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Autocrine Loop Through Cholecystokinin-B/Gastrin Receptors Involved
in Growth of Human Leukemia Cells
By Nobuko Iwata, Tohru Murayama, Yoshinobu Matsumori, Mitsuhiro lto, Aki Nagata, Taizo Taniguchi,
Kazuo Chihara, Yoshinobu Matsuo, Jun Minowada, and Toshimitsu Matsui
The cholecystokinin (CCK)-Blgastrin receptor binds two
brain-gut hormones, CCK and gastrin, with high affinities.
These peptides have a trophic effect on gastrointestinal cells
expressing the receptor in vivo as well as in vitro. Recently,
this receptor mRNA was reported t o be expressed in immunocytes localized in the lamina propria of normal rat stomach mucosa. Here, we studied the receptor expression in
human hematopoietic cells in order t o determine whether
they play a role in cell growth. The CCK-B/gastrin receptor
mRNA was detectable in the polymorphonuclear (PMN) cells
but not in the mononuclear cells of normal peripheral white
blood cells by reverse transcription-polymerase chain reaction. The receptor transcript was, however, expressed in human leukemia cell lines (14 of 18 cell lines tested) derived
from not only myeloid, but also T- and B- lymphoid lineages.
The CCK-B/gastrin receptors on several leukemia cell lines
were shown t o be biologically active by demonstrating ligand-dependent cell proliferation in serum-deprived medium. Interestingly, a human CCK-Blgastrin receptor specific
antagonist, YM022, but not its stereotype isoform, selectively inhibited the DNA synthesis of THP-1, MOLT-16,
MOLT-14, and CCRF-CEM in the absence of exogenous peptide ligands. Further investigation revealed that these leukemia cell lines and normal PMN cells also expressed gastrin
mRNA. These results suggest that growth of human leukemia cells is promoted by an autocrine mechanism through
the CCK-Blgastrin receptors.
0 1996 b y The American Society of Hematology.
T
designated YM022, and an enantiomer of YM022, YM022-S, were
kindly provided by Yamanouchi Pharmaceutical Co, Ltd (Tsukuha,
Japan). YM022-S is about three orders of magnitude less potent in
the competitive binding for ‘251-laheledCCK-8 or gastrin I than
YM022.“
Separation of normal human white blood cell fractions. Peripheral blood was obtained from five healthy volunteers and collected
in heparinized tubes. Polymorphonuclear cells (PMN) were separated by gradient sedimentation over Mono-Poly Resolving Medium
(ICN Biomedicals Inc, Aurora, OH). The cells were pelleted to 1.5
X 10’ cells per donor. Mononuclear cells (MNC) were separated
by gradient sedimentation over Ficoll-Hypaque (Pharmacia Biotech,
Uppsala, Sweden). The cells were washed once by calcium and
magnesium-free phosphate-buffered saline (PBS), resuspended in
RPMI 1640 medium (GIBCO, Grand Island, NY), disseminated over
petri-dishes and incubated in a 95% air, 5% C0,atmosphere at 37°C
for 1 hour. Then, the nonadherent MNC, the majority of which were
lymphocytes, were collected and washed once by PBS. The cells
were pelleted to 1.3 to 3.3 X IO’ cells per individual. The adherent
cells, the majority of which were related to macrophages, were removed from dishes with a cell scraper, washed once in PBS, and
collected in another tube. The cells were pelleted at 1.5 to 4.2 X
lo7 cells per donor.
Cell culture. Leukemia-lymphoma cell lines used were HL-60,
U-937, THP-I, ML-I, MOLM-I, NALM-6, NALM-26, Daudi, HairM, Jurkat, HPB-MLT, MOLT- 16, MOLT-3, HPB-ALL, DND-41,
MOLT-14, CCRF-CEM, and RPMI-8402.’7 These cell lines were
HE PEPTIDE HORMONES, cholecystokinin (CCK)
and gastrin, play important roles in the function of the
central nervous system and digestive organs.’.’ Both CCK
and gastrin have a trophic effect on gastrointestinal cells in
vitro as well as in v ~ v o . ~
Recently
.~
we showed that both
peptide hormones could promote cell growth through a single guanine nucleotide regulatory protein (G protein)-coupled CCK-B/gastrin receptor.’,6 Abundant expression of the
receptor transcript was demonstrated in the normal human
brain cortex and stomach m u c o ~ a . ~Unexpectedly,
~’~~
the receptor mRNA was found to be expressed in the human pancreas.’~’ By using reverse transcription-polymerase chain reaction (RT-PCR), we also showed the ectopic expression of
the CCK-B/gastrin receptor gene in human small cell lung
cancers.’
By means of in situ hybridization histochemistry, immune
cells in the lamina propria of normal rat stomach mucosa
were recently shown to express the CCK-B/gastrin receptor
mRNA, as well as the transcripts of histamine, muscarine
and dopamine receptor genes.” The CCK-B receptor has
been pharmacologically shown to be expressed on a T-cell
leukemia cell line, Jurkat.” Receptors for neuropeptides such
as vasoactive intestinal peptide, somatostatin and substance
P are expressed in human immuno-hematopoietic cells. ’*-”
These data raise questions on whether the CCK-B/gastrin
receptors are expressed in normal human blood cells, what
sort of neoplastic cells derived from immuno-hematopoietic
systems express the receptor gene, and whether such receptors are physiologically relevant.
In this study, we show the expression of CCK-B/gastrin
receptor mRNA in normal human peripheral white blood
cells and in various lineages of leukemia cell lines by RTPCR. We also investigated the pathophysiological roles of
the CCK-B/gastrin receptor by using a novel receptor antagonist.
MATERIALS AND METHODS
Chemicals. Cholecystokinin (residues 26-33 [CCK-81) and gastrin I were purchased from Peninsula Laboratories, Inc (Belmont,
CA). A specific and strong benzodiazepine-derived CCK-Blgastrin
receptor antagonist, (R)-l-[2,3-dihydro-l-(2’-methylphenacyl)-2oxo-5-phenyl- 1H - 1,4-benzodiazepin-3-y1]-3-(
3methylphenyl)urea,
Blood, Vol 88,No 7 (October 1). 1996:pp 2683-2689
From the Third Division, Department ?f Medicine, Kobe University School of Medicine, Kobe; and Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan.
Submitted October 2, 1995; accepted May 28, 1996.
Supported by a Grants-in-Aidfor Scientific Research on Priority
Areas from the Ministry of Education, Science and Culture of Japan
and by research grants from Uehara Memorial Foundation and
Hyogo Science and Technology Association.
Address reprint requests to Toshimitsu Matsui, MD, Third Division, Department o f Medicine, Kobe University School of Medicine,
Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8807-0020$3.00/0
2683
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IWATA ET AL
2684
maintained in suspension culture using RPMI 1610 medium supplemented with 10%fetal calf serum (ICN Flow, Costa Mesa, CA) and
antibiotics in a 95% air, 5% COz atmosphere at 37°C.
RNA preparation and RT-PCR. Total cellular RNA was extracted from normal human white blood cells (PMN, adherent and
nonadherent MNC) and leukemia cell lines by using an acid guanidinium thiocyanate-phenol-chloroform method as described.’xThe
cDNA templates for PCR analyses were prepared by RT of total
RNA (2 fig) using the Superscript Preamplification System (GIBCOBRL, Life Technologies, Inc, Gaithersburg, MD).”’ PCR reactions
were performed in a 25 ,uLreaction mixture containing 5 ,uL of the
cDNA, 20 mmol/L Tris-HCI (pH 8.4). SO mmollL KCI, 2.5 mmoll
L MgClz, 0.1 &/fig bovine serum albumin, 200 ,umol/L each deoxynucleotide triphosphate, 20 pmol/L each specific primer (CCK-B/
gastrin receptor: sense primer, 5’-TCACCAATGCCTTCCTCCTCTCACTGGCAG-3’; antisense primer, 5‘-TTGGCTGTCGCTGTCACTGTCGCCGTCAAA-3’; human gastrin: sense primer,
S’-TGCAGCGACTATGTGTGTATGTGCTGATCT-3’;
antisense
primer, S’-GGCCGAAGTCCATCCATCCATAGGCTTC-3’: p-actin: sense primer, 5‘-ACCACACCTTCTACAATGAGCTGCGTG3’; antisense primer, 5‘-CACAGCTTCTCCTTAATGTCACGCACG-3’) and 1.25 units of Taq DNA polymerase (Promega,
Madison, WI).*.”’.” These primer pairs are designed to amplify genomic fragments including one or two introns. Thus, the size of fragments amplified from the contaminating genomic DNA is different
from that of the cDNA bands. The reaction conditions were as
follows: denaturation at 94°C for 30 seconds, annealing at 72°C for
1 minute and extension at 72°C for I minute. The reaction was
performed for 35 cycles in a GeneAmp PCR system 9600 (PerkinElmer Cetus, Norwalk. CT)?
Southern hlor analysis. To confirm amplification of the appropriate DNA fragments, the RT-PCR products (8 ,uL of a 25 ,uL
reaction mixture) were electrophoresed in a I % agarose gel and
transferred onto nitrocellulose filters. The filters were hybridized at
42°C in a buffer containing 50% (wt/vol) formamide and full-length
”P-labeled cDNA probes. After a 16-hour hybridization, the filters
were washed twice for 15 minutes in 2 X SSC ( I X SSC is 0.15
molL NaCI-0.015 m o l L sodium citrate, pH 7.0). 0.1% sodium dodecyl sulfate (SDS) at room temperature, and twice for 30 minutes
in 0.1 X SSC, 0.1% SDS at W C , and then subjected to autoradiography.”
Measurement of DNA swhesis. The DNA synthesis of the HL60, U-937, THP-I, Hair-M, Jurkat, HPB-MLT, MOLT-16, MOLT3, HPB-ALL, MOLT-14, and CCRF-CEM cell lines were measured
by [methyl-’Hlthymidine incorporation. Cells (2 X IO5 cells per well)
were preincubated in serum-deprived medium for 24 hours, and then
cultured in 96-well plates in the presence or absence of various
concentrations of peptide ligands with or without receptor antagonists for 44 hours, followed by a further 4-hour incubation with
[methy/-’H]thymidine( I mCilmL, 25 mcilmmol: Amersham International plc, Buckinghamshire, UK). The radioactivity of incorporated [methyL3H]thymidinein 5% trichloroacetic acid-insoluble precipitates was counted by using a scintillation counter (Top Count.
Packard, Meriden, CT).
Rudioimmunoassuy of gastrin. Cells were grown to confluence
over a 4-day period in 9 cm diameter petridishes. Fresh growth
medium was then added. After 48 hours, culture supernatants were
collected and assayed for immunoreactive gastrin levels using a
radioimmunoassay kit (Dainabot, Inc, Tokyo, Japan).
The statistical significance of the difference between
antagonist-treated and control cell DNA synthesis was assessed by
analysis of variance followed by Student’s t-test (P < .01).
RESULTS
Lineage specijc expression of CCK-B/gastrin receptor
mRNAs in normal human peripheral white blood cells. To
RT
-+ -+ -+
-
CCK-B/gastrin
receptor
II
-
922bp
528bp
Fig 1. Expression of CCK-Blgastrin receptor mRNA in normal human white blood cell fractions. Total RNAs of polymorphonuclear
cells, macrophages and lymphocytes were incubated with ( + I or
without (-1 RT. RT-PCR using the CCK-Blgastrin receptor cDNA specific primers generated a 528-bp band from mRNA and a 922-bp fragment from genomic DNA. The primers specific for human p-actin
generated a 365-bp band. Representative autoradiographs of Southern blot analysis using the CCK-Blgastrin receptor cDNA probe (upper panel) and ethidium bromide staining of the human p-actin fragment (lower panel) are shown. &X174-Haelll fragments were used
as size markers.
examine the expression of CCK-B/gastrin receptor mRNA
in normal human peripheral white blood cells, RT-PCR was
performed using specific paired primers in the presence or
absence of RT (Fig I). A 528-bp sequence was amplified
from CCK-B/gastrin receptor mRNA. Southern blot analysis
of RT-PCR products showed the expression of this receptor
mRNA only in PMN among normal peripheral white blood
cell fractions. No signal was detected in reactions without
RT, indicating that the observed signals were due specifically
to the presence of receptor transcripts. Contaminating genomic DNA did not confound the analysis, because the primers
amplified the 922-bp fragment from the genomic I O C U S . ~ ~ ’ ~
Primers specific for human &actin generated the expected
365-bp specific band and confirmed the presence of intact
mRNAs in all samples (Fig 1). The results were reproducible
in all RNA samples extracted from five individuals.
Expression of CCK-B/gastrin receptor mRNA in various
human leukemia cell line lineages. To elucidate the lineage
specificity, we next examined the expression of the CCK-B/
gastrin receptor transcript in various human leukemia cell
lines by RT-PCR followed by Southern blot analysis. Surprisingly, it became clear that the CCK-B/gastrin receptor
was expressed in all sorts of leukemia lineages in widely
varying amounts (HL-60, THP- I , ML- I , MOLM- 1 in myeloid, NALM-6, NALM-26, Daudi, Hair-M in B lymphoid
and Jurkat, MOLT- 16, MOLT-3, MOLT- 14, CCRF-CEM,
RPMI-8402 in T lymphoid cells) (Fig 2). No signal was
detected in reactions without RT. Expression was undetectable in U-937, HPB-MLT, HPB-ALL, and DND-41.
We have tried to quantify the expression levels of CCK-
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CCK-B/GASTRIN RECEPTORS ON LEUKEMIA CELLS
2685
CCK-B/gastrin receptor
Myeloid cell
RT
-
+
-
+
-
+
I
Megakaryo.
+
-
+
Btell
-+ -+-+ -+
--
922bp
Fig 2. Expression of CCK-BI
gastrin receptor mRNA in various lineages of human leukemia
cell lines. Total RNAs of myeloid
(HL-60, U-937, THP-1, ML-1, and
MOLM-1). B lymphoid (NALM-6,
NALM-26, Daudi, and Hair-M)
and T lymphoid (Jurkat, HPBMLT, MOLT-16, MOLT-3, HPBALL, DND-41, MOLT-14, CCRFCEM, and RPMI-84021cells were
incubatedwith (+)or without RT
(-1. RT-PCR using the CCK-BI
gastrin receptor cDNA specific
primers generated a 528-bp
band from mRNA and a 922-bp
fragment from genomic DNA.
Representative autoradiographs
of Southern blot analysis using
human CCK-Blgastrin receptor
cDNA probe are shown.
528bp
T-cell
RT
-+ -+-+-+ -+-+
.- + - + - +
--.
.
B/gastrin receptor mRNA in these leukemia cell lines by
RNA blot analysis.' However, most of them except for Jurkat
and MOLT-I4 were under the detectable level by the analysis (data not shown).
Inhibition of leukemia cell growth by a specific antagonist
for human CCK-B/gastrin receptors. We previously
showed that the peptide ligands, CCK-8 and gastrin I could
promote DNA synthesis and cell growth of Chinese hamster
ovary or mouse NIH 3T3 fibroblast cells transfected with a
human CCK-B/gastrin receptor cDNA expression vector.s.h.X
Thus, we next examined the effect of CCK-8 on DNA synthesis of 11 human leukemia cell lines (Table I). In serumfree medium, CCK-8 (IO-' mol/L) did significantly increase
[methyl-'Hlthymidine incorporation of HL-60, THP- I , Jurkat, MOLT-3, MOLT-14, and CCRF-CEM, all of which
express CCK-B/gastrin receptor mRNA. In U-937, HPBMLT, and HPB-ALL cells, lacking the mRNA expression,
CCK-8 did not increase [methyl-'Hlthymidine incorporation
(Table I). The receptor mRNA was expressed on Hair-M
and MOLT-16, but exogenous CCK-8 (IO-' mol/L) did not
stimulate their DNA synthesis.
DNA synthesis of MOLT-14 was stimulated by CCK-8
in a dose-dependent manner. It was completely blocked by
a specific CCK-B/gastrin receptor antagonist, YM022, but
not by its stereoisomer, YM022-S (Fig 3). Gastrin I similarly
promoted DNA synthesis (data not shown). These results
indicate that the CCK-B/gastrin receptors expressed on
MOLT- 14 are biologically functional.
1
--
922bp
528bp
Even in the absence of exogenous CCK-8, DNA synthesis
of MOLT-I4 was inhibited by YM022, but not by YM022S (Fig 3 legend). Thus, we further examined the effect of
YM022 on DNA synthesis of several leukemia cell lines.
Surprisingly, YM022, but not YM022-S, selectively inhibited not only DNA synthesis of MOLT-I4 but also that of
THP-I, MOLT-I6 and CCRF-CEM cells in the absence of
exogenous peptide ligands (Table 2). The inhibitory effect of
YM022 for DNA synthesis of MOLT-I4 in serum-deprived
medium was concentration-dependent (Fig 4). No obvious
effect of YM022 or YM022-S was seen in Hair-M or HPBALL cells. This implies that YM022 inhibited proliferation
of THP-I, MOLT-16, MOLT-I4 and CCRF-CEM cells by
specific interaction with the CCK-B/gastrin receptor. These
results raise the possibility that leukemia cell growth is promoted by an autocrine mechanism through CCK-B/gastrin
receptors.
Coexpression of gastrin and its receptor mRNAs in several
human leukemia cell lines. To support the presence of an
autocrine mechanism, we examined the expression of the
gastrin gene in human leukemia cell lines. By RT-PCR using
a specific pair of primers for the human gastrin gene, a
279-bp sequence was amplified from gastrin mRNA. The
contaminating genomic DNA did not interfere with the analysis. The primers amplify a 409-bp fragment from the genomic IOCUS.~" Southern blot analysis revealed that this ligand
mRNA was expressed in almost all human leukemia cell
lines (17 of 18 cell lines) tested (Fig 5 ) . Both gastrin and its
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2686
IWATA ET AL
Table 1. Effect of CCK-8 on Leukemia Cell Growth
Exp 1
Cell Lines
Control
HL-60
u-937
THP-1
Hair-M
Jurkat
HPB-MLT
MOLT-16
MOLT-3
HPB-ALL
MOLT-I4
4,550
2,695
5,790
2,180
627
3,988
9,760
2,120
8,260
1,557
3,210
CCRF-CEM
Exp 2
CCK-8
t 194
i 192
t 78
i 175
t 24
t 136
2 990
t 71
t 79
t 111
t 139
6,260
2,761
9,340
2,110
859
3,952
9,400
3,150
8,220
3,113
5,150
i 498*
t 132
i 62 1*
I
131
t 35*
i 203
-t 1,200
t 89*
2 230
i 79*
? 320*
Control
7,050
1,570
4,860
3,410
810
2,600
8,540
1,400
6,920
2,830
3,100
Exp 3
CCK-8
t 276
t 67
F 477
t 310
i 24
t 405
t 866
i 14
2 194
t 90
2 49
8,610
1,550
6,010
3,500
959
2,540
8,740
1,710
6,670
4,740
3,950
t 493'
i 48
t 435*
i 180
t 17*
i 160
t 1,400
t 103*
i 292
t 400*
f 425*
Control
6,910
1,860
5,040
3,550
1,220
1,930
7.120
1,030
9,150
1,390
2,770
i 194
2 202
i 395
2 314
i 40
t 58
i 586
i 52
f 1,160
i 129
i 195
CCK-8
8,520
1,840
7,300
3,590
1,500
1,940
7,220
1,250
9,330
2,590
3,390
F
292*
i 109
i 827*
i 283
i 24*
5
390
f 995
i 48*
f 1,840
i 33*
i 116*
Data of three independent experiments (Exp 1, Exp 2, and Exp 3) are shown as the mean t SD (cpm) of quadruplicate samples.
* P < .01 compared with control
receptor genes were coexpressed in HL-60, THP-1, ML1, MOLM- 1, NALM-6, Daudi, Hair-M, Jurkat, MOLT-16,
MOLT-3, MOLT-14, CCRF-CEM, and RPMI-8402. Other
cell lines expressed either receptor (NALM-26) or ligand
n
Q)
2
2.0
-c 1.8
0
LLO
5 1.6
.-
DISCUSSION
W
c.
!
'
-8
0
1.4
c
Q)
S
.z 1.2
E
>
r
!I
= 1.0
E
Y
mRNA (U-937, HPB-MLT, HPB-ALL, and DND-41).
Among normal human peripheral white blood cells, only
PMN expressed gastrin mRNA (Fig 6). No signal was detected in reactions without RT.
The mRNA expression of the gastrin gene in the leukemia
cell lines was also under the detectable levels by Northern
blot analysis as that of the CCK-B/gastrin receptor gene
(data not shown). However, we could detect gastrin-like immunoreactivity in the conditioned media of several leukemia
cell lines. Jurkat, HPB-ALL and MOLT-14 produced 20,
26, and 27 pg gastrin per 10" cells over a 48-hour period,
respectively.
I
0
10-10
10-9
4
10'8
CCK-8 (M)
Fig 3. Effect of CCK-8 and a CCK-Blgastrin receptor antagonist on
DNA synthesis by MOLT-14cells. Serum-deprivedcells were cultured
in the presence of various concentrations of CCK-8 without IO) or
with lo-' mol/L YM022 (0)or YM022-S IO) for 44 hours, followed by
a further 4-hour incubation with [methyk3Hlthymidine(means f SD
of four different experiments, each performed in triplicate).The radioactivities of [methykJHlthymidine incorporated into control cells
without addition of CCK-8 were 1,670 2 101 cpm in the absence of
YM022 or YM022-S, 1,070 + 31 cpm in the presence of lo-' mol/L
YM022 and 1,620 k 30 cpm in the presence of YM022-S.
In the last two decades, experimental evidence has suggested that neuropeptides play important roles in the modulation of the immune system through receptor-mediated
events. For example, sensory neuropeptides such as somatostatin, vasoactive intestinal peptide and substance P have
been reported to modulate T lymphocyte function.'*," The
CCK-A (peripheral type CCK) receptor has been reported to
mediate inhibition of cell growth in MOLT-4 lymphoblasts."
Receptors for substance P, opiate peptides and benzodiazepine have been shown to be involved in chemotaxis of human
monocytes.'4.2'-26
In addition, substance P has been shown to
stimulate hematopoiesis for both erythroid and granulocytic
progenitors in short-term bone marrow ~u1tures.l~
Furthermore, the receptor transcript of nerve growth factor, trk, is
functionally expressed in human immune and hematopoietic
cells.*'-'" On the other hand, erythropoietin receptors have
been shown in the central nervous system3' Many receptors
for peptide hormones, growth factors and cytokines previously thought to be limited to the brain or the immunohematopoietic system are now known to be expressed by
both."
The CCK-B receptor that binds CCK-8 as well as gastrin
I with high affinities, has been pharmacologically shown to
be expressed on a human Jurkat T lymphocyte cell line."
But, its biological function is unknown. Recently, Mezey et
all" reported that immune cells in the lamina propria are the
only cells that express detectable mRNA for gastrin receptors
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2687
CCK-WGASTRIN RECEPTORS ON LEUKEMIA CELLS
Table 2. Inhibition of Leukemia Cell Growth by YM022
Cell Lines
Exp 1
THP-1
Control
YM022
YM022-S
Control
YM022
YM022-S
Control
YM022
YM022-S
Exp 2
Exp 3
5,281
3,095
4,736
7,840
5,340
7,560
6,060
4,530
5,990
t 842
t 224*
t 194
t 163
t 100'
t 178
t 528
t 446*
t 829
Heir-M
2,038
2,557
2,500
2,730
2,730
2,670
3,550
3,680
3,480
MOLT-16
t 176
2 367
t 127
2 192
t 257
t 173
t 157
2 195
t 212
8,214
5,565
8,056
6,790
5,030
6,670
12,200
7,640
11,500
t 779
t 270*
t 1,092
t 162
t 67'
t 202
t 1,140
t 500*
t 2,430
HPB-ALL
9,783
9,536
9,525
9,520
9,850
9,358
7,090
7,070
6,940
t 298
t 382
t 296
t 356
t 666
2 280
t 104
t 246
2 339
MOLT-14
1,666
815
1,699
1,780
787
1,680
2,130
1,370
2,070
t 101
2 26'
t 84
t 100
t 65*
100
t 127
t 19'
t 58
CCRF-CEM
3,577
2,591
3,693
1,710
915
1,710
2,210
1,430
2,320
t 310
t 412'
2 289
t 193
2 69'
t 166
t 186
2 218*
t 118
Data of three independent experiments (Exp 1, Exp 2, and Exp 3) are shown as the mean t SD (cpm) of quadruplicate samples.
P < .01 compared with control and YM022-S.
in the rat gastric mucosa by in situ hybridization histochemistry. Our present study revealed that the CCK-B/gastrin
receptor mRNA was expressed in normal human PMN cells
and in both the myeloid and lymphoid lineages of leukemia
cell lines. These results give rise to the importance of further
elucidation of the physiological interaction between the neuroendocrine and immuno-hematopoietic systems.
CCK-B/gastrin receptor mRNA is not detectable in nonadherent normal MNC but is in several tumor cell lines including T- and B- lymphoid lineages. It is of interest to determine
T
Y
0
10-11
10-10
10-9
10-8
Antagonist (M)
Fig 4. Inhibitory concentration-response curve of YM022 for
MOLT-14. Serum-deprived cells were cultured in the presence of various concentrations of YM022 (0)
or YMOU-S (01for 44 hours, followed by a further Chour incubation with [methyC'Hthymidine. Results ere expressed as means 2 SE of three independent experiments,
each performed in quedruplicate.
whether the leukemia cells ectopically express the peptide
hormone receptor. If so, the receptor could be a novel molecular probe for the diagnosis of neoplasms derived from the
immuno-hematopoietic system as well as that of human
small cell lung cancers.' It would be important to examine
the expression in the progenitor cells of the human bone
marrow, lymph node, or thymus.
In the present study, we also showed for the first time that
the CCK-B/gastrin receptor mediates cell proliferation of
several human leukemia cells in a ligand dependent manner.
Moreover, YM022 but not its stereoisomer specifically inhibits the proliferation of THP-1, MOLT-16, MOLT-14, and
CCRF-CEM cells in serum-deprived medium. The transcripts of a peptide ligand, gastrin, and its receptor genes
are coexpressed in these leukemia cell lines. The expression
level of gastrin mRNA in the leukemia cell lines was too
low to be detected by RNA blot analysis. However, the
presence of gastrin-like immunoreactivity was confirmed in
the conditioned media of several leukemia cells. These results are consistent with previous reports of a human colon
carcinoma cell line, HCT 1 16.33.34
Thus, our present results
imply that leukemia cell growth is accelerated by an autocrine mechanism through the CCK-B/gastrin receptor. Data
suggesting that gastrin could participate in an autocrine loop
driving tumor cell growth have been reported for other human neoplastic cell lines derived from the colon, stomach,
and kidne~.'~.~'
Our current data are the first to suggest the
presence of a gastrin autocrine system in human neoplastic
cells derived from nonepithelial (mesoderm) origin.
Very recently, posttranslational processing intermediates
of gastrin, specifically glycine-extended gastrin, as well as
gastrin itself have been reported to stimulate the proliferation
of a rat pancreatic tumor cell line and mouse Swiss 3T3
fibroblast^.^^,^^ Glycine-extended gastrin was at least four
orders of magnitude less potent than gastrin in its ability to
induce acid secretion. Moreover, CCK-B/gastrin receptorselective antagonists did not inhibit the proliferation induced
by glycine-extended gastrin. This implies that there could
be two gastrin receptors that mediate the proliferative action
of peptides derived from the gastrin gene. Although immunoreactive gastrin was detected in the conditioned media of
leukemia cell lines, we could not distinguish between amidated gastrin and glycine-extended form by the assay. This
may help to explain the varying inhibitory effects of a CCK-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2688
gastrin
Myeloid cell
RT
Megakaryo.
i n
-+ -+ -+ -+ -+
-
IWATA ET AL
Bell
-+ -+ -+ -+
T-cell
RT
-+ -+ - + - + - + - + - + - + - +
--
409bp
B/gastrin receptor antagonist on the leukemia cell lines coexpressing the gastrin and CCK-B/gastrin receptor genes.
In conclusion, the CCK-B/gastrin receptor mRNA is expressed in normal human PMN cells and various lineages
279bp
Fig 5. Expression of gastrin
mRNA in human leukemia cells.
The same cDNA samples as used
in Fig 2 were amplified using
primers specific for human gastrin generating a 279-bp band
from mRNA and a 409-bp fragment from genomic DNA. Representative autoradiographs of
Southern blot analysis using
gastrin cDNA are shown.
of hematopoietic tumor cell lines. Moreover, in certain leukemia cell lines such as THP-1, MOLT-16, MOLT-14, and
CCRF-CEM, an autocrine mechanism seems to promote cell
growth. To elucidate the physiological and pathological significance of the CCK-B/gastrin receptor in immuno-hematopoietic systems, further studies are necessary.
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RT
gastrin
-+-+I+
--
409bp
279bp
Fig 6. Expression of gastrin mRNA in normal human peripheral
white blood cells. The same cDNAs of PMN, macrophages and lymphocytes as used in Fig 1 were amplified using the primers specific
for the gastrin gene generating a 279-bp band from mRNA and a 409bp fragment from genomic DNA. A representative autoradiograph
of Southern blot analysis using the human gastrin cDNA probe is
shown.
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1996 88: 2683-2689
Autocrine loop through cholecystokinin-B/gastrin receptors involved
in growth of human leukemia cells
N Iwata, T Murayama, Y Matsumori, M Ito, A Nagata, T Taniguchi, K Chihara, Y Matsuo, J
Minowada and T Matsui
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