Bystander Effect in Herpes Simplex Virus-Thymidine …...[CANCER RESEARCH 60, 3989–3999, August 1,...

[CANCER RESEARCH 60, 3989–3999, August 1, 2000]

Review

Bystander Effect in Herpes Simplex Virus-Thymidine Kinase/Ganciclovir CancerGene Therapy: Role of Gap-junctional Intercellular Communication1

Marc Mesnil 2 and Hiroshi Yamasaki3

Unit of Multistage Carcinogenesis, IARC, F-69372, Lyon cedex 08, France

Abstract

Antitumor suicide gene therapy is one of the emerging strategiesagainst cancer. It consists of the introduction into cancer cells of a genecapable of converting a nontoxic prodrug into a cytotoxic drug. Becausethis therapeutic gene cannot be easily introduced into the whole cellpopulation of a tumor, the successful eradication of tumors depends on aphenomenon called the “bystander effect,” by which the introduced genecan affect even cells in which it is not itself present. From a therapeuticpoint of view, it may be crucial to enhance this phenomenon throughvarious means to achieve tumor eradication. One such suicide gene, thethymidine kinase gene from the herpes simplex virus, in combination withthe prodrug ganciclovir, has been extensively and successfully used insome animal models exhibiting a strong bystander effect. Among themechanisms involved in this phenomenon, gap junctional intercellularcommunication (GJIC) is directly involved in the transfer of the toxicmetabolites of ganciclovir, which pass directly from herpes simplex virusthymidine kinase-expressing cells to surrounding cells that do not expressit. Because GJIC appears to be a mediator of the bystander effect bothinvitro and in vivo, here we review possible molecular strategies for enhanc-ing the extent of tumor cell death by increasing the intratumoral GJICcapacity.

Introduction

Despite clear progress, conventional radiotherapeutic and chemo-therapeutic treatments against cancer still place a burden on patients;their systemic administration can even lead to the appearance ofsecondary cancers (1, 2). Local strategies and better targeted weaponsto destroy only tumor cells without affecting neighboring tissues canbe developed using knowledge of carcinogenic processes. Some suchemerging strategies make use of pharmaceutical drugs designed toblock molecular pathways that are activated in cancer cells (3), thesteps of carcinogenesis that make cancer cells invasive (4), or angio-genesis (5, 6). Strategies aimed at suppressing the expression of anoncogene or restoring the function of a defective tumor suppressorgene have already been studied experimentally, with the transfer oftherapeutic genes into cancer cells to correct their genetic errors (7, 8).However, this kind of gene therapy can target one of several genesresponsible for the malignant phenotype. Because no single gene hasbeen shown to be altered in all human tumors, approaches to genetherapy that can operate independently of the genetic background ofthe cancer cells are therefore required. Such alternative gene therapiesmust focus on the eradication of the tumor cells. In particular, local

and intratumoral chemotherapy can be achieved through gene therapyby inserting genes conferring drug sensitivity into the tumor cells, thusavoiding the systemic toxicity that occurs during conventional che-motherapeutic treatments. One such strategy involves “suicide genetherapy,” in which a gene coding for an enzyme that converts anontoxic prodrug into a lethal compound is inserted into the tumorcells. Thus far, the most successful enzyme/prodrug combinationtestedin vitro and in animal models is HSV-tk,4 which has a highaffinity for the prodrug GCV (9–12). This is an attractive approachbecause it targets the cell division process that is a characteristic of alltumor cells (13, 14).

To accomplish complete killing of a tumor, it was initially thoughtthat the suicide gene HSV-tk would have to enter each individual cell.Given the low efficiency of current methods of gene delivery, this wasnot a realistic prospect. Fortunately, the HSV-tk/GCV strategy resultsin the death not only of the recipient (HSV-tk1) tumor cells but alsoof surrounding nonrecipient (HSV-tk2) tumor cells. This phenome-non is called the “bystander effect,” and it compensates for the lowefficacy of vectors (viral or liposomal) in transferring genes into thetumor cells (15). Because gene transfer will probably remain a lim-iting factor for suicide gene therapy, it is crucial to induce a potentbystander effect. One approach is to enhance the local transfer of toxicmetabolites from the transduced cells to the surrounding untransducedones. A promising strategy is mediation of this transfer through GJIC.Propagation of toxic metabolites through GJIC was first observed 30years ago (16), and we have shown its possible use in cancer therapyusing anin vitro model (17).

Here we review recent progress made in the use of GJIC to enhancethe bystander effect during cancer gene therapy mediated by theHSV-tk/GCV strategy.

The Molecular Mechanisms of the HSV-tk/GCV Therapy

HSV-tk (18) has a high affinity for acyclic analogues of deox-yguanosine such as ACV and GCV; this affinity may explain theantiherpetic and anticytomegalovirus activities of these drugs (19–21). The growth of cells infected with attenuated herpes simplex virusis inhibited by exposure to ACV or GCV, an action mediated by theHSV-tk gene of the viral genome (22–24). GCV is a better inhibitorof the growth of cells transfected with the HSV-tk gene than ACV,and, in particular, it is 10-fold more potent than ACV as an inhibitorof the growth of HSV-tk gene-transfected tumor cells (25).

The cytotoxic effect of guanosine analogues on cells expressingHSV-tk was demonstrated two decades ago (22). Its application tocancer therapy was first tested by transfecting BALB/c murine sar-coma cell lines with the HSV-tk gene. GCV treatment was sufficientto induce complete regression of palpable tumors that formed afterinjection of the transfected cells into mice (26). The effect of GCV

Received 12/23/99; accepted 5/31/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by United States National Cancer Institute Grant R01-CA-40534, Asso-ciation for International Cancer Research Grant No. 96-27, and the Association pour laRecherche sur le Cancer.

2 Present address: Department of Neuropharmacology, INSERM U114, Colle`ge deFrance, 11 place M. Berthelot, F-75231, Paris cedex 05, France.

3 To whom requests for reprints should be addressed. Present address: Faculty ofSciences, Kwansei Gakuin University, 1-1-155 Uegahara, Nishinomiya, Hyogo 662,Japan.

4 The abbreviations used are: HSV-tk, herpes simplex virus thymidine kinase; cAMP,cyclic AMP; Cx, connexin; GCV, ganciclovir; GJIC, gap junctional intercellular com-munication; GJ, gap junction; HGPRT, hypoxanthine guanine phosphoribosyltransferase;ACV, acyclovir; GCV-P, phosphorylated GCV.

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treatment was specific and did not induce resorption of HSV-tk2

tumors. After similar GCV treatment, this strategy also led to theresorption of palpable tumors after s.c. injection of murine K2 sar-coma or Ly18 lymphoma cells transduced with retroviral vectorsbearing the HSV-tk gene (9). Similar results were obtained with rat C6glioma cells that were transduced with the HSV-tk gene beforeinjection into nude mice (27).

The cytotoxic effect of GCV results from its incorporation intoDNA by a process involving several steps, starting with its phospho-rylation to form a monophosphate (ganciclovir monophosphate). Be-cause its affinity for eukaryotic thymidine kinases is 1000 timeslower, GCV is essentially phosphorylated only by the viral enzymeHSV-tk (23, 28). The ultimate product of further phosphorylation bycellular kinases, GCV-triphosphate, competitively inhibits incorpora-tion of the endogenous DNA precursor dGTP into DNA (29–31).Moreover, the GCV-terminated strands of DNA are poor substratesfor DNA chain elongation (29), therefore the elongation of DNAstrands is prevented (30, 32, 33), leading to cell death. Apart from afew possible exceptions with certain cell types (34), the death isapoptotic and is probably independent of the p53 pathway (35–37).Indeed, tumor cell lines exhibiting mutated p53 are also sensitive toHSV-tk vectors, hence this therapeutic strategy can be applied irre-spective of the p53 status of the tumors (38).

Other mechanisms of HSV-tk/GCV cytotoxicity also appear tooperate. Recent studies suggest that the high toxicity of GCV com-pared with that of other substrates of HSV-tk such as ACV or1-b-D-arabinofuranosylthymine is due not only to GCV triphosphatebut also to the incorporation of GCV monophosphate into the wholegenome (39). Moreover, independent of cell replication, some toxicityof GCV to quiescent cells expressing HSV-tk, such as thyrocytes andhepatocytes, has been reported (37, 40). The mechanism underlyingthis phenomenon is not yet clearly understood but could be theconsequence of inhibition of mitochondrial DNA polymerase (37).However, replicating DNA is considered to be the major target incancer cells for GCV activated by HSV-tk.

The HSV-tk/GCV Bystander Effect

The first in vivo experiments showed that systemic GCV treatmentcan eradicate a tumor in which all cells express HSV-tk (9, 26, 27).These experiments represented an ideal situation in the sense that allof the malignant cells injected into the animals expressed the suicidegene. However, in practical therapeutic approaches, only some of thetumor cells can be reachedin situ with a transgene.

One way to overcome the limited transfer of the suicide gene intothe tumor cells is to continuously producein situ retroviral particlescarrying the HSV-tk gene. Such an approach, which is closer topossible therapeutic applications, was tested by coinjecting fibrosar-coma cells and 3T3 fibroblasts producing retroviral vectors carryingthe neomycin resistance (NeoR) gene (10). Analysis of the tumorsinvitro demonstrated that theNeoRgene was transferred to some (9%)of the tumor cells, which thus became resistant to neomycin treatment(10). The same strategy was used, replacing theNeoRgene with theHSV-tk gene. Surprisingly, although the suicide gene transfer to thetumor cells was again much less than 100%, the tumors rapidlyregressed in most of the animals. The growth of s.c. injected tumorcells of which 50% or even only 10% expressed HSV-tk could beprevented by i.p. injections of GCV (10). This observation meant that,contrary to the original expectation,in situ transfer of the HSV-tkgene into a tumor could lead to its regression, even if the suicide genewas not introduced into all of the tumor cells. It also suggested thepossibility of eliminating an established tumor byin situ injection ofa HSV-tk producer cell line. This was indeed observed in Fischer 344

rats carrying a 9L glioma previously implanted in a cerebral hemi-sphere. Five days later, cells producing the HSV-tk retroviral vectorwere injected intratumorally by stereotactic guidance. Subsequent i.p.administrations of GCV induced complete tumor regression in nearlyall animals (10). When theb-galactosidase gene was used as atransduction marker,in situobservation of the tumors showed that theextent of transduction of the gene into the glioma cells varied greatly(10–70%). This implies that cells that did not express the HSV-tkgene became sensitive to GCV treatment even if 30–90% of the tumorcells lacked the suicide gene (41). This phenomenon was not specificto this cell line because the transfer of sensitivity to GCV from cellsinfected with a retrovirus bearing the HSV-tk gene to neighboring“naive” cells has also been observed bothin vitro andin vivo with ratC6 glioma cells (42).

GJIC as a Major Mechanism of the Bystander Effect

All of the in vivo studies described above have demonstratedpropagation of sensitivity to GCV from cells expressing HSV-tk tonearby tumor cells that do not express it. Such a bystander tumor-killing phenomenon (10) has been termed the “bystander effect” (43).

The involvement of GJs in the bystander effect was suggested (44,45) because of the analogy between this phenomenon and the meta-bolic cooperation assay that was described three decades ago (16).This was an assay to estimate the extent of the GJIC capacity of cellsin culture by mixing cells deficient in HGPRT (HGPRT2 cells),which are unable to metabolize hypoxanthine, with normal cells (46).The transfer of the toxic metabolite from HGPRT1 cells to HGPRT2

cells was proven to be mediated by GJs (47), and the extent of toxicity(“kiss of death”) in the coculture reflected the capacity of the cells tocommunicate through GJs. The analogy between this kiss of deathphenomenon and the bystander effect was clear to Moolten (26), whomade the first observation of this effectin vitro, which was “presumedto reflect transfer of GCV-P by metabolic cooperation betweenHSV-tk positive and HSV-tk negative cells” (Ref. 26; see Fig. 1).

The hypothesis of metabolic cooperation to explain the bystandereffect was supported by direct observation of the transfer of toxicderivatives of radiolabeled GCV from HSV-tk1 cells to HSV-tk2

cells (48). The implication of GJs in this transfer was based on theknown cell-to-cell transfer of phosphorylated nucleotides (16, 49).GJs are intercellular channels that are made up from two juxtaposedtransmembrane hemichannels (connexons) provided by the adjacentcells (reviewed in Ref. 50; see Fig. 2A). Each connexon is composedof six Cx protein subunits with a central pore through which GCV-Phas appropriate size (Mr ,1000) (51) and properties to pass, as hasbeen shown for other nucleotides such as cAMP and ADP or othersecond messengers (52, 53).

The mediation of the bystander effect by GJs is particularly inter-esting because GJs seem to transfer toxic metabolites very efficiently.For instance, it has been shown in three-dimensional cultures that only1% of a tumor cell mass can significantly alter the growth of a mixtureof cells (HGPRT2 and wild-type cells) in collagen gel containing6-thioguanine, if they are extensively coupled (54).

In Vitro GJIC-mediated Bystander Effect

The first in vitro study to exploit GJIC to spread a cancer thera-peutic effect was conducted in our laboratory over a decade ago (17).This study demonstrated that diffusion of cytotoxic agents via GJICcan improve chemotherapy efficiency. Because of the lack of GJICbetween normal and transformed cells, the killing was selective, andonly the transformed cells were eliminated (17). We later demon-strated that GJs can also mediate a bystander effect in cancer genetherapyin vitro by using HeLa cells, which exhibit very poor GJIC

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HSV-tk/GCV BYSTANDER EFFECT AND GJs



Fig. 1. In a tumor cell population, only a few cells can be reached by vectors carrying the HSV-tk gene. When those cells do express the tk gene (red nucleus), they become sensitiveto GCV, which diffuses through the cell membrane. The HSV-tk1 cells are killed by the GCV-P they produce.A, because the GCV-P molecules cannot pass through the cell membrane,theoretically, only the HSV-tk1 cells (red cells) should be killed by GCV treatment.B, a direct diffusion from cytoplasm to cytoplasm of GCV-P could induce a bystander effectsufficient to eradicate a tumor cell population, even if only a few cells are HSV-tk1.

Fig. 2. Bystander effect in cancer gene therapy due to GJIC.Toxic GCV-P molecules can pass from a cell expressing theHSV-tk (tk1) gene to neighboring HSV-tk2 tumor cells.A, thetoxic GCV-P molecules (red arrows) pass from HSV-tk1 cellsto HSV-tk2 cells through the GJs.B, in HeLa cells, the by-stander killing effect is observed only when cells expressing theHSV-tk gene communicate (as shown by the dye transfer assayon theleft) through GJs. Cells expressing HSV-tk were mixedwith their HSV-tk2 counterparts at a 1:1 ratio. Similar resultswere obtained when only 10% of the cells were HSV-tk1.

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capacity (Fig. 2B). When HeLa cells were transfected with an expres-sion vector carrying the HSV-tk gene (HSV-tk1 cells), they becamehighly sensitive to GCV. If these cells were mixed with untransfectedHeLa cells (1:1 ratio), GCV failed to eliminate the whole cell popu-lation. However, the outcome was quite different when we used HeLacells that were able to communicate through GJs as a result oftransfection of a gene coding for a common Cx, Cx43. Under theseconditions, not only the HSV- tk1 cells but also the HSV-tk2 cellswere killed by GCV, even if the culture contained only 10% HSV-tk1

cells (Fig. 2B). This bystander effect, by which the toxicity of GCVwas transmitted to the remaining 90% of the culture, was clearlymediated by GJs because it was inhibited either by a lack of contactbetween the two cell types or by treating the cells with a long-terminhibitor of GJIC (55). These findings are supported by those fromother laboratories reporting that the level of GJIC is predictive of (orcorrelated with) the extent of the bystander effectin vitro, whateverthe origin of the cancer cell lines (Refs. 56–60; see Table 1). Indeed,it has been shown that cell lines exhibiting the bystander effectin vitroare able to transfer radiolabeled GCV between them if they do expressCxs (67). The type of Cx expressed (by transfection or otherwise)does not appear to be crucial for the bystander effect because similarresults were obtained with HeLa cells expressing various Cxs such asCx43 or Cx26 (55, 68) and in a mouse neuroblastoma cell lineexpressing Cx37 (Ref. 69; see Table 2).

Nevertheless, somein vitro work did not exhibit a close relationshipbetween GJIC capacity and the extent of the bystander effect. Thiswas observed on lung cancer cell lines in which the dye transferassays exhibited a low GJIC capacity. The authors concluded fromtheir results that basal GJIC would be sufficient to obtain an extensivebystander effect. However, the fact that they could not decrease thebystander effect by inhibiting the GJIC may suggest that the bystandereffect was not completely mediated by GJs, even if cell-to-cell con-tacts were necessary (71). In some rare cases, the lack of intercellularcontacts did not prevent the occurrence of the bystander effect. It ispossible that in such cases, as with some rat colon adenocarcinomacells, a diffusible factor is involved in the death propagation (61).Similar conclusions were obtained with rat glioma cells in culture(72), even if GJIC was clearly involved in thein vivo situation (64).

In Vivo GJIC-mediated Bystander Effect

The bystander effect mediated by GJs that we reportedin vitro hasalso been observedin vivo. The major conclusion from thesein vivostudies is the same as that from thein vitro studies: that GJIC, as amediator of the bystander effect, can compensate for a low number ofHSV-tk1 cells in propagating the killing effect of GCV in an inducedtumor. For instance, transfection of the Cx37 gene reduced the weightof tumors induced by mouse neuroblastoma cells by up to 60% when50% of the cells expressed the HSV-tk gene (69). Expression of theCx43 gene in rat C6 glioma cells induced a similar phenomenon innude mice, even when only 5% of the injected cells were HSV-tk1

cells (64). However, the tumor-suppressive role of endogenous Cx43,which is expressed in parental nontransfected C6 glioma cells, mayhave interfered with the observed bystander effect (73). Moreover, inthe above-mentioned studies, the mice were killed soon after the endof GCV treatment, preventing an estimation of the duration of thebystander effect mediated by Cxs in down-regulating or preventingthe growth of the remaining part of the tumors.

We found that the growth of 106 injected HeLa cells, which arehighly tumorigenic, was prevented by 10% HSV-tk1 cells only if thecells expressed Cx43. These results indicate that Cx43 expression canefficiently induce the bystander effect such that the growth of tumorsis inhibited even when 90% of the injected cells are resistant to GCV,reproducingin vivo what we observedin vitro. Even when GCV wasadministered when the tumors were already palpable, tumor growthwas reduced by 66% or 77% (for mixtures containing 10% or 50%HSV-tk1 cells, respectively) compared with the growth of tumorsinduced by communicating but HSV-tk2 cells (65). The GJ-depend-ent bystander effect was so efficient that some of the mice were stillalive more than 2 months after the GCV treatment. In contrast, withCx432 HeLa cells, GCV decreased the size of the tumors only in thesame proportion as the percentage of HSV-tk1 cells present in theoriginal mixtures.

Increasing the GJIC Bystander Effect

All of the data we have presented thus far show that GJIC mediatesa strong bystander effect. Moreover, in most cases, there is a corre-

Table 1 Correlation between GJIC and in vitro/in vivo bystander effects in various tumor cell types

Species Tumor Cell lineBystander

effect GJIC Ref. no.

In vitroMouse Colon adenocarcinoma MCA38 1 1 60

Neuroblastoma N2a 2 2 60Lymphoma S49-1 2 2 60

Rat Gliosarcoma 9L 11 11 6061

Osteosarcoma UMR106 11 1 60Mesothelioma MesII20 1 11 60Colon adenocarcinoma DHD/K12 1 1/2 61

Human Pancreas BXPC-3 11 11 59PANC-3 1 1 59MIAPAC2 2 2 59

Mesothelioma JMN 1 11 60Colon adenocarcinoma HT29 2 2 62

HCT-8 2 2 62HCT-116 1 1 62

Erythroleukemia K562 2 2 60Cervical carcinoma HeLa 2 2 55Breast adenocarcinoma MDA-MB435 2 2 61

In vivo (transplanted tumors)In rats

Rat Colon tumor DHDProB 1/2 1/2 63In mice

Rat Glioma C6 2 1/2 64Human Cervical carcinoma HeLa 2 2 65

In vivo (intrinsic tumors)In mice

Neu transgenic Mammary tumor 1/2 1/2 66

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lation between a lack of GJIC capacity and the absence of a bystandereffect (Table 1). When GJIC is present, the intercellular transfer ofGCV occurs within a few hours (58). This rapid intercellular spread ofthe toxic metabolites within a tumor mass helps to increase theefficiency of a suicide gene transferred into a tumor and points toapplications of GJIC in therapy of localized cancers (67). The extentof GJIC in tumors then becomes a decisive factor for the bystanderefficacy. Unfortunately, most of the cancer cell lines we have testedexhibit such a low GJIC capacity that we considered this to be aphenotypic characteristic of cancer cells (Ref. 74; see Table 3). Sucha lack of GJIC would decrease the bystander effect and thus preventan efficient eradication of the tumors. In a few cases, we have indeedobserved that cancer cells that did not exhibit a satisfactory bystandereffect in vivo were GJIC-deficientin vitro. This was the case forGJIC-deficient mammary tumor cells obtained fromneu transgenicmice; when the HSV-tk gene was transduced into 10% of the tumorcells, the bystander effect was insufficient to allow the eradication ofthe tumors by GCV (66). Similarly, with colon cancer cells injectedi.p. into rats, the bystander effect was so low because of their poorGJIC capacity that 75% of the injected cells had to contain thetransduced HSV-tk gene before any curative effect of GCV treatmentwas seen (63). The low expression of Cx43 was also related to thelimited bystander effect observedin vivo for human medulloblastomacells (81). It is therefore imperative to screen tumors for their GJICand/or induce GJIC to obtain successful gene therapy with the help ofthe bystander effect.

Prediction of Bystander Effect by Screening GJIC-proficientHuman Tumors

As a crucial parameter for HSV-tk gene therapy, the level of GJICin situ in tumors should be predictive of the efficacy of the bystandereffect (59). Although most cancer cell lines are defective for GJIC, westill know very little about GJIC capacity inside human tumors. Fewin situ functional studies of GJIC in human tumors have been carriedout, but the available data show that GJIC was absent or low instomach carcinomas and hepatocellular carcinomas (Refs. 79 and 80;see Table 3). To assess whether certain tumor types would be moresusceptible to HSV-tk/GCV therapy, we propose to screen humantumors for their GJIC ability. The directex vivoestimation of GJIC ontumor biopsies would be the best way of performing such screening(82). However, this approach would be limited by the availability offresh tumor samples and the technical difficulty of performing suchanalyses in tissues that are difficult to microinject with dyes and/or areheterogeneous in their cell populations. A broader study could be

performed by screening for the presence of Cxs in fixed tumor tissues,which are already available in existing banks of biopsies, usingantibodies directed against some common Cxs (Cx26, Cx32, andCx43), which have recently become commercially available. Suchscreening would also provide valuable information about the putativerole of GJIC in human carcinogenesis (74). This indirect screening oftumors by immunolocalization of Cxs in precisely identified cancercells has previously been carried out with some human samples (83,84), and the results suggested that some human tumor types would bemore likely to exhibit a GJ-mediated bystander effect than others. Thedeficiency of originally expressed Cx43 GJs (85, 86) in breast ductalcarcinomas and infiltrating lobular carcinomas (87) and also in high-grade prostatic adenocarcinomas (88) suggests that these tumorswould not be a good target for the HSV-tk/GCV strategy. Similarobservations have been made on human ovarian cystadenocarcinomas(89). The situation appears to be different for primary human braintumors, in which both Cx26 and Cx43 are highly expressed (90).However, generalization of the results from one biopsy may bedifferent for others taken from the same tissue because Cx expressionmay vary greatly from one sample to another, as observed for Cx43 inhigh-grade astrocytomas (91). However, according to ourin vitro andin vivodata, the localization of GJs at cell-to-cell contact areas is moreimportant than the Cx expressionper sein predicting their function.In human hepatocellular carcinomas as well as in rat liver, we ob-served that defective GJIC capacity was related to the cytoplasmiclocalization of the Cxs in the cells (79, 92), and this has been observedto be associated with a poor bystander effect, independent of the levelof expression of the Cxs (62).

Table 2 Increase of bystander effect by expressing exogeneous (transfected) Cx genes in different cell lines

Species Tumor of origin Cell line Transfected Cx gene GJIC Bystander effect Ref. no.

In vitroMouse Hepatoma Hepa 1-6 Cx43 1 1 70

Neuroblastoma N2A Cx37 1 1 69Rat Adrenal pheochromocytoma PC12 Cx43 1 1 69

Glioma C6 Cx43 NTa 1 64Human Hepatoma SKHep J Cx32 1 1 57

Cx43 1 1 57Colon carcinoma HT-29 Cx43 NT 7b 62

HCT-8 Cx43 NT 7b 62HCT-116 Cx43 NT 1 62

Cervical carcinoma HeLa Cx43 1 1 55Cx26 1 1 68

In vivo (transplanted tumors)c

Mouse Neuroblastoma N2A Cx37 1 1 69Rat Glioma C6 Cx43 NT 1 64Human Cervical carcinoma HeLa Cx43 1 1 65

a NT, not tested.b No increased bystander effect due to the cytoplasmic localization of Cx43.c The host animals were nude mice in all cases.

Table 3 Examples of human carcinoma cell lines and tumors in situ with lowGJIC capacity

Tumors GJIC

Cx

Ref. no.mRNA Protein Localization

Cell linesPancreas None 1 2 75Hypopharynx None 2 2 75Epidermis 2a 1/2 1/2 Cytoplasm 76Cervix None 2 2 75Liver None NDb ND ND 77Bladder 2a 2a ND ND 78

Tumorsin situLiver Loss 11 11 Cytoplasm 79Stomachc None ND ND ND 80

a Decreased GJIC capacity or Cx mRNA correlated with the malignant phenotype.b No data.c GJIC capacity was estimated by electrical coupling.

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Biochemical Induction of GJIC in Tumors

The HSV-tk/GCV strategy may still be applicable to GJIC-deficienttumors, if one can increase the GJIC capacityin situ,e.g.,by specificchemical treatments (93). cAMP (94, 95), retinoic acids (96, 97),carotenoids (98), glucocorticoids (96, 99), and flavanoids (100) havesuch an effectin vitro. This effect may be cell type specific, as withcarotenoids, which increase the dye coupling of human dermal fibro-blasts but not of human keratinocytes (98). It may also be Cx typespecific; for example, glucocorticoids (dexamethasone and hydrocor-tisone) enhance the GJIC capacity of primary rat hepatocytes byincreasing the expression of Cx32 but not Cx43 (99).

Some evidence already suggests that a chemically induced increaseof GJIC (or Cx expression) can lead to an enhanced bystander effect.This has been observed bothin vitro andin vivoby treating tumor celllines with retinoids, apigenin, or lovastatin (101, 102). Similarly,treatment of human breast adenocarcinoma cells with cAMP signifi-cantly decreased the percentage of HSV-tk-transduced cells necessaryfor a 50% bystander effect (103). Likewise, doubling the GJIC ofCx43-transfected HeLa cells by cAMP treatment also resulted indoubling of the bystander effect.5 This type of biochemical increase ofGJIC needs to be further studiedin vitro and in vivo to determinewhether it is a Cx- and/or cell type-specific phenomenon (Table 4).However, the present evidence indicates that a basal GJIC mediatedby Cx43 will be increased by treatment with cAMP or retinoids(Table 4).

The full induction of GJIC depends on the mechanisms that regu-late the synthesis of the Cxs in tumors. However, these mechanisms

are far from being well understood, mainly because the noncodingregions upstream of the Cx genes, which regulate Cx gene expression,have not been fully analyzed. On the other hand, it appears that Cxexpression can be modified because these genes have specific pro-moter regions. For instance, cAMP consensus responsive elementshave been found in the Cx32 gene (123, 124). This is in agreementwith the Cx32 gene expression induced in hepatocytes by cAMPtreatment (125). The promoter region appears to be complex; sites forhepatocyte nuclear factor 1, nuclear factorkB, and glucocorticoidshave been identified in the 59-untranslated region of the Cx32 gene(124). Such a complex promoter region is probably a common featureof most Cx genes because they are expressed in a time- and tissue-specific manner in organisms (126–128). However, this complexity,in turn, enhances the possibilities for inducing the expression of aspecific Cx in a targeted cell population.

Even if we were able to target the expression of a specific Cx, theoverall effect, as with many other genes, would depend greatly on themethylation status of the promoter region. Silencing of Cx geneexpression due to hypermethylation has been observed for the Cx26,6

Cx43, and Cx32 genes (129). Silencing of Cx gene expression byhypermethylation could explain why only a few cases of induction ofCx expression and function by biochemical treatments have beenreported.

Another critical aspect is whether the newly induced Cx functionsnormally in a tumor. Cx proteins have to migrate through the cyto-plasm to be inserted into the cell membrane as connexons, which must

5 M. Mesnil, unpublished data.

6 R. Singal, Z. J. Tu, J. M. vanWert, G. D. Ginder, and D. T. Kiang, personal com-munication.

Table 4 Examples of up-regulation of GJIC by chemicals

Chemicals Cell types Cx regulation Ref. no.

cAMP (and derivatives 8-bromo-cAMP, dbcAMP) Choriocarcinoma cells (human) Cx40 up-regulation 104Thyroid cells (porcine) NDa 105Hepatoma cells (rat) Cx43 up-regulation 95Mammary tumor cells (mouse) “ “ “ “ “ 106Glioma cells (rat) “ “ “ “ “ 107Ventricular myocytes (rat) Cx43, Cx45 up-regulation 108Lung epithelial cells (mouse) Cx43 up-regulation 109

Forskolin Normal and malignant prostate epithelial cells (human) Cx43 up-regulation 110Corticoids

Dexamethasone Hepatocytes (rat) Cx32, Cx26 up-regulation 99111

Hepatoma cells (rat) Cx32 up-regulation 99Pancreatic tumor cells (rat) GJ increase 112

Hydrocortisone Hepatocytes (rat) Cx26, Cx32 up-regulation 113Glucagon

Hepatocytes (rat) Cx26, Cx32 up-regulation 113Retinoids

All-trans-retinoic acid Kidney cells (dog) Cx43 up-regulation 114Liver epithelial cells (rat) “ “ “ “ “ 115Skin (rat) Cx26, Cx43 up-regulation 116Teratocarcinoma cells (mouse) Cx43 up-regulation 117

Retinol (vitamin A) Fibroblasts (mouse) ND 118“ “ “ “ “ “ “ “ “ “ 119

Carotenoidsb-Carotene Fibroblasts (mouse) Cx43 up-regulation 120

“ “ “ “ “ ND 121Echinenone “ “ “ “ “ Cx43 up-regulation 121Canthaxanthin “ “ “ “ “ “ “ “ “ “ 121

“ “ “ “ “ ND 121Cryptoxanthin “ “ “ “ “ “ “ “ “ “ 1214-Hydroxy-b-carotene “ “ “ “ “ “ “ “ “ “ 121Lutein “ “ “ “ “ ND 121Lycopene “ “ “ “ “ “ “ “ “ “ 121a-Carotene “ “ “ “ “ “ “ “ “ “ 121

FlavanoidsFlavanone Liver epithelial cells (rat) Cx43 up-regulation 122Apigenin “ “ “ “ “ “ “ “ “ “ 100Tangeretin “ “ “ “ “ “ “ “ “ “ 100

a ND, no data at the molecular level.

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then dock efficiently with the connexons of adjacent cells to makeintercellular channels that link the cytoplasms. In some cells, thismechanism may not function because we have observed accumulationof Cxs in the cytoplasm (79, 92, 130). Such Cx transport and dockingis mediated by cell-to-cell recognition molecules such as E-cadherin(130, 131). Indeed, prevention of cell-to-cell recognition mediated bycadherins seems to be sufficient to prevent GJIC (132). The loss ofGJIC caused by appropriate treatments was also associated with lossof cadherin function (133, 134). More recent studies suggest that theCOOH-terminal domain of Cx43 binds to the zona occludens-1 pro-tein, a component of tight junctions (135). It is possible that zonaoccludens-1 anchors Cx43 at the cytoplasmic membrane orvice versa,a phenomenon that may lead to the polarized colocalization of Cx43and tight junctions in some cells such as thyrocytes (136). Othermolecular mechanisms responsible for the induction of Cx functionmay include increased stability of the mRNA and phosphorylation ofthe protein (95).

Usually, a few hours are necessary to induce the expression of Cxgenes; because of the rapid metabolism of GCV in HSV-tk-expressingcells, Cx-enhancing treatment would have to be performed before theinitiation of the suicide therapy or, at the latest, at the beginning of theGCV treatment, as was done with apigenin or lovastatin injected i.p.(102). Another aspect to consider is the possible modulation ofHSV-tk activity by the pharmacological treatment used for the GJICinduction. Although forskolin inhibited the activity of HSV-tk inmurine C3H10T fibroblasts (60), the induction of GJIC and of thebystander effect by cAMP in a murine mammary carcinoma cell linedid not affect HSV-tk activity (103). This inconsistency may be dueto the use of different cell types but needs to be considered whenattempting to apply the GJIC-based bystander effect.

Intratumoral Cx Gene Transfer

A more direct approach to local induction of GJIC would be totransfer a Cx gene into the tumor cells. As for the HSV-tk gene,transfer efficacy would be a limiting factor, as has been observed ina test in vivo (137). We have recently observed that the bystandereffect is more efficient when a HeLa cell population consists of cellscarrying HSV-tk or a Cx gene alone than when each cell contains avector carrying both genes (bigenic vector). The bigenic vector in-duced a weak bystander effect. Our preliminary results suggest thatthis could be a consequence of induced GJIC in the non-Cx-express-ing cells simply through contact with cells freshly transfected with aCx gene by an unknown mechanism.7 As discussed earlier for thebiochemical treatments, the success of this strategy is dependent onthe operation of mechanisms necessary for the function of the newlysynthesized Cxs. These mechanisms would need to be ready to func-tion in the recipient cells, and fortunately, in most reported cases, theintroduction of a Cx gene in a cell has been shown to restore GJs(Refs. 138–141; see Table 2).

Posttranslational modification of Cxs may, in some cases, decreasetheir ability to function for GJIC. One possible way to avoid this is tocreate a gene for an artificial Cx that will efficiently form GJs but willnot be vulnerable to endogenous modification. Candidate gene con-structs include a modified Cx26 gene, which already has a shortCOOH-terminal, and innexin gene, both of which are functionalhomologues of Cxs that are specific to invertebrates (142, 143) andtherefore may not be liable to modification in mammalian cells.

Cxs Not Only Mediate the Bystander Effect But Are AlsoTumor Suppressors

Most communication-defective cancer cells lose their tumorigeniccapacity or exhibit down-regulated growth once they are transfectedwith the appropriate Cx genes. This has been observed with cell linesof many different origins, such as rat hepatoma, murine fibroblasts(chemically transformed C3H10T1/2 cells), HeLa cells, and rat gli-oma (C6 cells), human breast carcinoma, and rhabdomyosarcoma(138–140, 144–146). Interestingly, the tumor-suppressive effect issometimes dependent on the Cx type (144) but is not necessarilydependent on the induction of GJIC (147, 148).

A priori, the induction of Cx expression in tumor cells could leadto two beneficial effects on tumor control: (a) bystander effect medi-ation; and (b) tumor suppression. However, a strong tumor-suppres-sive effect resulting from the induction of Cx expression mightactually prevent efficient DNA incorporation of GCV-derived nucle-otides by slowing down the cell cycle. The results from our studiesinvitro suggest that these two phenomena are not entirely antagonisticbecause we have shown that Cx26, a tumor suppressor in HeLa cells,can mediate a satisfactory bystander effectin vitro (68). However,such antagonism may need to be considered in relation to GCV killingefficacy in the case of a strongin situ induction of Cx expression.

This is also an important issue for the pharmaceutical manipulationof GJs. Some chemical compounds known to induce GJIC may act asgrowth regulators. For instance, apigenin induces not only GJIC butalso G1 cell cycle arrest (149). Such activity could limit the efficacyof GCV incorporation into DNA, although a recentin vivo studysuggests that this is not the case (102).

Conclusions and Perspectives

GJIC increases the extent of the bystander effect observed withHSV-tk/GCV gene therapy bothin vitro and in vivo. This anticancerstrategy may therefore be best adapted to treatment of tumors in whichthe cells have a significant basal level of GJIC. GJIC cannot easily bemeasuredin situ, but a predictive estimation can be made by screeningtumors for the presence of GJs correctly localized at cell-to-cellcontact areas.

Tumors that may be suitable targets for HSV-tk/GCV therapy canbe chosen according to a few characteristics (Fig. 3). They must beaccessible for the intratumoral injections of vectors carrying theHSV-tk gene. Their cellular structure should be homogeneous, withcells firmly in contact with each other. Depending on the Cx typesexpressed, it should be possible to increase the basal level of GJIC byappropriate treatments. We believe that this kind of therapy could beextended in the future to GJ-deficient tumors by artificially enhancingthe in situ communication capacity of the tumor cells through appro-priate chemical treatments, once the promoter regions have beenclearly identified. Artificial induction of GJIC by direct transfer of Cxgenes may be inefficient but should be further studied in view of thesurprising bystander effect that was induced through this strategyinvitro.

The results ofin vitro studies indicate that GJIC may have variousbenefits for HSV-tk-mediated gene therapy. Some studies have shownthat the strength of the bystander effect is less dependent on the levelof HSV-tk activity than the cell-to-cell communication capacity (59).Other studies have also shown that HSV-tk activity can increase Cx43function in hepatoma cells (70). Enhanced communication mightenable the HSV-tk-expressing cells to resist the GCV treatment forlonger time periods by permitting efficient diffusion of the GCV-Pmetabolites to the surrounding cells. This phenomenon has beentermed the “Good Samaritan effect” and, if confirmed, should in-crease the efficiency of such therapy (150).7 T. Tanaka, H. Yamasaki, and M. Mesnil, unpublished data.

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Cxs themselves not only mediate the bystander effect but also havea separate tumor-suppressive action (151). Thus, we have proposedthat Cxs have a dual effect on tumor control (68). Whereas it is clearthat Cx genes suppress the growth of many tumor cells, there isincreasing evidence to suggest that increased Cx gene expression atlater stages of tumor development may facilitate metastasis (152).Therefore, for tumor control, it may be prudent to use Cx genestogether with therapeutic suicide genes.

Moreover, the Cxs may work synergistically with other mecha-nisms that may be involved in the bystander effect. For instance, theCxs may help to amplify the disruption of angiogenesis that is ob-servedin vivo. Indeed, transduction experiments carried outin situwith cells producing retroviral particles bearing ab-galactosidasegene in a 9L glioma have shown that some blood vessel endothelialcells were infected with the viruses and expressed the gene (41). Cellslocated within or adjacent to a tumor are the most mitotically activeendothelial cells responding to angiogenesis factors released by thetumor, and they should be sensitive to GCV if they express theHSV-tk gene. In rats, a decrease in tumor vasculature has beenobserved after the initiation of GCV therapy in HSV-tk-transducedtumors (153). The disruption of angiogenesis was accompanied bydiffuse necrotic changes associated with intratumoral hemorrhage(153). Therefore, the elimination of these transduced endothelial cellswith GCV may result in ischemia of the tumor mass (41, 153).Because the endothelial cells are GJIC proficient and express partic-ular types of Cxs [Cx37, Cx43, and Cx40 (154)], they are potentialtargets for a “vascular” bystander effect that should contribute totumor eradication.

Another consequence of the induction of the bystander effect byGJs may be the increased inflammatory infiltrations that have beenobserved in a range of target organs of both rats and mice (155–159).This antitumor response, leading to centralized hemorrhagic tumornecrosis, follows the killing of neoplastic cells as a result of treatmentwith GCV (160).

This immune response is probably one of the mechanisms involvedin the bystander effect because the bystander effect is usually ineffi-cient in athymic nude mice, being only a transitory phenomenon if nomore than 50% of the injected cells express HSV-tk and are thussensitive to GCV (161). In contrast to athymic mice, total tumorregression can be obtained with normal mice even if only 10–20% ofthe cells express HSV-tk (162). In addition, the more tumor cellskilled by HSV-tk/GCV, the better the immune response should be-come. Therefore, the induction of the bystander effect by GJs shouldin turn lead to an increase in the immune response.

The local involvement of the immune system in the bystander effectraises the possibility that distant- and long-term systemic antitumorresponses may also be induced. It has been reported that the growth ofuntreated tumors (tumors with no cells expressing HSV-tk) in oneflank of a mouse can decrease when tumors with cells expressingHSV-tk are treated on the other flank (163, 164). This distant by-stander effect seems to be efficient enough to prevent the growth ofcolonies of both HSV-tk1 and HSV-tk2 rat colon cancer cells in thewhole peritoneal cavity of rats. These results suggest that such localsuicide gene therapy could be used as a kind of cancer immunotherapybecause it may have an effect not only against the HSV-tk-expressingtumors but also against distant tumors that do not express the trans-gene and are disseminated in the same organ (155, 165, 166). Thisdistant bystander effect suggests that a local HSV-tk/GCV genetherapy may also prevent the growth of disseminated tumors andpossibly metastases (167).

In conclusion, we consider that a strongly induced local bystandereffect mediated by GJs, by enhancing the death of the cells in theprimary tumor and, in turn, enhancing the local inflammation process,should increase the systemic immune response that may also actefficiently against metastases.

Acknowledgments

We thank Dr. John Cheney for editing the manuscript and Chantal Dechauxfor secretarial help.

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2000;60:3989-3999. Cancer Res Marc Mesnil and Hiroshi Yamasaki

1 Gap-junctional Intercellular CommunicationKinase/Ganciclovir Cancer Gene Therapy: Role of Bystander Effect in Herpes Simplex Virus-Thymidine

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