[CANCERRESEARCH54, 2055—2059,April 15,1994J

Abstract

Transferof the herpessimplexvirus thymidinekinase(H5Vtk)geneinto tumor cells using retroviral vectors followed by administration ofgandclovir provides a potential strategy for the treatment of malignancy.Because of the limitations of using retroviral vectors for clinical applicadon, the feasibility of using a recombinant adenovirus containing HsVtkwas examined. Cell lines derived from human malignant mesotheiomasand non-small cell lung cancers Infected with a recombinant adenoviruscontaining HSVtk showed strong expression of HSVtk protein as determined by immunohistothemlcal staining. Infection with a recombinantadenovirus containing HSVtk rendered cells sensitive to doses of ganciclovir that were 2-3 logs lower than uninfected cells or those infected witha control virus. A strong “bystandereffect―was noted In mesotheliomalines; there was no diminution In the efficacy of ganciclovir treatmentuntil the ratio of infected:uninfected cells fell below 1:10. This study thusdemonstratesin vitmefficacyof an adenovlrus-transducedHSVIIIdrugsensitization gene therapy system in thoracic malignancies. Recombinant

adenovirus transfer of the HSVtk gene followed by ganciclovir may havepromise as an in situ treatment for tumors.

Introduction

Malignant mesothelioma is a tumor arising primarily from thenormal mesothelium lining the thoracic cavity. Although mesothelioma is relatively uncommon compared to a number of other neoplasms, it is a frustrating clinical problem due to its extremely poorresponse to treatment. Aggressive multimodality therapies, includingradical resection with adjuvant chemotherapy and radiation, result insurvivals of only 18—24months from diagnosis (1). Despite this, thereare a number of characteristics that make mesothelioma an attractivetarget for gene therapy. Residing in the potential space of the thoraciccavity, the tumor is easily accessible, facilitating administration ofsubstances such as recombinantgenetic material. In addition, localextension of disease ratherthan distantmetastasis is responsible forthe majority of the morbidity and mortality associated with thisneoplasm. Thus, even small increments of improvement in localcontrol could lead to significant improvement in survival or palliation.NSCLC@is a much more common malignancy, and combined che

Received 1/14/94; accepted 3/4/94.The costsof publicationof this articleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertIsement in accordance with18U.S.C.section1734solelyto indicatethisfact.

1 This study was partially supported by a development grant from the University of

Pennsylvania Cancer Center.2 To whom requests for reprints should be addressed, at Thoracic Oncology Research

Laboratury, Hospital of the University of Pennsylvania, Maloney Building, Room 809,36th and Spruce Streets, Philadelphia, PA 19104.

3 The abbreviations used are: NSCLC, non-small cell carcinoma of the lung; HSVIJC,

herpes simplex virus type 1 thymidine. kinase; GCV, ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine]; Ad.RSVtk, recombinant adenovirus containing HSVtk; IC50,concentration required to kill 50% of cells.

motherapy/radiationtherapyfor advancedstage disease may not significantly improve survival beyond what is offered by supportive care(2). Local complications of advanced disease such as malignant effusion and bronchial obstruction may also be reasonable targets forsomatic gene therapy.

Varioussystems of somaticgene therapyfor malignancyhave beeninvestigated, including transfer of drug susceptibility genes. Thisparadigm allows recipient cells to selectively produce a novel substance, usually an enzyme, which sensitizes them to a normallynontoxic drug. One such combination is transferof the HSVtkgenefollowed by administrationof GCV (3). GCV is an acyclic nucleosidethat is normallymetabolized at very low levels by mammaliancells(4). GCV is transformed by viral thymidine kinase into a monophosphate form that is subsequently converted by endogenous mammaliankinases to GCV triphosphate, a potent inhibitor of viral DNA polymerase and a purine analogue competing with normal nucleotides forviral DNA replication (4). In addition, GCV triphosphate also cornpetes with normal nucleotides for DNA replication in mammaliancells and can cause inhibition of cell growth and cell death. By thesemechanisms, tumor cells expressing the HSVtk enzyme become susceptible to the toxic effects of GCV. One of the more promisingaspects of the use of HSVtk for gene therapy is the existence of a“bystandereffect.―Tumor cells not actually expressing the HSVtkgene, but in proximity to neighboring HSVtk-transfected cells, arekilled after exposure to GCV (5, 6). The presence of the bystandereffect means that effective tumor treatment may then occur aftertransfer of genetic material to a minority of the tumor cells.

Recent reports describing the use of the HSVtk-GCV system for thetreatment of tumors have used retroviral vectors for gene transfer(6—8).Although retroviruses have the advantage of mediating stablegene transfer with a low potential for immunogenicity, this vectordelivery system has a numberof characteristicswhich make in vivoapplication problematic, including: (a) difficulties in producing hightiters of retrovirus for in vivo use; (b) the fact that only activelydividing cells are infected; and (c) the possibility of insertionalmutagenesis (9).

For these reasons, an alternativegene delivery system would bedesirable. In contrast to retroviruses, adenoviral vectors can be experimentallyproducedin very high titersand have no significant knownpotential for integration and insertional mutagenesis (10). A variety ofdividing and nondividing cells have been successfully infected in vivowith recombinant adenovirus allowing the transfer of either marker ortherapeutic genes into both animal and human tissues (1 1—14).Inaddition, we have recently shown that a recombinant adenovirusvector can transfer a marker gene into human mesothelioma andNSCLC cells in vitro, as well as in an animal model of human

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Advances in Brief

Use of Recombinant Adenovirus to Transfer the Herpes Simplex Virus ThymidineKinase (HSVtk) Gene to Thoracic Neoplasms: An Effective in Vitro Drug

Sensitization System'

w. RoySmythe,HarryC.Hwang,KunjiataM.Amin,StephenL. Eck,BeverlyL. Davidson,JamesM.Wilson,Larry it Kaiser, and Steven M. Albelda2

ThoracicSurgerySection,Departme,uofSurgery [W R. S.,K M. A., L R. K], and Pulmonary/CriticalCareSection,DepartmentofMedicine [H. C. H., S. M. A.J, UniversityofPennsylvania Thoracic Oncology Research Labora:oiy; Hematology Oncology Section, Department of Medicine [S. L. E.J, Department of Molecular and Cellular Engineering(I. M. W.J, and Institute for Human Gene Therapy [S. L E., J. M. W, L. R. K, £M. A.J, University of Pennsylvania Medical Center, Philadelphin, Pennsylvania 19104; andRheumatology Section, Department of Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109 [B. L D.J

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ADENOVIRUSTRANSFEROF HSVt1c

B

FIGURE 1. Ad.RSVtk-infected and uninfected REN humanmesothelioma cells stained in vitro with a polyclonal antiHSVtk antibody.(A) Ad.RSVtk-infectedREN cells exhibitingpositive cellular staining with the anti-H5Vtk antibody. Approximately 40% of cells exhibit clear nuclear or cytoplasmic staining (large arrows) with a number of other cells exhibiting faintstaining (small arrows) which may represent lower levels ofHSVtk expression. (B) Uninfected REN cells exhibiting nopositive anti-HSVtkantibodystaining.

I4

•1@

mesotheliomain immunodeficientmice.4The purposeof this reportisto describe experiments demonstrating that transfer of the HSVtk geneto human NSCLC and malignant mesothelioma cells can be accompushed using a recombinant adenovirus. In addition, cellular killingwith bystander effect occurs in vitro following GCV administration.These results suggest that an adenovirus-HSVtk system may be aneffective alternative for gene therapy of malignant mesothelioma andNSCLC, as well as other humantumors.

Materials and Methods

Cell Lines. The 1-45 malignant mesothelioma cell line was the generousgift of Dr. Joseph Testa (Fox Chase Institute, Philadelphia,PA). Humanmesothelioma cell lines REN and HeMe-B were isolated in our laboratoryfrom patient tumor specimens with development and characterization described elsewhere.4 Cells were grown in RPM! containing 10% fetal bovineserum, nonessential amino acids, and penicillin/streptomycin (RPM! 10). Allthree mesothelioma cell lines were furthercharacterizedby conventionalimmunohistochemistry and exhibited strong expression of the Wilms tumorgene (15).@The human non-small cell carcinoma cell lines A549 (carcinoma)and Calu-6 (anaplastic adenocarcinoma) were obtained from the AmericanType Culture Collection (Rockville, MD).

RecombinantAdenovirus.The productionof recombinantreplicationdeficient adenoviral vectors has been described in detail elsewhere (13).6Briefly Ad.RSVtk was constructedfrom an adenovirustype 5 mutant,thatlacks most of the viral sequence from the Ela, Elb, and a portion of the E3region. With the use of homologous recombination techniques, the herpessimplex type I thymidinekinasegene andthe Rous sarcomavirus3' promoterregion were insertedinto the viral genome. Viral stocks were propagatedin293 cells and titerswere quantifiedby 293 plaque assays and spectrophotometric density.

4 w. R. Smythe, L R. Kaiser, K. M. Amin, J. M. Pilewski, S. Eck, J. M. Wilson, and

S. M. Albelda. Successful adenovirus-mediatedgene transferin an in vivo model ofhuman malignant mesothelioma, Ann. Thoracic Surg., in press.

S K. M. Amin, W. R. 5mythe, F. J. Rauscher, M. Daise, H. Pass, U. Rodeck, L R.

Kaiser, and 5. M. Albelda. The Wilma tumor 1 (W'Fl) gene and protein are expressed athigh levels in malignant mesothelioma, submitted for publication.

6 D. S. Shewach, L K. Zerbe, T. L Hughes, B. J. Roessler, X. 0. Breakefield, and

B. L Davidson. Enhanced cytotoxicity of antiviral drugs mediated by adenoviral directedtransfer of the herpes simplex virus thymidine kinase gene in rat glioma cells, submittedfor publication.

Cell CUltUreInfectiOnwith ACLRSVtk.For initial in vitro experiments,all cell lines were platedand grown to confluence in T25 cell cultureflasks.Stock Ad.RSVtk in 5% glycerol was diluted to 5 X 1O@particles/ml in RPM!10 and 2.0 ml (corresponding to —100viral particles/tumor cell) of viralsuspension were placed on cell monolayers. Flasks were then incubated at

37°Cand5% CO2for 3 h, afterwhich time theviral suspensionwasremovedand replacedwith fresh media.

In Vitro Ganciclovir Sensitivity Assay. Initial studies examining the effect of Ad.RSVtk were performed in 24-well culture plates. Infected anduninfected cells were plated at a density of 20,000 cells/well. After incubationfor 2 days, the cell culture medium in each well was removed and replacedwith RPM! 10 with varyingconcentrations(0—2000@M)of ganciclovir(Syntex Laboratories,Inc., Palo Alto, CA). The cells were incubatedat 37°Cand5% CO2 for 5 days. Sensitivity to ganciclovir treatment was measured by direct

cell counts using a Coulter Counter (Coulter, Inc., Hialeah, FL). Cell number

was also assessed using a colorimetriccell proliferationassay that measuresviablecell dehydrogenaseactivity(16)(CellTiter 96 AqueousNon-radioactiveMTF Cell ProliferationAssay, Promega,Madison,WI). Infectedand/oraninfected cells were plated at a concentration of 6000 cells/well in 96-wellcultureplates (100 @.tlmedia/well). After a 24-h incubationperiod, mediumwas removedandreplacedwith RPM!10 containingvaryingconcentrationsofganciclovir (0—2000ELM).After 5 days of incubation at 37°C,20 pJ of assay

mix were added to each well and the plates were incubated for 3 additional hand absorbanceat 490 am was measured.

The bystander effect was determined by mixing infected and uninfectedcells at varying ratios. Cells were then plated into 96-well plates at 20,000cells/well to ensurecell-cell contact.Twenty-fourh later,thecells were treatedwith 20 @LMGCV for 5 days. Cell numberwas then determinedusing the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described above.

Immunohistochemistry. Confluent cell monolayers on coverslips wereinfected with either 100 or 500 viral particles/tumor cell of Ad.RSVtk. Fortyeight h later, the monolayers were fixed in 3% paraformaldehyde, permeabilized with 0.5 % NP4O,and blocked with 0.1 Mglycine. The monolayers werethen treatedwith 5% goat serum,washed in phosphate-bufferedsaline, andreacted with a polyclonal rabbit anti-HSVtk antibody (courtesy of W. C.Summers, Yale University) at room temperature for 1 h. After a washing inphosphate-buffered saline, the bound primary antibody was detected with theVectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA) using3-amino-9-ethylcarbazole as chromogen. An anti-human FVII!-related antigen

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ADENOVIRUSTRANSFEROFHSVtk

Percent ofUninfectedControlGrowth

Percent ofUninfectedControlGrowth

.01 .1 1 10 100 1000 10000GanciclovirConcentratIon(@tM)

PercentofUnlnfectedControlGrowth

Percent ofUnlnfectedControlGrowth

Percent ofUninfectedControlGrowth

Fig. 2. Comparison of ganciclovir dose response in three human mesotheioma and two human non-small cell lung carcinoma cell lines. Ad.RSVtk-infected cells are compared touninfected cells or cells infected with Ad.RSV1aCZ. Points on the ordinate correspond to the percentage of cells present at each dose compared to cells treated with no ganciclovir(untreatedcells).1C51,,doserequiredto eliminate50%of cellspresentcomparedto untreatedcells.(A) RENmesotheliomacell line.ICreforAd.R5Vtk-infectedcellsis -@2.O@LM,with uninfected cells having an ICre of -4509 pi@.AdR5V1acZ-infectedcells do not exhibit increased sensitivity. (B) 1-45 mesotheioma cell line. IC@ for Ad.RSVtk-infected cellsis —0.08gLM,with Uninfected cells having an IC@ of —500pi.i. AdRSV1acZ-infected cells do not exhibit increased sensitivity. (C) HeMe-B mesothelioma cell line. IC50 forAd.RSVtk-infected cells is —2.0pi@i,with uninfected cells having an ICre of >2000 @M.(D) Calu-6 non-small cell lung carcinoma cell line. IC50for Ad.RSVIk-infected cells is —1.0ELM, with uninfected cells having an ICR, of —1000 psi. (E) A549 non-small cell lung carcinoma cell line. Uninfected cells exhibit an ICre of >2000 @i,and Ad.R5Vtk-infected cells

at ratios of 500, 1000, and 2000 viral particles/tumor cell exhibit an ICse of —100jIM.

rabbit polyclonal antibody (Dako Corp., Carpenteria, CA) was used as a made of the efficiency of the HSVtk gene transfer by extrapolation

negative control. from data using the lacZ marker gene (5). In this study, however, weResults determined the efficiency of HSVtk gene transfer directly by staining

coverslips containing Ad.RSVtk-infected mesothelioma cells with aAd.RSV.tk Transfers the Thymidine Kinase Gene to Human rabbit polyclonal antibody specifically recognizing the HSVtk protein.

Thoracic Tumor Cells. We have previously shown the Ad.RSVIacZ At an infection ratio of 100 viral particles/tumor cell, approximatelyvector infects 90—100% of human thoracic tumor cells in vitro at 40% of both REN (Fig. IA) and 1-45 (data not shown) cells expressed50—100viral particles/tumor cell.4 Previously, estimates have been clearly detectable HSVtk enzyme in the nucleus and/or cytoplasm by

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ADENOVIRUSTRANSFEROF HSVtk

immunohistochemical staining. Faint staining was seen in many othercells and may representgene transferat lower levels. Increasingthevirus concentration to 500 particles/cell did not appear to increase thepercentage of cells strongly expressing HSVtk protein. Uninfectedcells from both lines did not stain with HSVtk antibody (Fig. 1B).Neither infected nor uninfected mesothelioma cells stained positivelywith the FVIIIraantibody(data not shown).

Recombinant Adenovirus-mediated Transfer of the HSVtkGene to Mesothelioma and Non-Small Cell Carcinoma Cell LinesSensitizes Them to Ganciclovir Treatment in Vitro. To test theability of Ad.RSVtk to confer sensitivity to GCV, all cell lines wereinfected at a concentration of 100 viral particles/tumor cell with both

Ad.RSVtk and Ad.RSV1acZvectors and exposed to varying doses ofGCV continuously for 4 days. The IC50of GCV for the mesotheliomacell lines 1-45, REN, and HeMe-B was determinedby either directcell counting (data not shown) or by using a chromogemc assay thatmeasures the number of viable cells in each group at the end of thetreatment period (Fig. 2, A—C).Both counting methods were in closeagreement. Cells of all three lines infected with Ad.RSVtk wererenderedsensitive to doses of GCV that were 3—4logs lower thanuninfected cells, with an IC50 reached at concentrations between@—0.08and 2.0 SAM.Fig. 2, D and E show the IC50data for the lungcancer lines Calu-6 and A549 infected with Ad.RSVtk followingexposure to GCV. The Calu-6 cells exhibited a sensitivity profilesimilar to those of the mesothelioma lines with an IC50 at a GCV doseof -—2.0,.LM;however, the A549 NSCLC line had a much higherIC50of approximately 100 p.MGCV. This comparativelyhigh IC50wasalso noted in the A549 cells at higher concentrations of viral particles/tumorcell of 500, 1000, and 2000 (Fig. 2E) andthus does not appearto be due to inefficient infection.

Inadditionto cellulardeath,HSVtk-infectedresidualviable cells ofall lines treated with GCV were noted to exhibit increased size,vacuolization, and granularcytoplasm. These morphologicalchangeswere notnotedin uninfectedorAd.RSVIacZ-infectedcells at identicalGCV doses. Of note is the fact thatinfectionwith an adenoviralvectorcontaininga controlmarkergene (AdJ?.SV1acZ)did not sensitize cellsto GCV (Fig. 2, A and B). GCV sensitivity of 1-45 cells infected withAd.RSV1acZ appeared to be similar to uninfected control cells, whileREN cells infected with this marker virus actually appeared lesssensitive.

A Bystander Effect Occurs in Ad.RSV.tk-infected Mesothelioma Cells in Vitro.To determineif Ad.RSVtk-infectedmesothelioma cells would exhibit a bystandereffect (where uninfected cellsadjacentto those expressing the HSVt/Cgene were killed after exposure to CCV), varying ratios of Ad.RSVtk-infected(100 viral pattides/cell) to uninfected cells were prepared and treated with 4 days ofcontinuousGCV in vitro.A dose of 20 ,.@MGCV was chosen becausethis approximates plasma concentrations of patients receiving GCVtherapy. Fig. 3, A and B display the results from experiments performed with both the 1-45 and REN lines. There was no diminutionin the efficacy of GCV treatmentuntil the infected:uninfectedcellratio fell below 1:10 (i.e., a maximum of 10% of cells Ad.RSVtkinfected). A partial but significant effect was seen at an infected:uninfected cell ratio of up to 1:100 (Fig. 3).

Discussion

In this report,we demonstratetransferof a therapeuticgene construct and successful in vitro drug sensitization of human malignantthoracic tumor cell lines using a recombinantreplication-deficientadenovirusvector. In addition,we characterizetransferof the HSVtkgene by use of immunohistochemicaltechniques and illustrate thepresence of a bystandereffect. These data suggest that recombinant

PercentofUnlnfectedControlGrowth

PercentofUnlnfectedControlGrowth

1.@ 3:1 2:1 1:1 12 13 15 1:101:100 0:1RatiO Ad.RSV.tk Infected : Unlntected REN Cells

Fig. 3. Mixing experimentsdemonstratean in vitro bystandereffect in humanmesotheiomacells.X-axis,ratiosof infected:uninfectedcells;Y-axis,growthof thesecellscompared to UnInfCCtCdcontrol cells after exposure to 20 @siganciclovir for 5 days. (A)1-45 cell line mixing experiment illustrating comparable cellular killing up to a ratio of1:10 (maximum of 10% of cells Ad.RSVtk infected). (B) REN cell line mixing experiment illustrating comparable cellular killing up to a ratio of 1:5 to 1:10 (maximum of10-20% ofcells Ad.RSVIk infected). In both cell lines, a partial but significant effect wasnoted at ratios of up to 1:100. Columns, sample means ±SEM (bars).

1.@3:1 2:1 1:1 1:2 1:3 15 1:101:201:501:1000:1RatioAd.RSV.tkInfected: Unlnfected1-45 Cells

adenovirustransferof a drugsusceptibilitygene offers an alternativeto recombinant retroviruses.

Applicationof the HSVtkdrugsusceptibilitysystem to malignancywas pioneeredby Moolten et a!. (7, 8) andothers(5, 6). A numberofreportshave recently been published illustratingefficacy in both invitro and in vivo tumormodels. To date, however, all of these studieshave used recombinant retroviral vector gene transfer technology(6—8,17, 18). Although the ability to infect rapidly dividing cells maybe a useful feature of retroviralvectors, this potential advantage islimited by the fact thatdirect inoculationof tumortissue in situ mayrequire that retroviral “producer―cells be used. Ram et a!. (17)showed that experimentalbrain tumors could be eliminated by administration of GCV to rats following injection of 3T3 cells activelyproducingrecombinantretroviruscarryingtheHSVt/Cgene. However,no effect was noted with intratumoralinjection of infectious viralsupernatant alone. Because recombinant adenovirus has the ability toprimarily infect mesothelioma cells in vivo after intratumoral injectionandafterinstillationintotheperitonealcavity of mice with establishedtumor,4 we have evaluated recombinant adenovirus as an alternativevector for transfer of the HSVtk gene.

The in vitro sensitivity of three Ad.RSVtk-transfectedmesothelioma cell lines to GCV reportedin this study is similarto reportsusingthe retrovirus.The IC50of CCV was less than2.0 @.LM.These doses arecertainlyacceptablewith regardto considering eventual in vivo efficacy, as nontoxic peak serum levels for patients receiving GCVtherapy for viral infections reach 15—18 @M(19). Although oneNSCLC lung cancer line was highly sensitive to this Ad.RSVtk-GCV

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ADENOVIRUSTRANSFEROF HSVtk

system, the relative insensitivity of the A549 NSCLC line to thistreatmentsystem suggests thatsome degree of variabilityof responsecan be anticipated when treating in situ tumors. The explanation forthis HSVtk insensitivity is not evident but is probably not due to aninabilityof the adenovirusto infect the cells since this line expresseslarge amounts of the (3-galactosidaseafter infection with Ad.RSVlacZ.4 Resistance to HSVtk has also been noted in some neoplasticcells in an in vitro retrovirus-transduced system (8).

Perhaps the most important characteristic of the HSVtk-GCV drugsusceptibility paradigm is the ability of HSVtk-transfected cells to killadjacent untransfected tumor cells after exposure to GCV, the socalled bystander effect (6). This becomes an extremely importantconsideration in the treatmentof preexisting solid tumors by thissystem since adenoviral transfer of new genetic material into 100% ofcells in such lesions is not likely. Other groups, using rat fibrosarcoma, murine sarcoma, and rat glioma tumors arising from cellsinfected ex vivo with retroviralHSVtk, have shown marked tumorgrowth inhibition, and even elimination, after systemic GCV therapy,even when as few as 10% of cells were originally transduced (5, 6,17). Explanations offered for this phenomenon include continuedretroviralinfection in situ, transferof an integratedretroviralHSVtkgene during mitosis, transfer of the toxic purine analogue GCVtriphosphate via gap junctions or apoptotic vesicles, or immune mediated effects (5, 6, 17, 18). The data from this study argue againstretroviral-specific or immune mechanisms, since a strong bystandereffect was demonstrated in this adenovirus HSVtk-transduced in vitrotumor system.

In summary, this is the first report, to our knowledge, that theHSVtk gene can be efficiently transferred to human tumor cells usinga recombinant adenovirus vector with impressive cytotoxicity following GCV exposure. Transfer is associated with a strong bystandereffect, suggesting that recombinant adenovirus transfer of this genecould potentially be effective for the in situ treatment of solid neoplasms.

References

1. Sugarbaker,D. J., Heher,E C., Lee, 1. H., Couper,0., Mentzer,S., Corson,J. M.,ci al. Extrapleural pneumonectomy, chemotherapy and radiotherapy in the treatmentof diffuse malignantmesotheioma. J. Thorac.Cardiovasc.Surg.,102: 10—15,1991.

2. Leung, W. T., Shiu, W. C. T., Pang, J. C. K., Lau, J., Tao, M., Leung, S. F., and Teo,P. Combined chemotherapy and radiotherapy versus best supportive care in thetreatment of inoperable non-small cell lung cancer. Oncology, 49: 321—326,1992.

3. St. Clair, M. H., Lambe, C. U., and Furman, P. A. Inhibition by ganciclovir of cellgrowth and DNA synthesls of cells biochemically transformed with herpesvirusgenetic information. Antimicrob. Agents Chemother., 31: 844—849, 1987.

4. Matthews, T., and Boehme, R. Antiviral activity and mechanism of action of ganciclovir. Rev. Infect. Dis., 10: S490—S494, 1988.

5. Freeman,S. M., Abboud, C. N., Whartenby, K. A., Packman,C. H., Koeplin, D. S.,Moolten, F. L, and Abraham, G. N. The “bystandereffect―:tumor regression whena fraction of the tumor mass is genetically modified. Cancer Res., 53: 5274—5283,1993.

6. Qilver, K. W., Ram, Z., Wallbridge, Ishii, H., Oldfield, E. H., and Blaese, R. M. Invivo gene transfer with retroviral vector-producer cells for treatment of experimentalbrain tumors. Science (Washington DC), 256: 1550—1552,1992.

7. Moolten, F. L., and Wells, J. M. Curability oftumors bearing herpes thymidine kinasegenes transferred by retroviral vectors. J. NaIl. Cancer Inst., 82: 297—300,1990.

8. Moolten, F. S., Wells, J. M., and Mroz, P. J. Multiple transduction as a means ofpreserving ganciclovir chemosensitivity in sarcoma cells carrying retrovirally transduced herpes thymidine kinase genes. Cancer Lett., 64: 257—263,1992.

9. Mulligan, R. C. The basic science of gene therapy. Science (Washington DC), 260:926—932,1993.

10. Kozarsky, K. F., and Wilson, J. M. Gene therapy: adenovirus vectors. Cuff. Opin.Genet. Dev., 3: 499—503,1993.

11. Roessler, B. J., Allen, E. D., Wilson, J. M., Hartman, J. W., and Davidson, B. LAdenoviral-mediated gene transfer to rabbit synovium in vivo. J. Clin. Invest., 92:1085—1092,1993.

12. Ezurum, S. C., Lemarchand, P., Rosenfeld, M. A., Yoo, J-H., and Crystal, R. 0.Protection of human endothelial cells from oxidant injury by adenovirus-mediatedtransfer of the human catalase cDNA. Nucleic Acids Res., 21: 1607—1612,1993.

13. Englehardt, J. F., Yang, Y., Stratford-Perricaudet, L D., Allen, E. D., Kozarsky, K.,Pericaudet, M., Yankaskas, J. R., and Wilson, J. M. Direct gene transfer of humanCvrR into humanbronchialepitheliaof xenograftswith El-deletedadenoviruses.Nature Genet., 4: 27—34,1993.

14. Davidson, B. L, Allen, E. D., Kozarsky, K. F., Wilson, J. M., and Roessler, B. J. Amodel system for in vivo transfer into the central nervous system using an adenoviralvector. Nat. Genet., 3: 219—233,1993.

15. Park, S., Schalling, M., Bernard, A., Maheswaran, S., Shipley, 0. C., Roberts, D.,et a!. The Wilma tumor gene 1471 is expressed in murine mesoderm derived tissuesand mutated in a human mesothelioma. Nat. Genet., 4: 415—420,1993.

16. Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L, Czerwinski, M. J., Fine, LF., ci al. Feasibilityof drugscreeningwith panelsof humantumorcell lines using amicroculture tetrazolium assay. Cancer Rca., 48: 589—601,1988.

17. Ram, Z., Culver, K. W., Waibridge, S., Blaese, R. M., and Oldfield, E. H. In situretroviral-mediated gene transfer for the treatment of brain tumors in rats. CancerRes., 53: 83—88,1993.

18. Caruso, M., Panis, Y., Gagandeep, S., Houssin, D., Salzmann, J-L, and Klatzmann,D. Regression of established macroscopic liver metastases after in situ transduction ofa suicide gene. Proc. Natl. Acad. Sci. USA, 90: 7024—7028, 1993.

19. Paul, S., and Dummer, S. Topics in clinical pharmacology: ganciclovir. Am. J. Med.Sci., 4: 272—277,1992.

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1994;54:2055-2059. Cancer Res   W. Roy Smythe, Harry C. Hwang, Kunjlata M. Amin, et al.  

Drug Sensitization Systemin VitroAn Effective ) Gene to Thoracic Neoplasms:HSVtkVirus Thymidine Kinase (

Use of Recombinant Adenovirus to Transfer the Herpes Simplex

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