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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1952-1960
Production of Thrombopoietin by Human Carcinomas and Its Novel Isoforms
By
Yutaka Sasaki,
Takayuki Takahashi,
Hiroshi Miyazaki,
Atsushi Matsumoto,
Takashi Kato,
Kishiko Nakamura,
Sumiko Iho,
Yoshiaki Okuno, and
Kazuwa Nakao
From the Department of Medicine and Clinical Science, Kyoto
University Graduate School of Medicine, Kyoto, Japan; the Department of
Hematology and Clinical Immunology, Kobe City General Hospital, Kobe,
Japan; the Pharmaceutial Reseach Laboratory, Kirin Brewery Co,
Takasaki, Japan; the College of Medical Technology, Kyoto University,
Kyoto, Japan; and the Department of Immunology and Medical Zoology,
Faculty of Medicine, Fukui Medical University, Fukui, Japan.
 |
ABSTRACT |
Thrombocytosis is occasionally seen in patients with carcinomas and
has been assumed to be attributable to interleukin-6 or granulocyte-macrophage colony-stimulating factor produced by carcinoma cells. In this study, we clarified whether thrombopoietin (TPO) is
involved in carcinoma-associated thrombocytosis. Expression of TPO mRNA
was observed in the majority of 27 carcinoma cell lines as determined
by reverse transcriptase-polymerase chain reaction (RT-PCR). There were
6 PCR products differing in size; sequence analysis showed the
full-length TPO mRNA (TPO-1), 12- and 116-bp deleted variants (TPO-2
and TPO-3, respectively), and 3 novel isoforms (197- and 128-bp deleted
forms and a 60-bp insert form of TPO-3; named TPO-4, TPO-5, and TPO-6,
respectively). Of 27 lines, 24 expressed TPO-1 mRNA with various other
isoforms. Culture supernatants of COS-1 cells transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive cells,
whereas Western blot analysis on the cell lysates demonstrated TPO-5
but not TPO-6 protein, suggesting poor extracellular secretion (TPO-5)
or poor protein synthesis (TPO-6). TPO protein was detected in 10-fold
concentrated culture supernatants of cells of these carcinoma lines,
with a median concentration of 0.38 fmol/mL as evaluated by
enzyme-linked immunosorbent assay. High blood TPO levels were observed
with a median value of 3.46 fmol/mL (range, 0.34 to 8.67 fmol/mL) in
patients with advanced carcinomas associated with thrombocytosis. These
results indicate that thrombocytosis in patients with carcinomas might
be caused, at least in part, by TPO produced by carcinoma cells.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THROMBOCYTOSIS IS occasionally seen in
patients with carcinomas.1-3 The cause of
malignancy-associated thrombocytosis has been assumed to be
attributable to interleukin-6 (IL-6),4 which potently
promotes megakaryocyte maturation and induces platelet production
(megakaryocyte potentiating [Meg-Pot] activity),5,6 although the cellular source of IL-6 may be different from patient to
patient, being carcinoma cells, bystander cells, or both. In our
previous study on 14 patients with tumors producing colony-stimulating factor (CSF), IL-6 produced by tumors was shown to be responsible for
the thrombocytosis.7 When the type of CSF produced was granulocyte-macrophage colony-stimulating factor (GM-CSF),
thrombocytosis was caused by the megakaryocyte colony-stimulating
factor (Meg-CSF) activity of GM-CSF and the Meg-Pot activity of GM-CSF
plus IL-6.7 In our additional series of patients with
CSF-producing tumors, the semisolid culture of normal bone marrow cells
with plasmas from patients with tumors producing granulocyte
colony-stimulating factor (G-CSF) occasionally generated small
megakaryocytic colonies in addition to granulocytic colonies
(unpublished data). This fact suggested the presence of
Meg-CSF activity in the plasmas examined, because neither G-CSF nor
IL-6 induces megakaryocytic colonies in vitro. Furthermore, Estrov et
al8 showed that thrombocytosis in patients with metastatic
cancer was not totally attributable to IL-6 or GM-CSF and subsequently
suggested the presence of unidentified thrombopoietic activity in
plasmas from these patients.
In recent years, thrombopoietin (TPO), the c-mpl ligand, was cloned
from several species.9-14 TPO has both Meg-CSF and Meg-Pot activity in vitro.15 It stimulates platelet production in
vivo in mice and markedly increases the number of megakaryocytes in the
spleen and bone marrow.16 TPO mRNA is expressed primarily in the liver and, to a lesser degree, kidney, smooth muscle, spleen, and bone marrow.9,10,12,17 Regarding malignant tumors,
constitutive TPO mRNA expression has been demonstrated in a human
hepatoma cell line (HepG2)18 and a number of leukemic cell
lines.19 However, there has been no report regarding TPO
production by human carcinomas other than HepG2 and HEK20
cells. In addition, the relationship between thrombocytosis and
TPO production by malignant cells has not been studied, although TPO
production by tumor cells was suspected in patients with hepatoblastoma
associated with thrombocytosis.21
To date, 3 different forms of human TPO mRNA have been identified from
the liver, ie, full-length mRNA and its 2 alternative spliced
forms.22 The alternative forms lack 12 and 116 bp in exon
6.22 The 2 isoforms may correspond to murine TPO isoforms, TPO-2 and TPO-3, respectively.23,24 In addition, TPO-4 has been identified in mice24 but not in humans. In this study, we determined the expression of TPO mRNA by human carcinoma cell lines
producing or not producing CSF using reverse transcriptase-polymerase chain reaction (RT-PCR). Most of these cell lines expressed TPO-1, TPO-2, and TPO-3 mRNAs, and novel isoforms (TPO-4, TPO-5, and TPO-6)
were identified in some lines. Furthermore, we examined the
biological activity of TPO-5 and TPO-6 proteins. TPO protein was detected in the culture supernatants of carcinoma cells
as evaluated by enzyme-linked immunosorbent assay
(ELISA). We also examined the relationship between blood TPO
levels and platelet counts in patients with advanced carcinomas.
| |
MATERIALS AND METHODS |
Human carcinoma cell lines.
Twenty-seven cell lines from human carcinomas were tested. KHC287 (lung
cancer),7,25 Lu-Y1 (lung),7 UT-M1
(uterus),7 HTC/C3 (thyroid),26 ST-Y1 (stomach;
unpublished data), and ES-O (esophagus; unpublished data)
were established in our institution. CHU-2 (oral cavity),27
T24 (urinary bladder),28 Lu65 (lung),29 Lu99
(lung),30 SK-HEP-1 (liver),31 HeLa (uterus),
PC-3 (lung),32 A-549 (lung),33 ABC-1
(lung),34 Lc-1sq (lung),35 LK-2 (lung), RERF-LC-MS (lung), RERF-LC-OK (lung), and VMRC-LCD (lung) were provided
by the Japanese Cancer Research Resources Bank (Osaka, Japan). HepG2 (liver)36 was obtained from the
American Type Culture Collection (Rockville, MD). HLC-1
(lung)37 and HL111783 (lung) were provided by the Riken
Cell Bank (Tsukuba, Japan). Sq-19 (lung), 86-2 (lung),38
LK-79 (lung), and 11-18 (lung) were provided by the Research Institute
for Tuberculosis and Cancer, Tohoku University (Sendai, Japan). All of
the lung carcinoma cell lines were established from non-small-cell
lung carcinomas. Of the 27 cell lines, KHC287,7
Lu-Y1,7 UT-M1,7 HTC/C3,7 ST-Y1,
ES-O, CHU-2,39 T24,39 Lu65,25 Lu99,
and SK-HEP-1 produce G-CSF or GM-CSF (HTC/C3) and IL-6, being regulated
by coproduced IL-1. A-549, RERF-LC-MS, RERF-LC-OK, PC-3, Sq-19, 86-2, 11-18, and HepG2 do not produce IL-1 and produce G-CSF and IL-6 only
when they are cultured with exogenous IL-1.40,41
Cell culture and culture supernatant.
All cells were cultured in RPMI 1640 (Nissui, Tokyo, Japan) containing
10% fetal calf serum (GIBCO, Grand Island, NY) at an initial cell
density of 2 × 105/mL. After 48 hours, culture
supernatants were collected, concentrated 10-fold by ultrafiltration
using a PM-10 membrane (Amicon, Tokyo, Japan), and kept frozen
( 20°C) until use. In some experiments, recombinant human
IL-1 was added at the initiation of culture at a concentration of 4 ng/mL. IL-1 was provided by Dainippon Pharmaceutical Co (Osaka, Japan).
RNA extraction, RT-PCR, and nested-PCR.
After 48 hours of incubation, cells were harvested and total RNA was
extracted using TRIzol (GIBCO) by a modification of the acid phenol
method. One microgram of total RNA was reverse transcribed by
Superscript II (GIBCO) in a reaction volume of 10 µL [50 mmol/L Tris-HCl, 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L
dithiothreitol (DTT), 1.25 mmol/L oligo(dT), 1 mmol/L
dNTPs, and 100 U RTase]. Reverse transcription was performed for 10 minutes at 30°C and for 60 minutes at 42°C. PCR and nested-PCR
reactions were performed in a reaction volume of 25 µL (1 µL of
cDNA pool or PCR products, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.1% TritonX-100, 0.2 mmol/L dNTPs, 0.4 µmol/L sense and antisense primers, and 0.5 U rTaq DNA polymerase
[Toyobo, Osaka, Japan]). Primers used for PCR and nested-PCR are
shown in Fig 1 and
Table 1. First-strand cDNA pool (PCR) or
PCR product (nested-PCR) was amplified for 35 cycles using the GeneAmp
PCR System 2400 (Perkin Elmer, Norwalk, CT). To determine the existence
of an isoform that corresponds to mouse TPO-4, we fractionated the
RT-PCR products (primers S3 and A3) at approximately 450 to 500 bp by
electrophoresis and then performed the second PCR using a set of S7 and
A2 primers. The amplification procedure consisted of denaturation at
94°C for 30 seconds; annealing at 62°C (primers S3 and A3),
65°C (S7 and A2), or 63°C (S2 and A4) for 30 seconds; and
extension at 72°C for 30 seconds.
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| Fig 1.
A schematic of the human TPO gene from intron 4 to exon 6 and mRNA isoforms. Corresponding portions exhibit the same patterns.
TPO-1, TPO-2, and TPO-3 were reported previously. TPO-4, TPO-5, and
TPO-6 are newly identified forms. TPO-3, TPO-4, and TPO-5 possess a
stop codon that is different from that of TPO-1 and TPO-2 due to the
frameshift. TPO-6 contains another stop codon in the inserted sequence
that is part of intron 5, which is not spliced out. *End of the
erythropoietin-like domain. Horizontal arrows show the regions
amplified by RT-nested PCR. The sizes of the PCR products and the sets
of primers used are written beside the arrows.
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Sequencing of PCR products.
Representative PCR products were electrophoresed in a 4% agarose gel
and separated to single bands. The isolated bands were sequenced
directly or after subcloning into pGEM-T vector (Promega, Madison, WI)
by Dye Deoxy terminator cycle sequencing, using a 373A DNA sequencer
(Applied Biosystems, Foster City, CA). The results were confirmed by
reading both DNA strands.
Construction of expression vectors for TPO-5 and TPO-6.
PCR product of S9 (5'-GGAGCCACGCCAGCCAGACA-3') and A1
(5'-TCCAACAATCCAGAAGTGGT-3') using the UT-M1 cDNA as a
template were subcloned into pGEM-T plasmid (pGEM-T/TPOS9A1). PCR
products of S10 (5'-CCCTGCAGAGCCTCCTTGGA-3') and A6
(5'-GGGCTTTGGGTTTCAGGAGA-3') using the same cDNA were
electrophoresed, and the band corresponding to the 3' half of the
coding region of TPO-5 was purified. pGEM-T/TPOS9A1 was digested with
Pst I and HincII and then ligated with the purified S10/A6 product digested with Pst I and Rsa I, resulting
in a construction of pGEM-T/TPO-5. pGEM-T/TPO-6 plasmid was constructed
with the PCR product of S9 and A10
(5'-CTCAGGCCTCCCTTGTCTGGGTTC-3') and then subcloned into
pGEM-T vector. Sequence analyses were performed to confirm that these
plasmids contained the entire coding regions of these TPO isoforms.
Expression of the hexahistidine (His)-tagged TPO-1, TPO-5, and
TPO-6.
TPO-1, TPO-5, and TPO-6 cDNAs were regenerated by PCR using the
subcloned cDNAs to introduce an EcoRI site at the 5' end
and a His residue and Not I site at the 3' end. Each PCR
product was digested with EcoRI and Not I and subcloned
into pEF18S vector derived from pEFneo,42 a mammalian
expression vector containing the elongation factor 1 (EF1) promoter and
the SV40 polyadenylation signal. The resulting expression vectors
(pEF18S-hTPO-1/His, pEF18S-hTPO-5/His, and pEF18S-hTPO-6/His) were
transiently transfected into COS-1 cells using the diethyl aminoethyl
(DEAE)-Dextran method. After 3 days of culture, the
conditioned medium was collected, sterilized by filtration, and
subjected to a TPO-dependent cell proliferation assay, as described
below. The expression levels of TPO were determined by Western blot
analysis. His-tagged TPOs in the culture supernatants and cell lysates
of COS-1 cells were first purified and concentrated by a Ni-NTA resin
column (QIAGEN Inc, Valentica, CA). The purified materials were
immunoprecipitated with the Ni-NTA resin, and the precipitates were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred onto a polyvinilidene difluoride membrane
(Millipore, Bedford, MA), and probed with anti-His(C-term) antibody
(Invitrogen, Carlsbad, CA). Antibody reactions were detected by an
enhanced chemiluminescence (ECL) method according to the
manufacturer's instructions (Amersham, Arlington Heights, IL).
Bioassay of TPO.
The in vitro biological activity of the His-tagged TPO-1, TPO-5, and
TPO-6 were determined using FDCP-hMp15 cells,43 which were
genetically engineered to express human c-mpl constitutively. The cells
were cultured with serially diluted culture supernatants of COS-1 cells
that expressed TPO-1, TPO-5, or TPO-6 in 200 µL of Iscove's modified
Dulbecco's medium (GIBCO) supplemented with 10% fetal calf serum in a
96-well tissue culutre plate (2.5 × 103
cells/well) for 3 days at 37°C. Each concentration was tested in
duplicate. Cell growth was determined by a colorimetric assay using a
tetrazolium salt, WST-1 (Dojindo Laboratories, Kumamoto, Japan).44
ELISA.
TPO concentrations in the culture supernatants of cells from each cell
line were measured in triplicate using an ELISA kit devised by Tahara
et al45,46 in the Pharmaceutical Research Laboratory, Kirin
Brewery Co (Takasaki, Japan). The lower limit of detection
for TPO both in the human plasma and the culture supernatant was 0.2 fmol/mL. Stem cell factor (SCF) concentrations in the culture
supernatants were measured by use of an ELISA kit (Amersham). The lower
limit of detection for SCF was 31.3 pg/mL.
Patients with advanced carcinomas.
Citrated plasmas were obtained with informed consent from patients with
advanced carcinomas associated with thrombocytosis more than 500 × 109/L. TPO concentrations in the plasmas were
determined with the same ELISA kit. Patients with infection, palpable
spleen, or abnormal blood pictures, such as leukoerythroblastosis,
giant platelets, or circulating megakaryocytes, were excluded in this
study. As a control, TPO concentrations in the plasmas from patients
with advanced carcinomas without thrombocytosis (<370 × 109/L) were measured.
Statistical analysis.
The differences in the plasma TPO concentrations were analyzed using
the Wilcoxon signed rank test.
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RESULTS |
Expression of TPO mRNA and sequencing of PCR products.
TPO mRNA expression by carcinoma cells was investigated using RT-PCR
and nested-PCR. There were 6 bands observed at 304, 292, 188, 176, and 248 bp (Fig 2A) and 1 band at 107 bp (Fig 2C) using a set of S7 and A2 primers. By sequencing each band,
the latter 5 were shown to be parts of the full-length TPO (TPO-1)
mRNA, ie, 12-bp (position 397 to 408), 116-bp (478 to 593), and 128-bp (397 to 408, 478 to 593) deleted forms in exon 6, a 60-bp insert form
between exon 5 and 6 in combination with the 116-bp (478 to 593)
deletion in exon 6, and a 197-bp (397 to 593) deleted form in exon 6. The former 2 variant mRNAs correspond to previously reported human and
mouse isoforms of TPO (TPO-2 and TPO-3, respectively)22,23 and the latter 3 are novel forms (TPO-5, TPO-6, and TPO-4; Fig 1).
Because the band representing TPO-2 was weak for many of the cell
lines, we constructed another set of primers (S2 and A4) that
specifically amplify TPO-2 (Fig 2B). Table
2 summarizes the TPO mRNAs expressed by carcinoma cells. Of the 27 cell
lines examined, 24 lines expressed TPO-1 mRNA, 15 expressed TPO-2, 23 expressed TPO-3, 6 expressed TPO-4, 7 expressed TPO-5, and 9 expressed TPO-6. Expression of TPO mRNA was not correlated with the production of
IL-1, IL-6, G-CSF, or GM-CSF by the carcinoma cells examined. In
addition, TPO mRNA expression was not correlated with the origin of the
carcinomas or the histological subtype of lung carcinomas.
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| Fig 2.
TPO mRNA isoforms detected by RT-nested PCR. (A and B).
Total RNA was reverse-transcribed and the first PCR (primer set S3 and
A3) was performed. PCR products were subjected to nested-PCR using a
set of S7 and A2 primers for TPO-1, TPO-2, TPO-3, TPO-5, and TPO-6 (A)
or a set of S2 and A4 primers for TPO-2 (B). (C) Approximately 450 to
500 bp of the first PCR products was fractionated by electrophoresis,
and then the second PCR using a set of S7 and A2 primers for TPO-4 was
performed.
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Biological activity of TPO-5 and TPO-6 proteins.
As shown in Table 3, culture supernatants of COS-1 cells
transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive FDCP-hMp15 cells, whereas a culture supernatant of
COS-1 cells that expressed TPO-1 stimulated the growth of FDCP-hMp15 cells in a dose-dependent manner.
Western blot analysis.
Western blot analysis was performed on immunoprecipitated His-tagged
TPOs both in the culture supernatants and cell lysates of COS-1 cells
transfected with TPO cDNAs. As shown in
Fig 3, only TPO-1 protein was
detected in the culture supernatants. On the other hand, in the cell
lysates, TPO-5 protein as well as that of TPO-1, but not TPO-6 protein,
was demonstrated. Similar results were obtained when anti-TPO
polyclonal antibody was used as a probe (data not shown).
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| Fig 3.
His-tagged TPOs in the culture supernatants and cell
lysates of COS-1 cells were adsorbed on a Ni-NTA resin column. The
eluates were precipitated with the Ni-NTA resin, and the precipitates
proteins were separated by SDS-PAGE and probed with anti-His(C-term)
antibody. Lanes 1 and 5, TPO-1; lanes 2 and 6, TPO-5; lanes 3 and 7, TPO-6; lane 4, untransfected COS-1 cells. In lane 5, the band
corresponding to TPO-1 appears to be masked by nonspecific bands.
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Detection of TPO and SCF proteins by ELISA.
We chose representative cell lines classified based on the intensity of
TPO-1 PCR bands (Fig 2A). TPO protein was detected in 10-fold
concentrated culture supernatants of the cells in 8 of 10 cell lines
tested, with a median concentration of 0.38 fmol/mL, ranging from 0.24 to 5.86 fmol/mL (Table 4). A hepatoma cell line (HepG2) showed a high level of TPO production. IL-1 exogenously added to the culture did not enhance the TPO production by any of the
cell lines examined. The SCF levels were determined in the culture
supernatants of T24, UT-M1, 86-2, LK-79, and VMRC-LCD cells and were
found to be 82.0, 85.3, 84.8, less than 31.3, and less than 31.3 pg/mL,
respectively.
Blood TPO levels in patients with advanced carcinomas with or without
thrombocytosis.
In Table 5, clinical and laboratory
findings and plasma TPO concentrations in individual patients are
shown. The plasma TPO concentrations in patients with thrombocytosis
were 3.46 ± 2.02 fmol/mL (mean ± SD, n = 13) and were
significantly (P = .0003) higher than those in patients without
thrombocytosis (0.91 ± 0.57 fmol/mL, n = 14). These blood TPO
levels in cancer patients with thrombocytosis were comparable to those
reported in patients with hepatoblastoma associated with
thrombocytosis.21 The plasma TPO levels in patients without
thrombocytosis were slightly higher than those in healthy volunteers
(0.40 ± 0.28 fmol/mL, n = 99); however, the difference
was not statistically significant.
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Table 5.
Blood TPO Levels and Laboratory Data in Patients With
Advanced Carcinomas With or Without Thrombocytosis
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| |
DISCUSSION |
In this study, we demonstrated TPO production by carcinoma cells
originating from various tumors both at the mRNA and protein levels.
Regarding the isoforms of TPO mRNA, we identified 3 novel isoforms
(TPO-4, TPO-5, and TPO-6) as well as previously reported forms (TPO-1,
TPO-2, and TPO-3). TPO-3, TPO-4, and TPO-5 yield frameshifts and TPO-6
contains a stop codon in the inserted sequence (Figs 1 and 4). Regarding biological
activity of TPO-5 and TPO-6, culture supernatants of COS-1 cells
transfected with TPO-5 or TPO-6 cDNA did not stimulate the growth of
TPO-responsive cells. Western blot analysis showed no detectable
protein of TPO-5 or TPO-6 in the culture supernatants of transfected
COS-1 cells, whereas the analysis demonstrated TPO-5 but not TPO-6
protein in cell lysates of the COS-1 cells. These results suggest poor extracellular secretion of TPO-5 protein and poor protein synthesis of
TPO-6. Therefore, biological activity of TPO-5 and TPO-6 remains unclear and to be examined. However, it has been reported in humans that the biological activity of TPO isoforms is abolished if the truncation extends into the region that shares homology with the erythropoietin (EPO) mRNA (EPO-like domain).47 The EPO-like domain (amino acid residues 1-154) activates mpl and the structure of
the domain is supposed to be stabilized by disulfide bonds between
cysteins 1 and 4 and cysteins 2 and 3. Replacement of either cystein 1 or 4 completely abrogates biological activity.48 In TPO-5,
the position of cystein 4 is changed from 151 to 145 due to frameshift.
This may prevent cysteins 1 and 4 to form the bonds. TPO-6 lacks
cystein 4 because of the stop codon in the insert between exons 5 and 6 (Figs 1 and 4). These findings suggest poor biological activity of
TPO-5 and TPO-6, although the possibility that these isoforms,
especially TPO-5, act as a membrane-bound ligand for mpl still remains.
Regarding the activity of TPO-4, mouse TPO-4 protein has comparable
biological activity to TPO-1 and the apparently low activity of
TPO-449 results from the poor extracellular secretion of
the truncated protein.50 Therefore, human TPO-4 should be
characterized with respect to its thrombopoietic activity.
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| Fig 4.
Deduced amino acid sequences of TPO-4, TPO-5, and TPO-6.
5' terminal sequences down to Gln-111 are common with TPO-1
(top). TPO-4 and TPO-5 contain the same sequence derived due to the
frameshift (underlined).
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It has been reported that the level of TPO mRNA expression in the liver
or kidney is unchanged, regardless of the platelet count.24
Therefore, it is assumed that the blood TPO level is regulated by
platelets expressing abundant TPO receptors that absorb
TPO.51,52 On the other hand, the cytokine regulation of TPO
production is poorly understood. Only 1 study regarding this was
performed using a human hepatoma cell line HepG2; however, all
cytokines tested did not have any effect on the TPO mRNA expression by
this cell line.18 In the present study, exogenously added proinflammatory cytokine, IL-1, which is a potent inducer of IL-6,
IL-8, G-CSF, or GM-CSF in normal macrophages or in some carcinomas,39,40 did not affect the TPO production by the
carcinoma cells examined. Furthermore, TPO production was not
correlated with the production of IL-1, IL-6, or CSFs by these
carcinoma cells. Therefore, TPO production in carcinomas may be
independent of the expression of these cytokines. Thus, it would be
very interesting and important to identify regulatory factors for TPO production.
In the present study, the amount of TPO produced by carcinoma cells,
except for HepG2 cells, appears to be minute. Higher levels of
production of TPO by HepG2 may reflect the potency of normal
hepatocytes with respect to the production of TPO. Several reports have
described marked thrombocytosis in patients with hepatocellular
carcinomas21,53,54; however, TPO production by tumor cells
was not determined. On the other hand, even for a relatively low level
of production, the amount of TPO may not be negligible when a patient
has a high tumor burden. Indeed, in this study, we observed high blood
TPO levels in almost all patients with advanced carcinomas associated
with thrombocytosis greater than 500 × 109/mL.
Whereas blood TPO levels in patients with advanced carcinomas without
thrombocytosis were almost equal to those in healthy controls, these
results indicate that TPO is involved in the carcinoma-associated thrombocytosis, although the exact contribution of TPO produced by
carcinomas to the thrombocytosis remains to be determined. We also
detected SCF in the culture supernatants in 3 of 5 TPO-producing cell
lines examined. SCF acts synergistically with TPO in the proliferation
of megakaryocyte progenitors.52,55 Therefore, the action of
TPO may be amplified by SCF produced by tumors in some patients. This
appears to further support the hypothesis of TPO-induced thrombocytosis
in carcinoma patients.
In conclusion, some carcinomas produce TPO that might be involved, in
part, in the carcinoma-associated thrombocytosis. However, the cause of
thrombocytosis is rather complicated, because certain carcinomas
produce multiple cytokines that exhibit Meg-Pot and/or Meg-CSF
activities, such as SCF, IL-6, IL-11, and GM-CSF, as well as TPO.
Indeed, in this study, blood TPO levels were not correlated with
platelet counts in cancer patients with thrombocytosis. This fact
suggests that other thrombopoietic factors were involved besides TPO in
the thrombocytosis in these cancer patients. Thus, further studies are
required to clarify the exact roles of individual cytokines in
carcinoma-associated thrombocytosis.
| |
ACKNOWLEDGMENT |
The authors are grateful to Kyoko Tanaka and Eiko Yamashita (Kobe City
General Hospital, Kobe, Japan) for preparing plasmas from patients with carcinomas.
| |
FOOTNOTES |
Submitted April 21, 1998; accepted May 17, 1999.
Supported in part by the Sasaki Foundation for the Promotion of
Leukemia Research, by the Smoking Research Foundation, and by the Kobe
City Foundation for the Promotion of Medical Research (to T.T.).
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 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Takayuki Takahashi, MD, Department of
Hematology and Clinical Immunology, Kobe City General Hospital, 4-6 Minatojima Nakamachi, Chuo-ku, Kobe 650-0046, Japan.
| |
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