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[CANCER RESEARCH56, 4614-4619, October 15, 19961

Advances in Brief

Osteocalcin Promoter-based Toxic Gene Therapy for the Treatment ofOsteosarcoma in Experimental Models

Song-Chu Ko,' Jun Cheon,' Chinghai Kao, Akinobu Gotoh, Toshiro Shirakawa, Robert A. Sikes, Gerard Karsenty,and Leland W. K. Chune

Molecular Urology and Therapeutics Program, Department of Urology, Box 422 University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 [S-C. K., J. C.,C. K., A. G., T. S., R. A. S., L W. K. CI, and Department ofMolecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 fG. K.]

Abstract

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed inhigh levels by osteoblasts. To determine whether the OC promoter mcdlates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OCpromoter that drives the expression of herpes simplex virus thymidineklnase (TK). We tested the expression ofTK by this virus in osteoblast celllines as well as in non-osteoblastic cell lines by assessing the enzymeactivity of TK in vitro. Whereas the OC promoter failed to drive theexpression of the 7K gene in several non-osteoblastic cell lines such as WH,a human bladder transitional carcinoma, and NIH 3T3, an embryonicmouse fibroblast cell line, the OC promoter mediated high levels ofexpression in osteoblast cell lines including murine ROS and humanMG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibilionofcell proliferation,resultedin theinductionofosteoblast-specificcelldeath in vitro. Intratumoral Injection of Ad-OC-TK into murine ROSosteosarcoma abolished tumor growth in a host treated with subsequent1.p. ACV Injection in vivo. The Ad-OC-TK virus plus ACV treatmentappears to be highly selective in blocking the growth of both murine andhuman osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.

Introduction

Osteosarcoma, a bone cancer occurring primarily in teenagers andyoung adults, affects approximately 2i00 individuals yearly in theUnited States (1). This malignancy accounts for as many as 5% of allchildhood malignancies and 60% of all malignant childhood bonetumors (2). Despite radical surgical resection of the primary tumor andaggressive adjuvant chemotherapy, the overall 2-year metastasis-freesurvival rate approaches only 66%. More than 30% of patients with

this disease develop lung metastasis within the first year (3, 4). Thesurvival rate among those affected with osteosarcoma has not changed

significantly over the past 10 years, despite changes in adjuvantchemotherapy (5). Because of the poor response rate of previouslytreated patients with relapsed osteosarcoma to second-line chemotherapy, it is important to develop new therapeutic approaches that can beapplied either separately or in conjunction with current treatmentmodalities. The goals of this study are: (a) to design a new genetherapy treatment modality by demonstrating effective delivery andexpression of a therapeutic toxic TK3 gene in a tissue-specific manner(e.g., osteoblast-like osteosarcoma cells) using an OC promoter; and

Received 7/29/96; accepted 8/28/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I These authors contributed equally to this study.

2 To whom requests for reprints should be addressed, at Department of Urology,

Molecular Urology and Therapeutics Program, The University of Virginia Health ScienceCenter,Box422,Charlottesville,VA22908.

3 The abbreviations used are: TK, thymidine kinase; OC, osteocalcin; Ad, adenovirus;

ACV, acyclovir; FBS, fetal bovine serum; RSV, Rous sarcoma virus; (3-Gal, (3-galactosidase; CMV, cytomegalovirus; GCV, ganciclovir; X-Gal, 5-bromo-4-chloro-3-indolVl-B-D-Ralactoovranoside: MOL, multiplicity of infection(s); ROS, rat osteoblastic

(b) to determine if the Ad-OC-TK (a recombinant Ad vector containing the TK gene driven by the OC promoter) plus acyclovir treatmentwill exhibit a cytotoxic effect on the growth of osteosarcoma cells invitro and tumors in vivo.

The concept of delivery and expression of therapeutic toxic genesto tumor cells through the use of tissue-specific promoters has beenwell recognized. This approach could decrease the toxic effect oftherapeutic genes on neighboring normal cells when virus-mediatedgene delivery results in the infection of the normal cells. Examplesinclude the uses of the albumin or a-fetoprotein promoter to targethepatoma cells (6), the bone morphogenic protein promoter for brainto target glioma cells (7), the tyrosinase promoter to kill melanomacells (8), and the carcinoembryonic antigen promoter for gastriccarcinoma cells (9). To date, the best studied therapeutic gene is

herpes simplex virus TK gene. Herpes simplex virus-TK converts thepro-drug ACV to a phosphorylated form that is cytotoxic to dividingcells (10). Critical to successful results is the â€œbystanderâ€•effect,which confers cytotoxicity on neighboring nontransduced cells; effective tumor cell kill can be achieved without the delivery to andexpression of suicide genes in every tumor cell in vivo. This approachhas been demonstrated recently to be efficacious in causing regressionof many solid tumors, including metastatic colon carcinoma in the ratliver (11), gastric carcinoma (9), and malignant mesothelioma (12).

OC, a noncollagenous Gla protein produced specifically in osteoblasts, is synthesized, secreted, and deposited at the time of bonemineralization (13). A recent study showed that immunohistochemicalstaining of OC was positive in primary osteoblastic osteosarcoma andchondroblastic osteosarcoma specimens as well as in five of sevenfibroblastic osteosarcomas (14). In an effort to develop a new treatment modality for osteosarcoma, we used an osteoblast-like osteosarcoma-specific OC promoter to drive toxic TK gene expression in the

form of a recombinant Ad, Ad-OC-TK. Our results demonstrate thatAd-OC-TK, when delivered concurrently with ACV, is highly effective in inhibiting murine and human osteoblast tumor cell proliferationin vitro and murine osteosarcoma growth in vivo.

Materials and Methods

Cells and Cell Culture. ROS 17/2.8, a rat osteoblastic osteosarcoma cellline, was generously provided by Dr. Cindy Farrach-Carson (University ofTexas Dental Branch, Houston, TX). MG-63, a human osteoblast-derivedosteosarcoma cell line; 293, a transformed human embryonic kidney cell line;and NIH 3D, an embryonic mouse fibroblast cell line, were purchased fromAmerican Type Culture Collection (Rockville, MD). WH, a human bladdertransitional cell carcinoma, was established by our laboratory (15). The ROS17/2.8 and MG-63 cell lines are considered the osteoblast lineage because of

their morphological, biochemical, and molecular characteristics (16, 17); theywere incubated in DMEM (Life Technologies, Inc., Grand Island, NY) and20% F12K (Irving Scientific, Santa Ana, CA) supplemented with 100 units/mi

penicillin, 100 @Lg/mlstreptomycin, and 10% FBS (Sigma Chemical Co., St.Louis, MO). The WH and NIH 3T3 cell lines were maintained in T medium(18) containing 5% FBS. The 293 cells were maintained in MEM (Lifeosteosarcoma.

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Technologies, Inc.) with 10% FBS and 1% tryptose phosphate broth (LifeTechnologies, Inc.). The cells were fed three times a week with fresh growth

medium unless otherwise indicated.Construction and Large-scale Production of a Recombinant Ad Vector

Containing the OC Promoter-TK. Construction of the recombinant AdOC-TK virus was accomplished as shown in Fig. 1. All plasmids wereconstructed according to standard protocols. Briefly, [email protected] (a gift from Dr.Frank Graham, McMaster University, Hamilton, Ontario, Canada) was digested with Hindffl (New England Biolabs, Beverly, MA) and treated withalkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN)according to the supplier's protocols. The Hindlil-digested 3.1-kb fragmentcontaining the murine OC promoter (1.3 kb)-driven TK (1.8 kb) construct wasligated into [email protected] vector using T4 ligase (New England Biolabs). Anexpression clone, p@E1SPl-OC-TK, was obtained with TK gene expressiondriven in a 3' to 5' orientation of an adenoviral DNA. Five @gof CsCl2-purified p@ElSPl-OC-TK and pJM17 were cotransfected into 293 cells bythe N-[l-(2,3-dioleoyloxyl)propyll-N,N,N-trimethylammoniummethyl sulfate(Boehringer Mannheim Biochemicals)-mediated transfection method (19). Theculture medium of the 293 cells showing the completed cytopathic effect wascollected and centrifuged at 1000 X g for 10 mm. The pooled supernatantswere aliquoted and stored at â€”80Â°Cas primary viral stock. Viral stocks werepropagated in 293 cells, and selected clones of Ad-OC-TK virus were obtainedby plaque purification according to the method of Graham and Prevec (20).One of the viral clones was propagated in 293 cells; cells were harvested 36 to40 h after infection, pelleted, resuspended in PBS, and lysed. Cell debris wasremoved by subjecting the cells to centrifugation, and the virus in the celllysate was purified by CsCl2 gradient centrifugation. Concentrated virus was

dialyzed, aliquoted, and stored at â€”80Â°C.The viral titer was determined byplaque assay. The control viruses used in this study, Ad-RSV-(3-Gal andAd-CMV-/3-Gal, were constructed in a similar manner (18).

X-Gal Staining of Ad-RSV4J-Gal Virus-infected Cells. ROS, MG-63,WH, and NIH 313 cells, after infected with various doses of recombinantAd-RSV-@3-Gal, were grown in 60-mm dishes to about 80â€”90%confluency for2 days, then the medium was discarded and fixed with 0.05% glutaraldehyde.After discarding the fixative solution, cells were rinsed thoroughly three timesin PBS at room temperature. An X-Gal solution mixture [0.5 ml; 35 mM

K3Fe(CN)6, 35 mM K4Fe(CN)63H20, 1 mM MgSO4, and 1 mg/mI X-Gal(Sigma Chemical Co.)] was added to cover cells. Cells were then incubated 1 hto overnight at 37Â°C.Positive cells were stained blue, and the percentage ofblue cells was calculated.

Determination ofTK Activity in Target Cells Infected with Ad-OC-TK.Crude cell extracts containing TK were prepared from Ad-OC-TK-infectedROS, MG-63, WH, and NIH 3T3 cells. Briefly, cells were cultured in 100-mmdishes to 60 to 70% confluency, at which point they were exposed to 20 MO!of Ad-OC-TK overnight. The culture medium was then replaced with fresh

medium after overnight infection, and the replaced culture medium was removed after an additional 48 h incubation. The infected cells were washed withPBS, trypsinized, and then counted; the expressed TK enzymatic activity wasrecovered from cells by the repeated freeze and thaw method. The resultant cellsuspension was centrifuged at 300 X g for 10 rain, and the supernatant fraction

was frozen at â€”80Â°Cfor TK activity assay. TK activity was assayed byphosphorylation of [3H]GCV, as described previously (20), with modification.

Briefly, the supernatant fraction harvested from approximately 1 X 106cellscontaining TK was mixed with an equal volume of TK assay buffer containing

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into 293 cells, and then recombinant Ad-OC-TK was generated through homologous recombination.

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OSTEOCALCIN PROMOTER-BASEDTOXIC GENE THERAPY

0.2 @&Ciof[3H]GCV (10 @xM;Moravek Biochemicals, Brea, CA), 3 mMMgCl2,3 mM ATP, 10 @g/@.dBSA, and 50 mM sodium phosphate buffer (pH 6.5). The

reaction mixture was incubated in a 36Â°Cwater bath for 90 mm, and thereaction mixture was transferred onto DE-8l discs (Whatman, Hillsboro, OR),air-dried, and washed thoroughly with 50% ethanol. Phosphorylated [3H]GCVbound to the filter discs was determined with a scintillation counter (BeckmanInstruments, Inc., Schaumburg, IL). GCV, a substrate for TK, differs fromACV (BurroughsWellcome, ResearchTriangle,NC) structurallyonly by theaddition of a hydroxymethyl group. Because of this one side chain difference,GCv is administered only through an iv. mute, whereas ACV can be administered p.o., i.v., and topically to humans. ACV and GCV are both commonlyused clinically against herpes virus infection. In this communication, becauseof the availability and rate of absorbency, [3H]GCV is used to assay TKactivity assay, whereas ACV is used for both the in vitro and in vivo studies.

Acyclovir in Vitro Cytotoxicity Assay. To determine ACV dose-responsecurves, ROS, MG-63, WH, and NIH 3T3 cells were plated in 24-well Falcontissue culture plates (Falcon Products, Franklin Lakes, NJ) and exposed tovarious concentrations of ACV ranging from 10 to 1000 @xg/mlmedia. Cell

numbers were measured daily by the crystal violet assay using an automated Emax spectrophotometric plate reader (Molecular Devices Corp., Sunnyvale,

CA).Ad-OC-TK plus ACV-induced in Vitro Cytotoxicity Assay. ROS, MG

63, WH, and NIH 3T3 cells were seeded onto 24-well plates at a density of5.0 X lO@cells/well. After 24 h, the cells were infected with PBS or AdOC-TK at a concentration of 20 MOI for 8 h. Following infection, the mediumcontaining Ad-OC-TK was replaced with fresh medium containing 0 or 10lLg/ml ACV with daily medium changes for an accumulated period of 7-9days. The cell number per well was assessed by crystal violet assay asdescribed above.

Ad-OC-TK and ACV-induced Inhibition of Tumor Growth in Vivo.Congenitally athymic nude (nu/nu) mice (Harlan Co., Houston, TX), 5 to 6weeks of age, were inoculated s.c. with ROS (3 X 106)cells, a rat osteosarcoma cell line. When the tumor became palpable (4â€”5mm in diameter), theanimals were randomly assigned to three experimental groups: group 1, ACVonly; group 2, Ad-OC-TK only; and group 3, Ad-OC-TK plus ACV. ForAd-OC-TK injection, a microliter syringe fitted with a 28-gauge needle wasused to deliver 75 pJ of Ad-OC-TK (1 X l0@ plaque-forming units). TheAd-OC-TK was injected intratumorally along both the long and short axes ofthe tumor: one injection parallel to the long axis, and one perpendicular to theaxis. The needle point was then rotated within the tumor to maximize the areaof Ad delivery. Ad-OC-TK was injected every other day for a total of threedoses. Tumor volume was calculated by the following formula: volume (arotational ellipsoid) M1 X M22X 0.5236 (M1,long axis; M2,short axis; Ref.18). Acyclovir treatment only or Ad-OC-TK plus ACV experimental groupswere treated with an i.p. injection of ACV at a dose of 40 mg/kg body weightdaily for 7 days. Ad-OC-TK and/or ACV treatment did not adversely affect thebody weight of experimental animals. Tumor volume was calculated everyother day for the first 8 days on day 20 and day 32.

Results

Adenoviral Transduction Efficiency to ROS and MG-63 Cellsand TK Expression in Vitro. To test the efficiency of Ad infectionin ROS, a rat osteogenic sarcoma cell line, and in MG-63, a humanosteosarcoma cell line, we used a recombinant adenoviral vectorcontaining the RSV-f3-Gal expression cassette (Ad-RSV-@3-Gal).Based upon X-Gal staining of @3-Galactivity, a dose-dependent increase of Ad infection was noted in ROS and MG-63 cells, with thepercentage of cells infected increasing gradually 60, 75, and 100% asthe adenoviral MOI increased 20, 40, and 60 MO! per target cell (datanot shown). A similar efficiency of adenoviral infection was observedin WH and NIH 3T3 cells. In comparison to uninfected control cells,mild cytotoxicity was noted through the application of Ad up to 60MO! (data not shown).

To assess whether Ad-OC-TK may drive the expression of the TKgene in a cell type-specific manner, ROS, MG-63, WH, and NIH 3T3cells were exposed to Ad-OC-TK (20 MOL/target cell), and their crudecellular lysates were obtained and assayed for TK activity. TK activity

was indirectly determined by measuring the amount of phosphorylated [3H]GCV mediated by TK enzyme present in cell lysate. Themean TK-mediated [3H]GCV phosphorylation (i.e., TK activity) per106 cells was significantly higher in osteosarcoma ROS and MG-63cells than in the WH and NIH 3T3 cell lines (Fig. 2). TK activity inROS cells following infection with 20 MO! of Ad-CMV-@3-Gal(18)serves as a negative control.

Cytotoxicity of ACV in Ad-OC-TK-transduced ROS andMG-63 Cells in Vitro. To determine whether Ad-mediated transduction with the OC-TK gene would render ROS or MG-63 cells sensitiveto cell killing by ACV, we first tested the toxicity of ACV (range,0â€”1000ILg/ml) in noninfected ROS, MG-63, WH, and NIH 3T3 cellsand observed that ACV at doses below 40 @tg/midid not appreciablyinhibit the growth of all cell lines tested (data not shown). The growthof ROS (Fig. 3A) and MG-63 (Fig. 3B) cells, infected with 20 MO! ofAd-OC-TK, was significantly inhibited by the addition of ACV (10

@g/ml);cells infected with Ad-OC-TK (20 MOl/target cell) or treatedwith ACV (10 @.tg/ml)alone did not exhibit altered growth or morphology during a 7â€”9day observation period. Consistent with the lowlevels of TK activity, the growth of WH (Fig. 3C) and NIH 3T3 (Fig.3D) cells after Ad-OC-TK infection was not affected by the addition

of pro-drug ACV in the cell culture medium. Similarly, Ad-OC-TK orACV alone did not affect proliferation in these cells.

Cytotoxicity of ACV in Ad-OC-TK-infected ROS Tumor

Growth in Vivo. ROS sarcoma xenografts were induced by s.c.injection of ROS cells (1 X 106 cells/site) in athymic mice. Aftertumor formation, animals were treated with either ACV alone, AdOC-TK alone, or Ad-OC-TK plus daily ACV i.p. injection. After

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were exposed to Ad-CO-TK (20 MOl/target cell), and their crude cellular lysates wereobtained and assayed for TK activity. The mean TK-mediated [3H)GCV phosphorylation(i.e.,TK activity)per l0@cellswassignificantlyhigherin osteosarcomaROSandMG-63cells than in the WH and NIH 3T3 cell lines. TK activity in ROS cells following infectionwith Ad-CMV-(3-GaIwas used as a negative control.

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highly infectious Ad as the gene delivery vehicle for OC-TK. In thisstudy, we have demonstrated that: (a) Ad-OC-TK-infected osteosarcoma cell lines of the osteoblast cell lineage (e.g., ROS and MG-63cells) expressed high levels of TK activity, and cell lines not of theosteoblast lineage, such as WH and NIH 3T3, expressed low TKactivity; and (b) consistent with measured TK activity, the addition of

ACV, a pro-drug substrate for TK, followed by Ad-OC-TK infectioninhibited the growth of osteosarcoma cell lines in vitro and osteosarcoma xenografts in vivo. The growth of WH and NIH 3T3 cells wasnot affected by Ad-OC-TK either with or without ACV administration. Therefore, Ad-OC-TK constitutes a tumor-specific toxic genetherapy that inhibits the growth of proliferating osteosarcoma cellsand spares significant tissue damage in the surrounding nonproliferating normal tissues and cells that are of nonosteoblastic lineage. TheOC promoter-mediated gene delivery system is superior to conventional gene therapy in which TK expression is driven by universalpromoters such as the CMV promoter (22) and the long terminalrepeat promoters from RSV or Moloney marine leukemia virus (23).These promoters could potentially lead to the expression of nonspecific TK activation in all cells, resulting in the eradication of prolifcrating normal and neoplastic cells.

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Fig. 3. Cytotoxicity of ACV in Ad-OC-TK transduced osteoblastic ROS and MG-63 cells in vitro. The growth of mw-inc osteogenic ROS (A) and human osteosarcoma MG-63 (B)cells, infected with 20 MOl of Ad-OC-TK, was significantly inhibited by the addition of ACV (10 p.g/ml). Consistent with low levels of TK activity, the growth of WH (C) and NIH3T3 (D) cells after Ad-OC-TK infection was not affected by the addition of ACV in the tissue culture medium. Acyclovir treatment or Ad-OC-TK infection alone did not affectproliferation in any cell lines tested.

completion of a 7-day course of ACV, the Ad-OC-TK plus ACVtreated group demonstrated no ROS tumor growth for up to 8 days anda significantly decreased growth rate for up to 20 days (Fig. 4). After1 month, the normal growth rate of ROS tumor resumed, and theexperiment was terminated. Ad-OC-TK infection or ACV treatmentalone did not affect the rate of tumor growth.

Discussion

Osteosarcomas, of all pediatric tumors, continue to challenge pediatric oncologists. Despite aggressive therapy, a significant numberof osteosarcoma patients relapse locally and systemically. For thesepatients, second-line chemotherapy is of limited efficacy (2). Todevelop new therapeutic modalities for osteosarcoma, we have cxplored the possibility of targeting this tumor with toxic gene therapymediated through the osteosarcoma-specific OC promoter. Since OCis the protein most commonly secreted by osteosarcoma cells of theosteoblastic lineage and is also a marker of osteoblastic differentiation(21), we have chosen to use the OC enhancer element to achievetissue-specific expression of the toxic TK gene in rat and humanosteogemc sarcoma cell lines. Because sarcoma cells are normallyvery resistant to conventional DNA transfection, we have used a



OSTEOCALCIN PROMOTER-BASEDTOXIC GENE THERAPY

such as prostate or breast tumors; (b) Ad-OC-TK may be used inconjunction with conventional chemotherapy and radiation therapy in

reducing tumor burden and pain associated with osseous metastases; and(c) long-lasting antitumor immunity might be elicited against the remaining osteoblastic cells from 1K-induced killing of tumor cells (29).

In this communication, we have explored the possibility of developing a new therapeutic agent that can selectively target and induce

the killing of osteosarcoma cells by using the tissue-specific OCpromoter. The new recombinant Ad-OC-TK virus was generated, andwe report, for the first time to our knowledge, the use of Ad-OC-TKas a novel therapeutic agent that can selectively target and induce thekilling of osteoblast lineage cells. Ad-OC-TK may become a potentialgene therapy agent for osteosarcoma patients as well as for patientswith melanoma, breast cancer, or prostate cancer who have symptomatic osseous metastases.

Acknowledgments

We thank Drs. Jonathan W. Simons (Johns Hopkins University), SusanMiesfeldt, and Thomas A. Gardner (University of Virginia) for their helpfuldiscussion and reading of the manuscript. We are very grateful to CarolChowdhry for excellent editorial help.

References

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7. Shimizu, K. Selective gene therapy of malignant glioma using brain-specific promoters: its efficacy and basic investigation. Nippon Rinsho, 52: 3053-3058, 1994.

8. Vile, R. G., Nelson, J. A., Castleden, S., Chong, H., and Hart, I. R. Systemic genetherapy of murine melanoma using tissue specific expression of the HSVtk geneinvolves an immune component. Cancer Res., 54: 6228â€”6234, 1994.

9. Tanaka, T., Kanai, F., Okabe, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori,Y., Lan, K-H., Ishitobi, M., and Ornate, M. Adenovirus-mediated prodrug genetherapy for carcinoembryonic antigen-producing human gastric carcinoma cells invitro. Cancer Res., 46: 1341â€”1345,1996.

10. Moolten, F. L. Tumor chemosensitivity conferred by inserted herpes thymidine kinasegenes: paradigm for a prospective cancer control strategy. Cancer Res., 46: 5276â€”5281,1986.

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12. Smythe, W. R., Hwang, B. S., Elshami, A. A., Amin, K. M., Eck, S., Davidson, B. L.,Wilson, J. M., Kaiser, L. R., and Albelda, S. M. Treatment of experimental humanmesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.Ann. Surg., 222: 78â€”86,1995.

13. Price, P. A. Vitamin-K dependent formation of bone GLA protein (osteocalcin) andits function. Vitam. Horns., 42: 65â€”108,1985.

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Fig. 4. Cytotoxicity of ACV in Ad-OC-TK-infected ROS tumor growth in vivo. Aftercompletion of a 7-day course of ACV, the Ad-OC-TK plus ACV treated group demonstrated no tumor growth for up to 8 days and a significantly decreased growth rate for upto 20 days. After I month, the normal growth rate of ROS tumor resumed. Ad-OC-TKinfection or ACV treatment alone did not affect the rate of tumor growth. Key: numbersin the brackets indicate the total number of animals used in each group.

Historically, the most challenging osteosarcoma patients are theones who develop unresectable pulmonary metastases after a curativeattempt with first-line chemotherapy. For these patients, the treatmentoptions include palliative radiation therapy or high-dose second-linechemotherapy. Patients developing recurrent disease usually have avery poor prognosis and die within 1 year of the development ofmetastatic disease (24). When clinicians encounter such patients, newAd-OC-TK may be used as a separate or adjuvant therapeutic agentwith second-line chemotherapy because these recombinant Ads carlying toxic genes might be easily aerosolized, delivered locally via

pulmonary artery catheterization, or injected directly into or sprayedonto lung metastases with a flexible thoracoscope. These treatmentoptions are under active investigation.

Another potential therapeutic application of Ad-OC-TK is to targetcancers that may metastasize to the skeleton. Prostate cancer (25),breast cancer (26), and rare ocular melanomas (26) metastasize prey

alently to the bone and elicit either an osteoblastic or an osteolyticreaction. A possible mechanism for malignant cell recruitment to boneis that osteogenic cells may synthesize and secrete products that areable to stimulate the growth, adhesion, and migration of these cancercells (27). Conversely, the proliferating and migrating tumor cellsmay secrete paracrine growth factors that stimulate osteoblast cellgrowth at sites of bone metastases (28). Since tumor cell growth isintimately affected by the surrounding stroma and reciprocal interactions exist between the growth of certain tumors (e.g., prostate andbreast) and bone stroma, the development of Ad-OC-TK as a newgene therapy modality for osteosarcoma patients could have additional therapeutic implications: (a) Ad-OC-TK could block not onlythe growth of osteosarcoma but also eradicate osteoblastic cells thatmay be required to maintain the survival of osseous metastatic tumors

Days

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Kao, C., and Chung, L W. K. Molecular therapy with recombinant p53 adenovirus inan androgen independent, metastatic human prostate cancer model. Hum. Gene Ther.,7: 1683â€”1691,1996.

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1996;56:4614-4619. Cancer Res Song-Chu Ko, Jun Cheon, Chinghai Kao, et al. Treatment of Osteosarcoma in Experimental ModelsOsteocalcin Promoter-based Toxic Gene Therapy for the

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