doi:10.1182/blood.V99.4.14192002 99: 1419-1427   

 AndersonYu-Tzu Tai, Klaus Podar, Deepak Gupta, Boris Lin, Gloria Young, Masaharu Akiyama and Kenneth C. secretion in human multiple myeloma cellsCD40 activation induces p53-dependent vascular endothelial growth factor

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NEOPLASIA

CD40 activation induces p53-dependent vascular endothelial growth factorsecretion in human multiple myeloma cellsYu-Tzu Tai, Klaus Podar, Deepak Gupta, Boris Lin, Gloria Young, Masaharu Akiyama, and Kenneth C. Anderson

It was previously demonstrated that p53status in human multiple myeloma (MM)cells regulates distinct cell cycle re-sponses to CD40 activation. In this study,the production of vascular endothelialgrowth factor (VEGF) and migration inMM cells triggered by CD40 activationwas examined, and the influence of p53status in regulating this process was de-termined. Two human MM cell lines thatexpress wild-type p53 at permissive (28°C)and mutant p53 at restrictive (37°C) tem-peratures were used as a model system.CD40 activation induces a 4-fold (RPMI8226) and a 6-fold (SV) increase in VEGFtranscripts, respectively, under restric-

tive, but not permissive, temperatures.VEGF expression is significantly inducedafter CD40 activation in patient MM cellsexpressing mutant p53. Increased VEGFtranscripts result in increased protein andsecretion levels, as evidenced by immuno-blotting and enzyme-linked immunosor-bent assay. In a double-chamber transmi-gration assay, CD40 activation of MMcells induced a 3-fold (RPMI 8226) and a5-fold (SV) increase in migration underrestrictive, but not permissive, condi-tions. A 2- to 8-fold induction in migrationof patient MM cells expressing mutantp53 was similarly observed. Transductionof MM cells with a luciferase reporter

under the control of a human VEGF pro-moter further indicated that CD40-in-duced VEGF expression was mediatedthrough a transcriptional control mecha-nism. Finally, adenovirus-mediated wild-type p53 overexpression down-regulatedCD40-induced VEGF expression andtransmigration in MM cells expressingmutant p53. These studies demonstratethat CD40 induces VEGF secretion andMM cell migration, suggesting a role forCD40 in regulating MM homing and angio-genesis. (Blood. 2002;99:1419-1427)

© 2002 by The American Society of Hematology

Introduction

CD40 plays an important role in the proliferation and differentia-tion of B lymphocytes. Ligation of cell surface CD40 on primary Bcells induces homotypic adhesion, proliferation, immunoglobulinisotype switch–secretion, and production of cytokines that coordi-nate their activation and expansion. Multimerization of CD40molecules by CD40L or anti-CD40 antibodies enhances theexpression 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 onmultiple myeloma (MM) patient cells and in MM cell lines3-6 andthat the ligation of CD40 on MM cells up-regulates the expressionof cell surface accessory molecules, transforming growth factor(TGF)–b1, and interleukin-6 (IL-6) secretion.6,7We further demon-strated an induction of Ku86 autoantigen on the cell surface ofCD40-activated MM cells that enhanced homotypic tumor celladhesion and heterotypic binding of tumor cells to bone marrowstromal cells and fibronectin.8 Interestingly, CD40 activationinduces the proliferation of some MM cells but triggers growtharrest and apoptosis in others.3,4,9,10 Specifically, we recentlydemonstrated that the cell cycle response to CD40 activation wasp53 dependent because CD40 ligation of MM cells results in eithergrowth arrest and apoptosis or proliferation, depending on thepresence or absence of wild-type (wt) p53 function.10

Vascular endothelial growth factor (VEGF) is a key mediator oftumor angiogenesis, and overexpression of VEGF is associated

with increased angiogenesis, growth, and metastasis in solidtumors. It may also play a role in the pathogenesis of hemato-logic malignancies, including MM.11,12 VEGF is expressed andsecreted by MM cells and bone marrow stromal cells; moreover,it stimulates IL-6 secretion in bone marrow stromal cells andthereby augments paracrine IL-6–mediated MM cell growth.12 Itmay also account for the increased microvessel density observedin the BM of patients with MM, which correlates with diseaseprogression and poor prognosis.13 VEGF binds to mononuclearcells, resulting in activation responses and chemotactic activi-ty.14 We recently demonstrated that VEGF directly inducesproliferation and triggers trans-filter migration of human MMcells expressing Flt-1 (VEGF receptor 1), suggesting an auto-crine VEGF loop in MM.15 VEGF expression can be regulatedby cytokines (eg, TGF-b, tumor necrosis factor-a, IL-1b, IL-6)in MM. IL-6 triggers tumor cell growth and survival, and it maypromote angiogenesis through the induction of VEGF.16 Mostrecently, CD40L–CD40 interactions have been shown to induceVEGF expression by endothelial cells and monocytes17 and alsoby synovial fibroblasts.18 In addition, activation of CD40induces migration, survival, growth, and neovascularization ofKaposi sarcoma cells in a SCID mouse model, promoting tumorcell growth and vascularization.19 Up-regulation of CD40 wasobserved in tumor vessels, further supporting a role for CD40 intumor angiogenesis.

From the Department of Adult Oncology, Jerome Lipper Multiple MyelomaCenter, Dana-Farber Cancer Institute, and the Department of Medicine,Harvard Medical School, Boston, MA.

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.).

Reprints: Kenneth C. Anderson, Dept of Adult Oncology, Dana-Farber CancerInstitute, M557, 44 Binney St, Boston, MA 02115; e-mail:kenneth_anderson@dfci.harvard.edu.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2002 by The American Society of Hematology

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Oncogenes and tumor suppressor genes may control the expres-sion and function of VEGF in human tumors. It has been found thattransient transfection of the wt p53 tumor suppressor and of thev-src oncogene exert opposing effects on human VEGF genepromoter activity in human glioblastoma and transformed fetalkidney cells.20 Mutant p53 potentiates the protein kinase C-mediated induction of VEGF expression,21 and p53 mutationsoccur frequently in human MM cell lines22 and are associated withadvanced disease.23,24Most p53 mutations found in human MM aresingle 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 andclinically aggressive MM.23 Gross rearrangements of the p53 geneare infrequent in this disease.25 The role of mutant p53 inpromoting tumor angiogenesis in many human solid tumors isestablished26-28; conversely, the antiangiogenic effect of wt p53 incancer gene therapy mediates transcriptional repression of VEGFexpression.29-32To date, however, p53-mediated regulation of bonemarrow angiogenesis in MM is not well studied.

In this study, we examined whether CD40 activation triggersVEGF secretion and migration in human MM cells, suggesting arole for CD40 in regulating MM homing and angiogenesis. Ourstudies further demonstrate that p53 status regulates CD40-inducedVEGF production and transmigration in human MM cells.

Materials and methods

Cell culture

RPMI 8226 (American Type Culture Collection [ATCC], Manassas, VA)10

and SV33 MM cell lines were cultured in culture medium (10% fetal calfserum [FCS]–RPMI 1640 medium supplemented with 2 mM L-glutamine,25mg/mL (mM) Pen/Strep). The p53 gene in the RPMI 8226 line is mutatedGlu-285 (GAG) to Lys (AAG).10,22 p53 sequencing was performed on SVMM cell line and patient MM samples, as previously described.10 Both celllines express wt p53 at permissive (28°C), but not at restrictive (37°C),temperatures. Freshly isolated MM cells (more than 90% CD381CD45RA2)obtained after informed consent were prepared by antibody (Ab)-mediatednegative selection, as previously described,33 using RosetteSep (StemCellTechnologies, Vancouver, BC, Canada). Normal B cells were isolated fromhealthy donor bone marrow, after informed consent, as previously de-scribed.34 MM cells expressed CD40, assessed either by flow cytometricanalysis or immunoblot analysis. An anti-CD40 monoclonal antibody(mAb; G28.5; ATCC) and a neutralizing antihuman CD40L mAb (5C8;ATCC) were prepared and purified using standard methods.

Northern blot analysis

Northern blot analysis was performed as previously described.34 Total RNAfrom 107 cells was prepared using RNeasy Mini Kit (Qiagen, Valencia,CA). Each RNA sample was resolved in 1.2% to 1.4% formaldehyde–agarose gels, transferred to Nitropure membrane (MSI, Westborough, MA),and probed with random primer-labeled VEGF cDNA. VEGF cDNA wasamplified by reverse transcription–polymerase chain reaction (408bp,VEGF121; 541bp, VEGF165) from total RNA of RPMI 8226 cells usingprimer pairs 59-GAAGTGGTGAAGTTCATGGATGTC-39 and 59-CGATCGTTCTGTATCAGTCTTTCC-39.11 These VEGF cDNAs werepurified using agarose gel electrophoresis and were recovered usingQIAEX II gel extraction kit (Qiagen, Chatsworth, CA), and they detectedVEGF transcripts of approximately 4.4 and 3.7 kb. An oligo probe forhousekeeping gene 18S rRNA was used for control loading. VEGF and18S rRNA expression were quantitated by densitometry of autoradio-grams using the Image Quant software program (Molecular Dynamics,Sunnyvale, CA).

Immunoblot analysis

Cell lysates were prepared using standard methods; immunoprecipitationand immunoblot analysis were performed as previously described.35

Reagents included anti-CD40 Ab C-20 (Santa Cruz Biotechnology, SantaCruz, CA); Ab-5 anti-wt p53 (pAb 1620) mAb recognizing wt p53 and Ab-3antimutant (mt) p53 (PAb 240) mAb recognizing mt p53 (OncogeneScience, Cambridge, MA); DO-1 horseradish peroxidase (HRP)–conju-gated antipantropic p53 mAb (Santa Cruz Biotechnology); anti-hVEGFmAb (R&D Systems, Minneapolis, MN); and DM1A anti-a–tubulinmAb (Sigma, St Louis, MO).

Enzyme-linked immunosorbent assay

Cells (33 105/mL) were seeded onto 24-well plates in control medium(RPMI 1640 medium with 2% fetal bovine serum) or CD40-containingmedium. Supernatants harvested from control and CD40-activated cellcultures were tested for soluble VEGF by enzyme-linked immunosorbentassay (ELISA) (R&D Systems), according to the manufacturer’s instruc-tions. VEGF was expressed as picograms VEGF protein per 53 106 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 diameterTranswell cluster plates (Corning Costar, Cambridge, MA). Briefly, cells(105/75mL) were loaded onto fibronectin (5mg/mL [mM])–coated polycar-bonate membranes (8-mm pore size) separating 2 chambers of a transwell.Medium–1% FCS (600mL) containing agonist anti-CD40 mAb (G28.5) orsCD40L (Immunex, Seattle, WA), with or without blocking anti-CD40mAb (M3; Genzyme, Cambridge, MA), neutralizing anti-CD40L mAb(5C8), or anti-VEGF mAb (R&D Systems), was added to the lowerchamber of the Transwell cluster plates. Antagonist anti-CD40 mAbM3 blocks CD40-CD40L interaction. After 8 to 16 hours, cells migratingto the lower chamber were counted using a Coulter counter ZBII(Beckman Coulter) and by hemacytometer. Results were comparable usingboth methods.

Recombinant adenovirus

Adwtp53 expressing wt p53 (kindly provided by Dr Bert Volgestein) andcontrol Adb-gal recombinant adenoviruses35,36were produced in 293 cellsand were purified by 2 runs of ultracentrifugation through CsCl gradient, aspublished previously.35 Adenoviruses expressing luciferase cDNA con-trolled by full-length human VEGF promoter or control luciferase cDNAwithout any VEGF promoter sequences were generated to allow hightransduction efficiency in MM cells. To obtain specific multiplicity ofinfection (MOI) achieving greater than 85% transduction efficiency foreach MM cell line and patient MM sample, control Adb-gal recombinantadenoviruses were first infected into MM cells at different MOIs (100-1000), and theb-galactosidase activity was quantified by an enhancedb-galactosidase assay kit (Gene Therapy System, San Diego, CA). Toobtain transduction efficiencies of more than 85%, MOIs of 800, 200, and400 for each adenovirus were used to infect RPMI 8226, SV, and patientMM5 cells, respectively.

Luciferase assay

A 2.4-kb KpnI-NheI genomic fragment (22274 to1745 relative totranscription start site) containing the human VEGF promoter was clonedupstream of firefly luciferase cDNA in a pGL2-basic vector (Promega,Madison, WI), resulting in a pVEGF-Luc reporter construct.37 The DNAfragment containing sequences derived from the human VEGF promoterdriving expression of luciferase was excised from pVEGF-Luc, purified,and subcloned into an adenoviral Ad5 left shuttle vector, as previouslydescribed.35 Briefly, the DF3-LacZ fragment in the adenoviral shuttle vectorpDF3-LacZ was replaced with a DNA fragment containing a VEGFpromoter upstream of luciferase cDNA. The resultant adenoviral shuttlevector pAdVEGF-Luc was cotransfected with the adenoviral packagingplasmid JM17 into 293 cells. Recombinant AdVEGF-Luc adenovirus was

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isolated from a single plaque, propagated in 293 cells, and purified.35 Toallow more than 85% infection efficiency, MOIs of 800 and 200 were usedfor RPMI 8226 and SV MM cells, respectively. The Ad-Luc adenoviruscontaining luciferase cDNA without any VEGF promoter sequences wasgenerated and used as a control. Twenty-four hours after adenovirusinfection, transduced MM cells were left intact or were activated withsCD40L. Sixteen hours after CD40 activation, control and CD40-activatedcells were harvested, cells were lysed in buffer, and the protein concentra-tion was determined by Bradford assay. Total protein content was used forthe normalization of luciferase activity. Luciferase activity using equalamounts of total cellular protein from each sample was measured using theMonolight 2010 Luminometer (Analytical Luminescence Laboratory, Fred-erick, MD) at room temperature and was expressed as relative light units(Luciferase Assay System; Promega). Experiments were performed intriplicate, and results were expressed as mean6 SE normalized lucif-erase activity.

Results

CD40 activation enhances VEGF production by MM cellsexpressing mutated p53

Because many tumors constitutively express VEGF, we firstdetermined the expression of VEGF transcripts by Northern blotanalysis in normal B cells from BM aspirates of healthy donors(NBM1-2), in purified MM cells from patients with MM (morethan 90% CD381CD45RA2) (MM1-5), and in MM cell linesRPMI 8226 and SV. Using immunoblot analysis (Figure 1A) anddirect immunofluorescence flow cytometry (Figure 1B), we con-firmed the expression of CD40 in these patient MM cells, MM celllines, and NBM samples. As shown in Figure 2A, VEGF mRNA isexpressed in MM cell lines RPMI 8226 and SV and in patient MMcells (MM1, MM2, MM4, MM5), but not in B cells from 2 healthydonors. Northern blotting for 18S rRNA was used as a loadingcontrol. The expression of VEGF transcripts in 3 additional NBMwas undetectable (data not shown). These results are consistentwith previous reports using reverse transcription–polymerase chainreaction to assay VEGF expression.12

Because loss or inactivation of the p53 tumor suppressor gene isassociated with VEGF overexpression in solid tumors,28,38,39 wenext characterized p53 status in these MM samples using conforma-tion-specific mAbs directed against wtp53 (PAb 1620) and mtp53(PAb 240) for immunoprecipitation, followed by immunoblottingwith 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 andMM3 express wt p53 at 37°C. Sequencing experiments on genomicDNA from these MM samples confirmed p53 mutations: Arg-248(CGG) to Trp (TGG) in MM2; His-179 (CAT) to Arg (CGT) inMM4; 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 wtp53 gene. The p53 mutation in RPMI 8226 has been previouslyreported.10,22The expression of mutated p53 was easily detected inMM cells that have higher expression of VEGF (MM2, MM4,MM5, RPMI 8226, SV MM) (Figure 2A). In contrast, VEGFexpression in patient MM1 and MM3 cells is weaker than that inpatient MM samples with mutant p53 (MM2, MM4, MM5).

We have previously characterized the effect of CD40 activationon p53 transactivation in the RPMI 8226 MM line,10 whichcontains a temperature-sensitive p53 mutation. The SV MM linealso contains a previously described temperature-sensitive mis-sense mutation.40 These MM cell lines were, therefore, used as amodel system to investigate the effect of CD40 on VEGF expres-sion in human MM. As shown in Figure 3A, VEGF expression wasinduced 6 hours after CD40 activation with sCD40L (10mg/mL[mM]) at restrictive (37°C), but not at permissive (28°C), tempera-tures in RPMI 8226 and SV cells. Because hypoxia easily inducesVEGF transcripts in human umbilical vein endothelial cells andfibroblasts, we also exposed MM cells to hypoxia for 24 hours; a 2-to 4-fold increase in VEGF transcripts was observed in thesesamples (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), consistentwith weaker VEGF expression in human lung and breast cancersthat express wt p53.28,41After CD40 activation, a decrease in VEGF

Figure 1. CD40 expression in patient MM cells andcell lines. CD40 expression on normal B cells (NBM1-2),patient MM cells (MM1-5), RPMI 8226, and SV MM celllines was determined by (A) immunoblotting and (B) flowcytometry. Solid histogram, isotype control; open histo-gram, CD40.

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mRNA expression was observed only at the permissive tempera-ture when wt p53 was expressed (Figure 3A, 28°C). CD40activation (24 hours at 37°C) also triggered a 2- to 8-fold inductionin VEGF mRNA in patients MM2, MM4, and MM5 with mutantp53 (Figure 3B). When an agonist anti-CD40 G28.5 mAb (at 10g/mL) was used to activate CD40, similar results were obtained(data not shown).

We next determined whether the induction of VEGF mRNA inCD40-activated MM cells was associated with increased VEGFprotein production, confirmed by immunoblotting and ELISA. Ascan be seen in Figure 4A, MM cells express the secreted 28- and42-kd VEGF protein isoforms encoded by the predominantlyexpressed mRNA isoforms VEGF121 and VEGF165, and CD40activation increases the expression of both isoforms. CD40 activa-tion, using either sCD40L or anti-CD40 G28.5 mAb (0-10mg/mL[mM]), triggered VEGF production in a dose-dependent fashion(Figure 4B). In these experiments, CD40-induced VEGF produc-tion in MM cells was not attributable to cellular proliferationbecause VEGF production was normalized for cell number. Incuba-tion with sCD40L and anti-CD40 G28.5 induced equivalentamounts of VEGF. CD40 activation triggered VEGF secretion in adose-dependent fashion. For example, the VEGF levels (n5 3)were 1203-1399/53 106 cells per pg/mL (pM) (RPMI 8226) and226-238/53 106 cells per pg/mL (pM) (SV) in control cellsupernatants versus 3018-3759/53 106 cells per pg/mL (pM)(RPMI 8226) and 1021-1205/53 106 cells per pg/mL (pM) (SV) inCD40-activated (10mg/mL [mM]) cell culture supernatants (Figure4B, P , .05). VEGF levels were significantly increased by theaddition of sCD40L or anti-CD40 G28.5 mAb in MM cells early (6hours), and they persisted to 72 hours, compared with those ofcontrol cultures with media alone or with isotype control MOPC-21immunoglobulin (Ig)G (Figure 4C). In contrast, the medium orisotype control MOPC-21 IgG-treated controls demonstrated nosignificant change in VEGF production.

Figure 2. MM cells express VEGF mRNA. (A) TotalRNA (5 mg) from control NBM1-2, patient MM1-5, RPMI8226, and SV MM cell lines was size separated on a1.4% agarose-formaldehyde gel, blotted, and probedwith a 32P-labeled VEGF cDNA probe that recognizesVEGF transcripts of approximately 3.7 to 4.4 kb. A 32Pend-labeled oligonucleotide probe for 18S rRNA wasused as the loading control. (B) Patient MM1-5 cells and2 MM cell lines were lysed, immunoprecipitated with wtp53 and mutant (mt) p53 mAbs, and subjected to immu-noblotting with an anti-p53-HRP mAb.

Figure 3. CD40 activation induces VEGF transcripts in human MM cellsexpressing mutant p53. (A) CD40-activated RPMI 8226 (upper panel) and SV(lower panel) MM lines were cultured for indicated intervals (0-48 hours), and totalRNA (5 mg) was subjected to Northern blotting using human VEGF cDNA, asdescribed in Figure 1. After probing for VEGF mRNA, the blot was stripped andhybridized with an 18S rRNA probe. The fold induction relative to 18S rRNA as acontrol was calculated by densitometry. Both cell lines express mt p53 at 37°C(restrictive temperature) and wt p53 at 28°C (permissive temperature). (B) Total RNA(5 mg) from patient MM cells without (2) or with (1) CD40 activation (24 hours) at37°C was subjected to Northern blotting for VEGF expression. Fold induction ofVEGF relative to an 18S rRNA probe was calculated by densitometry.

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Functional effect of CD40 activation on MM cell transmigration

We recently demonstrated that VEGF induces migration of MMand plasma cell leukemia (PCL) patient cells; moreover, themarkedly enhanced migration in PCL cells compared with that inMM cells suggests an important role for VEGF in the transition ofMM to PCL.15 Having shown that CD40 activation significantlyinduces VEGF expression at the mRNA and protein levels in MMcells with mutant p53, we next asked whether CD40 activationinduced MM cell migration and, if so, whether CD40-inducedtransmigration of MM cells is p53 dependent. Cell migration wasassayed by measuring the trans-filter migration activity of RPMI8226 and SV MM cells and of patient MM cells, seeded on

membranes precoated with fibronectin (5mg/mL [mM]). As shownin Figure 5A-C, the addition of sCD40L or agonist anti-CD40G28.5 mAb to medium containing 1% FCS in the lower chamberinduced the dose-dependent migration of MM cells at restrictivetemperature (37°C), when mutated p53 was functional. For ex-ample, the addition of sCD40L or agonist anti-CD40 G28.5 mAb at10mg/mL (mM) in the lower chamber for 8 and 16 hours induced a3-fold and a 4.2-fold activation of migration in RPMI 8226 and SVMM cells, respectively. In contrast, when either sCD40L oranti-CD40 mAb G28.5 was added at the lower chamber of thetranswell, a dose-dependent decrease in the migration of MM cellswas observed at permissive temperature (28°C), when wt p53 was

Figure 4. CD40 induces VEGF secretion in a dose-and time-dependent fashion. (A) RPMI 8226 and SVMM cell lysates (15 mg) at intervals (0-48 hours) afterCD40 activation were subjected to 12% sodium dodecylsulfate–polyacrylamide gel electrophoresis and weretransferred to a polyvinylidene difluoride membrane.VEGF protein was detected with anti-VEGF mAb andwas visualized by enhanced chemiluminescence. Anti-a–tubulin mAb was used as a loading control. Upper panel,RPMI 8226; lower panel, SV. Similar results were ob-tained in 2 independent experiments. (B) Cells werecultured at 37°C in triplicate with 0 to 10 mg/mL (mM)sCD40L (circle) or anti-CD40 mAb G28.5 (triangle). (C)Cells were cultured with sCD40L (10 mg/mL [mM]) (circle),anti-CD40 mAb G28.5 (10 mg/mL [mM]) (triangle), me-dium alone (diamond), or MOPC-21 isotype control IgG(square) at 37°C. VEGF levels in the supernatants atintervals were determined by ELISA. Data are expresseda means 6 SE of culture triplicates. Open symbol, RPMI8226; solid symbol, SV.

Figure 5. CD40 induces migration in human MM cellsexpressing mutant p53. MM cells were plated on apolycarbonate membrane (8 mm pore size) precoatedwith fibronectin (5 mg/mL [mM]) in the transwell clusterplate and were activated using 0 to 10 mg/mL (mM)sCD40L (A) or anti-CD40 mAb (G28.5) (B) added to thelower chamber. Cells were cultured at restrictive tempera-ture (37°C) with mt p53 or at permissive temperature(28°C) with wt p53. Migrated cells in the lower chamberwere counted using a Coulter counter ZBII. (C) Isotypecontrol antibody MOPC 21 (10 mg/mL [mM]) did notinduce transmigration in MM cells at restrictive tempera-ture. Results are representative of 2 independent experi-ments. (M) RPMI 8226 at 37°C; (z) RPMI 8226 at 30°C;(f) SV at 37°C; ( ) SV at 30°C. (D) Patient MM1-5 cellswere subjected to transmigration assay, using sCD40L(10 mg/mL [mM]) in the lower chamber at 37°C. (f) MM1;( ) MM2; (M) MM3; (u) MM4; ( ) MM5. Increasedtransmigration is observed in MM cells expressing mu-tated p53 (MM2, MM4, MM5), but not in those with wt p53(MM1, MM3), after CD40 activation.

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activated. When patient MM cells were seeded on the filtersprecoated with fibronectin in the upper chamber of the transwellplate, the addition of sCD40L or anti-CD40G28.5 mAb to thelower chamber for 16 hours induced significant transmigration ofMM cells—10-, 1.8-, and 12-fold for patients MM2, MM4, andMM5 with mutant p53, respectively. In contrast, similar treatmentresulted in a decrease in patient MM1 and MM3 cells expressing wtp53 (Figure 5D). These results indicate that CD40 significantinduced the motility of MM cells expressing mutant, but notwt, p53.

Induction of VEGF is associated with transmigration of MMcells after CD40 activation

We next tested whether the induction of VEGF is associatedwith the transmigration activity of MM cells after CD40activation. RPMI 8226 or SV MM cells were plated on thefibronectin-coated filters separating 2 chambers in the transwell,and sCD40L (10mg/mL [mM]), with or without a neutralizinganti-VEGF mAb (0-5mg/mL [mM]), was added to the lowerchamber. Eight to 16 hours later, cells in the lower chamber werecollected and counted. As shown in Figure 6, anti-VEGF mAbalone (0.05-5mg/mL [mM]) did not affect the mobility ofunstimulated MM cells, but it did inhibit the transmigration ofCD40-activated MM cells.

We next asked whether the inhibition of CD40-CD40Linteraction could block VEGF expression and transmigrationinduced by the CD40 activation of MM cells. Multiple myelomacells were treated with sCD40L (10mg/mL [mM]) for 24 hours,with or without a blocking anti-CD40L mAb 5C8, and totalRNA (5 mg) from treated MM cells was subjected to Northernblotting for VEGF expression. As shown in Figure 7A, 4- and6-fold inductions of VEGF expression were observed in CD40-activated RPMI 8226 and SV MM cells, respectively. Con-versely, anti-CD40L mAb (1mg/mL [mM]) inhibited VEGFtranscript induction by sCD40L (10mg/mL [mM]) (1-foldreduction for RPMI 8226, 1.4-fold reduction for SV). Higherconcentrations of anti-CD40L (10mg/mL [mM]) completelyblocked the induction of VEGF transcripts after CD40 activa-tion. In the transmigration experiments in which sCD40L, withor without a blocking anti-CD40L mAb 5C8, was added to thelower 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 mediumcontrol, the addition of anti-CD40L (5C8) or anti-CD40 (M3)mAbs alone at the lower chamber of the transwell did not inducemigration. 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 afterCD40 activation, and the transmigration was observed later (8hours to overnight), suggesting that the specific induction ofVEGF by CD40 is associated with the transmigration ofMM cells.

CD40 activation increases VEGF promoter activity in MM cells

Given that VEGF transcript expression was significantly en-hanced by CD40 activation in MM cell lines and patient MMcells, we wanted to determine whether CD40 activation inducesincreased VEGF transcription. To test the transcriptional activ-ity of VEGF induced by CD40 activation, a 2.4-kb genomicfragment containing human VEGF promoter upstream of lucif-erase reporter was transduced into RPMI 8226, SV, and patientMM5 cells. The transduction of VEGF-Luc reporter wasperformed using adenovirus infection at MOI 800, 200, and 400for RPMI 8226, SV, and patient MM5 cells, respectively, toachieve greater than 80% transduction efficiency. Luciferaseactivity was assayed using equal amounts of cell lysates 6 hoursafter CD40 activation. As shown in Figure 8, the baselineluciferase activity by VEGF-Luc reporter was higher in RPMI8226 cells than in SV cells. The higher baseline VEGF-Lucactivity correlates with higher expression of constitutive VEGFtranscript (Figure 1). Luciferase activity was significantlyincreased (approximately 4-fold for RPMI 8226, 6-fold for SVcells, and 8-fold for patient MM5) in the presence of sCD40L(10 mg/mL [mM]) (Figure 8). Similar induction of luciferaseactivity by VEGF-Luc reporter in the presence of sCD40L wasobtained on cell lysates of MM cells 36 hours after CD40

Figure 6. Neutralizing anti-VEGF mAb blocks CD40-induced MM cell migration.MM cells were subjected to transmigration assay and exposed for 8 hours (RPMI8226 cells) or 16 hours (SV cells) to CD40 activation by sCD40L (0 or 10 mg/mL [mM]),in the presence or absence of neutralizing anti-VEGF mAb (0-5 mg/mL [mM]) added tothe lower chamber of the transwell. Migrated cells collected from the lower chamberwere counted. Enhanced transmigration of cells induced by sCD40L was abrogatedby neutralizing anti-VEGF mAb. (M) RPMI 8226; (f) SV.

Figure 7. Effect of CD40 activation on VEGF production and migration isinhibited by 5C8 blocking anti-CD40L mAb. MM cells were cultured in thepresence or absence of sCD40L (10 mg/mL [mM]) and a 5C8 blocking anti-CD40LmAb (0-10 mg/mL [mM]) to confirm the specificity of sCD40L-triggered VEGF mRNAexpression and cell migration. Blocking mAbs were added to the wells at the initiationof cultures at 37°C. (A) Twenty-four hours after indicated treatments, cells werecollected and total RNA was prepared for Northern blotting (upper panel, RPMI 8226;lower panel, SV). Total RNA (5 mg) was used for analysis, and 18 rRNA served as aninternal control. All autoradiographs are representative of 2 experiments with similarresults. (B) MM cells were subjected to transmigration assay with the addition ofsCD40L in the presence or absence of 5C8 blocking anti-CD40L mAb in the lowerchamber of the transwell. (M) RPMI 8226 at 37°C; (f) SV at 37°C. Identical effects ofCD40 activation on the induction of VEGF were obtained with anti-CD40 mAb G28.5(1 to 10 mg/mL [mM]) (data not shown).

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activation (data not shown). These data, therefore, show thatsCD40L induces VEGF promoter activity, indicating that in-creased transcription accounts, at least in part, for the increaseof VEGF transcripts.

Overexpression of wt p53 in MM cells expressing mutated p53abrogates 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 inMM cells expressing mutated p53 down-regulates CD40-inducedVEGF production. MM cells were first infected with controlAdb-gal or Adwtp53 adenoviruses; 6 hours later, cell lysates weresubjected to immunoblotting for p53 expression. As shown inFigure 9A, the overexpression of p53 was induced only byAdwtp53 adenovirus transduction, whereas control Adb-gal trans-duction did not change p53 expression. Next, MM cells wereincubated with sCD40L (10mg/mL [mM]), with or withoutAdwtp53 or control Adb-gal adenoviruses. Twenty-four hourslater, total RNA was prepared and subjected to Northern blottingfor VEGF expression. As shown in Figure 9B, VEGF expressionwas decreased in the Adwtp53-transduced MM cells, consistentwith previous studies in other human cancer cell lines.28-31 Al -though VEGF expression was induced by CD40 in the medium orAdb-gal–transduced MM cells (4-fold for RPMI 8226; 5.5-fold forSV), no VEGF expression was induced in the presence of Adwtp53adenoviruses. The level of VEGF transcript expression in CD40-activated MM cells transduced with Adwtp53 was approximatelythe same as that in control untreated MM cells, and the addition ofcontrol Adb-gal adenoviruses did not result in any further changesin 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 simi-larly inhibited by the overexpression of Adwtp53 (data not shown).These results provide direct evidence that CD40-induced VEGFexpression 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

Figure 9. Adenovirus-mediated wt p53 overexpression down-regulates CD40-induced VEGF mRNA expression. (A) Six hours after adenovirus infection into MMcells, whole cell lysates (15 mg) were subjected to immunoblotting using HRP-conjugated antipantropic p53 (anti-p53-HRP) mAb. Enhanced expression of p53 wasobserved in RPMI 8226 and SV MM cell lines. Control adenoviruses expressing theb-gal reporter gene (Adb-gal) were used at the same MOIs as Adwtp53 (MOIs of 800and 200 for RPMI 8226 and SV cells, respectively). As an additional control,nontransduced MM cells were used as an internal control. (B) MM cells were culturedwith control (Adb-gal) or wt p53 (Adwtp53) adenoviruses in the presence or absenceof sCD40L for 48 hours. At the end of treatment, total RNA (5 mg) was subjected toNorthern blotting, and an 18S rRNA transcript was used as a loading control. Steadystate mRNA expression was quantitated by densitometry of autoradiograms. (M)RPMI 8226; (f), SV. (C) Adwtp53-transduced MM cells were subjected to transmigra-tion assay in the presence or absence of sCD40L (10 mg/mL [mM]) in the lowerchamber of the transwell.

Figure 8. CD40 activation enhances VEGF promoter activity. MM cells were firstincubated overnight in 2% fetal bovine serum containing RPMI 1640 medium at 37°C.A 2.3-kb full-length human VEGF promoter-luciferase reporter (VEGF-Luc) wastransduced into MM cells by adenoviruses. MM cells were cultured for 6 hours in thepresence or absence of sCD40L, in duplicate wells for each condition. Luciferaseactivity was analyzed using equal amounts of total protein for each sample.Mock-transduced MM cells with Luc reporter without any VEGF promoter sequences(Luc) served as a negative control. Luciferase activity was consistently induced(4-fold for RPMI 8226, 5.5-fold for SV, and 6-fold for patient MM5 cells) insCD40L-activated cells compared with unstimulated cells. Results are representativeof 2 independent experiments performed in duplicate cultures. (M) RPMI 8226; (f)SV; and ( ) patient MM5 cells.

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filter in the upper chamber of the transwell, decreased transmigra-tion of transduced MM cells was observed (RPMI 8226, from3.4-fold to 1.8-fold; SV, from 4.7-fold to 2.2-fold) in response tosCD40L (10 mg/mL) in the lower chamber when compared withthe transmigration of untransduced or control Adb-gal–transducedMM cells (Figure 9C). Similarly, the overexpression of wt p53 inpatient MM5 cells expressing mutated p53 reduced (by 2-fold) thetransmigration induced by sCD40L (data not shown). These dataindicate that induced VEGF expression and transmigration of MMcells after CD40 activation is p53 dependent.

Discussion

In this study, we defined the role of CD40-CD40L interactions inthe regulation of VEGF expression in human MM cell lines andfreshly isolated patient MM cells and the influence of p53 statuson this process. Our results show that CD40 activation signifi-cantly increases VEGF production and transmigration in MMcells expressing mutant p53, but not in MM cells expressing wtp53. MM cell lines RPMI 8226 and SV, with temperature-sensitive p53 mutations, were used to demonstrate VEGFinduction after CD40 activation at restrictive (37°C) mutant p53but not at permissive (28°C) wt p53 temperatures. Significantly,VEGF transcript expression was also induced after CD40activation in patient MM cells (MM2, MM4, MM5) expressingmutant p53, but not in patient MM cells (MM1, MM3)expressing wt p53. We used luciferase reporter assays withfull-length human VEGF promoter to demonstrate that theinduction of VEGF after CD40 activation is controlled, at leastin part, by a transcriptional mechanism. Using an adenovirus-mediated transfer of wt p53, we further showed that theoverexpression of wt p53 down-regulates CD40-induced VEGFproduction and transmigration in MM cells. These studies helpto identify a VEGF role in MM pathophysiology. Because p53mutations occur more frequently in patients with MM duringadvanced stages of the disease, our demonstration of enhancedVEGF expression and migration after CD40 activation in MMcells expressing mutant p53 supports the role of p53 in MMtumor progression. Furthermore, the association of mutant p53and CD40 activation on VEGF production also suggests a rolefor VEGF in MM progression.

Although CD40 activation generates multiple biologic se-quelae in human MM cells, the induction of VEGF has notpreviously been explored. Most reports thus far are in favor of aparacrine action by VEGF in MM, whereby VEGF secreted byMM cells augments IL-6 production in stromal cells.12 How-ever, 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 ontumor cell transmigration and survival. Our current studydemonstrating CD40 induction of VEGF in MM cells alsosuggests autocrine effects of VEGF on MM cells. In addition, itwas shown recently that CD40 induces the cell motility ofKaposi sarcoma and human umbilical vein endothelial cells invitro, which may enhance tumor cell invasion of tissues andendothelial cell organization.19 Because cell migration involvesa dynamic regulation of cell adhesion and our prior studies showan up-regulation of cell surface adhesion molecules on MM cellsafter CD40 activation,6,8 we investigated here whether CD40activation modulated MM cell motility using the transmigrationassay. Interestingly, we observed increased MM cell transmigra-

tion induced by CD40, correlating with tumor cell p53 status.These data, coupled with the clinical observation that patientswith advanced stages of MM express high CD40 levels,5 suggesta role for CD40 in MM homing or invasion.

It has been postulated that p53 dysfunction may play animportant role in MM tumor progression. Specifically, wt p53regulates MM cell growth and apoptosis and attenuates IL-6production, whereas loss of p53 function deregulates IL-6production. In addition, the presence of a mutated p53 allelemay potentiate autonomous MM cell growth and secretion ofangiogenic factors (ie, VEGF), which enhance the emergence ofMM cell clones bearing only the mutated p53 allele. Ourfindings that MM cells expressing mutated p53 produced moreVEGF after CD40 stimulation supports this view.

It has been shown that wt p53 protein down-regulates VEGFpromoter activity in cell lines in a dose-dependent manner.20

Recently, it was reported that the introduction of wt p53 intosarcoma cells repressed the transcription and decreased theexpression of VEGF.32 Endothelial cell growth and migrationwere also decreased when cells were treated with conditionedmedium from sarcoma cells expressing wt p53 compared withmedia from sarcoma cells expressing mutant p53.32 In thecurrent study, the overexpression of wt p53 in MM cell lines andfreshly isolated patient MM cells expressing mutant p53 down-regulated CD40-induced VEGF production, indicating thatVEGF secretion by MM cells can be regulated by wt p53. Thisresult suggests the potential usefulness of adenovirus-mediateddelivery of wt p53 in gene therapy for MM.42 Our ongoingstudies are examining the growth-inhibiting and potentiallytherapeutic benefits of CD40 activation of MM cells heterozy-gous for p53. In addition, because wt p53 suppresses angiogen-esis by the regulation of thrombospondin (TSP-1) expression inhuman fibroblasts43 and because preclinical studies have shownthat wt p53 protein enhances the expression of TSP-1, thedown-regulation of TSP-1 may be observed when alterations ofthe p53 protein occur. Studies are ongoing to determine whetherTSP-1 expression is increased in MM cells expressing wt p53after CD40 activation to further define the role of CD40 inangiogenesis and progression of MM.

CD40 ligation is known to regulate multiple genes throughtranscriptional mechanisms, and our study demonstrates thatVEGF expression in MM cells after CD40 activation is alsoprimarily controlled by a transcriptional mechanism. Specifi-cally, we showed that sCD40L increased luciferase activity inMM cells transduced with the VEGF-Luc reporter. Thus far, theCD40 responsive element in the promoter region of CD40-induced genes has not been identified. However, in a previousreport, the induction of VEGF expression by IL-6 was found tobe mediated by specific DNA motifs located on the putativepromoter region of VEGF and by specific elements located inthe 59-UTR.16 One DNA control element mediating IL-6 re-sponse has been identified between2796 and2804 in humanVEGF promoter.16 Given that the VEGF-Luc reporter used inthe current study contains these regions, the observed inductionof luciferase activity by CD40 activation could have beenindirectly regulated by IL-6. We have shown that CD40activation also triggers IL-6 secretion in MM cell lines andpatient MM cells.6 Therefore, we cannot rule out the possibilitythat the effects of sCD40L on the transmigration of MM cellsmay, at least in part, be a result of indirect (non-VEGF)mechanisms. Because CD40 activation results in the expressionof multiple cytokines, chemokines, and adhesion molecules,

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which themselves may have effects on VEGF expression andcell motility, studies are under way to delineate the direct role ofCD40 in regulating these processes.

In conclusion, we have demonstrated that CD40 activationinduces p53-dependent VEGF expression in human MM cells.Specifically, CD40 activation enhanced VEGF expression andtransmigration in MM cells with mutant p53. Our current study

validates the important role of VEGF and p53 status in MM diseaseprogression.

Acknowledgments

We thank Dr Bert Vogelstein and Dr Robert Taylor for reagents.

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