appears that h u m a n fibroblasts with a
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 23:41:07
470
Short communication
1
2
3
4
5
200 - -
97 m
68 m
43--
25--
W i
18--
~t
14--
:i
~~:~~~!:I
I~I~~I~
Fig. 3. Immunoblot of pRSV-BKV- and pRSV-SV40-transformed del (11) human fibroblasts. Total
cellular lysates (50 ~tg protein per lane) of del (11) SH human fibroblasts were electrophoresed on a 10
SDS-polyacrylamide gel according to Laemmli (1970) and blotted onto nitrocellulose according to
Dunn (1986). The large T and small t proteins of BKV or SV40 were detected using the monoclonal
antibody PAB1614 (Ball et al., 1984) and peroxidase-labelled rabbit anti-mouse Ig as second antibody.
Lanes 1 to 3, BKV-transformed del (11) SH cells (three separate colonies); lane 4, del (11) SH cells
(untransformed); lane 5, SV40-transformed del (11) SH cells. The positions of the large T and the small t
antigens are indicated (the BKV small t migrates somewhat more slowly than SV40 small t). MT are
shown at the left ( x t0-3).
deletion in the short arm of one chromosome 11 are more susceptible to morphological
transformation by BKV than any other human cell described so far by others and ourselves. This
difference in susceptibility might be explained by the loss of a putative 'transformation
suppressor' locus situated in the deleted region of the short arm of chromosome 11.
We are indebted to Dr J. W. E. Oorthuys for assistance in obtaining biopsy material. We would also like to
thank M. E. A. M. Overbeeke-Melkert, F. Koperdraad and M. T. A. van den Kroonenberg for their technical
assistance, and Dr R. K. Ball for kindly providing monoclonal antibody PAB1614.
REFERENCES
BALL, R. K., SIEGL, B., QUELLHORST,S., BRANDNER,G. & BRAUN,G. (1984). Monoclonal antibodies against simian
virus 40 nuclear large T tumour antigen: epitope mapping, papovavirus cross-reaction and cell surface
staining. E M B O Journal 3, 1485-1491.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 23:41:07
Short communication
471
BISHOP, J. M. (1987). The molecular genetics of cancer. Science 235, 305-311.
COMINGS, n. E. (1973). A general theory of carcinogenesis. Proceedingsof the National Academy of Sciences, U.S.A.
70, 3324-3328.
DE RONDE, A., MACDONALD,M., SOL, C., TER SCHEGGET, J., VAN STRIEN, A., WOUTERS, E. & VAN DER NOORDAA, J.
(1987). The BKV early enhancer-promoter and the host range for transformation. Intervirology 27, 38-44.
DUNN, S. D. (1986). Effects of the modification of transfer buffer composition and the renaturation of proteins in
gels on the recognition of proteins on western blots by monoclonal antibodies. Analytical Biochemistry 157,
144-153.
FEARON, E. R., VOGELSTEIN,B. & FEINBERG, A. P. (1984). Somatic deletion and duplication of genes on chromosome
11 in Wilms' tumors. Nature, London 309, 176-179.
FRIEND, S. H., BERNARDS,R., ROGELJ, S., WEINBERG, R. A., RAPAPORT,J. M., ALBERT, D. M. & DRYJA, T. P. (1986). A
h u m a n D N A segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma.
Nature, London 323, 643-646.
FROST, E. & WILLIAMS,J. (1978). M a p p i n g temperature-sensitive and host-range mutations of adenovirus type 5 by
marker rescue. Virology 91, 39-50.
GORMAN,C., PADMANABHAN,R. & HOWARD,B. H. (1983). High efficiency D N A - m e d i a t e d transformation of primate
cells. Science 221, 551-553.
GRAHAM,F. L. & VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of h u m a n adenovirus 5 D N A .
Virology 52, 456-457.
GROSSI, M. P., CAPUTO,A., MENEGUZZI, G., CORALLINI,A., CARRA, L., PORTOLANI,M., BORGATTI,M., MILANESI, G. &
BARBAtCrI-BRODANO,G. (1982). Transformation of h u m a n embryonic fibroblasts by B K virus, BK virus D N A
and a subgenomic BK virus D N A fragment. Journal of General Virology 63, 393-403.
KLINGER, H. P. & KAEBLING,M. (1986). Suppression of tumorigenicity in somatic cell hybrids. Cytogeneticsand Cell
Genetics 42, 225-235.
KNUDSON, A. G., JR (1985). Hereditary cancer, oncogenes, and anti-oncogenes. Cancer Research 45, 1437 1443.
KOUFOS, A., HANSEN, M. F., LAMPKIN, B. C., WORKMAN,M. L., COPELAND, M. G., JENKINS, N. A. & CAVENEE, W. K.
(1984). LOSSof alleles at loci on h u m a n chromosome 11 during genesis of Wilms' tumor. Nature, London 309,
170-172.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 227, 680-685.
MANIATIS, T., FRITSCH, E. F. & SAMBROOK,J. (1982). Molecular Cloning. A Laboratory Manual. New York: Cold
Spring Harbor Laboratory.
ORKIN, S. H., GOLDMANN,D. S. & SALLAN,S. E. (1984). Development of homozygosity for chromosome 1 lp markers
in Wilms' tumors. Nature, London 309, 172-174.
PURCHIO, A. F. & FAREED, G. C. (1979). Transformation of h u m a n embryonic kidney cells by h u m a n papovavirus
BK. Journal of Virology 29, 763-769.
RICCARD1,V. M., HITTNER, H. M., FRANCKE,U., YUNIS, J. J., LEDBETTER,D. & BORGES, W. (1980). The aniridia-Wilms'
tumor association: the critical role of chromosome band 11 p 13. CancerGeneticsand Cytogenetics 2, 131-137.
SAXON, P. J., SRIVATSAN,E. S. & STANBRIDGE, E. J. (1986). Introduction of a h u m a n chromosome 11 via microceU
transfer controls tumorigenic expression of HeLa cells. EMBO Journal 5, 3461 3466.
TAKEMOTO, K. K., LINKE, H., MIYAMURA, T. & FAREED, G. C. (1979). Persistent BK papovavirus infection of
transformed h u m a n fetal brain cells. Journal of Virology 29, 1177-1185.
TOOZE, J. (editor) (1981). DNA Tumor Viruses, 2rid edn. New York: Cold Spring Harbor Laboratory.
VAN DER NOORDAA,J., VAN S'I'RIEN,A. & SOL, C. J. A. (1986). Persistence of B K virus in h u m a n foetal pancreas cells.
Journal of General Virology 67, 1485-1490.
WEISSMAN,B. E., SAXON,P. J., PASQUALE,S. R., JONES, G. R., GEISER, A. G. & STANBRIDGE,E. J. (1987). Introduction of a
normal h u m a n chromosome 11 into a Wilms' tumor cell line controls its tumorigenic expression. Science 236,
175-180.
YOSHIIKE, K. & TAKEMOTO,K. K. (1986). Studies with B K virus and monkey lymphotropic papovavirus. In The
Papovaviridae, vol. 1, The Polyomaviruses, pp. 295-326. Edited by N. P. Salzman. New York: Plenum Press.
(Received 26 October 1987)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 23:41:07