Tohoku
J. exp.
Med.,
Alterations
1978,
in
125, 177-183
Surface
Sialyltransferase Kidney
Glycoproteins
Activity
Cells
Infected
Adenovirus
Type
and
of Human
with
Level
of
Embryo
Oncogenic
12
YOSHITAKA AOI and
MASANORI YOKOTA
The Institute of Medical Science, the University of Tokyo, Tokyo 108
AOI,
Y.
and
YOKOTA,
Sialyltransferase
Activity
Adenovirus
Type
embryo
kidney
mutants at
the
(Cyt) early
mutants in
the
12.
Tohoku
(HEK)
cells
synthesized stage
of
synthesized
between
type the oncogenic
Alterations
Human J.
exp.
of 12.
It
sialyltransferase adenovirus
Med.,
1978,
was
that in 12;
of
microsomes
microsomal
Human
and on
its
the
cytocidal
cell
surface
tumorigenic
sialyltransferase were a
of
Oncogenic
177-183 12
cells was
Level
with
a highly
when there
the
and
level
increased
confirmed
type
less
The
activity
(2), type
respectively a
and
Infected
125
glycoproteins Furthermore,
cells was
Glycoproteins Cells
adenovirus
glycoproteins.
HEK
Surface
Kidney
with
different infection.
in
Embryo
infected
different
microsomes
adenovirus
M. of
positive and
sialyltransferase
Cyt activity
infected
with
correlation tumorigenicity.„Ÿ activity;
tumorigenicity
It is generally events leading to 1973). However, of tumor in vivo
accepted that cell surface alterations are the primary biochemical the loss of growth regulation in virus transformed cells (Tooze what surface alterations are directly related to the development still remains to be clarified.
Several approaches have been adopted to find a correlation between the cell surface changes and tumorigenicity (Glick et al. 1973; Glick et al. 1974; Onodera et al. 1976). One of the most important problems may be to isolate the tumor specific transplantation antigen (TSTA) and to elucidate its chemical structure. Human adenovirus type 12 (H12) is highly oncogenic in hamsters. However, most H12 Cyt mutants are less tumorigenic in hamsters and less efficient in trans formation of cells (Takemori et al. 1968; Takemori et al. 1969). These mutants, which do not show the typical cytopathic effect in adenovirus-infected cells, were named on the basis of their cytocydal effect.
The comparison of less tumorigenic H12 Cyt mutants with highly tumorigenic H12 wild type (WT) showed that they differed only in their capacity to induce cell surface changes, such as lectin-agglutinability (Yamamoto et al. 1972; Salzberg and Raskas 1972) and TSTA (Yamamoto et al. 1972). This close cor relation between tumorigenicity and the capacity to induce cell surface changes Received
for publication,
November
1, 1977. 177
178
Y. Aoi and M. Yokota
provides an efficient system which is useful transformation and membrane alteration.
for analysis
of the relationship
between
In the previous paper (Aoi 1977), the author suggested that the level of sialic acid content in the plasma membrane may be under the control of the microsomal sialyltransferase activity. There may be a link between sialic acid content in the plasma membrane and the transformed phenotype in the cell lines. This membrane and
paper describes a comparative study of glycoproteins in the plasma and the sialyltransferase activity in the microsomes of H12WT-infected
Cyt mutant-infected
tumorigenicity
and
cells.
The results
alteration
in the
also suggest
glycoproteins
a close correlation
of the
plasma
between
membrane.
MATERIALS AND METHODS Secondary cultures of human embryo kidney (HEK) cells and H12WT, Cyt 31 and Cyt 66 mutants were used. These mutants were named by N. Takemori according to the proposal made by Ginsberg et al. (1973). Most Cyt mutants are less tumorigenic in hamsters with an exception of Cyt 66, which is highly tumorigenic (Takemori et al. 1968) and not defective in inducing cell surface change (Yamamoto et al. 1972). The procedures for cell culture, virus production and virus assay were described previously (Yamamoto et al. 1972). The plasma membrane and microsomes were prepared according to a modified method of Ray (Ray 1970). The membrane isolation procedure is summarized in Fig. 1. Protein determination was carried out by the method of Lowry (Lowry et al. 1951), using bovine serum albumin as a standard. The activity of sialyltransferase in the microsomes was measured according to a modified method of Grimes (Grimes 1970) . Successful applica tion of this method to the microsomes used in this experiment was reported previously (Aoi 1978). Cells and
were
grown
plated
in
serum.
When
were
infected
after
infection
ƒÊ Ci/ml
above,
method
run
with the
gel.
of
sample
estimation ,000)
(‡U),
indicated
as
gave
slices
0.3ml
of
radioactivity
Fairbanks
10
with for
following
(‡V)
and
(‡W)
in
sample
were
were
was
counted
until
and
were
with
10%
calf
the
cells
Fifteen
labeled 25 ƒÊCi/mmol
hr
with
2.5 ,
radioactive
the R as
sugar the
use
(SDS-PAGE)
was
carried
. Polyacrylamide cytochrome c
gels migrated
(7.5%) to the
(without
molecular
weight
The slices
10ml by
markers
(160
,000)
cytochromec
.
The
added
as
chain
figure
width.
vial
1971)
used
H
(‡V) the
labeled
and
respectively.
cells activity
policeman
al.
mA/gel
globulin
1.0mm each
5
(25,500)
,
the
cells/dish
with
discarded
electrophoresis et
proteins
human
,
rubber
4 •~ 105
of
10min. gel
of
was
PFU/cell
pulse
(Fairbanks
current
peaks:
each
medium
at
of
supplemented
synthesis)
being
g
concentration
specific
After
200 •~
a
(MEM)
31
DNA
harvested at
approximately
in
at
the
Cyt
viral
hr.
were
The
To
and
of
chymotrypsinogen
H2O2.
monolayer,
66
four
constant
(‡T), (‡U),
dishes medium
polyacrylamide of
a
Petri
(D-6-[3H]-glucosamine,
cells
dodecylsulfate
the
of
Cyt
centrifuged
by
a
onset
for
Sodium
bottom
formed
the
the
then
Falcon essential
3H-glucosamine
and
were
mm
H12WT,
(before
Centre)
described
out
cells
with
of
65 minimum
the
adiochemical
trypsin)
on
Eagle's
of
Beckman
gels
were
were
for (I), (11 cut
swollen
the bovine
,700)
serum (‡W).
with
a gel
scintillation
(SL-200)
liquid
were
slicer
overnight
dioxane
albumin (68 They
at fluid
scintillation
which 55•‹
with
and
the
counter
.
RESULTS The Ray yield:
(Ray after
isolation 1970)
procedure except
centrifugation
plasma
membrane
that
of
the
the
following
steps
at
5,000 •~
g
for
10min,
according were the
reformed precipitates
to
the to
method imp
were
of
rove
the
taken
up
Surface
Fig.
1.
Glycoproteins
Isolation
after
procedure
Adenovirus
of the plasma
Infection
179
membrane.
in a small volume of distilled water (1-2ml), and then this suspension was layered on the 37% sucrose solution (w/w). SDS-PAGE patterns of glycoproteins in the plasma membrane newly synthesized by HEK cells are shown in Fig. 2(a). Major components were observed in fraction No. 10 and in fractions Nos. 18-19. Cells infected with H12WT newly synthesized fraction No. 10 glycoprotein, but there was no glycoprotein peak at fractions Nos. 18-19 (Fig. 2(b) ). On the other hand, cells infected with either Cyt 31 or Cyt 66 produced a different pattern from that of mock infected or WTinfected cells. The major peak (fraction No. 10) was not detected in either case (Fig. 2 (c), (d) ). The newly synthesized glycoproteins in the virus infected HEK cells were different in the SDS-PAGE patterns. The microsomal sialyltransferase activity involved in the elongation of the terminal carbohydrate (sialic acid) of glycoproteins in the plasma membrane was examined. Cells infected with H12WT and Cyt mutants were harvested at 19 hr after infection and microsomes were prepared. The transfer of CMP-[14C]-sialicacid to desialized fetuin was measured (Table 1). The activity of sialyltransferase increased by 230% in WT-infected HEK cells than that of uninfected cells. The level of sialyltransferase in Cyt 66 (highly tumorigenic) infected cells was 1.23 times higher than that of Cyt 31 (less tumori genic) infected cells.
180
Fig.
Y. Aoi and M. Yokota
2. SDS-PAGE membrane.
patterns
(a) mock-infected cells. 66-infected cells.
of
3H-glucosamine
(b) H12WT-infected
labeled
cells.
glycoproteins
in the
(c) Cyt 31-infected cells.
plasma
(d) Cyt
DISCUSSION
Concerning the molecular defects of Cyt 31 (less tumorigenic) mutant, two interesting facts have been reported (Yamamoto et al. 1972). First, cells infected with H12WT or Cyt 66 (highly tumorigenic) demonstrated marked lectin agglutinability at 24 hr to 32 hr after infection. In contrast, cells infected with Cyt 31 mutant showed much less marked lectin agglutinability during the infection process. This observation, indicating that Cyt 31 mutant does not induce surface alterations, is in contrast to the marked surface changes induced by H12WT or Cyt 66. Secondly, Cyt 31 does not induce transplantation immunity in hamsters. However, the correlation between the surface alterations induced by infection in vitro and the lack of induction of TSTA in vivo still remains to be clarified.
Surface
Glycoproteins
after
Adenovirus
Infection
181
TABLE 1. Level of sialyltransferase activity in the microsomalfraction after infection of human adenovirus 12 and Gyt mutants
*
Results and
are as
expressed
cpm/mg
The study in this paper for the phenomena described
as
of
the
microsomal
mean•}s
.E.
of
3 to
5 determinations,
protein.
was performed to find above. It was found
out biochemical that the newly
mechanisms synthesized
glycoproteins characteristic the
level
and the level of sialyltransferase activity in the microsomes to the cells infected with each mutant . It is worthy of note of sialyltransferase activity was raised by oncogenic virus infection
that the degree of activation was different depending on the These results seemed to indicate that the level of sialyltransferase microsomes
is related
with
the
were that and
infected mutants. activity in the
tumorigenicity.
Sialic acid content in the plasma membrane has been reported to correspond well with the microsomal sialyltransferase activity (Aoi 1978). Sialic acid in the cell surface glycoproteins has been reported to be involved in the determination of MN antigenicity (Springer and Ansell 1958) and tumor antigen (Sanford 1967; Currie and Bagshawe 1968; Simons et al. 1971; Bekesi et al. 1971). Moreover, the presence or absence of sialic acid on the surface has a considerable effect on the fluidity of the plasma membrane, e.g., Con A agglutinability (Novogradsky and Katchalski 1973; Vaheri et al. 1972; Kemp 1968). In this context, it is considered that sialic acid content in the plasma membrane of HEK cells seemed to be raised by infection of those viruses and altered surface glycoproteins. On the other hand, it has been known that adenovirus types 1, 2 and 5, although classified as non-oncogenic, do transform rodent cells in vitro (Freeman et al. 1967; McAllister et al. 1969; Williams and Ustacebeli 1971) and transformed rat cells are non-tumorigenic (Freeman et al. 1967) unless the animals are im munosuppressed (Gallimore 1972). Whether cells transformed by non-oncogenic adenovirus are related to the level of sialyltransferase activity in the microsomes, is not explored at this moment. Research along this problem is now in progress. Acknowledgment
We would like to express thanks to Prof. H. Shimojo of this encouragement and the supply of experimental materials.
Institute
for his
References
1)
Aoi, Y. (1978) Biosynthesis of glycoprotein-glycosyl transferases during the cell cyc le. Tohoku J. exp. Med., 124, 139-144. 2) Aoi, Y. (1978) Sialic acid content and growth control of mouse cells transformed
182
Y. Aoi and M. Yokota by
3)
a
temperature-sensitive
Bekesi,
J.G.,
mutant
St-Arneault,
munogenicity
by
G.
vibrio
of
SV40.
& Holland,
cholerae
FEBS
J.F.
letters,
(1971)
in
Increase
neuraminidase
press.
in
treatment.
leukemia
Cancer
L Res.,
1210 31,
im 2130
- 2132. 4)
Currie,
G.A.
&
munogenicity Cancer, 5)
Bagshawe, of
22,
the
K.D.
(1968)
Landuschutz
The
ascites
effect
tumor;
of
Site
neuraminidase
and
on
mode
of
the
action
.
im
Brit.
J.
588-594.
Fairbanks,
G.,
the
polypeptides
major
Stock,
T.L. of
&
Wallach,
the
human
D.H.F.
(1971)
Electrophoretic
erythrocyte
analysis
membranes.
of
Biochemistry
,
10,
2606-2617. 6)
Freeman,
A.E.,
Huebner, type 7)
8)
Black,
R.J. 2.
Proc. P.H.
transformed
in
9)
Acad.
H.S.,
M.C.,
which
coincide
by
(Wash.),
2.
J.
Doerfler,
&
Sachs
Austin, by
cells
immunosuppressed gen.
Virol.,
W.H.
&
J. ,
P.H.,
J.R. adenovirus
&
1205-1212.
adenoviruses.
Z., virus
Henry, rat embyro
in
type
J.F., of
Rabinovitz, with
58,
production
adenovirus
mutants
, E.A., primary
of
Tumor
Williams, for
Glick,
Sci.
(1972) vitro
nomenclature
Vanderpool
Transformation
nat.
Gallimore,
Ginsberg,
B.H.,
(1967)
L.
with
cells
.
H.
12,
(1973)
Proposed
663-664.
Surface
and
99-102
Shimojo,
Virol.,
(1974)
transformation
rats
16,
membrane
tumorigenesis.
glycopeptides
J.
Virol
.,
13,
967
- 974. 10)
Glick,
M.C.,
Rabinovitz,
correlated 11)
with
Grimes,
G.W.
(1970)
transformed 12)
tion 13)
Sachs,
R.B.
Sialic
acid
E.J.,
Lowry,
218,
O.H.,
McAllister, (1969) T
the
R.M.,
48641869.
and
sialic
acid
glycopeptides
levels
in
normal
and
.
Jourdian
of the
membrane
, G.W. glycoproteins
removal
of
cell
&
Roseman,
.
J.
biol.
surface
S.
(1966)
Incorpora
Chem.,
241,
5735-5737.
sialic
acid
on
cell
aggregation
1255-1256.
Rosebrough with
Surface
12,
5083-5092
into
Effect
Nature,
(1973)
transferases 9,
McGuire,
(1968)
vitro.
L.
Biochemistry,
N-acetyl-n-glucosamine
measurement 15)
&
Biochemistry,
I.R.,
of
Kemp, in
14)
cells.
Johnston,
Z .
tumorigenesis.
, folin
Nicolson,
ransformation
N.J., phenol
M .D., of
rat
Farr,
A.L.
reagent.
J.
Lewise,
embyro
A.M., cells
&
Randall,
biol Jr.,
by
R.J.
. Chem.,
193,
MacPherson,
adenovirus
type
(1951)
Protein
265-275. I.
&
Huebner
1 .
J.
gen.
, R.J. Viol.,
4,
29-36. 16)
Novogrodsky,
A.
lymphocytes nodera,
K.,
sensitive
and of
, N., of
simian
(1970)
, E.
A
(1973)
agglutinin
level
mutant T.K.
Katchalski
soybean
Yamaguchi
glycoproteins
ay,
&
by
Transformation
. Proc.
nat.
Kuchino,
T.
&
sialyltransferase virus
modified
Acad. Aoi,
of
40 .
Proc.
method
Y.
cells
nat.
for
neuraminidase
(Wash.)
(1976)
70
Sci.
isolation
of
treated
, 2515
Alterations
transformed
Acad.
the
of Sci.
by
(Wash.) the
in a
73
2518.17) O surface
temperature
, 4090-4094.18) R membrane
plasma
f rom
rat 19)
liver.
Biochim.
Salzberg,
S.
biophys.
Raskas,
J.
Acta
(1972)
(Amst Surface
.),
196,
1-9.
changes
of
human
cells
productively
inf ected
with 20)
21)
22)
human
adenoviruses.
Sanford, E.H. b y neuraminidase. Springer,
alteration
631-637. tumor
histocompatibility
antigen
ind uced
&
(Wash.)
Takemori,
N.,
M
and
36, N.,
adenovirus. ‡U.
5,
Ansell, N
44, Riggs,
1.
Takemori,
An
, 48, in
Transplantation,
G.F.
rology, 23)
(1967)
agglutinogen A cad. Sci.
adenovirus.
Virology
by
N .J. influenza
1273-1279.
(1958) virus
Inactivation and
receptor
of destroying
human
erythrocyte
enzyme
.
Proc.
nat.
182-189. J.L.
Isolation
&
Aldrich
of
, C.D.
(1968) (cyt)
cytocydal
Genetic mutants
studies with of adenovir
tumorigenic us
type
12. Vi
575-586. Riggs,
J.L.
&
Aldrich
Heterogeneity
of
, C.D. (1969) cyt mutants
of
Genetic adenovirus
studies
with type
tumorigenic 12 .
Virology, 38
, 8-15. 24)
Tooze, S pring
J.
(1973)
Harbor
The Laboratory,
Molecular New
Biology York.
of
Tumor
Viruses
. edited
by
J.
Tooze,
Cold
Surface
25)
Glycoproteins
after
Adenovirus
Infection
183
Vaheri, A., Rouslahti, E. & Nordling, S. (1972) Neuraminidase stimulates division and sugar uptake in density inhibited cell cultures. Nature New Biol., 238, 211-212. 26) Williams, G.F., & Ustacelebi, S. (1971) Complementation and recombination with temperature sensitive mutants of adenovirus type 5. J. gen. Virol., 13, 345 - 348. 27) Yamamoto, H., Shimojo, H. & Hamada, C. (1972) Less tumorigenic (cyt) mutants of adenovirus 12 defective in induction of cell surface change. Virology, 50, 743-752.
J. exp.
Med.,
Alterations
1978,
in
125, 177-183
Surface
Sialyltransferase Kidney
Glycoproteins
Activity
Cells
Infected
Adenovirus
Type
and
of Human
with
Level
of
Embryo
Oncogenic
12
YOSHITAKA AOI and
MASANORI YOKOTA
The Institute of Medical Science, the University of Tokyo, Tokyo 108
AOI,
Y.
and
YOKOTA,
Sialyltransferase
Activity
Adenovirus
Type
embryo
kidney
mutants at
the
(Cyt) early
mutants in
the
12.
Tohoku
(HEK)
cells
synthesized stage
of
synthesized
between
type the oncogenic
Alterations
Human J.
exp.
of 12.
It
sialyltransferase adenovirus
Med.,
1978,
was
that in 12;
of
microsomes
microsomal
Human
and on
its
the
cytocidal
cell
surface
tumorigenic
sialyltransferase were a
of
Oncogenic
177-183 12
cells was
Level
with
a highly
when there
the
and
level
increased
confirmed
type
less
The
activity
(2), type
respectively a
and
Infected
125
glycoproteins Furthermore,
cells was
Glycoproteins Cells
adenovirus
glycoproteins.
HEK
Surface
Kidney
with
different infection.
in
Embryo
infected
different
microsomes
adenovirus
M. of
positive and
sialyltransferase
Cyt activity
infected
with
correlation tumorigenicity.„Ÿ activity;
tumorigenicity
It is generally events leading to 1973). However, of tumor in vivo
accepted that cell surface alterations are the primary biochemical the loss of growth regulation in virus transformed cells (Tooze what surface alterations are directly related to the development still remains to be clarified.
Several approaches have been adopted to find a correlation between the cell surface changes and tumorigenicity (Glick et al. 1973; Glick et al. 1974; Onodera et al. 1976). One of the most important problems may be to isolate the tumor specific transplantation antigen (TSTA) and to elucidate its chemical structure. Human adenovirus type 12 (H12) is highly oncogenic in hamsters. However, most H12 Cyt mutants are less tumorigenic in hamsters and less efficient in trans formation of cells (Takemori et al. 1968; Takemori et al. 1969). These mutants, which do not show the typical cytopathic effect in adenovirus-infected cells, were named on the basis of their cytocydal effect.
The comparison of less tumorigenic H12 Cyt mutants with highly tumorigenic H12 wild type (WT) showed that they differed only in their capacity to induce cell surface changes, such as lectin-agglutinability (Yamamoto et al. 1972; Salzberg and Raskas 1972) and TSTA (Yamamoto et al. 1972). This close cor relation between tumorigenicity and the capacity to induce cell surface changes Received
for publication,
November
1, 1977. 177
178
Y. Aoi and M. Yokota
provides an efficient system which is useful transformation and membrane alteration.
for analysis
of the relationship
between
In the previous paper (Aoi 1977), the author suggested that the level of sialic acid content in the plasma membrane may be under the control of the microsomal sialyltransferase activity. There may be a link between sialic acid content in the plasma membrane and the transformed phenotype in the cell lines. This membrane and
paper describes a comparative study of glycoproteins in the plasma and the sialyltransferase activity in the microsomes of H12WT-infected
Cyt mutant-infected
tumorigenicity
and
cells.
The results
alteration
in the
also suggest
glycoproteins
a close correlation
of the
plasma
between
membrane.
MATERIALS AND METHODS Secondary cultures of human embryo kidney (HEK) cells and H12WT, Cyt 31 and Cyt 66 mutants were used. These mutants were named by N. Takemori according to the proposal made by Ginsberg et al. (1973). Most Cyt mutants are less tumorigenic in hamsters with an exception of Cyt 66, which is highly tumorigenic (Takemori et al. 1968) and not defective in inducing cell surface change (Yamamoto et al. 1972). The procedures for cell culture, virus production and virus assay were described previously (Yamamoto et al. 1972). The plasma membrane and microsomes were prepared according to a modified method of Ray (Ray 1970). The membrane isolation procedure is summarized in Fig. 1. Protein determination was carried out by the method of Lowry (Lowry et al. 1951), using bovine serum albumin as a standard. The activity of sialyltransferase in the microsomes was measured according to a modified method of Grimes (Grimes 1970) . Successful applica tion of this method to the microsomes used in this experiment was reported previously (Aoi 1978). Cells and
were
grown
plated
in
serum.
When
were
infected
after
infection
ƒÊ Ci/ml
above,
method
run
with the
gel.
of
sample
estimation ,000)
(‡U),
indicated
as
gave
slices
0.3ml
of
radioactivity
Fairbanks
10
with for
following
(‡V)
and
(‡W)
in
sample
were
were
was
counted
until
and
were
with
10%
calf
the
cells
Fifteen
labeled 25 ƒÊCi/mmol
hr
with
2.5 ,
radioactive
the R as
sugar the
use
(SDS-PAGE)
was
carried
. Polyacrylamide cytochrome c
gels migrated
(7.5%) to the
(without
molecular
weight
The slices
10ml by
markers
(160
,000)
cytochromec
.
The
added
as
chain
figure
width.
vial
1971)
used
H
(‡V) the
labeled
and
respectively.
cells activity
policeman
al.
mA/gel
globulin
1.0mm each
5
(25,500)
,
the
cells/dish
with
discarded
electrophoresis et
proteins
human
,
rubber
4 •~ 105
of
10min. gel
of
was
PFU/cell
pulse
(Fairbanks
current
peaks:
each
medium
at
of
supplemented
synthesis)
being
g
concentration
specific
After
200 •~
a
(MEM)
31
DNA
harvested at
approximately
in
at
the
Cyt
viral
hr.
were
The
To
and
of
chymotrypsinogen
H2O2.
monolayer,
66
four
constant
(‡T), (‡U),
dishes medium
polyacrylamide of
a
Petri
(D-6-[3H]-glucosamine,
cells
dodecylsulfate
the
of
Cyt
centrifuged
by
a
onset
for
Sodium
bottom
formed
the
the
then
Falcon essential
3H-glucosamine
and
were
mm
H12WT,
(before
Centre)
described
out
cells
with
of
65 minimum
the
adiochemical
trypsin)
on
Eagle's
of
Beckman
gels
were
were
for (I), (11 cut
swollen
the bovine
,700)
serum (‡W).
with
a gel
scintillation
(SL-200)
liquid
were
slicer
overnight
dioxane
albumin (68 They
at fluid
scintillation
which 55•‹
with
and
the
counter
.
RESULTS The Ray yield:
(Ray after
isolation 1970)
procedure except
centrifugation
plasma
membrane
that
of
the
the
following
steps
at
5,000 •~
g
for
10min,
according were the
reformed precipitates
to
the to
method imp
were
of
rove
the
taken
up
Surface
Fig.
1.
Glycoproteins
Isolation
after
procedure
Adenovirus
of the plasma
Infection
179
membrane.
in a small volume of distilled water (1-2ml), and then this suspension was layered on the 37% sucrose solution (w/w). SDS-PAGE patterns of glycoproteins in the plasma membrane newly synthesized by HEK cells are shown in Fig. 2(a). Major components were observed in fraction No. 10 and in fractions Nos. 18-19. Cells infected with H12WT newly synthesized fraction No. 10 glycoprotein, but there was no glycoprotein peak at fractions Nos. 18-19 (Fig. 2(b) ). On the other hand, cells infected with either Cyt 31 or Cyt 66 produced a different pattern from that of mock infected or WTinfected cells. The major peak (fraction No. 10) was not detected in either case (Fig. 2 (c), (d) ). The newly synthesized glycoproteins in the virus infected HEK cells were different in the SDS-PAGE patterns. The microsomal sialyltransferase activity involved in the elongation of the terminal carbohydrate (sialic acid) of glycoproteins in the plasma membrane was examined. Cells infected with H12WT and Cyt mutants were harvested at 19 hr after infection and microsomes were prepared. The transfer of CMP-[14C]-sialicacid to desialized fetuin was measured (Table 1). The activity of sialyltransferase increased by 230% in WT-infected HEK cells than that of uninfected cells. The level of sialyltransferase in Cyt 66 (highly tumorigenic) infected cells was 1.23 times higher than that of Cyt 31 (less tumori genic) infected cells.
180
Fig.
Y. Aoi and M. Yokota
2. SDS-PAGE membrane.
patterns
(a) mock-infected cells. 66-infected cells.
of
3H-glucosamine
(b) H12WT-infected
labeled
cells.
glycoproteins
in the
(c) Cyt 31-infected cells.
plasma
(d) Cyt
DISCUSSION
Concerning the molecular defects of Cyt 31 (less tumorigenic) mutant, two interesting facts have been reported (Yamamoto et al. 1972). First, cells infected with H12WT or Cyt 66 (highly tumorigenic) demonstrated marked lectin agglutinability at 24 hr to 32 hr after infection. In contrast, cells infected with Cyt 31 mutant showed much less marked lectin agglutinability during the infection process. This observation, indicating that Cyt 31 mutant does not induce surface alterations, is in contrast to the marked surface changes induced by H12WT or Cyt 66. Secondly, Cyt 31 does not induce transplantation immunity in hamsters. However, the correlation between the surface alterations induced by infection in vitro and the lack of induction of TSTA in vivo still remains to be clarified.
Surface
Glycoproteins
after
Adenovirus
Infection
181
TABLE 1. Level of sialyltransferase activity in the microsomalfraction after infection of human adenovirus 12 and Gyt mutants
*
Results and
are as
expressed
cpm/mg
The study in this paper for the phenomena described
as
of
the
microsomal
mean•}s
.E.
of
3 to
5 determinations,
protein.
was performed to find above. It was found
out biochemical that the newly
mechanisms synthesized
glycoproteins characteristic the
level
and the level of sialyltransferase activity in the microsomes to the cells infected with each mutant . It is worthy of note of sialyltransferase activity was raised by oncogenic virus infection
that the degree of activation was different depending on the These results seemed to indicate that the level of sialyltransferase microsomes
is related
with
the
were that and
infected mutants. activity in the
tumorigenicity.
Sialic acid content in the plasma membrane has been reported to correspond well with the microsomal sialyltransferase activity (Aoi 1978). Sialic acid in the cell surface glycoproteins has been reported to be involved in the determination of MN antigenicity (Springer and Ansell 1958) and tumor antigen (Sanford 1967; Currie and Bagshawe 1968; Simons et al. 1971; Bekesi et al. 1971). Moreover, the presence or absence of sialic acid on the surface has a considerable effect on the fluidity of the plasma membrane, e.g., Con A agglutinability (Novogradsky and Katchalski 1973; Vaheri et al. 1972; Kemp 1968). In this context, it is considered that sialic acid content in the plasma membrane of HEK cells seemed to be raised by infection of those viruses and altered surface glycoproteins. On the other hand, it has been known that adenovirus types 1, 2 and 5, although classified as non-oncogenic, do transform rodent cells in vitro (Freeman et al. 1967; McAllister et al. 1969; Williams and Ustacebeli 1971) and transformed rat cells are non-tumorigenic (Freeman et al. 1967) unless the animals are im munosuppressed (Gallimore 1972). Whether cells transformed by non-oncogenic adenovirus are related to the level of sialyltransferase activity in the microsomes, is not explored at this moment. Research along this problem is now in progress. Acknowledgment
We would like to express thanks to Prof. H. Shimojo of this encouragement and the supply of experimental materials.
Institute
for his
References
1)
Aoi, Y. (1978) Biosynthesis of glycoprotein-glycosyl transferases during the cell cyc le. Tohoku J. exp. Med., 124, 139-144. 2) Aoi, Y. (1978) Sialic acid content and growth control of mouse cells transformed
182
Y. Aoi and M. Yokota by
3)
a
temperature-sensitive
Bekesi,
J.G.,
mutant
St-Arneault,
munogenicity
by
G.
vibrio
of
SV40.
& Holland,
cholerae
FEBS
J.F.
letters,
(1971)
in
Increase
neuraminidase
press.
in
treatment.
leukemia
Cancer
L Res.,
1210 31,
im 2130
- 2132. 4)
Currie,
G.A.
&
munogenicity Cancer, 5)
Bagshawe, of
22,
the
K.D.
(1968)
Landuschutz
The
ascites
effect
tumor;
of
Site
neuraminidase
and
on
mode
of
the
action
.
im
Brit.
J.
588-594.
Fairbanks,
G.,
the
polypeptides
major
Stock,
T.L. of
&
Wallach,
the
human
D.H.F.
(1971)
Electrophoretic
erythrocyte
analysis
membranes.
of
Biochemistry
,
10,
2606-2617. 6)
Freeman,
A.E.,
Huebner, type 7)
8)
Black,
R.J. 2.
Proc. P.H.
transformed
in
9)
Acad.
H.S.,
M.C.,
which
coincide
by
(Wash.),
2.
J.
Doerfler,
&
Sachs
Austin, by
cells
immunosuppressed gen.
Virol.,
W.H.
&
J. ,
P.H.,
J.R. adenovirus
&
1205-1212.
adenoviruses.
Z., virus
Henry, rat embyro
in
type
J.F., of
Rabinovitz, with
58,
production
adenovirus
mutants
, E.A., primary
of
Tumor
Williams, for
Glick,
Sci.
(1972) vitro
nomenclature
Vanderpool
Transformation
nat.
Gallimore,
Ginsberg,
B.H.,
(1967)
L.
with
cells
.
H.
12,
(1973)
Proposed
663-664.
Surface
and
99-102
Shimojo,
Virol.,
(1974)
transformation
rats
16,
membrane
tumorigenesis.
glycopeptides
J.
Virol
.,
13,
967
- 974. 10)
Glick,
M.C.,
Rabinovitz,
correlated 11)
with
Grimes,
G.W.
(1970)
transformed 12)
tion 13)
Sachs,
R.B.
Sialic
acid
E.J.,
Lowry,
218,
O.H.,
McAllister, (1969) T
the
R.M.,
48641869.
and
sialic
acid
glycopeptides
levels
in
normal
and
.
Jourdian
of the
membrane
, G.W. glycoproteins
removal
of
cell
&
Roseman,
.
J.
biol.
surface
S.
(1966)
Incorpora
Chem.,
241,
5735-5737.
sialic
acid
on
cell
aggregation
1255-1256.
Rosebrough with
Surface
12,
5083-5092
into
Effect
Nature,
(1973)
transferases 9,
McGuire,
(1968)
vitro.
L.
Biochemistry,
N-acetyl-n-glucosamine
measurement 15)
&
Biochemistry,
I.R.,
of
Kemp, in
14)
cells.
Johnston,
Z .
tumorigenesis.
, folin
Nicolson,
ransformation
N.J., phenol
M .D., of
rat
Farr,
A.L.
reagent.
J.
Lewise,
embyro
A.M., cells
&
Randall,
biol Jr.,
by
R.J.
. Chem.,
193,
MacPherson,
adenovirus
type
(1951)
Protein
265-275. I.
&
Huebner
1 .
J.
gen.
, R.J. Viol.,
4,
29-36. 16)
Novogrodsky,
A.
lymphocytes nodera,
K.,
sensitive
and of
, N., of
simian
(1970)
, E.
A
(1973)
agglutinin
level
mutant T.K.
Katchalski
soybean
Yamaguchi
glycoproteins
ay,
&
by
Transformation
. Proc.
nat.
Kuchino,
T.
&
sialyltransferase virus
modified
Acad. Aoi,
of
40 .
Proc.
method
Y.
cells
nat.
for
neuraminidase
(Wash.)
(1976)
70
Sci.
isolation
of
treated
, 2515
Alterations
transformed
Acad.
the
of Sci.
by
(Wash.) the
in a
73
2518.17) O surface
temperature
, 4090-4094.18) R membrane
plasma
f rom
rat 19)
liver.
Biochim.
Salzberg,
S.
biophys.
Raskas,
J.
Acta
(1972)
(Amst Surface
.),
196,
1-9.
changes
of
human
cells
productively
inf ected
with 20)
21)
22)
human
adenoviruses.
Sanford, E.H. b y neuraminidase. Springer,
alteration
631-637. tumor
histocompatibility
antigen
ind uced
&
(Wash.)
Takemori,
N.,
M
and
36, N.,
adenovirus. ‡U.
5,
Ansell, N
44, Riggs,
1.
Takemori,
An
, 48, in
Transplantation,
G.F.
rology, 23)
(1967)
agglutinogen A cad. Sci.
adenovirus.
Virology
by
N .J. influenza
1273-1279.
(1958) virus
Inactivation and
receptor
of destroying
human
erythrocyte
enzyme
.
Proc.
nat.
182-189. J.L.
Isolation
&
Aldrich
of
, C.D.
(1968) (cyt)
cytocydal
Genetic mutants
studies with of adenovir
tumorigenic us
type
12. Vi
575-586. Riggs,
J.L.
&
Aldrich
Heterogeneity
of
, C.D. (1969) cyt mutants
of
Genetic adenovirus
studies
with type
tumorigenic 12 .
Virology, 38
, 8-15. 24)
Tooze, S pring
J.
(1973)
Harbor
The Laboratory,
Molecular New
Biology York.
of
Tumor
Viruses
. edited
by
J.
Tooze,
Cold
Surface
25)
Glycoproteins
after
Adenovirus
Infection
183
Vaheri, A., Rouslahti, E. & Nordling, S. (1972) Neuraminidase stimulates division and sugar uptake in density inhibited cell cultures. Nature New Biol., 238, 211-212. 26) Williams, G.F., & Ustacelebi, S. (1971) Complementation and recombination with temperature sensitive mutants of adenovirus type 5. J. gen. Virol., 13, 345 - 348. 27) Yamamoto, H., Shimojo, H. & Hamada, C. (1972) Less tumorigenic (cyt) mutants of adenovirus 12 defective in induction of cell surface change. Virology, 50, 743-752.
