[EUGIC (Extension of the Use of Gametes in Intra-Conjugal): New uses of gametes within the couple]. / EUGIC (Extension de l'Utilisation de Gamètes en IntraConjugal) : les nouveaux usages des gamètes au sein du couple.

OBJECTIVE: New possibilities for using gametes within a couple were created by the French law of August 2, 2021 related to bioethics by opening Assisted Reproductive Technics (ART) to all women. It concerns previously self-preserved gametes, thus avoiding the need for gamete donation. The objective of our study is to evaluate the perception of these new uses by ART practitioners. METHOD: A questionnaire of twelve short questions was sent to professionals concerned with gamete donation. RESULTS: One hundred and ten professionals answered the questionnaire. The majority of them approve of the Reception of Oocytes from the Partner (ROPA), notably if there is a medical indication. Requests are rarer for the care of trans* people, and raise more questions. Although less favorable to the use of eggs from trans* men, more of them support the practice when it is an alternative to oocyte donation. CONCLUSION: The acronym EUGIC (Extension of the Use of Gametes in Intra-Conjugal) makes it possible to group together these new situations generated by the change in the French law.

Serotonin and cancer: what is the link?

Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic monoamine that acts as a neurotransmitter in the central nervous system, local mediator in the gut and vasoactive agent in the blood. Serotonin exerts its multiple, sometimes opposing actions through interaction with a multiplicity of receptors coupled to various signalling pathways. In addition to its well-known functions, serotonin has been shown to be a mitogenic factor for a wide range of normal and tumoral cells. Serotonin exhibits a growth stimulatory effect in aggressive cancers and carcinoids more often through 5- HT1 and 5-HT2 receptors. In contrast, low doses of serotonin can inhibit tumour growth via the decrease of blood supply to the tumour, suggesting that the role of serotonin on tumour growth is concentration-dependent. Data are also available on serotonin involvement in cancer cell migration, metastatic processes and as a mediator of angiogenesis. Moreover, the progression of some tumours is accompanied by a dysregulation of the pattern of serotonin receptor expressions. Serum serotonin level was found to be suitable for prognosis evaluation of urothelial carcinoma in the urinary bladder, adenocarcinoma of the prostate and renal cell carcinoma. In some cases, antagonists of serotonin receptors, inhibitors of selective serotonin transporter and of serotonin synthesis have been successfully used to prevent cancer cell growth. This review revaluates serotonin involvement in several types of cancer and at different stages of their progression.

Deletion of a single allele of Cx43 is associated with a reduction in the gap junctional intercellular communication and increased cell proliferation of mouse lung pneumocytes type II.

OBJECTIVES: Connexins (Cx) are proteins that form the gap junctional channels at neighbouring plasma membranes between adjacent cells. Cxs are involved in cell communication, which is reportedly correlated with cell proliferation and differentiation. Alterations in connexin expression and/or gap junctional intercellular communication (GJIC) capacity have long been postulated to be important in a number of pathological conditions including cancer. This study was performed to determine the consequences of the deletion of a single allele of Gja1 (Cx43 gene) in Alveolar Type II cells (APTIIs), and its impact on GJIC and cell proliferation. MATERIAL AND METHODS: In order to do so, APTIIs from wild type (Cx43(+/+)) and heterozygous (Cx43(+/-)) mice were harvested and cultured for 4 days. The GJIC capacity was evaluated by scrape-loading method, with the transfer of lucifer yellow dye. The expression of Cx43 was evaluated by immunofluorescence method and Western blotting. Cell proliferation was evaluated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: It was observed that GJIC capacity was significantly reduced and cell proliferation index was significantly higher in Cx43(+/-) cells compared to Cx43(+/+) cells. CONCLUSIONS: These results show that knocking out one allele of Cx43 leads to a lower cell to cell communication capacity, and consequently induces a higher cell proliferation. Because chemically induced lung adenomas in mice are known to originate from APTIIs, these alterations may play a critical role in their susceptibility to lung carcinogenesis.

NBT-II carcinoma behaviour is not dependent on cell-cell communication through gap junctions.

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.

Induction of a bystander effect in HeLa cells by using a bigenic vector carrying viral thymidine kinase and connexin32 genes.

We previously showed that gap junction intercellular communication mediates the bystander effect in anticancer gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir. Because most cancer cell lines have lost their ability to communicate through gap junctions, we investigated whether we could induce such a communication by transferring a gene for a gap junction. We transfected a vector carrying the HSV-tk (tk) and gap junction (connexin (Cx) 32) genes (Cx32(+)tk(+)) into noncommunicating HeLa cells. We compared the cytotoxicity of ganciclovir with mixtures of these cells and HeLa cells that expressed (Cx32(+)) or did not express (Cx32(-)) the Cx32 gene. The bystander effect was strong when the two mixed cell types expressed Cx32 (i.e., Cx32(+)tk(+) cells and Cx32(+)tk(-) cells). Only 25% of cells survived in this communicating mixture, even when only 10% of the cells were Cx32(+)tk(+). There was also a moderate bystander effect when the Cx32(+)tk(+) cells were mixed with noncommunicating HeLa cells in a 50% ratio. These results demonstrated that the bystander effect is enhanced by Cx32 and suggested that expression of Cx in only one cell type in a mixture can cause a bystander effect. Mol. Carcinog. 30:176--180, 2001.

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro.

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.

Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication.

Antitumor suicide gene therapy is one of the emerging strategies against cancer. It consists of the introduction into cancer cells of a gene capable of converting a nontoxic prodrug into a cytotoxic drug. Because this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the "bystander effect," by which the introduced gene can affect even cells in which it is not itself present. From a therapeutic point of view, it may be crucial to enhance this phenomenon through various means to achieve tumor eradication. One such suicide gene, the thymidine kinase gene from the herpes simplex virus, in combination with the prodrug ganciclovir, has been extensively and successfully used in some animal models exhibiting a strong bystander effect. Among the mechanisms involved in this phenomenon, gap junctional intercellular communication (GJIC) is directly involved in the transfer of the toxic metabolites of ganciclovir, which pass directly from herpes simplex virus thymidine kinase-expressing cells to surrounding cells that do not express it. Because GJIC appears to be a mediator of the bystander effect both in vitro and in vivo, here we review possible molecular strategies for enhancing the extent of tumor cell death by increasing the intratumoral GJIC capacity.

A cell type-specific and gap junction-independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.

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.

Internalization of gap junctions in benign familial pemphigus (Hailey-Hailey disease) and keratosis follicularis (Darier's disease).

Hereditary skin disorders involving acantholysis, such as Hailey-Hailey disease and Darier's disease, have been genetically linked to distinct chromosomal parts which do not code for known structural proteins. Such evidence suggests that the genomic abnormalities underlying these dermatoses may concern functional/regulatory mechanisms of keratinocyte cohesion. Epidermal communication junctions (gap junctions) are responsible for direct coupling of cells and, thus, co-ordinate the behaviour of keratinocytes within the tissue. Consequently, they remain one of the potential, and poorly studied, elements in the pathogenesis of hereditary acantholytic diseases. We have investigated the distribution and fate of gap junctions during non-immune acantholysis, using fine immunolocalization methods at the light and electron microscopic levels. Our results demonstrate normal expression of epidermal gap junction proteins, connexins 26 and 43, in non-lesional skin of Hailey-Hailey and Darier's diseases. The gap junctions were not primarily dismantled during acantholysis, typical of both of the studied dermatoses, but underwent internalization and subsequent cytoplasmic dispersion in the portions of cells which were no longer attached to the rest of the tissue. In Darier's disease, perifollicular acantholysis did not specifically concern epithelium of appendages coexpressing connexin 26 in addition to connexin 43, further indicating that the observed changes in gap junction localization were secondary to the loss of cell-cell contact. We demonstrated that the sequence of changes was identical in both diseases and that the previously described putative differences were apparently related to the degree of acantholysis present in the studied biopsies. The fate of the junctional structures and proteins, documented in the present study, is most probably a form of recycling process also used by normal keratinocytes during organogenesis and tissue differentiation.

Genetic and epigenetic changes of intercellular communication genes during multistage carcinogenesis.

During multistage carcinogenesis, the functions of several key genes involved in cell growth control must be damaged. Such genes include not only those involved in cell cycle control of individual cells, but also those involved in the coordination of cell growth throughout a given tissue through cell-cell communication. The most intimate form of intercellular communication is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is known to transfer small water soluble molecules, including cAMP and IP3, from the cytoplasm of one cell to that of its neighbors; the growth of a given GJIC-associated cell is thus kept in check by other GJIC-connected cells. Most tumor cells have a reduced ability to communicate among themselves and/or with surrounding normal cells, confirming the importance of intact GJIC in growth control. When connexin (gap junction protein) genes are transfected into such cells, normal cell growth control is often recovered. Certain dominant-negative mutant connexin genes can reverse such tumor suppression. While these results suggest that connexin genes form a family of tumor suppressor genes, so far we have found no connexin gene mutations in human tumors; only two connexin gene mutations were found in chemically induced rat tumors. On the other hand, our recent studies suggest that connexin genes may be inactivated by hypermethylation of their promoter regions, suggesting that epigenetic inactivation of connexin genes may be a mechanism of GJIC disturbance in certain tumors. However, in many tumor cells connexins are normally expressed but aberrantly localized. The mechanisms of aberrant localization of connexins include lack of an appropriate cell-cell recognition apparatus and aberrant phosphorylation of connexins. These results suggest that GJIC disorders may occur not only because of aberrant expression of connexin genes themselves, but also as a result of disruption of various control mechanisms of the protein functions.

Connexins in tumour suppression and cancer therapy.

Malignant cells usually show altered gap junctional intercellular communication and are often associated with aberrant expression or localization of connexins. Transfection of connexin genes into tumorigenic cells restores normal cell growth, suggesting that connexins form a family of tumour suppressor genes. Some studies have also shown that specific connexins may be necessary to control growth of specific cell types. Although we have found that genes encoding connexin32 (Cx32; beta 1), Cx37 (alpha 4) and Cx43 (alpha 1) are rarely mutated in tumours, our recent studies suggest that methylation of the connexin gene promoter may be a mechanism by which connexin gene expression is down-regulated in certain tumors. We have produced various dominant negative mutants of the genes encoding Cx26 (beta 2), Cx32 and Cx43, some of which prevent the growth control exerted by the corresponding wild-type genes. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through gap junctions. Recently, we and others have shown that gap junctional intercellular communication is responsible for the bystander effect seen in herpes simplex virus thymidine kinase/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

Role of connexin (gap junction) genes in cell growth control and carcinogenesis.

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

Role of connexin (gap junction) genes in cell growth control: approach with site-directed mutagenesis and dominant-negative effects.

Evidence is accumulating that connexin (Cx) genes form a family of tumor-suppressor genes. Our long-standing study revealed that, in almost all tumors, some abnormality in gap junction is observed, including loss or reduction of expression, aberrant localization of gap junction. In this study, we have examined the dominant-negative effects of mutant (prepared by site-directed mutagenesis) Cx43 constructs in C6 glioma cells, and of mutant Cx26 constructs in HeLa cells, on tumorigenicity. The mutant Cx43 A253V (Ala 253 to Val) inhibited the tumor-suppressive function exerted by wild-type Cx43 in C6 cells. Similarly, the mutant Cx26 P87L (Pro 87 to Leu) manifested dominant-negative inhibition of connexin-mediated cell growth control in HeLa cells. These results suggest that mutations of connexin genes can affect the tumor-suppressive function of gap junction and that gap junctional intercellular communication can be regulated by not only non-genotoxic but also genotoxic activities of environmental carcinogens.

Ultrastructural and biochemical evidence for gap junction and connexin 43 expression in a clonal Sertoli cell line: a potential model in the study of junctional complex formation.

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.

In vitro and in vivo effects of retrovirus-mediated transfer of the connexin 43 gene in malignant gliomas: consequences for HSVtk/GCV anticancer gene therapy.

In tumors, gap junctional intercellular communication (GJIC) is usually down-regulated and the expression of connexins, membrane proteins constituting gap junction channels, is often low or altered. GJIC, allowing the intercellular diffusion of ganciclovir (GCV) triphosphate, is also one mediator of the 'bystander effect', the phenomenon by which herpes simplex virus thymidine kinase (HSVtk)-transduced, neoplastic cells kill surrounding HSVtk-negative cells when treated with GCV. We set up experiments to evaluate the effects of retrovirus-mediated in vivo gene transfer of connexin 43 in malignancies with low GJIC capacity. We found that U-87 human glioblastoma cells transfected in vitro by the human Cx43 cDNA grow significantly more slowly than control U-87 cells and lose their tumorigenicity when injected subcutaneously in nude mice. When the Cx43 gene was transduced in vitro in U-87 cells by a retroviral producer cell line (N3.2.ii, titer 1.5 x 10(6) c.f.u./ml) in vivo results were similar. However, only when U-87 cells were co-injected with N3.2.ii cells in nude mice in a 1:5 ratio, a 50% reduction in tumor size was obtained during the first 3 weeks. Moreover the coinjection of U-87 cells with N3.2.ii and SBA cells (a retroviral producer cell line expressing the HSVtk gene), was not able to potentiate the effects of GCV administration, suggesting that Cx43 gene transfer requires more efficient vectors to increase the bystander effect in vivo.

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy.

Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk- HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43(+)-tk+ and Cx43(+)-tk- while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, i.e. Cx43(+)-tk+/Cx43(-)-tk-. When GCV was given after the appearance of tumors, the size of the tumors from Cx43- cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.

Dominant-negative abrogation of connexin-mediated cell growth control by mutant connexin genes.

Connexin genes exert negative growth control when transfected into various types of tumor cell lines. We previously demonstrated that connexin 26 (Cx26) suppresses in vitro and in vivo growth of HeLa cells. In this study, we have examined whether certain Cx26 mutants can abrogate cell growth control and the gap junctional intercellular communication (GJIC) capacity of such Cx26-transfected HeLa cells. For this purpose, we transfected three mutated Cx26 genes (C60F, P87L and R143W) into HeLa cells already containing the wild-type Cx26 gene, which are GJIC-competent and non-tumorigenic. Transfection of P87L and R143W mutants enhanced the tumorigenicity of the HeLa Cx26 cells in nude mice without any change in GJIC capacity. On the other hand, transfection of the C60F mutant reduced the GJIC capacity of HeLa Cx26 cells without affecting their growth in vivo. Immunostaining studies demonstrated that the Cx26 proteins were localized mainly at cell-cell contact areas in the HeLa Cx26 cells both before and after transfection of mutated Cx26 genes. These results suggest that certain mutant Cx26 proteins exert a dominant-negative effect on Cx26-regulated growth of HeLa cells and that such effects may be independent of the effect on GJIC ability. It is proposed that wild-type and mutant Cx26 proteins produce heteromeric connexons and that such heteromeric connexons may exert different effects on growth control from those of homomeric connexons.

A tumor suppressor gene, Cx26, also mediates the bystander effect in HeLa cells.

The connexin 26 (Cx26) gene suppresses the growth of HeLa cells in vitro and in vivo. We explored the possibility that the Cx26 gene not only suppresses growth but can also mediate the bystander effect that is observed in some gene therapy. In gene therapy mediated by the herpes simplex virus thymidine kinase, the toxicity of ganciclovir affects not only the cells transduced with the gene but also affects neighboring tumor cells; it has been suggested that gap junctional intercellular communication (GJIC) may play a role in such a bystander effect. HeLa cells expressing the Cx26 gene (Cx26+) or not expressing the Cx26 gene were transfected with the herpes simplex virus thymidine kinase (tk+) gene, producing Cx26(-)-tk-, Cx26(-)-tk+, Cx26+-tk-, and Cx26+-tk+ cells. By making different kinds of cocultures of these cells, we observed a clear bystander killing effect, assessed by the neutral red toxicity test, in the coculture of Cx26+-tk-/Cx26+-tk+ cells. The bystander effect was markedly prevented by a long-term inhibitor of GJIC, 18-alpha-glycyrrhetinic acid, demonstrating that a major part of the bystander effect seen occurred through Cx-mediated GJIC. These data suggest the possibility of using of Cxs as both tumor suppressor genes and as diffusers of ganciclovir toxicity in therapeutic approaches.

Long-term survival of immunocompetent rats with intraperitoneal colon carcinoma tumors using herpes simplex thymidine kinase/ganciclovir and IL-2 treatments.

We evaluated the gain in long-term survival of BDIX rats bearing DHDProB colon cancer developed in the peritoneal cavity after in vivo therapy with the tk gene and GCV. The sensitivity and the bystander effect of DHDProB cells stably transduced with the tk gene evaluated in vitro were low, as one tk+ cell killed two tk- cells. This correlated with the low ability of a fluorescent dye to diffuse through gap junctions. In vivo, more than 75% of tk-transduced cells were required and at least 100 mg/kg/day of GCV had to be injected no later than day 5 after tumor implantation to obtain a curative effect. A partial protection of the cured animals against rechallenge with the parental cells was also observed. Based on these results, a protocol of in vivo gene therapy was designed in which the tk/GCV treatment was combined with IL-2 gene expression. When the tk- and IL-2 encoding plasmids were injected twice i.p. with DOTAP and the animals treated with GCV, three of five rats were cured. This antitumoral activity resulted from the combined toxic effects of DNA/DOTAP and tk/GCV plus a potential immune response mediated by IL-2.

Partial regression, yet incomplete eradication of mammary tumors in transgenic mice by retrovirally mediated HSVtk transfer 'in vivo'.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.