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Stem Cells, Vol. 18, No. 6, 415-421, November 2000
© 2000 AlphaMed Press

In Vivo Demethylation of a MoMuLV Retroviral Vector Expressing the Herpes Simplex Thymidine Kinase Suicide Gene by 5' Azacytidine

Mauro Di Iannia, Adelmo Terenzia, Sabrina Di Florioa, Gigliola Vendittia, Roberta Benedettia, Antonella Santuccia, Andrea Bartolib, Katia Fettucciarib, Pierfrancesco Marconib, Ruggero Rossib, Massimo F. Martellia, Antonio Tabilioa

a Haematology and Clinical Immunology and
b Pathology Sections, Department of Clinical and Experimental Medicine, Perugia University, Perugia, Italy

Key Words. 5' Azacytidine • Retroviral vector • SCID mice • lacZ

Antonio Tabilio, M.D., Haematology and Clinical Immunology Section, Department of Clinical and Experimental Medicine, Perugia University, Policlinico Monteluce, V. le Brunamonti, 06122 Perugia, Italy. Telephone: 39-075-5783990; Fax: 39-075-5726449; e-mail: medemat{at}unipg.it


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 
We constructed a functional MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type I thymidine kinase gene, which induces sensitivity to the prodrug ganciclovir (gcv), and the reporter ß-galactosidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduced with this vector, and a clone (VB71) with high-level transgene expression was selected. Severe combined immunodeficient (SCID) mice were injected with VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5' aza-C) demethylating agent prevented it.

We found 5' aza-C maintained gene expression at high level in vitro. In vivo, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5' aza-C, and gcv compared with animals treated with either 5' aza-C or gcv alone. The number of injected tumor cells influences tumor onset time and the efficacy of 5' aza-C and gcv treatment. The standard gcv treatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusion, the results extend our previous findings that transgene methylation in vivo may be prevented with an appropriate schedule of 5' aza-C and gcv.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 References
 
Gene therapy is a new therapeutic approach with the potential to treat monogenic congenital deficiencies of hematopoietic stem cells or their progeny and acquired neoplastic diseases [1, 2]. Major problems lie in obtaining an efficient gene transfer, high transgene expression, adequate in vivo regulation, and long-term transgene expression [3]. Several mechanisms, such as histone acetylation and deacetylation [4], retroviral integration near DNAase I-hypersensitive sites in chromatin [5], and DNA methylation [6] have been implicated in regulating transgene expression. Methylation of CpG doublets of the enhancer sequences in retroviral long terminal repeats (LTRs) or of the coding regions are involved in gene silencing in vitro [7-9] and in vivo [10].

The DNA demethylating agent, 5' azacytidine (5' aza-C), an irreversible inhibitor of DNA methyltransferases, is reported to reactivate transgene expression in vitro [7] and in vivo [10, 11]. Using a MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type 1 thymidine kinase (tk) gene, which induces sensitivity to the prodrug ganciclovir (gcv) and the reporter gene ß-galactosidase (lacZ), we recently reported that silencing caused by retroviral LTR hypermethylation can be efficiently prevented in vivo by 5' aza-C administration [12].

To define the factors regulating, preventing, and reversing in vivo gene silencing due to methylation, we analyzed the in vivo effects of 5' aza-C under different experimental conditions. We report the number of injected tumor cells determines tumor onset time and the efficacy of 5' aza-C treatment. On the other hand, escalating doses of gcv do not significantly influence tumor onset times when compared with a standard gcv schedule (10 mg/kg from d + 1 until tumor onset). These results extend our previous findings that transgene methylation may be prevented in vivo with appropriate 5' aza-C and gcv schedules.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 References
 
Retroviral Vector and Producing Cell Line
The vector used in this study derives from an MFG retroviral backbone in which the herpes simplex virus-thymidine kinase (HSV-tk) and Escherichia coli lacZ genes were linked by an IRES sequence as previously described [13]. GP + E86 cells and derivatives [14], and GP + envAM 12 cells and derivatives [15], were grown in complete Dulbecco's modified Eagle's medium (BioWhittaker, Inc.; Walkersville, MD; http://www.biowhittaker.com) with 10% newborn calf serum (GIBCO BRL; Gaithersburg, MD), 2 mM glutamine, and 1% antibiotic. A packaging cell line, GP + envAM12/tk-IRES-lacZ [13], producing 1 x 105 colony-forming units/ml supernatant, was obtained using a "ping pong amplification" procedure according to a previously published protocol [16].

Transduction and Selection Procedure
Untransduced and transduced U937 histyocytic cell lines were grown in RPMI 1640 with 10% fetal calf serum (GIBCO), 2 mM glutamine, and antibiotics.

Gene transfer was performed by cocultivating U937 cells on a layer of mitomycin-treated producing cells for 96 h. Nonadherent U937 cells were harvested and examined for ß-galactosidase activity by 5-bromo-4-chloro-3-indolyl ß-D galactopyranoside (X-gal) staining as described below. Transduced cell populations were enriched in lacZ positive cells by fluorescein-di-ß-D-galactopyranoside (FDG) (Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com) immunofluorescent staining, followed by a fluorescence-activated cell sorting procedure as described below. Sorted cells were cloned by end point dilution. One clone (VB71), expressing lacZ/tk genes in >90% cells, was employed in this study for in vitro and in vivo experiments.

Cytochemical and Immunofluorescent Stainings
Cell aliquots were placed in 96-well plates, centrifugated and fixed for 15 min at room temperature in 0.2% glutaraldehyde, 1% formaldehyde, 0.02 NP40 in phosphate buffered saline (PBS), washed with PBS, and overlaid with a reaction mixture containing 0.5 mg/ml X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCL2, 0.01% sodium deoxycholate, and 0.02% NP40, 0.1 M phosphate buffer (pH 7.3). All reagents were purchased from Sigma.

FDG staining was performed as previously described [17]. Briefly, 1 x 107 cells were placed in polystyrene tubes and the cell suspension was brought to 37°C for 5 min. After adding 100 µl of 2 mM FDG, solution tubes were placed back at 37°C for 1 min and then on ice. Then, 1,800 µl of ice-chilled isotonic incubation medium and 1 µM propidium iodide were added. Stained cells were analyzed and sorted by a fluorescence-activated cell sorter (Epics Elite; Coulter Corp.; Hialeah, FL; http:/www.coulterpharm.com).

The U937/transduced cell cycle during 5' aza-C treatment was determined by flow cytometry analysis of cellular DNA content using DNA-Prep Reagents (Coulter Corp.). Briefly, 1 x 106 cells, after permeabilizing and DNA staining (50 µg/ml propidium iodide), were determined on an Epics Profile II (Coulter Corp.) and then analyzed with the Multicycle Software program (Phoenix Flow Systems; San Diego, CA). The proliferative activity was defined as the sum of all cells in the S + G2/M phases of the cell cycle.

The cell surface phenotypes of untransduced, transduced, and tumor-derived U937 cells were determined using a panel of monoclonal antibodies (mAbs) and an indirect fluorescence labeling method. Briefly, the cells were washed and incubated for 15 min at room temperature in PBS containing 2% human antibody serum. Then, 50 µl aliquots of 1 x 106 cells were placed in tubes for incubation with the mAbs directed against the following antigens: CD11a, CD11c, CD18, CD29, CD34, CD36, CD54, CD58, CD71 (Immunotech; Marseille, France), CD11b and CD38 (Ylem; Rome, Italy), CD13, CD14, CD33, and HLA-DR (Coulter Corp.), and CD45 (Dako; Glostrup, Denmark; http://www.dako.dk). After 30 min incubation at 4°C, cells were washed twice with PBS and resuspended with fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse antibody. The negative control was assessed using isotype-matched, irrelevant mAbs. Stained cells were analyzed on an Epics XL-MCL (Coulter Corp.).

In Vitro 5' Aza-C Treatment
For 1 mo, a 4-µM 5' aza-C concentration was added to the culture medium every 72 h. The percentage of X-gal + cells was assessed with X-gal and FDG stainings every week.

In Vivo 5' Aza-C Treatment
Tumors were induced in 35-day-old severe combined immunodeficient (SCID) mice by a single intraperitoneal injection of VB71 cells.

In experiment A (nine mice per group), group I mice (controls) were injected with 3 x 106 VB71 cells; group II with cells followed by 5' aza-C (2 mg/kg every 48 h until tumor onset); group III cells followed by gcv (10 mg/kg from d + 1 after VB71 injection and prolonged until the onset of tumor); group IV cells plus 5' aza-C as in group II plus gcv as in group III.

In experiment B, group I (10 mice, controls) was injected with 4 x 106 VB71 cells; group II (five mice) with cells followed by 5' aza-C (2 mg/kg every 48 h until tumor onset); group III (seven mice) with cells followed by gcv (10 mg/kg from d + 1 after VB71 injection and prolonged until the onset of tumor); group IV (nine mice) with cells plus 5' aza-C as in group II plus gcv as in group III.

In experiment C, group I mice (10 mice, controls) were injected with 4 x 106 VB71 cells; group II (five mice) with cells followed by 5' aza-C (2 mg/kg every 48 h until tumor onset); group III (seven mice) with cells followed by gcv (150 mg/kg/daily from d + 1 to d + 5 after VB71 injection); group IV (eight mice) with cells plus 5' aza-C as in group II plus gcv as in group III.

LacZ was revealed in imprints of tumor masses by X-gal staining as described above.

Statistical Analysis
Estimates of tumor onset distributions were calculated using the method of Kaplan and Meier [18] and comparisons among curves were performed by the log-rank test [19]. Contrasts between curves were obtained applying the Cox regression model [20].


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 References
 
In Vitro 5' Aza-C Treatment
LacZ expression was monitored by X-gal staining and by FDG staining over a one-month period. In the cell population treated continuously with 5' aza-C, lacZ expression remained at 90% while it decreased slowly and continuously in untreated controls (Fig. 1Go).



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  		Figure 1. LacZ+ cells evaluated by FDG (A) and by X-gal (B) stainings.

 
Analyses of S phase and proliferative activity after 15 and 30 d 5' aza-C treatment revealed no differences between wild-type U937, transduced U937, and transduced U937/5' aza-C (46%, 47%, and 48% of S-G2M phases, respectively). The number of U937 clones obtained in end point dilution assay overlapped in the three different populations (data not shown).

Immunophenotyping of untransduced and transduced cells, treated or untreated with 5' aza-C, performed to analyze the effects of 5' aza-C treatment on the selected cell surface markers, showed no variations in the surface expression.

In Vivo 5' Aza-C Treatment
No significant blood or organ toxicity was observed with either gcv or 5' aza-C at the administered dosages. Toxicity related mortality of <10% overlapped in all groups. Table 1Go shows three different experiments were carried out varying the number of VB71 cells and gcv dosage.


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  		Table 1. Experimental schedules
 
In experiment A (Fig. 2AGo), tumors developed at a median of 29 d (range 28-37) after VB71 injection alone (controls, group I), after a median of 32 d (range 17-45) in group II (VB71 cells plus 5' aza-C), after a median of 35 d (range 28-50) in group III (VB71 cells plus gcv), and after a median of 75 d (range 64-95) in group IV (VB71 cells, gcv and 5' aza-C; p < 0.0001). In particular, tumor onset was significantly delayed (p = 0.02) in mice treated with gcv (group III) compared with controls (group I). The delay in tumor onset was significantly longer (p < 0.0001) when mice were treated with gcv and 5' aza-C (group IV) compared with mice treated with gcv (group III) or with 5' aza-C (group II).



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  		Figure 2. Tumor onset probability in each set of experiments.

 
In experiment B (Fig. 2BGo), tumors developed at a median of 20 d (range 13-21) after VB71 injection alone (controls, group I), after a median of 25 d (range 16-29) in group II (VB71 plus 5' aza-C), after a median of 29 d (range 23-38) in group III (VB71 plus gcv), and after a median of 51 d (range 21-96) in group IV (VB71, gcv, and 5' aza-C) (p < 0.001). Again, the delay in tumor onset was significantly longer when animals were treated with both gcv and 5' aza-C (group IV) compared with mice treated with only gcv (group III) (p < 0.01) and 5' aza-C (group II) (p < 0.0005).

In experiment C (Fig. 2CGo), tumors developed at a median of 20 d (range 13-21) after VB71 alone (controls, group I), after a median of 25 d (range 16-29) in group II (VB71 plus 5' aza-C), after a median of 25 d (range 22-57) in group III (VB71 plus gcv), and after a median of 40 d (range 29-67) in group IV (VB71, gcv, and 5' aza-C) (p < 0.0001). The delay in tumor onset was significantly longer when animals were treated with both gcv and 5' aza-C (group IV) compared with mice treated only with gcv (group III) (p < 0.03) or with 5' aza-C (group II) (p < 0.004).

The delay in tumor onset was significantly longer in group I animals treated according to schedule A compared with group I mice in schedules B and C (p < 0.0001). The delay in tumor onset was significantly longer in group II treated according to schedule A compared with group II mice in schedules B and C (p < 0.02). No significant differences emerged in tumor onset times in any group III mice (Fig. 3AGo). The delay in tumor onset was significantly longer (p < 0.01) in group IV mice treated according to schedule A compared with group IV mice in schedules B and C (Fig. 3BGo).



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  		Figure 3. A) Tumor onset probability in each group III. B) Tumor onset probability in each group IV. n.s. = not significant.

 
LacZ Staining of Tumor Masses
Examination of tumor masses showing lacZ expression was weak (1%-2% lacZ positive cells) in group I in all schedules. It was maintained at baseline levels in group II in all schedules. LacZ expression was absent in groups III and IV of all schedules.

Immunophenotyping Analysis of Tumor-Derived Cells
Immunophenotype studies were carried out on the tumor-derived cells. No variations in the surface expression of any of the antigens were found.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 References
 
DNA methylation in the differential regulation of gene expression is a common eukaryotic DNA modification and is one of the many epigenetic phenomena [21, 22]. It is implicated in developmental gene regulation [23, 24], tissue-specific differentiation [25], and cancer [26, 27].

Several observations in mice [28, 29] and humans [30] suggest methylation is a strong transcriptional repressor of transposed genes and could play a role in protecting the genome from the potentially harmful effects of inserted sequences as part of the genome defense system. Methylation of virally transduced genes could be an obstacle to gene therapy. LTR enhancer sequences are targets for repressing factors and de novo methylation of CpG doublets. Unmethylated LTRs are fully functional but methylated LTRs are inefficient as promoters [7-10].

LTR methylation can be reversed by the demethylating agent 5' aza-C, which is an irreversible inhibitor of cytosine 5-methyltransferases. Hoeben et al. [7] showed in vitro inactivation of MoMuLV LTRs is associated with de novo methylation of cytidine residues in murine fibroblast cell lines. Treating a cell clone with the DNA-demethylating agent 5' aza-C raised the number of ß-gal-positive cells from 8% to over 60% [7]. Jaenisch et al. [11] demonstrated 5' aza treatment of Mov-7 and Mov-10 mice efficiently reactivated a premethylated provirus with a mutation in the coding region causing high methylation and biological inactivity.

The MoMuLV vectors and the HSV-tk gene are commonly used as backbone and suicide gene respectively, in animal experiments [31] and clinical protocols [32]. Several reports have shown HSV-tk expression is inhibited by either methylation of the promoter or of the coding region [8, 9]. However, we have recently shown transgene methylation can be partially prevented by in vivo administration of 5' aza-C [12].

In this report we attempt to define the best conditions for preventing in vivo transgene methylation in order to increase median survival of tumor-bearing mice.

We analyzed the impact of the number of injected tumor cells on tumor onset times. Our results show a one-log increase in the number of infused neoplastic cells (3 x 106 versus 4 x 106 VB71 cells) significantly reduced median survival (from approximately 28 d to 20 d). This shortening of the tumor onset time was independent of the gcv administration schedule. 5' aza-C administration confirmed it exerts a poor antineoplastic activity at the dosage used in this study.

We also compare two different gcv dosages and administration schedules. Franken et al. [33] reported short-term high gcv dosages killed an elevated number of HSV-tk transduced cells. Changing the gcv dosage from 10 mg/kg daily until tumor onset to 150 mg/kg daily for 5 d (from d + 1 to d + 5), in an attempt to kill most tk-expressing cells before the silencing phenomenon started, shortened tumor onset times.

These data show transgene hypermethylation is a slow process that continuously recruits new cells and cannot be prevented by increasing the gcv dosage, but which may be prevented by long-term administration. In a rat model, a retroviral vector carrying genes encoding human adenosine deaminase and neomycin phosphotransferase gradually lost expression over a one-month period [34]. On the other hand, Gram et al. [35] reported gene silencing occurred rapidly after transduction in a murine leukemia virus-derived retroviral vector encoding the autofluorescent humanized green fluorescent protein and neomycin phosphotransferase. The time divergence in these results may be explained by the following observations: A) the propensity to methylation may not be the same in all target cell types [1], and B) different vectors may be methylated in different ways.

As many clinical trials use MoMuLV-based vectors, the results of the present study could have an impact on gene therapy protocols. Clinical reports [36] showed 5' aza-C, infused at a dosage of 2 mg/kg body weight daily for 4 d at two- to four-week intervals, provided long-term benefits in patients with end-stage ß-thalassemia. Neutropenia, the major adverse side effect, was avoided by reducing the dosage and adjusting the timing of administration. As 5' aza-C is well tolerated in vivo under experimental conditions and in humans [36], it might be proposed as a valid way of preventing methylation phenomena.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
References
 
Gene-silencing retroviral vectors by DNA methylation may constitute a serious problem for the success of gene therapies. Our in vivo studies on animal models clearly show transgene hypermethylation inhibits therapeutical effects and reduces the median survival of tumor-bearing mice. Many ongoing clinical trials use the MoMuLV-tk-based retroviral vectors in several diseases (solid tumors, acute leukemia). Transgene inactivation may make antitumoral therapy inefficacious in patients with solid tumors and may cause severe graft-versus-host disease in leukemia patients. Our data show the combination of 5' aza-C and gcv significantly enhances the efficacy of the procedure without being associated with any significant additional toxicity. Finally, our observations may have important implications for future clinical applications of retroviral-mediated gene transfer into somatic cells, where persistent gene expression is crucial for an enduring therapeutic effect.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 
The authors gratefully thank Dr. Geraldine Anne Boyd for her helpful criticism and comments.

This work was supported by the Italian Ministry of Scientific and Technological Research (MURST) and the Italian Association for Cancer Research (AIRC).


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 

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Received June 5, 2000; accepted for publication August 8, 2000.




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