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The effect on cell phenotype of the mutagenic peptide of herpes simplex virus type-1
Oral Oncol, EurJ Cancer, Vol. 31B, No. 4, pp. 267-274, 1995 Elsevier Science Ltd Pruned in Great Britain 0964-1955/95$9.50+0.00
0964-1955(95)00020-S
The Effect on Cell Phenotype of the Mutagenic Peptide of Herpes Simplex Virus Type-l C.M. Das, V.A. Murrah, S. Zhang, G. Wong, and E.J. Shillitoe
E.P. Gilchrist
The transforming region of the genome of herpes simplex virus type-l (HSV-1) encodes a peptide that raises the mutation frequency of cells. To find the effect of this peptide on cell phenotype, three types of cells were transfected with a shuttle vector plasmid that expressed the peptide. When immortalised rat fibroblasts were transfected they rapidly became anchorage-independent with high efficiency, but were not tumorigenic in nude mice. When monkey kidney cells were transfected, five clonal cell lines were isolated, of which one became anchorage-independent but was not tumorigenic in nude mice. When human oral keratinocytes were transfected they did not become immortalised. The peptide therefore induced some of the features of transformation in different cell types, but did not induce a malignant phenotype in any cell. This suggests that interaction with co-factors would be necessary for the peptide to contribute to the development of oral cancer. Keywords:
herpes simplex, transformation,
Oral 0~1~01, Eur J Cancer, Vol. 31B,
cancer
No. 4, pp. 267-274,
1995.
INTRODUCTION THE ORAL mucosa is regularly exposed to agents which are mutagenic or carcinogenic under experimental conditions. These include herpes simplex virus type-l (HSV-l), human papillomaviruses, and components of tobacco smoke [l]. Although these agents can transform cells in culture, the mechanisms by which each one operates is not fully understood. This makes it difficult to analyse a tumour and decide which, if any, particular agent was responsible for its induction. In particular, the relationship between the herpes simplex virus and oral cancer is obscure, and rests on studies of antibodies and tissues performed over a decade ago [2, 31. It is known that HSV- 1 can transform cells into a malignant phenotype, using a ‘hit-and-run’ mechanism in which viral DNA is not retained by the transformed cells [4]. Transformation does not require the entire viral genome, but can be accomplished by a region known as mtr-1. Within mtr-1 we have located an open reading frame within the UL26 gene which encodes a peptide that is expressed in virus-infected cells [5]. The peptide raises the mutation frequency of cells [6,
71 and sequence
analysis shows that it has some characteristics
proteins of other tumour viruses [ 11. The peptide is therefore a candidate for a transforming protein of HSV-1. The present study was performed to find if this peptide could indeed transform cells into a malignant phenotype, with the eventual goal of using the peptide in later studies of human oral cancers. of the transforming
MATERIALS AND METHODS Cells Immortalised rat fibroblasts (NRK536 cells) were kindly provided by Dr U. Rapp (NIH, Bethesda, Maryland, U.S.A.). They were maintained in Eagle’s minimum essential medium containing 5% fetal bovine serum (FBS) supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 0.25 pg/ml amphotericin B and 280 pg/ml glutamine. Vero cells, Cos-1 cells, HeLa cells and SV-T2 cells were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.). They were maintained in Ml99 containing 100/bFBS, penicillin, streptomycin and amphoteritin B. Transfected Vero cells were maintained in the same medium containing 400 lg/ml G418. Primary human oral keratinocytes were obtained from biopsy specimens obtained from the Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center, San Antonio, Texas, U.S.A., or from cadaveric donors at autopsy. Tissues were processed by dissecting away the connective tissue and dicing the epithelium into 0.5-1.0 mm cubes. Tissue pieces were rinsed in minimum essential
Correspondence to E. J. Shillitoe.
C.M. Das, S. Zhang and G. Wong are at the Department of Basic Sciences, Dental Branch, University of Texas Health Science Center, Houston, Texas 77225, U.S.A.; V.A. Murrah and E.P. Gilchrist are at the Department of Pathology, Emory University College of Medicine, Atlanta, Georgia 30322, U.S.A.; E.J. Shillitoe is at the Department of Microbiology and Immunology, State University of New York College of Medicine, Syracuse, New York 13210, U.S.A. Received 19 Dec. 1994; provisionally accepted 20 Feb. 1995; revised manuscript received 16 Mar. 1995. 267
268
C.M.
medium
with
antibiotics
RBC-lysing under
buffer.
conditions
epithelial
and incubated
They
were
to suppress
cell growth
again
Das et al.
for 10 min in sterile
flasks containing
rinsed
l”,, FBS.
fibroblasts
and
while
cultured
encouraging
Human
[8-IO].
the same medium
oral
Lipofectin
keratinocyte
reagent
with 800 ug/ml G418
cultures
(BRL,
were
Gaithersburg,
transfected
Maryland,
[ 121. For each culture well, 10 ug of appropriate in 500 ~1 of serum
Plasmids A 378
base pair
HSV-1
genome vector
Inc,
Alto,
start
UL26
codon
open
promoter
and pDB339
orientation.
The
acid
Laboratories
cloned
frame.
The
fragment
pDB337
orientation
sequence
contains to the
in the opposite
described
that can be expressed
was
relative
has the viral sequences were
the
and a eukaryotic
in the construct.
constructs
of the
and included
previously
from pDB337
medium
(KDM)
15 ug of
[7].
has the
IPTGGIQRGGRGPCQRQQRSTR-
[9] was mixed
Lipofectin
and
allowed
to
Cultures
were
rinsed
three
times
remove
residual
serum,
after
which
the
were
cultures Lipofectin.
12-16
1 ml
an additional containing
were confluent
cultures continued
to
Control
with
were rinsed with
5 uM dexamethasone
When
cultures.
15 ltg of
antibiotic-free
and then re-fed
The
for
KDM
KDM
was added to each culture.
24 h the cultures
KDM
of the peptide. This
of
serum
containing incubate
with
vehicle-treated
h
1“,) fetal bovine
with complete KDM
or
DNA defined
DNA/Lipofectin
on the appropriate
untreated
After
containing After
placed
were
plasmid
with 500 ul KDM
reagent
with
U.S.A.)
keratinocyte
15 min. solutions
fragment
orientation)
promoter,
in the correct
viral peptide
amino
reading
was included
sequences
The
The
of HSV-1
to the MMTV
the viral
pUC-9-G-B
(Clontech
U.S.A.).
G of the genome
adjacent
of the
into the Eco RV site of the
of the 5’ end (in the prototype
3’ end of the
region
the plasmid
pMAMneo-BLUE California,
Barn HI fragment cloned
from
in both orientations
expression consisted
of the transforming
was removed
[6] and cloned Palo
region
and antibiotic-free
and
three
to induce
cells were observed
times
1 ml of complete expression
daily and fed weekly.
they were split into two flasks.
until the cultures
became
senescent
and were
lost.
GR. Confirmation Synthetic The
peptide
and antisera
synthetic
sequence
peptide
shown
obtained
above
as described
the same animals
was and
synthesized rabbit
previously
according
and
goat
to the
antisera
[5]. Pre-immune
were
sera from
to ensure carried
pDB337
passage after
oxidase
10 and 30. Cells
24 h, while
fected that
still
by calcium had
been
Chatsworth, herpes
phosphate
purified virus
(Marine
complete well tissue
above
were
directly
a bottom
FBS
and G418
at a final medium colonies
lo-12
days.
7 x 10m6,
pMAMneo-BLUE, were
flasks. From dilutions 96-well
pDB337
Gene
code.
darker
brown
in 6-
at 37 ‘C
Pulser For
a viable transfected
plates with an average
DNA
(Bio-
pDB339
2-3
were
pZ189
days
the
subcultured
cell culture
Col-
in 75 mm*
a series of limiting into wells of
of less than one cell per well and by transferring
them
SV40 York, selected
fields.
stained,
since all of these
endogenous
by two observers,
positive
if they showed to the cell
PMAMneo-BLUE
Vero cells reacted
under
regularly
or
the same
at passages
up
assays had been completed.
efficiency were
of the human
transfected
the
with
keratino-
the
plasmid
immunoperoxidase
with a monoclonal
T antigen (Oncogene U.S.A.), and stained
in methanol
quench
plasmids
3 days
was performed
staining
antibody
to the
Science Inc., Uniondale, New cells were counted in randomly-
As a positive
Enzyme-linked
plasmids
respectively.
cultures
California,
H,O,
of the cells relative
in loo”,,
lines were examined
[ 141. After
technique
to
examined
lines were scored
time all other
out with an
Burlingame, with 0.5”,,
were
of peptide.
was carried
temperature
with control
The
for 24 h with 1 uM
control,
SV-T2
cells express
cells
were also
the T antigen.
at
were calculated
3 x 10m6 for
staining
and untransfected
parallel
induced
to assay for expression
To find the transfection cytes,
500 cells were apparent efficiencies
cell lines was obtained
lines transfected
each
of 400 ug/ml. every
[ 131. Individual on both chambers
Cells
staining
for
immunoper-
described
Laboratories,
room
Clonal
to 15, at which
at 200 V and 500 uF
was added
at
that
were used.
by an indirect
preincubation
activity.
under
in
gradient-purified
concentration
and
30 min
peroxidase
U.S.A.)
U.S.A.).
and pDB339,
picked
for
(Vector
following
Sea Plaque agarose
were made in the same media and plated
a set of clonal
as
cells containing plasmid resistance in Ml99 with
ofover
5 x 10mh, and
trypsinised,
light
stain
Cells
that the peptide
and 10” cells plated
immunoperoxidase
conditions.
CsCl
was performed
Transfection
onies
to UV
Maine,
California,
around be
U.S.A.)
into 0.3”,,
with a Bio-Rad
until G418-resistant to
ABC
after 2 weeks.
The electroporated selected for neomycin
G418containing
Indirect
with
3 x 10’ Vero cells and 100 ug plasmid
capacitance. DNA were
DNA Inc.,
peptide
slide and one chamber
or 25 uM dexamethasone
infected
were maintained
with
of a LabTek
to ensure
techniques
as previously
lines were subcultured
(Qiagen
layer of 0.5”,,
Cultures
Richmond,
used and electroporation
lo”,,
exposed
Rockland,
by electroporation
experiment
column
and
repeatedly
plasmid.
staining. Cell lines were examined
of the HSV-1 technique
tested
different
trans-
48 h the cells were harvested
transfected
Rad Laboratories,
Fresh
had been
plates.
with plasmid
[ 1 l] or were
[7]. After
were counted
cells
plasmids
U.S.A.)
Biocolloids,
culture
and colonies Vero
on a Qiagen
were seeded
medium
in 100 mm2 dishes they were either
precipitation
that
described
and 2 x 10’ cells agarose
were plated
California,
simplex
previously
when the cells were between
sub-confluent,
Three
Zmmunohistochemical
Transfection cells were transfected
the appropriate
were further
was expressed.
were used as controls.
from Vero cells was tested
that it carried
expression NRK536
of transfectiolz
Each cell line derived
to
immunosorbent
assay was also used to confirm Cells
were grown
absence
were harvested without
to confluent
of dexamethasone
monolayers
J The ELISA
of the peptide
[5,15].
in the presence
for 2 days in 150 mm dishes
24 h after the addition
dexamethasone.
O’C, rinsed
assay f ELISA
expression
Briefly,
twice with ice cold PBS
0.6 uM phenylmethylsulphonyl
of fresh medium
the dishes with 0.001
fluoride
were
with or
chilled
M EDTA
and removed
or and to and
with a
Effect
rubber
scraper.
Cells were recovered
for 5 min, washed
vigorously
The
by three
cells were disrupted
and ethanol
by centrifugation
once and finally
same buffer by vortexing and thawing
suspended
ELISA.
tube
The
entire
procedure
was collected
for
concentrations
the
protein
(Pierce
Illinois,
U.S.A.)
for the
preparation
was
were determined
Chemical
Co.,
and 100 ug of the total proteins
coat the wells of a 96-well
at
in a fresh
of antigen
extract
at 4°C. Protein assay
tube.
in dry ice
by centrifugation
and used as a source
performed BCA
of freezing
followed
12 000 g for 5 min and the supernatant microcentrifuge
at 200 g
in 0.5 ml of the
in a microcentrifuge cycles
at 37°C
on Cell Phenotype
plate overnight
by
of the Mutagenic
U.S.A.).
This
vector
was
were used to
chain reaction.
appropriate
plasmid,
To confirm
the polymerase
employed.
Primers
consisted
and reverse
primers
(USB,
either
had been
inserted.
plasmid PCR
DNA
DNA
extracts
of appropriate
Ohio,
mutation
of Vero
as previously
cells,
pDB337
described
[16].
Reaction
rates
were
determined
in an agarose from pK189
in newly
expanded
clones
test
cell lines 1 x 10’
groups
of six nude mice.
as well as two non-expressing
lines
were injected
339.
SV-T2
presence
duplicate
and the cells Growth
were
curves
immediately
of
wells
25 uM
counted
for
culture
dexamethasone. cell type
At
cell
24-h
were trypsinised,
in a haemocytometer
individual
after clonal
or with PDB
into wells of a 24-well
of each
lines
months, organs
chamber.
were
transfected
2 days, but
suspended
60 mm dishes
coated
ability
greater
than
included
Vero
The
sterile
cells
were for
was
agarose
with
determine
were
counted
to
and incubated
having
[17].
on
for colony a diameter
HeLa
cells
were
control.
if the peptide in M199/G418
serum,
0.2,
0.6, 0.8,
peptide
was induced
counted
were
increased
in low serum,
100 mm dishes
the dishes
the ability
500 cells were plated on to
medium
1, or lo”,,
containing
serum.
either
Expression
by 25 PM dexamethasone.
examined
of Vero
and the number
After
no
after
with
of the
of colonies
were
vector
frequency
a shuttle
gene
vector
cassette
and 0.9 Kb.
The
as an insertion
to make
injected
As a positive numbers
mutation if the total
tumours,
either
subcutaneously
into
control,
were were
at autopsy
tumorigenic
injected
were examined
they
some animals
of the known
animals
time
frequencies
and the different
with
non-
daily for up to 3
sacrificed.
The
for the presence
major
of tumours,
was examined
were compared
proportions
histologically
system
in cell lines [14].
by removal
and its replacement (Pharmacia
The
Inc.,
was determined
mutagenesis
of the region
shuttle that codes
with a kanamycin-resistance Piscataway,
New
by Fisher’s
of different
were compared
by a Chi square
Transformation
of NRKS36
types
exact test
of mutations
test.
RESULTS The cells
spontaneous was
observed
low,
cells
transformation
with
only
per 2 x 10” cells,
two
frequency
transformed
and the number
by the addition
of 10 mM dexamethasone.
pDB339
raised
the number
pDB337
produced
when
being
was not increased Transfection
of colonies
15 colonies
of NRK536 colonies
to five. The
in the absence
dexamethasone
with plasmid
of dexametha-
was added
to the
cultures. HSV-1
that had been irradiated
to 53 colonies,
while irradiation
by UV light for 10 min led
of the virus for 15 min led to 3 1
colonies. Transformation
cells of mutations
pS 189 was modified
for ampicillin
which
of Vero cells
A total of seven clonal
frequency
as a
with sizes that
analysis
continuously The
was classified
12 days,
[ 171.
Mutation
were
other
were examined
sone and 32 colonies
cells to form colonies 0.4,
and
G418
plated
dishes were then examined and colonies
anchor-
dependence
To
sizes and the
of the numbers
for any foci of tumours.
dexamethasone
methylcellulose
with 0.8”,,
100 mm
induced 1 x lo5
suspension
on soft agar,
as a positive
cells,
of 25 PM
in 1.5”,,
at 37-C for 21 days. The
Serum
in
dexamethasone.
forming
of the peptide
in the presence
then
no
to be
than 5.2 Kb.
Vero cells. Animals
Mutation
growth
pre-incubated
were
was confirmed
expansion.
if expression
age-independent
bacterial
fragments
if the total size of the fragments
and the skin at the site of injection
determined
independence
To determine
cells
with equal
cells,
Statistical Anchorage
the
of fragment
for the ability
or
with the vector,
in the
that had occurred, overnight
mutation
4.3
was greater
1 x lo6 the peptide
intervals,
from
was
Tumorigenicity
transfected
plate
recovered.
were obtained
to be a deletion
size of fragments
colonies
by examination The
and transfer frequency
mutant
if two fragments
with a change
mutation
and
by examination
indistinguishable
that had been
cells (1 x 104) were plated
gel. Each
Plasmids
by a colour
from
HI
Bum
was assessed
was considered
that expressed The
with
by
plasmids,
by hybridisa-
probe.
rate
Growth
recovered
was less than 5.2 Kb and classified
gels and bands
7070.
for
followed
by recovery
of white
and sizes of the fragments.
To Growth
digested
to replicate
any non-replicated
The
The
phosphate
lysis,
the types of mutation
were
type of mutation
were
and the
and allowed
culture.
as the proportion
point mutation
and pDB337
as viral DNA
DNA
to anneal sequences
controls,
on 2”,, agarose
size were confirmed
tion to a 32P-labelled
U.S.A.)
calcium
by alkaline
to be mutants
bacterial
plasmids
derived
by
into E. Coli MBM
In order to classify
was
was named pK189.
cells
in the sup F gene were recognised
another
resolved
vector
shock
recovered
and were confirmed into
the
forward
the HSV
and positive
were electrophoresed
(PCR)
universal
site into which
acted as negative
was performed
mixtures
chain reaction
of the Ml3
Cleveland,
side of the cloning
that cells retained
and glycerol
and was introduced
cultures, Polymeruse
into
with Dpn I to remove
mutant
at 4’C.
shuttle
48 h. It was then
expressed
Rockford,
modified introduced
precipitation digestion
269
Peptide
Jersey,
that
BLUE
in medium had been
alone,
transfected
The
was
transfected
pDB337
number
with G418.
transfected
with pDB339,
that had been contained
one
cell lines were developed
from
with
the vector
Vero
and the other with
were named
pDB337. H1l.l,
which grew
One line was from Vero cells
pMAMneo-
that
had
five were from The
cell lines
B7, BlO,
of cells per 75 cm2 flask ranged
been cells that
F7, and F9.
from
1 x 10’ to
C.M. Das et al.
270
2 x lo7 in these
seven lines. Cell morphology was normal fibroblast-like and was very similar from clone to clone. However, the cells in the Hl 1.1 line were much larger than the others. Expression
Vero cell line. By this time only the Hl 1 .l cell line expressed the peptide, as shown by both assays, in the presence of 1 or 25 PM dexamethasone, but not in the absence of dexamethasone. The other lines B-7, B-10, F-7 and F-9 had become peptide-negative at this time. The inducibility of peptide expression was also confirmed in H11.1 at passages 15-19 using various doses of dexamethasone (Table 2). At passage 40 the H 11.1 cell line was examined by the PCR to ensure that it still retained the viral sequences. A band corresponding to 410 bp was seen, and hybridised to the probe derived from the pDB337 DNA (not shown). In the human keratinocyte cultures no non-transfected cells were stained with anti-T antibody, but in the cells transfected with pZ189 the proportion of stained cells was 48/4760 ( lqO). In the SV-T2 cell cultures, every cell showed nuclear staining.
of viral peptide
To confirm expression of the viral peptide, all cell lines were examined by immunoperoxidase staining with antiserum to the peptide within two passages after being established. Staining was done on cultures that were maintained without dexamethasone or with 1 or 25 PM dexamethasone. No stain was seen in non-transfected Vero cells, Vero cells transfected with pMAMneo-BLUE or in Vero cells transfected with pDB339. Each of the Vero cell lines transfected with pDB337 showed positive staining when cultured in the presence of 1 or 25 PM dexamethasone but not in the absence of dexamethasone (Fig. 1). Staining was intense, restricted to the cytoplasm, and seen in almost 100% of the cells. The appearance was similar with each of the cell lines. At passages 10-12 the immunoperoxidase staining was repeated, and the ELISA (Table 1) was performed on each
Fig.
1. Photomicrographs
showing staining of different (b) Vero/pMAMneo-BLUE;
Mutation
frequency
in cell lines
The mutation frequency of pK189 in Cos-1 cells was O.O24qi,, which is very similar to that obtained previously with pS189. Thus, the replacement of the ampicillin-resistance
cell lines with antiserum to mutagenic (c) Vero/pDB339; (d) Hll.1.
Table 1. Induction of peptide in cell lines by dexamethasone as measured by an ELISA
assay
Concentration of dexamethasone None
25 FM
Increase
VerolpMAMneo-BLUE Vero/pDB339 H1l.l
0.463 (f0.014)* 0.380 (f0.004) 0.440 (i 0.020)
0.445 (kO.067) 0.373 (kO.011) 0.949 (f0.014)
-0.02
B7
0.464 ( k 0.060)
BlO
0.511 (+0.060)
F7 F9
0.668 ( * 0.054) 0.480 ( k 0.097)
0.452 (+0.090) 0.592 (f 0.010) 0.519 (kO.120) 0.418 (kO.020)
Cells
-0.01 0.51 -0.01
0.08 -0.15 -0.06
*A414 values expressed as meanskstandard error of triplicate readings. The assay was
performed at passage 12.
peptide.
(a) Vero;
271
Effect on Cell Phenotype of the Mutagenic Peptide Table 2. Induction of peptide in H11.1 cells by dexamethasone measured by an ELISA assay Dexamethasone None 1pM 5pM 25 FM
A,,, values* 0.150 0.551 0.375 0.291
as
Increase?
(+ 0.04) (+0.18) (f0.07) (kO.09)
5 4 3
*4,4 values are expressed as means&standard error of triplicate readings. tFold-increase was obtained in comparison with the A4,4 value obtained in the absence of dexamethasone. All A,,, values were corrected by subtraction of the reading with pre-immune serum (0.486* 0.074). The assay was performed at passage 17. gene with a kanamycin-resistance gene had no significant effect on the mutagenesis plasmid. In the presence of 25 FM dexamethasone the spontaneous mutation frequency in Vero cells was 0.210/h which is higher than in Cos-1 cells. The highest mutation frequency was seen in the Hl 1.1 cells, while the other peptide-expressing cells showed lower mutation frequencies (Table 3). However, these tests were done at relatively high passage levels, by which time some of the lines
were no longer expressing peptide. In the Hl 1 .l cells the mutation frequency increased with increasing concentrations of dexamethasone in the medium although other cells showed no change in the frequency with added dexamethasone (Table 4). The types of mutations that occurred in the Vero and H 11.1 cells were analysed by examination of digested fragments of the plasmid that were recovered from the cells. In no case had plasmid pDB337 been recovered in place of pK189. In Vero cells, point mutations accounted for 53:/b of mutations. Of the 47 plasmids with altered mobility, 37 showed slight reductions in size of one fragment and eight showed an increase in size of one fragment. Two plasmids were linearised by Bum HI and the single fragment was larger than 5.2 Kb; they were classified as insertions. In the H1l.l cells 54’$, of mutant plasmids were classified as having point mutations. Of the 46 with altered gel mobility, 39 showed a slight reduction in size of one fragment and one showed an increase in size of one fragment. Six plasmids showed neither simple increases nor decreases in one or the other fragments, but five of those were classified as insertions and one as a deletion based on the total size of all fragments. The types of mutant plasmids derived from each cell type are summarised in Table 5 and did not differ significantly between Vero and Hl 1.1 cells.
Table 3. Frequency of mutations in cells lines in the presence of dexamethasone Colonies Cells
White
Mutations Blue
Frequency*
Pt
Vero
45
26 500
0.17%
n.s.
VerolpMAMneo-BLUE Vero/pDB339
40 34
19000 11800
0.124; 0.29%
n.s. <0.05
148 21 44 59 71
19 200 12 400 18 500 19 800 18 300
0.770, 0.17 o/o 0.24:,, 0.30% 0.39%
H1l.l B7 BlO F7 F9
*Mutation frequency expressed as a percentage, derived from counting at least 15 000 colonies. The assay was performed at passages 10-12. tThe frequency of mutations was compared with that in the same cells in the absence of dexamethasone, using Fisher’s exact test. n.s.: not significant.
Table 4. Mutation frequency
in cells in the presence of different concentrations of dexamethasone Concentration of dexamethasone
Cells
None
1 KM
5pM
10 FM
25 pM
Vero VerolpMAMneo-BLUE Vero/pDB339 H1l.l
0.21* 0.20 0.12 0.19
0.20 0.14 0.11 0.377
0.19 0.17 0.10 0.40f
0.13 0.21 0.11 0.621
0.19 0.26 0.291 0.76$
*Mutation frequency expressed as a percentage, derived from counting at least 15 000 colonies. The assay was performed at passages 10-12. isignificantly higher than mutation frequency of same cells in the absence of dexamethasone (P
C.M.
272
cells
Table 5. Types of mutatiorrs observed in Vera and H1 1.1
Type of muration
Vero cells*
Poinr muration Deletion Insertion
H1l.l
53 37 10
Das et al.
cells
showed
cells
showed
numbers
of serum.
a maximum
of
However,
number
foci
with
the Hll
of foci
with
54 40 6
F7
cells
showed
pMAMneo-BLUE concentration larger
a pattern cells,
of serum.
in size than
most
0.6”,,
with
HI 1.1 cells formed
any other
lower serum concentration,
lines,
0.2”,,,
serum,
3). The
consistent
although
increasing
.l and B7 cells
fewer foci with either more or less serum (Fig.
*One hundred mutant plasmids were examined from Vero cells and another 100 from Hll, 1 cells.
with
fewer
and could
the Vera/
foci
colonies form
and
F9 and at each
that were foci at a
than any other line (Fig.
4).
A Verolvector
.-..,”
n
increasing
concentrations
Vero
A Vera/vector
. VerolpDB339
1
l Hl1.1
0
Days Fig. 2. Growth of cell lines. Cells were plated into wells of 24-well culture plates at a concentration of 1 x 1O’cells per well in the presence of 25 pM dexamethasone. At daily intervals duplicate wells were harvested and cells counted.
rates were measured
0.8
I.0
(%)
Fig. 3. Serum dependence of cell lines. Cells at the 10th to 12th passage were plated in 100 mm dishes in media containing different concentrations of serum. Growing colonies were counted after 12 days and expressed as a percentage of cells that had been plated.
for all cell lines that had been
derived
from Vero cells at pasage 8 or earlier
shown
in Fig.
expressed
2. At this
the peptide.
passage,
cells
express
as determined
the peptide
Non-transfected concentration
Vero
and cells
Vero/pDB339
BlO
became
plated.
grew
while Vera/ cells
rapidly
up to day 4. However, lines H 11.1,
rapidly. apparently
They
are
and F9 did
not
by immunostaining.
on a plateau
of cells declined
cell lines grew more slowly,
when
plated their
at a peak
for a few days and then Each
of the transfected
since a smaller
were very similar
from day 5 onwards
number
to each other
the peptide-positive
B7, B 10 and F9 grew at only half the rate of the
peptide-negative peak densities the pattern
B7,
of 1 x 10” cells per well and reached
on day 5, stayed
the number
and the results
H1l.l,
F7 showed weak expression
pMAMneo-BLUE
of them
0.6
rate
Growth
number
0.4
Serum concentration
2
Growth
0.2
VerolpDB339
line.
All these
on day 8 or later. The growth close
were measured
to the VeroipDB339
three
times
using
lines
reached
rate of F7 followed
line.
Growth
each cell line,
curves
with similar
results.
Serum dependence When serum,
cells
were
two distinct
pMAMneo-BLUE
plated patterns cells,
with
different
of survival
the VerolpDB339
concentrations
were seen. The
of Veroj
cells and the BlO
Fig. 4. Appearance of cell colonies from experiments shown in Fig. 3. (a) Vero/pDB339 cells, 10% serum; (b) VerolpDB339 cells, 0.5% serum; (c) H1l.l cells, 10% serum; (d) H1l.l cells, 0.5% serum.
Effect
on Cell Phenotype
of the Mutagenic examination received
of the
injection
the control,
1 x 10’ cells,
273
Peptide sites.
tumorigenic
all developed
However,
animals
cell line SV-T2,
tumours
that
at a dose of
with an average
diameter
of 1.5 x 0.7 cm by the 6th week.
DISCUSSION It is clear
that HSV-1
phenotype
since
repeatedly acts
genome raises
DNA
have
The
been
serum
antibody
be anticipated malignant
that
the peptide
the mutagenic by HSV-1
transformed in
induced
Vero
expressed
were
Vero
colonies
cell
passage
as anchorage-independent of foci per dish as HeLa each
focus
was
the same time did not develop
All cultures smaller,
by 1%20”,,
but
differences
no
transfected nally using
by
cultures
However,
received
of the peptide frequency
to express
sequences
than the non-transfected The
tumours
in nude
or induce
had only induced
the
to express
up to at least
cells when expression
cells did not form foci in soft mice,
indicating
some of the features
we
half
the
in parallel
the
diameter
cultures
for
to form foci.
with
between
days
each were cultures
All cultures and
died.
plasmid
and
consistently cultures, that
were
were termi-
Transformation
cells were performed five different
appeared
any dose of transformed tumour
foci
five times,
cell donors,
by 3 months NRK536 were
no phenotypic
to the viral peptide.
can be extended
of non-transfected
plasmids.
from
oral cells showed
keratinocytes
diameters
seen 22
Human
be attributed
that transfection
that
the
of transforma-
with
in animals cells,
detected
that
Vero cells or
by histological
found
that
the viral peptide The
results
HSV-1
could
However, require
mtr-1 . The
In other
human
other
The activities
reported
of this virus peptide
other pheno-
that are also encoded
that transformation
to this viral peptide
the cell phenotype
of
for cell in
to a malignant
viral proteins
data suggest
peptide
or to the anchorage-
been
transformation
but
such changes.
that is responsible
has
oral
[ 191. There-
modifications,
of inducing
phenotype
that
be attributed
for changing
of
to phenotypic
to an immortal
experiments.
cells cannot
lifespan
be the viral protein
controls
successfully.
of this study show that the mutagenic
phenotype
within
the
that could
was despite
by N-nitrosamines
was not capable
independent type must
changes
This
was achieved
fore the cells were susceptible
of human
alone.
that might be responsible
are unknown.
However,
it is
known that the mutagenic peptide represents the carboxy terminus of one member of a family of virus-encoded proteins. proteins
DNA-binding
No
that
to show
the fact that in
and the mutation
was induced.
experiments
producing
Tumorigenicity
Hl 1 .l cells.
slower
was despite
concentration
These
had
not
had not
the plasmid
were
the expression
the viral
foci,
results.
tumours
with
of the peptide
[20], protease No
were
10, but the H 11.1 line continued
and retained
transformation
those
with the human
primary
similar
as the virus
cells
lines lost the ability
showing
about
transfection
than
with different
experiments
also
by others
that the peptide
they
This
30 they were capable
the ability
Monolayer were
differentiated
make
cells
survived
to grow.
could
cells (200 colonies/105
5). Vero cells that were maintained
of human
lines
at early passages.
after the H 11.1 cells had reached
continued
were
tion.
transfected
anchorage-independent
Transformation
to a
passage 40. The Hl 1.1 cells were able to grow in a lower serum
agar
although
cells
the
transfected
3). Four
by passage
peptide
(Fig.
cells
as reported
was as efficient
at early passages
(Table
the peptide
These
However,
peptide
of transformation.
was raised
cells),
Since
it might
transformation.
cells
could be induced
same number
[18].
transform
particles,
each of the five lines examined
of growing
which
did lead to morphologi-
indicating
the mutagenic
features
the
of the
with oral
carcinogens,
cells.
foci.
in nude mice,
complete
When
independence
the peptide
would
pDB337
producing
tumorigenic
of
and patients
peptide
readily
[l 11, and the plasmid
None
a peptide
of many
of NRK536
particle
Anchorage
region
to encode
against
cal transformation
peptide
that the
phenotype.
As expected,
Fig. 5. Anchorage independence of transfected cells. Cells were seeded in methylcellulose and anchorage-independent colonies were photographed after 21 days. HeLa cells were included for comparison. No colonies were seen with cells that were non-transfected, or were transfected with pDB339. (a) HeLa cells; (b) H1l.l cells.
but the
implies
cells
of cells,
is a characteristic
demonstrated
obscure,
transforming
shown
frequency
to a malignant
been
basis of this remains
as a mutagen.
has recently
mutagenesis
cells
has
in the transformed
the mutation
cancer
can transform
phenomenon
[l, 41. The
lack of viral virus
the
involved
activities
properties
that
include
activities
[21,22],
virion
assembly
and kinase and
[5]. Any of these functions
in cell transformation.
that the peptide suggested
have
and substrate
is antigenic,
by the observation
The
present
might
study
and its role in oral cancer that
patients
with
be
confirms has been
oral cancer
274
C.M. Das el
increased levels of IgM antibody to the mutagenic peptide. This suggests that it is expressed at some point in the development of the tumour [ 181. The peptide, therefore, might play several different roles during viral infection, as do most of the transforming proteins of DNA viruses [23]. It is now important to test the effects of the mutagenic peptide on oral cells that have been immortalised, or partially transformed in other ways. Oral cells that have been immortalised by papillomaviruses can become fully transformed by subsequent exposure to an alkylating chemical carcinogen [24], and so might also be transformed by the HSV-1 peptide. Alternatively, our oral cells whose survival was extended by exposure to tobacco-related carcinogens [ 191 might be susceptible to transformation by the peptide. These studies are in progress.
for the maintenance of the transformed state. Int J 1992,51, 754-760. Barbosa MS, Vass WC, Lowy DR, Schiller JT. In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential. Virology 1991,65,292-298. Hwang CBC, Greenspan JS, Shillitoe EJ. Detection of herpes simplex virus proteins in cultured cells by monoclonal antibodies and the avidin-biotin-immunoperoxidase complex method. 3 Oral Path01 1986, IS, 179-184. Hwang CBC, Shillitoe EJ. DNA sequence of mutations induced in cells by herpes simplex virus type-l. Virology 1990, 178, 180-188. Shillitoe EJ, Greenspan D, Greenspan JS, Silverman Jr S. Antibody to early and late antigens of herpes simplex virus type 1 in patients with oral cancer. Cancer 1984, 54, 266-273. Woods KV, Shillitoe EJ, Spitz MR, Schantz SP, Adler-Storthz K. Analysis of human papillomavirus DNA in oral squamous cell carcinomas. 3 Oral Pathol Med 1993, 22, 101-108. Ray FA, Peabody DS, Cooper JL, Cram LS, Kraemer PM. SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts. 3 Cell Biochem 1990, 42, 13-31. Das CM, Schantz SP, Shillitoe EJ. Antibody to a mutagenic peptide of herpes simplex virus in young adult patients with cancer of the head and neck. Oral Surg Oral Med Oral Path01 required
have
1. Steele C, Shillitoe EJ. Viruses and oral cancer. Crit Rev Oral Biol Med 1991,2, 153-175. 2. Shillitoe EJ, Greenspan D, Greenspan JS, Silverman S. The immunoglobulin class of antibody to herpes simplex virus in patients with oral cancer. Cancer 1983, 51, 65-71. 3. Eglin RI’, Scully C, Lehner T, Ward-Booth P, McGregor IA. Detection of RNA complementary to herpes simplex virus in human oral squamous cell carcinoma. Lancet 1983, ii, 766-768. 4. Macnab JCM. Herpes simplex virus and human cytomegalovirus: their role in morphological transformation and genital cancers. r Gen Viral 1987, 68, 2525-2550. 5. Das CM, Shillitoe EJ. Expression of the mutagenic peptide of herpes simplex virus type-l in virus-infected cells. Virus Res 1994,34,97-l 14. 6. Shillitoe EJ, Matney T, Conley AJ. Induction of mutations in bacteria by a fragment of DNA from Herpes simplex virus type 1. Virus Res 1986, 6, 181-191. 7. Shillitoe EJ, Zhang S, Wang G, Hwang CBC. Functions and proteins ofherpes simplex virus type-l that are involved in raising the mutation frequency of infected cells. Virus Res 1993, 27, 239-251. 8. Green M, Wold WSM, Mackey JK, Rigden P. Analysis of human tonsil and cancer DNAs and RNAs for DNA sequence of group C (serotypes 1, 2, 5, 6) human adenoviruses. Proc Nat1 Acad Sci USA 1979, 7X,6606-6610. adhesion during 9. Adams JC, Watt FM. Changes in keratinocyte terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cells. Cell 1990, 63, 425-435. DK, Lillie JH, Jepsen A, Arenholt-Bindsley D. The 10. MacCallum culture of oral epithelium. Int Rev Cytol 1987, 109, 313-329. 11. Bauer G, Kahl S, Singh Sawheney I, Hofler P, Gerspach R, Matz B. Transformation of rodent fibroblasts by herpes simplex virus: presence of morphological transforming region 1 (mtr 1) is not
al.
Cancer
12.
13.
14.
15.
16.
17.
18.
1993,
75, 610-614.
and growth 19. Murrah VA, Gilchrist El’, Moyer Ml’. Morphologic effects of tobacco-associated chemical carcinogens and smokeless tobacco extracts on human oral epithelial cells in culture. Oral Surg Oral Med Oral Path01 1993, 75, 323-332. and character20. Preston VG, Coates JA, Rixon FJ. Identification ization of a herpes simplex virus gene product required for encapsidation of virus DNA. 3 Viral 1983, 45, 1056-1064. 21. Liu F, Roizman B. The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate. 3 Viral 1991, 65, 5149-5156. IM, McGregor M, El Kobiasi 22. Preston VG, Rixon FJ, McDougall MF. Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame. Virology 1992, 186, 87-98. by viruses In: Fields 23. Benjamin T, Vogt PK. Cell transformation BN, Knipe DM, eds. Fundamental Virology. New York, Raven Press 1991, 291-341. 24. Shin KH, Min BM, Cherrick HM, Park NH. Combined effects of human papillomavirus-18 and N-Methyl-N’-nitro-N-nitrosoguanidine on the transformation of normal human oral keratinocytes. Mol Carcinogen 1994, 9, 76-86.
Acknowledgements-The authors wish to thank Dr M.P. Moyer for helpful advice with the human cell cultures. This study was supported by PHS grant DE07007. CD. was the recipient of NRS award F32 DE05577. The synthetic peptide was made in the Synthetic Antigen Laboratory, M.D. Anderson Cancer Center, which is supported by PHS grant CA 16672.
0964-1955(95)00020-S
The Effect on Cell Phenotype of the Mutagenic Peptide of Herpes Simplex Virus Type-l C.M. Das, V.A. Murrah, S. Zhang, G. Wong, and E.J. Shillitoe
E.P. Gilchrist
The transforming region of the genome of herpes simplex virus type-l (HSV-1) encodes a peptide that raises the mutation frequency of cells. To find the effect of this peptide on cell phenotype, three types of cells were transfected with a shuttle vector plasmid that expressed the peptide. When immortalised rat fibroblasts were transfected they rapidly became anchorage-independent with high efficiency, but were not tumorigenic in nude mice. When monkey kidney cells were transfected, five clonal cell lines were isolated, of which one became anchorage-independent but was not tumorigenic in nude mice. When human oral keratinocytes were transfected they did not become immortalised. The peptide therefore induced some of the features of transformation in different cell types, but did not induce a malignant phenotype in any cell. This suggests that interaction with co-factors would be necessary for the peptide to contribute to the development of oral cancer. Keywords:
herpes simplex, transformation,
Oral 0~1~01, Eur J Cancer, Vol. 31B,
cancer
No. 4, pp. 267-274,
1995.
INTRODUCTION THE ORAL mucosa is regularly exposed to agents which are mutagenic or carcinogenic under experimental conditions. These include herpes simplex virus type-l (HSV-l), human papillomaviruses, and components of tobacco smoke [l]. Although these agents can transform cells in culture, the mechanisms by which each one operates is not fully understood. This makes it difficult to analyse a tumour and decide which, if any, particular agent was responsible for its induction. In particular, the relationship between the herpes simplex virus and oral cancer is obscure, and rests on studies of antibodies and tissues performed over a decade ago [2, 31. It is known that HSV- 1 can transform cells into a malignant phenotype, using a ‘hit-and-run’ mechanism in which viral DNA is not retained by the transformed cells [4]. Transformation does not require the entire viral genome, but can be accomplished by a region known as mtr-1. Within mtr-1 we have located an open reading frame within the UL26 gene which encodes a peptide that is expressed in virus-infected cells [5]. The peptide raises the mutation frequency of cells [6,
71 and sequence
analysis shows that it has some characteristics
proteins of other tumour viruses [ 11. The peptide is therefore a candidate for a transforming protein of HSV-1. The present study was performed to find if this peptide could indeed transform cells into a malignant phenotype, with the eventual goal of using the peptide in later studies of human oral cancers. of the transforming
MATERIALS AND METHODS Cells Immortalised rat fibroblasts (NRK536 cells) were kindly provided by Dr U. Rapp (NIH, Bethesda, Maryland, U.S.A.). They were maintained in Eagle’s minimum essential medium containing 5% fetal bovine serum (FBS) supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 0.25 pg/ml amphotericin B and 280 pg/ml glutamine. Vero cells, Cos-1 cells, HeLa cells and SV-T2 cells were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.). They were maintained in Ml99 containing 100/bFBS, penicillin, streptomycin and amphoteritin B. Transfected Vero cells were maintained in the same medium containing 400 lg/ml G418. Primary human oral keratinocytes were obtained from biopsy specimens obtained from the Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center, San Antonio, Texas, U.S.A., or from cadaveric donors at autopsy. Tissues were processed by dissecting away the connective tissue and dicing the epithelium into 0.5-1.0 mm cubes. Tissue pieces were rinsed in minimum essential
Correspondence to E. J. Shillitoe.
C.M. Das, S. Zhang and G. Wong are at the Department of Basic Sciences, Dental Branch, University of Texas Health Science Center, Houston, Texas 77225, U.S.A.; V.A. Murrah and E.P. Gilchrist are at the Department of Pathology, Emory University College of Medicine, Atlanta, Georgia 30322, U.S.A.; E.J. Shillitoe is at the Department of Microbiology and Immunology, State University of New York College of Medicine, Syracuse, New York 13210, U.S.A. Received 19 Dec. 1994; provisionally accepted 20 Feb. 1995; revised manuscript received 16 Mar. 1995. 267
268
C.M.
medium
with
antibiotics
RBC-lysing under
buffer.
conditions
epithelial
and incubated
They
were
to suppress
cell growth
again
Das et al.
for 10 min in sterile
flasks containing
rinsed
l”,, FBS.
fibroblasts
and
while
cultured
encouraging
Human
[8-IO].
the same medium
oral
Lipofectin
keratinocyte
reagent
with 800 ug/ml G418
cultures
(BRL,
were
Gaithersburg,
transfected
Maryland,
[ 121. For each culture well, 10 ug of appropriate in 500 ~1 of serum
Plasmids A 378
base pair
HSV-1
genome vector
Inc,
Alto,
start
UL26
codon
open
promoter
and pDB339
orientation.
The
acid
Laboratories
cloned
frame.
The
fragment
pDB337
orientation
sequence
contains to the
in the opposite
described
that can be expressed
was
relative
has the viral sequences were
the
and a eukaryotic
in the construct.
constructs
of the
and included
previously
from pDB337
medium
(KDM)
15 ug of
[7].
has the
IPTGGIQRGGRGPCQRQQRSTR-
[9] was mixed
Lipofectin
and
allowed
to
Cultures
were
rinsed
three
times
remove
residual
serum,
after
which
the
were
cultures Lipofectin.
12-16
1 ml
an additional containing
were confluent
cultures continued
to
Control
with
were rinsed with
5 uM dexamethasone
When
cultures.
15 ltg of
antibiotic-free
and then re-fed
The
for
KDM
KDM
was added to each culture.
24 h the cultures
KDM
of the peptide. This
of
serum
containing incubate
with
vehicle-treated
h
1“,) fetal bovine
with complete KDM
or
DNA defined
DNA/Lipofectin
on the appropriate
untreated
After
containing After
placed
were
plasmid
with 500 ul KDM
reagent
with
U.S.A.)
keratinocyte
15 min. solutions
fragment
orientation)
promoter,
in the correct
viral peptide
amino
reading
was included
sequences
The
The
of HSV-1
to the MMTV
the viral
pUC-9-G-B
(Clontech
U.S.A.).
G of the genome
adjacent
of the
into the Eco RV site of the
of the 5’ end (in the prototype
3’ end of the
region
the plasmid
pMAMneo-BLUE California,
Barn HI fragment cloned
from
in both orientations
expression consisted
of the transforming
was removed
[6] and cloned Palo
region
and antibiotic-free
and
three
to induce
cells were observed
times
1 ml of complete expression
daily and fed weekly.
they were split into two flasks.
until the cultures
became
senescent
and were
lost.
GR. Confirmation Synthetic The
peptide
and antisera
synthetic
sequence
peptide
shown
obtained
above
as described
the same animals
was and
synthesized rabbit
previously
according
and
goat
to the
antisera
[5]. Pre-immune
were
sera from
to ensure carried
pDB337
passage after
oxidase
10 and 30. Cells
24 h, while
fected that
still
by calcium had
been
Chatsworth, herpes
phosphate
purified virus
(Marine
complete well tissue
above
were
directly
a bottom
FBS
and G418
at a final medium colonies
lo-12
days.
7 x 10m6,
pMAMneo-BLUE, were
flasks. From dilutions 96-well
pDB337
Gene
code.
darker
brown
in 6-
at 37 ‘C
Pulser For
a viable transfected
plates with an average
DNA
(Bio-
pDB339
2-3
were
pZ189
days
the
subcultured
cell culture
Col-
in 75 mm*
a series of limiting into wells of
of less than one cell per well and by transferring
them
SV40 York, selected
fields.
stained,
since all of these
endogenous
by two observers,
positive
if they showed to the cell
PMAMneo-BLUE
Vero cells reacted
under
regularly
or
the same
at passages
up
assays had been completed.
efficiency were
of the human
transfected
the
with
keratino-
the
plasmid
immunoperoxidase
with a monoclonal
T antigen (Oncogene U.S.A.), and stained
in methanol
quench
plasmids
3 days
was performed
staining
antibody
to the
Science Inc., Uniondale, New cells were counted in randomly-
As a positive
Enzyme-linked
plasmids
respectively.
cultures
California,
H,O,
of the cells relative
in loo”,,
lines were examined
[ 141. After
technique
to
examined
lines were scored
time all other
out with an
Burlingame, with 0.5”,,
were
of peptide.
was carried
temperature
with control
The
for 24 h with 1 uM
control,
SV-T2
cells express
cells
were also
the T antigen.
at
were calculated
3 x 10m6 for
staining
and untransfected
parallel
induced
to assay for expression
To find the transfection cytes,
500 cells were apparent efficiencies
cell lines was obtained
lines transfected
each
of 400 ug/ml. every
[ 131. Individual on both chambers
Cells
staining
for
immunoper-
described
Laboratories,
room
Clonal
to 15, at which
at 200 V and 500 uF
was added
at
that
were used.
by an indirect
preincubation
activity.
under
in
gradient-purified
concentration
and
30 min
peroxidase
U.S.A.)
U.S.A.).
and pDB339,
picked
for
(Vector
following
Sea Plaque agarose
were made in the same media and plated
a set of clonal
as
cells containing plasmid resistance in Ml99 with
ofover
5 x 10mh, and
trypsinised,
light
stain
Cells
that the peptide
and 10” cells plated
immunoperoxidase
conditions.
CsCl
was performed
Transfection
onies
to UV
Maine,
California,
around be
U.S.A.)
into 0.3”,,
with a Bio-Rad
until G418-resistant to
ABC
after 2 weeks.
The electroporated selected for neomycin
G418containing
Indirect
with
3 x 10’ Vero cells and 100 ug plasmid
capacitance. DNA were
DNA Inc.,
peptide
slide and one chamber
or 25 uM dexamethasone
infected
were maintained
with
of a LabTek
to ensure
techniques
as previously
lines were subcultured
(Qiagen
layer of 0.5”,,
Cultures
Richmond,
used and electroporation
lo”,,
exposed
Rockland,
by electroporation
experiment
column
and
repeatedly
plasmid.
staining. Cell lines were examined
of the HSV-1 technique
tested
different
trans-
48 h the cells were harvested
transfected
Rad Laboratories,
Fresh
had been
plates.
with plasmid
[ 1 l] or were
[7]. After
were counted
cells
plasmids
U.S.A.)
Biocolloids,
culture
and colonies Vero
on a Qiagen
were seeded
medium
in 100 mm2 dishes they were either
precipitation
that
described
and 2 x 10’ cells agarose
were plated
California,
simplex
previously
when the cells were between
sub-confluent,
Three
Zmmunohistochemical
Transfection cells were transfected
the appropriate
were further
was expressed.
were used as controls.
from Vero cells was tested
that it carried
expression NRK536
of transfectiolz
Each cell line derived
to
immunosorbent
assay was also used to confirm Cells
were grown
absence
were harvested without
to confluent
of dexamethasone
monolayers
J The ELISA
of the peptide
[5,15].
in the presence
for 2 days in 150 mm dishes
24 h after the addition
dexamethasone.
O’C, rinsed
assay f ELISA
expression
Briefly,
twice with ice cold PBS
0.6 uM phenylmethylsulphonyl
of fresh medium
the dishes with 0.001
fluoride
were
with or
chilled
M EDTA
and removed
or and to and
with a
Effect
rubber
scraper.
Cells were recovered
for 5 min, washed
vigorously
The
by three
cells were disrupted
and ethanol
by centrifugation
once and finally
same buffer by vortexing and thawing
suspended
ELISA.
tube
The
entire
procedure
was collected
for
concentrations
the
protein
(Pierce
Illinois,
U.S.A.)
for the
preparation
was
were determined
Chemical
Co.,
and 100 ug of the total proteins
coat the wells of a 96-well
at
in a fresh
of antigen
extract
at 4°C. Protein assay
tube.
in dry ice
by centrifugation
and used as a source
performed BCA
of freezing
followed
12 000 g for 5 min and the supernatant microcentrifuge
at 200 g
in 0.5 ml of the
in a microcentrifuge cycles
at 37°C
on Cell Phenotype
plate overnight
by
of the Mutagenic
U.S.A.).
This
vector
was
were used to
chain reaction.
appropriate
plasmid,
To confirm
the polymerase
employed.
Primers
consisted
and reverse
primers
(USB,
either
had been
inserted.
plasmid PCR
DNA
DNA
extracts
of appropriate
Ohio,
mutation
of Vero
as previously
cells,
pDB337
described
[16].
Reaction
rates
were
determined
in an agarose from pK189
in newly
expanded
clones
test
cell lines 1 x 10’
groups
of six nude mice.
as well as two non-expressing
lines
were injected
339.
SV-T2
presence
duplicate
and the cells Growth
were
curves
immediately
of
wells
25 uM
counted
for
culture
dexamethasone. cell type
At
cell
24-h
were trypsinised,
in a haemocytometer
individual
after clonal
or with PDB
into wells of a 24-well
of each
lines
months, organs
chamber.
were
transfected
2 days, but
suspended
60 mm dishes
coated
ability
greater
than
included
Vero
The
sterile
cells
were for
was
agarose
with
determine
were
counted
to
and incubated
having
[17].
on
for colony a diameter
HeLa
cells
were
control.
if the peptide in M199/G418
serum,
0.2,
0.6, 0.8,
peptide
was induced
counted
were
increased
in low serum,
100 mm dishes
the dishes
the ability
500 cells were plated on to
medium
1, or lo”,,
containing
serum.
either
Expression
by 25 PM dexamethasone.
examined
of Vero
and the number
After
no
after
with
of the
of colonies
were
vector
frequency
a shuttle
gene
vector
cassette
and 0.9 Kb.
The
as an insertion
to make
injected
As a positive numbers
mutation if the total
tumours,
either
subcutaneously
into
control,
were were
at autopsy
tumorigenic
injected
were examined
they
some animals
of the known
animals
time
frequencies
and the different
with
non-
daily for up to 3
sacrificed.
The
for the presence
major
of tumours,
was examined
were compared
proportions
histologically
system
in cell lines [14].
by removal
and its replacement (Pharmacia
The
Inc.,
was determined
mutagenesis
of the region
shuttle that codes
with a kanamycin-resistance Piscataway,
New
by Fisher’s
of different
were compared
by a Chi square
Transformation
of NRKS36
types
exact test
of mutations
test.
RESULTS The cells
spontaneous was
observed
low,
cells
transformation
with
only
per 2 x 10” cells,
two
frequency
transformed
and the number
by the addition
of 10 mM dexamethasone.
pDB339
raised
the number
pDB337
produced
when
being
was not increased Transfection
of colonies
15 colonies
of NRK536 colonies
to five. The
in the absence
dexamethasone
with plasmid
of dexametha-
was added
to the
cultures. HSV-1
that had been irradiated
to 53 colonies,
while irradiation
by UV light for 10 min led
of the virus for 15 min led to 3 1
colonies. Transformation
cells of mutations
pS 189 was modified
for ampicillin
which
of Vero cells
A total of seven clonal
frequency
as a
with sizes that
analysis
continuously The
was classified
12 days,
[ 171.
Mutation
were
other
were examined
sone and 32 colonies
cells to form colonies 0.4,
and
G418
plated
dishes were then examined and colonies
anchor-
dependence
To
sizes and the
of the numbers
for any foci of tumours.
dexamethasone
methylcellulose
with 0.8”,,
100 mm
induced 1 x lo5
suspension
on soft agar,
as a positive
cells,
of 25 PM
in 1.5”,,
at 37-C for 21 days. The
Serum
in
dexamethasone.
forming
of the peptide
in the presence
then
no
to be
than 5.2 Kb.
Vero cells. Animals
Mutation
growth
pre-incubated
were
was confirmed
expansion.
if expression
age-independent
bacterial
fragments
if the total size of the fragments
and the skin at the site of injection
determined
independence
To determine
cells
with equal
cells,
Statistical Anchorage
the
of fragment
for the ability
or
with the vector,
in the
that had occurred, overnight
mutation
4.3
was greater
1 x lo6 the peptide
intervals,
from
was
Tumorigenicity
transfected
plate
recovered.
were obtained
to be a deletion
size of fragments
colonies
by examination The
and transfer frequency
mutant
if two fragments
with a change
mutation
and
by examination
indistinguishable
that had been
cells (1 x 104) were plated
gel. Each
Plasmids
by a colour
from
HI
Bum
was assessed
was considered
that expressed The
with
by
plasmids,
by hybridisa-
probe.
rate
Growth
recovered
was less than 5.2 Kb and classified
gels and bands
7070.
for
followed
by recovery
of white
and sizes of the fragments.
To Growth
digested
to replicate
any non-replicated
The
The
phosphate
lysis,
the types of mutation
were
type of mutation
were
and the
and allowed
culture.
as the proportion
point mutation
and pDB337
as viral DNA
DNA
to anneal sequences
controls,
on 2”,, agarose
size were confirmed
tion to a 32P-labelled
U.S.A.)
calcium
by alkaline
to be mutants
bacterial
plasmids
derived
by
into E. Coli MBM
In order to classify
was
was named pK189.
cells
in the sup F gene were recognised
another
resolved
vector
shock
recovered
and were confirmed into
the
forward
the HSV
and positive
were electrophoresed
(PCR)
universal
site into which
acted as negative
was performed
mixtures
chain reaction
of the Ml3
Cleveland,
side of the cloning
that cells retained
and glycerol
and was introduced
cultures, Polymeruse
into
with Dpn I to remove
mutant
at 4’C.
shuttle
48 h. It was then
expressed
Rockford,
modified introduced
precipitation digestion
269
Peptide
Jersey,
that
BLUE
in medium had been
alone,
transfected
The
was
transfected
pDB337
number
with G418.
transfected
with pDB339,
that had been contained
one
cell lines were developed
from
with
the vector
Vero
and the other with
were named
pDB337. H1l.l,
which grew
One line was from Vero cells
pMAMneo-
that
had
five were from The
cell lines
B7, BlO,
of cells per 75 cm2 flask ranged
been cells that
F7, and F9.
from
1 x 10’ to
C.M. Das et al.
270
2 x lo7 in these
seven lines. Cell morphology was normal fibroblast-like and was very similar from clone to clone. However, the cells in the Hl 1.1 line were much larger than the others. Expression
Vero cell line. By this time only the Hl 1 .l cell line expressed the peptide, as shown by both assays, in the presence of 1 or 25 PM dexamethasone, but not in the absence of dexamethasone. The other lines B-7, B-10, F-7 and F-9 had become peptide-negative at this time. The inducibility of peptide expression was also confirmed in H11.1 at passages 15-19 using various doses of dexamethasone (Table 2). At passage 40 the H 11.1 cell line was examined by the PCR to ensure that it still retained the viral sequences. A band corresponding to 410 bp was seen, and hybridised to the probe derived from the pDB337 DNA (not shown). In the human keratinocyte cultures no non-transfected cells were stained with anti-T antibody, but in the cells transfected with pZ189 the proportion of stained cells was 48/4760 ( lqO). In the SV-T2 cell cultures, every cell showed nuclear staining.
of viral peptide
To confirm expression of the viral peptide, all cell lines were examined by immunoperoxidase staining with antiserum to the peptide within two passages after being established. Staining was done on cultures that were maintained without dexamethasone or with 1 or 25 PM dexamethasone. No stain was seen in non-transfected Vero cells, Vero cells transfected with pMAMneo-BLUE or in Vero cells transfected with pDB339. Each of the Vero cell lines transfected with pDB337 showed positive staining when cultured in the presence of 1 or 25 PM dexamethasone but not in the absence of dexamethasone (Fig. 1). Staining was intense, restricted to the cytoplasm, and seen in almost 100% of the cells. The appearance was similar with each of the cell lines. At passages 10-12 the immunoperoxidase staining was repeated, and the ELISA (Table 1) was performed on each
Fig.
1. Photomicrographs
showing staining of different (b) Vero/pMAMneo-BLUE;
Mutation
frequency
in cell lines
The mutation frequency of pK189 in Cos-1 cells was O.O24qi,, which is very similar to that obtained previously with pS189. Thus, the replacement of the ampicillin-resistance
cell lines with antiserum to mutagenic (c) Vero/pDB339; (d) Hll.1.
Table 1. Induction of peptide in cell lines by dexamethasone as measured by an ELISA
assay
Concentration of dexamethasone None
25 FM
Increase
VerolpMAMneo-BLUE Vero/pDB339 H1l.l
0.463 (f0.014)* 0.380 (f0.004) 0.440 (i 0.020)
0.445 (kO.067) 0.373 (kO.011) 0.949 (f0.014)
-0.02
B7
0.464 ( k 0.060)
BlO
0.511 (+0.060)
F7 F9
0.668 ( * 0.054) 0.480 ( k 0.097)
0.452 (+0.090) 0.592 (f 0.010) 0.519 (kO.120) 0.418 (kO.020)
Cells
-0.01 0.51 -0.01
0.08 -0.15 -0.06
*A414 values expressed as meanskstandard error of triplicate readings. The assay was
performed at passage 12.
peptide.
(a) Vero;
271
Effect on Cell Phenotype of the Mutagenic Peptide Table 2. Induction of peptide in H11.1 cells by dexamethasone measured by an ELISA assay Dexamethasone None 1pM 5pM 25 FM
A,,, values* 0.150 0.551 0.375 0.291
as
Increase?
(+ 0.04) (+0.18) (f0.07) (kO.09)
5 4 3
*4,4 values are expressed as means&standard error of triplicate readings. tFold-increase was obtained in comparison with the A4,4 value obtained in the absence of dexamethasone. All A,,, values were corrected by subtraction of the reading with pre-immune serum (0.486* 0.074). The assay was performed at passage 17. gene with a kanamycin-resistance gene had no significant effect on the mutagenesis plasmid. In the presence of 25 FM dexamethasone the spontaneous mutation frequency in Vero cells was 0.210/h which is higher than in Cos-1 cells. The highest mutation frequency was seen in the Hl 1.1 cells, while the other peptide-expressing cells showed lower mutation frequencies (Table 3). However, these tests were done at relatively high passage levels, by which time some of the lines
were no longer expressing peptide. In the Hl 1 .l cells the mutation frequency increased with increasing concentrations of dexamethasone in the medium although other cells showed no change in the frequency with added dexamethasone (Table 4). The types of mutations that occurred in the Vero and H 11.1 cells were analysed by examination of digested fragments of the plasmid that were recovered from the cells. In no case had plasmid pDB337 been recovered in place of pK189. In Vero cells, point mutations accounted for 53:/b of mutations. Of the 47 plasmids with altered mobility, 37 showed slight reductions in size of one fragment and eight showed an increase in size of one fragment. Two plasmids were linearised by Bum HI and the single fragment was larger than 5.2 Kb; they were classified as insertions. In the H1l.l cells 54’$, of mutant plasmids were classified as having point mutations. Of the 46 with altered gel mobility, 39 showed a slight reduction in size of one fragment and one showed an increase in size of one fragment. Six plasmids showed neither simple increases nor decreases in one or the other fragments, but five of those were classified as insertions and one as a deletion based on the total size of all fragments. The types of mutant plasmids derived from each cell type are summarised in Table 5 and did not differ significantly between Vero and Hl 1.1 cells.
Table 3. Frequency of mutations in cells lines in the presence of dexamethasone Colonies Cells
White
Mutations Blue
Frequency*
Pt
Vero
45
26 500
0.17%
n.s.
VerolpMAMneo-BLUE Vero/pDB339
40 34
19000 11800
0.124; 0.29%
n.s. <0.05
148 21 44 59 71
19 200 12 400 18 500 19 800 18 300
0.770, 0.17 o/o 0.24:,, 0.30% 0.39%
H1l.l B7 BlO F7 F9
*Mutation frequency expressed as a percentage, derived from counting at least 15 000 colonies. The assay was performed at passages 10-12. tThe frequency of mutations was compared with that in the same cells in the absence of dexamethasone, using Fisher’s exact test. n.s.: not significant.
Table 4. Mutation frequency
in cells in the presence of different concentrations of dexamethasone Concentration of dexamethasone
Cells
None
1 KM
5pM
10 FM
25 pM
Vero VerolpMAMneo-BLUE Vero/pDB339 H1l.l
0.21* 0.20 0.12 0.19
0.20 0.14 0.11 0.377
0.19 0.17 0.10 0.40f
0.13 0.21 0.11 0.621
0.19 0.26 0.291 0.76$
*Mutation frequency expressed as a percentage, derived from counting at least 15 000 colonies. The assay was performed at passages 10-12. isignificantly higher than mutation frequency of same cells in the absence of dexamethasone (P
C.M.
272
cells
Table 5. Types of mutatiorrs observed in Vera and H1 1.1
Type of muration
Vero cells*
Poinr muration Deletion Insertion
H1l.l
53 37 10
Das et al.
cells
showed
cells
showed
numbers
of serum.
a maximum
of
However,
number
foci
with
the Hll
of foci
with
54 40 6
F7
cells
showed
pMAMneo-BLUE concentration larger
a pattern cells,
of serum.
in size than
most
0.6”,,
with
HI 1.1 cells formed
any other
lower serum concentration,
lines,
0.2”,,,
serum,
3). The
consistent
although
increasing
.l and B7 cells
fewer foci with either more or less serum (Fig.
*One hundred mutant plasmids were examined from Vero cells and another 100 from Hll, 1 cells.
with
fewer
and could
the Vera/
foci
colonies form
and
F9 and at each
that were foci at a
than any other line (Fig.
4).
A Verolvector
.-..,”
n
increasing
concentrations
Vero
A Vera/vector
. VerolpDB339
1
l Hl1.1
0
Days Fig. 2. Growth of cell lines. Cells were plated into wells of 24-well culture plates at a concentration of 1 x 1O’cells per well in the presence of 25 pM dexamethasone. At daily intervals duplicate wells were harvested and cells counted.
rates were measured
0.8
I.0
(%)
Fig. 3. Serum dependence of cell lines. Cells at the 10th to 12th passage were plated in 100 mm dishes in media containing different concentrations of serum. Growing colonies were counted after 12 days and expressed as a percentage of cells that had been plated.
for all cell lines that had been
derived
from Vero cells at pasage 8 or earlier
shown
in Fig.
expressed
2. At this
the peptide.
passage,
cells
express
as determined
the peptide
Non-transfected concentration
Vero
and cells
Vero/pDB339
BlO
became
plated.
grew
while Vera/ cells
rapidly
up to day 4. However, lines H 11.1,
rapidly. apparently
They
are
and F9 did
not
by immunostaining.
on a plateau
of cells declined
cell lines grew more slowly,
when
plated their
at a peak
for a few days and then Each
of the transfected
since a smaller
were very similar
from day 5 onwards
number
to each other
the peptide-positive
B7, B 10 and F9 grew at only half the rate of the
peptide-negative peak densities the pattern
B7,
of 1 x 10” cells per well and reached
on day 5, stayed
the number
and the results
H1l.l,
F7 showed weak expression
pMAMneo-BLUE
of them
0.6
rate
Growth
number
0.4
Serum concentration
2
Growth
0.2
VerolpDB339
line.
All these
on day 8 or later. The growth close
were measured
to the VeroipDB339
three
times
using
lines
reached
rate of F7 followed
line.
Growth
each cell line,
curves
with similar
results.
Serum dependence When serum,
cells
were
two distinct
pMAMneo-BLUE
plated patterns cells,
with
different
of survival
the VerolpDB339
concentrations
were seen. The
of Veroj
cells and the BlO
Fig. 4. Appearance of cell colonies from experiments shown in Fig. 3. (a) Vero/pDB339 cells, 10% serum; (b) VerolpDB339 cells, 0.5% serum; (c) H1l.l cells, 10% serum; (d) H1l.l cells, 0.5% serum.
Effect
on Cell Phenotype
of the Mutagenic examination received
of the
injection
the control,
1 x 10’ cells,
273
Peptide sites.
tumorigenic
all developed
However,
animals
cell line SV-T2,
tumours
that
at a dose of
with an average
diameter
of 1.5 x 0.7 cm by the 6th week.
DISCUSSION It is clear
that HSV-1
phenotype
since
repeatedly acts
genome raises
DNA
have
The
been
serum
antibody
be anticipated malignant
that
the peptide
the mutagenic by HSV-1
transformed in
induced
Vero
expressed
were
Vero
colonies
cell
passage
as anchorage-independent of foci per dish as HeLa each
focus
was
the same time did not develop
All cultures smaller,
by 1%20”,,
but
differences
no
transfected nally using
by
cultures
However,
received
of the peptide frequency
to express
sequences
than the non-transfected The
tumours
in nude
or induce
had only induced
the
to express
up to at least
cells when expression
cells did not form foci in soft mice,
indicating
some of the features
we
half
the
in parallel
the
diameter
cultures
for
to form foci.
with
between
days
each were cultures
All cultures and
died.
plasmid
and
consistently cultures, that
were
were termi-
Transformation
cells were performed five different
appeared
any dose of transformed tumour
foci
five times,
cell donors,
by 3 months NRK536 were
no phenotypic
to the viral peptide.
can be extended
of non-transfected
plasmids.
from
oral cells showed
keratinocytes
diameters
seen 22
Human
be attributed
that transfection
that
the
of transforma-
with
in animals cells,
detected
that
Vero cells or
by histological
found
that
the viral peptide The
results
HSV-1
could
However, require
mtr-1 . The
In other
human
other
The activities
reported
of this virus peptide
other pheno-
that are also encoded
that transformation
to this viral peptide
the cell phenotype
of
for cell in
to a malignant
viral proteins
data suggest
peptide
or to the anchorage-
been
transformation
but
such changes.
that is responsible
has
oral
[ 191. There-
modifications,
of inducing
phenotype
that
be attributed
for changing
of
to phenotypic
to an immortal
experiments.
cells cannot
lifespan
be the viral protein
controls
successfully.
of this study show that the mutagenic
phenotype
within
the
that could
was despite
by N-nitrosamines
was not capable
independent type must
changes
This
was achieved
fore the cells were susceptible
of human
alone.
that might be responsible
are unknown.
However,
it is
known that the mutagenic peptide represents the carboxy terminus of one member of a family of virus-encoded proteins. proteins
DNA-binding
No
that
to show
the fact that in
and the mutation
was induced.
experiments
producing
Tumorigenicity
Hl 1 .l cells.
slower
was despite
concentration
These
had
not
had not
the plasmid
were
the expression
the viral
foci,
results.
tumours
with
of the peptide
[20], protease No
were
10, but the H 11.1 line continued
and retained
transformation
those
with the human
primary
similar
as the virus
cells
lines lost the ability
showing
about
transfection
than
with different
experiments
also
by others
that the peptide
they
This
30 they were capable
the ability
Monolayer were
differentiated
make
cells
survived
to grow.
could
cells (200 colonies/105
5). Vero cells that were maintained
of human
lines
at early passages.
after the H 11.1 cells had reached
continued
were
tion.
transfected
anchorage-independent
Transformation
to a
passage 40. The Hl 1.1 cells were able to grow in a lower serum
agar
although
cells
the
transfected
3). Four
by passage
peptide
(Fig.
cells
as reported
was as efficient
at early passages
(Table
the peptide
These
However,
peptide
of transformation.
was raised
cells),
Since
it might
transformation.
cells
could be induced
same number
[18].
transform
particles,
each of the five lines examined
of growing
which
did lead to morphologi-
indicating
the mutagenic
features
the
of the
with oral
carcinogens,
cells.
foci.
in nude mice,
complete
When
independence
the peptide
would
pDB337
producing
tumorigenic
of
and patients
peptide
readily
[l 11, and the plasmid
None
a peptide
of many
of NRK536
particle
Anchorage
region
to encode
against
cal transformation
peptide
that the
phenotype.
As expected,
Fig. 5. Anchorage independence of transfected cells. Cells were seeded in methylcellulose and anchorage-independent colonies were photographed after 21 days. HeLa cells were included for comparison. No colonies were seen with cells that were non-transfected, or were transfected with pDB339. (a) HeLa cells; (b) H1l.l cells.
but the
implies
cells
of cells,
is a characteristic
demonstrated
obscure,
transforming
shown
frequency
to a malignant
been
basis of this remains
as a mutagen.
has recently
mutagenesis
cells
has
in the transformed
the mutation
cancer
can transform
phenomenon
[l, 41. The
lack of viral virus
the
involved
activities
properties
that
include
activities
[21,22],
virion
assembly
and kinase and
[5]. Any of these functions
in cell transformation.
that the peptide suggested
have
and substrate
is antigenic,
by the observation
The
present
might
study
and its role in oral cancer that
patients
with
be
confirms has been
oral cancer
274
C.M. Das el
increased levels of IgM antibody to the mutagenic peptide. This suggests that it is expressed at some point in the development of the tumour [ 181. The peptide, therefore, might play several different roles during viral infection, as do most of the transforming proteins of DNA viruses [23]. It is now important to test the effects of the mutagenic peptide on oral cells that have been immortalised, or partially transformed in other ways. Oral cells that have been immortalised by papillomaviruses can become fully transformed by subsequent exposure to an alkylating chemical carcinogen [24], and so might also be transformed by the HSV-1 peptide. Alternatively, our oral cells whose survival was extended by exposure to tobacco-related carcinogens [ 191 might be susceptible to transformation by the peptide. These studies are in progress.
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Acknowledgements-The authors wish to thank Dr M.P. Moyer for helpful advice with the human cell cultures. This study was supported by PHS grant DE07007. CD. was the recipient of NRS award F32 DE05577. The synthetic peptide was made in the Synthetic Antigen Laboratory, M.D. Anderson Cancer Center, which is supported by PHS grant CA 16672.