[CANCER RESEARCH 43, 4233-4238,
September 1983]
Effect of Cellular Phospholipid Modification on Phorbol Diester Binding1
Myles C. Cabot
Medical and Health Sciences Division, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830
ABSTRACT
The influence of cellular lipid composition on the specific
binding of [20-3H]phorbol-12,13-dibutyrate
to intact human promyelocytic leukemia cells was investigated. Cellular phospholipid
composition could be manipulated by culturing cells in serumfree, chemically defined media containing base analogues of
phospholipid polar head groups. Human promyelocytic leukemia
cells grown in the presence of dimethylethanolamine, monomethylethanolamine, 3-aminopropanol, or isopropylethanolamine
assimilated these natural and unnatural base moieties into en
dogenous phospholipids to the extent that 22 to 52% of the cell
glycerophospholipids contained the base analogue. The forma
tion of the phospholipid analogues was accompanied by a pro
nounced reduction in the levels of intracellular choline and ethanolamine glycerophospholipids.
Analogue-supplemented
cul
tures exhibited a reduced growth rate compared to control cells
maintained in choline-containing medium. Specific [20-3H]phorbol-12,13-dibutyrate binding was examined in lipid-altered cells
and shown to be markedly higher (approximately 200% of con
trol) in cells grown with dimethyl- or monomethylethanolamine.
In contrast, exposure of cells to 3-aminopropanol or isopropyle
thanolamine resulted in a major reduction in [20-3H]phorbol12,13-dibutyrate binding. Only minimal changes in nonspecific
29, 40, 48). Methods for modifying the lipid composition of
cultured mammalian cells have been reviewed by Spector et al.
(44). One approach that has been used to manipulate the phos
pholipid composition of cells involves the use of polar-headgroup analogues. Glaser e? al. (21) and Blank et al. (3) have
shown that L-M cells, cultured in chemically defined medium
supplemented with base analogues, such as DMEA, MEA, IPE,
or 3-AP, synthesize phospholipids containing the natural or un
natural groupings. Subsequent studies have revealed that these
modified phospholipids are localized in plasma membranes, microsomes, and mitochondria (40) and are thus integrated into
the functional membrane systems of the cell. The present study
was designed to determine the effects of modified phospholipid
composition on phorbol diester binding in cells that are target
specific for TPA-induced differentiation. This report shows that
HL-60 cells readily incorporate polar-head-group analogues into
cellular phospholipids. The assimilation is accompanied by a
decrease in the endogenous, naturally occurring phospholipids
(choline and ethanolamine glycerophospholipids) and significantly
modifies the specific binding of [3H]phorbol diesters to intact
cells. The cells used in this study were cultured in serum-free
medium, and we have since shown that, although their fatty acid
composition is altered compared with serum-grown HL-60 cells,
this does not alter TPA-induced differentiation (47).
binding occurred between control and experimental cells. Be
cause phorbol esters are highly membrane targeted, it is possible
that phospholipid modification or the resulting changes in mem
brane organization influence receptor dynamics.
MATERIALS AND METHODS
INTRODUCTION
purchased from New England Nuclear, Boston, Mass. Unlabeled PBD
was a product of Chemicals for Cancer Research, Eden Prairie, Minn.
DMEA, MEA, and 3-AP were obtained from Aldrich Chemical Co.,
Tumor-promoting
phorbol diesters, most notably TPA,2 exert
diverse cellular and biochemical responses in numerous biologi
cal systems (6,16,17,46).
In vitro, these agents can induce (26,
39) or inhibit (38, 49) cellular differentiation; exposure of cultured
HL-60 cells to TPA induces terminal differentiation to a macrophage-like cell. Although the mechanism of tumor promoterinduced differentiation is unknown, the facts that cell surface
membranes, of which lipids are integral components, contain
TPA-binding receptors (18, 20, 41-43) and that phospholipid
metabolism is altered in cells after short time exposure to TPA
(10,11, 28, 36) suggest that membranes are directly involved in
the cellular responses elicited by phorbol diesters.
The lipid composition of cells maintained in culture can be
readily modified by serum removal (1, 47) or by the addition of
glycerolipid precursors to the diet or growth medium (2,3,9,27,
1This work was supported
by the Office of Energy Research, United States
Department of Energy (Contract DE-AC05-760R00033).
2 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate:
HL-
60 cells, human promyelocytic leukemia cells; DMEA, dimethylethanolamine; MEA,
monomethylethanolamine;
IPE, isopropylethanolamine;
3-AP, 3-aminopropanol;
fH]PDB, [20-3H]phorboM2,13-dibutyrate;
PDB, unlabeled phorbol-12,13-dibutyrate; RPMI, Roswell Park Memorial Institute; P-DMEA, P-MEA, P-IPE, P-3-AP, intact
glycerophospholipids containing the base group analogue.
Received September 27,1982; accepted June 8,1983.
SEPTEMBER
1983
Chemicals.
[3H]PDB (specific activity, 15.3 and 20 Ci/mmol) was
Milwaukee, Wis., and IPE was a gift from Dr. C. Piantadosi (Department
of Medicinal Chemistry, University of North Carolina, Chapel Hill, N. C.).
üpidstandards for thin-layer chromatography included egg phosphatidylA/,A/-dimethylethanolamine (Avanti Polar Lipids. Birmingham, Ala.) and La-phosphatidyl-A/-monomethylethanolamine
(Sigma Chemical Co., St.
Louis, Mo.). Peanut phospholipase D was purchased from Sigma.
Cell Culture. HL-60 cells (13) were provided by Dr. R. C. Gallo
(National Cancer Institute, Bethesda, Md.). For routine passage, cells
were cultured in the serum-free RPMI Medium 1640 containing insulin
and transferrin as described by Breitman ef al. (7); cell passages 70 to
90 were used for these experiments. To grow cells in the presence of
phospholipid base analogues, stock cultures were washed once in
phosphate-buffered saline (pH 7.2; sodium chloride, 7.6 g/liter; disodium
phosphate, 1.3 g/liter; monosodium phosphate, 0.1 g/liter; monopotassium phosphate, 0.2 g/liter) at room temperature and once in special
formulation choline-free RPMI Medium1640 (Grand Island Biological Co.,
Grand Island, N. Y.) and seeded in 9.5 ml of choline-free medium with
insulin and transferrin (4 x 106 cells/75-sq cm Costar tissue culture flask).
Analogue solutions were prepared in phosphate-buffered saline (800 ng
analogue per ml). The pH was adjusted to 7.2 with HCI, and a 0.5-ml
aliquot was added to cell cultures to achieve a final concentration of 40
tig analogue per ml culture medium. Cells were grown in the presence
of the analogues for 1 to 3 days.
[3H]PDB Binding Assay. [3H]PDB was used in the binding experi
ments because this diester, which is also active in vivo, is less lipophilic
4233
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M. C. Cabof
than is TPA and thus favorably suited for specific binding studies (18).
Control and experimental cells were harvested by centhfugation and
washed twice in RPMI Medium 1640. [3H]PDB binding to intact cells was
Table 1
General structure of phospholipids
earned out essentially as detailed by Solanki er al. (43). Cells (1 to 2 x
106 per 0.25 ml medium per tube) were incubated with [3H]PDB (27 nw
H2C-O-C-R,
final concentration) in an oscillating ice bath for 45 min. Polypropylene
test tubes (1.5 ml) were used in all binding assays (Brinkmann Instrument
Co., Inc., Westbury, N. Y.). Labeled and unlabeled PDB was added to
the reaction in dimethyl sulfoxide so that the final concentration of solvent
was always 1.2%, and the samples were dispersed on a vortex mixer.
The reaction was terminated by adding 0.5 ml of ice-cold phosphatebuffered saline, and the cells were harvested by centrifugation and
washed (Eppendorf Model 5414 centrifuge; Brinkmann Instrument Co.
Inc.). All harvesting and cell washing was carried out at 4°.The final cell
pellets were dissolved in 1 ml of 1% sodium dodecyl sulfate: 10 HIM
dithiothreitol and assayed for radioactivity using Aquasol (New England
Nuclear). Cells were counted before each assay to ensure that approxi
mately equal cell numbers (control and analogue supplemented) were
used within each experiment. Specific [3H]PDB binding, which ranged
between 52 and 83% of the total, was calculated as the difference
between [3H]PDB bound in the absence and in the presence of 30 UM
and bas>eanalogues
O
u
R2-C-O-C
O
n
H2C-0-P-X
cr
Structure
Choline
DMEA
MEA
Ethanolar-.ne
3-AP
IPE
*(CH3)3
_O— CHr-CHz— NHCH3
—O—
CHz—CHz—CHz—NHj
CH,
I
O—CHj—CH?—
NH—C —H
I
CH,
* X, phospTK)tipkJbase.
unlabeled POB. PDB was soluble at 30 MMas evidenced by radioassay
counting of replicate aliquots of a cell-free reaction mixture.
choline; cell viability in choline-free medium fell sharply between
24 and 48 hr of incubation; however, the addition of choline,
DMEA, or MEA (40 fÃ-g/m\culture medium) sustained cell viability,
supplemented cells were extracted by a modified method of Bligh and
although cell growth was variably influenced, depending on the
Dyer (4) in which the met hano I contained 2% glacial acetic acid. Aliquots
analogue supplemented. IPE and 3-AP appeared to be toxic over
of the lipid extract were applied to Silica Gel G or Silica Gel H thin-layer
a 3-day exposure period; thus, cells supplemented with these
Chromatographie plates and resolved in the solvent systems described
previously (21 ). P-IPE was separated from total phospholipids on Silica
analogues were analyzed after a 40- to 42-hr incubation. Chart
1 depicts the effect of polar-head-group analogues on HL-60 cell
Gel H plates developed in either chloroform:methanol:ammonium
hy
droxide (65:35:5, v/v) or chloroform:methanol:glacial
acetic acid (50:25:8,
growth. HL-60 cells maintained in serum-free medium (control)
v/v). Commercial standards were used to identify P-DMEA and P-MEA;
grew at a reduced rate compared to cells grown in medium
the identification of P-3AP and P-IPE was based on standards synthe
containing 16% fetal bovine serum (data not shown). Over a 3sized via transphosphatidylation using phospholipase D (50). Verification
day growth period, the cell population of serum-free cultures
of cell-associated P-DMEA was also accomplished by HPLC on a Becktripled, wheres the cell number in serum-rich cultures increased
man Model 324 M system fitted with a 250- x 4.6-mm Ultrasphere-Si (5
6-fold. As shown in Chart 1, base analogue supplementation
urn) column using 96% isopropyl alcohohhexane (1:1) and 4% water with
a linear gradient to 8% water over a 15-min postinjection period. The
resulted in reduced cellular growth rates. IPE had the most
fraction eluting with a retention time similar to commercial egg P-DMEA
profound influence, and by 1 day cessation of growth occurred.
3-AP and MEA exposure for 2 days and DMEA supplementation
was collected and rechromatographed on thin layer, and the phosphorus
was measured (37). For quantitative analysis of cellular phospholipids,
over a 3-day period resulted in growth, which was 83, 81, and
total lipids were resolved in the appropriate solvent system and, after
72% of control cells, respectively. MEA supported cell growth
visualizing on the chromatogram by H2SO4 charring, the individual spots
for approximately 2 days, although growth was diminished
were scraped into test tubes for direct phosphorus analysis (37).
thereafter. Percentage of cell viability was determined in the 3Phorbol Ester Degradation Assay. [20-3H]Phorbol-12-myristate-13day cultures (control [94.3 ±0.9 (S.D.)], DMEA [92.1 ±1.3],
acetate (6.5 Ci/mmol; New England Nuclear) was used to determine the
MEA [90.1 ±1.7]) and 2-day-exposed cultures (3-AP [90.1 ±
stability of phorbol esters exposed to HL-60 cells. In vitro incubations
0.9], IPE [82.6 ±4.9]). Thus, cell viability in analogue-supple
were carried out in 1-dram glass vials at 37°in a total volume of 0.2 ml
and contained 0.1 ml 0.25 M Tris-HCI buffer (pH 7.6) and either whole
mented cultures, for the periods designated, was only 2.2 to
11.7% lower than control. Cell morphology in DMEA cultures
cells or a cell sonicate as the enzyme source (0.5 to 1.0 mg protein). The
was identical with that of control populations; however, mainmixture was preincubated for 3 min before the reaction was initiated by
injecting 5 pi of acetone containing [3H]TPA (10 pmol) and was incubated
tainence of cells in MEA, 3-AP, and IPE media produced cells of
for 30 min. Details of this assay procedure have been described by
irregular shape, deviating from the roundness characteristic to
Lackey and Cabot (30). Radiolabeled reaction products (TPA, phorbolnonsupplemented
cells. The data of Chart 2 show that, when
12-monomyristate, phorbol-13-monoacetate)
were resolved by thin-layer
HL-60 cells are grown in the presence of base analogues, they
chromâtography and quantitated as described by O'Brien and Diamond
readily incorporate these compounds into cellular phospholipids.
(33, 34).
DMEA was utilized to the greatest extent; after a 3-day exposure
period, 52% of the cellular phospholipids contained the DMEA
RESULTS
base. Exposure of cells to MEA, 3-AP, or IPE resulted in the
The analogues shown in Table 1 were incubated with HL-60 synthesis of cell-associated P-MEA, P-3AP, and P-IPE, account
cells in serum-free, choline-free medium to determine if the ing for 44, 35, and 22.5% of the total cellular phospholipids,
compounds would support cell growth in the absence of choline respectively. Interestingly, the formation of the phospholipid an
and be utilized by the cell for the synthesis of intracellular alogues was accompanied by a concomitant, pronounced reduc
phospholipid. HL-60 cells showed a definite requirement for tion in the levels of intracellular choline and ethanolamine glycerIdentification of Cellular Phospholipids. Cells were washed twice in
ice-cold phosphate-buffered saline. Lipids from control and analogue-
4234
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VOL. 43
Modulation of Phorbol Diester Binding
60
7 -
oÃ-—L
o e
Incubation Time (days)
Chart 1. HL-60 cell growth as influenced by base group analogue supplemen
tation. Cells were seeded (2.5 x ICC/dish) in 5 ml of serum-free RPMI Medium
1640 (control) or in 5 ml of serum-free, choline-free RPMI Medium 1640 containing
analogue (40 fig/ml). At the times indicated, the cells were harvested, washed, and
counted using a hemocytometer. Cell viability was determined by trypan blue
exclusion. Cell number refers to viable cells only.
24
48
72
Incubation Time (hr)
Charts. Alteration of HL-60 cell phospholipid composition during increased
periods of incubation with DMEA. Cells, seeded at 4 x lO'/flask, were cultured in
the presence of DMEA (40 »»g/ml
in choline-free medium) for the times indicated.
Total lipids were resolved by thin-layer chromatography, and the phospholipids
were quantitated by P, determination as stated in "Materials and Methods." Exper
iments were carried out in duplicate, and values differed <5% from the mean. PE,
ethanolamine glycerophospholipids; PC, choline glycerophospholipids; SPM, sphin
gomyelin.
99BO4540i
occurred from 48 to 72 hr. The data of Chart 3 provide additional
information on the disappearance rates of the naturally occurring
choline- and ethanolamine-containing phospholipids. A significant
decline in endogenous choline glycerophospholipids ensued dur
ing the first 24 hr, paralleling the increase in cellular P-DMEA,
38I
whereas the decline in ethanolamine glycerophospholipids was
3DS
less pronounced from 6 to 72 hr. Total cellular lipids were
separated and analyzed by high-performance liquid chromatog
25|203,610B
raphy as an alternate method to verify the presence of P-DMEA.
P-DMEA eluted with a retention time of 16 min, identical to
commercial P-DMEA; this fraction was collected, and phospho
1I£o.£-O.1£r~uE£-O.!•ï\\;;'ift\£%-a-\1f£C£-ïi¿—__£.£-Õ_*_£i—r-.£~Õ-----_ri!
lipid Pi analyses showed that it accounted for 51 % of the total
phospholipid P,.
Having shown that HL-60 cells utilize base group analogues
0-----__i_
Control
DMEA
MEA
3-AP
IPE
for the synthesis of cellular lipid, experiments were carried out
Chart 2. Phospholipid composition of HL-60 cells cultured in the presence of
to determine the effect of this modification on phorbol diester
base group analogues. Cells (4 x I06/flask) were maintained in the absence or
binding. The data of Table 2 show that cells grown in either
presence of analogue (40 pg/ml in choline-free medium) for 3 days (OMEA, MEA)
DMEA- or MEA-rich medium bind significantly more [3H]PDB
or 2 days (3-AP, IPE). Control cells were cultured in choline-containing medium.
The cells were harvested, and the lipids were extracted and analyzed for phosphothan do control cells. Specific [3H]PDB binding was 214 and
-li
-
lipid P, as described in "Materials and Methods." The data represent the average
of duplicate determinations from 2 independent experiments; values differed <10%
from the mean. LPC, lysophosphalidylcholine; SPM, sphingomyelin; PC, choline
glycerophospholipids; PS, serine glyccrophospholipids; PI, inositol glycerophospholipids: PE, ethanolamine glycerophospholipids.
ophospholipids. This effect was most evident in cells grown with
DMEA, MEA, and 3-AP and resulted in a 3- to 2.1-fold decrease
in the amount of endogenous choline glycerophospholipids and
a 2.0- to 1.7-fold decrease in cellular ethanolamine-containing
phospholipids.
In order to determine the length of time necessary to permit
maximal incorporation of base group analogues into HL-60 lipids,
cells were exposed to DMEA for varying time periods and
analyzed for P-DMEA. The data of Chart 3 show increased
formation of P-DMEA through 48 hr; however, high amounts of
analogue were incorporated at early times. After a 6-hr exposure
to DMEA, approximately 30% of the cellular phospholipids con
tained the base analogue. A plateau in DMEA incorporation
SEPTEMBER
1983
197% higher than control binding in DMEA- and MEA-exposed
cells, respectively. Specific binding of [3H]PDB to cells grown
with the unnatural polar-head-group analogues, 3-AP and IPE,
was greatly reduced compared to control cells. Cells containing
P-3-AP showed the greatest suppression, with binding account
ing for only 27% of that of the nonsupplemented cells, and cells
exposed to IPE over a 2-day incubation period showed specific
[3H]PDB binding that was 37% of control values. Because phor
bol diesters are lipophilic, changes in cellular lipid composition
could influence the degree of specific versus nonspecific [3H]PDB binding. Therefore, it was important to examine whether
base group analogue assimilation effected any change in non
specific binding. In control and supplemented cells, specific
[3H]PDB binding ranged from 52 to 83%; between experimental
groups, some differences in the amount of specific binding were
noted (Table 3). Specific [3H]PDB binding was highest in MEAtreated cells and lowest in 3-AP- and IPE-treated cells; however,
4235
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M. C. Cabot
Table 2
Effect of base analogue supplementation on specific [3H]PDB binding to HL-60
220
cells
Cells were grown for 3 days in serum-free medium or in serum-free, cholinefree medium plus DMEA or MEA (40 ^g/ml). [3H]PDB binding was carried out on
the washed cells as detailed in "Materials and Methods."
Specific [3H]PDB
Culture additions
binding
(cpm/106 cells)
None
DMEA
MEA
a Mean ±S.D. of 3 to 6 separate experiments.
731 ±36a
1564 ±77
1438 ±79
Table 3
Erfecf of base analogue supplementation on the degree of specific [3H]PDB
binding to HL-60 cells
Values for specific binding were calculated on a percentage basis using cpm
[3H] per 10" cells. Specific binding is defined as the difference between [3H]PDB
bound in the absence and in the presence of 30 UM unlabeled PDB.
Culture additions
None
DMEA
MEA
3-AP
IPE
67.8 ±4.7" (10)"
63.9
79.3
57.2
59.8
±5.5
±4.5
±4.4
±0.8
(5)
(5)
(3)
(3)
" Mean ±S.D.
" Numbers in parentheses, number of experiments.
the changes in specific binding that accompany altered phospholipid composition were not major.
Exposing HL-60 cells to polar-head-group analogues produces
analogue-containing phospholipids with a concomitant decrease
in the endogenous natural phospholipids (Charts 2 to 3). To
distinguish between the effects of cell-associated phospholipid
analogue and natural phospholipid modification on [3H]PDBbind
ing, an analogue-free culture system that would modify the levels
of choline- and ethanolamine-containing glycerophospholipids
was sought. As noted previously, HL-60 cells display a definite
requirement for choline and could not be cultured in choline-free
medium for more than 24 to 48 hr. Maintenance of HL-60 cells
in choline-free medium for only 24 hr caused a 23% reduction in
cellular choline glycerophospholipids and a 27% increase in the
ethanolamine glycerophospholipid component. When tested for
specific binding, these cells bound [3H]PDB in amounts identical
with those for control cells maintained in choline-containing me
dium. Additionally, HL-60 cells did not grow during the 24-hr
period in the absence of choline.
The data of Chart 3 show that a rapid assimilation of DMEA
occurs in HL-60 cells after only 6 hr of exposure. Thus, it was
of interest to correlate the time of DMEA exposure with the
effects on specific [3H]PDB binding. As shown in Chart 4, the
binding capacity of the cells was increased with increased ex
posure time to DMEA; the most pronounced effect occurred
between 6 and 24 hr (60% > control values). Although, by 6 hr,
the cells contain high amounts of P-DMEA (Chart 3), the specific
binding of [3H]PDB was not altered at early times. Exposure of
cells to 3-AP or IPE (40 MQ/ml)for 18 hr did not significantly
modify [3H]PDB specific binding compared to control nonsupplemented cells.
The binding parameters of phorbol diesters could likely be
influenced by the stability of ligand in the presence of cells or
cellular material. In order to determine if HL-60 cells degrade
phorbol esters, [3H]TPA was incubated with whole-cell or cellfree homogenates. Neither cell preparation, after a 30-min incu4236
24
% of specific binding
48
Incubation Time (hr)
Chart 4. Effect of increased exposure time to DMEA on the specific binding of
[3H]PDB to HL-60 cells. Cells were seeded either in choline-containing medium
(controls) or choline-free medium plus DMEA (40 jig/ml) and incubated for the times
indicated. At each time point, both control and DMEA-treated cells were harvested,
and specific binding of | 'H |PBD was determined as described. Experimental values,
from duplicate experiments, were within 10% of the mean.
bation at 37°,demonstrated esterase or lipase activity as shown
by the absence of labeled products (phorbol-12-monomyristate,
phorbol-13-monoacetate) in the reaction. Using the same assay
system, we have recently demonstrated the presence of serum
enzymes that hydrolyze TPA (30).
DISCUSSION
Both natural and unnatural analogues of phospholipid-polarhead groups are transported across the plasma membrane and
utilized by cultured HL-60 cells for the synthesis of intracellular
phospholipids. This experimental model was used to investigate
the influence of lipids on phorbol diester binding to intact cells.
Previous studies have shown that ligand binding to cell receptors
is sensitive to changes in the lipid composition of membranes,
suggesting that lipids are important constituents of binding sites
or share in the government of ligand-receptor interaction. Treat
ment of cells and membrane preparations with phospholipases,
enzymes specific for the catabolism of phospholipids, alters the
receptor binding of insulin and glucagon (14) and abolishes the
specific binding of gonadotropin-releasing hormone agonist and
antagonist (22). From the work of McCaleb and Donner (31), it
was concluded that the polar head groups of phospholipids
within the plasma membrane influence the binding properties of
the hepatic insulin receptor. In a like manner, binding of phorbol
diesters to paniculate preparations from rat brain is modified by
the action of phospholipase A2 (19). The present study shows
that culturing HL-60 cells with analogues of the phospholipid
polar head groups produces altered [3H]PDB binding compared
to control, nonsupplemented cells. DMEA and MEA base groups
were used in this study because their incorporation into lipids
would produce P-DMEA and P-MEA; these phospholipids are
intermediates of phosphatidylcholine biosynthesis and are syn
thesized during the stepwise methylation of phosphatidylethanolamine (8). It has further been shown that the process of
phospholipid methylation alters membrane-related biological sig
nal transmission, as in the interaction of cell surface receptors
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VOL. 43
Modulation of Phorbol Diester Binding
with spécifiemolecules (23,24,45), and that phospholipid methylation is stimulated in HL-60 cells after exposure to phorbol
diesters (25). It was thus of primary interest to test the effects
of membrane-associated methylated phospholipids on [3H]PDB
binding to intact cells. The results show that stimulated binding
of [3H]PDB occurred only in those cells enriched with the DMEAand MEA-containing phospholipids.
To demonstrate a modulatory role for phospholipids as regu
lators of phorbol diester binding, this work sets forth the initial
observation that maintenance of HL-60 cells in a culture system
devised to alter cellular lipid composition influences the specific
binding of [3H]PDB. The growth state of analogue-supplemented
HL-60 cells could account for some of the differences noted in
PDB binding, although growth cessation did not correspond with
either increased or decreased PBD binding (relative to control).
Nontransformed and transformed AKR-2B and C3H/MCA-58
cells exhibit a marked reduction in epidermal growth factor
binding when arrested due to nutrient deficiency (35). Further,
analogue addition for the periods indicated did not promote
cytotoxicity as evidenced by viability data.
It has recently been demonstrated that the phorbol diester
receptor (rat brain particulate fraction) copurifies with protein
kinase C (32) and that phospholipid-dependent protein kinase C
(human platelets) is activated by phorbol esters (12). Although it
has not been shown that the phorbol esters receptor is plasma
membrane associated, studies strongly suggest that TPA acts
directly on cell surface membranes (5). Both live and glutaraldehyde-fixed cells bind [3H]PDB similarly (41 ), and phorbol esters
bind to artificial phospholipid membranes in a manner close to
that for the parameters observed for biological membranes (15).
The involvement of phospholipid-dependent protein kinase C in
phorbol ester interaction lends significance to the role of lipids in
TPA-elicited reactions. These data, using living cells, describe
the potential involvement of phospholipids in the regulation of
phorbol ester binding. It remains to be determined if the observed
alterations in binding can be related to changes in cellular sen
sitivity to TPA.
12.
13.
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VOL. 43
Effect of Cellular Phospholipid Modification on Phorbol Diester
Binding
Myles C. Cabot
Cancer Res 1983;43:4233-4238.
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