Induction of Sensitivity to Ganciclovir in Human Hepatocellular Carcinoma Cells by Adenovirus-Mediated

Gene Transfer of Herpes Simplex Virus Thymidine Kinase

CHENG QIAN, ROBERTO BILBAO, O S C A R BRUNA, AND JESUS PRIETO

We have analyzed the ability of a recombinant replica- tion-defective adenovirus to transfer the thymidine ki- nase gene of herpes simplex virus (HSV-tk) into hepato- cellular carcinoma (HCC) cells to confer sensitivity to ganciclovir. Three HCC cell lines (Hep3B, PLC/PRF/5, and HepG2) were efficiently infected in uitro by a recom- binant adenovirus carrying lacZ reporter gene (Ad- CMVlacZ). Expression of HSV-tk in HCC cells infected with a recombinant adenovirus carrying HSV-tk gene (AdCMVtk) induced sensitivity to ganciclovir in a dose- dependent manner. A bystander killing effect was ob- served when 90% of uninfected tumor cells were mixed with only 10% of AdCMVtk-infected cells. These data show that recombinant adenoviruses are efficient vec- tors for transduction of drug-sensitizing genes to HCC cells in uitro. We suggest that a gene therapy approach to hepatocellular carcinoma can be established using adenoviral transfer of HSV-tk to tumor cells and subse- quent administration of ganciclovir. (HEPATOLOGY 1995; 22:118-123.)

Hepatocellular carcinoma (HCC) is a common human malignancy that resists conventional chemotherapy and radi~therapy.'.~ Modifications of chemotherapy, such as intrahepatic artery infusion of cytotoxic drugs, improve tumor responses but do not substantially af- fect patient The best chance for cure is com- plete surgical ablation of the tumor by partial hepatec- tomy or by total hepatectomy combined with liver transplantation, but these procedures are appropriate only in a minority of Thus, HCC remains one of the most difficult tumors to treat.

Progress in molecular biology has recognized a num-

Abbreviations: HCC, hepatocellular carcinoma; HSV-tk, thymidine kinase of herpes simplex virus; GCV, ganciclovir; CMV, cytomegalovirus; moi, multi- plicity of infection.

From the Department of Internal Medicine, School of Medicine, University of Navarra, Pamplona, Spain.

Received January 5, 1995; accepted February 15, 1995. Supported by Fundacion Ramon Areces, Madrid, Spain, and Dr Cervera's

Gene Therapy Grant. Address reprint requests to: Jesus Prieto, MD, PhD, Department of Internal

Medicine, Clinica Universitaria, University of Navarra, 31080 Pamplona, Spain.

Copyright 0 1995 by the American Association for the Study of Liver Diseases.

0270-9139/95/2201-0018$3.00/0

ber of mechanisms that control cell growth and differ- entiation. Targeting these pathways using gene ther- apy is rapidly becoming the fifth procedure in cancer treatment, in addition to surgery, radiotherapy, chemo- therapy, and imm~notherapy.~ Various technologies of somatic gene therapy for cancer treatment have been investigated, including drug sensitization, genetic im- munomodulation, normal tissue protection, gene re- placement, and antisense s t r a t eg ie~ .~ .~ One of the most promising methods is drug sensitization, which in- cludes the transfer of suicide genes into tumor cells to render them sensitive to a type of chemotherapy that is relatively nontoxic to the rest of the body. Examples of suicide gene are thymidine kinase of herpes simplex virus (HSV-tk) and cytosine deaminase, which convert the nontoxic drugs ganciclovir and 5-fluorocytosine to the toxic products ganciclovir-tryphosphate and 5-flu- orouracil, re~pectively.~.' HSV-tk is the most often studied suicide gene.1°'16 It converts the protoxic nucle- oside analog ganciclovir (GCV) into a highly toxic phos- phorylated GCV that acts as a chain terminator of DNA synthesis and an inhibitor of DNA polymerase, selec- tively killing dividing cells. In animal experiments, complete regression of established tumors was shown even when only a relatively small proportion of tumoral cells was transduced with the HSV-tk gene. This phe- nomenon has been called bystander effect.17 Transfer of HSV-tk gene into the tumoral tissue followed by sys- temic administration of GCV is being used in clinical trials of some cancers.6

Recent results from Macri and Gordon'' have shown suppression of tumor growth and increased animal sur- vival in transgenic mice that expressed HSV-tk in HCC tumor cells and were treated with GCV. Huber et all' have used a retrovirus vehicle to transfer varicella- zoster virus thymidine kinase into HCC tumor cells to confer susceptibility to 6-methoxypurine arabino- nucleoside. Adenoviruses have been used to deliver suc- cessfully exogenous genes into a variety of cells and tissues.20 This vector has the advantage of producing high-titer virus capable of efficient infection and subse- quent foreign gene expression in target cells. These data prompted us to explore the adenovirus-mediated gene transfer of HSV-tk into HCC cells to induce sus- ceptibility to GCV.

118

HEPATOLOGY Vol. 22, No. 1, 1995 &IAN ET AL 119

1 mu=360 bp

1.25 mu 9.24 mu 100 mu

CMV HSV-tk Poly A

AdCMVtk

AdCMVlacZ

CMV LacZ Poly A

FIG. 1. Construction of AdCMVtk and AdCMVlacZ vectors. A recombinant replication-defective adenovirus lacking the viral E l region and part of the E3 region and containing HSV-tk under the control of CMV promoter was constructed. Ad5, human adenovirus type 5; mu, map unit; CMV, cytomegalovirus major immediate early promoter.

MATERIALS AND METHODS

Construction of Recombinant Adenouiruses. The recombi- nant replication-defective adenoviruses of HSV-tk (AdCMVtk) and lacZ reporter gene (AdCMVlacZ) (Fig. 1) were con- structed according to the methodology described pre- viously.21,22 The 2.8 kb Bgl IIIBam HI fragment of pMK con- taining the HSV-tk gene was inserted into a transient expression vector pMVlOO under the control of the cytomega- lovirus (CMV) major immediate early promoter and upstream of a polyadenylation signal. The orientation was checked by restriction mapping. The CMV immediate early promoter/ HSV-tk cassette was excised from the transient expression vector on a Hind I11 fragment and inserted into the adenovi- rus transfer vectors pMV60 to generate pMV6Okk. To pro- duce a recombinant adenovirus, pMV6O/tk and pJMl7 were cotransfected into the 293 cells by calcium phosphate precipi- tation. Recombinant adenovirus was isolated from a single plaque, expanded in the 293 cells, and purified by double cesium chloride ultracentrifugation. The purified virus was extensively dialyzed against 10 mmol/L of Tris per 1 mmoU L of MgC1, and stored in aliquots at -80°C. The virus titer was determined by spectrophotometry and plaque assay.

Cell Lines. Human HCC cell lines (Hep3B, PLC/PRF/5, and HepG2) and human embryonal kidney cell line 293 were obtained from European Collection of Animal Cell Culture (Salisbury, UK). Hep3B and PLC/PRF/5 were grown in Dul- becco’s modified Eagle medium supplemented with 10% heat- inactivated fetal bovine serum and penicillidstreptomycin, and HepG2 cells were grown in RPMI 1640 medium supple- mented with 10% heat-inactivated fetal bovine serum and penicillidstreptomycin. 293 Cells were grown in Glasgow’s modified minimal essential medium containing 10% fetal bo- vine serum and penicillidstreptomycin.

Gene Transfer and Cytopathicity of GCV. The exponen- tially growing cells were plated in 60-mm petri dishes a t 5 x lo5 cells in 2 mL of medium 24 hours before AdCMVtk was added. Immediately before transduction, culture medium was removed from the dishes, and suspensions of AdCMVtk or AdCMVlacZ at a multiplicity of infection (moi) of 10

plaque-forming units per cell were placed onto cell mono- layers, distributed over the cell monolayer, and adsorbed at room temperature for 30 minutes. The viral suspension was then removed and replaced with fresh medium.

The second day of transduction, cells were typsinized and plated onto 6-well plates at a density of 2 x lo5 cells/well. After incubation for 1 day, the culture medium was removed from each well and replaced with new medium containing different concentration of GCV (0 to 500 pmol/L). The cells were incubated at 37°C and 5% CO, for 5 days and the condi- tioned medium was changed every day. After culture for 5 days, the cells were trypsinized and the viable cells were counted directly by trypan blue exclusion. Values of cell via- bility are given as mean t SE of four separate experiments.

RESULTS Adenovirus-Mediated Gene Transfer Emciency in

HCC Tumor Cell Lines. To test the adenovirus-medi- ated gene transfer efficiency in HCC cells, three HCC cell lines (Hep3B, PLC/PRF/5, and HepG2) were in- fected with a control adenovirus AdCMVlacZ a t differ- ent moi (1,000, 100, 10, and 1). As shown using 5- bromo-4-chloro-3-indolyl-~-~-galatoside staining of lacZ gene product, we observed that AdCMVlacZ was able to infect all the previously mentioned cell lines with 90% to 100% efficiency at the moi of 10 or more. Figure 2 is an example of Hep3B cells infected with AdCMVlacZ at a moi of 10.

Tumor Cells Expressing HSV-tk Are Sensitive to GCV In Vitro. To assess if the expression of the HSV-tk gene in HCC cells would render them susceptible to treat- ment with GCV, we infected Hep3B cells, PLC/PRF/5 cells, and HepG2 cells with AdCMVtk or AdCMVlacZ (as a control) a t a moi of 10. Forty-eight hours later, the cells were cultured in different concentrations of GCV for 5 days. The growth rate of cells infected with either AdCMVlacZ (doubling time; 1.42 & 0.16, 1.07 ? 0.11 and 1.22 ? 0.07 days for HepSB, PLC/PRF/5,

FIG. 2. Transduction efficiency of adenovirus in Hep3B HCC cells in uitro. Hep3B cells (2 X lo5) were plated on 6-well plate and infected with AdCMVlacZ at a moi of 10. Forty-eight hours later, cells were stained with 5-bromo-4-chloro-3-indolyl-~-~-galatoside. Size bar = 50 pn.

120 QIAN ET AL HEPATOLOGY July 1995

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and HepG2, respectively) or AdCMVtk (doubling time; 1.42 t- 0.18, 1.08 ? 0.06, and 1.24 ? 0.10 days for Hep3B, PLC/PRF/5, and HepG2, respectively) was found to be similar to that of uninfected cells (doubling time; 1.40 ? 0.22, 1.08 ? 0.12, and 1.20 ? 0.12 days for Hep3B, PLC/PRF/5, and HepG2, respectively). As shown in Fig. 3, expression of HSV-tk in Hep3B cells increased their sensitivity to GCV, by decreasing the 50% inhibitory concentration of GCV from 3.07 x 10' ? 73.1 pmoVL in cells infected with the control vector AdCMVlacZ to 3.00 X ? 0.017 pmoVL in cells infected with AdCMVtk. The other two HCC cell lines, PLC/PRF/5 and HepG2, had a sensitivity profile simi- lar to Hep3B cells, being the 50% inhibitory concentra- tion of GCV 3.30 X lop1 ? 0.069 pmol/L and 1.70 X lop1 ? 0.052 pmol/L respectively in the cells infected with AdCMVtk, while it was 5.18 X lo2 ? 49.6 pmoV L, and 1.52 X lo3 t- 392 prnoVL, respectively, in cells infected with AdCMVlacZ. These results show that ex- pression of HSV-tk in HCC cells confers susceptibility to GCV, by declining the 50% inhibitory concentration three to four logs below cells infected with a control vector.

In Vitro Analysis of the Bystander Effect. To deter- mine if AdCMVtk-transduced HCC cells would exhibit a bystander killing effect (the ability of infected cells to kill neighboring uninfected cells), experiments were performed mixing AdCMVtk-infected and uninfected cells. The cells were replated at various proportions and cultured in medium containing GCV (50 pmol/L) for 5 days. Figure 4 shows the results from experiments performed with Hep3B, PLC/PRF/5, and HepG2 cells. A marked decline in cell survival occurred when unin- fected tumor cells were mixed with only 10% of AdCMVtk-infected cells. These results indicate that AdCMVtk-infected tumor cells were toxic to adjacent uninfected cells.

DISCUSSION In this study, we have successfully used the recombi-

nant adenovirus vehicle to transfer HSV-tk, a suicide gene, to HCC cell lines and to confer susceptibility to GCV. When AdCMVtk-infected cells were mixed with uninfected cells, a bystander killing effect in vitro was also observed. Our findings suggest that adenovirus- mediated gene transfer methodology could be used in the induction of sensitivity to GCV in HCC by transfer

Application of drug sensitization with the HSV-tW of HSV-tk.

i

FIG. 3. GCV dose response of AdCMVtk-infected HCC cells. HCC cells were infected with AdCMVtk or AdCMVlacZ and cultured in different doses of GCV (0.05, 0.5, 5, 50, and 500 pmoVL) for 5 days. The surviving cells were counted and percentages of cell survival were calculated by comparing cell counts with those of cells cultured without GCV. Data are shown as mean i SE of four separate experi- ments. (A) Hep3B cells. (B) PLC/PRF/5 cells. (C) HepG2 cells.

HEPATOLOGY Vol. 22, No. 1, 1995

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QIAN ET AL 121

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FIG. 4. Bystander killing effect of HCC cells expressing HSV-tk in uitro. HCC cells were infected with AdCMVtk at a moi of 10 and then mixed with uninfected cells in the proportions indicated. After exposure to 50 pmol/L of GCV for 5 days, the surviving cells were counted and percentages of cell survival were calculated by compar- ing cell counts with those of uninfected cells treated without GCV. Values are represented as mean 2 SE of four separate experiments.

GCV system in cancer was pioneered by MootlenZ3 and Mootlen and Wells24 and has been used in clinical trials of brain cancer, leptomeningeal carcinomatosis, ovary tumor, head and neck carcinoma, and mesothelioma. Previous studies using retrovirus gene transfer meth- odology have shown that HCC cells selectively express- ing varicella-zoster virus thymidine kinase become selectively sensitive to 6-methoxypurine arabinonu- cleoside based on the selective anabolism of 6-me- thoxypurine arabinonucleoside to adenine arabinonu- cleoside tr iph~sphate. '~ Recently, Macri and Gordon" have used a transgenic animal model to study the ther- apeutic efficacy of HSV-tk insertion in HCC cells fol- lowed by GCV treatment. These investigators found that subsequent treatment with GCV leads to suppres- sion of tumor growth and delayed animal mortality. Our results also show that HCC tumor cells transfected with a defective recombinant adenovirus containing the HSV-tk gene become sensitive to GCV. In addition, we have observed an important bystander killing effect in our i n uitro system. This bystander effect may be important in the therapy of solid tumors, because none of the available gene transfer techniques is able to transfer foreign genes into all the cells of these tumors. Thus, the toxic effect exerted by the cells expressing HSV-tk on the neighboring tumoral tissue may en- hance the efficacy of this therapy. The mechanisms of the bystander killing effect, which has been reported to occur both in vitro and in viuo in gene therapy strate- gies based on suicide genes, are not well known. It has been suggested that this effect might be caused by the transfer of dying material as apoptotic vesicles phago- cytized by adjacent unmodified tumor cells,17 or to the transfer of GCV triphosphate through gap junctions to cells in the ~icinity. '~ I n viuo this effect might also be explained by the stimulation of the immune response against the tumor.26 However, i n viuo studies are needed to ascertain whether the bystander effect af- fects only tumor cells or neighboring normal hepato- cytes as well.

In most studies, the application of the HSV-tWGCV system to malignancies has been performed using re- combinant retrovirus.7~10~11~13.19.24.25 Although this vehi- cle possesses the ability to infect rapidly dividing cells, its application in vivo seems problematic because of difficulties in producing high titers of retrovirus and because of the possibility of inducing insertion muta- genesis. On the other hand, recent studies from Rich- ards and HuberZ7 using the retrovirus vehicle found difficulties expressing varicella-zoster virus thymidine kinase in transgenic mice because of extensive methyl- ation of entire transgene, including the 5' long-termi- nal repeat, the neomycin and tk genes, and the albumin promoter. Moreover, Kimura et alZ8 have found that hepatocarcinogenesis could enhance retroviral delivery of foreign genes into the liver, but the expression of the foreign gene was very low under the control of different promoters (long-terminal repeat and MDRlb). For these reasons, an alternative gene delivery system

122 QIAN ET AL HEPATOLOGY July 1995

seems desirable. As shown in this article and in other ~tudies , ’~-~l recombinant adenoviruses can deliver for- eign genes to parenchymal liver cells in vitro and in vivo with high efficiency, and some studies have shown that they can successfully transduce the HSV-tk gene into tumor cells from gliomas, mesotheliomas, and non-small cell carcinoma of the lung making them sus- ceptible to GCV.14-16 Using this system, regression of experimental gliomas was also reported in animals.16

In this study, we have administered GCV 2 days after transduction of HCC cells with HSV-tk, because i n vivo studies have shown that foreign genes in recombinant adenoviruses have a maximum expression at 1 to 6 days after infection; gene expression starts declining after 1 week and becomes very low 3 to 4 weeks after i n f e ~ t i o n . ’ ~ ~ ~ ~ This transient expression of adenoviral genes should be taken into consideration for therapeu- tic purposes.

Unlike retroviral vectors, which only infect dividing cells, adenoviruses can infect both dividing and resting cells when they are introduced i n vivo. In principle, only dividing cells are damaged by phosphorylated GCV. Hepatocytes are mature cells and should not be killed by this form of therapy. However, phosphory- lated GCV may be toxic for extrahepatic dividing cells and therefore the use of ubiquitous CMV promoter to control HSV-tk expression might induce side effects such as bone marrow depression. Toxicology studies should be performed to determine if adenoviral-trans- duced HSV-tk can be of value in the treatment of HCC. Different strategies could be used to avoid extrahepatic toxicity. One strategy would be to employ a tumor-spe- cific promoter, such as alpha-fetoprotein promoter. An- other possible method uses procedures for selective de- livery of the virus to the tumor.

It should be considered that although the efficiency of transfection and the bystander effect are high i n uitro, as shown in this study, they may not be so high in vivo. Thus, studies are needed to assess in vivo in the animal model the efficacy of recombinant adenovi- ruses to apply the HSV-tWGCV system in liver cancer and to evaluate its possible toxicity, the extent of the bystander effect, and the ability of this system to in- duce tumor regression and prolonged survival.

Acknowledgment: We thank Dr Gavin Wilkinson for use of the plasmids pMVlOO and pMV60, Dr Frank Graham for plasmid pJMl7, and Dr Xiaozhou Chen for plasmid pMK.

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