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Aggregation of human lymphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocidin B
Vol. 95, No. 2, 1980
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1980
Pages 842-848
AGGREGATION OF HUMAN LYMPHOBLASTOID CELLS BY TUMOR-PROMOTING PHORBOL ESTERS AND DIHYDROTELEOCIDIN Hiro-o National
Hoshino,
Cancer
Received
Masanao
Center
Miwa,
Research
Hirota
Institute,
B
Fujiki
and Takashi
Tsukiji,
Chuo-ku,
Sugimura Tokyo
104 (Japan)
June 17,198O
SUMMARY: Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation In amounts of a few ng, they induced significant aggregation. for 6 hours. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors. INTRODUCTION Tumor-promoting induce
tumors
The tumor
in animals
promoters
--in vitro (4)
agents
phorbol cells
esters
actions
through
their
interactions
phorbol
esters
induce
aggregation
manner.
This
the assay
system
compounds
with
effects,
not
on lymphoid
cells
but
with
also
rapidly
the cell
described
is easy in
tumor-promoting
this
membrane
paper
cells
dihydroteleocidin
only
B
on epidermal
(3,9-10). to be mediated
(3,11,12).
We found
that
B and teleocidin, rapidly
to be measured should
and
Tumor-promoting
and thought
dihydroteleocidin
alkaloids,
aggregation
(5).
can
of carcinogens.
--in vivo
that
to have various
of human lymphoblastoid
cell
recently promoter
are expressed
and indole
amounts
tumor
(3,6-g),
Some of their
to limited
but they
most extensively
We found
potent
reported
and fibroblasts
exposed
(l-3).
occurring
are
noncarcinogenic,
have been studied
esters
was a naturally
themselves
previously
that
are phorbol
are
in a dose-dependent quantitatively
be useful
and so
in screening
activity.
: EB, Epstein-Barr : ODC, ornithine decarboxylase, : PBS, phosAbbreviations phate buffered saline (135mM NaCl, 2.7mM KCl, 5.3mM NanHPO+, and 1.45mM KHzPOt, 13-didecanoate ; ) ; PDD, phorbol-12, 13-didecanoate ; 4a-PDD, 4a-phorbol-12, TPA, 12-Q-tetradecanoyl-phorbol-13-acetate ; 4-Q-Me-TPA, 4-g-methyl-TPA. 0006-291X/80/140842-07$01.00/0 Copyright 0 1980 by Academic Press, Inc. Al[ rights of reproduction in any form reserved.
842
Vol.
95, No.
2, 1980
BIOCHEMICAL
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
: NL-3 and XPL-17 cells are human lymphoblastoid cell lines trans--Cells formed by EB virus. They were established from peripheral lymphocytes of a normal adult and a patient with xeroderma pigmentosum, respectively, and were kindly supplied by Dr. A. Oikawa of Tohoku University (13). They were grown in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum in a humidified incubator at 37°C under 5% COZ. In order to decrease spontaneous aggregation, cells were seeded at a concentration of 3-4 x 105/ml and passaged every other day. Chemicals : Phorbol, TPA, 4-0-Me-TPA, PDD and 4a-PDD were purchased from Consolidated Midland Corporation,Brewster, New York. They were dissolved in distilled water containing 30% acetone at a concentration of lmg/ml and then diluted with PBS to 100 pg/ml. Dihydroteleocidin B and teleocidin (4) were gifts from Dr. M. Takashima, Fujisawa Pharmaceutical Industries, Ltd., Osaka, Japan. Dihydroteleocidin B is a hydrogenated compound of teleocidin, which is isolated from Streptomyces. Dihydroteleocidin B and teleocidin were dissolved in acetone at a concentration of 1 mg/ml and diluted with PBS to 10 ug/ ml. Hydrolysed dihydroteleocidin B was prepared by heating dihydroteleocidin B in 6 N HCl containing 4% thioglycolic acid at 110°C for 20 hours. Under these conditions, the amide bond of the lactam ring was completely hydrolysed. Croton oil was obtained from Sigma Chemical Co., St. Louis, Mo. All chemicals were stored at -20°C and further diluted with PBS just before use. Estimation of cell aggregation : Lymphoblastoid cells grew in suspension and formed aggregates of loosely associated cells (13). Since these aggregates were readily dispersed, a suspension consisting almost entirely of single cells could be obtained by pipetting the culture. Cells were collected by centrifugation at 350 x 2 for 5 minutes and resuspended by pipetting at a concentration of 7-9 x 10 single cells/ml in fresh medium containing 2 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid buffer (pH 7.2). Samples of 1 ml of cell suspension were seeded onto Falcon tissue culture plates No. 3001 (35 x 10 mm) containing 0.1 ml PBS with various amounts of chemicals. The cells were cultivated for 6 hours in a humidified incubator at 37°C under 5% CO>. The degree of cell aggregation was estimated by counting the numbers of single cells in the dishes ; for this, the dishes were rocked back and forth several times to obtain a homogeneous cell suspension and then aliquots were removed and the numbers of single cells in them were determined in a hemocytometer. Duplicate dishes were used for each concentration of each compounds. EDs0 was taken as the concentration of the test compound that decreased the number of single cells in the control culture by 50%.
RESULTS Time
course
of cell
aggregation
TPA and dihydroteleocidin remarkable
aggregation
The aggregates dissociated
formed by pipetting
formed
in the
in the
presence
control
B at a concentration
of NL-3 cells in the culture and were culture.
of TPA (100
after
The time rig/ml),
cultivation
containing larger
phorbol
843
of 100 rig/ml for
them could
and more spherical course
of aggregation
(1 ug/ml)
induced
6 hours not
(Fig.
readily
than
1). be
those
was examined
dihydroteleocidin
B
Vol.
95, No.
2, 1980
6lOCHEMlCAL
AND
BIOPHYSKAL
RESEARCH
COMMUNICATIONS
Fig. 1. Aggregation of NL-3 cells after incubation for 6 hours in the presence of (A) PBS, (B) TPA (lOOng/ml),and (C) dihydroteleocidin B (100 rig/ml) (magnification, x 28). (100
rig/ml),
and teleocidin
teleocidin after
B, and teleocidin
seeding
80-100%
and after
of the cell
significant
but
XPL-17
cell
cells
examined treatment
line
showed
whether with
(WC-2),
leukemia
Quantification NL-3 cells dihydroteleocidin
The effects
aggregation
were
most of the cells
single
2).
in the
Teleocidin
activity
a similar
more aggregates
and the cell
AKR lymphoma Moloney
(Fig.
2).
control
apparent
within
formed
aggregates,
culture.
Phorbol
is not yet
of teleocidin
of TPA, dihydro-
proven
is
2 hours whereas had no
to be a tumor
similar
to that
of aggregation
in the
of
B (4,5).
of TPA and formed
after
6 hours,
ODC inducing
dihydroteleocidin
ng/ml)(Fig.
on cell
remained
effects
promoter,
We also
(100
virus
other
course
spontaneously cell
lines
lines
of murine
radiation-induced (YAC).
seeded
origin,
cell
of tumor
The lines
e.g.,
lines
L1210,
(RLal) were
cell
presence
(data
not shown).
aggregation
tested
were
the Raji
plasmacytoma
and lymphoma
(X5563),
induced
by
not aggregated
remarkably.
concentrations
of TPA,
promoters
in the presence
B, and teleocidin
NL-3 cells
increased
B.
lymphoma
These
than
showed
TPA and dihydroteleocidin
of the effects were
time
and the
844
of various numbers
of single
cells
were
counted
Vol.
95, No.
BIOCHEMICAL
2, 1980
! b-
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PBS
PhOl-bOl
\
10
A
1
w
after
Time
addition
( h )
Concentration(ng/ml)
w
Fig. 2. Time course of cell aggregation. NL-3 cells were seeded into aishesin the presence of (A) TPA (100 rig/ml)(O), phorbol (1 pg/ml)(A), or PBS alone (0) and (B) dihydroteleocidin B (100 rig/ml)(m), teleocidin (100 rig/ml)(A), or PBS alone (0). At various times, numbers of single cells were counted. Ranges of values are shown by bars. Dose response of cell aggregation. NL-3 cells were seeded into Fig. 3. dishescontaining indicated concentrations of TPA (O), dihydroteleocidin B (m), and teleocidin (A). After 6 hours, numbers of single cells were counted. The arrows indicate EDSo's. Ranges of values are shown by bars.
after
6 hours
cidin
B,
of
viable
to
the
were
(Fig.
and cells
as
The
EDso
values
the
same
way
rig/ml
, respectively. even
could
not
less
effective
cells PDD, 1).
more
than
alkaloids
The
or
by
EDs0
The 10
of
than
induce
ODC in
inducing
of
cells
values
for
of
11.2
and
croton
EDs0
of
to
induce
Hydrolysed
mouse
skin
aggregation
845
and
1 rig/ml.
3.9
oil has
not
cell
adhere
teleocidin
well
two
than
unhydrolysed
with
6.5
(Table and
were
3.1
similar B,
three
the
abilities
determined were
to
in
Then
dihydroteleocidin was
respectively.
tumor-promoting
aggregation
(5),
experiments
determined
(l-3,10). were
numbers
aggregation
rig/ml, were
no
cells
cell
The
did
correlated
vivo
NL-3
B and
as
dihydroteleo-
independent
values --in
TPA,
several
which
compounds
TPA. in
EDsa
values
These
these
of
3,
phorbol,
abilities
cell
most
TPA,
aggregation
activity
and
4cr-PDD,
ug/ml.
that
low
With
dihydroteleocidin
Their
greater
ED 5o
EDso
induce
as
Fig.
arrows.
of
with
decrease
From
4-0-Me-TPA, -
to
values
not
gave
activities
2).
to
indicated
(Table
was
indole
shown).
detectable
a concentration did
not
of
was
at
culture
XPL-17
tumor-promoting of
the
(data
and
activity,
even
in
dishes
NL-3
Aggregation
teleocidin
obtained
using
3).
hundred
dihydroteleocidin
which times B
Vol.
95, No.
2, 1980
Table
BIOCHEMICAL
1.
Quantification
of
inducing
AND
BIOPHYSICAL
activities
aggregation
of of
EDSO
NL-3
RESEARCH
TPA and and
related
XPL-17
compounds
TPA
11.2
f
(w/ml)
Tumor-promoting activity
PDD
50
*
4-0-Me-TPA -
8,500
XPL-17 6.7a
(6)b
23
(2)
jz 4,600
(2)
a : Mean EDs0 f SEM b : Number of independent
*
3.8
(6)
+
(3)
f
(3)
-
(2)
-
(2)
+
*
'10,000 49
+
4,200
Phorbol
f.
(11)
f
(2)
69
0.4
2,700
'10,000
oil
15
*
(2)
4cl-PDD
Croton
3.9
290 1,900
>10,000
(2)
67
in
cells
Compound NL-3
COMMUNICATIONS
f
11
experiments
DISCUSSION In
cells,
this
work
esters
that
found
NL-3 and XPL-17,
phorbol
esters
effects
a good correlation
rapidly
alkaloids,
of phorbol
respective
EB virus-transformed
aggregated
and indole
The abilities their
we
Table
2.
and
related
of in
cells
tumors
between
Quantification
in the presence
dihydroteleocidin
in promoting
compounds
and firmly
to aggregate
was observed
human lymphoblastoid
the
activities
inducing
B and teleocidin. correlated
in mouse skin abilities
of
well (Table
of
NL-3
with 1).
of indole
Also
alkaloids
dihydroteleocidin
aggregation
of
B cells -
Compound
Eb0
Dihydroteleocidin
B
dihydroteleocidin
HCl/dihydroteleocidin
Bc Bd
)
6.5
k
2.3a
(5)b
+
3.1
f
0.1
(5)
t
(2)
-
(2)
t
Teleocidin Hydrolysed
(w/ml
ODC induction
460
rt 1.7
a and b : as in Table 1 c : After hydrolysis in 6N HCl containing for 20 hours, the mixture was dried d : After6N HCl containing 4% thioglycolic teleocidin B was added.
846
rt
140 0.8
4% thioglycolic up. acid was
acid dried
up,
at
-
110°C
dihydro-
Vol.
95,
No.
2, 1980
to aggregate
BIOCHEMICAL
cells
was hydrolysed, (5)
it
and also
cells
by pipetting
increased
the intercellular
structure
of lymphoid
promoters
and induce
leukemia
cells
tells(4).
Thus the
non-phorbol simple
ineffective
in inducing
aggregation
with
tumor
and rapid.
within
formed
dihydroteleocidin
2 hours
in the
cell
cells. ODC activity
and inhibition system
promoters
that
in mouse skin of lymphoblastoid
Unlike could
developed
promoters the membrane potent
differentiation work
be
tumor
of human promyelocytic
would
activities.
be used as a screening
readily
B are
adhesion
in this
had TPA-like
not
by changing
of terminal
spontaneous
tumor
TPA and dihydroteleocidin in mouse skin,
assay
2).
Thus these possibly
B
B, and teleocidin,
culture
suspension.
Both
could
(Fig.
treated
adhesiveness,
So it
of tumor-promoting
TPA,
COMMUNICATIONS
when dihydroteleocidin
ODC activity
the
(HL-60),
is,
in inducing
those
dissociated
RESEARCH
That
ODC activity.
was detectable
of cells,
BIOPHYSICAL
ineffective
On treatment
2).
aggregation
aggregates
became
became quite
(Table
cell
and to induce
AND
of Friend detect
This test
for
system certain
some of is types
agents.
ACKNOWLEDGEMENTS This from Health
work
was supported
the Ministry and Welfare,
of Education,
in part
by Grants-in-Aid
Science
and Culture,
for
Cancer
Research
and the Ministry
of
Japan.
REFERENCES 1.
R. (1974) Prog. Chem. Org. Nat. Prod., 31, Hecker, E. , and Schmidt, 377-566 2. Slaga, T. J., Sivak, A., and Boutwell, P. K. (Editors) Carcinogenesis ___ A comprehensive survey Vol. 2. (1978) Raven Press, New York. Sivak, A. (1979) Biochim, Biophys. Acta., 560, 67-89. 43: Fujiki, H., Mori, M., Nakayasu, M., Terada, M., and Sugimura, T. (1979) Biochem. Biophys. Res. Comnun., 90 , 976-983. 5. Fujiki, H., Mori, M., Nakayasu, M., Terada, M., Sugimura, T., and Moore, R. E. (manuscript in preparation). 6. Weinstein, I. B., Wigler, M. and Pietropaolo, C. (1977) Origins of Human Cancer, pp. 751-772, Cold Spring Harbor Laboratory 7. Wigler, M., and Weinstein, I. B. (1976) Nature, 259, 232-233. 8. Yuspa, S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J., Colburn, N., and Kelsey, W. (1976) Nature, 262, 402-404. 9. Mastro, A. M., and Meuller, G. C. (1974) Exp. Cell Res., 88, 40-46.
847
Vol.
95, No.
10. 11. 12. 13.
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
zur Hausen, H., Bornkamm, G. W., Schmidt, R., and Hecker, E. (1979) Proc. Natl. Acad. Sci. USA., 76, 782-785. I. B. (1978) Science 202, 313-315. Lee, L-S., and Weinstein, Wenner, C. E., Hackney, J., Kernelberg, H. K., and Mayhew, E. (1974) Cancer Res., 34, 1731-1737. Tohda, H., Oikawa, A., Katsuki, T., Hinuma, Y., and Seiji, M. (1978) Cancer Res., 38, 253-256.
848
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1980
Pages 842-848
AGGREGATION OF HUMAN LYMPHOBLASTOID CELLS BY TUMOR-PROMOTING PHORBOL ESTERS AND DIHYDROTELEOCIDIN Hiro-o National
Hoshino,
Cancer
Received
Masanao
Center
Miwa,
Research
Hirota
Institute,
B
Fujiki
and Takashi
Tsukiji,
Chuo-ku,
Sugimura Tokyo
104 (Japan)
June 17,198O
SUMMARY: Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation In amounts of a few ng, they induced significant aggregation. for 6 hours. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors. INTRODUCTION Tumor-promoting induce
tumors
The tumor
in animals
promoters
--in vitro (4)
agents
phorbol cells
esters
actions
through
their
interactions
phorbol
esters
induce
aggregation
manner.
This
the assay
system
compounds
with
effects,
not
on lymphoid
cells
but
with
also
rapidly
the cell
described
is easy in
tumor-promoting
this
membrane
paper
cells
dihydroteleocidin
only
B
on epidermal
(3,9-10). to be mediated
(3,11,12).
We found
that
B and teleocidin, rapidly
to be measured should
and
Tumor-promoting
and thought
dihydroteleocidin
alkaloids,
aggregation
(5).
can
of carcinogens.
--in vivo
that
to have various
of human lymphoblastoid
cell
recently promoter
are expressed
and indole
amounts
tumor
(3,6-g),
Some of their
to limited
but they
most extensively
We found
potent
reported
and fibroblasts
exposed
(l-3).
occurring
are
noncarcinogenic,
have been studied
esters
was a naturally
themselves
previously
that
are phorbol
are
in a dose-dependent quantitatively
be useful
and so
in screening
activity.
: EB, Epstein-Barr : ODC, ornithine decarboxylase, : PBS, phosAbbreviations phate buffered saline (135mM NaCl, 2.7mM KCl, 5.3mM NanHPO+, and 1.45mM KHzPOt, 13-didecanoate ; ) ; PDD, phorbol-12, 13-didecanoate ; 4a-PDD, 4a-phorbol-12, TPA, 12-Q-tetradecanoyl-phorbol-13-acetate ; 4-Q-Me-TPA, 4-g-methyl-TPA. 0006-291X/80/140842-07$01.00/0 Copyright 0 1980 by Academic Press, Inc. Al[ rights of reproduction in any form reserved.
842
Vol.
95, No.
2, 1980
BIOCHEMICAL
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
: NL-3 and XPL-17 cells are human lymphoblastoid cell lines trans--Cells formed by EB virus. They were established from peripheral lymphocytes of a normal adult and a patient with xeroderma pigmentosum, respectively, and were kindly supplied by Dr. A. Oikawa of Tohoku University (13). They were grown in RPM1 1640 medium supplemented with 10% heat-inactivated fetal calf serum in a humidified incubator at 37°C under 5% COZ. In order to decrease spontaneous aggregation, cells were seeded at a concentration of 3-4 x 105/ml and passaged every other day. Chemicals : Phorbol, TPA, 4-0-Me-TPA, PDD and 4a-PDD were purchased from Consolidated Midland Corporation,Brewster, New York. They were dissolved in distilled water containing 30% acetone at a concentration of lmg/ml and then diluted with PBS to 100 pg/ml. Dihydroteleocidin B and teleocidin (4) were gifts from Dr. M. Takashima, Fujisawa Pharmaceutical Industries, Ltd., Osaka, Japan. Dihydroteleocidin B is a hydrogenated compound of teleocidin, which is isolated from Streptomyces. Dihydroteleocidin B and teleocidin were dissolved in acetone at a concentration of 1 mg/ml and diluted with PBS to 10 ug/ ml. Hydrolysed dihydroteleocidin B was prepared by heating dihydroteleocidin B in 6 N HCl containing 4% thioglycolic acid at 110°C for 20 hours. Under these conditions, the amide bond of the lactam ring was completely hydrolysed. Croton oil was obtained from Sigma Chemical Co., St. Louis, Mo. All chemicals were stored at -20°C and further diluted with PBS just before use. Estimation of cell aggregation : Lymphoblastoid cells grew in suspension and formed aggregates of loosely associated cells (13). Since these aggregates were readily dispersed, a suspension consisting almost entirely of single cells could be obtained by pipetting the culture. Cells were collected by centrifugation at 350 x 2 for 5 minutes and resuspended by pipetting at a concentration of 7-9 x 10 single cells/ml in fresh medium containing 2 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid buffer (pH 7.2). Samples of 1 ml of cell suspension were seeded onto Falcon tissue culture plates No. 3001 (35 x 10 mm) containing 0.1 ml PBS with various amounts of chemicals. The cells were cultivated for 6 hours in a humidified incubator at 37°C under 5% CO>. The degree of cell aggregation was estimated by counting the numbers of single cells in the dishes ; for this, the dishes were rocked back and forth several times to obtain a homogeneous cell suspension and then aliquots were removed and the numbers of single cells in them were determined in a hemocytometer. Duplicate dishes were used for each concentration of each compounds. EDs0 was taken as the concentration of the test compound that decreased the number of single cells in the control culture by 50%.
RESULTS Time
course
of cell
aggregation
TPA and dihydroteleocidin remarkable
aggregation
The aggregates dissociated
formed by pipetting
formed
in the
in the
presence
control
B at a concentration
of NL-3 cells in the culture and were culture.
of TPA (100
after
The time rig/ml),
cultivation
containing larger
phorbol
843
of 100 rig/ml for
them could
and more spherical course
of aggregation
(1 ug/ml)
induced
6 hours not
(Fig.
readily
than
1). be
those
was examined
dihydroteleocidin
B
Vol.
95, No.
2, 1980
6lOCHEMlCAL
AND
BIOPHYSKAL
RESEARCH
COMMUNICATIONS
Fig. 1. Aggregation of NL-3 cells after incubation for 6 hours in the presence of (A) PBS, (B) TPA (lOOng/ml),and (C) dihydroteleocidin B (100 rig/ml) (magnification, x 28). (100
rig/ml),
and teleocidin
teleocidin after
B, and teleocidin
seeding
80-100%
and after
of the cell
significant
but
XPL-17
cell
cells
examined treatment
line
showed
whether with
(WC-2),
leukemia
Quantification NL-3 cells dihydroteleocidin
The effects
aggregation
were
most of the cells
single
2).
in the
Teleocidin
activity
a similar
more aggregates
and the cell
AKR lymphoma Moloney
(Fig.
2).
control
apparent
within
formed
aggregates,
culture.
Phorbol
is not yet
of teleocidin
of TPA, dihydro-
proven
is
2 hours whereas had no
to be a tumor
similar
to that
of aggregation
in the
of
B (4,5).
of TPA and formed
after
6 hours,
ODC inducing
dihydroteleocidin
ng/ml)(Fig.
on cell
remained
effects
promoter,
We also
(100
virus
other
course
spontaneously cell
lines
lines
of murine
radiation-induced (YAC).
seeded
origin,
cell
of tumor
The lines
e.g.,
lines
L1210,
(RLal) were
cell
presence
(data
not shown).
aggregation
tested
were
the Raji
plasmacytoma
and lymphoma
(X5563),
induced
by
not aggregated
remarkably.
concentrations
of TPA,
promoters
in the presence
B, and teleocidin
NL-3 cells
increased
B.
lymphoma
These
than
showed
TPA and dihydroteleocidin
of the effects were
time
and the
844
of various numbers
of single
cells
were
counted
Vol.
95, No.
BIOCHEMICAL
2, 1980
! b-
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PBS
PhOl-bOl
\
10
A
1
w
after
Time
addition
( h )
Concentration(ng/ml)
w
Fig. 2. Time course of cell aggregation. NL-3 cells were seeded into aishesin the presence of (A) TPA (100 rig/ml)(O), phorbol (1 pg/ml)(A), or PBS alone (0) and (B) dihydroteleocidin B (100 rig/ml)(m), teleocidin (100 rig/ml)(A), or PBS alone (0). At various times, numbers of single cells were counted. Ranges of values are shown by bars. Dose response of cell aggregation. NL-3 cells were seeded into Fig. 3. dishescontaining indicated concentrations of TPA (O), dihydroteleocidin B (m), and teleocidin (A). After 6 hours, numbers of single cells were counted. The arrows indicate EDSo's. Ranges of values are shown by bars.
after
6 hours
cidin
B,
of
viable
to
the
were
(Fig.
and cells
as
The
EDso
values
the
same
way
rig/ml
, respectively. even
could
not
less
effective
cells PDD, 1).
more
than
alkaloids
The
or
by
EDs0
The 10
of
than
induce
ODC in
inducing
of
cells
values
for
of
11.2
and
croton
EDs0
of
to
induce
Hydrolysed
mouse
skin
aggregation
845
and
1 rig/ml.
3.9
oil has
not
cell
adhere
teleocidin
well
two
than
unhydrolysed
with
6.5
(Table and
were
3.1
similar B,
three
the
abilities
determined were
to
in
Then
dihydroteleocidin was
respectively.
tumor-promoting
aggregation
(5),
experiments
determined
(l-3,10). were
numbers
aggregation
rig/ml, were
no
cells
cell
The
did
correlated
vivo
NL-3
B and
as
dihydroteleo-
independent
values --in
TPA,
several
which
compounds
TPA. in
EDsa
values
These
these
of
3,
phorbol,
abilities
cell
most
TPA,
aggregation
activity
and
4cr-PDD,
ug/ml.
that
low
With
dihydroteleocidin
Their
greater
ED 5o
EDso
induce
as
Fig.
arrows.
of
with
decrease
From
4-0-Me-TPA, -
to
values
not
gave
activities
2).
to
indicated
(Table
was
indole
shown).
detectable
a concentration did
not
of
was
at
culture
XPL-17
tumor-promoting of
the
(data
and
activity,
even
in
dishes
NL-3
Aggregation
teleocidin
obtained
using
3).
hundred
dihydroteleocidin
which times B
Vol.
95, No.
2, 1980
Table
BIOCHEMICAL
1.
Quantification
of
inducing
AND
BIOPHYSICAL
activities
aggregation
of of
EDSO
NL-3
RESEARCH
TPA and and
related
XPL-17
compounds
TPA
11.2
f
(w/ml)
Tumor-promoting activity
PDD
50
*
4-0-Me-TPA -
8,500
XPL-17 6.7a
(6)b
23
(2)
jz 4,600
(2)
a : Mean EDs0 f SEM b : Number of independent
*
3.8
(6)
+
(3)
f
(3)
-
(2)
-
(2)
+
*
'10,000 49
+
4,200
Phorbol
f.
(11)
f
(2)
69
0.4
2,700
'10,000
oil
15
*
(2)
4cl-PDD
Croton
3.9
290 1,900
>10,000
(2)
67
in
cells
Compound NL-3
COMMUNICATIONS
f
11
experiments
DISCUSSION In
cells,
this
work
esters
that
found
NL-3 and XPL-17,
phorbol
esters
effects
a good correlation
rapidly
alkaloids,
of phorbol
respective
EB virus-transformed
aggregated
and indole
The abilities their
we
Table
2.
and
related
of in
cells
tumors
between
Quantification
in the presence
dihydroteleocidin
in promoting
compounds
and firmly
to aggregate
was observed
human lymphoblastoid
the
activities
inducing
B and teleocidin. correlated
in mouse skin abilities
of
well (Table
of
NL-3
with 1).
of indole
Also
alkaloids
dihydroteleocidin
aggregation
of
B cells -
Compound
Eb0
Dihydroteleocidin
B
dihydroteleocidin
HCl/dihydroteleocidin
Bc Bd
)
6.5
k
2.3a
(5)b
+
3.1
f
0.1
(5)
t
(2)
-
(2)
t
Teleocidin Hydrolysed
(w/ml
ODC induction
460
rt 1.7
a and b : as in Table 1 c : After hydrolysis in 6N HCl containing for 20 hours, the mixture was dried d : After6N HCl containing 4% thioglycolic teleocidin B was added.
846
rt
140 0.8
4% thioglycolic up. acid was
acid dried
up,
at
-
110°C
dihydro-
Vol.
95,
No.
2, 1980
to aggregate
BIOCHEMICAL
cells
was hydrolysed, (5)
it
and also
cells
by pipetting
increased
the intercellular
structure
of lymphoid
promoters
and induce
leukemia
cells
tells(4).
Thus the
non-phorbol simple
ineffective
in inducing
aggregation
with
tumor
and rapid.
within
formed
dihydroteleocidin
2 hours
in the
cell
cells. ODC activity
and inhibition system
promoters
that
in mouse skin of lymphoblastoid
Unlike could
developed
promoters the membrane potent
differentiation work
be
tumor
of human promyelocytic
would
activities.
be used as a screening
readily
B are
adhesion
in this
had TPA-like
not
by changing
of terminal
spontaneous
tumor
TPA and dihydroteleocidin in mouse skin,
assay
2).
Thus these possibly
B
B, and teleocidin,
culture
suspension.
Both
could
(Fig.
treated
adhesiveness,
So it
of tumor-promoting
TPA,
COMMUNICATIONS
when dihydroteleocidin
ODC activity
the
(HL-60),
is,
in inducing
those
dissociated
RESEARCH
That
ODC activity.
was detectable
of cells,
BIOPHYSICAL
ineffective
On treatment
2).
aggregation
aggregates
became
became quite
(Table
cell
and to induce
AND
of Friend detect
This test
for
system certain
some of is types
agents.
ACKNOWLEDGEMENTS This from Health
work
was supported
the Ministry and Welfare,
of Education,
in part
by Grants-in-Aid
Science
and Culture,
for
Cancer
Research
and the Ministry
of
Japan.
REFERENCES 1.
R. (1974) Prog. Chem. Org. Nat. Prod., 31, Hecker, E. , and Schmidt, 377-566 2. Slaga, T. J., Sivak, A., and Boutwell, P. K. (Editors) Carcinogenesis ___ A comprehensive survey Vol. 2. (1978) Raven Press, New York. Sivak, A. (1979) Biochim, Biophys. Acta., 560, 67-89. 43: Fujiki, H., Mori, M., Nakayasu, M., Terada, M., and Sugimura, T. (1979) Biochem. Biophys. Res. Comnun., 90 , 976-983. 5. Fujiki, H., Mori, M., Nakayasu, M., Terada, M., Sugimura, T., and Moore, R. E. (manuscript in preparation). 6. Weinstein, I. B., Wigler, M. and Pietropaolo, C. (1977) Origins of Human Cancer, pp. 751-772, Cold Spring Harbor Laboratory 7. Wigler, M., and Weinstein, I. B. (1976) Nature, 259, 232-233. 8. Yuspa, S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J., Colburn, N., and Kelsey, W. (1976) Nature, 262, 402-404. 9. Mastro, A. M., and Meuller, G. C. (1974) Exp. Cell Res., 88, 40-46.
847
Vol.
95, No.
10. 11. 12. 13.
2, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
zur Hausen, H., Bornkamm, G. W., Schmidt, R., and Hecker, E. (1979) Proc. Natl. Acad. Sci. USA., 76, 782-785. I. B. (1978) Science 202, 313-315. Lee, L-S., and Weinstein, Wenner, C. E., Hackney, J., Kernelberg, H. K., and Mayhew, E. (1974) Cancer Res., 34, 1731-1737. Tohda, H., Oikawa, A., Katsuki, T., Hinuma, Y., and Seiji, M. (1978) Cancer Res., 38, 253-256.
848