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Effect of n-3 and n-6 Fatty Acids on Proliferation and Differentiation
of Promyelocytic Leukemic HL-60 Cells
By Hanne S. Finstad, Svein 0. Kolset, Jdrn A. Holme, Richard Wiger, Ann-Kristin Ostlund Farrants,
Rune Blomhoff, and Christian A. Drevon
Flow cytometric analyses showed
that in cultures incubated
Promyelocytic leukemic HL-60 cells
were incubated with difwith AA or EPA, the proportion of cells in the G, phase of
ferent fatty acids. Arachidonic acid
(AA; 20:4,n-6) andeicosathe cell cycle increased. Similar effects were observed with
pentaenoic acid(EPA; 20:5,n-3) were the most potent inhibiRA. By flow cytometry and light scatter analysesit could be
tors of proliferation in a dose-dependentway. Retinoic acid
shown that AA made 8% of the cells apoptotic and 7% ne(RA) was used as a positive control. Inhibitors of cyclooxycrotic. The corresponding numbers were 21% and 10% for
genase and lipoxygenase oraddition of antioxidantsdid not
The
RA-treatedcells, and 19% and 32% for EPA-treated cells.
influence the effect ofEPAor AA on cell proliferation. Inpresent study showsthat AA and EPA reduce the proliieracreased capacity to generate superoxide anions after phoris mediated bymechanisms
bo1 ester treatment and a reduced serglycin messenger RNA tion rate ofHL-60cells.This
independent of eicosanoids or lipid peroxidation products
level in cells treated with AA orEPA indicated that these
and is due to effects both on apoptosislnecrosis and cell
fatty acids induced differentiation in HL-60 cells similar to
differentiation.
that induced byRA. However, down-regulationof the c-myc
0 1994 by The American Society of Hematology.
mRNA level, also typical for differentiation with RA in HL60 cells, was not observed in cells incubatedwith AA or EPA.
S
TRONG EVIDENCE exists that a high intake of polyunsaturated fatty acids of the n-3 series may have beneficial health effects, particularly with reference to coronary
heart di~ease."~
A series of studies have demonstrated that
these fatty acids have many biological effects, ranging from
decreasing the levels of serum triacylgly~erols~
and reducing
blood p r e ~ s u r e to
~,~
modulating symptoms in patients with
inflammatory disease^."^ Effects of polyunsaturated fatty
acids on tumor development have recently been evaluated
in experimental studies. Fatty acids from the n-6 series may
enhance tumor growth.'"," On the other hand, n-3 fatty acids
may inhibit the growth of tumor cells both in vivo and in
vitro,'*." decrease metastasi~'~
and cachexia in animals with
tumors,Is and increase cytotoxic effects of some chemotherapeutic agents.IbIn the future, dietary intake of n-3 fatty acids
may be recognized as an inhibitor of tumor growth and may
be used to increase the efficiency of chemotherapy.
Several explanations exist for how n-3 fatty acids may
affect cell proliferation; docosahexaenoic acid (DHA) may
increase membrane permeability in tumor cells, and render
the cells more susceptible to anticancer drugs or to destruction by the immune system.17 Conversion of fatty acids to
eicosanoids or lipid peroxidation products may influence cellular processes such as proliferation, chemotaxis, and motility.'"20 Influence by fatty acids on signal transduction
through interactions with protein kinases, lipases, or G proteins have become a subject of
Furthermore, influence on gene transcription by fatty acids andor
their derivatives through interaction with nuclear receptors
in the peroxisomal proliferator activated receptor (PPAR)
family, may be of importance.*3
The human promyelocytic cell line HL-6024.25
has been
used to study the effect of different agents on differentiation
and proliferation of transformed myeloid cells.26When the
cells are exposed to agents such as retinoic acidz7(RA) or
dimethyl sulfoxide,2Rthey differentiate into granulocytelike
cells. Exposed to phorbol esters, the cells differentiate to
macrophagelike cells.29In the present study, we used HL60 cells to investigate possible mechanisms responsible for
the inhibition of cell proliferation by very long chain polyunsaturated fatty acids. We describe the effects of arachidonic
Blood, Vol 84, No 11 (December l ) , 1994: pp 3799-3809
acid (AA; n-6)and eicosapentaenoic acid (EPA; n-3) on
proliferation, differentiation, necrosis, and apoptosis and
compare these to the effects of RA. Results show that polyunsaturated fatty acids inhibit the proliferation rate and induce differentiation, necrosis, and apoptosis in HL-60 cells,
but todifferent extents and possibly through different mechanisms.
MATERIALSANDMETHODS
Materials. Stearic acid (SA), oleic acid, (OA), linoleic acid
(LA), AA, adrenic acid (AdA), a-linolenic acid (LnA), EPA, DHA,
all-trans RA, bovine serum albumin (BSA; essentially fatty acid
free), nitroblue tetrazolium (NBT), trypan blue, phorbol 12-myristate
13-acetate (PMA), guanidinium isothiocyanate, 3(N-morpholino)
propane-sulfonic acid, formamide, formaldehyde, Hoechst 33258,
cytochrome c, superoxide dismutase, indomethacin, nordihydroguaiaretic acid (NDGA), vitamin E, and butylated hydroxytoluene (BHT)
were all purchased from Sigma Chemical CO,St Louis, MO. Agarose
was obtained from FMC Bioproducts, Rockland, MN, and (3H)thymidine and (a"P)dCTP from DuPont-NEN Research Products, Du
Pont Scandinavia AB, Stockholm, Sweden. The probe for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was supplied from
Clontech Laboratories, Palo Alto, CA. The third exon of the c-myc
gene was obtained from Dr Kiil Blomhoff. The open reading frame
of serglycin was obtained via polymerase chain reaction from a U-
From the Section for Dietary Research, Institute of Nutrition Research, University of Oslo, andthe Department of Environmental
Medicine, National Institute of Public Health, Oslo, Norway.
Submitted November IO, 1993; accepted August I , 1994.
Supported by grants from the Norwegian Cancer Society, Nansen
Foundation,Anders Jahres Foundation, Pronova Biocare A/S, FamilienBlix forskningsfond, andFreia Medicinske Fond. H.S.F. is a
fellow of the Norwegian Cancer Society.
Address reprint requests to Christian A . Drevon, Section for Dietary Research, Institute for Nutrition Research, University of Oslo,
PO Box I 1 17, Blindern, 0317 Oslo, Norway.
The publication costs of this article were defrayed in part by page
charge puyment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/84/ 1-0035$3.00/0
3799
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3800
937 library.'" A leukotriene B,-kit was obtained from Amersham,
UK.
Cells. Allhuman cell lines used were purchased from ATCC,
Rockville, MD, except for the monocytic cell line U937-clone I ,
which was obtained from Dr K. Nilsson, University of Uppsala,
Sweden. U937-1 is the most mature of the U937 sublines available.
HL-60 cells were cultured in Iscove's medium from Whittaker Bioproducts, Walkersville, MD. The medium was supplemented with
10% heat-inactivated fetal calf serum (FCS; GIBCO, Paisley, Scotland), l-glutamine (2 mmol/L), and gentamycin (0.1 mg/mL). U9371 cells and the monocytic cell line THP-l were cultured inRPM1
1640 with 10%FCS, 100 IUlmL penicillin, 100 pg/mL streptomycin,
and 2 mmoVL l-glutamine (all from Whittaker). The human colon
carcinoma cell line CaCo-2 was cultured in Dulbecco's modified
Eagle's medium with 20% FCS, 10 pg/mL of insulin, 1% nonessential amino acids and antibiotics, and l-glutamine as for U937- 1 and
THP-l cells. Fatty acids were presented to the cells complexed to
BSA in a molar ratio of 2.5:1 .'l HL-60 cells were routinely kept in
a logarithmic phase at 0.2 to 0.8 X lo6 cells/mL, and all experiments
were performed before passage 50. For experiments, cells were
seeded at a density of 0.3 X lo6 cells/mL and counted in Burkner
chambers. Exclusion of trypan blue was used asa criterion for
viability.
(-'H)thymidine incorporation. Cells were incubated for different
incubation periods in the absence and presence of fatty acids in 96well microtiter plates from Costar, Cambridge, MA. An aliquot of
0.4 pCi ("H)thymidine was added to each 200-pL culture. After 1
hour of incubation, cells were harvested and lysed, and DNA was
transferred to filters by use of a cell-harvesting system from Flow
Laboratories/Skatron A / S , Lier, Norway. After transferring filters
to scintillation vials and adding scintillation fluid, ('H)thymidine
incorporated into DNA was measured in a Packard Tri-Carb 1900
TR scintillation counter (Packard, Meriden, CT). Results, presented
as the percentage of ('H)thymidine incorporation in untreated cells,
were calculated from the mean of triplicates, and the standard errors
of the means (SEMs) were 10% to 15%.
NBTand respiratory burst. The capacity of differentiated HL60 cells to phagocytose and reduce NBT and to perform respiratory
burst was used as a criterion of differentiation. These activities were
initiated as described
with minor modifications. Cells
(0.7 X lo6) were sedimented via centrifugation at 1,OOO rpm for 10
minutes and resuspended in 200 pL ice-cold PBS containing 1.5
mg/mL of cytochrome c. After addition of 2 pL phorbol-12-myristate-13-acetate (20 pg/mL), the suspension was incubated at 37°C
for 15 minutes and stopped by transfer to ice. The suspension was
centrifuged at 1,000 rpm for I O minutes. Absorbance of the supematant at 550 nm was measured in a Shimadzu UV-2100 spectrophotometer (Kyoto, Japan), and the increase in absorption was compared
with that of a solution of cytochrome c kept on ice.
Flow cyrometry. Blue fluorescence from Hoechst 33258 was
measured at SS0 nm in an Argus 100 flow cytometer (Skatron .W
S). The percentage of cells in different phases of the cell cycle was
estimated from the Hoechst 33258 histograms using the Multicycle
program (Phoenix Flow Systems, San Diego, CA). The percentages
of viable, apoptotic, and necrotic cells were calculated via measurements of fonvard and side light scatte?' proportional to cell diameter
and internal granularity, respectively.
Northern blots. Total RNAwas extracted via the guanidinium
isothiocyanate method with centrifugation through a CsCl gradient
and subsequent phenol/chloroform extraction^.'^ Concentration of
RNA was determined by absorption at 260 nm. Twenty micrograms
total RNA was denatured for 15 minutes at 50°C in 50% formamide
and 6% formaldehyde, followed by15 minutes on ice, and then
transferred to a 1% agarose gel with 2.2 molL formaldehyde dissolved in 1 X MOPS (lox MOPS = 0.5 molL 3(N-morpholino)
FINSTAD ET AL
propane-sulfonic acid, pH 7.0, and 0.01 mol& EDTA, pH 7.5) for
size fractionation. The presence of equal amounts of RNA in each
lane was ensured by inspection after ethidium bromide staining.
RNA was then transferred using a Pharmacia vacuum blotter (Pharmacia Biotechnology, Oslo, Norway) to a Zeta-Probe membrane
from Bio-Rad. Membranes were prehybridized at 65°C for 2 hours
under standard hybridization conditions (2 g/L polyvinylpyrrolidone,
2 g/L Ficoll-400, 2 g/L BSA, 0.05 m o a Tris-HCI(pH 7.5). 1
m o m sodium chloride, 2.2 mmol/L sodium pyrophosphate. and I 8
sodium dodecyl sulfate) with 100 pg/mL of denaturated salmon
testis DNA. The probe was labeled with Megaprime DNA labeling
system 1606 from Amersham, Buckinghamshire, England. and hybridized to the membrane over night under the same conditions as
described above. The membranes werewashedfor IS minutes at
room temperature with 2X SSC/O.l% SDS, 0.SX SSC/O. 1 5 % SDS,
and 0.1 X SSC/O.l % SDS successively. The size of the messenger
RNA (mRNA) was determined with reference to 18s and 28s recombinant RNA (rRNA), which were visualized by ethidium bromide
staining. After autoradiography (Fuji medical X-ray film, Japan) the
densities of the signals were determined by scanning the film with
a Bio-Image scanner and analyzing it with a programfrom BioImage (Millipore, Bedford, MA). To calibrate themRNA signal
levels with an internal standard in addition to total RNA, filters were
stripped and rehybridized with a probe for G3PDH, whichisregarded as a "housekeeping gene" because expression of this gene
often remains refractory to many common gene transcription-inducing agents."
RESULTS
A
concentration of 60 pmoVL was chosen for all fatty acids,
and the incorporation of ("H)thymidine in HL-60 cells was
measured after 1, 2, and 3 days of exposure. RA ( 1 pmol/
L) was used as a positive control because it inhibits proliferation and induces differentiation in HL-60 cells.27After I day
of incubation, RA reduced the incorporation of ('H)thymidine by almost 60% (Fig 1 A), and after 3 days of exposure,
the incorporation was less than 20% of that observed in
untreated cells. SA(18:O) inhibited (3H)thymidineincorporation approximately 20% after both 2 and 3 days of incubation. DNA synthesis in cells incubated with the monounsaturated OA (18:1) was not different from that observed in
untreated cells.
Polyunsaturatedfatty acids fromthe
n-6 series-LA
(18:2), AA (20:4), and AdA(22:4)-werealso
tested for
their ability to inhibit DNA synthesis (Fig IB). LA did not
inhibit incorporation of (")thymidine after 1 and 3 days of
exposure. In contrast, AdA reduced (3H)thymidine incorporation by 40% after day 1 and by 60% at days 2 and 3. AA
was the strongest inhibitor among the n-6 fatty acids tested:
after 1 and 3 days of incubation, (3H)thymidineincorporation
was reduced by 85% and 90%, respectively.
Of the fatty acids in the n-3 series (Fig 1C), LnA (18:3)
appeared to be the weakest inhibitor of (3H)thymidineincorporation, which was 80% of that observed in untreated cells.
However, both DHA (22:6)and EPA (205) caused a marked
reduction in theincorporation of (3H)thymidine: 60% and
70% after 1 dayand 85% and 95%, respectively, after 3
days.
The amount of BSA used to present the fatty acids to the
cells, reduced (3H)thymidine incorporation by 10% to 30%.
Effect of fatty acids on (3H)thymidine incorporation.
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3801
FATTY ACIDS AND HL-60 CELLS
n-3 Fatty acids
n-6 Fatty acids
Controls
B
m
I
I
I
I
1
2
3
1
AA
2
3
Incubation time (days)
1
2
3
Fig 1. Effect of fatty acids on ('H)thymidine incorporation. HL-60 cells were incubated in the presence of 60 pmol/L of the following fatty
acids: oleic acid (OA) and stearic acid (SA) (A); linoleic acid (LA), adrenic acid (AdA), and arachidonic acid (AA) (B); and a-linolenic acid (LnA),
docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) (C). Retinoic acid (RA) was used as a positive control (A). After 1,2, and 3 days
of incubation, ('Hlthymidine incorporation was measured after 60 minutes exposure t o ('Hlthymidine. Similar results were obtainedin four
separate experiments.
During cell-counting studies, BSA alone, in concentrations
from 6 to 96 pmol/L, reduced cell numbers by up to 20%
(not shown). However, the effects observed with BSA were
at all times far less than those observed with the very long
chain polyunsaturated fatty acids. Reduction in (3H)thymidine incorporation was thus observed in cultures incubated
with 60 pmoVL AdA, AA, EPA, and DHA. For further
studies, we chose to use AA and EPA because they were
the strongest inhibitors of (3H)thymidineincorporation.
Effects of EPA and AA on ('H)thymidine incorporation
and cell number. HL-60 cells were incubated with AA and
EPA in concentrations ranging from 15 to 240 pmol/L for
24 hours (Fig 2).At 15 pmol/L, no inhibitory effect on
(3H)thymidine incorporation was observed with either of
these two fatty acids. However, a 10%to 20% reduction of
the incorporation was observed with 30 pmol/L of both EPA
and AA, andthis effect increased further with higher concentrations. At 240 pmoVL, AA inhibited (3H)thymidineincorporation by 67%, whereas the corresponding decrease in
EPA-treated cells was 83%.
The effect of AA and EPA was also evaluated by cell
counting for 5 days. A 75% decrease was noted in the number of cells incubated with 1 pmol/L RA after 5 days as
compared withday 1 (Fig 3A). Cells incubated with 15
pmol/L AA (Fig 3B) or EPA (Fig 3C) had a growth curve
similar to that observed for untreated cells. However, when
cells were exposed to 30 and 60 pmol/L of the respective
fatty acids, a 40% to 50% reduction in the number of cells
was observed as compared with untreated cells after 5 days.
Exposure to 120 and 240 pmol/L AA for 5 days reduced
the number of cells by 71% and 82%, respectively. The
corresponding figures with EPA were 93% and 97%. Results
obtained by cell counting were thus in agreement with
(3H)thymidine incorporation studies; AA as well as EPA
inhibited (3H)thymidine incorporation and the proliferation
rate in a concentration-related manner. In both types of ex-
15
30
60240 120
Fatty acids @M)
Fig 2. The effect of AA and EPA on 13H)thymidineincorporation.
HL-60 cells were incubated with different concentrations of AA and
('Hlthymidine was
EPA for 24 hours, whereafter the incorporation of
measured. Experiments were performed four timeswith similar results.
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FINSTAD ET AL
3802
1
5
3
1
3
5
Incubation time (days)
1
3
5
Fig 3. The effect of AA and €PA on cell number. HL-60 cellswere incubatedin thepresence of 1 pmollL RA, 96 pmollL BSA (A), or different
concentrations of AA (B) and EPA IC), rangingfrom 15 to 240 pmollL, for 5 days. Each day cells
were taken out for counting in Bllrker chambers
after the addition of trypan blue. Experimentswere done twice with similar results.
periments, EPA was a more potent inhibitor than was AA
at the highest concentrations tested.
Effects of EPA and AA on other cell types. To evaluate
whether the inhibitory effects of polyunsaturated fatty acids
on cell proliferation were a general phenomenon, the monocytic cell lines U937-1, THP-l, and the colon carcinoma cell
line CaCo-2 were exposed to OA, AA, and EPA. From Table
1 it is evident that no decrease was seen in the incorporation
of (3H)thymidineafter 1 day of exposure to 60 pmol/L OA,
EPA, or AA in the three human cell lines tested. In contrast,
the proliferation of HL-60 cells was inhibited in the presence
of EPA and AA but not in the presence of OA. Pilot studies
with cell count experiments on the human acute myelogenous leukemia cell lines KG-l and KG-la showed no inhibition of proliferation using 60 pmol/L AA and EPA (results
not shown).
Flow cytometry. To study the inhibition of proliferation
byflow cytometry, cells were incubated with 120 pmollL
AA or EPA or 1 pmollL RA for 5 days. Cells were analyzed
daily for the percentage distribution in the various phases of
the cell cycle. Among untreated cells, 48% were in the GI
phase, 39% in the S phase, and 13% in the G2/M phase after
24 hours of incubation (Fig 4A). Only minor changes in this
distribution could be observed in control cultures during the
Table 1. Effect of Fatty Acids on ('H)Thymidine Incorporation
W of control) in Different Cell Types
Cell Line
CaCo-2
HL-60
THP-1
U-937-1
OA (60pmol/L)
107 103
100
99
98
103
AA (60 pmol/L)
106
52
107
97
EPA (60 prnol/L)
48
95
Abbreviations: OA, oleic acid;AA, arachidonic acid;EPA, eicosapentaenoic acid.
incubation period. Similar results were observed with cells
exposed to 48 p m o m BSA for 5 days (not shown). Treatment of HL-60 cells with 1 pmol/L RA (Fig 4B), increased
the percentage of cells in the GI phase to 83% after 1 day.
Concomitantly, the percentage of cells in the S and GJM
phases decreased to 9.6% and 7.5%, respectively. Through
the rest of the incubation period, an elevated proportion of
cells in the GI phase was observed in RA-treated cells.
An increased proportion of cells in the G, phase was also
observed after treating cells with AA or EPA (Fig 4C and
D). After exposure to 120 pmol/L AA for 1 day, the number
of cells in the GI phase was 58% and after 5 days 79%.
During the same period, the percentage of cells in the S
phase decreased from 30% to 10%.Similarly, after 1 day of
incubation with 120 pmol/L EPA, the percentage of cells in
the GI and S phases was 57% and 34%, respectively. After
3 days, 76% of the cells were in the G, phase compared
with only 62% with AA. After 5 days of exposure to EPA,
80% of the cells were in the GI phase. EPA was thus most
effective in causing an increase of cells in the G,phase with
a concomitantly reduced number of cells in the S and G2/M
phases.
Decreased proliferation and cell death. It is possible
that a decrease in the proliferation of HL-60 cells incubated
in the presence of AA or EPA could be attributable to initiation of apoptosis or necrosis. To investigate this possibility
further, cells treated with RA, AA, or EPA for 3 days were
analyzed by flow cytometry. By analyzing the light-scattering properties of the cells, it is possible to distinguish between apoptotic and necrotic cells. Little apoptosis or necrosis was observed in untreated cells (Fig 5A). When HL-60
cells were exposed to 1 pmollL RA for 3 days, a portion of
the cells went into apoptosis. The position of the apoptotic
cells is indicated for the RA-treated cells in Fig 5B.In contrast, both apoptosis and necrosis could be observed in HL-
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3803
FA‘TPI ACIDS AND HL-60CELLS
Untreated
100
B
A
80 m s
G2+M
60
40
20
0
1
2
3
5
4
1
2
Time (days)
3
4
Time (days)
5
EPA
AA
100
D
C
80
60
40
20
0
1
2
3
Time (days)
4
5
1
2
3
Time (days)
4
5
Fig 4. Flow cytometry. Symbol explanation is shown in the panel for untreated cells (A). HL-60 cells were incubated in the premnce of 1
pmol/L RA (B), 120 prnol/L AA (C), or 120 pmol EPA (D) for 1 to 5 days and analyzed for percentage distribution of cells in different phasee
of the cell cycle. Similar rerults were obtained in three separate experiments.
60 cells treated with 120 pmoVL AA or EPA. After AA
treatment only a minor portion of the cells were necrotic or
apoptotic (Fig K ) , whereas a larger percentage of HL-60
cells responded to EPA by going into either apoptosis or
necrosis. Data from Fig 5 were used to calculate the percentage of necrotic and apoptotic cells. From Table 2 it is evident
that after RA treatment 21% of the cells were apoptotic and
10% were necrotic, whereas the corresponding values for
cells treated with AA were 8% and 7%, respectively. However, when cells were exposed to EPA, 19%were apoptotic
and 32% were necrotic. There are, accordingly, large differences in the effects of RA, AA, and EPA on the induction
of apopotosis and necrosis in HL-60 cells.
CeZZ diflerentiution. HL-60 cells are bipotent cells and
can differentiate to either monocytedmacrophages or granul o c y t e ~ . ~The
” ~ ~differentiated monocytes and granulocytes
have the ability to reduce NI3T and to produce superoxide
anion upon stimulation with PMA. Cells were incubated with
120 p m o m AA or EPA or 1 pmoVL RA for 3 days. Among
untreated cells, 5% were NBT positive, and in cells treated
with 1 pmol/L of R A , the corresponding number was 14%.
Exposure to 120 pmoVL AA or EPA yielded 24% or 19%
NBT-positive cells, respectively (Fig 6A).
The same cultures were also tested for their ability to go
through respiratory burst, assessed by reduction of cytochrome c, after stimulation with PMA (Fig 6B). Incubation
with 120 pmoVLAA or EPA enhanced the reduction of
cytochrome c 4.2 and 4.7 times, respectively, when compared with untreated cells, whereas RA treatment increased
respiratory burst twofold. Addition of superoxide dismutase
resulted in a complete abrogation of cytochrome c reduction
both with fatty acids and RA (not shown).
Antioxidants and (3H)thymidine incorporation. To examine whether the reduced incorporation of (3H)thymidine
induced by AA and EPA wasdue to the generation of peroxidation products inhibiting DNA replication, the effect of
antioxidants in combination with fatty acid was investigated.
Increasing concentrations of vitamin E or BHT were added
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FINSTAD ET A L
3804
RA
Untreated
1
G2+M
AA
the cultures before thcadditionof
60 pmol/L AA or
EPA. Neither o f the two antioxidants affected the rcduccd
('H)thymidinc incorporation i n HL-60 cells exposed t o AA
and EPA for 24 hours (Fig 7A and B).
Eknsmoitlt a ~ t('H)lhmidirIc
l
inc,or/,orcltion. The ability o f AA and EPA to inhibit proliferation of HL-60 cells
nay be duc t o thc gcncration of their rcspcctivc cicosanoid
products, which may interfere with regulation of cell division. Two key en/,ymes i n the generation o f eicosanoids
(lipoxygenitse and cyclooxygenase) may be inhibited with
NDCA and indomethacin, respectively. Neither indornethacinnorNDGA
alleviated the inhibition of ('H)thymidine
to
Table 2. Percentage of Normal, Apoptotic, and Necrotic Cells After
3 Days of Incubation as Determined by Flow Cytometry
~~~
Normal
Apoptotic
Necrotic
Untreated
94
2
4
RA ( 1 pmol/L)
AA (120 pmol/L)
69
21
10
85
8
EPA (120 pmol/L)
49
19
7
32
~
Abbreviations: RA, retinoic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid.
incorporation by A A or EPA (Fig 8A and B). In contrast,
an additive effect of both AA and EPA with NDGA could
be observed. Furthermore, NDCA alone promoted a dosedependent inhibition of ('H)thymidinc incorporation. Control experimentsshowed that in both AA-trcatcdand untreated HL-60 cells the release of leukotriene B,, was inhibited when cultured in thepresence of NDCA (result not
shown). An increase in the level of eicosanoids after AA or
EPA treatment does not seem to be a rnechanisrn by which
thesefatty acidsdecrease theproliferationrate
of HL-60
cells.
Exp,cssiorl o/' c.-nlyc crrzd sc~rg/ycirzmRNA. HL-60 cells
contain amplified copieso f the c-myc gene,"' which is dowm
regulatcd during in vitro diffcrcntiation. We wanted to investigate whether the decrease in proliferation rate and increase
in capacity for oxidative burst was correlated to a decrease
in thc cxprcssion of c-myc in cells exposed to AA and EPA.
However,thelevel
of c-myc expressionwasnotreduced
alter treatment with 60 pnol/L AA or EPA for 2 days (Fig
9A). In contrast, the level o f c-myc was reduced to an undetectable level after exposure of the cells to 1 pmol/L RA for
the same time period. Using 120 pmol/L concentrations of
AA and EPA for 3 days did not lead to any down-regulation
of the c-myc mRNA.
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3805
FATTY ACIDS AND HL-60 CELLS
NBT-test
0
5
10
15
20
25
% NBT positives
4
30
Respiratory burst test
B
0
0.2
0.4
1
0.8
0.6
Reduction of cyt c (Aabs,)
Fig 6. Col1differentiation. HL-60 cells were treated with 1 pmol/L RA, 120 pmol/L AA, or 120 pmol/L €PA for 3 days and subjectedto the
NBT tost (A) or the respiratory burst teat (B]. Similar results were obtained with both tests in three separate expdmentt.
calibrated with that of the G3PDH transcript, the serglycin
mRNA observed after treatment with60 pmol/L AA or EPA
or 1 pmolL RA was reduced by 6570, 80%, and 95%, respectively (Fig 9B).
Serglycin is the major proteoglycan in hematopoietic cells,
including HL-60 ~ e l l s . ~ Proteoglycan
~,~'
biosynthesis decreases in differentiated HL-60
and the expression
of serglycin has recently beenshown to decrease in the
monocytic cell line U-937 after differentiati~n.~'
We therefore investigated whether the expression of serglycin was
affected after treatment of HL-60 cells with AA or EPA for
2 days. Both fatty acids and RA promoted reduced expression of the serglycin transcript. When the signal levels were
DISCUSSION
Our findingsdemonstrate that verylong chain polyunsaturatedfattyacidsfromthen-6andthe
n-3 series hadthe
ability to reduce (3H)thymidine incorporation and prolifera-
60 .
40 .
20
-
D
-D
I
m
AA
-
-
0
EPA
n
-
0
5
20
Vitamin E (FM)
a
I
?
100
0
W
I
I
0.1
1
Bi"r
AAD
EPA*
10
(PM)
Fig 7. Effect of antioxidants and fatty acids on ('Hlthymidino incorporation. HL-60 cells were treated with 80 pmol/L AA or EPA and the
indcsted concentrations of vitamin E (A) and BHT (B). After 24 hours, the incorporation of (aH)thymidinewas measured. Exporimenta were
performed three times with similar results.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FINSTAD ET AL
3806
6
i
0
0.1
1
10
0
0.1
1
10
U
Indomethacin (PM)
NDGA (PM)
Fig 8. Effect of eicosanoid inhibitors andfatty acids on ('Hithymidine incorporation. HL-60 cells were incubated with 60 pmollL AA or €PA
and the indicated concentrationsof indomethacin (AI and NDGA (B). After 24 hours, the incorporation of ('Hfthymidine was measured.Results
are representativesof three separate experiments.
tion rate of HL-60 promyelocytic leukemic cells. Reports on
the effects of fatty acids on cancer cell proliferation are
contradictory. In some studies, only n-3 fatty acids inhibited
tumor cell proliferation, whereas n-6 fatty acids had noeffect
or even stimulated cell proliferation.",39 Other reports
showed inhibition of cancer cell proliferation with both n-3
and n-6fatty acids similar to our finding^.^' These discrepancies may be due to the use of different model systems, all
expressing different sets of proteins. Some systems may be
sensitive to n-3 fatty acids only, whereas others respond to
both n-3 and n-6 fatty acids. It has been reported that the
proliferation of T-lymphocytes is inhibited by n-3 as well
as n-6 polyunsaturated fatty acids4' Similar to the results
presented here, inhibitors of enzymes involved in eicosanoid
synthesis did not abrogate the reduced proliferation rate observed with long polyunsaturated fatty acids from both the
n-3 and the n-6 family, nor did the addition of antioxidants.
This finding is in contrast to several studies in which antioxidants or inhibitors of eicosanoid synthesis abrogated effects
observed with n-6 and n-3 fatty a ~ i d s . ' ~ . ~We
' . ~were
'
furthermore able to show thatthe inhibitory effects of AA and EPA
were restricted to HL-60 cells and not seen in two different
monocytic cell lines and one colon carcinoma cell line.
However, the data presented also showed that RA, AA,
and EPA induced both apoptosis and necrosis in HL-60cells.
After 3 days of RA treatment, 21% of the cells became
apoptotic and 10% necrotic, whereas AA treatment shifted
8% to apoptosis and 7% to necrosis. In contrast, EPA-treated
HL-60 cells contained 19% apoptotic and 32% necrotic cells.
The decrease in cell number observed both after EPA and
RA treatment (Fig 3) can to a large extent be ascribed to
the induction of cell death by these two different pathways.
However, it is interesting to note that HL-60 cells treated
withAA contained few necrotic cells and fewer apoptotic
cells than seen in the RA-treated cultures. Although all three
agents inhibited proliferation and induced differentiation to
a certain extent, their effects on cellular death processes
differed considerably. The differences in effects on apoptosis
and necrosis suggest that theyoperate, at least partly, through
different signal pathways.
Several experiments were performed to investigate
whether AA and EPA induced differentiation in HL-60 cells
similar to that of RA. Reduction of NBT and generation of
oxidative burst were higher in cells incubated with 120 pmol/
L AA or EPA for 3 days than in cells treated with 1 pmol/
L RA, indicating a higher degree of differentiation in cells
treated with fatty acids. Furthermore, the expression of serglycin, which is almost exclusively restricted to hemopoietic
cells, is a useful marker for differentiation of these cells.
Proteoglycan synthesis has been demonstrated to decrease
when HL-60 cells differentiate,3xand the mRNA level for
serglycin in HL-60 cells is similar to that found in cells from
patients with acute myelogenous leukemia.& If decrease in
the serglycin mRNA level is usedas a marker for differentiation, HL-60 cells differentiated in the presence of RA, EPA,
and AA. DHA combined with RA, has been found to increase the rate of differentiation in HL-60
supporting
the notion that very long chain polyunsaturated fatty acids
can influence the differentiation process in HL-60 cells.
Down-regulation of c-myc mRNA in differentiating HL60 cells is well documented and seems to be important in
the differentiation p r o c e ~ s . *Use
~ ~ of
~ ~c-myc antisense is
sufficient to induce differentiation in this cell line.47 Our
results show that only RA induced differentiation of HL-60
cells by that criteria. This is in contrast to what we observe
when respiratory burst, NBT reduction, and decrease in serglycin mRNA expression are used as parameters; all three
agents induce differentiation. However, the role of c-myc in
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
F A T W ACIDS AND HL-60 CELLS
3807
serglycin mRNA
c-myc mRNA
m
0.7
0.6
Fig 9. Effact of AA and EPA
on themRNA level of c-myc and
serglycin. HL-60 cells were incubatedwith
l pmollL RA, 60
pmollL AA, orEPA for 2 days,
whereafter the cells were harvested and mRNA isolated.
Northern blotanalyses were performed with cDNA probes for cmyc IA) and serglycin (B). The
autoradiograms displayed in the
upper panel were subject to
scanninganalyses. These data
were related to those of GBPDH
in the same blots, and the relative signal intensity plottedin
the lower part of each panel.
Experiments were performed
twice, with similar results.
I
0.35
i
0.3
0.5
0.25
0.4
0.2
0.3
0.15
0.2
0.1
0.1
0.05
0
0
a
a:
L
c
c
3
the regulation of differentiation and apoptosis is complex
and dependent on cell type and specificity of stimuli. Expression of c-myc also seems to be necessary for several, but
not all, forms of apoptosis?* It has been demonstrated that
activation-induced apoptosis in T-cell hybridomas was inhibited by c-myc antisense, whereas glucocorticoid-induced
apoptosis was unaffected by such treatment?’ In order to
define more clearly the differences between RA, AA, and
EPAon the differentiation and apoptosis of HL-60 cells,
studies are now being performed withflow cytometry together with measurements of mRNA levels of several genes
shown to be involved in these processes.
Several reports have appeared on the importance of activation of protein kinase C (PKC) during HL-60 differentiation.”.” Unsaturated fatty acids may also stimulate PKC,’*
and down-regulation of serglycin in HL-60 cells can be
caused by activation of PKC,” suggesting that this may be a
mechanism by which fatty acid reduced the level of serglycin
mRNA reported here. In pilot studies using a peptide inhibitor of PKC,we were unable to detect any effects on the
inhibition of proliferation byAA andEPA. There is also
another possible mechanism throughwhich AA and EPA
can regulate the serglycin mRNA level. In the promoter of
the serglycin gene, a response element for two types of nuclear receptors was fo~nd’~.’~:
PPAR and the retinoid X receptor (RXR). These two types of receptors recognize a
consensus sequence of six base pairs repeated twice with
one intervening base pair.Fatty acids can activate PPAR
and PPAR-like receptors found in humans, such as the NUCI
or thehumanperoxisomeproliferatoractivated
The reduced level of serglycin mRNA observed with AA
and EPAmay thus be caused by an interaction between a
fatty acid, a human analog to PPAR, and the serglycin gene
promoter. Similarly, RA may, if converted to the 9-cis form,
activate the RXR receptor and down-regulate serglycin transcription.
Fatty acids may exert their effects at several levels, both
through signal transduction pathways and on gene transcription. At present it is not possible to outline the molecular
basis for the differences between RA, AA, and EPA in con-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
3808
FINSTAD ET AL
trolling apoptosis and necrosis in HL-60 cells while at the
same time differently affecting the level of c-myc. It has
been demonstrated that there is a close link between differentiation and apoptosis in HL-60 cells.60Further use of AA
and EPA in studies of cell proliferation and apoptosis should
provide useful experimental tools to gain further insight into
control mechanisms at the molecular level. Before polyunsaturated fatty acids canbe considered for usein cancer
therapy, or as a dietary support for conventional cancer treatment, these processes must be outlined in further detail
ACKNOWLEDGMENT
We thank Man C. Myhrstad, Amaha Amote, Linda Kvam, and
Anne-Randi Alvestad for excellent technical assistance.
REFERENCES
I . Bang HO, Dyerberg J, Hjorne N: The composition of food
consumed by Greenland Eskimos. Acta Med Scand 200:69, 1976
2. Bang HO, Dyerberg J, Sinclair HM: The composition of the
Eskimo food in North Western Greenland. Am J Clin Nutr 33:2657,
1980
3. Dyerberg J, Bang HO, Stoflersen HO, Moncada S, Vane J:
Eicosapentaenoic acid and the prevention of thrombosis and atherosclerosis. Lancet 2:117, 1978
4. Harris WS: Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J Lipid Res 30:785, 1989
5 . Knapp HR, Fitzgerald GA: Theantihypertensive effects of fish
oil. A controlled study of polyunsaturated fatty acid supplementation
in essential hypertension. N Engl J Med 320:1037, 1989
6. Benaa KH, Bjerve KS, Straume B, Gram IT, Thelle D: Effect
of eicosapentaenoic acid and docosahexaenoic acid on blood pressure in hypertension. N Engl J Med 322:795, 1990
7. Bjerneboe A, Seyland E, Bjerneboe GEA, Rajka G, Drevon
CA: Effect of dietary supplementation with eicosapentaenoic acid
in the treatment of atopic dermatitis. Br J Dermatol I 17:463, 1987
8. Drevon CA, Baksaas I, Krokan HE: Omega-3 Fatty Acids:
Metabolism and Biological Effects. Basel, Switzerland, Birkhauser
Verlag, 1993
9. De Caterina R, Endres S, Kristensen SD, Schmidt EB: Current
topics i n cardiovascular disease. n-3 fatty acids and vascular disease.
Heidelberg, Germany, Springer Verlag, I993
IO. Rag0 CA, Abraham S: Enhanced growth rate of transplanted
mammary adenocarcinoma induced in C'H mice by dietary linoleate.
J Natl Cancer Inst 56:431, 1976
l I . Sauer LA, Dauchy RT: The effect of omega-6 and omega 3
fatty acids on 'H-thymidine incorporation in hepatoma 7288CTC
perfused in situ. Br J Cancer 66:297, 1992
12. Gabor H, Abraham S: Effect of dietary menhaden oil on
tumor cell loss and the accumulation of mass of a transplantable
mammary adenocarcinoma in Balbk mice. J Natl Cancer Inst
76:1223, 1986
13. Krokan HE, Rudra PK, Schenberg S, Slettahjell W, Solum
K, Mollerup S, Mzhle L, Haugen A: Effects of N-3 fatty acids on
the growth of human tumor cell lines in culture and in nude mice,
in Drevon CA, Baksaas I, Krokan HE (eds): Omega-3 Fatty Acids:
Metabolism and Biological Effects. Basel, Switzerland, Birkhauser
Verlag, 1993, p 327
14. Reich R, Royce L, Martin GR: Eicosapentaenoic acid reduces
the invasive and metastatic activities of malignant tumor cells. Biochem Biophys Res Commun 160:559, 1989
15. Beck SA, Smith KL, Tisdale MJ: Anticachetic and antitumor
effects of eicosapentaenoic acid and its effect on protein turnover.
Cancer Res S1 :6089, 1991
16. Guffy MM, North JA, Burns CP: Effect of cellular fatty acid
alteration on adriamycin sensitivity in cultured L 1210 murine leukemia cells. Cancer Res 44:1863, 1984
17. Stillwell W, Ehringer W, Lenski LJ: Docosahexaenoic acid
increases permeability of lipid vesicles and tumor cells. Lipids
28:2.103. 1993
18. Drevon CA: Sources, chemistry and biochemistry of dietary
lipids, in Drevon CA, Baksaas I, Krokan HE (eds): omega-3 Fatty
Acids: Metabolism and Biological Effects. Basel, Switzerland, Birkhauser Verlag 1993, p 1
19. Cornwell DC, Zhang H: Fatty acid metabolism and cell proliferation, in Sevanian A (ed): Lipid Peroxidation in Biological Systems. Champaign, IL, American Oil Chemists Society, 1988, p 163
20. Wickremasinghe RC, Khan MA, Hoffbrand AV: Do leukotrienes play a role in the regulation of proliferation of normal and
leukemic hematopoietic cells? Prostaglandins Leukot Essent Fatty
Acids 48: 123, 1993
21. Sumida C, Graber R, Nunez E: Role of fatty acids in signal
transduction: modulators and messengers. Prostaglandins Leukot Essent Fatty Acids 48:117, 1993
22. Sellamyer A, Obermeier H, Weber C, Weber PC: Modulation
of cell activation by n-3 fatty acids, in De Catermina R, Endres S,
Kristensen SD, Schmidt EB (eds): n-3 Fatty Acids and Vascular
Disease. Heidelberg, Germany, Springer Verlag, 1993
23. Auwerx .I: Regulation of gene expression by fatty acids and
fibric acid derivatives: an integrative role for peroxisome proliferator
activated receptors. Horm Res 38:269, 1992
24. Collins SJ, Gallo RC, Gallagher RE: Continuous growth and
differentiation of human myeloid leukemia cells in suspension culture. Nature 270:347, 1977
25. Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M,
Tsai S, Metzgar R, Aulakh G, Ting R, Ruscetti F, Gallo R: Characterization of the continuous differentiating myeloid cell line (HL-60)
from a patient with acute promyelocytic leukemia. Blood S4:713,
1979
26. Collins SJ: The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood
70: 1233, I987
27. Breitman TR, Selonick ES, Collins SJ: Induction of differentiation of the human promyelocytic cell line (HL-60) by retinoic acid.
Proc Natl Acad Sci USA 77:2936, 1980
28. Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC: Terminal
differentiation of human promyelocytic leukemic cells induced by
dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci
USA 75:2458, 1978
29. Rovera G, Santoli D, Damsky C: Human promyelocytic leukemia cells in culture differentiation and into macrophage-like cells
when treated with a phorbol diester. Proc NatlAcadSciUSA
75:2779, 1979
30. Uhlin-Hansen L, Wik T, KjellCn L, Berg E, Forsdahl F, Kolset
SO: Proteoglycan metabolism in normal and inflammatory human
macrophages. Blood 82:2880, 1993
3 I . Rustan AC, Nossen J 0 , Christiansen EC, Drevon CA: Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A. J Lipid Res
29:1417, 1988
32. Collins SJ, Ruscetti F W , Gallagher RE, Gallo RC:Normal
functional characteristics of cultured human promyelocytic leukemia
cells (HL-60) after induction of differentiation by dimethylsulfoxide.
J Exp Med 149:969, 1979
33. Darzynkiewicz Z, Bruno S, Del Bin0 G, Gorczyca W, Hotz
MA, Lassota P, Traganos F: Features of apoptotic cells measured
by flow cytometry. Cytometry 13:795-808, 1992
34. Chirwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isola-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
F A T W ACIDS AND HL-60 CELLS
tion of biologically active ribonucleic acid from sources enriched in
ribonuclease. Biochemistry 18:5294, 1979
35. Arcari P, Martinelli R, Salvatore F: The complete sequence
of a full length cDNA for human liver glyceraldehyd-3-phosphate
dehydrogenase: Evidence for multiple mRNA species. Nucleic Acids
Res 12:9179, 1984
36. Kolset SO, Gallagher JT: Proteoglycans in haemopoietic cells.
Biochim Biophys Acta 1032:191, 1990
37. Humphries DE, Nicodemus CF, Schiller V, Stevens RL: The
human serglycin gene. Nucleotide sequence and methylation pattern
in human promyelocytic leukemia HL-60 cells and T-lymphoblast
Molt-4 cells. J Biol Chem 267:13558, 1992
38. Luikart SD, Maniglia CA, Sartorelli AC: Glycosaminoglycan
synthesis during differentiation of HL-60MGPRT- leukemia cells
induced by dimethyl sulfoxide and 12-0-tetradecanoylphorbol-13acetate. Cancer Res 44:2907, 1984
39. Fritsche KL, Johnston PV: Effect of dietary a-linolenic acid
on growth, metastasis, fatty acid profile and prostaglandin production
of two murine mammary adenocarcinomas. J Nutr 120:1601, 1990
40. Mengeaud V, Nano JL, Fournel S, Rampal P: Effects of eicosapentaenoic acid, gamma-linoleic acid and prostaglandin El on
three human colon carcinoma cell lines. Prostaglandins Leukot Essent Fatty Acids 47:313, 1992
41. Seyland E, Nenseter MS, Braathen L, Drevon CA: Very long
chain n-3 and n-6 polyunsaturated fatty acids inhibit proliferation
of human T-lymphocytes in vitro. Eur J Clin Invest 23:112, 1993
42. Miller JS, Gavino VC, Ackerman GA, Sharma HM, Milo GE,
Geer JC, Cornwell DG: Triglycerides, lipid droplets, and lysosomes
in aorta smooth muscle cells during the control of cell proliferation
with polyunsaturated fatty acids and vitamin E. Lab Invest 42:495,
1980
43. Fox PL, Dicorleto PE: Fish oils inhibit endothelial cell production of platelet-derived growth factor-like protein. Science
241:453, 1988
44. Stellrecht CM, Mars W,
Miwa H, Beran M, Saunders GF:
Expression pattern of a hematopoietic proteoglycan core protein
gene during hematopoiesis. Differentiation 48: 127, 1991
45. Burns CP, Petersen ES, North JA, Ingraham LM: Effect of
docosahexaenoic acid on rate of differentiation of HL-60 human
leukemia. Cancer Res 49:3252, 1989
46. Grosso LE, Pitot HC: Transcriptional regulation of c-myc
during chemically induced differentiation of HL-60 cultures. Cancer
Res 45347, 1985
47. Holt JT, Redner RL, Nienhuis AW: An oligomer complemen-
3809
tary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic
cells and induces differentiation. Mol Cell Biol 8:963, 1988
48. Williams GT, Smith CA: Molecular regulation of apoptosis:
genetic controls on cell death. Cell 74:777, 1993
49. Shi Y, Glynn JM, Guilbert W, Cotter TG, Bissonnette RP,
Green DR: Role for c-myc in activation-induced apoptotic cell death
in T-cell hybridomas. Science 257:212, 1992
50. Aihara H, Asaoka Y, Yoshida K, Nishizuka Y: Sustained
activation of protein kinase C is essential to HL-60 cell differentiation to macrophage. Proc Natl Acad Sci USA 88:11062, 1991
51. Ebeling JG, Vandenbark GR, KuhnLJ, Ganong BR,Bell
RM, Niedel JE: Diacylglycerols mimic phorbol diester induction of
leukemic cell differentiation. Proc Natl Acad Sci USA 82:815, 1985
52. Shinomura T, Asaoka Y, Oka M, Yoshida K, Nishizuka Y:
Synergistic action of diacylglycerol and unsaturated fatty acid for
protein kinase C activation: its possible implication. Proc Natl Acad
Sci USA 885149, 1991
53. Whyzmuzis CA, Wu JM, Danishefsky I: Changes in proteoglycan core protein (PCP) mRNA levels during HL-60 cell differentiation. Biochem Biophys Res Commun 194:118, 1993
54. Issemann I, Green S: Activation of a member of the steroid
hormone receptor superfamily by peroxisome proliferators. Nature
347545, 1990
55. Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Chen JY, Staub A, Gamier JM, Mader S, Chambon
P: Purification, cloning, and RXR indentity of the HeLa cell factor
withwhichRAR or TR heterodimerizes to bind target sequences
efficiently. Cell 68:377, 1992
56. Gettlicher M, Widmark E, Li Q, Gustafsson JA: Fatty acids
activate a chimera of clofibric acid-activated receptor and the glucocorticoid reseptor. Proc Natl Acad Sci USA 89:4653, 1992
57. Issemann I, Prince R, Tugwood J, Green S : A role for fatty
acids and liver fatty acid binding protein in peroxisome proliferation.
Biochem SOCTransact 20:824, 1992
58. Schmidt A, Endo N, Rutledge SJ, Vogel R, Shinar D, Rodan
GA: Indentification of a new member of the steroid hormone receptor
superfamily that is activated by a peroxisome proliferator and fatty
acids. Mol Endocrinol 6:1634, 1992
59. Sher T, Yi HF, McBride OW, Gonzales FJ: cDNA cloning,
chromosomal localization and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry
32:5598, 1993
60. Martin SJ, Bradley JG, Cotter TG: HL-60 cells induced to
differentiate toward neutrophils subsequently die via apoptosis. Clin
Exp Immunol 79:448, 1990
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1994 84: 3799-3809
Effect of n-3 and n-6 fatty acids on proliferation and differentiation of
promyelocytic leukemic HL-60 cells
HS Finstad, SO Kolset, JA Holme, R Wiger, AK Farrants, R Blomhoff and CA Drevon
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