An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice

An Optimal Therapeutic Expression Level Is Crucial for Suicide Gene Therapy for Hepatic Metastatic Cancer in Mice Yasuhiro Terazaki,1 Shojiro Yano,1 Kentaro Yuge,2 Satoshi Nagano,2,3 Mari Fukunaga,1 Z. Sheng Guo,4 Setsuro Komiya,3 Kazuo Shirouzu,1 and Ken-ichiro Kosai2 The most serious problem in current gene therapy is discrepancies between experimental data and actual clinical outcomes, which may be due to insufficient analyses and/or inappropriate animal models. We have explored suicide gene therapy by using various clinically relevant animal models and doubt the clinical use of maximal suicide gene expression, which has been generally recommended. To explore this subject further, we studied what expression level of suicide gene and what promoter led to the maximal clinical benefit in the case of hepatic metastatic cancer in mice. Therapeutic and adverse side effects of 4 adenoviral vectors that express herpes simplex virus thymidine kinase (HSV-tk) under different promoters were scrupulously investigated in 2 mouse models of hepatic metastasis of gastric cancer that possess clinical characteristics. Surprisingly, increases in HSV-tk expression beyond a certain point, achieved by the Rous sarcoma virus long terminal repeat promoter, not only enhanced the adverse side effects of lethal hepatotoxicity and ganciclovirindependent cytotoxicity but also failed to further increase therapeutic potential. Moreover, the carcinoembryonic antigen (CEA) tumor-specific promoter, the therapeutic potential of which had been underestimated, was much more useful—even in the case of low CEA-producing cancer—than had been previously reported. In conclusion, the optimal therapeutic expression level of a suicide gene is a novel concept and a crucial factor for successful cancer gene therapy. The present results, which contradict those of previous studies, alert researchers about possible problems with ongoing and future clinical trials that lack this concept. (HEPATOLOGY 2003;37:155-163.)

A

n important issue in cancer gene therapy is that in vivo transfer of a therapeutic gene to every cancer cell by using any current vector is impossible in human patients or animals with orthotopic cancer. In this

Abbreviations: HSV-tk, herpes simplex virus thymidine kinase; GCV, ganciclovir; ADV, adenoviral vector; HMGC, hepatic metastasis of gastric cancer; CMV, human cytomegalovirus immediate-early gene; CAG promoter, a modified chicken ␤-actin promoter with human cytomegalovirus immediate-early enhancer; RSV, Rous sarcoma virus long terminal repeat; CEA, carcinoembryonic antigen; MOI, multiplicity of infection; x-gal, o-nitrophenyl-␤-D-galactophyranoside; PBS, phosphate-buffered saline; ALT, alanine aminotransferase. From the 1Department of Surgery, Kurume University School of Medicine, Kurume, Japan; 2Department of Gene Therapy and Regenerative Medicine, Gifu University School of Medicine, Gifu, Japan; 3Department of Orthopedic Surgery, Kagoshima University School of Medicine, Kagoshima, Japan; and the 4Department of Surgery and Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, PA. Supported in part by Uehara Memorial Foundation and in part by a Grant-inAid for Scientific Research from the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Address reprint requests to: Ken-ichiro Kosai, M.D., Ph.D., Department of Gene Therapy and Regenerative Medicine, Gifu University School of Medicine, 40 Tsukasamachi, Gifu 500-8705, Japan. E-mail: [email protected]; fax: (81) 58-267-2390. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3701-0023$35.00/0 doi:10.1053/jhep.2003.50018

regard, suicide gene therapy, a representative of which is transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene, followed by administration of ganciclovir (GCV), has the practical advantage of causing a potent bystander effect that confers cytotoxicity to the neighboring nontransduced cells.1-6 The normally nontoxic prodrug GCV is phosphorylated by HSV-tk and then converted to GCV-triphosphate, which inhibits DNA synthesis by acting as a chain terminator, leading to the killing of predominantly dividing tumor cells.2,7 This putative cytotoxic mechanism may constitute another safety advantage in HSV-tk gene therapy because normal nondividing cells will theoretically have low cytotoxicity in the case of undesirable gene transduction to organs proximal to the tumor. Generally, the most serious problem in biomedical research, including gene therapy, is that despite promising experimental laboratory data, clinical application has often led to unsatisfactory results.8-13 This may be, at least in part, caused by the use of inappropriate animal models and/or insufficient preclinical analyses, in which neither the therapeutic potential nor the adverse side effects have been well assessed.14,15 Many previous studies on cancer 155

156

TERAZAKI ET AL.

gene therapy by other investigators have represented tumor regression as the sole therapeutic parameter in very simple subcutaneous tumor models in mice, but this is characterized by a number of limitations. First, macroscopic tumor size is not sufficient as a therapeutic parameter because after treatments, macroscopically recognized tumor nodules often consist of a mixture of viable and dead tumor cells and other cells with some inflammatory reaction.3,16 Second, subcutaneous tumor models do not possess tissue characteristics or native microenvironments that could conceivably influence tumor growth and therapeutic effects.14,15 More importantly, some of the most critical factors in a clinical situation, gene transduction efficiency in tumors and undesirable gene transduction in other intact tissues, cannot be evaluated in such models. For example, chronic brain inflammation after HSV-tk gene therapy for glioma is an apparently serious complication that was not anticipated until a recent study using an orthotopic mouse model.17 As another example, HSV-tk plus GCV-dependent liver injury has been observed both in animal models of orthotopic cancer (our unpublished data) and in human patients,18 but the cytotoxic mechanism in the liver, in which more than 99% of hepatocytes are nondividing,19,20 has not been completely elucidated. In addition, we have recently shown that the therapeutic effects of HSV-tk gene therapy assessed according to their in vitro cytotoxicity are in appearance much better than those actually shown in the orthotopic lung cancer model in mice.15 Thus, many aspects of any new therapeutic strategy must be tested by using animal models that possess clinical characteristics.14,15 It seems, however, that many previous studies have preferentially used any animal models that can show the data at its best, leading to inconsistent, unsatisfactory, or even catastrophic outcomes (a sorrowful death of a human patient resulting from inappropriate clinical gene therapy itself) in actual clinical trials.8-10,13 The questionable value of earlier studies encouraged us to try to determine crucial factors, which have been to date missing, for the successful clinical application of suicide gene therapy. Obviously, it is quite important to choose the best vector and to administer the optimal dose of that vector for successful gene therapy21; however, the concept of the optimal therapeutic expression levels of suicide genes has not yet emerged. Instead, the investigators of previous studies simply investigated promoter strength and the resulting cytotoxicity and often recommended that the strongest promoter be used.22,23 Many of them noted that tissue-specific promoters might be too weak to be useful.22,23 However, it is quite uncertain whether such data are in fact applicable to the clinical situation because these investigators’ experimental design and animal models

HEPATOLOGY, January 2003

were not clinically relevant.22,23 Because of this we doubt the clinical use of maximal suicide gene expression for clinical gene therapy. Metastatic cancer in the liver, including metastatic gastric cancer in the liver, in which the poor prognosis indicates an urgent need for the development of novel therapies,24 may be a suitable model for assessing the clinical use of increasing suicide gene expression. This is caused by a possible hepatotoxicity that may be an obstacle in clinical application of adenovirus-mediated suicide gene therapy,9,13 and can be maximally explored in them. To that end, we carefully and extensively analyzed the therapeutic and adverse side effects of using 4 adenoviral vectors (ADVs) expressing HSV-tk under different promoters in 2 clinical-relevant animal models of hepatic metastasis of gastric cancer (HMGC). As a result, the present study revealed a novel concept and the need for serious caution in the ongoing and future clinical trials of suicide gene therapy.

Materials and Methods Recombinant ADV and Cell Lines. The replicationdefective Ad.RSV-tk and Ad.CMV-tk, and Ad.CMVLacZ and Ad.CAG-LacZ, which express HSV-tk or LacZ gene, respectively, under the transcriptional control of Rous sarcoma virus long terminal repeat (RSV) promoter, human cytomegalovirus immediate-early gene enhancer/ promoter (CMV promoter) and a modified chicken ␤-actin promoter with human cytomegalovirus immediateearly enhancer (CAG promoter), were constructed as described previously.3 Ad.CEA-tk (Adex1CEAprTK) encoding HSV-tk gene and carcinoembryonic antigen (CEA) promoter, Ad.CAG-tk (Adex1CA HSVTK) and Ad.CEA-LacZ (Adex1CEAprZ) were provided through Riken Gene Bank (Tsukuba, Japan). CEA Radioimmunoassay. In in vitro experiments, the supernatant was collected from 1 ⫻ 106 cells after 48 hours of culture, and then the cell lysate was prepared in phosphate-buffered saline by the freeze-thaw method. CEA levels were quantified by radioimmunoassay according to the manufacturer’s protocol (Dainabot Co., Tokyo, Japan). In in vivo experiments, the serum collected from peripheral blood of HMGC mice was directly qualified for CEA levels, as in a clinical examination in human patients.25,26 ␤-Galactosidase Enzyme Reporter Assay. The activity of ␤-galactosidase enzyme in gastric cancer cells was measured according to the manufacturer’s protocol (Promega, Tokyo, Japan) at 48 hours after infection with each Ad-LacZ at a multiplicity of infection (MOI) of 30. Briefly, the cell lysate was incubated for 30 minutes with

HEPATOLOGY, Vol. 37, No. 1, 2003

the substrate of o-nitrophenyl-␤-D-galactophyranoside (x-gal), and the absorbance was read at 420 nm with a spectrophotometer. The activity of ␤-galactosidase was calculated according to the standard curve of the predetermined concentration of ␤-galactosidase enzyme. Viability of Gastric Cancer Cell Lines. Gastric cancer cells in 96-well plates (1 ⫻ 103 cells/well) were infected with each ADV at an MOI of 30 and were cultured in the media with or without 10 ␮g/mL GCV for 6 consecutive days. The viability was determined by WST-1 assay (Dojindo Laboratories Co., Mashiki, Japan) at 7 days after ADV infection. Animal Experiments. Animal models of HMGC-28 and HMGC-1 were generated by implanting 1 ⫻ 106 MKN-28 cells and 5 ⫻ 105 MKN-1 cells into the tip of the left lateral liver lobe of 10-week-old male BALB/c athymic nude mice, respectively. At day 7, 3 ⫻ 108 pfu of each ADV in 70 ␮L of 10 mmol/L Tris-HCl, pH 7.4/1 mmol/L MgCl2/10% (vol/vol) glycerol/hexadimethrine bromide (Sigma, St. Louis, MO) (20 ␮g/mL) was injected directly into the hepatic tumors. Twelve hours after ADV injection the animals were treated intraperitoneally with GCV at 10 mg/kg twice daily for 12 consecutive days and then killed. All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Research Committee of Kurume University. Histopathology, CEA Immunohistochemistry, and X-Gal Staining. Formalin-fixed and paraffin-embedded tissues were stained with hematoxylin-eosin to evaluate the morphology. Other paraffin-embedded tissues were stained with rabbit anti-human CEA antibody (DAKO, Carpinteria, CA) by the labeled streptavidin-biotin method (DAKO LSAB Kit, DAKO) in accordance with the manufacturer’s protocols to detect the cytoplasmic CEA expression. For x-gal staining, OCT-frozen tissues were cut in 10-␮m sections, which were fixed in phosphate-buffered saline (PBS) containing 1.25% glutaraldehyde and 2% formaldehyde for 30 minutes at room temperature. The slides were stained with x-gal overnight at 37°C and counterstained with hematoxylin-eosin. Tumor Volume and Morphometric Analysis of Residual Tumors. After various gene therapy treatments, tumor volumes were calculated as (the largest diameter) ⫻ (the smallest diameter)2. To quantify the residual viable tumor accurately, computer-assisted morphometric analysis of the largest cross-sectional area of the residual tumors was performed using Mac SCOPE software (Mitani Co., Maruoka, Japan). The functional area of viable tumor cells among the groups was compared statistically.

TERAZAKI ET AL.

157

Biochemical Analyses. Serum alanine aminotransferase (ALT) levels were measured using a standard clinical automatic analyzer (Hitachi 736, Tokyo, Japan). Statistic Analysis. All results are expressed as mean ⫾ standard deviation. Statistical comparisons were made using Student’s t test.

Results Endogenous CEA Levels in Gastric Cancer Cell Lines. MKN-45 cells secreted about 200-fold more CEA in the media and produced about 10,000-fold more CEA in the cell lysates than MKN-28 cells, whereas the level of CEA in MKN-1 cells was below the detectable level of radioimmunoassay (Table 1). Thus, MKN-45, MKN-28, and MKN-1 cells showed high, low, and no (below the detectable level of ) CEA expression, respectively. The following in vitro experiments were performed by infecting with each of the ADVs at MOI of 30, leading to 70% or slightly higher gene transduction efficiencies with less variability in all 3 gastric cancer cell lines. Promoter Strength in ADV In Vitro. The RSV, CMV, and CAG promoters have each been widely used as a ubiquitously strong promoter3,17,22; however, no studies comparing the activity of the 3 promoters have been performed. To investigate the promoter strength in ADV strictly, we performed a ␤-galactosidase enzyme reporter assay after infection with each of the 4 Ads-LacZ (Table 2). The differences in promoter strength among these 3 were relatively large with the RSV, CMV, and CAG promoters showing moderate, medium, and strong expression in all 3 cell lines, respectively. The maximal differences in ␤-galactosidase activity between Ad.RSVLacZ and Ad.CMV-LacZ and between Ad.RSV-LacZ and Ad.CAG-LacZ were 18- and 363-fold, respectively, both of which were seen in MKN-28 cells. On the other hand, the strength of the CEA promoter was well correlated with the endogenous CEA level in each cell line. Interestingly, the activity of ␤-galactosidase after Ad.CEA-LacZ infection was only a little lower than that after Ad.RSV-LacZ infection in low CEA-producing

Table 1. Endogenous CEA Levels in Three Gastric Cancer Cell Lines Cell Line

Supernatant (ng/mL/106 Cells/48 h)

Lysate (ng/mg Protein)

MKN-1 MKN-28 MKN-45

⬍1.0 ⫻ 10⫺3 (1.6 ⫾ 0.3) ⫻ 10⫺2 3.3 ⫾ 0.2

⬍1.0 ⫻ 10⫺4 (5.9 ⫾ 1.3) ⫻ 10⫺3 (5.6 ⫾ 4.6) ⫻ 10

NOTE. The supernatant was collected from 1 ⫻ 106 cells after 48 hours of culture, and then the cell lysate was prepared. CEA levels were quantified by RIA. Results are mean ⫾ SD for 3 independent experiments.

158

TERAZAKI ET AL.

HEPATOLOGY, January 2003

Table 2. ␤-Galactosidase Enzyme Reporter Assay Promoters (mIU) Cell Line

CEA

RSV

CMV

CAG

MKN-1 MKN-28 MKN-45

0.0 1.2 2.8 ⫻ 10

3.6 1.9 1.5

4.9 ⫻ 10 3.4 ⫻ 10 1.4 ⫻ 10

3.9 ⫻ 102 6.9 ⫻ 102 5.2 ⫻ 102

NOTE. The cell lysate was prepared at 48 hours after infection with the Ad-LacZ at MOI of 30. Results are mean for 3 independent experiments.

MKN-28 cells, and was higher than those after Ad.RSVLacZ or Ad.CMV-LacZ infections in high CEA-producing MKN-45 cells. In addition, Ad.CEA-LacZ infection in MKN-1 cells did not result in detectable levels of ␤-galactosidase activity, indicating the tight tissue specificity of CEA promoter in ADV. In addition, HSV-tk messenger RNA levels analyzed by reverse-transcription polymerase chain reaction after infection of each Ad-tk revealed similar results (data not shown), despite certain obstacles due to the semiquantitative nature of the polymerase chain reaction assay and the appearance of cell death after Ad.CAG-tk infection without GCV (Fig. 1). Cytotoxic Effects After Ads-tk With Different Promoters In Vitro. To analyze the cytotoxic effects of Ads-tk with different promoters, we examined cell viability after infection of Ad-tk with or without GCV (Fig. 1). Ad.RSV-tk and Ad.CMV-tk induced a significant toxicity in all 3 cell lines in a strictly GCV-dependent manner. Unexpectedly, Ad.CAG-tk induced a significant cytotoxicity not only with GCV but also without GCV. On the other hand, Ad.CEA-tk with GCV rendered not only high CEA-producing MKN-45 cells but also low CEAproducing MKN-28 cells susceptible to killing, but exerted no cytotoxic effect in MKN-1 cells that did not produce CEA. In addition, the small difference in cytotoxic effects observed between Ad.CEA-tk/GCV and Ad.RSV-tk/GCV in MKN-28 cells was consistent with the small differences in transgene expression levels between them (Table 2). Thus, relatively low expression levels of HSV-tk may be sufficient to induce some GCVdependent cytotoxicity, and excessively high levels may further induce GCV-independent cytotoxicity. Mouse HMGC Models and ADV Gene Transduction. We generated mouse models of low and no CEA-producing HMGCs, designated HMGC-28 and HMGC-1, by intrahepatic implantation of MKN-28 and MKN-1 cells, respectively. Seven days after cell implantation, tumor nodules of a variety of sizes were macroscopically visible in both HMGCs. CEA levels in the serum were below the detectable levels (0.5 ng/mL) by regular clinical examination of radioimmunoassay not

only in HMGC-1 mice but also in HMGC-28 mice. Interestingly, about half of the tumor cells in HMGC-28 mice were immunohistochemically CEA-positive heterogeneously, as observed in about 50% of human patients,25 whereas all tumor cells in HMGC-1 mice were immunohistochemically CEA negative. We also tried to establish a mouse model of high CEA-producing HMGC by using MKN-45 cells, but high mortality 1 or 2 days after tumor implantation was an obstacle to further animal experiments. Next, we investigated adenoviral in vivo gene transduction efficiencies in tumors and undesirable gene transduction in the liver and other organs by x-gal staining after an intratumor injection of Ad.CMV-LacZ in HMGC-28 mice (Fig. 2). The gene transduction efficiency in the tumor was variable. Connective tissues and some hepatocytes adjacent to the tumor nodule were also x-gal posi-

Fig. 1. Cytotoxic effects after infection of Ads-tk containing different promoters in vitro. Gastric cancer cells were infected with each ADV and were cultured with GCV or PBS for 6 consecutive days. The viability was determined by WST-1 assay. Data represent 3 separate experiments (5 samples/group/experiment). *P ⬍ .001 (groups treated with each adenovirus plus PBS or GCV vs. controls treated with Ad.CMV-LacZ plus PBS or GCV, respectively).

HEPATOLOGY, Vol. 37, No. 1, 2003

Fig. 2. In vivo ADV gene transduction efficiencies in tumor nodules and the liver. (A) Tumor and (B and C) liver sections in HMGC-28 mice that received an intratumoral injection of Ad.CMV-LacZ were stained with x-gal. (A) Representative picture of tumor and surrounding connective tissues. (B) Representative picture of hepatocytes far from the tumor nodules. Most of the hepatocytes were x-gal negative. (C) About 5% of the hepatocytes far from the tumor nodules were x-gal positive in a few mice despite the correct injection administered into the tumor nodules (original magnification ⫻100).

tive, whereas all other organs were x-gal negative (Fig. 2A and B). Unexpectedly, less than 5% of x-gal–positive hepatocytes far from the tumor nodule were seen in a few mice even when the correct injection of Ad.CMV-LacZ into

TERAZAKI ET AL.

159

the tumor nodule was confirmed by x-gal staining (Fig. 2C). Therapeutic and Adverse Side Effects in Diverse Ads-tk Gene Therapy for Low CEA-Producing HMGC-28 Mice. Seven days after tumor inoculation, the size of the tumor nodules varied, with an average size of 8.2 ⫻ 6.6 mm and a maximum size of 15 ⫻ 12 mm. HMGC-28 mice having a visible tumor larger than 5 ⫻ 5 mm (89% of all HMCG-28 mice) were injected randomly with one of the 5 ADVs. In the case of all 4 Adstk/GCV treatments, the small tumor nodules consisted of a mixture of viable and dead tumor cells, dead hepatocytes, and infiltrating inflammatory cells (Fig. 3C). The therapeutic effects of all 4 Ads-tk/GCV were significant to a similar degree with respect to not only regression of the macroscopic tumor volume but also the exact area of remaining viable tumor cells and the percentage of mice being histologically free from viable tumor cells (Fig. 3A). Interestingly, GCV-independent tumor death was observed only in mice treated with Ad.CAG-tk, in agreement with the in vitro results. Treatment with Ad.CAG-tk/GCV resulted in various degrees of pathologic liver injury and ALT level elevation up to 2,205 IU/L, whereas mice treated with Ad.RSV-tk/ GCV and Ad.CMV-tk/GCV exhibited histologically mild liver injury and moderate elevations of ALT levels (Fig. 3B). Apparent hepatotoxicity was not caused by Ad.CEA-tk/GCV or by ADV and/or GCV themselves. No apparent histopathologic findings were seen in organs other than the liver after any treatment (data not shown). One of the major causes of ADV-derived hepatotoxicity is known to be cytotoxic immune reaction against ADV-infected hepatocytes.27 Several attempts to establish an immunocompetent animal model of HMGC using the syngeneic mouse gastric cancer cell line MGT-4028 (kindly provided by Dr. M. Tatematsu) have been unsuccessful. Therefore, to elucidate the possible maximal hepatotoxicity in Ad-tk gene therapy for HMGC, we injected each ADV into the tip of the left lateral lobe of an intact liver without tumor in an immunocompetent mouse, which was then treated with GVC according to the schedule used earlier (Fig. 4). All mice treated with Ad.CAGtk/GCV or Ad.CMV-tk/GCV died of severe liver injury within 9 and 12 days after ADV injections, respectively. The histopathologic examination showed that infiltration of inflammatory cells and hepatocyte death, including apoptosis and necrosis, were apparently seen in the whole liver parenchyma and such necroinflammatory reaction was more prominent in the mice treated with Ad.CAGtk/GCV than those with Ad.CMV-tk/GCV. In contrast, none of the mice treated with Ad.RSV-tk/GCV, Ad.CEA-tk/GCV or Ad.CMV-LacZ/GCV died of liver

160

TERAZAKI ET AL.

HEPATOLOGY, January 2003

Taken together, these data show that hepatotoxicity during Ad-tk gene therapy was more severe and more lethal with increasing HSV-tk expression, and that Ad.RSV-tk and Ad.CEA-tk showed a great safety advantage without any compromise in therapeutic potential. Therapeutic and Adverse Effects of Ad-tk Gene Therapy in HMGC Mice Not Expressing CEA. To verify the clinical use and tissue specificity of Ad.RSV-tk/ GCV and Ad.CEA-tk/GCV, therapeutic and adverse effects were investigated in non–CEA-expressing HMCG-1 mice according to the schedule used for CEA-expressing mice. Treatment with Ad.RSV-tk/GCV resulted in sig-

Fig. 3. Therapeutic and adverse side effects in Ads-tk gene therapy for HMGC-28 mice. (A) Therapeutic effects were compared among treatment groups from the tumor volume, the viable tumor area that was accurately measured by computer-assisted morphometric analysis, and by the percentage of no viable tumor cells obtained by histologic examination. Data represent 3 separate experiments (9 or more mice/group). *P ⬍ .05 (each group vs. Ad.CMV-LacZ/GCV control). (B) Serum ALT levels in individual mice were plotted. (C) Representative histopathology of the tumor nodule in the liver after each treatment. The squared area in the upper picture, which is the boundary area between the tumor nodule and the liver, corresponds to the lower picture (Original magnification ⫻10 for upper picture; ⫻100 for lower picture). Macroscopically recognized tumor nodules were histologically revealed to consist of dead tumor cells and dead hepatocytes, but not viable tumor cells in these representative pictures of Ad.RSV-tk/GCV or Ad.CAG-tk/GCV treatment; in contrast, they consist of viable tumor cells in this representative picture of Ad.CMVLacZ/GCV treatment.

injury before the termination of experiments on day 13. The histologic examination showed the minimal necroinflammatory reaction (i.e., moderate hyperplasia of Kupffer cell and a sparse single apoptosis) in the mice treated with Ad.RSV-tk/GCV, whereas the livers were histologically intact in the mice treated with Ad.CEA-tk/ GCV or Ad.CMV-LacZ/GCV. In addition, none of the mice treated with any ADV plus PBS died of liver injury (data not shown).

Fig. 4. Hepatotoxicity after intrahepatic ADV injection with GCV treatment in immunocompetent mice. (A) Immunocompetent mice received an intrahepatic injection of ADV, followed by intraperitoneal injection of GCV. A survival study was performed (5 mice/group). (B) Representative histopathology of the liver in the survival mice treated with Ad.CMV-LacZ/ GCV or Ad.RSV-tk/GCV on day 12, or in the dead mice treated with Ad.CMV-tk/GCV on day 12 or with Ad.CAG-tk/GCV on day 6 (original magnification ⫻100).

HEPATOLOGY, Vol. 37, No. 1, 2003

Fig. 5. Therapeutic effects in Ads-tk gene therapy for HMGC-1 mice. Therapeutic effects were compared between treatment (Ad.CEA-tk/GCV or Ad.RSV-tk/GCV) and control (Ad.RSV-tk/PBS) groups from the tumor volume. Data represent 2 separate experiments (9 or more mice/group). *P ⬍ .05 (Ad.CEA-tk/GCV or Ad.RSV-tk/GCV treatment vs. Ad.RSV-tk/ PBS control).

nificant tumor regression (Fig. 5) and mild liver injury (ALT levels: 80 ⫾ 45 IU/L in Ad.RSV-tk/GCV vs. 29 ⫾ 35 IU/L in Ad.RSV-tk/PBS) in HMGC-1 mice much as in HMGC-28 mice. On the other hand, treatment with Ad.CEA-tk/GCV did not result in any therapeutic effects or hepatotoxicity (Fig. 5).

Discussion The present study reveals that the optimal therapeutic (i.e., not the highest) expression level of a suicide gene is a crucial factor for successful clinical cancer gene therapy. An important feature of the present study is the suggestion that a relatively low expression of HSV-tk may be sufficient to render cancer cells highly susceptible to killing, which leads to a number of conclusions concerning the use of individual Ads-tk that contradict those of previous reports.9,10,22,23 In Ad-tk gene therapy for HMGC, therapeutic effects do not change, whereas adverse side effects become more severe as HSV-tk expression levels are increased beyond a certain point. This point may be the optimal therapeutic expression level of HSV-tk,

TERAZAKI ET AL.

161

which may exist around the expression level after Ad.RSV-tk infection. Thus, Ad.RSV-tk may be the first and the best choice for the treatment of metastatic cancer in the liver. On the other hand, Ad.CAG-tk, which showed the highest expression level of HSV-tk, may not be appropriate for clinical gene therapy for metastatic cancer in the liver because it showed no increase in therapeutic advantage but it did show a significant increase in potential risk for lethal hepatotoxicity. In addition, the appearance of GCV-uncontrollable cell death may be another disadvantage because lethal hepatotoxicity or unexpected severe side effects could not be prevented by discontinuing GCV administration. Undesirable gene transduction in less than 5% of hepatocytes in the liver, probably due to the liver’s abundant blood circulation and high infectivity to ADV, and the resultant excessive expression of HSV-tk in these limited numbers of hepatocytes, may result in severe injury of most nontransduced and nondividing hepatocytes in the whole liver as a bystander effect. The mechanisms of these effects, especially the cytotoxicity in nondividing hepatocytes, the GCV-independent manner of the effects or the bystander effect in the intact liver, remain to be solved. Notably, lethal hepatotoxicity by Ad.CAG-tk was observed even in a clinically relevant dog model of primary gastric cancer (not adjacent to the liver), although the investigators describing this model did not significantly discuss the importance of this lethality but only emphasized the cytotoxicity to tumors.29 More generally, the CAG promoter has been preferentially used for biologic and biomedical studies, including gene therapy, because very strong expression readily leads to drastic changes in appearance.11,30,31 Based on our results, we caution against the clinical usage of promoters that are too strong for driving certain genes, the excessive expression of which may readily cause severe adverse effects. In contrast, Ad.CEA-tk may be more widely applicable— even for low CEA-producing cancers—than previously thought due to its impressive therapeutic effects and minimal side effects. To date, the clinical use of tumorspecific promoters, including the CEA promoter, for suicide gene therapy has been unknown or underestimated.22,23 Inappropriate experimental design, such as having the sole control of the CAG promoter for comparing the promoter activity, in addition to insufficient animal models and analyses in previous studies have given a misleading impression that tumor-specific promoters, including the CEA promoter, were too weak to be useful for suicide gene therapy.22,23 However, the present results clearly show that the CEA promoter exerted sufficient expression levels of transgene. The resulting therapeutic

162

TERAZAKI ET AL.

effect was similar to that of the RSV promoter even in low CEA-producing HMGC. Moreover, the CEA promoter was stronger than the RSV and the CMV promoters in high CEA-producing cancer, even though it was weaker than the clinically useless CAG promoter. In the case of clinical trials, Ad.CEA-tk may theoretically reveal the maximal therapeutic effect when the activity of the CEA promoter is stronger than that of the RSV promoter in an individual CEA-producing cancer. Before Ad.CEA-tk can be used to replace Ad.RSV-tk in clinical trials, however, it will be important to determine the proper level of endogenous CEA. Ad.CEA-tk revealed therapeutic effects even for low CEA-producing HMGC, in which serum CEA levels were not detected by regular clinical examination but were partially detected by CEA immunohistochemistry. We hypothesize that Ad.CEA-tk may be applicable at least for cancer patients who have detectable levels of CEA both in the serum and in tumor tissue.25,26 The resultant therapeutic effect for metastatic cancer in the liver was not increased in the case of the expression level being beyond that by the RSV promoter, probably because other factors such as limited gene transduction in a tumor nodule may have much more significant influences on therapeutic effects in a clinical situation than does excessive expression of the suicide gene itself. Furthermore, although it is preferable to kill all tumor cells, this has not been feasible with HSV-tk gene therapy.9,10 Rather, the goal has been to kill the majority of tumor cells in a tumor nodule and to sequentially induce systemic antitumor immunity with combined cytokine gene therapy to kill the remaining viable tumor cells.3,16,32,33 Thus, all these facts generally imply the necessity of reevaluating the clinical use of various tumor-specific promoters using individual clinically relevant animal models. It is noteworthy that the conclusion drawn from the present study contradicts those of previous analyses. Modern trends in biomedical research are showing spectacular results using novel methods in models that show the data at its best, whereas some fundamental but less spectacular studies might not get the attention they are due. We believe the present findings generally underscore the importance of relevant animal models and careful preclinical analyses in the field of biomedical research and translational science. In conclusion, this report reveals the novel concept of an optimal therapeutic expression level of a suicide gene for successful clinical cancer gene therapy. The present results, which contradict those of previous studies, provide crucial information reminding us to use caution in ongoing or future clinical trials that inappropriately use promoters that are too strong.

HEPATOLOGY, January 2003

Acknowledgment: The authors thank M. Uenosono and Dr. E. Mekada for their help; Dr. H. Hamada and F. Hoffmann-La Roche Ltd. (Basel, Switzerland) for kindly supplying some ADVs and GCV, respectively, and Mr. M. O’Malley for editing the manuscript.

References 1. Mesnil M, Yamasaki H. Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. Cancer Res 2000;60:3989-3999. 2. Kuriyama S, Nakatani T, Masui K, Sakamoto T, Tominaga K, Yoshikawa M, Fukui H, et al. Bystander effect caused by suicide gene expression indicates the feasibility of gene therapy for hepatocellular carcinoma. HEPATOLOGY 1995;22:1838-1846. 3. Chen SH, Chen XH, Wang Y, Kosai K, Finegold MJ, Rich SS, Woo SL. Combination gene therapy for liver metastasis of colon carcinoma in vivo. Proc Natl Acad Sci U S A 1995;92:2577-2581. 4. Bonnekoh B, Greenhalgh DA, Bundman DS, Kosai K, Chen SH, Finegold MJ, Krieg T, et al. Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma. J Invest Dermatol 1996;106:1163-1168. 5. Kwong YL, Chen SH, Kosai K, Finegold MJ, Woo SL. Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer. Cancer Gene Ther 1996;3:339-344. 6. Block A, Chen SH, Kosai K, Finegold M, Woo SL. Adenoviral-mediated herpes simplex virus thymidine kinase gene transfer: regression of hepatic metastasis of pancreatic tumors. Pancreas 1997;15:25-34. 7. Frank KB, Chiou JF, Cheng YC. Interaction of herpes simplex virusinduced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. J Biol Chem 1984;259:1566-1569. 8. Marshall E. Cancer therapy. Setbacks for endostatin. Science 2002;295: 2198-2199. 9. Galanis E, Russell S. Cancer gene therapy clinical trials: lessons for the future. Br J Cancer 2001;85:1432-1436. 10. McCormick F. Cancer gene therapy: fringe or cutting edge? Nature Rev Cancer 2001;1:130-141. 11. Kiwaki K, Kanegae Y, Saito I, Komaki S, Nakamura K, Miyazaki JI, Endo F, et al. Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter. Hum Gene Ther 1996;7:821830. 12. Ye X, Robinson MB, Batshaw ML, Furth EE, Smith I, Wilson JM. Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. J Biol Chem 1996;271:3639-3646. 13. Lehrman S. Virus treatment questioned after gene therapy death. Nature 1999;401:517-518. 14. Miyoshi T, Kondo K, Ishikura H, Kinoshita H, Matsumori Y, Monden Y. SCID mouse lymphogenous metastatic model of human lung cancer constructed using orthotopic inoculation of cancer cells. Anticancer Res 2000; 20:161-163. 15. Fukunaga M, Takamori S, Hayashi A, Shirouzu K, Kosai K. Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model. Ann Thorac Surg 2002;73:1740-1746. 16. Chen SH, Kosai K, Xu B, Pham-Nguyen K, Contant C, Finegold MJ, Woo SL. Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival. Cancer Res 1996;56:3758-3762. 17. Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJ, Southgate TD, et al. Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat Med 1999;5:1256-1263. 18. Herman JR, Adler HL, Aguilar-Cordova E, Rojas-Martinez A, Woo S, Timme TL, Wheeler TM, et al. In situ gene therapy for adenocarci-

HEPATOLOGY, Vol. 37, No. 1, 2003

19. 20.

21.

22.

23.

24. 25. 26.

noma of the prostate: a phase I clinical trial. Hum Gene Ther 1999;10: 1239-1249. Macdonald R. Lifespan of liver cells. Arch Intern Med 1961;107:335-343. Kosai KI, Finegold MJ, Thi-Huynh BT, Tewson M, Ou CN, Bowles N, Woo SL, et al. Retrovirus-mediated in vivo gene transfer in the replicating liver using recombinant hepatocyte growth factor without liver injury or partial hepatectomy. Hum Gene Ther 1998;9:1293-1301. Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001;7: 33-40. Kijima T, Osaki T, Nishino K, Kumagai T, Funakoshi T, Goto H, Tachibana I, et al. Application of the Cre recombinase/loxP system further enhances antitumor effects in cell type-specific gene therapy against carcinoembryonic antigen-producing cancer. Cancer Res 1999; 59:4906-4911. Nagayama Y, Nishihara E, Iitaka M, Namba H, Yamashita S, Niwa M. Enhanced efficacy of transcriptionally targeted suicide gene/prodrug therapy for thyroid carcinoma with the Cre-loxP system. Cancer Res 1999;59: 3049-3052. Roukos DH. Current status and future perspectives in gastric cancer management. Cancer Treat Rev 2000;26:243-255. Tabuchi Y, Deguchi H, Saitoh Y. Carcinoembryonic antigen levels of portal blood in gastric cancer patients. J Surg Res 1989;47:81-86. Santeusanio G, Peronace L, Castagna G, De Muro G, Santi D, D’Orazio A, Amanti C, et al. Immunohistochemical study of carcinoembryonic an-

TERAZAKI ET AL.

27.

28.

29.

30.

31. 32.

33.

163

tigen (CEA) in gastric tumors: correlation with preoperative serum levels, histologic type, and grade of anaplasia of the tumor. J Surg Oncol 1988; 37:13-19. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A 1994;91:4407-4411. Ichinose M, Nakanishi H, Fujino S, Tatematsu M. Establishment and characterization of two cell lines from N-methyl-N-nitrosoureainduced mouse glandular stomach carcinomas. Jpn J Cancer Res 1998; 89:516-524. Matsukura N, Hoshino A, Igarashi T, Hasegawa H, Okino T, Onda M, Iijima O, et al. In situ gene transfer and suicide gene therapy of gastric cancer induced by N-ethyl-N⬘-nitro-N-nitrosoguanidine in dogs. Jpn J Cancer Res 1999;90:1039-1049. Ishii A, Hagiwara Y, Saito Y, Yamamoto K, Yuasa K, Sato Y, Arahata K, et al. Effective adenovirus-mediated gene expression in adult murine skeletal muscle. Muscle Nerve 1999;22:592-599. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 1991;108:193-199. O⬘Malley BW Jr, Sewell DA, Li D, Kosai K, Chen SH, Woo SL, Duan L. The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer. Mol Endocrinol 1997;11:667-673. Kwong YL, Chen SH, Kosai K, Finegold M, Woo SL. Combination therapy with suicide and cytokine genes for hepatic metastases of lung cancer. Chest 1997;112:1332-1337.