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NEOPLASIA
From the Department of Adult Oncology, Jerome Lipper
Multiple Myeloma Center, Dana-Farber Cancer Institute, and the
Department of Medicine, Harvard Medical School, Boston, MA.
It was previously demonstrated that p53 status in human multiple
myeloma (MM) cells regulates distinct cell cycle responses to CD40
activation. In this study, the production of vascular endothelial
growth factor (VEGF) and migration in MM cells triggered by CD40
activation was examined, and the influence of p53 status in regulating
this process was determined. Two human MM cell lines that express
wild-type p53 at permissive (28°C) and mutant p53 at restrictive
(37°C) temperatures were used as a model system. CD40 activation
induces a 4-fold (RPMI 8226) and a 6-fold (SV) increase in VEGF
transcripts, respectively, under restrictive, but not permissive,
temperatures. VEGF expression is significantly induced after CD40
activation in patient MM cells expressing mutant p53. Increased VEGF
transcripts result in increased protein and secretion levels, as
evidenced by immunoblotting and enzyme-linked immunosorbent assay. In a
double-chamber transmigration assay, CD40 activation of MM cells
induced a 3-fold (RPMI 8226) and a 5-fold (SV) increase in migration
under restrictive, but not permissive, conditions. A 2- to 8-fold
induction in migration of patient MM cells expressing mutant p53 was
similarly observed. Transduction of MM cells with a luciferase reporter
under the control of a human VEGF promoter further indicated that
CD40-induced VEGF expression was mediated through a transcriptional
control mechanism. Finally, adenovirus-mediated wild-type p53
overexpression down-regulated CD40-induced VEGF expression and
transmigration in MM cells expressing mutant p53. These studies
demonstrate that CD40 induces VEGF secretion and MM cell migration,
suggesting a role for CD40 in regulating MM homing and angiogenesis.
(Blood. 2002;99:1419-1427) CD40 plays an important role in the proliferation
and differentiation of B lymphocytes. Ligation of cell surface CD40 on
primary B cells induces homotypic adhesion, proliferation,
immunoglobulin isotype switch-secretion, and production of cytokines
that coordinate their activation and expansion. Multimerization of CD40
molecules by CD40L or anti-CD40 antibodies enhances the expression of
cell surface molecules such as CD80 and CD86, LFA-1, VLA-4, and ICAM-1 on normal and leukemic B cells.1,2 We and others have
demonstrated that CD40 is expressed on multiple myeloma (MM) patient
cells and in MM cell lines3-6 and that the ligation of
CD40 on MM cells up-regulates the expression of cell surface accessory
molecules, transforming growth factor (TGF)- Vascular endothelial growth factor (VEGF) is a key mediator of tumor
angiogenesis, and overexpression of VEGF is associated with
increased angiogenesis, growth, and metastasis in solid tumors. It may
also play a role in the pathogenesis of hematologic malignancies, including MM.11,12 VEGF is expressed and secreted by MM
cells and bone marrow stromal cells; moreover, it stimulates IL-6
secretion in bone marrow stromal cells and thereby augments paracrine
IL-6-mediated MM cell growth.12 It may also account for
the increased microvessel density observed in the BM of patients with
MM, which correlates with disease progression and poor
prognosis.13 VEGF binds to mononuclear cells, resulting in
activation responses and chemotactic activity.14 We
recently demonstrated that VEGF directly induces proliferation and
triggers trans-filter migration of human MM cells expressing Flt-1
(VEGF receptor 1), suggesting an autocrine VEGF loop in
MM.15 VEGF expression can be regulated by cytokines (eg,
TGF- Oncogenes and tumor suppressor genes may control the expression and
function of VEGF in human tumors. It has been found that transient
transfection of the wt p53 tumor suppressor and of the v-src
oncogene exert opposing effects on human VEGF gene promoter activity in
human glioblastoma and transformed fetal kidney cells.20 Mutant p53 potentiates the protein kinase C-mediated induction of VEGF
expression,21 and p53 mutations occur frequently in human
MM cell lines22 and are associated with advanced
disease.23,24 Most p53 mutations found in human MM are
single nucleotide substitutions that result in an amino acid change.
For example, point mutations of p53 were detected in 7 of 52 (13%)
patients and were specifically associated with more advanced and
clinically aggressive MM.23 Gross rearrangements of the
p53 gene are infrequent in this disease.25 The role of
mutant p53 in promoting tumor angiogenesis in many human solid tumors
is established26-28; conversely, the antiangiogenic effect
of wt p53 in cancer gene therapy mediates transcriptional repression of
VEGF expression.29-32 To date, however, p53-mediated regulation of bone marrow angiogenesis in MM is not well studied.
In this study, we examined whether CD40 activation triggers VEGF
secretion and migration in human MM cells, suggesting a role for CD40
in regulating MM homing and angiogenesis. Our studies further
demonstrate that p53 status regulates CD40-induced VEGF production and
transmigration in human MM cells.
Cell culture
Northern blot analysis
Immunoblot analysis Cell lysates were prepared using standard methods; immunoprecipitation and immunoblot analysis were performed as previously described.35 Reagents included anti-CD40 Ab C-20 (Santa Cruz Biotechnology, Santa Cruz, CA); Ab-5 anti-wt p53 (pAb 1620) mAb recognizing wt p53 and Ab-3 antimutant (mt) p53 (PAb 240) mAb recognizing mt p53 (Oncogene Science, Cambridge, MA); DO-1 horseradish peroxidase (HRP)-conjugated antipantropic p53 mAb (Santa Cruz Biotechnology); anti-hVEGF mAb (R&D Systems, Minneapolis, MN); and DM1A anti- -tubulin mAb (Sigma, St Louis, MO).
Enzyme-linked immunosorbent assay Cells (3 × 105/mL) were seeded onto 24-well plates in control medium (RPMI 1640 medium with 2% fetal bovine serum) or CD40-containing medium. Supernatants harvested from control and CD40-activated cell cultures were tested for soluble VEGF by enzyme-linked immunosorbent assay (ELISA) (R&D Systems), according to the manufacturer's instructions. VEGF was expressed as picograms VEGF protein per 5 × 106 cells. The minimum detectable level of VEGF was 10.0 pg/mL (pM).Transwell migration assay Cell migration was conducted in 24-well, 6.5-mm internal diameter Transwell cluster plates (Corning Costar, Cambridge, MA). Briefly, cells (105/75 µL) were loaded onto fibronectin (5 µg/mL [µM])-coated polycarbonate membranes (8-µm pore size) separating 2 chambers of a transwell. Medium-1% FCS (600 µL) containing agonist anti-CD40 mAb (G28.5) or sCD40L (Immunex, Seattle, WA), with or without blocking anti-CD40 mAb (M3; Genzyme, Cambridge, MA), neutralizing anti-CD40L mAb (5C8), or anti-VEGF mAb (R&D Systems), was added to the lower chamber of the Transwell cluster plates. Antagonist anti-CD40 mAb M3 blocks CD40-CD40L interaction. After 8 to 16 hours, cells migrating to the lower chamber were counted using a Coulter counter ZBII (Beckman Coulter) and by hemacytometer. Results were comparable using both methods.Recombinant adenovirus Adwtp53 expressing wt p53 (kindly provided by Dr Bert Volgestein) and control Ad -gal recombinant
adenoviruses35,36 were produced in 293 cells and were
purified by 2 runs of ultracentrifugation through CsCl gradient, as
published previously.35 Adenoviruses expressing luciferase
cDNA controlled by full-length human VEGF promoter or control
luciferase cDNA without any VEGF promoter sequences were generated to
allow high transduction efficiency in MM cells. To obtain specific
multiplicity of infection (MOI) achieving greater than 85%
transduction efficiency for each MM cell line and patient MM sample,
control Ad-gal recombinant adenoviruses were first infected into MM
cells at different MOIs (100-1000), and the -galactosidase activity
was quantified by an enhanced -galactosidase assay kit (Gene Therapy
System, San Diego, CA). To obtain transduction efficiencies of more
than 85%, MOIs of 800, 200, and 400 for each adenovirus were used to
infect RPMI 8226, SV, and patient MM5 cells, respectively.
Luciferase assay A 2.4-kb KpnI-NheI genomic fragment ( 2274 to +745 relative to transcription start site) containing the
human VEGF promoter was cloned upstream of firefly luciferase cDNA in a
pGL2-basic vector (Promega, Madison, WI), resulting in a pVEGF-Luc
reporter construct.37 The DNA fragment containing
sequences derived from the human VEGF promoter driving expression of
luciferase was excised from pVEGF-Luc, purified, and subcloned into an
adenoviral Ad5 left shuttle vector, as previously
described.35 Briefly, the DF3-LacZ fragment in the
adenoviral shuttle vector pDF3-LacZ was replaced with a DNA fragment
containing a VEGF promoter upstream of luciferase cDNA. The resultant
adenoviral shuttle vector pAdVEGF-Luc was cotransfected with the
adenoviral packaging plasmid JM17 into 293 cells. Recombinant
AdVEGF-Luc adenovirus was isolated from a single plaque, propagated in
293 cells, and purified.35 To allow more than 85%
infection efficiency, MOIs of 800 and 200 were used for RPMI 8226 and
SV MM cells, respectively. The Ad-Luc adenovirus containing luciferase
cDNA without any VEGF promoter sequences was generated and used as a
control. Twenty-four hours after adenovirus infection, transduced MM
cells were left intact or were activated with sCD40L. Sixteen hours
after CD40 activation, control and CD40-activated cells were harvested,
cells were lysed in buffer, and the protein concentration was
determined by Bradford assay. Total protein content was used for the
normalization of luciferase activity. Luciferase activity using equal
amounts of total cellular protein from each sample was measured using
the Monolight 2010 Luminometer (Analytical Luminescence Laboratory, Frederick, MD) at room temperature and was expressed as relative light
units (Luciferase Assay System; Promega). Experiments were performed in
triplicate, and results were expressed as mean ± SE normalized
luciferase activity.
CD40 activation enhances VEGF production by MM cells expressing mutated p53 Because many tumors constitutively express VEGF, we first determined the expression of VEGF transcripts by Northern blot analysis in normal B cells from BM aspirates of healthy donors (NBM1-2), in purified MM cells from patients with MM (more than 90% CD38+CD45RA ) (MM1-5), and in MM cell lines
RPMI 8226 and SV. Using immunoblot analysis (Figure
1A) and direct immunofluorescence flow
cytometry (Figure 1B), we confirmed the expression of CD40 in these
patient MM cells, MM cell lines, and NBM samples. As shown in Figure
2A, VEGF mRNA is expressed in MM cell
lines RPMI 8226 and SV and in patient MM cells (MM1, MM2, MM4, MM5),
but not in B cells from 2 healthy donors. Northern blotting for 18S
rRNA was used as a loading control. The expression of VEGF transcripts
in 3 additional NBM was undetectable (data not shown). These results
are consistent with previous reports using reverse
transcription-polymerase chain reaction to assay VEGF
expression.12
Because loss or inactivation of the p53 tumor suppressor gene is associated with VEGF overexpression in solid tumors,28,38,39 we next characterized p53 status in these MM samples using conformation-specific mAbs directed against wtp53 (PAb 1620) and mtp53 (PAb 240) for immunoprecipitation, followed by immunoblotting with a pantropic p53 mAb (DO-1) (Figure 2B). Patients MM2, MM4, and MM5, as well as RPMI 8226 and SV MM cell lines, express predominantly mutant p53, whereas patients MM1 and MM3 express wt p53 at 37°C. Sequencing experiments on genomic DNA from these MM samples confirmed p53 mutations: Arg-248 (CGG) to Trp (TGG) in MM2; His-179 (CAT) to Arg (CGT) in MM4; Arg-273 (CGT) to His (CAT) in MM5; and Val-143 (GTG) to Ala (GCG) in SV MM cell line. MM1 and MM3 contain the wt p53 gene. The p53 mutation in RPMI 8226 has been previously reported.10,22 The expression of mutated p53 was easily detected in MM cells that have higher expression of VEGF (MM2, MM4, MM5, RPMI 8226, SV MM) (Figure 2A). In contrast, VEGF expression in patient MM1 and MM3 cells is weaker than that in patient MM samples with mutant p53 (MM2, MM4, MM5). We have previously characterized the effect of CD40 activation on p53
transactivation in the RPMI 8226 MM line,10 which contains
a temperature-sensitive p53 mutation. The SV MM line also contains a
previously described temperature-sensitive missense mutation.40 These MM cell lines were, therefore, used as a
model system to investigate the effect of CD40 on VEGF expression in human MM. As shown in Figure 3A, VEGF
expression was induced 6 hours after CD40 activation with sCD40L (10 µg/mL [µM]) at restrictive (37°C), but not at permissive
(28°C), temperatures in RPMI 8226 and SV cells. Because hypoxia
easily induces VEGF transcripts in human umbilical vein endothelial
cells and fibroblasts, we also exposed MM cells to hypoxia for 24 hours; a 2- to 4-fold increase in VEGF transcripts was observed in
these samples (data not shown). In contrast, at permissive temperature
(28°C), VEGF expression in both cell lines harboring
temperature-sensitive mutant p53 was reduced (Figure 3A, 28°C),
consistent with weaker VEGF expression in human lung and breast cancers
that express wt p53.28,41 After CD40 activation, a
decrease in VEGF mRNA expression was observed only at the permissive
temperature when wt p53 was expressed (Figure 3A, 28°C). CD40
activation (24 hours at 37°C) also triggered a 2- to 8-fold induction
in VEGF mRNA in patients MM2, MM4, and MM5 with mutant p53 (Figure 3B).
When an agonist anti-CD40 G28.5 mAb (at 10 g/mL) was used to activate CD40, similar results were obtained (data not shown).
We next determined whether the induction of VEGF mRNA in CD40-activated
MM cells was associated with increased VEGF protein production,
confirmed by immunoblotting and ELISA. As can be seen in Figure
4A, MM cells express the secreted 28- and
42-kd VEGF protein isoforms encoded by the predominantly expressed mRNA
isoforms VEGF121 and VEGF165, and CD40
activation increases the expression of both isoforms. CD40 activation,
using either sCD40L or anti-CD40 G28.5 mAb (0-10 µg/mL [µM]),
triggered VEGF production in a dose-dependent fashion (Figure 4B). In
these experiments, CD40-induced VEGF production in MM cells was not
attributable to cellular proliferation because VEGF production was
normalized for cell number. Incubation with sCD40L and anti-CD40 G28.5
induced equivalent amounts of VEGF. CD40 activation triggered VEGF
secretion in a dose-dependent fashion. For example, the VEGF levels
(n = 3) were 1203-1399/5 × 106 cells per pg/mL (pM)
(RPMI 8226) and 226-238/5 × 106 cells per pg/mL (pM)
(SV) in control cell supernatants versus 3018-3759/5 × 106 cells per pg/mL (pM) (RPMI 8226) and
1021-1205/5 × 106 cells per pg/mL (pM) (SV) in
CD40-activated (10 µg/mL [µM]) cell culture supernatants (Figure
4B, P < .05). VEGF levels were significantly increased by
the addition of sCD40L or anti-CD40 G28.5 mAb in MM cells early (6 hours), and they persisted to 72 hours, compared with those of control
cultures with media alone or with isotype control MOPC-21
immunoglobulin (Ig)G (Figure 4C). In contrast, the medium or isotype
control MOPC-21 IgG-treated controls demonstrated no significant change
in VEGF production.
Functional effect of CD40 activation on MM cell transmigration We recently demonstrated that VEGF induces migration of MM and plasma cell leukemia (PCL) patient cells; moreover, the markedly enhanced migration in PCL cells compared with that in MM cells suggests an important role for VEGF in the transition of MM to PCL.15 Having shown that CD40 activation significantly induces VEGF expression at the mRNA and protein levels in MM cells with mutant p53, we next asked whether CD40 activation induced MM cell migration and, if so, whether CD40-induced transmigration of MM cells is p53 dependent. Cell migration was assayed by measuring the trans-filter migration activity of RPMI 8226 and SV MM cells and of patient MM cells, seeded on membranes precoated with fibronectin (5 µg/mL [µM]). As shown in Figure 5A-C, the addition of sCD40L or agonist anti-CD40 G28.5 mAb to medium containing 1% FCS in the lower chamber induced the dose-dependent migration of MM cells at restrictive temperature (37°C), when mutated p53 was functional. For example, the addition of sCD40L or agonist anti-CD40 G28.5 mAb at 10 µg/mL (µM) in the lower chamber for 8 and 16 hours induced a 3-fold and a 4.2-fold activation of migration in RPMI 8226 and SV MM cells, respectively. In contrast, when either sCD40L or anti-CD40 mAb G28.5 was added at the lower chamber of the transwell, a dose-dependent decrease in the migration of MM cells was observed at permissive temperature (28°C), when wt p53 was activated. When patient MM cells were seeded on the filters precoated with fibronectin in the upper chamber of the transwell plate, the addition of sCD40L or anti-CD40G28.5 mAb to the lower chamber for 16 hours induced significant transmigration of MM cells 10-, 1.8-, and 12-fold for patients MM2, MM4, and MM5 with
mutant p53, respectively. In contrast, similar treatment resulted in a
decrease in patient MM1 and MM3 cells expressing wt p53 (Figure 5D).
These results indicate that CD40 significant induced the motility of MM
cells expressing mutant, but not wt, p53.
Induction of VEGF is associated with transmigration of MM cells after CD40 activation We next tested whether the induction of VEGF is associated with the transmigration activity of MM cells after CD40 activation. RPMI 8226 or SV MM cells were plated on the fibronectin-coated filters separating 2 chambers in the transwell, and sCD40L (10 µg/mL [µM]), with or without a neutralizing anti-VEGF mAb (0-5 µg/mL [µM]), was added to the lower chamber. Eight to 16 hours later, cells in the lower chamber were collected and counted. As shown in Figure 6, anti-VEGF mAb alone (0.05-5 µg/mL [µM]) did not affect the mobility of unstimulated MM cells, but it did inhibit the transmigration of CD40-activated MM cells.
We next asked whether the inhibition of CD40-CD40L interaction
could block VEGF expression and transmigration induced by the CD40
activation of MM cells. Multiple myeloma cells were treated with sCD40L
(10µg/mL [µM]) for 24 hours, with or without a blocking anti-CD40L mAb 5C8, and total RNA (5 µg) from treated MM cells was
subjected to Northern blotting for VEGF expression. As shown in Figure
7A, 4- and 6-fold inductions of VEGF
expression were observed in CD40-activated RPMI 8226 and SV MM cells,
respectively. Conversely, anti-CD40L mAb (1 µg/mL [µM]) inhibited
VEGF transcript induction by sCD40L (10 µg/mL [µM]) (1-fold
reduction for RPMI 8226, 1.4-fold reduction for SV). Higher
concentrations of anti-CD40L (10 µg/mL [µM]) completely blocked
the induction of VEGF transcripts after CD40 activation. In the
transmigration experiments in which sCD40L, with or without a blocking
anti-CD40L mAb 5C8, was added to the lower chamber of the transwell,
the transmigration activity triggered by sCD40L was inhibited in the
presence of anti-CD40L mAb (Figure 7B). When compared with the medium
control, the addition of anti-CD40L (5C8) or anti-CD40 (M3)
mAbs alone at the lower chamber of the transwell did not induce
migration. When an anti-CD40 mAb M3 that blocked CD40-CD40L interaction
was used, a similar inhibition of CD40-induced VEGF expression and
migration activity was observed (data not shown). VEGF was induced as
early as 6 hours after CD40 activation, and the transmigration was
observed later (8 hours to overnight), suggesting that the specific
induction of VEGF by CD40 is associated with the transmigration of
MM cells.
CD40 activation increases VEGF promoter activity in MM cells Given that VEGF transcript expression was significantly enhanced by CD40 activation in MM cell lines and patient MM cells, we wanted to determine whether CD40 activation induces increased VEGF transcription. To test the transcriptional activity of VEGF induced by CD40 activation, a 2.4-kb genomic fragment containing human VEGF promoter upstream of luciferase reporter was transduced into RPMI 8226, SV, and patient MM5 cells. The transduction of VEGF-Luc reporter was performed using adenovirus infection at MOI 800, 200, and 400 for RPMI 8226, SV, and patient MM5 cells, respectively, to achieve greater than 80% transduction efficiency. Luciferase activity was assayed using equal amounts of cell lysates 6 hours after CD40 activation. As shown in Figure 8, the baseline luciferase activity by VEGF-Luc reporter was higher in RPMI 8226 cells than in SV cells. The higher baseline VEGF-Luc activity correlates with higher expression of constitutive VEGF transcript (Figure 1). Luciferase activity was significantly increased (approximately 4-fold for RPMI 8226, 6-fold for SV cells, and 8-fold for patient MM5) in the presence of sCD40L (10 µg/mL [µM]) (Figure 8). Similar induction of luciferase activity by VEGF-Luc reporter in the presence of sCD40L was obtained on cell lysates of MM cells 36 hours after CD40 activation (data not shown). These data, therefore, show that sCD40L induces VEGF promoter activity, indicating that increased transcription accounts, at least in part, for the increase of VEGF transcripts.
Overexpression of wt p53 in MM cells expressing mutated p53 abrogates CD40-induced VEGF production Because the overexpression of wt p53 has been shown to down-regulate transcript and promoter activity of VEGF in a dose-dependent manner in human glioblastoma, colon cancer cell lines, leiomyosarcoma, and synovial sarcoma in vitro and in vivo,20,31,32 we sought to determine whether the overexpression of wt p53 in MM cells expressing mutated p53 down-regulates CD40-induced VEGF production. MM cells were first infected with control Ad-gal or Adwtp53 adenoviruses; 6 hours later,
cell lysates were subjected to immunoblotting for p53 expression. As
shown in Figure 9A, the overexpression of
p53 was induced only by Adwtp53 adenovirus transduction, whereas
control Ad-gal transduction did not change p53 expression. Next, MM
cells were incubated with sCD40L (10 µg/mL [µM]), with or without
Adwtp53 or control Ad-gal adenoviruses. Twenty-four hours later,
total RNA was prepared and subjected to Northern blotting for VEGF
expression. As shown in Figure 9B, VEGF expression was decreased in the
Adwtp53-transduced MM cells, consistent with previous studies in other
human cancer cell lines.28-31 Although VEGF expression was
induced by CD40 in the medium or Ad-gal-transduced MM cells (4-fold
for RPMI 8226; 5.5-fold for SV), no VEGF expression was induced in the
presence of Adwtp53 adenoviruses. The level of VEGF transcript
expression in CD40-activated MM cells transduced with Adwtp53 was
approximately the same as that in control untreated MM cells, and the
addition of control Ad-gal adenoviruses did not result in any
further changes in VEGF expression either in the absence or the
presence of sCD40L. We performed similar experiments on patient MM
cells. CD40-induced VEGF expression in patient MM5 cells was similarly inhibited by the overexpression of Adwtp53 (data not shown). These results provide direct evidence that CD40-induced VEGF expression is
regulated by p53 in MM cells.
We also performed transmigration assays on adenovirus-transduced MM
cells. When MM cells transduced with Adwtp53 (for 6 hours) were
directly seeded on the fibronectin-precoated filter in the upper
chamber of the transwell, decreased transmigration of transduced MM
cells was observed (RPMI 8226, from 3.4-fold to 1.8-fold; SV, from
4.7-fold to 2.2-fold) in response to sCD40L (10 mg/mL) in the lower
chamber when compared with the transmigration of untransduced or
control Ad
In this study, we defined the role of CD40-CD40L interactions in the regulation of VEGF expression in human MM cell lines and freshly isolated patient MM cells and the influence of p53 status on this process. Our results show that CD40 activation significantly increases VEGF production and transmigration in MM cells expressing mutant p53, but not in MM cells expressing wt p53. MM cell lines RPMI 8226 and SV, with temperature-sensitive p53 mutations, were used to demonstrate VEGF induction after CD40 activation at restrictive (37°C) mutant p53 but not at permissive (28°C) wt p53 temperatures. Significantly, VEGF transcript expression was also induced after CD40 activation in patient MM cells (MM2, MM4, MM5) expressing mutant p53, but not in patient MM cells (MM1, MM3) expressing wt p53. We used luciferase reporter assays with full-length human VEGF promoter to demonstrate that the induction of VEGF after CD40 activation is controlled, at least in part, by a transcriptional mechanism. Using an adenovirus-mediated transfer of wt p53, we further showed that the overexpression of wt p53 down-regulates CD40-induced VEGF production and transmigration in MM cells. These studies help to identify a VEGF role in MM pathophysiology. Because p53 mutations occur more frequently in patients with MM during advanced stages of the disease, our demonstration of enhanced VEGF expression and migration after CD40 activation in MM cells expressing mutant p53 supports the role of p53 in MM tumor progression. Furthermore, the association of mutant p53 and CD40 activation on VEGF production also suggests a role for VEGF in MM progression. Although CD40 activation generates multiple biologic sequelae in human MM cells, the induction of VEGF has not previously been explored. Most reports thus far are in favor of a paracrine action by VEGF in MM, whereby VEGF secreted by MM cells augments IL-6 production in stromal cells.12 However, we recently showed that MM cells express Flt-1 receptor (VEGFR-1) and Flt-1 activation by VEGF on MM cell lines,15 suggesting that VEGF may have important autocrine effects on tumor cell transmigration and survival. Our current study demonstrating CD40 induction of VEGF in MM cells also suggests autocrine effects of VEGF on MM cells. In addition, it was shown recently that CD40 induces the cell motility of Kaposi sarcoma and human umbilical vein endothelial cells in vitro, which may enhance tumor cell invasion of tissues and endothelial cell organization.19 Because cell migration involves a dynamic regulation of cell adhesion and our prior studies show an up-regulation of cell surface adhesion molecules on MM cells after CD40 activation,6,8 we investigated here whether CD40 activation modulated MM cell motility using the transmigration assay. Interestingly, we observed increased MM cell transmigration induced by CD40, correlating with tumor cell p53 status. These data, coupled with the clinical observation that patients with advanced stages of MM express high CD40 levels,5 suggest a role for CD40 in MM homing or invasion. It has been postulated that p53 dysfunction may play an important role in MM tumor progression. Specifically, wt p53 regulates MM cell growth and apoptosis and attenuates IL-6 production, whereas loss of p53 function deregulates IL-6 production. In addition, the presence of a mutated p53 allele may potentiate autonomous MM cell growth and secretion of angiogenic factors (ie, VEGF), which enhance the emergence of MM cell clones bearing only the mutated p53 allele. Our findings that MM cells expressing mutated p53 produced more VEGF after CD40 stimulation supports this view. It has been shown that wt p53 protein down-regulates VEGF promoter activity in cell lines in a dose-dependent manner.20 Recently, it was reported that the introduction of wt p53 into sarcoma cells repressed the transcription and decreased the expression of VEGF.32 Endothelial cell growth and migration were also decreased when cells were treated with conditioned medium from sarcoma cells expressing wt p53 compared with media from sarcoma cells expressing mutant p53.32 In the current study, the overexpression of wt p53 in MM cell lines and freshly isolated patient MM cells expressing mutant p53 down-regulated CD40-induced VEGF production, indicating that VEGF secretion by MM cells can be regulated by wt p53. This result suggests the potential usefulness of adenovirus-mediated delivery of wt p53 in gene therapy for MM.42 Our ongoing studies are examining the growth-inhibiting and potentially therapeutic benefits of CD40 activation of MM cells heterozygous for p53. In addition, because wt p53 suppresses angiogenesis by the regulation of thrombospondin (TSP-1) expression in human fibroblasts43 and because preclinical studies have shown that wt p53 protein enhances the expression of TSP-1, the down-regulation of TSP-1 may be observed when alterations of the p53 protein occur. Studies are ongoing to determine whether TSP-1 expression is increased in MM cells expressing wt p53 after CD40 activation to further define the role of CD40 in angiogenesis and progression of MM. CD40 ligation is known to regulate multiple genes through
transcriptional mechanisms, and our study demonstrates that VEGF expression in MM cells after CD40 activation is also primarily controlled by a transcriptional mechanism. Specifically, we showed that
sCD40L increased luciferase activity in MM cells transduced with the
VEGF-Luc reporter. Thus far, the CD40 responsive element in the
promoter region of CD40-induced genes has not been identified. However,
in a previous report, the induction of VEGF expression by IL-6 was
found to be mediated by specific DNA motifs located on the putative
promoter region of VEGF and by specific elements located in the
5'-UTR.16 One DNA control element mediating IL-6 response
has been identified between In conclusion, we have demonstrated that CD40 activation induces p53-dependent VEGF expression in human MM cells. Specifically, CD40 activation enhanced VEGF expression and transmigration in MM cells with mutant p53. Our current study validates the important role of VEGF and p53 status in MM disease progression.
We thank Dr Bert Vogelstein and Dr Robert Taylor for reagents.
Submitted July 10, 2001; accepted October 11, 2001.
Supported by a Multiple Myeloma Research Foundation Fellowship (Y.-T.T.) and a Doris Duke Distinguished Clinical Research Scientist Award (K.C.A.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth C. Anderson, Dept of Adult Oncology, Dana-Farber Cancer Institute, M557, 44 Binney St, Boston, MA 02115; e-mail: kenneth_anderson{at}dfci.harvard.edu.
1. Nishioka Y, Lipsky PE. The role of CD40-CD40 ligand interaction in human T cell-B cell collaboration. J Immunol. 1994;153:1027-1036[Abstract].
2.
Ranheim EA, Kipps TJ.
Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.
J Exp Med.
1993;177:925-935 3. Westendorf JJ, Ahmann GJ, Armitage RJ, et al. CD40 expression in malignant plasma cells: role in stimulation of autocrine IL-6 secretion by a human myeloma cell line. J Immunol. 1994;152:117-128[Abstract].
4.
Tong AW, Zhang BQ, Mues G, Solano M, Hanson T, Stone MJ.
Anti-CD40 antibody binding modulates human multiple myeloma clonogenicity in vitro.
Blood.
1994;84:3026-3033
5.
Pellat-Deceunynck C, Bataille R, Robillard N, et al.
Expression of CD28 and CD40 in human myeloma cells: a comparative study with normal plasma cells.
Blood.
1994;84:2597-2603
6.
Urashima M, Chauhan D, Uchiyama H, Freeman GJ, Anderson KC.
CD40 ligand triggered interleukin-6 secretion in multiple myeloma.
Blood.
1995;85:1903-1912
7.
Urashima M, Ogata A, Chauhan D, et al.
Transforming growth factor-beta 1: differential effects on multiple myeloma versus normal B cells.
Blood.
1996;87:1928-1938 8. Teoh G, Urashima M, Greenfield EA, et al. The 86-kD subunit of Ku autoantigen mediates homotypic and heterotypic adhesion of multiple myeloma cells. J Clin Invest. 1998;101:1379-1388[Medline] [Order article via Infotrieve]
9.
Pellat-Deceunynck C, Amiot M, Robillard N, Wijdenes J, Bataille R.
CD11a-CD18 and CD102 interactions mediate human myeloma cell growth arrest induced by CD40 stimulation.
Cancer Res.
1996;56:1909-1916
10.
Teoh G, Tai YT, Urashima M, et al.
CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines.
Blood.
2000;95:1039-1046
11.
Bellamy WT, Richter L, Frutiger Y, Grogan TM.
Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies.
Cancer Res.
1999;59:728-733
12.
Dankbar B, Padro T, Leo R, et al.
Vascular endothelial growth factor and interleukin-6 in paracrine tumor- stromal cell interactions in multiple myeloma.
Blood.
2000;95:2630-2636
13.
Vacca A, Ribatti D, Presta M, et al.
Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma.
Blood.
1999;93:3064-3073
14.
Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D.
Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1.
Blood.
1996;87:3336-3343
15.
Podar K, Tai TY, Davies FE, et al.
Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration.
Blood.
2001;98:428-435
16.
Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ.
Interleukin 6 induces the expression of vascular endothelial growth factor.
J Biol Chem.
1996;271:736-741
17.
Melter M, Reinders ME, Sho M, et al.
Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo.
Blood.
2000;96:3801-3808
18.
Cho CS, Cho mL, Min SY, et al.
CD40 engagement on synovial fibroblast up-regulates production of vascular endothelial growth factor.
J Immunol.
2000;164:5055-5061
19.
Biancone L, Cantaluppi V, Boccellino M, et al.
Activation of CD40 favors the growth and vascularization of Kaposi's sarcoma.
J Immunol.
1999;163:6201-6208
20.
Mukhopadhyay D, Tsiokas L, Sukhatme VP.
Wild-type p53 and v-Src exert opposing influences on human vascular endothelial growth factor gene expression.
Cancer Res.
1995;55:6161-6165 21. Kieser A, Weich HA, Brandner G, Marme D, Kolch W. Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene. 1994;9:963-969[Medline] [Order article via Infotrieve]. 22. Mazars GR, Portier M, Zhang XG, et al. Mutations of the p53 gene in human myeloma cell lines. Oncogene. 1992;7:1015-1018[Medline] [Order article via Infotrieve].
23.
Neri A, Baldini L, Trecca D, Cro L, Polli E, Maiolo AT.
p53 Gene mutations in multiple myeloma are associated with advanced forms of malignancy.
Blood.
1993;81:128-135 24. Kanavaros P, Stefanaki K, Vlachonikolis J, et al. Immunohistochemical expression of the p53, p21/Waf-1, Rb, p16 and Ki67 proteins in multiple myeloma. Anticancer Res. 2000;20:4619-4625[Medline] [Order article via Infotrieve]. 25. Avet-Loiseau H, Li JY, Godon C, et al. P53 deletion is not a frequent event in multiple myeloma. Br J Haematol. 1999;106:717-719[CrossRef][Medline] [Order article via Infotrieve]. 26. Grant SW, Kyshtoobayeva AS, Kurosaki T, Jakowatz J, Fruehauf JP. Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. Cancer Detect Prev. 1998;22:185-194[CrossRef][Medline] [Order article via Infotrieve]. 27. Riedel F, Gotte K, Schwalb J, Schafer C, Hormann K. Vascular endothelial growth factor expression correlates with p53 mutation and angiogenesis in squamous cell carcinoma of the head and neck. Acta Otolaryngol. 2000;120:105-111[Medline] [Order article via Infotrieve].
28.
Linderholm BK, Lindahl T, Holmberg L, et al.
The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer.
Cancer Res.
2001;61:2256-2260
29.
Nishizaki M, Fujiwara T, Tanida T, et al.
Recombinant adenovirus expressing wild-type p53 is antiangiogenic: a proposed mechanism for bystander effect.
Clin Cancer Res.
1999;5:1015-1023
30.
Liu Y, Thor A, Shtivelman E, et al.
Systemic gene delivery expands the repertoire of effective antiangiogenic agents.
J Biol Chem.
1999;274:13338-13344
31.
Bouvet M, Ellis LM, Nishizaki M, et al.
Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer.
Cancer Res.
1998;58:2288-2292
32.
Zhang L, Yu D, Hu M, et al.
Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression.
Cancer Res.
2000;60:3655-3661 33. Tai YT, Teoh G, Shima Y, et al. Isolation and characterization of human multiple myeloma cell enriched populations. J Immunol Methods. 2000;235:11-19[CrossRef][Medline] [Order article via Infotrieve].
34.
Tai YT, Teoh G, Lin B, et al.
Ku86 variant expression and function in multiple myeloma cells is associated with increased sensitivity to DNA damage.
J Immunol.
2000;165:6347-6355
35.
Tai YT, Strobel T, Kufe D, Cannistra SA.
In vivo cytotoxicity of ovarian cancer cells through tumor-selective expression of the BAX gene.
Cancer Res.
1999;59:2121-2126
36.
Teoh G, Chen L, Urashima M, et al.
Adenovirus vector-based purging of multiple myeloma cells.
Blood.
1998;92:4591-4601
37.
Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, Taylor RN.
Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta.
Proc Natl Acad Sci U S A.
2000;97:10972-10977 38. Kondo Y, Arii S, Furutani M, et al. Implication of vascular endothelial growth factor and p53 status for angiogenesis in noninvasive colorectal carcinoma. Cancer. 2000;88:1820-1827[CrossRef][Medline] [Order article via Infotrieve].
39.
Mehta R, Kyshtoobayeva A, Kurosaki T, et al.
Independent association of angiogenesis index with outcome in prostate cancer.
Clin Cancer Res.
2001;7:81-88
40.
Friedlander P, Legros Y, Soussi T, Prives C.
Regulation of mutant p53 temperature-sensitive DNA binding.
J Biol Chem.
1996;271:25468-25478 41. Giatromanolaki A, Koukourakis MI, Kakolyris S, et al. Vascular endothelial growth factor, wild-type p53, and angiogenesis in early operable nonsmall cell lung cancer. Clin Cancer Res. 1998;4:3017-3024[Abstract]. 42. Liu Q, Gazitt Y. Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes. Exp Hematol. 2000;28:1354-1362[CrossRef][Medline] [Order article via Infotrieve].
43.
Dameron KM, Volpert OV, Tainsky MA, Bouck N.
Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1.
Science.
1994;265:1582-1584
© 2002 by The American Society of Hematology.
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  M. Luqman, S. Klabunde, K. Lin, G. V. Georgakis, A. Cherukuri, J. Holash, C. Goldbeck, X. Xu, E. E. Kadel III, S. H. Lee, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells Blood, August 1, 2008; 112(3): 711 - 720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-T. Tai, X.-F. Li, I. Breitkreutz, W. Song, P. Neri, L. Catley, K. Podar, T. Hideshima, D. Chauhan, N. Raje, et al. Role of B-Cell-Activating Factor in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment. Cancer Res., July 1, 2006; 66(13): 6675 - 6682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-T. Tai, X.-F. Li, L. Catley, R. Coffey, I. Breitkreutz, J. Bae, W. Song, K. Podar, T. Hideshima, D. Chauhan, et al. Immunomodulatory Drug Lenalidomide (CC-5013, IMiD3) Augments Anti-CD40 SGN-40-Induced Cytotoxicity in Human Multiple Myeloma: Clinical Implications Cancer Res., December 15, 2005; 65(24): 11712 - 11720. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hideshima, D. Chauhan, P. Richardson, and K. C. Anderson Identification and Validation of Novel Therapeutic Targets for Multiple Myeloma J. Clin. Oncol., September 10, 2005; 23(26): 6345 - 6350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-T. Tai, X. Li, X. Tong, D. Santos, T. Otsuki, L. Catley, O. Tournilhac, K. Podar, T. Hideshima, R. Schlossman, et al. Human Anti-CD40 Antagonist Antibody Triggers Significant Antitumor Activity against Human Multiple Myeloma Cancer Res., July 1, 2005; 65(13): 5898 - 5906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Connor, S. Subbaram, K. J. Regan, K. K. Nelson, J. E. Mazurkiewicz, P. J. Bartholomew, A. E. Aplin, Y.-T. Tai, J. Aguirre-Ghiso, S. C. Flores, et al. Mitochondrial H2O2 Regulates the Angiogenic Phenotype via PTEN Oxidation J. Biol. Chem., April 29, 2005; 280(17): 16916 - 16924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Podar and K. C. Anderson The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications Blood, February 15, 2005; 105(4): 1383 - 1395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. H. Lapchak, M. Melter, S. Pal, J. A. Flaxenburg, C. Geehan, M. H. Frank, D. Mukhopadhyay, and D. M. Briscoe CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island Am J Physiol Renal Physiol, September 1, 2004; 287(3): F512 - F520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hideshima, P. L. Bergsagel, W. M. Kuehl, and K. C. Anderson Advances in biology of multiple myeloma: clinical applications Blood, August 1, 2004; 104(3): 607 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-T. Tai, L. P. Catley, C. S. Mitsiades, R. Burger, K. Podar, R. Shringpaure, T. Hideshima, D. Chauhan, M. Hamasaki, K. Ishitsuka, et al. Mechanisms by which SGN-40, a Humanized Anti-CD40 Antibody, Induces Cytotoxicity in Human Multiple Myeloma Cells: Clinical Implications Cancer Res., April 15, 2004; 64(8): 2846 - 2852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Abe, K. Terada, H. Wakimoto, R. Inoue, E. Tyminski, R. Bookstein, J. P. Basilion, and E. A. Chiocca PTEN Decreases in Vivo Vascularization of Experimental Gliomas in Spite of Proangiogenic Stimuli Cancer Res., May 1, 2003; 63(9): 2300 - 2305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-T. Tai, K. Podar, N. Mitsiades, B. Lin, C. Mitsiades, D. Gupta, M. Akiyama, L. Catley, T. Hideshima, N. C. Munshi, et al. CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling Blood, April 1, 2003; 101(7): 2762 - 2769. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Lin, K. Podar, D. Gupta, Y.-T. Tai, S. Li, E. Weller, T. Hideshima, S. Lentzsch, F. Davies, C. Li, et al. The Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor PTK787/ZK222584 Inhibits Growth and Migration of Multiple Myeloma Cells in the Bone Marrow Microenvironment Cancer Res., September 1, 2002; 62(17): 5019 - 5026. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) RPMI 8226 at 37°C;
(
) RPMI 8226 at 30°C; (
) SV at 37°C;
(
) SV at
30°C. (D) Patient MM1-5 cells were subjected to transmigration assay,
using sCD40L (10 µg/mL [µM]) in the lower chamber at 37°C.
(
)
MM2; (
) MM4;
(
) MM5.
Increased transmigration is observed in MM cells expressing mutated p53
(MM2, MM4, MM5), but not in those with wt p53 (MM1, MM3), after CD40
activation.
