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Conney, March 27, 1987 (received for review November 18, 1986)
ABSTRACT Monocyte maturation markers were induced
in cultured human myeloblastic ML-2 leukemia cells after
treatment for
increases
were found in the percentages of cells exhibiting reactivity with
either the murine
monoclonal antibody or the Leu-MS
monoclonal antibody, staining positively for nonspecific
esterase activity, and displaying a promonocyte morphology.
The increases in these differentiation markers after treatment
with 0.03-l
THC were dose dependent. At this dose range,
THC did not cause an inhibition of cell growth. The
Sci. USA
Vol. 84, pp. 5414-5418, August 1987
Medical Sciences
cells)
G
ERALD
or longer treatment,
C
YNTHIA
B. H. C
HUBB
to
or
,S
AKAN
M
AEDA
, M. A
NNE
(GIBCO).
The cultures were incubated at 37°C in an atmosphere of 8%
it cell blood and ration ical nabinol about differentiation inducers, two cells a markers; terminal (CBN),
related (12, exhibits 13). these induced cannabinoids, cell and, Using differentiation.
a compounds, when phenotype the these expression treated cannabidiol cells, displays resembling
however, with we of appropriate useful showed monocyte (CBD) failed that markers of that and
to mature
chem-
matu-
bring
THC
of
MATERIALS AND METHODS
Triangle National Chemicals. Park, Institute THC, NC, of and
CBD, Drug and Abuse CBN repository were obtained in Research
from the
(National from leukemia (Falcon man monocytes five Plastics) cells individuals Cancer were were at 1.5
prepared inoculated as Institute, described from Bethesda, into
peripheral previously MD) Petri blood (21). (15). pooled
The
Hu-
medium lin (100 supplemented units/ml), and with streptomycin 20% fetal bovine (100
dishes
serum, penicil-
1640
sulfoxide treated One Labeling day cultures after of THC Cells was in 0.1%.
treatment and
the growth of ML-2 medium cells of incubated control and
in
(1420 Ci/mmol; 1 Ci
= 37
Petri dishes,
G
EMMELL
of ethanol at
,
AND
E
LIEZER
were dissolved in
(1987) 5415
were placed into a flat-bottomed multiwell plate. The cells in
the wells were then labeled with
LKB) at a
final
of 9.5. Two-dimensional electrophoresis was per-
formed with the 7
was obtained
from
butyrate (liquid,
N-8000; Sigma) dissolved in 4 ml of acetone. The second
solution was prepared by mixing 0.4 ml of 4% pararosaniline
(free base, P-7632; Sigma) in 2 M
at
various
(60 &i/ml)
and 18 hr later were harvested and solubilized in 50
mixture of Bio-Lyte
and autoradiographed
on Kodak XAR-2 film for 1 week.
Reactivity with
bodies. The
of the
monoclonal antibody and incubated for 30 min at
of a
solution
(Bio-Rad) used as
ampholyte. The gels were then
cells suspend-
ed in 200
9 M urea, 4% Nonidet
7 inch
5-7 and
Anti-Cell Surface
of FITC-conjugated goat
‘0.03
0.1 0.3
al.
with 0.4 ml of 4%
For morpho-
logical evaluation of cell differentiation, control and treated
ML-2 cells were spread on glass slides in the same manner as
the cells were prepared for detecting nonspecific esterase
activity, except that they were stained with Wright’s stain
(no. 840-100; Sigma). For each determination, 200-400 cells
from at least two preparations were evaluated.
RESULTS
Expression of Monocyte Markers in Human Myeloblastic
Leukemia Cells After Treatment with
During a six-day
period in culture,
the percentage of
cells staining positively for nonspecific esterase activity
increased by
and the percentage of cells exhib-
iting a promonocytic morphology increased by
to S-fold
(Fig. 1). Results obtained after either a
treatment
with THC were similar to those obtained after a
of control cells
reacted with B52.1.1, another monocyte-specific monoclonal
antibody (25). In addition to expressing these antigenic
determinants,
monoclonal
antibody, which detects a
surface antigen<
antibody increased by
to
Sci. USA
to
and monocytes
or a
(
A
), or CBN
or
and C) ML-2 cells treated
with either 1
(1987)
cells (data not shown), The THC-induced increase in the
expression of the differentiation markers was dose dependent
up to about 1
untreated peripheral blood monocytes. Each panel shows the same section of the respective
two-dimensional electrophoretic protein pattern, with acidic proteins to the left and higher molecular masses at the top. The
vertical scale shows
THC caused an
increase in the percentage of KG-1 cells staining for nonspe-
cific esterase activity and exhibiting a promonocyte morphol-
ogy; these percentages were increased from
in the
control to about 20% in the treated cultures. Again, as in
ML-2 cells, control and THC-treated KG-1 cells did not
exhibit a mature monocytic morphology, nor did they attach
F
IG
. 2. Two-dimensional electrophoretic patterns of cells labeled with
antibody and
or 10
as a result of THC treatment but are not detected in the monocytes are also indicated.
THC (C);
or
cells (Fig.
2). Among the ML-2 proteins that exhibited a decrease or low
level of synthesis after THC treatment were 5
Changes
or decrease
or 10
Natl.
Sci. USA
Proteins in
(1987) 5417
Table 1. Induction of cell differentiation markers by either
in ML-2 leukemia cells: Effect of pretreatment with
THC as compared with untreated controls
Inducer
Control THC
None
3.5 13 42 15 62 91 59 9 41 0 0
PMA 0.3 4.3 3.2 68 36 66 65 20 62 29 16 51 22
1.0 0.5 0.4 88
to THC, we
treated the ML-2 cells with two other cannabinoids, CBD and
CBN. Treatment of the ML-2 cells for 4
The
previous experiments in this study indicated that THC
induces an incomplete cell maturation in ML-2
How-
ever, the same ML-2 cells can acquire a mature phenotype
when treated with either PMA
which are common inducers of cell. differentiation.. As a
result, we tested these two agents for their ability to complete
the differentiation process initiated by THC in the ML-2 cells.
The results indicated that either PMA or
stage by
THC, to express a mature phenotype
to that induced
by PMA or
esterase activity
(Fig. 1) and in the percentage of ‘cells reacting with the
when
added 2 days after THC treatment, could cause the ML-2
cells, which were induced only to the
dose of PMA
(e.g., 0.3
84 87 9 29 i3 10 78 70
THC at 10
with Either
1).
72 82 17 21 17 14 66 65
100.0 4.0 3.0
was added to the cultures 1 day after cell seeding, PMA and
with the
11 30 17 25 72 45
3.0 0.5 0.4
Control THC Control THC Control THC Control THC Control THC
the
or
with
did not
number
or
nonspecific
namely, THC,
cell differentiation
affinity specific
1 13 6 22 93 65
receptors (27).
Sci. USA
can bind to
it is still
5418 Medical Sciences: Murison
(1987)
cells, whether untreated or induced for an “incomplete” cell
maturation by THC, can acquire a mature phenotype that
resembles that of monocytes/macrophages when treated
with either PMA or
THC
reduced to some degree the ability of PMA at its lower dose
(e.g., 0.3
(but not
257-353.
antibody and those achieving monocytic morphology.
These results suggest that THC not only induces in ML-2
cells an “incomplete” monocytic differentiation but also
induces in these cells conditions that reduce the transition of
the treated cells from their “incomplete” state to the more
mature monocytic/macrophage phenotype. More experi-
ments are needed, however, to clarify whether THC-induced
“incomplete” cell maturation is normal but incomplete or
whether THC induces, in the ML-2 cells, an aberrant type of
The cell system described here may be useful for deci-
phering critical biochemical events that lead to the
was
additive or synergistic with that of THC. Moreover,
Beach, FL),
pp. 257-310.
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