Download THP-1 - Cancer Research

[CANCER
RESEARCH 46. 1388-1394,
March 1986]
Expression of Tissue Transglutaminase
(THP-1) Cells during Differentiation1
in Cultured Monocytic Leukemia
Kapil Mehta2 and Gabriel Lopez-Berestein3
Department of Clinical Immunology and Biological Therapy, The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030
ABSTRACT
Retinole acid (RA) and 12-O-tetradecanoyl-phorbol-13-acetate
(TPA) induced differentiation of a human monocytic leukemia cell
line, THP-1. RA- or TPA-treated cells stopped proliferating, be
came adherent to plastic surfaces, and acquired the ability to
phagocytose yeast cells, plain sheep RBCs, and IgG-coated
sheep RBCs. The morphological and functional changes, induced
by RA or TPA, were associated with a 20-50-fold increase in
cellular transglutaminase activity. This increase in enzyme activ
ity was found to be due to the induction of a specific intracellular
transglutaminase, tissue transglutaminase. The induction of tis
sue transglutaminase was a specific response of THP-1 cells to
differentiation and was not observed with agents that did not
induce their morphological or functional differentiation. Dibutyryl
cyclic AMP potentiated the RA-induced expression of tissue
transglutaminase. A 15-min exposure to TPA was sufficient to
induce differentiation and expression of tissue transglutaminase
in THP-1 cells. In contrast, RA required a continuous exposure
(48 h) to induce similar changes in morphology or enzyme
activity. These results support the view that differentiation of
cells of the monocytic lineage is associated with an induction
and accumulation of the protein cross-linking enzyme tissue
membrane-related processes, studying these changes in mem
brane properties during the differentiation and functional matura
tion of macrophages is important.
Tissue TGase, an intracellular enzyme that catalyzes the covalent cross-linking of proteins via f-(7-glutaminyl)lysine isopeptide bonds (5), affects the physical properties of biomembranes
by cross-linking membrane proteins (6-8). Several laboratories
have indicated that in vivo activation of macrophages is associ
ated with increased levels of TGase activity (9-12). In vitro
differentiation of human blood monocytes to mature macro
phages and their activation by immune rlFN-7 and lipopolysaccharide is associated with large increases in TGase activity and
levels (13, 14). Similarly, the differentiation of myeloblastic cells
to mature macrophages is accompanied by a marked increase
in the enzyme's activity (15). Thus, an increase in TGase activity
seems to be a general phenomenon associated with the func
tional maturation and differentiation of macrophages.
In this report, we demonstrate that the TPA-induced differen
tiation of THP-1 cells to mature macrophages is associated with
the induction of the enzyme tissue TGase. Treatment of THP-1
cells with RA resulted in a similar induction and accumulation of
tissue TGase and was accompanied by morphological and func
tional differentiation of the cells.
transglutaminase.
MATERIALS AND METHODS
INTRODUCTION
Tumor-promoting
phorbol diesters, such as TPA,4 produce a
wide variety of biological, physiological, and biochemical changes
in cultured cells (for review, see Ref. 1) that have been associated
with cellular differentiation (2). However, little is known about the
mechanisms involved in the events leading to cellular differentia
tion. The establishment of a pure human monocytic leukemia cell
line, THP-1 (3), which can be induced by TPA to differentiate
from a nonfunctioning promonocyte to a cell displaying features
typical of a mature macrophage (4), has made it possible to
study the biochemical events that occur during monocytic differ
entiation. Since many of the physiological functions of macro
phages, such as phagocytosis, cytocidal activity against tumor
cells, and helper activity toward lymphocytes, involve cellular
Received 6/6/85; revised 8/26/85, 11/8/85; accepted 11/12/85.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported by grants from the NIH, BRSG-149308 and 5511,
and the Leukemia Society of America.
2 To whom requests for reprints should be addressed, at Department of Clinical
Immunology and Biological Therapy, Box 41, University of Texas M. D. Anderson
Hospital and Tumor Institute at Houston, 6723 Bertner Avenue, Houston, TX
77030.
3 Scholar of Leukemia Society of America.
'The abbreviations used are: TPA, 12-O-tetradecanoyl-phorbol-13-acetate;
DMFA, dimethyl-formamide; DMSO, dimethyl sulfoxide; rlFN-y, human recombinant
•y-interferon;SDS. sodium dodecyl sulfate; RA. all-frans-retinoic acid; ROH, allfrans-retinol; SRBCs, sheep red blood cells; TGase, transglutaminase.
CANCER
RESEARCH
Reagents. Dibutyryl cyclic AMP, all-frans-retinoic acid, and all-fransretinol (Sigma, St. Louis, MO), TPA (Consolidated Midland Corp., Brewster, NY), DMSO and DMFA (Fisher Scientific Co., Pittsburgh, PA), and
tissue culture medium, RPMI-1640, and fetal calf serum (Grand Island
Biological Co., Grand Island, NY) were used. [3H]Leucine (specific activ
ity, 48.5 Ci/mmol) and [3H]putrescine (specific activity, 33.1 Ci/mmol)
were purchased from New England Nuclear, Boston, MA. rlFN-7 was
kindly provided by Genentech, Inc., South San Francisco, CA, and goat
anti-guinea pig tissue TGase antibody used in these studies was kindly
provided by Dr. Peter Davies of The University of Texas Medical School
at Houston, Houston, TX. The characterization and preparation of this
antibody has been described earlier (11).
Stock solutions of RA and ROH were prepared in ethanol and stored
at -20°C for up to 10 days. A stock solution of TPA was prepared in
DMSO. In all experiments, the final concentrations of ethanol and DMSO
never exceeded 0.1 and 0.05%, respectively, concentrations that had
no detectable effect on the growth or differentiation of THP-1 cells.
Cell Line. Continuous cultures of the THP-1 cell line were established
from seed cultures kindly provided by Dr. Jim Klostergaard (Department
of Tumor Biology, The University of Texas, M. D. Anderson Hospital and
Tumor Institute at Houston, Houston, TX). These cells were originally
derived from a culture obtained from the laboratory of Dr. S. Tsuchiya,
Tohoku University School of Medicine, Sendai, Japan. The cells were
grown in RPMI-1640 medium supplemented with 10% fetal calf serum,
streptomycin (100 Mg/ml), and penicillin (100 units/ml) in T-75 ml flasks
in a 95% air-5% CO2 humidified incubator at 37°Cand subcultured once
per week at an initial density of 4 x 104 cells/ml. The cells stained 100%
positive for «-naphthyl butyrate esterase.
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TRANSGLUTAMINASE
Human peripheral-blood
EXPRESSION
monocytes were isolated by centrifugal elu-
triation as described earlier (16), and mouse peritoneal macrophages
were prepared by lavage of the peritoneal cavities of 6- to 8-week-old
Hale Stoner mice as described previously (17).
Phagocytic Activity. Adherent cells from the cultures treated with
TPA or RA were obtained by exposure to 10 rriM EDTA in Hanks'
balanced salt solution for 3 min followed by gentle scraping with a rubber
policeman. The cells so obtained were mixed with nonadherent cells,
and the mixed cell population (adherent plus nonadherent) was washed
twice with warm phosphate-buffered saline (pH 7.4). Candida albicane
(0.2 ml) (107 colony-forming units/ml) or 107 IgG-coated or plain SRBCs
IN THP-1 CELLS
differentiation of monocytic cells (Fig. 1). RA-treated cells became
adherent to the plastic surfaces and developed pseudopodia, a
kidney-shaped nucleus, dense chromatin, and 1-2 nucleoli. After
3 days of incubation of 1 pM RA, over 80% of THP-1 cells
became adherent (Table 1) and started spreading onto the plastic
surfaces. Treatment with TPA induced similar changes in the
morphology of THP-1 cells. However, the adherence to the
substratum was much stronger, and spreading was more prom
inent in TPA-treated cells than in RA-treated cells (Fig. 1). Also,
were added to the cell suspension so as to yield a 1:10 THP-1 celhyeast
cell or SRBC ratio. The cultures were incubated at 37°Cfor 90 min. At
the end of the incubation period, the cells were cytospun on the slides,
fixed, and stained with May-Grunwald-Giemsa stain. The percentage of
cells with ingested yeast cells or SRBC was counted by a direct micro
scopic examination by scoring at least 200 cells. (Cells were grouped
according to the number of particles ingested).
Assay of TGase Activity. TGase activity in cell extracts was measured
in triplicate as a Ca2+-dependent incorporation of [3H]putrescine into
dimethylcasein (18). Cells (0.5 x 106/ml) were cultured in 6-well Costar
»
tissue-culture plates in 4 ml medium. At the end of the incubation period,
the cells were washed twice with Tris-buffered saline (20 HIM, 7.6 pH)
containing 1 HIM EDTA and 2 mM DL-dithiothreitol and sonicated in 300
¡i\of the same buffer with a heat-systems ultrasonicator (W-225 model)
at the 3-output setting for 1 min. Lysed cells were assayed for enzyme
activity at 37°Cin culture tubes in a final volume of 100 M!as described
O
O,.,
-vi"
O o
earlier (13, 19). The enzyme activity in cell extracts was expressed as
pmol of putrescine covalently incorporated into dimethylcasein/min/mg
cell protein. Protein content in cell extracts was determined by the
method of Lowry ef al. (20).
Detection of Tissue TGase in Cell Extracts. The amount of tissue
TGase in cell lysates was measured by using a 125l-labeled, affinity-
¿•"
' ' "
'—
't'"!.-. *•. "
i '>'•*Hic :
'S*\'•$-fi'^
purified goat antibody produced against guinea pig liver TGase (11). Cell
lysates were adjusted to 1% SDS:0.75 M /3-mercaptoethanol:2.5% sucrose:0.001% bromophenol blue and solubilized for 3 min. The solubilized cell extracts were fractionated by SDS slab gel electrophoresis on
a 6.5% discontinuous polyacrylamide gel and then electroblotted onto a
nitrocellulose paper as described previously (11). The binding capacity
of the nitrocellulose paper was saturated with bovine serum albumin,
and the paper was treated with 125l-labeled anti-TGase antibody for 2 h.
Unbound radioactivity was removed by washing the paper six times in
Tris-HCI buffer (50 mM, pH 7.5) containing 200 mw NaCI, 5 mM EDTA,
0.5% Triton X-100, 0.1% SDS, and 0.25% gelatin. The washed paper
was dried and autoradiographed at -70°C, using Dupont Lightning plus
intensifier screens and Kodak X-OMAT XAR film.
Quantitation of DNA, RNA, and Protein Synthesis. Quadruplicate
samples of THP-1 cells were grown in 96-well microtiter plates (5x10"
cells'well in 0.2 ml medium) with or without TPA (50 ng/ml), RA (1 MM),
or ROH (1 MM). At various time intervals, the cultures were pulsed with
0.5 MCi/ml of [3H]thymidine, [3H]uridine, or [3H]leucine for 60 min at 37°C.
The cells were then collected onto glass-fiber filter-paper discs and
washed three times with deionized water, using a semiautomated microharvester (Flow Laboratories, Rockville, MD). The discs were dried at
room temperature and placed in 5 ml of scintillation cocktail; radioactivity
was determined by a liquid scintillation counter. The cells from a parallel
treated plate were used for protein determination.
RESULTS
Morphological Changes Induced by RA. Untreated THP-1
cells grew as rounded, ovoid-shaped cells in suspension. Treat
ment with RA, a known inducer of cell differentiation in various
normal and leukemia cells (21-26), resulted in a striking change
in the morphology of THP-1 cells, characteristic of terminal
CANCER
RESEARCH
Fig. 1. Morphology of untreated and differentiated THP-1 cells. Control cells (A)
and cells treated for 72 h with 1 /IM RA (B) and TPA (50 ng/ml) (C) are shown.
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1986
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TRANSGLUTAMINASE
EXPRESSION
IN THP-1 CELLS
Table 1
Effect of TPA and retinoids on the proliferation and adherence of THP- 7 ce//s
(x105)6Treatment3MediumTPARAROHConcentration1
Cells/dish
inhibition
cells
(% of untreated
controls)007984895882862530
(% oftotal)19294949838224Growth
0.8d11.6
±
1.40.6
±
±0.29.0
0.40.6
±
0.40.4
±
0.76.0
±
±0.122.0
0.52.2
±
0.58.6
±
0.31.8
±
/iM5.0
0.67.0
±
±0.61.5
0.142.0
+
^M1.0
0.61.0
±
±0.11.8
2.438.4
±
M"5.0
±0.1Nonadherent56.8±2.0Total57.512.29.66.424.210.48.543.040.2Adherent
MMAdherent0.7
8 THP-1 cells (1 x 10") were incubated for 72 h at 37°Cin 5% C02 atmosphere in 4 ml of culture medium at the specified concentrations of different inducers.
b Nuclei of both nonadherent and adherent cells were enumerated by means of a Coulter counter.
c Growth inhibition was calculated as a percentage of total number of cells recoveredfrom the treated dish as compared to those recoveredfrom the untreated control
ng/ml50.0
.0
ng/ml100.0
ng/ml0.1
MM1.0
dish.
d Mean ±SO from triplicate cultures.
50-60% of the cells became adherent within 3-4 h following
TPA treatment, whereas RA required much longer exposure
(>48 h) to induce such a change in cellular morphology. RAinduced morphological differentiation appeared to be optimal at
concentrations ranging between 0.5 and 5.0 UM. ROH, the
physiological analogue of RA, had no effect on THP-1 cells.
Treatment of THP-1 cells with ROH at concentrations up to 5
ßMinduced no adherence or differentiation, even after 8 days of
continuous exposure (Table 1).
Inhibition of Cell Growth by RA and TPA. The morphological
changes induced by RA or TPA were associated with a decrease
in the cell proliferation rate. At lower concentrations of RA (0.01
//M), the inhibition was partial and reversible. However, at higher
concentrations (1-5 UM),inhibition of cell proliferation was almost
complete. Thus, by day 3, RA-treated cultures contained 70%
less cells than controls (Table 1). Growth was totally inhibited in
cells treated with TPA; no net change in cell number occurred.
ROH showed a moderate growth-inhibitory effect, but only at
25
15
o
O)
e
x
12
O
showed no detectable band for tissue TGase (Fig. 4, lane 1),
suggesting that the cells have an enzyme level of less than 5 ng/
CANCER
RESEARCH
123
Days
in Culture
Fig. 2. Effect of ROH, RA, and TPA treatment on DNA(A), RNA (B),and protein
(C) synthesis of THP-1 cells. Five x 10* cells were cultured in 96-well microplates
with or without the inducers for indicated periods of time. At the end of each time
period, cells were pulsed with 0.5 ¿iCi/ml
of either [3H]thymidine,[3H]uridine,or
[3H]leucinefor 60 min and harvested as described in "Materials and Methods."
tured for 72 h in the presence of TPA or RA, however, the cells
became phagocytic (Fig. 3, Table 2) in a dose- and time-depend
1 cells, even after 96 h of continuous exposure.
Induction of Tissue TGase during Differentiation of THP-1
Cells. THP-1 cells in culture have undetectable levels of endog
enous tissue TGase. Immunoblots of untreated THP-1 cells
2
E
5 15
ment were evident within 24 h and persisted for at least 4 days.
RA also caused a significant inhibition of all three components,
but either the effect was slow or the cells partially recovered.
ROH showed a very transient effect on all three components,
and the cells seemed to recover fully from the effect with time.
Induction of Phagocytic Capability in TPA and RA-treated
Cells. THP-1 cells exhibit virtually no phagocytic capability to
ward yeast cells, plain SRBC, or IgG-coated SRBC. When cul
The optimal effect of TPA was observed at a
of 50 ng/ml after 24 h exposure and of RA (5 UM)
treatment. ROH at concentrations up to 5 ßMhad
effect on induction of phagocytic capability of THP-
e
CM
O
higher concentrations.
The effects of TPA, RA, and ROH treatment of DNA, RNA,
and protein synthesis in THP-1 cells are summarized in Fig. 2.
TPA caused a significant inhibition of thymidine, uridine, and
leucine incorporation in THP-1 cells. These effects of TPA treat
ent fashion.
concentration
after 72 h of
no significant
10
Control cells (•)
and cells treated with 5 MMROH (O), 1 MMRA (A), or TPA (50 ng/
ml) (•)
are shown. Each value is a mean of quadruplicate values.
ml cell protein, the limit of detection of the immunoblot assay.
Both RA (Fig. 4, lane 2) and TPA (Fig. 4, lane 3) induced a large
increase in the amount of tissue TGase, as evidenced by the
appearance of a single major immunoreactive band at M, 78,000.
ROH, however, failed to induce tissue TGase accumulation in
the cells, even when they were exposed to higher concentrations
(5 ßM;Fig. 4, lane 4). The amount of tissue TGase accumulated
in fully induced THP-1 cells was comparable to the levels of the
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1986
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TRANSGLUTAMINASE
EXPRESSION
IN THP-1 CELLS
Table 2
Effect of TPAand retinoids on the phagocytic ability of THP-1cells
particlesYeast
% of cells with ingested
SRBCs1-3
cellsTreatment3Medium
cells6.0" cells1.619.0cells2.6
cells2.3 cells1.618.6
cells1.3
55.3
30.0
21.0
0.3IgG-coated
9.0>3
3.0
a THP-1 cells (1 x 106),suspended in 4 ml of culture medium in presence or
TPA
RA
ROH1-3
55.6
50.6
15.3>3
496
17.3
30.6
4.6SRBCs1-3
8.0>3
5.616.0
absence of TPA (50 ng/ml), RA (1 /JM),or ROH (5 MM),were cultured for 72 h. At
the end of the incubation period, the cells were washed and incubated with yeast
cells, plain SRBCs, or IgG-coated SRBCs for 1 h at 37°C.cytospun. stained, and
counted for ingested cells.
0 Mean of triplicate determinations.
80K— «•
Std 1
Fig. 4. Immunospecific detection of tissue TGase in THP-1 cells. THP-1 cells,
cultured for 4 days in medium alone or medium containing 1 »JM
RA, TPA (50 ng/
ml), or 5 »MROH were solubilized and fractionated by electrophoresis on a 6.5%
discontinuous polyacrylamide gel Tissue TGase in untreated (lane 7), RA-treated
(lane 2), TPA-treated (lane 3). and ROH-treated (lane 4) THP-1 cell extracts (100
/jg cells protein) was detected by immunoblot analysis with 1S5l-anti-tissue TGase
antibody as described in "Materials and Methods." Sid represents 20 ng of guinea
pig liver tissue TGase. Lanes 5 and 6 contain cell extracts (100 /jg cell protein) of
freshly isolated mouse resident peritoneal macrophages and human peripheral
blood monocytes, respectively, cultured for 4 days in RPMI medium containing 5%
human AB serum. 80K, M, 80,000.
600
400
200
Fig. 3. Induction of phagocytic ability by RA and TPA treatment in THP-1 cells.
10ecells were cultured in medium alone (A), in medium containing 1 ^M RA (B), or
in medium containing TPA (50 ng/ml) (C) for 72 h. At the end of the incubation
period, IgG-coated SRBCs were added, and the cultures were incubated for an
additional 90 min; cytospun slide preparations were stained with May-GrunwaldGiemsa.
enzyme in normal macrophages (Fig. 4, lane 5 versus lanes 2
and 3) and human peripheral-blood monocytes (Fig. 4, lane 6
versus lanes 2 and 3).
The induction of tissue TGase by RA and TPA was dose- and
time-dependent (Fig. 5). RA at 1 ^M or TPA at 50 ng/ml resulted
in a linear induction of tissue TGase activity in THP-1 cells with
CANCER
RESEARCH
O
18
246
Days
9
in Culture
Fig. 5. Time course of induction of tissue TGase in THP-1 cells. THP-1 cells
(0.5 x 105)were cultured in medium alone (O) or medium containing 1 ^M RA (A)
or TPA (50 ng/ml) (•)
for the indicated periods of time. At each time point, cells
were harvested and assayed for tissue TGase activity as described in "Materials
and Methods." Inset, the dose-response curve for RA (nw) and TPA (ng/ml) for
tissue TGase induction in THP-1 cells cultured for 72 h in the presenceor absence
of different concentrations of the inducers.
time. The induction was evident after 24 h treatment and reached
a maximal level by day 7. No enzyme activity was detectable in
cells treated with ROH for up to 9 days. Since the morphological
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TRANSGLUTAMINASE
EXPRESSION
IN THP-1 CELLS
and functional differentiation of THP-1 cells was associated with
the induction of tissue TGase, we tested several other agents
that have been shown to induce differentiation in myeloid leu
kemia cells and macrophage-like cells (27-33) for their ability to
induce the enzyme activity in THP-1 cells. The cells were cultured
for 24, 48, or 72 h in presence of RA, TPA, human rlFN-7, ROH,
DMSO, or DMFA. The cells were assessed for morphological
changes and enzyme activity (Table 3). Agents such as rlFN-7,
ROH, DMSO, and DMFA did not induce the morphological
differentiation or tissue TGase activity in THP-1 cells. Results
shown in Table 3 were from cells exposed to the agents for 72
h, but similar results were obtained from cells exposed for 24 or
48 h. Cells exposed to RA and TPA showed increased adherence
and increased tissue TGase activity (Table 3).
The induction of tissue TGase by RA and TPA was blocked
by cycloheximide and actinomycin D (Table 4). Addition of RA
(10 U.M)or TPA (1 u.g/m\) to the cell lysates did not cause any
potentiation of the enzyme activity (data not shown).
Continuous presence of RA in the cultures seemed to be
essential to induce morphological differentiation and the expres
sion of enzyme activity in THP-1 cells. In contrast, only a brief
0 1530 60
120
Minutes
240
72Hr
Fig. 6. Effect of RA (1 /¿M)
or TPA (50 ng/ml) pretreatment on tissue TGase
induction.THP-1 cells were treated with RA (A) or TPA (•)
for the indicated periods
of time. At the end of each incubation period, cells were washed three times with
RPMI medium, resuspended in the same medium, and cultured for an additional
72 h without RA or TPA. Tissue TGase activity in cell lysates was determined at
the end of 72-h culture and was expressed as the mean of triplicate values. Values
were also determinedfor tissue TGase activity in THP-1 cells cultured continuously
for 72 h in medium alone P) or in medium containing 1 ^M RA (A) or TPA (50 ng/
ml) (O).Bars, SO.
showed no increase in enzyme activity on subsequent incubation
for 72 h in medium alone. The cells continued proliferating and
showed no changes in the morphology. However, preincubation
with TPA for a similar or much shorter time (15 min) caused a
RA or TPA for the indicated periods of times. At the end of each
full expression of tissue TGase in THP-1 cells; the enzyme levels
incubation period, cells were washed extensively, resuspended
were comparable to the cells cultured continuously with TPA
in fresh medium, cultured for an additional 72 h, and assayed for
(Fig. 6). Cell proliferation was completely blocked by even brief
tissue TGase activity. Cells preincubated with RA up to 4 h
preexposure (15 min) to TPA, and the cells were fully differen
tiated, as determined by their morphological appearance.
Table 3
Effects of Cyclic AMP of RA-induced Expression of Tissue
Effect of various agents on the induction of tissue TGase and differentiation of
TGase. We studied the effect of dibutyryl cyclic AMP on the RATHP-1 cells
induced expression of tissue TGase in THP-1 cells because
Tissue TGase activity
Adherent cells
Treatment8
Concentration
(pmol/min/mg)
(% of total)
previous work by Olsson et al. (34, 35) had suggested that cyclic
AMP
inducing agents potentiate the RA-induced differentiation
MediumTPARAROHrlFN-7DMSODMFA50
of monoblast-like cells, U-937. THP-1 cells cultured for 72 h in
ng/ml200
ng/ml1
medium alone or medium containing either RA, dibutyryl cyclic
;iM5
AMP, or RA + dibutyryl cyclic AMP. Both the enzyme activity
jiM1
and enzyme levels were determined in cell lysates. As seen in
»M5
M5u
Fig. 7, dibytyryl cyclic AMP induced a small increase (57.5 pmol/
units/ml500
min/mg)
in enzyme activity (Fig. 7A) as compared to cells cultured
units/ml0.5%
in medium alone (5.4 pmol/min/mg). This increase in enzyme
(v/v)1.0%0.5%1
activity was also detected by the appearance of a single faint
immunoreactive band at the M, 78,000 position in an immunoblot
.0%5.226228610212018.324.56.64.56.33.24.02.21.4929574818101.223.11.22.46.1
assay (Fig. 7ß,lane 2 versus lane 1). Treatment with RA induced
a THP-1 cells were cultured in medium for 72 h in the presence of various
a larger increase (112 pmol/min/mg) in the enzyme activity (Fig.
differentiating agents at the specified concentrations. Both the nonadherent cells
7A) and accumulation (Fig. IB, lane 3 versus lane 1). Further
and nuclei of adherent cells were enumerated by means of a Coulter counter.
more, a combined treatment with RA and dibutyryl cyclic AMP
caused a 60-fold increase in enzyme accumulation (Fig. 78, lane
Table 4
4 versus lane 1) and enzyme activity (336.6 pmol/min/mg) as
Effect of protein- and RNA synthesis-inhibitors on the TPA and RA induced tissue
TGase activity THP-1 cells
compared to the cells treated with RA or dibutyryl cyclic AMP
alone. Co-culture of THP-1 cells with RA or dibutyryl cyclic AMP
TGase activity
Treatment8Medium1
(pmol/min/mg)"0
together, however, did not augment the morphological or func
tional differentiation of the cells (data not shown) when compared
24.0
to those treated with RA alone. Similarly, dibutyryl cyclic AMP
fiM RA + actinomycin D (2.5 /ig/ml)
2.3
alone had not antiproliferative or differentiation-inducing effect
1 fiM RA + cycloheximide(5 //M)
7.8
on THP-1 cells.
TPA (50 ng/ml)
38.0
exposure to TPA was necessary to cause a full expression of
differentiation and tissue TGase activity in these cells. In the
experiment shown in Fig. 6, THP-1 cells were preincubated with
TPA (50 ng/ml) -I-actinomycin D (2.5 ^g/ml)Tissue
14.9
THP-1 cells were cultured in medium alone or medium containing RA (1 ^M)or
TPA (50 ng/ml) for 48 h and then for an additional 24 h in the presenceor absence
of actinomycin D or cycloheximide.
Cell lysates were assayed for tissue TGase activity, and results were ex
pressed as the net enzyme activity induced during the last 24 h.
CANCER
RESEARCH
DISCUSSION
The present study demonstrates that the RA-induced morpho
logical and functional differentiation of THP-1 cells is associated
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TRANSGLUTAMINASE
EXPRESSION
responsible for the large tissue TGase induction in these cells
(11,13,14,19,40).
Therefore, we elected to focus on the effects
of RA on tissue TGase expression and its induction of morpho
logical and functional differentiation in cultured THP-1 cells. RA
appeared to have a potentiating effect on tissue TGase accu
mulation, inducing an increase up to 180-fold in the level of the
enzyme in optimally treated THP-1 cells (1 /¿M
RA for 7 days).
As in TPA-treated cells, the enzyme induction in RA-treated cells
B
o
^
IN THP-1 CELLS
350
I
o
a
> 200
100
1234
Fig. 7. Effect of dibutyryl cyclic AMP on RA-induced expression of tissue TGase.
In A, tissue TGase activity of THP-1 cells cultured for 72 h in the presence of
medium alone (O) or medium containing 200 MMdibutyryl cyclic AMP (Db.C-AMP,
W), 1 MM(RA (È), or 200 MMdibutyryl cyclic AMP plus 1 MMRA (•)
was determined
as described in "Materials and Methods." The values shown represent the mean
of triplicate determinations. Bars, range. In B, tissue TGase accumulation in
untreated THP-1 cells and THP-1 cells treated for 72 h with either 1 MM RA, 200
MMdibutyryl cyclic AMP, or RA + dibutyryl cyclic AMP was determined by Western
blot technique as described in "Materials and Methods." Lanes 1-4 show the
autoradiograms obtained with extracts (60 Mg cell protein) of untreated THP-1 cells
(lane 7), dibutyryl cyclic AMP treated cells (lane 2), RA-treated cells (tene 3), and
cells treated with RA + dibutyryl cyclic AMP (tene 4).
with a 20-50-fold increase in the level of tissue TGase. TPA,
known to induce THP-1 cells to mature macrophage-like cells
(4), also induced tissue TGase. Other inducers of cell differentia
tion such as IFN-7, ROH, DMSO, and DMFA had no effect on
the enzyme activity or on the morphological differentiation of
THP-1 cells. Thus, the functional and morphological differentia
tion of human monocytic leukemia cells (THP-1) is associated
with an increase in tissue TGase activity.
In untreated THP-1 cells, no detectable levels of tissue TGase
were identified by enzymatic or immunoblot techniques. Birckbichler and Patterson (36) have reported that many transformed
cell lines have low levels of tissue TGase activity, and THP-1
cells certainly conform to this generalization. TPA treatment
induced a 200-fold increase in the level of TGase in optimally
treated cells (50 ng/ml for 7 days). This effect is probably due to
an increased rate of TGase gene expression, since TPA-induced
enzyme induction was blocked by actinomycin D, an inhibitor of
RNA synthesis.
In order to further investigate if the induction of tissue TGase
in THP-1 cells is linked to cellular maturation and differentiation,
we studied the effects of other agents that are known to induce
the differentiation and maturation of myeloid leukemia cells (2732). Compounds such as DMSO, DMFA, rlFN--v, and ROH, which
did not induce differentiation of THP-1 cells, were also unable to
induce tissue TGase activity. rlFN-7, which is known to induce
the expression of tissue TGase in cultured human blood monocytes (13) and to differentiate human leukemia myeloid cells and
monoblast cells toward a monocytic pathway (27, 30, 32, 33),
did not induce morphological or functional differentiation nor
enzyme activity in THP-1 cells. These results suggest that TGase
induction is a specific response to inducers of differentiation in
THP-1 cells.
RA induces tissue TGase activity in a variety of cultured cells.
Epidermal cells and malignant melanoma cells respond to RA
with increased tissue TGase activity (37-39). Previous studies
on the mechanisms involved in regulating tissue TGase in mac
rophage and monocytes suggest that serum retinoids are directly
CANCER RESEARCH
was blocked by RNA synthesis inhibitors such as actinomycin
D. THP-1 cells became adherent after 48-72 h of RA treatment
and were capable of phagocytosing yeast cells, plain SRBCs,
and IgG-coated SRBCs. One major difference between TPAand RA-treated cells was that the cells responded more quickly
to TPA (3-4 h) than to RA (usually more than 48 h). Also, a brief
exposure to TPA (30 min) was sufficient to induce the morpho
logical changes and enzyme accumulation in THP-1 cells. How
ever, continuous presence of RA was required to induce similar
changes. This difference might reflect the difference in properties
of TPA and RA. TPA binds rapidly to cells and in many cells is
not metabolized significantly. RA, on the other hand, binds less
avidly and is metabolized by most cells. Our previous studies
with mouse macrophages and human monocytes suggest that
SRBP bound retinoids play an important role in inducing tissue
TGase in these cells (11, 13, 14, 19, 40). However, in THP-1
cells both RA and TPA induced full expression of the enzyme's
activity and differentiation in serum-free medium (data not
shown).
Cyclic AMP or cyclic AMP inducing agents have been shown
to potentiate RA-induced differentiation of histiocytic lymphoma
(U-937) and myeloid leukemia (HL-60) cell lines (34, 35). We
observed that analogues of cyclic AMP such as dibutyryl cyclic
AMP potentiate RA-induced expression of tissue TGase. The
effects of RA and dibutyryl cyclic AMP were much more than
additive and suggest a true synergistic interaction between the
two agents. We do not know the mechanism of this synergism,
but the ability of cyclic AMP to promote RA-induced differentia
tion possibly reflects its ability to promote RA-induced gene
expression.
The functional significance of tissue TGase induction in differ
entiating THP-1 cells is not known. Several studies suggest that
tissue TGase is involved in the processing of ligand-receptor
complexes by receptor-mediated phagocytosis (9, 10). Whether
or not the TPA- or RA-mediated induction of tissue TGase in
differentiating THP-1 cells contributes to the acquisition of phag
ocytosis activity, however, remains to be established, although
the present studies show that unstimulated THP-1 cells, which
do not have any detectable levels of tissue TGase, are not
capable of phagocytosis.
It is equally possible that induction of tissue TGase may be
important to other macrophage functions. The enzyme can
cross-link membrane proteins such as /32-microglobulin (41) and
thus may play a role in the processing of cell-surface antigens.
Tissue TGase also catalyzes the covalent conjugation of polyamines to cellular proteins, including such important enzymes as
ornithine deoxycarboxylase (42). It is possible that the accumu
lation of tissue TGase is important, not only in protein crosslinking, but also for the post-translation modifications of regula
tory enzymes. Future studies to identify the specific substrates
of tissue TGase in differentiating THP-1 cells should clarify the
significance of its induction in these cells.
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1986
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TRANSGLUTAMINASE
EXPRESSION
ACKNOWLEDGMENTS
The authors would like to express their gratitude to Dr. Peter J. Davies for
providing the antibody to tissue TGase. We thank Genentech, Inc. (South San
Francisco, CA) for providing rIFN--). We would also like to express our appreciation
to Jennie Rayls for typing the manuscript.
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Expression of Tissue Transglutaminase in Cultured Monocytic
Leukemia (THP-1) Cells during Differentiation
Kapil Mehta and Gabriel Lopez-Berestein
Cancer Res 1986;46:1388-1394.
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