Inhibition of HHV-8/KSHV infected primary effusion lymphomas in NOD/SCID mice by azidothymidine and...

Leukemia Research 29 (2005) 545–555

Inhibition of HHV-8/KSHV infected primary effusion lymphomas inNOD/SCID mice by azidothymidine and interferon-�

William Wua, Rosemary Rochforda, Lan Toomeyb, William Harrington Jr.a, Gerold Feuera,∗a Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY 13210, USA

b Division of Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL 33101, USA

Received 26 July 2004; accepted 1 November 2004Available online 19 January 2005

Abstract

Kaposi’s sarcoma-associated herpesvirus/human herpesvirus type-8 (KSHV/HHV-8) is associated with primary effusion lymphomas (PEL),a rare form of B-cell lymphoma. PEL cell lines infected with HHV-8, but negative for Epstein-Barr virus (EBV), were analyzed for theirtumorigenic potential in a small animal model system. Inoculation of PEL cell lines into non-obese diabetic/severe combined immunodeficient( d malig-n thymidine( sisf -associatedl nduc-t . Thesed ms of newt©

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NOD/SCID) mice results in efficient engraftment and tumorigenesis in vivo. PEL-engrafted NOD/SCID (PEL/SCID) mice displayeant ascites development with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. AzidoAZT, zidovudine) and interferon-alpha (IFN-�) induce apoptosis in HHV-8+/EBV− PEL cells in culture, by induction of a tumor necroactor-related apoptosis inducing ligand (TRAIL) mediated suicide program and has been proposed as a therapy for herpesvirusymphomas. Daily injection of AZT and IFN-� significantly increased mean survival time (MST) of PEL/SCID mice suggesting that iion of apoptosis in PEL cells in vivo may be exploited as an effective relatively non-toxic therapy targeting HHV-8 infected PELata demonstrate that the PEL/SCID mouse is an important preclinical model to characterize efficacy and anti-tumor mechanis

herapeutic targets in vivo and will be useful in the design and testing of agents in viral lymphoproliferative diseases.2004 Elsevier Ltd. All rights reserved.

eywords:KSHV/HHV-8; Primary effusion lymphoma; SCID mice; Apoptosis; Antiviral therapy

. Introduction

Primary effusion lymphoma (PEL) is a unique recentlydentified non-Hodgkin’s lymphoma which arises predom-nantly in HIV-1 infected AIDS patients[1,2]. PEL re-ponds poorly to conventional chemotherapy and is almostnvariably fatal. PEL is associated with infection by the hu-

an gammaherpesvirus type-8 (HHV-8), also known as Ka-osi’s sarcoma-associated herpesvirus (KSHV) (reviewed in

3–5]). Although most cases of PEL display co-infectionith Epstein-Barr virus (EBV), EBV infection is not re-uired for the development of PEL as singly infected HHV-+/EBV− PEL cells have recently been identified[6–9]. PELsre distinct from most AIDS-related lymphomas such as

∗ Corresponding author. Tel.: +315 464 7681; fax: +315 464 7682.E-mail address:[email protected] (G. Feuer).

EBV-associated Burkitt’s lymphoma due, in many casethe absence of identifiable primary tumor masses, theof expression of B lymphocyte differentiation antigens,lack of c-myc gene rearrangement[10–12]. PEL and otheoncogenic herpesvirus-associated lymphomas are gendifficult to treat in patients as conventional chemotheis poorly tolerated and causes further immunosuppre[13,14]. The development and use of antiviral agents spically targeting virally induced tumors may be more advtageous and efficacious in immunocompromised indivals.

The survival of viral mediated lymphomas, includPEL, depends upon constitutive nuclear factor kappa B�B) activity [15]. It was recently reported that azidothymdine (AZT, zidovudine) and interferon-alpha (IFN-�) inducesapoptosis in HHV-8+/EBV− PEL cells in vitro[16,17]. In-cubation of PEL cells with either AZT or IFN-� individu-

145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2004.11.010

546 W. Wu et al. / Leukemia Research 29 (2005) 545–555

ally had no effect on the induction of apoptosis, suggestingthat AZT and IFN-� function synergistically to initiate pro-grammed cell death[16]. It has been demonstrated that IFN-�induces expression of TNF-related apoptosis inducing lig-and (TRAIL) expression and AZT interferes with the NF-�Bpathway by inhibiting the degradation of I�B and blockingnuclear entry of the p50/p65 NF-�B heterodimer in HHV-8+/EBV− PEL cells[17]. Notably, lymphoma cells that arenegative for gammaherpesvirus infection, such as RAMOS,are unaffected by AZT/IFN-� treatment[16], promptingspeculation that the viral thymidine kinase (TK) may playan important role in the conversion of AZT to an activatedform which inhibits NF-�B activity in PEL cells[18,19]. Therecent reports of clinical improvement in two PEL patientstreated with parenteral AZT and IFN-� prompted us to in-vestigate whether multiple PEL cell lines would respond tothis treatment protocol in vivo[17] (W.H., unpublished ob-servations).

NOD/SCID mice lack functional T and B lymphocyteswith accompanying defects in NK cell function, absence ofcirculating complement and a lack of functional antigen pre-senting cells. NOD/SCID mice have successfully been usedto propagate a variety of hematological malignancies, in-cluding acute lymphoblastic (ALL) and acute myeloblasticleukemia (AML) [20,21], EBV-infected B cell lymphomas[ )[ pleH mn cu-l ah cellsa ning1 ftedNs time( tantp iralt

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2.2. SCID mice

NOD.CB17-PrkdcScid/J (NOD/SCID) mice (Jackson Lab-oratory, Bar Harbor, ME) were kept under specific pathogen-free conditions at the SCID Mouse Core Facility at SUNYUpstate Medical University, Syracuse, NY. NOD/SCID micewere housed in micro-isolator cages, and all food, water, andbedding were autoclaved prior to use. NOD/SCID breedermice were originally purchased from the Jackson Labora-tory (Bar Harbor, ME). All experiments were approved bythe Institutional Animal Care and Use Committee of SUNYUpstate Medical University. Mice were humanely euthanizedwhen the ascites tumor mass reached 20% of the weight ofthe mouse or when the tumor burden was large enough to in-terfere with ambulation, eating, drinking, defecation and/orurination. Mice were weighed and monitored, and weightwas used as a criterion for ascites tumorigenesis and asa marker for AZT and IFN-� efficacy studies. The mock-treated PEL-5/SCID mouse presented inFig. 1 representsan animal which eluded visual detection and displayed anunforeseen rapid weight gain. Although this mouse did notappear to be distressed, it was immediately euthanized upondetection. NOD/SCID mice were anesthetized with isoflu-rane (Minrad Inc., Buffalo, NY) or methoxyflurane (Lan-caster Synthesis, Pelham, NH) prior to all manipulations.

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22,23] and HTLV-1 infected adult T cell leukemia (ATL24]. In this study, we demonstrate the ability of multiHV-8+/EBV− PEL cell lines to efficiently engraft and foreoplasms in NOD/SCID mice. Intraperitoneal (i.p.) ino

ation of NOD/SCID mice with PEL cell lines resulted inigh percentage of mice displaying engraftment of PELnd the development of lymphomatous effusions begin–3 weeks post-inoculation. Treatment of PEL-engraOD/SCID mice (PEL/SCID) with AZT and IFN-� demon-trated a significant improvement in the mean survivalMST). The PEL/SCID mouse model may be an imporre-clinical in vivo model to design and evaluate antiv

herapy strategies against PEL.

. Materials and methods

.1. Cell lines

PEL-5, BC-3, and BCBL-1 are PEL cell lines positor HHV-8 infection and negative for EBV. All PEL ceines were maintained in Iscove’s modified Dulbecco’sia (IMDM) (Gibco BRL, Grand Island, NY) supplementith 10% heat inactivated FBS (Gibco BRL, Grand IslaY), penicillin (100 U/ml) and streptomycin (100�g/ml)

Gemini, Calabasas, CA). Jurkat, Raji, and murine Eells were maintained in Dulbecco’s modified Eagle medGibco BRL, Grand Island, NY) supplemented with 1eat inactivated FBS, penicillin and streptomycin. All cere maintained in a humidified incubator at 37◦C, 5%O2.

.3. Engraftment of PEL-5, BCBL-1, and BC-3 cells inOD/SCID mice

PEL-5, BC-3, or BCBL-1 cells were inoculated intraponeally (i.p.) into NOD/SCID mice. Seventeen mice wnjected with PEL-5 cells at 5× 106 (n= 3), 2× 107 (n= 3),nd 5× 107 (n= 11) cells per mouse. Twenty mice wereculated with BC-3 cells at 2× 106 (n= 5), 3× 106 (n= 2),× 107 (n= 4), and 5× 107 (n= 9) cells per mouse. Eig

een mice were injected with BCBL-1 cells at 2× 106 (n= 5),× 106 (n= 2), 2× 107 (n= 3), and 5× 107 (n= 8) cells peouse. Mice were subjected to peritoneal lavage using 5

erum-free IMDM as previously described[24,25]. A frac-ion of the recovered cells from PEL-5, BCBL-1, and B

inoculated mice were injected i.p. into naı̈ve NOD/SCIDice to propagate and expand the cell lines in vivo. Mere sacrificed by cervical dislocation after administraf isoflurane and were visually analyzed for the presenross malignancies. Tissue samples were excised and fi0% buffered neutral formalin, sectioned at 5�m, and staineith hematoxylin and eosin (H&E) for histological analys described previously[25].

.4. AZT and IFN-� treatment of PEL-engraftedOD/SCID mice

PEL-5, BC-3, or BCBL-1 cells were inoculated intraponeally (i.p.) into NOD/SCID mice at a dose ensurymphomagenesis in a majority (≥85%) of animalsEL-5 inoculated mice (PEL-5/SCID) were injected w

W. Wu et al. / Leukemia Research 29 (2005) 545–555 547

Fig. 1. (A) AZT/IFN-� treatment of PEL-5/SCID mice delays onset of tumorigenesis. NOD/SCID mice were injected with PEL-5 cells (2× 107) on day 0.Daily inoculations of AZT (1.1 mg) and IFN-� (5300 U) were initiated at day 6 post PEL cell inoculation. Mock-treated PEL-5/SCID mice receiving PBSpresented with abdominal distention and were significantly heavier at 23 days post inoculation than PEL-5/SCID mice treated daily with AZT and IFN-�. Theuntreated PEL-5/SCID mouse represents an animal which displayed a rapid unforeseen weight gain and eluded visual monitoring. Although this mouse didnot appear to be in distress, it was immediately euthanized upon detection. Weights of the AZT/IFN-� treated (A01) and mock-treated (A04) mice shown were23.7 and 48.4 g, respectively, at 23 days post inoculation with PEL-5 cells. (B–D) Survival curves for AZT/IFN-�-treated and mock-treated PEL/SCID mice.NOD/SCID mice were inoculated with PEL-5 (2× 107 cells/mouse), BCBL-1 (2× 106 cells/mouse), or BC-3 cells (2× 106 cells/mouse). Daily i.p. injectionof AZT (1.1 mg) and IFN-� (5300 U) per mouse was initiated at 6 days post-inoculation for PEL-5/SCID mice and at 2 days post-inoculation for BCBL-1/SCIDand BC-3/SCID mice. Mice were sacrificed and cells recovered from peritoneal lavage and spleens were tested by PCR for the presence of HHV-8 and human�-globin sequences. Number of mice used per group is indicated in parentheses. Mice which died as a result of adverse reaction to anesthesia (A02, A08,A44) were excluded from the analysis. MST was increased by 70% (P= 0.0006), 38% (P= 0.029) and 46% (P= 0.106) for PEL-5/SCID, BCBL-1/SCID andBC-3/SCID mice, respectively. Statistical analysis was performed using single-tail Student’st-test. E. Histological analysis of lymphomas in NOD/SCID miceinoculated with PEL cells. Hematoxylin and eosin staining demonstrates infiltration of spleen by tumor cells (T) in a BCBL-1/SCID mouse (A35), 100×magnification. Mouse was sacrificed at 49 days after BCBL-1 cell inoculation. F. Spleen from an AZT and IFN-� treated BCBL-1/SCID mouse (A40) displaycharacteristic red (R) and white (W) pulp similar to the morphology of spleen from uninoculated NOD/SCID mice, 100× magnification. Mouse was sacrificed99 days post BCBL-1 cell inoculation.

2× 107 cells/mouse while BCBL-1 and BC-3 inoculatedmice (BCBL-1/SCID and BC-3/SCID, respectively) received2× 106 cells/mouse. Daily i.p. injections of AZT (CatalyticaPharmaceuticals Inc., Greenville, NC) (1.1 mg) and IFN-�(Schering Corporation, Kenilworth, NJ) (5300 U) were per-formed in 0.5 ml of PBS. PEL-5/SCID received AZT/IFN-�at 6 days post-PEL cell inoculation. BCBL-1/SCID and BC-3/SCID received drugs initiating at 2 days post-PEL cell inoc-ulation. Mock-treated mice were given daily i.p. injections of

0.5 ml PBS. Peritoneal lavage samples were recovered frommice at various time points and at the time of sacrifice for flowcytometry, PCR, and apoptosis analysis. Mice were sacri-ficed and tissue samples collected as described in Section2.3.Individual survival times of AZT/IFN-�-treated and mock-treated mice were averaged to calculate mean survival time(MST) of treated and mock mice for each PEL cell line used.Percent MST increase of AZT/IFN-�-treated mice was cal-culated by using the following formula: %MST increase=


MSTtreated−MSTmockMSTmock

× 100%. For PEL-5/SCID, BCBL-1/SCID, and BC-3/SCID, significant difference between thesurvival times of AZT/IFN-�-treated and mock-treated ani-mals was determined using single-tail Student’st-test. Statis-tical significance was assumed at aP value of less than 0.05.Mice which died as a result of adverse reaction to anesthesia(A02, A08, A44) were excluded from statistical analysis.

2.5. Polymerase chain reaction (PCR)

DNA and RNA were isolated from peritoneal lavageand spleen tissue samples using the urea lysis method,as described previously[24,26,27]. Briefly, cells werewashed in PBS, lysed in urea lysis buffer (4.7 Murea, 1.3% (w/v) sodium dodecyl sulfate (SDS), 0.23 MNaCl, 0.67 mM EDTA (pH 8.0)), and subjected tophenol–chloroform extraction and ethanol precipitation.PCR analysis was performed using primers specificfor HHV-8 (KS330233 fragment within ORF 26 mi-nor capsid protein) (5′-AGCCGAAAGGATTCCACCAT-3′, 5′-TCCGTGTTGTCTACGTCCAG-3’) [28], and hu-man �-globin (5′-ACACAACTGTGTTCACTAGC-3′, 5′-CAACTTCATCCACGTTCACC-3′) [24] using the OneStepRT-PCR kit (Qiagen Inc., Valencia, CA) or the SuperScriptIII one-step RT-PCR system with Platinum Taq DNA poly-m n aD A)waP 2%S , Inc.,R 1T li-fi nd1 10-f llela d cor-r

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2 min 72◦C, 1 min detection at 78◦C; 55–95◦C meltingcurve. Data was collected and HHV-8 and human�-globinDNA copy number per sample was calculated using theiCycler iQ Real-Time Detection System Software (BioradLaboratories, Inc., Hercules, CA). HHV-8 and human�-globin DNA copies were normalized to total DNA persample as determined by UV spectrophotometry. Statisticalanalysis comparing the HHV-8 and human�-globin copynumbers from AZT/IFN-�-treated and mock-treated micewas performed using single-tail Student’st-test. Statisticalsignificance was assumed at aP value of less than 0.05.

2.7. Flow cytometry

Cells were incubated with phycoerythrin (PE)-conjugatedmonoclonal antibodies (mAbs) directed against human CD45or CD23 and fluorescein isothiocyanate (FITC)-conjugatedmAbs directed against human CD19, CD30, or murineCD45.2 (all from BD Biosciences Pharmingen, San Diego,CA). Cells were washed twice with 3 ml PBS and then re-suspended in 4 ml PBS for analysis. Samples were run ona FACStarplus flow cytometer (Becton Dickinson, MountainView, CA). One- or two-color analysis was performed usingWinMDI 2.8 software.

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ateswR revi-o PAr ran-d edb p kit( pli-fi g-m adi-s d se-q sys-t fromE ibo-p d [3 is-c rmeda et 73),v b-

erase (Invitrogen, Carlsbad, CA). Cycle conditions oNA Thermal Cycler 480 (Perkin-Elmer, Wellesley, Mere set as follows: 15 min at 95◦C; 40 cycles of 1 mint 94◦C, 1 min at 60◦C, 2 min 72◦C; 10 min at 72◦C.CR products were electrophoresed at 210 V for 1 h oneaKem GTG agarose (Cambrex Bio Science Rocklandockland, ME) prestained with ethidium bromide in×BE buffer. Bands were visualized by UV light. Amped KS330233 and human�-globin fragments are 233 a10 bp, respectively. BCBL-1 cells were serially diluted

old, DNA was extracted and was PCR-amplified in paras a standard measure of the number of human cells anesponding HHV-8 sequences.

.6. Real-time PCR

Real-time PCR assay utilized the KS330233 primers28], human �-globin primers 5′-GAAGAGCCA-GGACAGGTAC-3′ (GH20) [29] and 5′-GCAAAGGTG-CCTTGAGGT-3′ [30], and the QuantiTech SYBR GreCR kit (Qiagen Inc, Valencia, CA). To prepare HHVNA standards, the KS330233 fragment was cloned in

he pCR2.1 plasmid using a TA cloning kit (Invitrogearlsbad, CA) to make the pCR2.1-KS330233. Similarly, the85 bp fragment amplified by the�-globin primers abovas cloned into pCR2.1 to make pCR2.1-huBGLOB5CR2.1-KS330233 and pCR2.1-huBGLOB585 were liarized with BamHI and serially diluted into a salmon spNA carrier (2�g/�l). Cycle conditions on an iCycler (Bio

ad Laboratories, Inc., Hercules, CA) were set as follo5 min at 95◦C; 55 cycles of 1 min at 94◦C, 1 min at 60◦C,

.8. Apoptosis analysis

Cells recovered by peritoneal lavage from a BC/SCID mouse were cultivated ex vivo in complete IMDupplemented with 10�g/ml AZT and 1000 U/ml of IFN-�.ells were stained with annexin V-FITC and propidiumide at 24, 46, and 68 h in triplicate and analyzed byytometry.

.9. RNase protection assay

RNA preparation and generation of riboprobe templas performed as previously described[31,32]. Briefly, totalNA was extracted from cell samples according to a pusly described method[33] and used as a template for Riboprobe generation. cDNA synthesis was initiated byom hexamer primers (Invitrogen, La Jolla, CA), followy polymerase chain reaction (PCR) using the GeneAmPerkin-Elmer Cetus) and appropriate primers for the amcation of HHV-8 specific sequences. Amplified DNA fraents were ligated into the pGEM-4 vector (Promega, M

on, WI). Subclone authenticity was verified by automateuencing on an ABI Model 3730 sequencer (Applied Bio

ems, Foster City, CA). Antisense RNA was generatedcoRI-linearized subclones using T7 polymerase, the Rrobe Gemini core system (Promega, Madison, WI), an�-2P]-UTP (3000 Ci/mmol; 1 Ci = 37 GBq; Amersham, Pataway, NJ) as the labeled nucleotide. RPA was perfos described previously[31,32]. A HHV-8 specific riboprob

emplate set was designed to examine LANA-1 (ORFIL-6 (ORF K2), and vFLIP (ORF 71) transcripts. A su


clone detecting the human ribosomal protein L32[34] wasused as a control for RNA loading. All riboprobe synthe-ses were driven by T7 bacteriophage RNA polymerase with[�-32P]UTP as the labeled nucleotide[31,32]. Probe purifica-tion, RNA-probe hybridization, RNase treatment, protectedRNA duplex purification, and resolution of protected probesby denaturing polyacrylamide gel electrophoresis were per-formed as described[31]. Probe bands were visualized byautoradiography on XAR film (Kodak, Rochester, NY) andquantified by using the Storm 400 PhosphorImager and Im-ageQuant software (Molecular Dynamics, Sunnyvale, CA).

3. Results

3.1. Engraftment and tumorigenesis of PEL cell lines inNOD/SCID mice

To characterize the ability of PEL cell lines to engraft inNOD/SCID mice, PEL-5, BCBL-1, or BC-3 cells were in-oculated intraperitoneally (i.p.) into NOD.CB17-Prkdcscid/J(NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME).BCBL-1 [7,35] and BC-3 cells[36] are PEL cell lines thathave been propagated in culture for at least 7 years. PEL-5 has been only recently established in culture (less than 2y withH Ra en-go la-t ase f en-g post-i asc ab-d vi-

ous reports of engraftment of PEL cell lines in SCID mice[37–39]. In addition, some BCBL-1/SCID mice also devel-oped subcutaneous tumors that infiltrated the abdominal wallnear the site of injection, as previously described[37].

Lack of expression of mature B cell markers is a char-acteristic of PEL cells[40]. Cells engrafted in SCID micewere recovered by peritoneal lavage at various time pointsfollowing inoculation, and flow cytometric analysis was per-formed to characterize PEL cell growth in vivo. The majorityof cells recovered from PEL-5, BCBL-1, and BC-3 inocu-lated mice by peritoneal lavage stained for cell surface ex-pression of human CD45, a pan-human lymphocyte marker,demonstrating expansion of PEL cells in the peritoneal cav-ity of NOD/SCID mice (Table 1). PEL-5 and BCBL-1 cellswere also positive for CD30 expression, a late stage B-cellmarker. Notably, PEL-5 cells displayed expression of CD23, amarker generally found on mature B-cells and not detected onBCBL-1 or BC-3 cells. Cells recovered by peritoneal lavagedisplayed phenotypes virtually identical to phenotypes exhib-ited by PEL cells grown in vitro[41,42]. All PEL lines testedwere negative for CD19, a mature B-cell marker, as well asmurine CD45.2. DNA was extracted from cells recovered byperitoneal lavage from PEL/SCID mice and analyzed by PCRusing primers specific for HHV-8 sequences (KS330233) andfor the human�-globin gene. PCR analysis demonstratedt incs la iced rphicm yto-p undt butd entn ighp of-

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ears). Each PEL cell line was confirmed to be infectedHV-8 and confirmed negative for EBV infection by PCnalysis (data not shown). All PEL cell lines efficientlyrafted in the peritoneal cavity of NOD/SCID mice (≥85%f total mice for each PEL line used) after i.p. inocu

ion (Table 1). Malignant ascites was observed in micearly as 1–3 weeks post-inoculation and the majority orafted mice developed malignant ascites 4–7 weeks

noculation. The gross morphology of PEL/SCID mice woncomitant with a notable increase in body mass andominal distention (Fig. 1A). These results extend on pre

able 1ngraftment and tumorigenesis of PEL cells in NOD/SCID mice

ell line Cells inoculated per mouse (×107) Tumor incidence (n

EL-5 0.5 1/3 (33%)2.0 3/3 (100%)5.0 11/11 (100%)

C-3 0.2 4/5 (80%)0.3 1/2 (50%)2.0 4/4 (100%)5.0 8/9 (89%)

CBL-1 0.2 5/5 (100%)0.4 1/2 (50%)2.0 3/3 (100%)5.0 8/8 (100%)

EL-5, BCBL-1, and BC-3 cells were inoculated intraperitoneally (i.p.he majority of mice injected with PEL cell lines, although some mice dor human lymphocyte markers was carried out on cells recovered byavage showed expression of human CD45. PEL-5 cells recovered bisplayed by PEL-5 cells grown in vitro.

he presence of HHV-8 and human�-globin sequencesells recovered by peritoneal lavage (Fig. 2A), and from thepleens of sacrificed PEL/SCID mice (Fig. 2B). Histologicanalysis of PEL/SCID mice revealed that many of the meveloped anaplastic neoplasms composed of pleomoononuclear cells with small amounts of basophilic clasm and indistinct cell borders. Nuclei were large, ro

o oval to indented, and somewhat vesicular with a thinistinct rim of marginated chromatin, one or more prominucleoli, and displayed a high mitotic index (5–10 per hower field). PEL cells formed solid infiltrating sheets,

e tumors/no. mice injected) Phenotype

CD45 CD23 CD30 CD1

+ + (low) + −

+ − − −

+ − + (low) −

OD/SCID mice. Ascites formation was detected 4–7 weeks post-inoion ind tumor engraftment as early as 1–3 weeks post-inoculation. Phenotnalysis

eal lavage at 3 weeks post-inoculation. A majority of cells recovered bitoneale also stained for CD30 and CD23, similar to patterns of cell surface


Fig. 2. PCR analysis of DNA from cells recovered from PEL/SCID mice. (A) PCR analysis of DNA isolated from peritoneal lavage cell samples collectedfrom BCBL-1/SCID (A35–A44), BC-3/SCID (A56–A65), and PEL-5/SCID (A01, A03–A07) mice. Lavage samples were collected 50 days post inoculationfrom mock-treated and from AZT (1.1 mg) and IFN-� (5300 U) treated PEL/SCID mice. Samples from animals which did not survive to the 50 day time pointwere collected at the time of death are as follows: A35, A36: 49 days post-inoculation (dpi); A44: 33 dpi; A06, A59: 40 dpi; A01, A03: 23 dpi; A04: 38 dpi;A05: 42 dpi; A07: 36 dpi. Amplified KS330233 (HHV-8 ORF26) and human�-globin sequences are 233 and 110 bp, respectively. Amplified products wereresolved on a 2% agarose gel in TBE buffer. BCBL-1 cells were serially diluted, DNA was extracted and these samples were used as a standard for the PCR.Note that BCBL-1 cells have been reported to contain approximately 70 copies of the HHV-8 genome per cell[51]. As a result, KS330233 primers are moresensitive and detect as few as 10 BCBL-1 cells per sample assayed, using these PCR conditions. In contrast, human�-globin primers were able to detect asfew as 1× 103 BCBL-1 cells. (B) DNA PCR analysis of cells recovered from spleen samples from PEL/SCID mice. Spleen samples were collected from thesame PEL/SCID mice used for the PCR analysis presented in (A) above.

ten perivascular, in many organs, including the spleen, liver,kidney, lung, and skin (Fig. 1E–F). Tumors that developed inPEL/SCID mice presented as a malignant ascites frequentlyaccompanied by significant abdominal distention, and theseobservations are consistent with the clinical manifestationsof PEL in humans, which include pericardial and/or peri-toneal effusions and abdominal distention[43,44]. These re-sults demonstrate that intraperitoneal inoculation of PEL-5,BCBL-1, or BC-3 cells into NOD/SCID mice results in highrates of engraftment of PEL cells in vivo and a malignantphenotype reminiscent of PEL pathogenesis in humans.

BCBL-1/SCID and PEL-5/SCID mice developed ma-lignancies which predominantly localized to the abdomi-nal organs and skin as determined by histological analysis(Fig. 1E–F). Evidence of neoplastic cells was also detectedin all BC-3/SCID mice (n= 5), irrespective of treatment withAZT and IFN-�. All mice which were scored positive for the

presence of neoplasms by histological analysis also scoredpositive for the presence of HHV-8 and human�-globin se-quences when analyzed by PCR. Histological analysis ofthree mice (A40, A63, and A65) demonstrated the presenceof PEL cells in the lung, kidney, liver, and intestine, eventhough PEL cells were not detected in the peritoneal cavityin these animals when analyzed by PCR, demonstrating thatPEL cells migrate and are sequestered in tissues and organsof mice resulting in the development of localized neoplasms.

3.2. AZT and IFN-α treatment of PEL-engraftedNOD/SCID mice extends latency to tumorigenesis

It was recently reported that AZT and IFN-� syner-gize to induce apoptosis of HHV-8+/EBV− PEL cell linesin vitro [16,17]. To evaluate AZT and IFN-� as a ther-apy for PEL in an in vivo preclinical model of PEL,


NOD/SCID mice were first engrafted with BCBL-1 (BCBL-1/SCID; 2× 106 cells/mouse;n= 10), BC-3 (BC-3/SCID;2× 106 cells/mouse; n= 10) and PEL-5 (PEL-5/SCID;2× 107 cells/mouse;n= 8) cells on day 0. PEL/SCID micewere subsequently treated with daily i.p. inoculations ofAZT and IFN-� (1.1 mg AZT and 5300 U IFN-�, respec-tively). AZT and IFN-� treatment was initiated on day 2 post-inoculation for BCBL-1/SCID and BC-3/SCID mice and onday 6 post-inoculation for PEL-5/SCID mice, and peritoneallavages were performed on days 10, 25, and 50. Since PEL-5cells demonstrated relatively lower levels of engraftment inNOD/SCID mice (Table 1), mice received a higher inocu-lum of PEL-5 cells and the treatment of PEL-5/SCID micewith AZT and IFN-� was delayed by an additional 4 daysto facilitate the engraftment of PEL-5 cells. The majority ofcells recovered by peritoneal lavage from BCBL-1/SCID andBC-3/SCID mice displayed human CD45, demonstrating theengraftment and expansion of PEL cells in the peritonealcavity at all time points analyzed (data not shown). Daily in-oculation of AZT and IFN-� treatment noticeably delayedascites formation and abdominal distention in PEL/SCIDmice in comparison to mock-treated mice (Fig. 1A). Injec-tion of AZT and IFN-� also markedly slowed the rapid weightgain associated with the development of lymphomatous effu-sion (data not shown). Flow cytometric analysis of peritoneall l bur-d -1 dbl 5.2c fP eanp ndB fP by7 nc N-tb , al-t ep op-t chd t theP aluef get-i

inN edf ficedP enceo u-m ted inaA neall dB rols

(Fig. 2A). Since recovery of cells from peritoneal lavages isvariable and semi-quantitative, real-time PCR was performedto quantify tumor cell burden in response to AZT and IFN-�treatment. When compared to mock-treated controls, therewere significant reductions in tumor cell number (as deter-mined by human�-globin copies) in the peritoneal lavagesamples from AZT/IFN-�-treated BCBL-1/SCID (22-fold,P= 9.9× 10−6) and BC-3/SCID (750-fold,P= 0.030) mice,when standardized to total DNA recovered. A concomitantdecrease in HHV-8 DNA levels was also observed in responseto AZT and IFN-� treatment. Real-time PCR demonstrated a13-fold and 2.6× 104-fold decrease in the HHV-8 copy num-ber in the peritoneal lavage samples from AZT/IFN-�-treatedBCBL-1/SCID (P= 0.002) and BC-3/SCID (P= 0.012) mice,respectively, in comparison to mock-treated mice when stan-dardized to the amount of DNA recovered. In addition, therewas a significant decrease (17-fold,P= 1.1× 10−4) in HHV-8 viral copies per PEL cell in recovered peritoneal lavage sam-ples from AZT/IFN-�-treated BC-3/SCID mice when com-pared to mock-treated BC-3/SCID mice. HHV-8 copies perneoplastic cell were not significantly altered in AZT/IFN-�-treated BCBL-1/SCID (P= 0.50) or PEL-5/SCID (P= 0.07)mice. Unexpectedly, HHV-8 and human�-globin copy num-bers were not significantly reduced in AZT/IFN-�-treatedPEL-5/SCID mice, even though drug treatment significantlyd nt ton N-� andt thesem

alg CIDm n ofr iso-l RF7 ro-t ry ofc RNAd nee eredfp atedm edi

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avage samples showed a notable reduction in tumor celen in the peritoneal cavities of AZT/IFN-�-treated BCBL/SCID (n= 5) and BC-3/SCID (n= 5) mice, as determiney the percentage of human CD45+ cells recovered from

avage samples relative to the number of murine CD4+

ells (data not shown). Notably, AZT and IFN-� treatment oEL-engrafted SCID mice significantly increased the most-inoculation survival time (MST) of PEL-5/SCID aCBL-1/SCID mice (Fig. 1B–D). AZT/IFN-� treatment oEL-5/SCID and BCBL-1/SCID mice increased the MST0% (P= 0.0006) and 38% (P= 0.029), respectively, wheompared to mock-treated PEL/SCID mice. AZT and IF�reatment also increased the MST of BC-3/SCID mice (n= 5)y 46% in comparison to mock-treated BC-3/SCID mice

hough this was not statistically significant (P= 0.106). Thesreliminary in vivo analyses suggest that induction of ap

osis by AZT and IFN-� may be a key mechanism whielays the onset of PEL tumorigenesis in vivo and thaEL/SCID mouse model may have a clinical predictive v

or evaluating therapeutic efficacy of antiviral agents tarng PEL cells.

As a semi-quantitative analysis of PEL cell retentionOD/SCID mice, DNA was purified from cells recover

rom the peritoneal exudates and from spleens of sacriEL/SCID mice, and was analyzed by PCR for the presf HHV-8 sequences (KS330233fragment in ORF26) and han�-globin sequences. HHV-8 sequences were detecll mice inoculated with PEL cell lines (n= 28 mice) (Fig. 2).relatively weaker PCR signal was observed in perito

avage samples from AZT/IFN-�-treated BCBL-1/SCID anC-3/SCID mice in comparison to mock-treated cont

elayed lymphomagenesis in these mice. It is importaote that all mice, including mice treated with AZT and IF, eventually succumbed to PEL-induced malignancies

hat HHV-8 sequences were detected in the spleens ofice by PCR (Table 2).To determine if AZT and IFN-� treatment modulates vir

ene expression in vivo, PEL cells recovered from PEL/Sice by peritoneal lavage were analyzed for expressio

epresentative HHV-8 latent viral transcripts. RNA wasated and analyzed for expression of HHV-8 LANA-1 (O3), vIL-6 (ORF K2), and vFLIP (ORF 71) by RNase p

ection assay (RPA). Although semi-quantitative recoveells from the peritoneal lavage made standardization ofifficult to achieve, no significant alterations in HHV-8 gexpression was detected in RNA isolated from cells recovrom PEL/SCID mice treated with AZT and IFN-� in com-arison to RNA isolated cells recovered from mock-treice or from RNA extracted from PEL cell lines cultivat

n vitro (data not shown).

.3. BCBL-1 cells recovered ex vivo retain sensitivity toZT and IFN-α-induced apoptosis

To test whether PEL cells are altered for resistancZT/IFN-�-mediated apoptosis during in vivo expansells were recovered by peritoneal lavage from a BC/SCID mouse treated with AZT and IFN-� at 40 days pos

noculation. Recovered cells were tested for sensitivitZT and IFN-� by cultivating cells in IMDM supplementeith 10�g/ml AZT and 1000 U/ml of IFN-� ex vivo andnalyzing by flow cytometry for staining with annexin


Table 2PEL tumorigenesis in NOD/SCID mice

ID PEL cell line Mouse no. AZT/IFN-� treatmenta Survival time (days)b Tumorc Peritoneal huCD45d Lavage PCRe Spleen PCRe

A35 BCBL-1 1 − 49 + + + +A36 BCBL-1 2 − 49 + + + +A37 BCBL-1 3 − 82 + + + +A38 BCBL-1 4 − 69 + + + +A39 BCBL-1 5 − 53 + + + +A40 BCBL-1 1 + 99 − − − +A41 BCBL-1 2 + 62 + + + +A42 BCBL-1 3 + 82 + − + +A43 BCBL-1 4 + 91 + − + -A44 BCBL-1 5 + 33 − − + +

A56 BC-3 1 − 80 nd + + +A57 BC-3 2 − 70 nd − − +A58 BC-3 3 − 100 nd − + +A59 BC-3 4 − 40 + + + +A60 BC-3 5 − 70 − + + +A61 BC-3 1 + 126 nd − − +A62 BC-3 2 + 95 nd − − +A63 BC-3 3 + 178 + − − +A64 BC-3 4 + 50 + − + −A65 BC-3 5 + 75 + − − +

A01 PEL-5 1 − 23 + + + +A02f PEL-5 2 − 10 nd nd nd ndA03 PEL-5 3 − 23 + + + +A04 PEL-5 1 + 38 nd + + +A05 PEL-5 2 + 42 + + + +A06 PEL-5 3 + 40 nd + + +A07 PEL-5 4 + 36 + + + +A08f PEL-5 5 + 7 nd nd nd nd

NOD/SCID mice were inoculated with PEL cell lines and the course of survival of mice was monitored. Cells recovered by peritoneal lavage were analyzedfor expression of human CD45. Mice were sacrificed and tissues analyzed by histology and DNA was tested by PCR. nd: no data.

a Daily i.p. injections of AZT/IFN-� was initiated 2 days post PEL cell inoculation for BCBL-1 and BC-3 cells, and at 6 days post inoculation for PEL-5.b Survival time of mice in days after i.p. inoculation of PEL cells.c Malignancy detected by histological analysis.d Flow cytometric analysis was performed using a monoclonal antibody for human CD45.e PCR analysis of DNA using primers specific for HHV-8 (KS330223).f Animals not analyzed due to undetected death and decomposition of tissue prior to analysis.

Fig. 3. BCBL-1 cells engrafted in vivo retain sensitivity to AZT and IFN-�.Cells (1× 106) recovered by peritoneal lavage from a BCBL-1/SCID mousetreated daily with AZT (1.1 mg) and IFN-� (5300 U) at 32 days post-PELcell inoculation were cultured in IMDM containing IFN-� (1000 U/ml) plusAZT (10�g/ml) or in IMDM alone. Cell sample aliquots (1.0× 105) weretested at 24, 46 and 68 h and subjected to staining with Annexin V-FITCand propidium iodide, followed by flow cytometric analysis to determinecell viability. The assay was performed in triplicate. Bars represent percentviable cells.

FITC and propidium iodide at 24, 46 and 68 h of in vitrocultivation. BCBL-1 cells recovered from NOD/SCID miceretained sensitivity to AZT and IFN-� induced apoptosis atlevels similar to those displayed by BCBL-1 cell lines culti-vated in vitro (Fig. 3), suggesting that AZT and IFN-� treat-ment of BCBL-1/SCID mice does not result in the selectionof apoptosis-resistant PEL cells in vivo.

4. Discussion

Primary effusion lymphoma is recognized as a uniqueclinical entity comprising of KSHV/HHV-8 infected trans-formed post-germinal center B cells[45]. In some cases theidentification of unmutated immunoglobulin gene sequencessuggests that PEL may represent transformation of B-cellsat different stages of ontogeny[46–48]. The engraftment ofPEL cells in NOD/SCID mice recapitulates characteristics ofthe human disease and this small animal system is an impor-


tant step towards developing an in vivo model system to studyHHV-8 pathogenesis. Understanding the oncogenic mecha-nisms involved in the pathobiology of gammaherpesvirus-associated lymphomas is important for the development andtesting of targeted therapeutics. We demonstrate that mul-tiple PEL cell lines efficiently engraft in NOD/SCID miceand maintain the phenotype displayed in vitro, demonstratingthat the SCID mouse provides an ideal microenvironment forpropagation and expansion of these lymphoma cells. The for-mation of malignant ascites displayed by PEL/SCID mice ischaracteristic of PEL disease in humans[42]. One molecularfeature of PEL that is common to other viral-mediated lym-phoproliferative diseases is elevated DNA-binding activity ofNF-�B. It was previously shown that AZT and IFN-� syn-ergistically induce apoptosis in HHV-8+/EBV− PEL cells invitro [16,17]. The concomitant induction of expression of thepro-apoptotic protein, TRAIL, and the inhibition of nucleartranslocation of NF-�B synergistically potentiates apopto-sis in PEL cells. These preliminary in vivo results demon-strate that AZT and IFN-� treatment significantly delays tu-morigenesis in PEL/SCID mice, suggesting that inductionof apoptosis may mechanistically modulate the progressionof PEL lymphomagenesis. Notably, the initial dose of PELcells used to inoculate SCID mice was at a level to insurethat a majority of mice developed lymphomas and representsa seo sisc g app f theo willb agep hichp gly,a ena meP e in-d se-q routb ani the-l andI is int

Lp ande pro-p reg-i teda eres tiveoA ilyp CIDm e toA e inM hort

time, in contrast to BCBL-1 and BC-3 cells, may have hadless time to selectively adapt to growth in vitro and may bemore representative of lymphoma cells from patients. More-over, engraftment and expansion of PEL cells directly frompatients in SCID mice may provide an in vivo model that ismore clinically relevant than tissue culture systems.

The combination of the antiviral thymidine analog AZTand IFN-� has been used to treat several viral associated can-cers, including HTLV-1 associated ATL/ATLL and AIDS-related Kaposi’s sarcoma[49,50]. Notably, the recent reportof a marked clinical improvement in a PEL patient with lym-phoma treated with parenteral AZT and IFN-� from oneof our laboratories (W.H.) suggests that this antiviral tar-geted therapy may be of important clinical benefit to patients.An additional patient diagnosed with a solid tumor variantof HHV-8 related lymphoma was treated with combinationchemotherapy AZT and IFN-� and has remained in remis-sion for 1 year. Refinement of the PEL/SCID mouse modeland evaluation of the expression of HHV-8 latent and lyticgenes will further characterize the anti-tumor mechanism ofAZT and IFN-� and will allow the development of targetedantiviral therapies to be evaluated in vivo.

Acknowledgements

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relatively high inoculum of tumor cells. Similarly, the dof AZT and IFN-� was formulated on a body-weight baomparable to the dosage used in humans and the drulication regimen employed may not be representative optimal dosage for chemotherapy. Further investigationse required to optimize inoculum of cells and drug dosaradigms to properly evaluate the treatment regimen wrevents lymphomagenesis in PEL/SCID mice. Interestinlthough AZT and IFN-� did reduce the tumor cell burdnd the HHV-8 DNA levels in the peritoneal cavity of soEL/SCID mice, these parameters were not a predictivicator of MST. The ability of PEL cells to migrate anduester into various tissues and organs suggests that they which AZT and IFN-� are administered may also be

mportant variable in the antitumorigenic therapy. Neveress, it is notable that intraperitoneal inoculation of AZTFN-� significantly extended latency to lymphomageneshe PEL/SCID mice.

Direct inoculation of NOD/SCID mice with primary PEatient samples may allow the preferential propagationxpansion of primary PEL cells in vivo and may be an apriate model to measure responsiveness to antiviral drug

mens. It was previously demonstrated that HTLV-1-infecdult T cell leukemia cells (ATL) which engrafted and whown to be tumorigenic in SCID mice were representaf lymphoma cells which predominated in ATL patients[24].TL cells do not proliferate when cultured in vitro, but readroliferate and are tumorigenic when injected into the Souse. Notably, PEL-5/SCID mice were most responsivZT and IFN-� and showed the most dramatic increasST. PEL-5 cells have been cultured for a relatively s

-

e

We thank Donna Kusewitt (Ohio State University) for porming the histological analysis and Mary Lutzke for teical assistance. This work was supported by the Nat

nstitutes of Health.Contributions: W. Wu performed in vivo experiments f

EL cell line engraftment in SCID mice and AZT/IFN�reatment; flow cytometry and PCR analyses of tissueles; participated in writing the manuscript. R. Rochford

ormed RNAse protection assays on RNA recovered fEL cells from SCID mice. L. Toomey performed ex-vpoptosis analyses presented inFig. 3. W. Harrington, Jrrovided PEL cell lines and advice on design of expents; provided AZT and IFN-�. G. Feuer designed exp

ments; analyzed data and outlined experiments; prepanuscript.

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