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Clinical Cancer Research Vol. 5, 3639-3644, November 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

A Cell Type-specific and Gap Junction-independent Mechanism for the Herpes Simplex Virus-1 Thymidine Kinase Gene/Ganciclovir-mediated Bystander Effect1

Frédéric Princen, Pierre Robe, Chantal Lechanteur, Marc Mesnil, Jean-Michel Rigo, Jacques Gielen, Marie-Paule Merville and Vincent Bours2

Laboratory of Medical Chemistry and Medical Oncology [F. P., C. L., J. G., M-P. M., V. B.] and Departments of Human Physiology [P. R., J-M. R.] and Neurosurgery [P. R.], University of Liège, 4000 Liège, Belgium, and Unit of Multistage Carcinogenesis, IARC, 69372 Lyon, France [M. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor cells expressing the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene are killed by nucleoside analogues such as ganciclovir (GCV). GCV affects not only the cells expressing HSV-tk but also neighboring cells that do not express the gene; this phenomenon commonly is called "bystander effect." GCV metabolites transfer via gap junctional intercellular communication (GJIC) accounts for the bystander effect in different cell lines, but other mechanisms have also been described. In this study, we analyzed the mechanisms of the bystander effect in two cell lines exhibiting different capacities of communication (DHD/K12 and 9L). The 9L cells exhibited a very good bystander effect, which was completely blocked by a long-term inhibitor of GJIC, 18 {alpha}-glycyrrhetinic acid. DHD/K12 cells exhibited a moderate bystander effect that was not abolished by 18 {alpha}-glycyrrhetinic acid or 1-octanol, another strong inhibitor of GJIC. Interestingly, we also observed a bystander effect in cultures where HSV-tk-expressing DHD/K12 cells were physically separated from their untransfected counterparts but grown in the same medium. Moreover, the transfer of filtered conditioned medium from GCV-treated HSV-tk-expressing DHD/K12 cells to DHD/K12 parental cells induced a decrease of survival in a concentration-dependent manner, suggesting that the bystander effect in this cell line was mediated by a soluble factor.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transfer of suicide genes into tumor cells constitutes a novel approach for the treatment of human cancers. The HSV-tk3 gene has been used successfully to eradicate a variety of tumors in animal models (1, 2, 3, 4, 5, 6, 7, 8) and has also been evaluated in several clinical protocols (9 , 10) . This strategy takes advantage of HSV-tk capacity to phosphorylate nucleoside analogues such as GCV into GCV-monophosphate. Subsequent phosphorylation of GCV-monophosphate by cellular kinases leads to the formation of cytotoxic GCV-triphosphate, which is incorporated into DNA and inhibits DNA polymerase (11, 12, 13) . Interestingly, untransduced adjacent cells are also sensitive to GCV treatment. Therefore, transduction of a limited number of cells with the suicide gene could be sufficient to eradicate a tumor (5 , 7 , 14) . This phenomenon has been called "bystander effect."

Several mechanisms have been proposed to explain the bystander effect. It could be linked to the level of TK activity in transduced cells (15) or to the uptake of apoptotic vesicles by the adjacent nontransduced cells (16) . Moreover, the transfer of GCV-triphosphate through gap junctions plays an important role in this mechanism both in vitro and in vivo (17, 18, 19, 20, 21, 22) . Finally, other phenomena such as immune reaction (23 , 24) or blood vessel destruction (25) seem to also be involved in the bystander effect in vivo.

In the present work, we studied the mechanisms of the bystander effect in vitro in two cell lines: a rat colon adenocarcinoma cell line (DHD/K12) and a rat gliosarcoma cell line (9L). These cell lines exhibit different capacities of communication and have been used as targets for in vivo HSV-tk gene therapy studies (5 , 25 , 26) . We showed that GJIC was essential for the bystander effect in 9L cells, as was described previously in numerous cell lines, but not in DHD/K12 cells where the release of soluble factors by the HSV-tk-expressing GCV-treated cells seems to play an important role.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Reagents.
Rat colon adenocarcinoma DHD/K12/Prob cells were grown in DMEM medium (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (20% FBS in the experiment described in Fig. 1Citation ), 1% 200 mM glutamine, 1% 1 M HEPES, 1% 0.55 mM arginine, and 1% 5 mg/ml penicillin-streptomycin. Rat gliosarcoma 9L cells were grown in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 1% nonessential amino acids, 1% 100 mM sodium pyruvate, and 1% 5 mg/ml penicillin-streptomycin. Cells were grown at 37°C in 5% CO2.



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  		Fig. 1. Comparison of the bystander effect in DHD/K12 (A) and 9L (B) cell lines. HSV-tk+ and HSV-tk- cells were mixed in different ratios in 96-well plates. These cocultures were treated with 20 µM GCV alone ({blacksquare}), 20 µM GCV + 70 µM AGA ({circ}), or 20 µM GCV + 1 mM 1-octanol ({blacktriangleup}). These cocultures were grown for 7 days (DHD/K12) or 4 days (9L). Cell viability was measured with the WST-1 test. Statistical analyses were performed in all culture conditions for the two cell lines. AGA and 1-octanol did not block the bystander effect in DHD/K12 cells. AGA blocked the bystander effect in the 9L cells at all ratios of HSV-tk+ and HSV-tk- cells (P < 0.05; ANOVA, Tukey post-tests).

 
9L-TK cells infected with a Moloney murine leukemia virus retrovirus containing the HSV-tk gene driven by an SV40 promoter were obtained from C. Grignet (Laboratory of Virology, University of Liège, Belgium). The DHD/K12-TK cell line was generated by infection with the same retrovirus (26) .

AGA and 1-octanol were obtained from Sigma-Aldrich NV/SA (Bornem, Belgium). AGA was dissolved in DMSO.

Estimation of the Bystander Effect.
To test the bystander effect, various proportions of HSV-tk-expressing cells were mixed (100, 50, 25, 15, 10, 5, and 0%) with corresponding untransduced cells. These cocultures were then treated daily with 20 µM GCV (Roche S.A., Brussels, Belgium) for 7 days (DHD/K12) or 4 days (9L). At the end of the treatment, cell viability was tested with WST-1 proliferation reagent as recommended by the manufacturer (Boehringer Mannheim, Mannheim, Germany). During the GCV treatment, 70 µM AGA (9L cells) and 70 µM AGA or 1 mM 1-octanol (DHD/K12 cells) were added to the medium.

Before the 1-octanol bystander experiment, DHD/K12 cells were pretreated for 5 days with 1 mM 1-octanol.

Dye Transfer.
GJIC was assessed by dye-transfer assay using Lucifer yellow as a tracer according to Stewart (27) . Briefly, cells were harvested in culture medium and seeded at low density on polyornithine coated glass coverslips. The cells were incubated in test medium and grown to confluence for 5–6 days. The medium was changed every 2 days. Coverslips were then placed in the perfusion chamber of a Zeiss fluorescence microscope and perfused with EA01 buffer ( 137 mM NaCl, 5.7 mM KCl, 1.8 mM CaCl2, 22.2 mMD-Glucose, 10 mM HEPES). A single cell in a confluent areas was microinjected with Lucifer yellow dye (sigma; 5% w/v in 0.1 M LiCl) by passing 0.5 Hz-500 ms-1 mA hyperpolarizing current pulses for 30 s through the electrode. The total number of colored adjacent cells was counted 60 s after the end of the injection.

Separated Culture Experiments.
HSV-tk-expressing cells (2 x 105 for DHD/K12-TK and 1 x 105 for 9L-TK) were seeded on a matrigel-precoated millicell (12 mm diameter; 0.4 µm pore size; Millipore Corporation, Bedford, MA). The millicells were placed in 24-well plates. Untransduced DHD/K12 (104 cells) or 9L (5 x 103 cells) were seeded in the wells. The two populations of cells (HSV-tk+ cells and HSV-tk- cells) thus were physically separated in the same culture medium. GCV (20 µM) was added to the medium, and the GCV-containing medium was replaced every day for 7 days (DHD/K12) or 4 days (9L). After the treatment, the HSV-tk- cells in the 24-well plates were harvested, and the total number of living cells was determined by the trypan blue exclusion method and compared to the HSV-tk- control cells, grown in separate wells.

Medium Transfer.
HSV-tk-expressing DHD/K12 cells were grown in 24-well plates (2 x 105 cells/well) in different volumes of medium (300, 500, 700, and 1000 µl) containing or not (control) 20 µM GCV. Twenty-four h later, the medium was collected, filtered on a Sartorius Minisart (pore size, 0.2 µm; Sartorius AG, Göttingen, Germany) and transferred into wells containing untransduced DHD/K12 cells (104 cells/well; 24-well plate). This operation was repeated daily for 7 days. After the treatment, HSV-tk- DHD/K12 cells were harvested, and the total number of living cells was determined by the trypan blue exclusion method and compared to HSV-tk- DHD/K12 cells, grown separately.

Statistical Analysis.
All statistical analyses were performed using a commercially available software (GraphPad Software Incorporation, San Diego, CA). Cell survival experiments involving 9L and DHD/K12 cells were analyzed using a parametric unpaired two-way ANOVA with Tukey post-tests. Dye-transfer experiments were compared using a nonparametric two-way ANOVA with Dunn’s post-tests for both DHD/K12 and 9L cells because it appeared that the results did not fit a gaussian distribution. Differences were considered significant when the probability (P) was <0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of the Bystander Effect in DHD/K12 and 9L Cell Lines.
We compared the extent of the bystander effect in 9L and DHD/K12 cells. In these experiments HSV-tk-expressing cells (HSV-tk+) and parental cells (HSV-tk-) were mixed at different ratios and treated with 20 µM GCV.

The bystander effect varied among the two cells lines (Fig. 1)Citation . 9L cells showed the highest bystander effect; in the presence of 5% HSV-tk+ cells, 52% of the cells were killed by the GCV treatment. DHD/K12 cells showed only a moderate bystander effect; 5 or 10% HSV-tk+ cells led to the killing of 15 or 40% of the cells, respectively. Because 9L cells were treated only for 4 days, a higher proportion of the cells were still viable at the end of the experiment when 100% TK+ cells were considered. Longer treatments led to a complete eradication of the TK+ 9L cells (data not shown).

To examine whether GJIC was responsible for the bystander effect observed in DHD/K12 and 9L cells, GCV treatments were performed in the presence of AGA, a selective and reversible inhibitor of GJIC (Fig. 1Citation ; Ref. 28 ). AGA alone did not have any toxicity on the different cell lines (data not shown). We observed that 70 µM AGA completely inhibited the bystander effect in 9L cells but not in DHD/K12 cells. To confirm these results, GCV treatments of DHD/K12 cells were also performed in the presence of 1-octanol, another selective and reversible inhibitor of cell-cell communication via gap junction (Fig. 1ACitation ; Refs. 29, 30, 31 ). 1-Octanol alone did not have any toxicity on DHD/K12 cells, but it had a strong cytotoxic effect, even at low concentrations, on 9L cells (data not shown). We observed that 1-octanol, like AGA, did not significantly inhibit the bystander effect in DHD/K12 cells.

These results suggested that the bystander effect was related to GJIC in 9L cells but not in DHD/K12 cells.

Effect of AGA and 1-Octanol on Intercellular Communications in DHD/K12 and 9L Cell Lines.
The effect of AGA on GJIC in DHD/K12 and 9L cells and of 1-octanol on GJIC in DHD/K12 cells was further studied by dye transfer. Cells were grown to confluence, Lucifer yellow was injected into a single cell, and the number of dye-colored cells was counted 60 s later. These experiments demonstrated that 9L cells communicated very efficiently because the injection of a single cell allowed dye transfer to an average of 102.1 cells. Conversely, DHD/K12 cells communicated poorly, the dye being transferred to 3.09 cells (Table 1)Citation .


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  		Table 1 Gap junctional communication after microinjection of Lucifer yellow

 
In both cell lines, AGA treatment significantly inhibited the number of dye-coupled cells per microinjection (3.4 communicating cells after AGA treatment in 9L cells and 1.05 after AGA treatment in DHD/K12 cells). In DHD/K12 cells, 1-octanol also significantly inhibited the number of dye-coupled cells (0.64 communicating cells after 1-octanol treatment; Table 1Citation ).

These data confirmed that the bystander effect was depen-dent on GJIC in 9L cells because it was blocked by AGA. Furthermore, the bystander effect in DHD/K12 cells was independent of GJIC because it was not affected by AGA or 1-octanol treatments. It should however be noted that the constitutive intercellular communication was much lower in DHD/K12 cells compared with that observed in 9L cells. This DHD/K12 GJIC might be too low to allow significant transfer of GCV metabolites, indicating that another mechanism is probably responsible for the bystander effect in these cells.

Role of Soluble Factors in the Bystander Effect in DHD/K12 and 9L Cell Lines.
We investigated whether soluble factors produced by dying HSV-tk+ cells might play a role in the bystander effect in DHD/K12 and 9L cells. HSV-tk- cells were first cocultivated with HSV-tk+ cells in the same medium but separated by a membrane filter as described in "Materials and Methods" (Fig. 2)Citation . Under these conditions, GCV treatment induced the killing of 77.5% of HSV-tk- DHD/K12 cells compared with control untreated cells. However, in similar conditions, we did not observe any toxicity on HSV-tk- 9L cells (Fig. 2)Citation or on HSV-tk- C6 cells (data not shown). These results showed that cell-cell contacts are required for bystander killing in 9L cells but not in DHD/K12 cells. The bystander effect in this cell line was probably due to the release of soluble factors from the dying HSV-tk+ cells in the medium.



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  		Fig. 2. Bystander effect between separated HSV-tk+ and HSV-tk- cells. HSV-tk- cells were plated in 24-well plates (104 DHD/K12 cells and 5 x 103 9L cells per well), and HSV-tk+ cells were seeded on a matrigel-precoated millicell (0.4 µm pore size) positioned in the same well over the HSV-tk- cells. The two populations were thus separated in the same well and cultivated in the same medium. Cells were untreated or treated with GCV (20 µM). The medium was replaced every day for 7 days (DHD/K12) or 4 days (9L). After treatment, survival was assessed by the trypan blue exclusion method and compared to the controls. Each column represents the mean of at least six independent experiments (bars, SD). The differences between the GCV-treated and untreated DHD/K12 cells were significant (P < 0.001; ANOVA parametric Tukey post-tests).

 
To confirm this observation, separate experiments were performed on DHD/K12 cells. HSV-tk+ DHD/K12 cells were grown in different volumes of medium (300, 500, 700, and 1000 µl) and treated with GCV. Conditioned medium from HSV-tk+ GCV-treated DHD/K12 cells was filtered and then transferred onto HSV-tk- DHD/K12 cells. The transfer of 300 µl of conditioned medium induced the killing of 69.3% of HSV-tk- DHD/K12 cells (Fig. 3)Citation , whereas filtered medium from untreated cells did not induce any cell death. Moreover, the toxicity of conditioned medium decreased when the volume increased (e.g., transfer of 700 µl of conditioned medium led to the killing of only 35.8% of HSV-tk- DHD/K12 cells). These results show that the cytotoxicity of the soluble factors contained in the conditioned medium was related to their concentration. Thus, these data confirm that the bystander killing was mediated by soluble factors released by the GCV-treated HSV-tk+ DHD/K12 cells.



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  		Fig. 3. Medium from GCV-treated HSV-tk+ DHD/K12 cells induced HSV-tk- DHD/K12 cell death in a concentration-dependent manner. Different volumes (300, 500, 700, or 1000 µl) of filtered (0.2 µm pore size) medium from 2 x 105 HSV-tk+ GCV-treated or untreated (control) cells were transferred into wells containing 104 HSV-tk- cells. This operation was repeated daily for 7 days. After GCV treatment, survival was assessed by the trypan blue exclusion method and compared to the cells cultivated in normal, unconditioned medium (100%). Each column represents the mean of at least six independent experiments (bars, SD). The P values from the ANOVA parametric Tukey post-tests are indicated.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The transfer of suicide genes to tumor cells might become an important tool for cancer gene therapy. The existence of a bystander effect in the HSV-tk/GCV system makes cancer gene therapy feasible because transduction of 100% of the cells is not required for tumor eradication. The understanding of the mechanisms responsible for the bystander effect may allow its modulation and could, therefore, lead to an enhanced therapeutic effect.

Numerous studies have shown that gap junctions between adjacent cells allow the transfer of phosphorylated GCV from HSV-tk+ cells to HSV-tk- cells both in vitro and in vivo (17, 18, 19, 20, 21, 22) . To investigate the role of gap junctions in the bystander effect in vitro, we compared two cell lines that exhibit different capacities of communication. After GCV treatment, 9L cells showed a strong bystander effect. The inhibition of both the bystander effect and the dye transfer after AGA treatment clearly showed the existence of a link between the two phenomena in this cell line. Separation of HSV-tk+ and HSV-tk- 9L cells by a filter membrane prevented any toxicity in HSV-tk- cells, confirming the necessity of direct intercellular contacts for bystander killing. Therefore, in 9L cells, the bystander effect is most likely linked to the presence of functional GJIC.

AGA and 1-octanol did not significantly inhibit the bystander effect in DHD/K12 cells, although they significantly inhibited gap junctional communication, suggesting that GJIC was not involved in this process. Separation of HSV-tk+ and HSV-tk- DHD/K12 cells by a filter membrane led the killing of HSV-tk- cells after GCV treatment. Moreover, the transfer of different volumes of filtered conditioned medium from HSV-tk+ GCV-treated cells to HSV-tk- cells also killed these cells in a concentration-dependent manner. These data indicated that direct intercellular contacts was not required for the bystander effect in DHD/K12 cells. This phenomenon could be explained by the phagocytosis by HSV-tk- cells of apoptotic vesicles released in the medium by dying HSV-tk+ cells as has been described by Freeman et al. (16) or by the transmission of soluble factors. However, the filtration of conditioned medium through 0.2 µm filters probably retained apoptotic bodies and thus supported the hypothesis of soluble factors mediating the bystander effect in DHD/K12 cells.

Our report as well as other reports indicate that the mechanism of the bystander effect is cell type specific. A recent report showed that the inhibition of GJIC by 1-octanol in lung cancer cell lines did not reduce the tumor cell killing through the bystander effect. However, the authors failed to detect any bystander effect after physical separation of HSV-tk+ and HSV-tk- cells, indicating that this effect still required intercellular contacts (29) . Different studies showed that phosphorylated GCV cannot pass through the cell membranes (17) , but it was also reported that rat hepatoma cells, which express connexin 43, were able to take up membrane-impermeable dye from the culture medium through connexon hemichannels that did not align with hemichannels from neighboring cells to form functional GJIC (32) . Another group reported that the bystander effect was not correlated with gap junctional communication in two human colon adenocarcinoma cell lines (SW620 and HT-29) and that GCV nucleotides can be transferred from HSV-tk-expressing to nonexpressing cells in cocultures even in cell lines exhibiting low levels of GJIC. If functional hemichannels were responsible for the uptake of GCV nucleotides from the extracellular environment, this mechanism would depend on the proximity of neighboring cells because extracellular nucleotides would be rapidly diluted in the medium or degraded if the cells were too far apart (33) . In contrast, our results suggested that cellular contacts or cellular proximity were not required for bystander effect in DHD/K12 cells and provide strong evidence for the existence of a novel mechanism: the release of soluble factors. The identification of these soluble factors will require additional experiments.

We think that multiple pathways rather than a single pathway may be involved in the mechanism of bystander effect in different cell types. Clearly, GJIC plays a crucial role in the bystander effect in numerous cell types as shown in many reports. Therefore, some studies investigated the possibility of enhancement of the bystander effect, in vitro and in vivo, by pharmacological modulation of gap junctions (34 , 35) . But as we and other reported, it is obvious that other mechanisms are also involved in distinct cell types. The understanding of these mechanisms will be a prerequisite to the development of new pharmacological tools aimed at the modulation of the bystander effect.


   ACKNOWLEDGMENTS
 
We thank Dr. C-M. Calberg for the retroviral vector and the tk-producing GP+EnvAm12 packaging cell line. We are most thankful to Prof. G. Moonen and Prof. A. Stevenaert for their support.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by grants from FNRS-Télévie, the "Center Anti-Cancéreux" (ULg, Liège, Belgium), "Concerted Action Program, Convention 97/02-214" Communauté Française de Belgique, and "Oeuvre Belge du Cancer" (Brussels, Belgium). F. P. is supported by a Fonds pour la formation a la Recherche dans L’Industrie et dans L’Agriculture F.R.I.A. fellowship, and C. L. is supported by a "Concerted Action Program" grant. P. R. is Research Assistant and V. B. and M-P. M. are Research Associates at the National Fund for Scientific Research (FNRS, Belgium). Back

2 To whom requests for reprints should be addressed, at Medical Oncology, CHU B35, University of Liège, Sart-Tilman, 4000 Liège, Belgium. Phone: 32-4-3662482; Fax: 32-4-3664534; E-mail: vbours{at}ulg.ac.be Back

3 The abbreviations used are: HSV-tk, herpes simplex virus type 1 thymidine kinase gene; GCV, ganciclovir; TK, thymidine kinase; GJIC, gap junctional intercellular communication; FBS, fetal bovine serum; AGA, 18-{alpha}-glycyrrhetinic acid. Back

Received 6/ 1/99; revised 8/13/99; accepted 8/16/99.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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