Vol. 181, No. 2, 1991 December 16, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 809-817
BIOCHEMICAL
THE MOBULATION OF GROWTH BY MBA, IN PKC 0VKRPROIXJCING BT29 COILIN CANCER CELLS
Paul
M. Choi
and I.
Bernard
Weinstein
Comprehensive Cancer Center and and Department of of Cancer Research, Columbia University, New York, NY 10032
Institute
Medicine,
Received September 27, 1991
To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses We found that although HMBA markedly high levels of PKCBl. inhibited the growth of the control cells, no inhibition was On the other hand the tumor seen with the HT29-PKC7 cells. promoter 12-O-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and These results may be relevant to the use of HMBA in TPA. combination with other agents in the therapy of specific cancers. Q1991Academic Press, Inc.
The polar-planar in
several
(MEL)
cell
cells
embryonal
systems,
(1,2), carcinoma
and MDCK kidney stimulated the
(4),
cancer
murine
embryonal or and groups
cells
of
however,
other
HL-60
in
growth,
Breitman
human
interest
appear
the
found
leukemia
(7).
potential
cells
cells
These
The effects
cell by
growth
(3),
HMBA
have
lines
can In
(3,10,11).
be
HL-60 by
in
HMBA on cell
The growth
enhancement
inhibition
(5,6),
compound
of
variable.
(4).
mouse
findings
use of this
to be quite
growth
erythroleukemia
neuroblastoma
(8,9).
unaffected
differentiation
murine
cells
carcinoma
He found
HMBA induces
including
epithelial
treatment
inhibited
compound
of
either cells,
HMBA,
while
Schroy
et
0006-291X/91$1.50 809
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
181, No. 2, 1991
a1.(12)
BIOCHEMICAL
found
that
the
diffferent
colorectal
stimulation
to
the
precise
mechanism
is
evidence
that
mediating
the
surprising
control
PKCBl
accelerates
the
that
PKC
ability
understand growth
that
of
the
determinants
level of
Although known,
role
MATERIALS
PKC by
HMBA,
the
HT29
the
is
in
not
signal and
expression
that
a phorbol
ester
colon
activation
play
studied
cancer PKCBl status
of
(16).
PKC might
we have
of
by HMBA
differentiation
overexpress
effects
in
induced
by
and
there
This
increased
role
stably
growth
differentiation
induce
the
of
particularly
differentiation
to
from
not
a role,
MEL cells
of HMBA to
was engineered
important
HMBA is
of
growth
no effect.
include
regulation
of
the
ranged
a central
that
on a derivative
demonstrate
play
plays
In
down
HMBA on
to of
RESEARCH COMMUNICATIONS
response.
the
modulation
effects
cells
differentiation
To better the
cancer
PKC may
(13).
and
(14,15),
of
action
pathways
growth
effects
inhibition, of
since
transduction
blocks
growth
AND BIOPHYSICAL
its
cell (17). of
in
line We
PKC are
HMBA on growth.
ANDMETHODS
The properties of the HT29-PKC7 cell line that expresses about a 15-fold increase in PKCBl, and a vector control cell line HT29-Cl, have been previously described HT29-Cl and HT29-PKC7 cells were grown as previously (17). described (17). Prior to analyses, the cells were grown in McCoy's 5A medium plus 10% fetal bovine serum for 48 hours to reach the exponential growth phase. Then, fresh growth medium containing 0.02% DMSO (solvent control), 5mM HMBA, 10 nM TPA, or medium containing 5mM HMBA plus 10nM TPA were added. The doses were selected for maximal biological effects (12,17,18 and unpublished observation). Replicate plates of cells were then counted (using a Coulter counter) as previously described (17). Only at 3-day intervals, exponential phase cultures could be analyzed since cells treated with the combination of HMBA and TPA started to come off the plate during the plateau phase of growth. The soft agar growth assays were performed as previously described Specific details are given in Figure 3. The results (17). presented represent the mean of triplicate plates (which varied by less than 15%). All experiments were repeated twice and gave similar findings. 810
Vol.
BIOCHEMICAL
181, No. 2, 1991
RESULTS
HT29-Cl lacked
the
growth
after
control
extent
parental
HT29
only
(Figure
that
growth
cells
minimal
1B).
in
PKC enzyme
A
with
the
cells
that
activity
when treated with
of
1A).
found
HT29-PKC7
treatment
but
a 40% reduction
was
inhibition
contrast,
vector
5mN HNBA (Figure
shown).
increase
growth
In
with
not
the
about
inhibition
(data
a 15-fold
contained
showed
treatment
of
about
showed
cells
a B-day
RESEARCH COMMUNICATIONS
AND DISCUSSION
PKC cDNA insert,
similar
express
AND BIOPHYSICAL
(17)
with
HMBA
TPA resulted
in
107
107
B
v
105-
1041
1041~ 0
2
5
2
0
a
5
-o-
Figure
+
TPA
curves
of
CONTROL
1.
Growth
monolayer plates,
cultures. the
Two
cultures
+
HMBA
HT29-Cl
days
were treated
and
after
then
changes determined
grown twice
repective
week.
triplicate
phase and
the
per
on
exponential Materials
in
with
of
growth.
every For
Methods.
811
HT29-7
10 nM TPA. with
counts
plates
TPA+HMBA
cells
in
a series
of
0.02% DMSO (Control),
medium, Cell
+
seeding
51nM MMBA, 1OnM TPA, or 51nM HMBA plus were
0
Days
Days
per 3 days
additional
The cells
fresh
medium
plate
were
during details
the see
Vol.
181, No. 2, 1991
marked in
BIOCHEMICAL
growth
growth)
on the
inhibition
(Figure
growth
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
lB),
HT29-PKC7
but
of HT29-Cl
there
cells
(Figure
in marked
HT29-Cl
and
cells,
growth
inhibition,
respectively
2 demonstrates of
the
the
cells,
growing
cells.
TPA treatment increase
treatment,
with
a slight
decline
change
in
increase
in
in
untreated than
intercellular
and
of the
combination
of
in The
colonies (Figure
3).
decrease
in
cell
3 of
numb,er. cells,
of
cell
and
appearance.
and
The
appearance
an
seen
change
the
resulted
similar
to that
an
some of
TPA treatment
HMBA
and
TPA plus
number
a rounder
a significant
in
increase in in
in
the cell
resulted
in
in slight
HT29-Cl number. a marked
number.
HT29-Cl
control
(>O.lmm)
when
Treatment the
by day
addition,
number
plus
and
was no apparent
resulted
TPA
cells
combination
a flat
typical
cell
HMBA treatment
cells,
without
decrease
cell
the
in
there
of
cells.
in
cells,
of
of the
In
had
the
of epithelioid
change
The
Figure
exponentially
spaces,
but
93%
morphology
showed
rounding
displayed
HT29-Cl
spaces.
elongation
in
spaces.
decrease
of
clusters
reduction
cells
untreated
a dramatic
control
a marked
elongated
on the
cells
spaces.
HT29-PKC7
the
drugs
treatment
number,
intercellular
were
1B).
some elongation
cell
intercellular
HMBA resulted
1A and
a significant
in
of both
(Figures
intercellular
HMBA caused
inhibition
80% and
displaying
the
without
Treatment
cells
in
The combination
about
these
resulted
effect
causing
control
cells,
1A).
growth
a 3-day
HT29-Cl
of HT29
a marked
of
following
cultures.
appearance
The
effects
(>85% reduction
was no significant
of HMBA and TPA resulted HT29-PKC7
cells
number
cells grown with
of
formed soft
agar
TPA resulted
in
colonies, 812
in
numerous
but
for only
treatment
large 4 weeks a slight with
HMBA
Vol.
181,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
HT29-Cl
Figure
after
HT29-PKC7
2. Morphologic
treatment.
Cells
seeding)
were
responses in
later.
the
treated
TPA, or 5mM HMBA plus 3 days
COMMUNICATIONS
exponential with
lOnM TPA.
Magnification
of the
x29. 813
cell
growth
lines
to
drug
phase
(2 days
0.02% DMSO, 5mM HMBA, 10 nM The photographs
were
taken
Vol.
181,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
HT29-Cl
w
Figure
3.
seeded
into
in
Colony
formation
60-mm
McCoy's
DMSO,
5mM HMBA,
plates
were
end
q
TPA
in
HMBA
soft
(diameter)
5A medium
10 nM TPA, with
of
4 weeks,
Colonies
greater
triplicate
per
independent
experiments
low
TPA + HMBA
agar.
Each
dishes with
or
in
cell
line
0.3%
soft
10 % FBS,
5 IIIM HMBA and
was agar
plus
1OnM
0.02%
TPA.
The
fresh
medium
every
7 days.
At
were
stained
with
a vital
stain.
colonies than
0
petri
supplemented
overlaid
COMMUNICATIONS
HT29-PKC7
q
CONTROL
RESEARCH
O.lmm
power
in
diameter
field
are
were
(40X).
given
The
the
counted
in
means
of
of
untreated
as percentage
two
cells.
resulted
Treatment in
about
in
with
colony
in
HT29-Cl
control
cells
reduction
of
than
in
HT29-PKC7
10% of
seen
that
with
counts, with
to
of
colonies,
with
the
HT29-Cl
with 814
formed to that
TPA plus
seen
resulted
less
in
the
numerous
than
HMBA resulted
number
cells
seen
TPA treatment
colony
the
decrease
similar
3).
of colonies.
a dramatic
also
an efficiency (Figure
number
in
cells
Treatment
30% inhibition
Treatment
less
in
cells.
the
HMBA resulted
with
reduction
untreated
in
HT29-PKC7
agar,
a dramatic
the
to
cells.
colonies
slight
TPA plus
counts,
untreated
the
a 30% reduction
in
10% of
in
only
contrast
control
in
a to
cells.
HMBA resulted
in
Vol.
181,
No.
a dramatic that
BIOCHEMICAL
2, 1991
decline
obtained
with
Taken that
the
modulating
the
surface
malignancy
in
cells.
cells
that
with
PKC in
cultures
presence
of
TPA,
did
HT29-PKC7
cells
cells
the
given of
with
cell
types
type.
in
between
(23).
Since
necessary
that
to
the
treatment of
the
induce
examine found
that
in
alkaline in
fluidcell possible
absence
or
phosphatase,
our
the
of
treatment
the
clones
of
studies). role
of
dome
HT29-Cl
and
further
Thus,
differentiation
that
level
is
possible
in
PKC in
growth
colon
cancer
in
in
in
that
PKC activity
identify
the
studies
specific
were
815
the
colon (13),
studied.
various
a
tumor it
using
cell
can
vary
samples
may also
be
present
in
We should out
in effects
PKC levels
and
is
(3,4,10,11,12)
PKC isoforms
carried
it present
studies
family
being
dealing
divergent
between
mucosa
a multigene
line
the
Indeed,
colon
studies
PKC activity
previous
PKC is
cell
future
and differentiation
of
studies.
normal
present
expression
however,
clarify
seen
these
particular
TGFSl
appearance
HMBA,
et
of a cell
goblet
with
in
Schroy
long-term
We have
the It
differences used
important
expression
the
of HMBA on growth
analyze
widely
the
to
evidence
mucin-producing
6 days not
10% of
HMBA.
HMBA on growth
reflect
not,
indicate
effects to
in
or
enterocyte
results
important
that
(unpublished
and
Our with
found
production
needed
treated
also
events.
up to
or mucin
inhibition
growth.
and
did
for
are
HMBA on cell
induce
these
formation,
studies
PKC are
marker
They
of
of
the
enterocyte
than
provide
HMBA can inhibit
also
less
results
status
HMBA resulted
variants(22).
to
COMMUNICATIONS
cells.
above
of
They
transporting
of
the
RESEARCH
count
untreated
effects
found
role
the
BIOPHYSICAL
colony
and activation
a1.(21)
HT29
the
together,
level
HT29
in
AND
stress
a derivative
Vol.
No. 2, 1991
181,
of
HT29
results isoforms
BIOCHEMICAL
cells
that
might
be obtained
of
suggest
activator
or
is
Studies
clinical
that
different
that
overexpress
other
may be of
clinical
relevance
since
patients the
and
highly
a novel
growth
tumor
HMBA in
activators
with of
colon
PKC may,
with
an
cancers.
compound
promoting
combination
of
HMBA plus
potent
exerting
employing
novel
of
inhibit
PKC without
other
and
cells
treatment
PKC might
Bryostatin
(24).
PKCBl
with
results
that
of
activates
overexpresses
PKC.
The present they
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
activity bryostatin
therefore,
be
of
interest.
ACKNOWLEDGNENTS
This study was supported by awards from the Aaron Diamond Foundation and the Markey Charitable Trust to I.B.W. P.M.C. is a recipient of an Advanced Research Training Award from the American Gastroenterology Association. P.M.C. is also indebted to Michael Field for providing valuable support, and Brenda Chicco and Sheila M. Washington for assistance in the preparation of this manuscript.
REFERENCES
1. Marks,P.A., Canc.Res. 47, Reuben &?ks,P.A.'
Sheffery,M., 659-666.
and
(1987)
Rifkind,R.A.
R.C ' Breslow,R., Rifkind,R.A., U.S.A. 73, 862-866. (19+~)w~~~~.~a~i:Acad.Sci.
3. Collins,S.J., Int.J.Canc.,
25,
Bodner,A., 213-218.
Ting,R.,
and Gall0,R.C.
4. McCue,P.A., (1983) Dev.Biol.
Matthaei,K.I., 96, 416-426.
Kimhi,Y., Littauer,U.Z.
Palfrey,C., Spector,I., (1976) Proc.Natl.Acad.Sci.
5.
Taketo,M.,
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and 464-
466.
Palfrey,C. &ks,P.A.
' ' Littauer (19i7f(i~~~~h?m~Biophys.R&.Co&un.,
7. Kennedy,B.G. 416.
and Lever,J.E.
(1985)
Kowinsky,E.K., Ettinger,D.S., Brundett,R.B., Cates,A.E., and J.Clin.Oncol., 4, 1835-1844.
8.
816
U.Z
Reuben,R.C and 76, 937'-$42.
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123,
410-
Grochow,L.B., Donehower,R.C. (1986)
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9. Egorin,M.J., Whitcare,M.U.,
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Sigman,L,M., and Aisner,J.
Bretman,T.R.
and He,R.
Van (1987) (1990)
11. Kong,X., Fanucchi,M.P., Res.12, 853-859.
12. Schroy,P.C., Friedman,E.A. (1988) 13.
Nishizuka,Y.
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Echo,D.A., Canc.Res.
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48,
Nature
334,
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47, 50,
Chou,T.-C.
Carnright,K., Canc.Res.
(1988)
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Forrest,A., 617-623. 6268-6273.
(1988)
Leukemia
Winawer,S.J., 5487-5494.
and
661-665.
14. Melloni,E., Pontremoli,S., Michetti,M., Sacco.O., Cakiroglu,A.G., Jackson,J.F., Rifkind,R.A., and Marks, P.A. (1987) Proc.Natl.Acad.Sci. U.S.A. 84, 5282-5286. 15. Melloni,E., Pontremoli,S., and Rifkind,R.A. Marks,P.A., 18418. 16. Melloni,E., Grossi,F., Proc.Natl.Acad.Sci. 17. Choi,P.M., Mol.Cell.Biol. 18,. Baron,P.L., Takarz,A.S.,
(1989)
Patrone,M., 264, 18414-
Pontremoli,S., Sparatore,B., Marks,P.A., and Rifkind, U.S.A. 87, 4417-4420. Tchou-Wong,K.M., 10, 4650-4657. Koretz,M.J., and Parker,G.A.
Gambari,R., Rifkind,R., U.S.A.
Schroy,P., Rkfkin,J., Friedman,E. (1990) Canc.Res.
Carchman,R.A., (1990) Arch.Surg.
23. Guillem,J.G., LoGerfo,P., Treat,M. 2036-2039.
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Nudel,U., Weinstein,I.B., Proc.Natl.Acad.Sci.,
Coffey,R.J., 261-265.
Winawer,S., 1, 617-626
O'Brian,C.A., and Weinstein,I.B.
24. Smith,J.A., Smith,L., Biochem.Biophys.Res.Commun.
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Shaw,P., ManiatiS,G., and Marks,P.A. 76, 1906-1910.
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22. Hafez,M.M., Infante,D., (1990) Cell Growth & Diff.
Patrone,M., R.A. (1990)
and Weinstein,I.B.
19. Yamasaki,H., Fibach,E., Rifkind,R.A., Marks,P.A. (1977) U.S.A. 74, 3451-3455. 20. Fibach,E., Sassa,S., Proc.Natl.Acad.Sci.
Viotti,P.L., J.Biol.Chem.
Reuben,R., (1979) Winawer,S.,
and
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and Pettit,G.R. 939-945.
Friedman,I. Forde,K., Canc.Res. 47, (1985)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 809-817
BIOCHEMICAL
THE MOBULATION OF GROWTH BY MBA, IN PKC 0VKRPROIXJCING BT29 COILIN CANCER CELLS
Paul
M. Choi
and I.
Bernard
Weinstein
Comprehensive Cancer Center and and Department of of Cancer Research, Columbia University, New York, NY 10032
Institute
Medicine,
Received September 27, 1991
To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses We found that although HMBA markedly high levels of PKCBl. inhibited the growth of the control cells, no inhibition was On the other hand the tumor seen with the HT29-PKC7 cells. promoter 12-O-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and These results may be relevant to the use of HMBA in TPA. combination with other agents in the therapy of specific cancers. Q1991Academic Press, Inc.
The polar-planar in
several
(MEL)
cell
cells
embryonal
systems,
(1,2), carcinoma
and MDCK kidney stimulated the
(4),
cancer
murine
embryonal or and groups
cells
of
however,
other
HL-60
in
growth,
Breitman
human
interest
appear
the
found
leukemia
(7).
potential
cells
cells
These
The effects
cell by
growth
(3),
HMBA
have
lines
can In
(3,10,11).
be
HL-60 by
in
HMBA on cell
The growth
enhancement
inhibition
(5,6),
compound
of
variable.
(4).
mouse
findings
use of this
to be quite
growth
erythroleukemia
neuroblastoma
(8,9).
unaffected
differentiation
murine
cells
carcinoma
He found
HMBA induces
including
epithelial
treatment
inhibited
compound
of
either cells,
HMBA,
while
Schroy
et
0006-291X/91$1.50 809
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
181, No. 2, 1991
a1.(12)
BIOCHEMICAL
found
that
the
diffferent
colorectal
stimulation
to
the
precise
mechanism
is
evidence
that
mediating
the
surprising
control
PKCBl
accelerates
the
that
PKC
ability
understand growth
that
of
the
determinants
level of
Although known,
role
MATERIALS
PKC by
HMBA,
the
HT29
the
is
in
not
signal and
expression
that
a phorbol
ester
colon
activation
play
studied
cancer PKCBl status
of
(16).
PKC might
we have
of
by HMBA
differentiation
overexpress
effects
in
induced
by
and
there
This
increased
role
stably
growth
differentiation
induce
the
of
particularly
differentiation
to
from
not
a role,
MEL cells
of HMBA to
was engineered
important
HMBA is
of
growth
no effect.
include
regulation
of
the
ranged
a central
that
on a derivative
demonstrate
play
plays
In
down
HMBA on
to of
RESEARCH COMMUNICATIONS
response.
the
modulation
effects
cells
differentiation
To better the
cancer
PKC may
(13).
and
(14,15),
of
action
pathways
growth
effects
inhibition, of
since
transduction
blocks
growth
AND BIOPHYSICAL
its
cell (17). of
in
line We
PKC are
HMBA on growth.
ANDMETHODS
The properties of the HT29-PKC7 cell line that expresses about a 15-fold increase in PKCBl, and a vector control cell line HT29-Cl, have been previously described HT29-Cl and HT29-PKC7 cells were grown as previously (17). described (17). Prior to analyses, the cells were grown in McCoy's 5A medium plus 10% fetal bovine serum for 48 hours to reach the exponential growth phase. Then, fresh growth medium containing 0.02% DMSO (solvent control), 5mM HMBA, 10 nM TPA, or medium containing 5mM HMBA plus 10nM TPA were added. The doses were selected for maximal biological effects (12,17,18 and unpublished observation). Replicate plates of cells were then counted (using a Coulter counter) as previously described (17). Only at 3-day intervals, exponential phase cultures could be analyzed since cells treated with the combination of HMBA and TPA started to come off the plate during the plateau phase of growth. The soft agar growth assays were performed as previously described Specific details are given in Figure 3. The results (17). presented represent the mean of triplicate plates (which varied by less than 15%). All experiments were repeated twice and gave similar findings. 810
Vol.
BIOCHEMICAL
181, No. 2, 1991
RESULTS
HT29-Cl lacked
the
growth
after
control
extent
parental
HT29
only
(Figure
that
growth
cells
minimal
1B).
in
PKC enzyme
A
with
the
cells
that
activity
when treated with
of
1A).
found
HT29-PKC7
treatment
but
a 40% reduction
was
inhibition
contrast,
vector
5mN HNBA (Figure
shown).
increase
growth
In
with
not
the
about
inhibition
(data
a 15-fold
contained
showed
treatment
of
about
showed
cells
a B-day
RESEARCH COMMUNICATIONS
AND DISCUSSION
PKC cDNA insert,
similar
express
AND BIOPHYSICAL
(17)
with
HMBA
TPA resulted
in
107
107
B
v
105-
1041
1041~ 0
2
5
2
0
a
5
-o-
Figure
+
TPA
curves
of
CONTROL
1.
Growth
monolayer plates,
cultures. the
Two
cultures
+
HMBA
HT29-Cl
days
were treated
and
after
then
changes determined
grown twice
repective
week.
triplicate
phase and
the
per
on
exponential Materials
in
with
of
growth.
every For
Methods.
811
HT29-7
10 nM TPA. with
counts
plates
TPA+HMBA
cells
in
a series
of
0.02% DMSO (Control),
medium, Cell
+
seeding
51nM MMBA, 1OnM TPA, or 51nM HMBA plus were
0
Days
Days
per 3 days
additional
The cells
fresh
medium
plate
were
during details
the see
Vol.
181, No. 2, 1991
marked in
BIOCHEMICAL
growth
growth)
on the
inhibition
(Figure
growth
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
lB),
HT29-PKC7
but
of HT29-Cl
there
cells
(Figure
in marked
HT29-Cl
and
cells,
growth
inhibition,
respectively
2 demonstrates of
the
the
cells,
growing
cells.
TPA treatment increase
treatment,
with
a slight
decline
change
in
increase
in
in
untreated than
intercellular
and
of the
combination
of
in The
colonies (Figure
3).
decrease
in
cell
3 of
numb,er. cells,
of
cell
and
appearance.
and
The
appearance
an
seen
change
the
resulted
similar
to that
an
some of
TPA treatment
HMBA
and
TPA plus
number
a rounder
a significant
in
increase in in
in
the cell
resulted
in
in slight
HT29-Cl number. a marked
number.
HT29-Cl
control
(>O.lmm)
when
Treatment the
by day
addition,
number
plus
and
was no apparent
resulted
TPA
cells
combination
a flat
typical
cell
HMBA treatment
cells,
without
decrease
cell
the
in
there
of
cells.
in
cells,
of
of the
In
had
the
of epithelioid
change
The
Figure
exponentially
spaces,
but
93%
morphology
showed
rounding
displayed
HT29-Cl
spaces.
elongation
in
spaces.
decrease
of
clusters
reduction
cells
untreated
a dramatic
control
a marked
elongated
on the
cells
spaces.
HT29-PKC7
the
drugs
treatment
number,
intercellular
were
1B).
some elongation
cell
intercellular
HMBA resulted
1A and
a significant
in
of both
(Figures
intercellular
HMBA caused
inhibition
80% and
displaying
the
without
Treatment
cells
in
The combination
about
these
resulted
effect
causing
control
cells,
1A).
growth
a 3-day
HT29-Cl
of HT29
a marked
of
following
cultures.
appearance
The
effects
(>85% reduction
was no significant
of HMBA and TPA resulted HT29-PKC7
cells
number
cells grown with
of
formed soft
agar
TPA resulted
in
colonies, 812
in
numerous
but
for only
treatment
large 4 weeks a slight with
HMBA
Vol.
181,
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
HT29-Cl
Figure
after
HT29-PKC7
2. Morphologic
treatment.
Cells
seeding)
were
responses in
later.
the
treated
TPA, or 5mM HMBA plus 3 days
COMMUNICATIONS
exponential with
lOnM TPA.
Magnification
of the
x29. 813
cell
growth
lines
to
drug
phase
(2 days
0.02% DMSO, 5mM HMBA, 10 nM The photographs
were
taken
Vol.
181,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
HT29-Cl
w
Figure
3.
seeded
into
in
Colony
formation
60-mm
McCoy's
DMSO,
5mM HMBA,
plates
were
end
q
TPA
in
HMBA
soft
(diameter)
5A medium
10 nM TPA, with
of
4 weeks,
Colonies
greater
triplicate
per
independent
experiments
low
TPA + HMBA
agar.
Each
dishes with
or
in
cell
line
0.3%
soft
10 % FBS,
5 IIIM HMBA and
was agar
plus
1OnM
0.02%
TPA.
The
fresh
medium
every
7 days.
At
were
stained
with
a vital
stain.
colonies than
0
petri
supplemented
overlaid
COMMUNICATIONS
HT29-PKC7
q
CONTROL
RESEARCH
O.lmm
power
in
diameter
field
are
were
(40X).
given
The
the
counted
in
means
of
of
untreated
as percentage
two
cells.
resulted
Treatment in
about
in
with
colony
in
HT29-Cl
control
cells
reduction
of
than
in
HT29-PKC7
10% of
seen
that
with
counts, with
to
of
colonies,
with
the
HT29-Cl
with 814
formed to that
TPA plus
seen
resulted
less
in
the
numerous
than
HMBA resulted
number
cells
seen
TPA treatment
colony
the
decrease
similar
3).
of colonies.
a dramatic
also
an efficiency (Figure
number
in
cells
Treatment
30% inhibition
Treatment
less
in
cells.
the
HMBA resulted
with
reduction
untreated
in
HT29-PKC7
agar,
a dramatic
the
to
cells.
colonies
slight
TPA plus
counts,
untreated
the
a 30% reduction
in
10% of
in
only
contrast
control
in
a to
cells.
HMBA resulted
in
Vol.
181,
No.
a dramatic that
BIOCHEMICAL
2, 1991
decline
obtained
with
Taken that
the
modulating
the
surface
malignancy
in
cells.
cells
that
with
PKC in
cultures
presence
of
TPA,
did
HT29-PKC7
cells
cells
the
given of
with
cell
types
type.
in
between
(23).
Since
necessary
that
to
the
treatment of
the
induce
examine found
that
in
alkaline in
fluidcell possible
absence
or
phosphatase,
our
the
of
treatment
the
clones
of
studies). role
of
dome
HT29-Cl
and
further
Thus,
differentiation
that
level
is
possible
in
PKC in
growth
colon
cancer
in
in
in
that
PKC activity
identify
the
studies
specific
were
815
the
colon (13),
studied.
various
a
tumor it
using
cell
can
vary
samples
may also
be
present
in
We should out
in effects
PKC levels
and
is
(3,4,10,11,12)
PKC isoforms
carried
it present
studies
family
being
dealing
divergent
between
mucosa
a multigene
line
the
Indeed,
colon
studies
PKC activity
previous
PKC is
cell
future
and differentiation
of
studies.
normal
present
expression
however,
clarify
seen
these
particular
TGFSl
appearance
HMBA,
et
of a cell
goblet
with
in
Schroy
long-term
We have
the It
differences used
important
expression
the
of HMBA on growth
analyze
widely
the
to
evidence
mucin-producing
6 days not
10% of
HMBA.
HMBA on growth
reflect
not,
indicate
effects to
in
or
enterocyte
results
important
that
(unpublished
and
Our with
found
production
needed
treated
also
events.
up to
or mucin
inhibition
growth.
and
did
for
are
HMBA on cell
induce
these
formation,
studies
PKC are
marker
They
of
of
the
enterocyte
than
provide
HMBA can inhibit
also
less
results
status
HMBA resulted
variants(22).
to
COMMUNICATIONS
cells.
above
of
They
transporting
of
the
RESEARCH
count
untreated
effects
found
role
the
BIOPHYSICAL
colony
and activation
a1.(21)
HT29
the
together,
level
HT29
in
AND
stress
a derivative
Vol.
No. 2, 1991
181,
of
HT29
results isoforms
BIOCHEMICAL
cells
that
might
be obtained
of
suggest
activator
or
is
Studies
clinical
that
different
that
overexpress
other
may be of
clinical
relevance
since
patients the
and
highly
a novel
growth
tumor
HMBA in
activators
with of
colon
PKC may,
with
an
cancers.
compound
promoting
combination
of
HMBA plus
potent
exerting
employing
novel
of
inhibit
PKC without
other
and
cells
treatment
PKC might
Bryostatin
(24).
PKCBl
with
results
that
of
activates
overexpresses
PKC.
The present they
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
activity bryostatin
therefore,
be
of
interest.
ACKNOWLEDGNENTS
This study was supported by awards from the Aaron Diamond Foundation and the Markey Charitable Trust to I.B.W. P.M.C. is a recipient of an Advanced Research Training Award from the American Gastroenterology Association. P.M.C. is also indebted to Michael Field for providing valuable support, and Brenda Chicco and Sheila M. Washington for assistance in the preparation of this manuscript.
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