Midkine Promoter-based Adenoviral Vector Gene Delivery for Pediatric Solid Tumors¹ Free

The liver is the predominant site of Ad³vector localization after systemic administration (1) and as a consequence is at risk when Ad vectors containing suicide genes ectopically localize to this site. In this regard, ectopic expression of HSV-tk has been shown to cause substantial hepatic toxicity and morbidity in animal models (2, 3). These results indicate that the restriction of tk gene expression to tumor cells is critical to the safe application of the HSV-tk/GCV system. Thus, a promoter with both tumor specificity and minimal transcriptional activity in hepatocytes would be ideal for cancer gene therapy. Whereas a number of promoters have been explored in the context of cancer gene therapy, few exhibit this optimal profile of inductivity and specificity.

MK is a heparin-binding, growth/differentiation factor that is induced by retinoic acid in embryonal carcinoma cells and is expressed temporarily during the mid-gestational period of mouse embryogenesis(4). MK induces mitogenic activity in fibroblasts, has neurotropic activity (5), and promotes survival of retinal cells in constant light-induced retinal degeneration (6). It has been reported recently that many types of malignant tumors highly express MK (7, 8, 9), especially Wilms’ tumors(8) and advanced neuroblastomas (9). Although inactivation of the Wilms’ tumor suppressor gene (WT1) has been documented as one of the reasons that Wilms’ tumors overexpress MK protein (10), the reason for MK expression observed in other tumors, including neuroblastomas, remains under investigation. The human MK promoter region contains an AP1 site, a DR5-type retinoic acid-responsive element, and consensus sequences for WT1(10, 11). Human MK and mouse MK are highly conserved, not only in cDNA structure but also in genomic organization and in discrete segments in the 5′ upstream region (11); however, tissue distribution of MK is slightly different; in normal human tissues, MK is expressed moderately in the small intestine and weakly expressed in the lung, colon, and thyroid. In contrast, in mouse normal tissues, MK is expressed moderately in the kidney and weakly in the brain, but importantly, MK expression is not observed in mouse or human liver(8, 12). On this basis, the MK promoter is a potential candidate promoter for having the desirable features of high activity in tumor and low activity in the liver. In addition, the MK promoter shows strong activity when transfected into the Wilms’ tumor G-401 cell line (10). We report herein the usefulness of MK promoter as a tumor-specific promoter for an Ad vector-based cancer gene therapy approach. A 2.3-kb upstream sequence of human MK gene (11), containing the promoter region,was inserted into an Ad vector and used to drive expression of either the luciferase reporter or HSV-tk genes. These vectors were then assessed in vitro and in vivo for activity and tumor specificity of transgene expression.

The Wilms’ tumor G-401, neuroblastoma SK-N-SH, colon cancer LS174T,and Burkitt’s lymphoma Daudi cell lines were purchased from the American Type Culture Collection (Manassas, VA). G-401 cells were cultured in McCoy 5A medium containing 10% FCS. SK-N-SH and LS174T cells were maintained in MEM with 10% FCS and 1% nonessential amino acid (Mediatech/Cellgro). Daudi cells were cultured in RPMI 1640 with 10% FCS. All media and FCS using this study were purchased from Mediatech/Cellgro (VA).

The MK cDNA probe corresponding to nucleotides 75–562, as reported by Tsutsui et al. (8), was used for this study. Total cellular RNA was extracted from 10⁷ cells using an RNeasy kit (Qiagen). Twentyμg of total RNA were denatured with formaldehyde, electrophoresed though a denaturing 1% agarose gel, and transferred to a Hybond nylon membrane (Amersham). Membranes were hybridized with a ³²P-labeled probe prepared using Ready To Go DNA labeling beads (Pharmacia Biotech) at 42°C for 4 h. Membranes were then washed twice with 2× SSC for 10 min and twice with 0.2× SSC at 56°C for 30 min. Membranes were exposed to BioMax film (Kodak) at−80°C with an intensifying screen for 2 days.

The recombinant Ad vectors AdMKLuc and AdMKTK, encoding firefly luciferase and HSV-tk, respectively, under the control of human MK promoter containing 27 bp of exon 1 and 2285 bp of the 5′ flanking region of the human MK gene, were constructed using the“AdEasy” method reported previously (13). Briefly, the MK promoter and either a luciferase gene (pGL3 basic vector; Promega)or tk gene (14) was inserted into a multicloning site in pShuttle vector. The resultant plasmid was linearized with PmeI digestion and subsequently cotransfected into Escherichia coli BJ5183 with pAdEasy-1 Ad backbone plasmid. After confirming recombination, the recombinants of interest were linearized with PacI digestion and transfected into 293 cells to generate AdMKLuc or AdMKTK. The recombinant adenoviruses were propagated in 293 cells and purified by double CsCl density centrifugation. Virus titers were determined by plaque assay in 293 cells. We also used Ad vectors containing luciferase or tk gene under the control of CMV enhancer/promoter as control vectors.

Tumor cells were plated in 24-well plates in triplicate at the concentration of 50,000/well 1 day prior to gene transfer. After overnight culture, the cells were infected with AdMKLuc or AdCMVLuc at a MOI of 50 pfu in medium containing 2% FCS for 1 h. The infecting medium was then replaced with complete medium. The infected cells were harvested and treated with 100 μl of lysis buffer 2 days after infection. A luciferase assay (Luciferase Assay System; Promega)and a luminometer (Lumat; Wallac, Inc.) were used for the evaluation of luciferase activities of Ad-infected cells. Luciferase activities were normalized by the protein concentration in cell lysate (Bio-Rad DC Protein Assay kit).

Tumor cells were plated in 96-well plates in triplicate at a density of 3000/well. After overnight culture, cells were infected with AdMKTK or AdCMVTK at MOIs of 0, 10, 50, and 100 for 5 h. The viral infection was followed by medium replacement including various concentrations of GCV ranging from 0 to 1000 μm. The number of surviving cells was determined by MTT assay (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega) after 5 days exposure of GCV. The MTT color development was measured and analyzed by an automated E max spectrophotometric plate reader (Molecular Device Corp., Sunnyvale,CA).

For determination of luciferase gene expression in mouse organs,C57/BL6 (Charles Rivers) mice received 1 × 10⁹ pfu of AdMKLuc or AdCMVLuc by tail vein injection. Three days later, livers, kidneys, lungs, and spleens were harvested to measure the luciferase gene expression.

s.c. tumors were induced by injection of 10⁷G-401 cells into the flank of the congenitally athymic nude mice(Charles Rivers). When tumor formation was confirmed (4–6 mm in diameter), AdMKLuc (2 × 10⁸ pfu)or AdCMVLuc (1 × 10⁸ pfu) were injected into the tumor. Two days later, mice were sacrificed, and tumors were resected, placed in the polypropylene tubes, and immediately frozen in ethanol/dry ice. Frozen tissues were ground to a fine powder using a pestle and mortar immersed in an ethanol/dry ice bath. Tissue powder was lysed using a tissue lysis buffer (Promega),and then luciferase activity was determined using a luciferase assay system kit (Promega). The luciferase activity was normalized by protein concentration in the tissue lysate.

To investigate the hepatic toxicity induced by the HSV-tk/GCV system,C57/BL6 mice received 1 × 10⁹ pfu of AdMKTK or AdCMVTK. The next day, i.p. GCV treatment was started (50 mg/kg, twice daily). After 7 days of continuous GCV administration,mice were sacrificed, and blood samples were taken to assess AST, ALT,total bilirubin, and LDH. Livers were fixed in 10% buffered formalin overnight and then processed into paraffin blocks, and 4-μm sections were cut, deparaffinized, and stained with H&E.

MK gene expression was examined in several human cell lines by Northern blotting (Fig. 1,A). As reported previously (8, 9), G-401 Wilms’tumor and SK-N-SH neuroblastoma cells highly expressed MK mRNA. In contrast, no MK expression was observed in the LS174T colon cancer cell line. Although Daudi lymphoma cells were used as a negative control of MK expression, this lymphoma cell line was not appropriate for the assessment of Ad gene infection, because these cells are refractory to infection by Ad. We have already confirmed that the MK promoter,extending from 27 bp of exon 1 to 2285 bp of the 5′ flanking region of the human MK gene, showed relatively high activity in G-401 cells in a plasmid context (10). We thus investigated whether the MK promoter in the adenoviral context would manifest high transcriptional efficiency in MK-positive cells. In a reporter gene experiment with firefly luciferase, tumor cells were infected with AdMKLuc or AdCMVLuc at a MOI of 50 (Fig. 1 B). In the G-401 cell line, luciferase activity induced by AdMKLuc showed comparatively high activity, approximately half the activity induced by AdCMVLuc infection. In the SK-N-SH cell line, the luciferase activity induced by AdMKLuc infection was less, ∼15% of that induced by AdCMVLuc. In comparison with tumor-specific promoters published previously(15, 16), this level was high. On the other hand,luciferase inducement by AdMKLuc was low in LS174T compared with AdCMVLuc. These results indicate that the MK promoter activity in the Ad context reflects the MK expression level of the cell lines.

To determine whether Ad-mediated infection with the MK-TK gene would render various cell lines sensitive to cell killing by GCV, the tumor cells were infected with AdMKTK or AdCMVTK at MOIs of 0, 10, 50,and 100. The recombinant Ad infection was followed by 5 days of GCV exposure at concentrations ranging from 0 to 1000 μm, and then the number of surviving cells was determined by MTT assay. AdMKTK successfully induced GCV sensitivity in the G-401 line (Fig. 2 A). Using AdMKTK at the dose of 50 MOI,IC₅₀s (the GCV concentration at which 50% cell survival is seen) are 4.5, 67, and 31 μm in G-401, SK-N-SH, and LS174T, respectively. Whereas for AdCMVTK at the same dose, the IC₅₀s are 3.6, 3.7, and 19μ m in G-401, SK-N-SH, and LS174T, respectively. In the G-401 cells, the effectiveness of this vector was comparable with AdCMVTK, as reflected by the similar IC₅₀s. AdMKTK/GCV also achieved cell killing in the SK-N-SH line, although this was less effective than the AdCMVTK/GCV combination. Although the luciferase study showed that the MK promoter activity was relatively low in the LS174T cells, this level of activity was sufficient to induce cell killing with AdMKTK/GCV. This may be a reflection of the sensitivity of the cell line to this particular suicide gene approach.

A key limitation to the use of HSV-tk/GCV approach for cancer therapy is the potential for toxicity to non-target organs. Because the Ad has particular tropism for the liver and because hepatic toxicity can be induced by HSV-tk/GCV, we were especially interested to determine whether the MK promoter would have low activity in the liver in vivo. Thus, AdMKLuc or AdCMVLuc (as a positive control) was injected i.v. into mice, and then the level of transgene expression at day 2 was determined (Fig. 3). In this assay, transgene expression induced by the MK promoter was a mean 200-fold less than that seen with the CMV promoter. This is consistent with previous studies that have shown no MK expression in mouse or human liver. Interestingly, the AdMKLuc-induced luciferase expression in the spleen was actually higher than that of AdCMVLuc. Although the possible reasons for this are speculative, it does indicate that the low luciferase results seen in the liver with AdMKLuc were not attributable to a fundamental problem with the efficacy of this vector in vivo per se. These results thus indicate the key property of MK promoter fidelity in the context of the Ad vector used in vivo.

As a correlate to the finding that the MK promoter had very low activity in the liver, we investigated the ability of AdMKLuc to transduce G-401 tumors in vivo. To assess this, tumors were implanted s.c. in nude mice and then injected with either AdCMVLuc or AdMKLuc (Fig. 4). Because the initial in vitro study had shown that luciferase expression using AdMKLuc was ∼50% of that seen with AdCMVLuc (Fig. 1 B), we used doses of 2 × 10⁸ pfu or 1 × 10⁸ pfu of AdMKLuc or AdCMVLuc, respectively. These studies confirmed the functionality of the MK promoter in MK-positive cells in vivo. Thus, these studies showed that the MK promoter in the context of an Ad vector possesses the two critical elements for consideration for use for cancer gene therapy, high-level expression in tumor and low-level expression in the liver.

As a final step, we investigated the effect on the liver of AdMKTK/GCV or AdCMVTK/GCV treatment. Mice were administered AdMKTK or AdCMVTK by tail vein injection, followed by either no further treatment or 7 days treatment with GCV. All of the mice that received 1 × 10⁹ pfu of AdCMVTK, and GCV administration manifested general weakening, weight loss, or reduced activity after 7 days of GCV injection. On the other hand, mice in other groups showed no abnormal features. The mice in this study were sacrificed after 7 days of GCV administration, and blood samples and livers were harvested to evaluate the liver dysfunctions. A prominent elevation of serum AST,ALT, total bilirubin, and LDH was observed in the AdCMVTK/GCV-treated mice as compared with the other groups (Fig. 5,A). In addition, the livers in this group appeared macroscopically yellowish, suggestive of fatty change, whereas the livers of mice that received AdMKTK/GCV appeared virtually normal (Fig. 5 B). Microscopically, the livers of the mice that received AdMKTK with or without GCV and of the animals that received GCV only did not show significant pathology; however, they did show some evidence of mild extramedullary hematopoiesis. In contrast, the AdCMVTK with GCV showed severe changes with microvesicular fatty change,individual cell necrosis, and acute inflammation, as well as extramedullary hematopoiesis. Minor inflammatory and fatty changes were noted in the AdCMVTK animals that did not receive GCV. Thus, these results demonstrate a key finding with respect to the use of the MK promoter for suicide gene therapy, i.e., prevention of hepatic toxicity, and indicate the potential benefits of using this promoter for cancer gene therapy.

In this study, we show for the first time that the MK promoter has tissue-specific fidelity in the Ad backbone. These findings indicate that this promoter may be an ideal candidate for tumor-specific suicide gene therapy. In this regard, the very low level of expression in hepatocytes should help to mitigate against the liver toxicity reported for the AdHSV-tk/GCV approach. AdMKTK did not cause liver dysfunction,despite GCV administration. This finding, coupled with the relatively high levels of expression seen in tumors in vivo, suggest this novel approach will have a significantly enhanced therapeutic window compared with the use of viral promoters such as CMV. The findings reported herein are important for several reasons. Although many tumor-specific promoters have been proposed for use in gene therapy vectors, these agents typically exhibit levels of activity much lower than viral promoters. This fact has lead to the development of amplification strategies to enhance the efficiency of a specific but weak promoter (15, 16). Thus, the finding that the AdMK viruses were capable of driving transgene expression at a level comparable with the CMV promoter is especially significant. Furthermore, many candidate tumor-specific promoters have been seen to have their specificity undermined when placed in the Ad backbone(17). This effect is poorly defined but may be attributable to cis- or trans-acting enhancing elements in the genome of the Ad vector. This finding has necessitated the use of insulator sequences to improve the fidelity of some promoters such as Erb B2 (18). In the current study,however, we demonstrate that the MK promoter retains its specificity in the Ad context. Levels of activity correlated with levels of MK expression in cell lines, and most importantly, the expression in the liver, where MK is not normally expressed, was very low. In addition,MK is expressed in certain tumor types for which current therapies are inadequate. In this regard, G-401 cells are properly regarded as a model for malignant rhabdoid tumor of the kidney (MRTK; Ref.19), a disease that shows poor response to conventional therapies (20). Thus, the high level of activity of AdMKTK in this line suggests that this approach may offer new therapeutic options for this disease. Furthermore, the treatment of advanced neuroblastomas, ordinarily occurring in older children and manifesting bone metastasis or aggressive local spread, is one of the crucial problems in pediatric oncology. In this context as well, various approaches, including high-dose chemotherapy combined with bone marrow transplantation, have not proven to be satisfactory. In this regard,Nakagawara et al. (9) reported that MK was expressed at high levels in almost all neuroblastomas, and aggressive neuroblastoma seemed to have a relatively increased amount of MK. Thus,the finding that AdMKTK has efficacy in a neuroblastoma line is significant with respect to the potential of applying gene therapy approaches, as we have proposed here.

In summary, we believe the MK promoter is a unique candidate tumor-specific promoter by virtue of its very low hepatic activity and toxicity, high tumor activity, and fidelity in the Ad vector. This new approach may have utility not only for Wilms’ tumors, neuroblastomas,but other MK-positive neoplasms (7, 8).

We thank Ludmila Kaliberova and Kathy Mercer for excellent technical support. We also wish thank Ramon Almany, Cristina Balague,Alex Pereboev, and Kaori Suzuki for excellent advice regarding recombinant Ad.