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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1715-1723
Heparin-Binding Epidermal Growth Factor-Like Growth
Factor/Diphtheria Toxin Receptor Expression by Acute Myeloid Leukemia
Cells
By
Fabrizio Vinante,
Antonella Rigo,
Emanuele Papini,
Marco A. Cassatella, and
Giovanni Pizzolo
From the Departments of Hematology and General Pathology, University
of Verona, Verona; and CNR Center for Biomembranes, University of
Padua, Padua, Italy.
 |
ABSTRACT |
Heparin-binding epidermal growth factor-like growth factor (HB-EGF)
is an EGF family member expressed by numerous cell types that binds to
EGF receptor 1 (HER-1) or 4 (HER-4) inducing mitogenic and/or
chemotactic activities. Membrane-bound HB-EGF retains growth activity
and adhesion capabilities and the unique property of being the receptor
for diphtheria toxin (DT). The interest in studying HB-EGF in acute
leukemia stems from these mitogenic, chemotactic, and receptor
functions. We analyzed the expression of HB-EGF in L428, Raji,
Jurkat, Karpas 299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562 cell
lines and in primary blasts from 12 acute myeloid leukemia (AML) cases,
by reverse-transcriptase polymerase chain reaction (RT-PCR) and
Northern blot and by the evaluation of sensitivity to DT. The release
of functional HB-EGF was assessed by evaluation of its proliferative
effects on the HB-EGF-sensitive Balb/c 3T3 cell line. HB-EGF was
expressed by all myeloid and T, but not B (L428, Raji), lymphoid cell
lines tested, as well as by the majority (8 of 12) of ex vivo AML
blasts. Cell lines (except for the K562 cell line) and AML blasts
expressing HB-EGF mRNA underwent apoptotic death following exposure to
DT, thus demonstrating the presence of the HB-EGF molecule on their membrane. Leukemic cells also released a fully functional HB-EGF molecule that was mitogenic for the Balb/c 3T3 cell line. Factors relevant to the biology of leukemic growth, such as tumor necrosis factor- (TNF-), 1,25-(OH)2D3, and
especially all-trans retinoic acid (ATRA), upregulated HB-EGF
mRNA in HL-60 or ML-3 cells. Granulocyte-macrophage colony-stimulating
factor (GM-CSF) induced HB-EGF mRNA and acquisition of sensitivity to
DT in one previously HB-EGF-negative leukemia case. Moreover, the U937
and Karpas 299 cell lines expressed HER-4 mRNA. This work shows that
HB-EGF is a growth factor produced by primary leukemic cells and
regulated by ATRA, 1,25-(OH)2D3, and GM-CSF.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEPARIN-BINDING epidermal growth
factor-like growth factor (HB-EGF) is a heavily glycosylated EGF
family member of approximately 22 kD capable of binding to heparin. It
was originally identified in human monocytes and U937 monocytic cell
line-conditioned medium.1-3 Subsequently, HB-EGF
expression was found in a wide range of cell types, including
monocytes,1 CD4+ lymphocytes,4
eosinophils,5 smooth muscle cells (SMC),6 and
endothelial7 and normal or neoplastic epithelial
cells.1,8 HB-EGF can be released from the cell membrane
through proteolytic mechanisms,9 but multiple spliced mRNAs
are likely to be produced and the cDNA corresponding to a short HB-EGF
form lacking intramembrane and intracytoplasmic domains has been
cloned.10 HB-EGF binds to EGF receptor 1 (HER-1) and 4 (HER-4)1,2,11 eliciting different biologic
responses.11 It is a potent mitogenic and a chemotactic
factor for fibroblasts4 and SMC,12 mitogenic factor for keratinocytes,8 and chemotactic factor for
endothelial cells13 and astrocytes.14 Moreover,
HB-EGF has been shown to participate in autocrine-paracrine loops,
which are active in a number of epithelial neoplasia,15 and
to be involved in stromal proliferation following
decidualization.16
Membrane-bound HB-EGF retains growth activity and adhesion
capabilities. Macrophages infiltrating atheromatous plaques actively induce SMC hyperplasia through HB-EGF.17 Rat blastocyst
implantation has been reported to be associated with HB-EGF expression
and adhesion activity.18 Finally, membrane-bound HB-EGF has
the unique property of acting as the receptor for the diphtheria toxin (DT),19 a protein translation inhibitor capable of
triggering apoptotic death.20 CD9 coexpression enhances the
mitogenic activity of membrane-bound HB-EGF,17 as well as
the sensitivity to DT.21
Attention has been devoted to the role of HB-EGF in reproductive
biology,16,18 wound healing,22 atheromatous
phenomena,17,23 angiogenesis,13 and epithelial
neoplastic proliferative events.15 The production of HB-EGF
by monocytes, CD4+ lymphocytes, and eosinophils suggests
that it may also be produced by other normal and neoplastic hematologic
cell types. In the present study, we analyzed the expression of HB-EGF
in a panel of human hematologic cell lines derived from different
lineages, and in primary blast cells from patients with acute myeloid
leukemia (AML). The interest in studying HB-EGF in AML stems from its
mitogenic, chemotactic, and receptor functions.
We found that HB-EGF was expressed by the human myeloid and T, but not
B, lymphoid cell lines tested, as well as by ex vivo blast cells in a
number of AML cases. Thus, HB-EGF is an additional growth
factor produced by primary leukemic cells.
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MATERIALS AND METHODS |
Cell lines.
The following cell lines available in our laboratory were studied:
B-cell lines L428 (Hodgkin-derived)24 and Raji
(non-Hodgkin's lymphoma)24; T-cell linesJurkat (acute
T-cell leukemia),25 Karpas 299 (anaplastic large-cell
lymphoma),24 and L540 (Hodgkin-derived)24; natural killer (NK)-cell line2C826; myeloid cell
linesHL-60 (AML)24, U937 (monocytic
leukemia),24 THP-1 (acute monocytic leukemia),27 ML-3 (acute myelomonocytic
leukemia),28 and K562 (blastic phase of chronic myeloid
leukemia).24
Patients and isolation of leukemic cells.
The main clinical characteristics of the patients whose leukemic cells
were investigated are listed in Table 1.
All cases were untreated AML patients at diagnosis with a high
percentage ( 90%) of blasts and minimal residual contamination by
normal cells. The diagnosis of AML and its French-American-British
(FAB) subtypes was based on clinical findings and on established
morphologic, cytochemical, and cytofluorimetric parameters of
peripheral blood and/or bone marrow cells. Viable leukemic
cells from freshly heparinized peripheral blood or bone marrow were
separated by centrifugation on Lymphoprep (Nycomed Pharma AS, Oslo,
Norway), washed twice with phosphate-buffered saline (PBS), and either
immediately used or stored frozen in liquid nitrogen. In all cases,
frozen cell samples contained greater than 95% blasts.
Cell storage.
The cell lines and aliquots of ex vivo blasts were stored frozen in
liquid nitrogen in 70% RPMI 1640 (Gibco-BRL Life Technologies, Paisley, UK), 20% dimethyl sulfoxide (DMSO), and 10% heat-inactivated fetal calf serum (FCS; Gibco-BRL). Frozen cells were thawed in 20%
FCS/80% RPMI 1640, immediately centrifuged, and washed once with
culture medium. Cell viability after thawing was always greater than
90%, as assessed by Trypan-blue staining. Freezing procedures did not
modify the expression of HB-EGF.
DT sensitivity assay.
Highly purified DT at 10 11, 1010,
109, and 108 mol/L concentration
was added to 1 × 106 cells/mL (cell lines or ex vivo
blasts) in a 96-well plate (Falcon, Lincoln Park, NJ) and incubated for
24 or 48 hours at 37°C in 5% CO2 in RPMI 1640 supplemented with 10% heat-inactivated FCS and penicillin (100 IU/mL)/streptomycin (100 µg/mL). The sensitivity to DT was evaluated
as cytotoxic activity assessed by the modified 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. Ten microliters per well of 5 mg/mL MTT solution was added and
the plates were incubated at 37°C for 4 hours. After adding 100 µL/well of 0.04N HCl in isopropanol and thoroughly mixing, the plates
were read (wavelengths: test, 570; reference, 630 nm) on an AutoReader
III (Ortho Diagnostic Systems, Raritan, NJ).29,30 Resting
cells were considered to be sensitive to DT when more than 50% of them
were killed with a typical dose-response curve in the
1011 to 108 mol/L DT
concentration range during a 48-hour period. When the acquisition of
sensitivity to DT was evaluated in stimulatory experiments on
previously insensitive cells, the appearance of statistically
significant cytotoxicity after stimulation was considered.
Assessment of apoptosis: DNA laddering and morphologic changes.
After 48 hours, 108 mol/L DT-incubated cells (1 × 106 cells/mL) and their controls were harvested and
subdivided into three aliquots to evaluate apoptotic death. (a) One
milliliter of cells was added with 200 µL of lysing buffer (2 mol/L
NaCl, 50 mmol/L Tris/HCl, 10 mmol/L EDTA, pH 8), 50 µL of 20% sodium
dodecyl sulfate (SDS), and 10 µL of proteinase K, and incubated for
45 minutes at 60°C and overnight at 37°C. Following the
addition of 400 µL of 5 mol/L NaCl. the cells were centrifuged for 30 minutes at 3,500 rpm. Supernatant (SN) was transferred to
a new tube, DNA precipitated for 3 hours with 5 mL ethanol 96% at
 80°C, and centrifuged at 4,000 rpm for 30 minutes at
4°C. The dried pellet was resuspended in Tris-EDTA
(TE)-RNAse (1 µg/µL) and incubated for 1 hour at 37°C. DNA was
loaded onto 1% agarose gel and stained with ethidium bromide. (b) Cell
shrinkage and acridine orange staining by phase contrast and
fluorescence microscopy were evaluated. (c) Cells were analyzed with a
FACScan cytometer (Becton Dickinson, San Jose, CA). A gate was depicted
including the living population in the forward and side light scatter
cytogram after acquiring 1 × 105 untreated cells, and
the percentage of DT-treated cells falling outside the defined gate due
to forward or side light scatter changes was calculated.
Transwell cultures and HB-EGF activity detection.
Transwell cultures of the Balb/c 3T3 cell line, proliferation of which
is induced by HB-EGF,2 and of leukemic cells were performed
to assess whether HB-EGF released by the ML-3 cell line and ex vivo AML
blasts retained its mitogenic capability on Balb/c 3T3 cells. Confluent
Balb/c 3T3 cells were trypsinized and resuspended at 5 × 104 cells/mL. Aliquots of 200 µL were plated in
Dulbecco's modified Eagle's medium (DMEM)/10% FCS in 24-well plates
(Falcon). The cells were incubated for 5 days after reaching confluence
to deplete the media of growth factors. Transwells (12-mm diameter,
0.4-µm pore size; Costar, Cambridge, MA) were introduced into the
wells of the 24-well plate containing Balb/c 3T3 cells and ML-3 or
myeloid blast cells (5 × 105 cells/mL) were
cocultured with or without 40 ng/mL phorbol myristate acetate (PMA) in
order to favor HB-EGF release from the cell membrane.31,32 A 100-µg/mL quantity of goat antihuman HB-EGF neutralizing antibody (R&D Systems, Minneapolis, MN) was used to block HB-EGF activity. Such
activity was evaluated after 72 hours by measuring the number of Balb/c
3T3 cells using the modified MTT assay,29,30 after constructing a standard curve based on the absorbance/cell number ratio. The results were expressed as percentage increase in cell number
as opposed to control.
Stimulation of cells in culture.
The cell lines and ex vivo blasts were cultured in RPMI medium
supplemented with 10% FCS at a concentration of 1 × 106 cells/mL. When indicated, cells were treated with PMA
(40 ng/mL), all-trans retinoic acid (ATRA,
105 mol/L), tumor necrosis factor- (TNF-, 100 U/mL), interferon-gamma (IFN- , 1,000 U/mL), granulocyte-macrophage
colony-stimulating factor (GM-CSF, 100 ng/mL),
1,25-(OH)2D3 (vitamin D3, 10 1 mol/L), DMSO (1.6%), or a combination of two of
these factors. After culture for the indicated times, the cells were
harvested and RNA was extracted, as previously
reported,33 and analyzed for HB-EGF gene expression by
reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot.
RT-PCR analysis of HB-EGF and HER-4; plasmid insertion of HB-EGF
cDNA.
Total cellular RNAs were isolated and 4 µg of RNA was
reverse-transcribed (universal primers, 1.25 U of AMV RT [Gibco-BRL Life Technologies]) as previously described.33,34 cDNA was PCR-amplified using the following primers (Genenco, m-medical, Florence, Italy). (1) HB-EGF sense
5'-TGGTGCTGAAGCTCTTTCTGG-3' and antisense 5'-GTGGGAA
TTAGTCATGCCCAA-3'; these primers were designed to span exons 1 to
5 of the gene giving a fragment of 605 bp (complete form of HB-EGF
cDNA)35 or a fragment of 605 + 94 bp (short form of HB-EGF
cDNA).10 (2) HB-EGF antisense 5'-TCAAGTAACATCTTTCTGCCCAGC-'3 specific for a sequence on
the 94-bp insert present in the short HB-EGF10 expected to
give a 407-bp fragment when associated with the above-specified sense primer. (3) HER-4 sense 5'-AGATGGAGGTTTTGCTGCTGAA CA-3' and
antisense 5'-TTACACCACAGTATTCCGGTGTCT-3' (726-bp
fragment)36; (4) vimentin sense
5'-GCTCAGATTCAGGAACAGCAT-3', and antisense
5'-TAAGGGCATCCACTTCACAGG-3' (266-bp fragment). The cDNA was
denatured for 5 minutes at 94°C before 35 runs in a
thermal cycler (GeneAmp PCR System 2400; Perkin Elmer,
Norwalk, CT) using 1.25 U of Taq polymerase (Perkin Elmer, Branchburg, NJ) in 50 µL (94°C 40 seconds,
57°C 40 seconds, 72°C 50 seconds) followed by 5 minutes at
72°C. PCR products were separated by electrophoresis on 1.5%
agarose gel. The HB-EGF RT-PCR product was analyzed for the
SmaI (Gibco-BRL) restriction site (which gave the expected
HB-EGF fragments of 388 and 217 bp), and was sequenced (Sequenase 2.0 sequencing kit; USB, Cleveland, OH) as a plasmid insert (TA cloning
kit; Invitrogen, San Diego, CA) from which the HB-EGF probe was
generated for Northern blot analysis.
Northern blot analysis.
Total RNA preparation and Northern blot analysis (10 µg of RNA per
lane) were performed as previously described.33 The RNA blots were hybridized with the 32P-labeled cDNA probe to
HB-EGF obtained as specified above and with a 32P-labeled
plasmid containing a cDNA probe to G6PDH or beta-actin.
Immunostaining.
Surface expression of HER-1 and CD9 was assessed by incubation of 1 × 106 cells with 10 µL fluorescein isothiocyanate
(FITC)-conjugated anti-HER-1 monoclonal antibody (mAb) (Medac,
Hamburg, Germany) and 10 µL phycoerythrin (PE)-conjugated anti-CD9
mAb (SBA, Birmingham, AL) for 30 minutes at 4°C. Cells were washed
twice in PBS. Irrelevant FITC- or PE-conjugated IgG2b mAbs (Immunotech,
Westbrook, MA) were used as a control. The analysis was performed with
a FACScan cytometer (Becton Dickinson).
Statistics.
Student's t-test, the Mann-Whitney U test, and
Kruskall-Wallis analysis of variance (ANOVA) by ranks were used.
When needed, a logarithmic transformation was performed.
Differences were considered statistically significant when the
P value was less than .05.
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RESULTS |
HB-EGF mRNA in cell lines and blasts.
We examined the presence of mRNA for HB-EGF in a panel of cell lines
and in ex vivo AML blasts. The main findings are listed in Tables 1 and
2 for patients and cell lines,
respectively. The results of mRNA analyses in AML blasts and cell lines
are detailed in Figs 1 and
2. HB-EGF mRNA expression by cell lines was
studied using RT-PCR. As shown in Fig 1, B-derived cell lines (L428,
Raji) were negative, whereas the remaining cell lines (Jurkat, Karpas
299, L540, 2C8, HL-60, U937, THP-1, ML-3, and K562) shared a band of
605 bp corresponding to cDNA encoding the complete form of HB-EGF. At
the resolution level adopted, only one clear-cut 605-bp band for HB-EGF
was amplified, corresponding to the complete HB-EGF molecule. In no
cell lines were we able to reamplify an HB-EGF cDNA corresponding to
the short form of the molecule.10 Restriction and base
sequence analysis of the PCR product confirmed that it was amplified
from HB-EGF mRNA. The 605-bp cDNA obtained from PCR was used as a probe
for HB-EGF in the Northern blot analysis of the leukemic
cells from patients. HB-EGF transcript, evaluated by Northern blot, was
present in 8 of 12 cases with a distribution apparently independent of
FAB subtype (Table 1 and Fig 2).
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| Fig 1.
(A) RT-PCR analysis of HB-EGF mRNA expression in a number
of leukemic, Hodgkin-, and NK-derived cell lines. HB-EGF: 605 bp;
vimentin: 266 bp; marker: 100-bp DNA ladder. (B) 48-hour DT
dose-sensitivity curve as evaluated by the MTT method. Only HB-EGF
mRNA-positive cell lines were sensitive to DT (ie, >50% death in the
range of tested concentrations), indicating membrane expression of the
HB-EGF molecule.
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| Fig 2.
(A) Northern blot analysis of HB-EGF mRNA expression in
different subtypes of ex vivo AML blasts. Human umbilical vein
endothelial cells (HUVEC) were used as positive control. (B) 48-hour DT
dose-sensitivity curve for 8 representative cases (2, 3, 4, 6, 7, 9, 10, 12 in A), as evaluated by the MTT method. Only HB-EGF mRNA-positive
leukemic cells (cases 2, 3, 7, 9) were sensitive to DT, indicating
membrane expression of the HB-EGF molecule.
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Membrane-bound HB-EGF assessed by sensitivity to DT.
To evaluate whether the HB-EGF molecule was expressed on cells positive
for HB-EGF mRNA, we tested their sensitivity to DT. We found that all
T-cell lines and patient blasts positive for HB-EGF mRNA were sensitive
to DT-induced cytolysis, as evaluated at 24 and 48 hours by a
dose-response curve comprising 1011 to
108 mol/L concentrations of DT. By contrast, B-cell
lines and four patients negative for HB-EGF mRNA expression were
insensitive to DT (Tables 1 and 2, Figs 1 and 2). The sole exception
was the myeloid cell line K562, which, though positive for HB-EGF mRNA,
was fairly resistant to DT (Fig 1). DT-related apoptosis was evaluated
by different techniques. Microscopy and flow cytometry analysis showed
morphologic changes usually associated with apoptotic death in the
majority of DT-sensitive cases. In the same cases, DNA laddering was
documented. No evidence of apoptosis in DT-insensitive cases was observed.
Expression of HER-1, HER-4, and CD9.
Since HB-EGF binds to HER-1 or HER-4, we evaluated whether these
receptors were expressed by cell lines and ex vivo AML cells. We failed
to detect HER-1 expression by such cells (Tables 1 and 2). HER-4 mRNA
was detectable in U937 and Karpas 299 cell lines in basal conditions,
whereas a very low expression was found in ML-3 and HL-60 (Table 2).
CD9, a coreceptor of membrane-bound HB-EGF,3,17,21 was
present on a minority of cell lines (Table 2) and ex vivo AML cells
(Table 1).
Regulation of HB-EGF expression in leukemic cells.
We analyzed whether the spontaneous expression of HB-EGF mRNA in HL-60
and ML-3 cell lines could be modified by exogenous agents. Cell lines
were stimulated with a panel of molecules, including DMSO, PMA, ATRA,
IFN-, 1,25-(OH)2D3, and TNF-, known to
induce biologic effects on HL-60 or ML-3 cells, such as proliferation or differentiation. PMA, DMSO, and, more interestingly, TNF-, 1,25-(OH)2D3, and especially ATRA and
costimulation with TNF- and ATRA induced an increase in transcripts
for HB-EGF (Figs 3 and
4). However, TNF- antagonized the
effects of ATRA in ML-3 cells (Fig 4). When we used GM-CSF to stimulate
AML blasts from a patient shown to be negative for HB-EGF mRNA and
insensitive to DT (patient no. 6 in Table 1), we induced both
expression of HB-EGF mRNA and sensitivity to DT (GM-CSF-treated
v untreated blasts, P = .002) (Fig
5).
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| Fig 3.
HB-EGF mRNA expression by HL-60 cell line in basal
conditions and after different stimuli. (A) After 24 hours, PMA, ATRA,
and ATRA + TNF- were the strongest inducers of HB-EGF mRNA. (B)
After 96 hours, PMA-induced band was more evident than at 24 hours.
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| Fig 4.
HB-EGF mRNA expression by ML-3 cell line in basal
conditions and after different stimuli. (A) After 24 hours, HB-EGF mRNA
was strongly induced by ATRA, 1,25-(OH)2D3
(vitamin D3), DMSO, and PMA. (B) After 96 hours, ATRA- and
1,25-(OH)2D3-induced bands were still
present.
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| Fig 5.
HB-EGF induction by GM-CSF treatment in AML blasts.
Resting blasts were negative for HB-EGF mRNA and insensitive to DT, but
after GM-CSF they acquired HB-EGF transcripts and sensitivity to
DT-induced cytolysis (P = .002). (A) Dose-response curves
testing the different sensitivity to DT in ex vivo myeloid blasts from
case 6 in Table 1 before and after treatment with GM-CSF. Results were
expressed as percentage of controls and represented the mean ± SD of
5 experiments. (B) The percentage of cell viability at the
108 mol/L DT concentration in two representative
experiments is associated with the corresponding pattern of RT-PCR
analysis, showing the induction of the transcripts for HB-EGF after
exposure to GM-CSF (HB-EGF: 605 bp; vimentin: 266 bp).
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HB-EGF proliferation assay.
We examined whether HB-EGF expressed by cell lines and leukemic cells
was released as a functional molecule mitogenic for Balb/c 3T3 cells in
coculture tests. As shown in Fig 6, the
stimulation of HB-EGF-positive ML-3 cells with PMA, which increases
HB-EGF expression and release from the cell membrane,31,32
induced a 1.67-fold increase in Balb/c 3T3 cell number as compared with controls (P < .01). In addition, antihuman HB-EGF antibody
inhibited this proliferative effect, as expected.8,37 Ex
vivo AML blasts presented a different pattern, characterized by a
higher HB-EGF proliferative effect on Balb/c 3T3 in basal conditions
than ML-3 cells, whereas PMA had no effect on HB-EGF activity (Fig 6).
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| Fig 6.
Mitogenic effect on Balb/c 3T3 cells of the HB-EGF
molecule released by the ML-3 cell line and by ex vivo myeloid blasts.
ML-3 and AML blasts were stimulated with PMA to induce both new
transcripts and release of HB-EGF (see text). Results were expressed as
percentage increase in Balb/c 3T3 cell number versus untreated
controls ± SD (P < .01). Each test was replicated
five times.
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DISCUSSION |
In this study, we found that all human hematologic cell lines
investigated, except for the B-derived ones, and blasts from a
substantial proportion of AML cases produce, bear on their membrane, and release a fully functional HB-EGF molecule. This evidence is based
on the following : (1) the demonstration of HB-EGF mRNA expression, (2)
the cytolytic effect of DT exposure exerted solely on HB-EGF
mRNA-expressing cells, and (3) the proliferative effect of HB-EGF
released by ML-3 cells or by AML blasts on the Balb/c 3T3 cell line. We
also found that factors relevant to the biology of leukemic growth
modified the expression of HB-EGF mRNA. TNF-, 1,25-(OH)2D3, and especially ATRA increased
the expression of HB-EGF mRNA in HL-60 and ML-3 cells. GM-CSF induced
HB-EGF mRNA and acquisition of sensitivity to DT in one previously
HB-EGF-negative AML case. At variance with another
report,10 we failed to demonstrate the expression of the
spliced short HB-EGF mRNA in the Raji and Daudi (the latter was studied
only for this purpose) cell lines, at least at the agarose gel
electrophoresis level. Finally, the U937 and Karpas 299 cell lines
expressed HER-4 mRNA.
HB-EGF has been shown to play a role in the context of proliferative
and chemotactic phenomena related to the inflammatory response.3 More recently, a number of reports have
demonstrated that HB-EGF is involved in epithelial neoplastic
proliferation15 and may stimulate angiogenesis through the
induction of vascular-endothelial growth factor (VEGF) in
vascular SMC.13 In such contexts, HB-EGF plays a relevant
part as a cytokine whose activity is mediated through paracrine,
cell-to-cell, or autocrine interactions.
The role, if any, played by HB-EGF in leukemic expansions is less
intuitive. The lack of HER-1 on AML cells suggests that this cytokine
is not directly involved in leukemic proliferation. However, it has
been shown that THP-1 cell line can be induced to express HER-4 upon
adequate stimulation36 and we found HER-4 mRNA at least in
resting U937 and Karpas 299 cell lines. Leukemia-derived HB-EGF may be
active on bystander cells via paracrine or juxtacrine mechanisms either
directly or through induction of secondary factors, including VEGF,
which is expressed by AML cells38 and, though in a
different context, has been shown to be induced by
HB-EGF.13 Transwell costimulatory experiments demonstrated
that HB-EGF was released by both the ML-3 cell line and ex vivo AML
blasts inducing the Balb/c 3T3 cell line to proliferate. Ex vivo blasts
released more HB-EGF as compared with ML-3 cells in basal conditions,
but PMA was less effective on AML blasts, possibly due to a direct cytotoxic effect (Fig 6). Interestingly, factors involved in
differentiation or proliferation of acute leukemia, such as ATRA,
1,25-(OH)2D3, TNF-, and GM-CSF, could
modify the expression of HB-EGF mRNA in leukemic cell lines. In HB-EGF
promoter, putative binding sites for NFkB and AP1 sites have been
identified.3,35 In addition, it has been shown that HB-EGF
could be induced through Ras pathway activation.3,39,40
Actually, TNF- has been reported to mobilize NFkB41; the
receptors for vitamins A and D (including
1,25-(OH)2D3) recognize common response
elements containing the AP1 site42; by binding to the beta
subunit of its receptor, GM-CSF activates Ras and Raf-1 and the MAP
kinase pathway.43 Thus, it is likely that TNF-, ATRA,
1,25-(OH)2D3, and GM-CSF had the HB-EGF gene as a downstream target. Whereas HB-EGF induction by TNF- has been
reported by others,7,35 to the best of our knowledge the
role of ATRA, 1,25-(OH)2D3, and GM-CSF has
yet to be characterized.
These findings may lead to a better understanding of a number of
biologic aspects of leukemic blasts, including their outgrowth capability in the context of skin or mucosal tissues or the induction of fibrosis, which characterize some FAB subtypes33,34 and are believed to play a part in limiting the effectiveness of the therapy. However, it is not clear how and to what extent AML cells may
be influenced by HB-EGF or by factors similar to, or induced by,
HB-EGF.
The presence of HB-EGF protein on the membrane of leukemic cells was
also assessed by evaluation of the degree of cytolysis induced by
DT.20 In general, DT cytolysis was associated with DNA
laddering. We found a clear-cut correlation between the expression of
HB-EGF mRNA and the susceptibility to death from DT in both cell lines
and ex vivo AML cells. Worthy of note is the fact that the K562 cell
line was the only case in which HB-EGF mRNA encoding for the complete
molecule was expressed, but DT did not induce cytotoxicity. Chang et al
have reported that actually DT is internalized by K562 cells in which
DT severely reduces protein synthesis.20 The lack of a
cytolytic effect in this cell line might be related to the expression
of antiapoptotic factors, such as the chimeric protein BCR/ABL being
translated as a consequence of t(9;22), which is known to confer
resistance to a variety of apoptotic signals.44,45 Hence,
our data suggest, albeit indirectly, that DT internalization after
binding to HB-EGF and inhibition of protein translation do not imply
straightforward apoptotic death in leukemic cell
lines.20,46 DT receptor density, coreceptors, efficiency of
internalization, and, in general, the status of pathways allowing transduction of apoptotic signals (ie, overexpression of antiapoptotic factors) should be considered in a given cell type. Yet, on the whole,
apoptotic mechanisms activated in response to DT were extremely efficient in the HB-EGF-positive leukemic cells tested.
In summary, HB-EGF was expressed in the majority of myeloid
blasts. It was inducible in HB-EGF-negative leukemic cells, at least
by TNF-, GM-CSF, ATRA, and 1,25-(OH)2D3.
It was released by leukemic blasts in a fully functional form mitogenic
for the Balb/c 3T3 cell line. The expression of at least HER-4 by a
number of leukemic cell lines adds significance to these findings.
Finally, membrane-bound HB-EGF was found to be a functional DT receptor efficiently mediating DT-induced cytotoxicity in ex vivo AML blasts. Since DT per se is not practical for therapeutic purposes, due to the
wide range of cell types expressing HB-EGF, DT fusion proteins binding
to GM-CSF and interleukin-3 (IL-3) receptors have been generated and
reported to be effective and more selective in targeting leukemic
cells.46-50
| |
FOOTNOTES |
Submitted August 10, 1998; accepted October 23, 1998.
Supported by grants from Associazione Italiana per la Ricerca sul
Cancro (AIRC, Milano), Progetto Sanità 96/97, Fondazione Cariverona (Verona), and Italy-USA Program on "Therapy of Tumors" ('96-'98).
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 Fabrizio Vinante, MD, Cattedra di
Ematologia, Ospedale Policlinico, 37134 Verona, Italy.
| |
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Abstract
Introduction