This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carystinos, G. D. Articles by Batist, G. Search for Related Content PubMed PubMed Citation Articles by Carystinos, G. D. Articles by Batist, G.

Cyclic-AMP Induction of Gap Junctional Intercellular Communication Increases Bystander Effect in Suicide Gene Therapy¹

McGill Centre for Translational Research in Cancer, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec H3T-1E2, Canada [J. G., H. C., M. A. A-J., G. B.]; Departments of Pharmacology and Therapeutics, Faculty of Medicine, McGill University [G. D. C., M. M. K.]; and Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5B8, Canada [D. W. L.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
One of the approaches of somatic gene therapy in cancer has been the attempt to sensitize tumor cells to prodrugs by incorporating suicide genes that activate the drug into a cytotoxic metabolite (1) . Present delivery systems, such as adenoviruses or retroviruses, have proven unable to reach the total cancer population (2 , 3) , and gene therapy trials were often considered inefficient in cell targeting and had a low overall success rate (2) . However, a striking phenomenon was observed, the BE³ , where cytotoxicity was seen not only in genetically altered cells, but as well in nontransfected or transduced neighboring cells (4 , 5) . This could then represent an important therapeutic opportunity, since not all of the tumor cells need be directly targeted. The mechanism of the BE may include a number of possibilities, however, there is compelling evidence that GJIC is important in at least some prodrug/suicide gene systems, including gene therapy using retrovirus/adenovirus gene-delivery systems (6, 7, 8, 9) . The evidence supports the hypothesis that GJs allow the passive diffusion of the activated metabolite to neighboring cells, thereby enabling the drug to target a greater number of the cell population.

GJIC is considered fundamental in cell-cell interactions, since GJs mediate the transfer of ions, nucleotides, and small regulatory molecules as well as drugs or their metabolites between adjacent cells (10) . They are made up of hemichannels (called connexons) in the membrane of one cell joined in mirror symmetry with the same number of hemichannels in the opposing cell membrane. Connexons are formed from members of a multigene family of distinct, but functionally related, proteins called Cxs (11 , 12) . In general, cancer cells have reduced or nonexistent levels of Cxs and greatly reduced GJIC as compared with normal cells (13 , 14) . Cx transfection and GJIC induction leads to a decreased rate of proliferation, increased differentiation, and reversal of cell-transformation phenotype (15 , 16) .

Cx43 is one of the major Cxs in breast tissue that is often lost or reduced in cancer (13 , 14) . Studies have shown that transfection or transduction of Cx43, resulting in increased GJIC, leads to an increased BE seen in suicide gene therapy (6 , 7) . Although the transfections were all performed in vitro, one study examined the effect of increased Cx43 by transfection in tumor cells subsequently grown in vivo and demonstrated that the BE is enhanced (17) . Together, these studies point to the potential use of increasing GJIC as a part of suicide gene therapy. Adding transfection of the Cx43 cDNA to the suicide gene therapy approach would be useful for enhancing the BE. Yet, chemical induction of Cx43 might have a more potent effect, since a greater proportion of the cancer cell population would be reached as compared with gene delivery. Using a chemical inducer of GJIC, therefore, has great potential to amplify the efficacy of suicide gene therapy.

There are a number of classes of chemicals that have been shown to increase Cx43 and, subsequently, GJIC including cAMP (18) , retinoids (19) , and carotenoids (20) . cAMP is an antiproliferative and differentiation agent that has a very wide range of action (21) . The use of cAMP in the clinical setting is limited by its toxicity, yet its properties have lead groups to work on the cAMP-dependent pathway in cancer in search of analogues and downstream factors to target.

This is the first study reporting the chemical induction of GJIC by cAMP and its resulting increase in the BE when cells are infected by the adenoviral vector Ad-RSV-vTK and treated with GCV.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Chemicals and Reagents
8-Bromo 3':5'-cAMP was obtained from Sigma Chemical Co. (St. Louis, MO). GCV was obtained from Hoffmann-LaRoche (Mississauga, Ontario, Canada) and dissolved in PBS before use. Calcein AM and DiI were obtained from Molecular Probes, Inc. (Eugene, OR).

Cell Culture
DA3 is a previously described and well established mouse mammary carcinoma cell line that grows both in vitro and as an s.c. tumor mass (22) . They are maintained in RPMI culture medium containing 10% FCS, 1% sodium pyruvate, 1% nonessential amino acids, 1% nucleosides, 1% glutamine, and 0.6% penicillin-streptomycin. Cells were maintained at 37°C under 5% CO₂ and passaged 1–2 times/week. When indicated, cAMP was added at a final concentration of 1 mM. This concentration, which has no toxic effect on DA3 cells, was established based on preliminary studies. The gene therapy system we are studying uses the adenoviral vector Ad-RSV-vTK (provided by Dr. S. Woo, Baylor University, Waco, TX), which is the type 5 adenoviral vector 5 containing the RSV constitutive promoter driving the expression of the vTK that activates the prodrug GCV. When indicated, DA3 cells were transduced with Ad-RSV-vTK at an MOI of 10.

DA3 cells were stably transduced with Moloney-based, replication-defective retrovectors encoding either vEGFP or vTKiEGFP. The retroviral promoter included in these constructs leads to stable constitutive expression of transgenes. The resulting polyclonal cell lines DA3-vEGFP and DA3-vTKiEGFP were 100% positive for GFP expression by FACS analysis (data not shown). The DA3-vTKiEGFP cell line is highly sensitized to GCV with an IC₅₀ approximately 10,000 lower than parental cells (data not shown). DA3-vEGFP, similarly to parental cells, are not sensitized to GCV.

Chemical Induction of GJIC
Immunofluorescent Studies for Cx43.
The presence of Cx43, one of the predominant GJ proteins expressed in breast cells and one shown to be reduced during carcinogenesis, was examined in the DA3 mouse mammary cancer cell lines (22 , 23) . Untreated or cAMP-treated cells (treated with 1 mM cAMP for 2 days) were grown onto coverslips and then fixed in 80 methanol- acetone (80:20 by volume) for 10–20 min at 20°C, blocked with 2% BSA in PBS for 1 h and incubated with a rabbit antibody directed against residues 360–382 of Cx43 (24) for 1 h. After washing, the samples were incubated with goat antirabbit antibody conjugated to rhodamine. The cells were mounted on coverslips and imaged with a 25 x 0.8 numerical aperture lens under identical conditions using a Zeiss LSM confocal microscope. Transmitted light and fluorescent images were printed on a Kodak XSL8300 printer.

Functional Assay of GJIC.
We also evaluated functionally the GJIC in the DA3 cells treated with cAMP, under the same conditions as in the mixing experiments below. We used FACS analysis, as described by Kiang et al. (25) , with minor modifications. Cells were trypsinized, washed in PBS, and stained for 15 min with either 4 µM DiI to mark the recipient cells or 0.5 µM Calcein AM to load the donor cells. Cells were then washed with PBS, and equal numbers of donor and recipient cells were mixed and incubated for 3.5 h in 12-well plates. To maintain the same degree of cell density and contact in different experiments, cells were always plated at the same density of 500,000/well in 24-well plates. After incubation, cells were washed with PBS, trypsinized, resuspended in PBS, and then analyzed by flow cytometry. Ten thousand cells were counted by two-color sorting in a Coulter Epics XL-MCL apparatus. The excitation wavelength used was 488 nm, and the emission wavelengths for Calcein AM and DiI were 525 nm and 575 nm, respectively. GJIC is defined as the percentage of recipient cells that received Calcein AM from donor cells (only possible through GJs; Ref. 23 ), corrected for background using a nonincubated control. Four replicates/condition were used, and the whole assay was performed twice.

Cytotoxicity/Mixing Assay.
A cytotoxicity assay using either DA3 infected with Ad-RSV-vTK or uninfected DA3 followed by exposure to the prodrug GCV was used to measure the effect of chemical induction of intercellular communication on suicide gene therapy in vitro. DA3 cells (6 x 10⁵) were plated in each of 4 x 75 cm² tissue culture flasks and allowed to adhere overnight. The following day, two flasks were treated with cAMP to a final concentration of 1 mM. Also, recombinant virus Ad-RSV-vTK was added to one flask of cAMP-treated cells and one flask of cells not treated with cAMP, at an MOI of 10 to ensure that all cells are infected. Forty eight hours later (when the GJIC of cells treated with cAMP was induced according to FACS analysis), the treated cells were harvested with trypsin, washed once with Dulbecco’s buffered saline solution, and resuspended in a final concentration of 10⁵ cells/ml. For each treatment, infected cells were mixed with uninfected DA3 cells to obtain a population of cells where 0, 1, 2, 5, 10, 20, 50, or 100% of the cells were transduced with Ad-RSV-vTK. The cells were then replated at 10⁴ cells/well in a 96-well tissue culture plate and treated with or without various amounts of GCV (one 96-well plate/cAMP treatment and GCV concentration was used, and final GCV concentrations were 10 and 20 µg/ml). The high cell density was selected to ensure cell-cell contact. After 3 days, cell survival was determined using a colorimetric assay, which measures the ability of viable cells to reduce a soluble yellow tetrazolium salt (MTT) to an insoluble purple formazan precipitate. The MTT assay was carried out as described (26) . Cell viability for each condition was determined as the ratio of cells (of the same infection-percentage and cAMP treatment) treated with GCV to GCV-untreated cells. To control for cAMP and GCV nonspecific effects on cell viability and proliferation, the cell viability of uninfected cells of the same cAMP treatment was used as a reference. The percentage of cell survival for each condition was scored as the ratio of cell viability divided by the cell viability of uninfected cells (0% infection) of the same treatment. Four replicates/condition, mixing percentage, and treatment were used, and the whole mixing experiment was repeated four times.

Effect of cAMP on Adenoviral Infectivity
The possibility that cAMP has an effect on adenovirus infectivity was approached in two ways.

Mixing Assays Using Stably Transduced DA3 Cells.
The mixing assays were performed as previously described, using DA3 cells stably transduced with a vTK-retrovector (DA3-vTKiEGFP) and control cells transduced with the retrovector (DA3-vEGFP). For each cAMP treatment, cells were mixed to a final percentage of 0, 1, 2, 5, 10, 20, 50, and 100% of DA3- vTKiEGFP cells and were treated with 10 or 20 µg/ml GVC for 3 days, in the same cell density and conditions as described above.

Adenoviral DNA Extraction and Analysis.
Adenoviral DNA was isolated using the Hirt lysis protocol. Cells were harvested by trypsinization, and pellets were washed twice in ice-cold PBS. After resuspension in TE buffer [10 mM Tris (pH 8)-1 mM EDTA], cell pellets were disrupted by three cycles of freeze haw. An equal volume of Triton-X100 was added, and samples were incubated at 0°C for 10 min. NaCl at a final concentration of 0.3 M was added, and samples were incubated at 0°C for 2 h. Samples were then centrifuged for 5 min at 12,000 x g, and supernatants were incubated with self-digested Pronase at 1 mg/ml for 1 h at 37°C. Finally, Rnase A was added at a final concentration of 20 µg/ml, and samples were incubated for 30 min at 37°C. DNA was resuspended in a minimal volume after phenol:chloroform extraction, followed by ethanol precipitation. Dot blots using equal amounts of DNA were probed with radiolabeled vTK. Two experiments were carried out. The first was to test the effect of the activated cAMP-dependent pathway on infectivity after cAMP treatment. DA3 cells (2 x 10⁵) were seeded in two 25 cm² plates and incubated overnight. The following day, cAMP was added in one of the two plates, at a final concentration of 1 mM. Cells were infected with Ad-RSV-vTK (MOI of 10) 48 h after treatment with cAMP. At 12 h after infection, cells were lysed as described above, and DNA was immobilized on a nitrocellulose and analyzed for vTK content. The second experiment also intended to see the effect of cAMP on infectivity and viral DNA stability and expression in the mixing assay conditions. The precise conditions of the cell mixing assay (described previously) were replicated, and cells were collected and lysed at 24 and 48 h after infection. DNA was immobilized on nitrocellulose and analyzed for vTK content.

Effect of cAMP on GCV Cytotoxicity
To test whether cAMP and GCV have a synergistic effect on cell survival, the mixing experiment conditions were repeated for uninfected cells. DA3 cells were seeded in two 75 cm² tissue culture plates and incubated overnight. The following day, 1 mM cAMP was added in one of the two plates, and both plates were further incubated for 2 days. Cells were then trypsinized, seeded in 96 well-plates at a density of 10⁴ cells/well, and incubated with or without GCV at the concentrations used in the mixing assay (10 or 20 µg/ml) for 3 days. Finally, the MTT survival assay was performed, and the percentage of cell survival was calculated as the cell viability of the GCV-treated cells divided by cell viability of GCV-untreated cells.

	Results

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
Increased GJIC by cAMP.
Cx43 is the predominant GJ protein in breast tissue. Fig. 1 shows the effect of cAMP on the spatial localization of Cx43 in DA3 breast adenocarcinoma cells. Untreated (A and B) or cAMP-treated (C and D) DA3 cells were immunolabeled for Cx43 and transmitted light (A and C) or fluorescent (B and D) images were collected under identical conditions for direct comparison. Cx43 assembly into GJ plaques is clearly more evident after cAMP treatment (D). We also did a functional assay of GJIC in the absence and presence of cAMP. As shown in Fig. 2 , there is a significant increase in the GJIC in DA3 cells after exposure to cAMP under these conditions. Together, these assays demonstrate that cAMP is effective at increasing the amount of functional Cx43 that results in induced GJIC.

View larger version (133K): [in this window] [in a new window]   Fig. 1. Effect of cAMP on the spatial localization of Cx43 in DA3 cells. Untreated DA3 cells (A and B) or DA3 cells treated with cAMP for 2 days (C and D) were immunolabeled for Cx43, and transmitted light (A and C) or fluorescent (B and D) images were collected under identical conditions for direct comparison. Bar, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Effect of cAMP on GJIC, as measured by FACS analysis. GJIC was estimated before and after exposure to cAMP under the conditions described in "Materials and Methods." The results represent the means of two separate experiments ± SE.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Mixing assays of cell cytotoxicity at various percentages of Ad-RSV-vTK-infected cells and drug treatment. DA3 cells infected with Ad-RSV-vTK were mixed with noninfected cells resulting in varying percentages of infected cells in the total cell population, as explained in "Materials and Methods." Cells were then exposed to 10 µg/ml (A) or 20 µg/ml (B) GCV for 3 days. Cell survival was measured in each condition, with or without prior exposure to cAMP and corrected to cell survival of the corresponding uninfected cell population. Data points, means ± SE of multiple separate experiments, each performed in quadruplicate.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Percentage of cells infected with Ad-RSV-vTK that are required to yield 50% cell killing. Estimates were extrapolated by the data points from Fig. 3 . Bars, means ± SE.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Cell cytotoxicity of various percentages of vTK-transduced stable cells and drug treatment. DA3-vTKiEGFP and DA3-vEGFP were combined at the same mixing percentages and cAMP treatments as in Fig. 3 . Cell survival was measured in each condition and cAMP treatment and corrected to the cell survival of the corresponding vTK-untransduced cell population (DA3-vEGFP). Data points, means ± SE.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Percentage of vTK-containing cells (DA3-vTKiEGFP) necessary to yield 50% killing. Estimates were extrapolated by the data points from Fig. 5 . Bars, means ± SE.

Effect of cAMP on Ad-RSV-vTK Infectivity and Viral DNA Stability.
Control experiments were performed to examine whether cAMP would affect infectivity, viral DNA stability, or GCV activity. DNA analyses show that there is no effect of cAMP on adenoviral infectivity, 48 h after cAMP treatment (Fig. 7A) . Also, cAMP exposure did not affect vTK levels or infectivity in the mixing assays described previously (Fig. 7B) . Finally, the possibility was examined that cAMP could have a synergistic effect in cell survival when coadministered with GCV. Fig. 8 shows that there are no cell survival differences between cAMP-treated and untreated cells after GCV administration.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Effect of cAMP on adenoviral infectivity. A, DA3 cells were treated with or without cAMP 48 h before infection with Ad-RSV-vTK at an MOI of 10. Cells were lysed 12 h after infection, and DNA was analyzed for the presence of the vTK gene. B, DA3 cells were seeded and treated according to the mixing assay protocol, as described in "Materials and Methods." Cells were lysed and analyzed at 24 and 48 h after infection. DA3 cells alone (uninfected and untreated) were also lysed to control for intracellular thymidine kinase. DNA was analyzed for the presence of the vTK gene.

View larger version (56K): [in this window] [in a new window]   Fig. 8. Effect of cAMP on GCV cytotoxicity. DA3 cells were treated with or without 1 mM cAMP for 2 days, as described in "Materials and Methods." Subsequently, cells were seeded at equal cell densities and treated with GCV for 3 days. MTT assays were performed to estimate cell survival, as described in "Materials and Methods." Bars, means ± SE.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

The BE observed in suicide gene therapy using the vTK/GCV system is a compelling example of an anticancer therapy in which not every tumor cell must be directly targeted. Therefore, efforts to enhance the effect may have a significant impact on the efficacy of this therapeutic approach. Studies on the BE using the vTK/GCV system were carried out using Cx43 stable transfectants (17) , Cx43 delivery systems, or cells with naturally higher levels of Cx43 (27) . Those studies were important in understanding the BE and establishing the role of GJIC as its main mediator. Although promising, introducing Cx43 DNA to induce the BE could have possible limitations in the therapeutic setting, since present gene delivery systems are ineffective in reaching a great proportion of the tumor population. An alternative approach would be to pharmacologically induce Cx43 and GJIC, and subsequently the BE. A chemical inducer can potentially access a greater number of the cancer population as compared with a virus delivery system. The first attempt to induce the BE pharmacologically was recently demonstrated using retinoic acid (28) . Although retinoids are known Cx43 and GJIC up-regulators, the study showed that they also have GJIC-unrelated effects on cell killing, because they affected cell survival even when all cells were infected with a vTK-expressing virus. Also, there was no examination of the BE on infectivity levels below 10%, which is likely a clinically relevant level of cell targeting when adenoviral delivery is used in vivo (2 , 3) .

We report here that 8-Bromo-cAMP clearly leads to increases in GJIC and Cx43-containing GJs, as previously reported for other cancer cell models (18) . This is associated with enhanced cell killing in a mixed population of breast cancer cells, most of which have not been transduced with vTK (vTK-transduced cells between 2 and 50% of the total cell population). We have shown this effect for both cell lines stably transduced with a retrovector and infected with an adenoviral vector. It has been proposed that activated GCV is able to pass through GJs. When a high percentage of the cell population already contains vTK and is, thus, able to intracellularly metabolize GCV, increased GJIC and transport of GCV to neighboring cells would not further enhance cell killing. Because the survival differences are only observed when a low proportion of cells is infected (between 2 and 50%), the most likely mechanism that could be involved is the BE via GJIC up-regulation. This reconfirms that cell killing is, in fact, due to modulation of GJIC and not another cAMP-dependent pathway. Subsequently, we have shown that cAMP does not affect virus infectivity, DNA stability, or GCV cytotoxicity. The reduction in cell survival, as enhanced through the BE, seems adequate to decrease by half the number of cells that need to be targeted to attain 50% killing.

Our study adds strength to the rationale to develop clinically tolerable potent GJ inducers, as well as to study their effects on other prodrug/suicide gene systems. Furthermore, there is evidence for a dose effect of the Cx43 expression, in that the enhanced BE observed in the Cx-transfection studies was most obvious in the clones expressing the highest concentration of Cx43 (17) . Previous work showing efficacy with retinoids suggests a number of avenues to pursue (19) . Although the study using retinoids seemed to also have a killing effect independent of the BE, we have previously shown synergy in Cx43 and GJIC induction using both retinoic acid and cAMP.⁴ Therefore, there may be merit to studying the retinoid-dependent pathway, in addition to the cAMP-dependent pathway, for Cx43 and GJIC induction in this context.

	ACKNOWLEDGMENTS

 
We thank Andre Paquin for technical assistance.

	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 Supported by grants from the Canadian Breast Cancer Research Initiative, the National Cancer Institute of Canada, the Medical Research Council of Canada, and the Natural Science and Engineering Research Council of Canada.

² To whom requests for reprints should be addressed, at McGill Centre for Translational Research in Cancer, Lady Davis Research Institute, Sir Mortimer B. Davis-Jewish General Hospital, Suite D127, 3755 Cote Ste. Catherine Road, Montreal, Quebec H3T-1E2, Canada. Phone: (514) 340-7915; Fax: (514) 340-7916; E-mail: gbatist{at}onc.jgh.mcgill.ca

³ The abbreviations used are: BE, bystander effect; GJ, gap junction; GJIC, GJ-mediated intercellular communication; Cx, connexin; cAMP, cyclic AMP; RSV, Rous sarcoma virus; vTK, viral thymidine kinase; vEGFP, enhanced green fluorescent protein; vTKiEGFP, herpes simplex vTK and EGFP; GCV, ganciclovir; FACS, fluorescence-activated cell sorting; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Calcein AM, Calcein acetoxymethylester; DiI, 1,1'-dioctadecyl-3,3,3,3'-tetramethylondocarbocyanine perchlorate; MOI, multiplicity of infection.

⁴ G. D. Carystinos, M. A. Alaoui-Jamali, J. Phipps, L. Yen, and G. Batist. Up-regulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells, submitted for publication.

Received 8/20/98; revised 10/19/98; accepted 10/27/98.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

1. Moolten F. L. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res., 46: 5276-5281, 1986.[Abstract/Free Full Text]
2. Kozarsky K. F., Wilson J. M. Gene therapy: adenovirus vectors. Curr. Opin. Genet. Dev., 3: 499-503, 1993.[Medline]
3. Putzer B. M., Bramson J. L., Addison C. L., Hitt M., Siegel P. M., Muller W. J., Graham F. L. Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer. Hum. Gene Ther., 9: 707-718, 1998.[Medline]
4. Freeman S. M., Abboud C. N., Whartenby K. A. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res., 53: 5274-5283, 1993.[Abstract/Free Full Text]
5. Fick J., Barker F. G. The extent of heterocellular communication mediated gap junctions is predictive of bystander tumor cytotoxicity in vitro. Proc. Natl. Acad. Sci. USA, 92: 11071-11075, 1995.[Abstract/Free Full Text]
6. Mesnil M., Piccoli C., Tiraby G., Willecke K., Yamasaki H. Bystander killing of cancer cells by herpes virus thymidine kinase gene is mediated by connexins. Proc. Natl. Acad. Sci. USA, 93: 1831-1835, 1996.[Abstract/Free Full Text]
7. Elshami A. A. Gap junctions play a role in the bystander effect of the herpes simplex thymidine kinase/ganciclovir system in vitro. Gene Ther., 3: 85-92, 1996.[Medline]
8. Wygoda M. R., Wilson M. R., Davis M. A., Trosko J. E., Rehemtulla A., Lawrence T. S. Protection of herpes simplex virus thymidine kinase-transduced cells from ganciclovir-mediated cytotoxicity by bystander cells: the Good Samaritan effect. Cancer Res., 57: 1699-1703, 1997.[Abstract/Free Full Text]
9. Lawrence T. S., Rehemtulla A., Ng E. Y., Wilson M., Trosko J. E., Stetson P. L. Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine. Cancer Res., 58: 2588-2593, 1998.[Abstract/Free Full Text]
10. Bruzzone R., White T. W., Paul D. L. Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem., 238: 1-27, 1996.[Medline]
11. Goodenough D. A., Goliger J. A., Paul D. L. Connexons, connexins, and intercellular communication. Annu. Rev. Biochem., 65: 475-502, 1996.[Medline]
12. Laird D. W., Castillo M., Kasprzak L. Gap junction turnover, intercellular trafficking and phosphorylation of Cx43 in brefeldin A-treated rat mammary tumor cells. J. Cell. Biol., 131: 1193-1203, 1995.[Abstract/Free Full Text]
13. Yamasaki H. Gap junctional intercellular communication and carcinogenesis. Carcinogenesis (Lond.), 11: 1051-1058, 1990.[Free Full Text]
14. Yamasaki H., Mesnil M., Omori Y., Mironov N., Krutovskikh V. Intercellular communication and carcinogenesis. Mutat. Res., 333: 181-188, 1995.[Medline]
15. Proulx A. A., Xiang Lin Z., Naus C. C. G. Transfection of Rhabdomyosarcoma cells with connexin43 induces myogenic differentiation. Cell Growth Differ., 8: 533-540, 1997.[Abstract]
16. Mehta P. P., Hotz-Wagenblatt A., Rose B., Shalloway D., Loewestein W. R. Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth. J. Membr. Biol., 124: 207-225, 1991.[Medline]
17. Dilber M. S., Abedi M. R., Christennson B., Kidder G., Naus C., Edvard Smith C. I. Gap junctions promote the bystander effect of herpes virus thymidine kinase in vivo. Cancer Res., 57: 1523-1528, 1997.[Abstract/Free Full Text]
18. Banoub R. W., Fernstrom M., Malkinson A. M., Ruch R. J. Enhancement of gap junctional intercellular communication by dibutyryl cyclic AMP in lung epithelial cells. Anticancer Res., 16: 3715-3719, 1996.[Medline]
19. Hossain M., Bertram J. S. Retinoids and gap junctions. Cell Growth Differ., 5: 1253-1261, 1994.[Abstract]
20. Stahl W., Nicolai S., Briviba K., Hanusch M., Broszeit G., Peters M., Martin H. D., Sies H. Biological activities of natural and synthetic carotenoids: induction of gap junctional communication and singlet oxygen quenching. Carcinogenesis (Lond.), 18: 89-92, 1997.[Abstract/Free Full Text]
21. Chen T. C., Hinton D. R., Zidovetzki R., Hofman F. M. Up-regulation of the cAMP/PKA pathway inhibits proliferation, induces differentiation, and leads to apoptosis in malignant gliomas. Lab. Invest., 78: 165-174, 1998.[Medline]
22. Monaghan P., Clarke C., Perusinghe N. P., Moss D. W., Chen X. Y., Evans W. H. Gap junction distribution and connexin expression in human breast. Exp. Cell Res., 223: 29-38, 1996.[Medline]
23. Lee S. W., Tomasetto J. C., Paul D. L., Sager R. Transcriptional down-regulation of gap junction proteins blocks junctional communication in mammary cell lines. J. Cell Biol., 118: 1213-1221, 1992.[Abstract/Free Full Text]
24. Laird D. W., Castillo M., Kasprzak L. Gap junction turnover, intercellular trafficking and phosphorylation of Cx43 in brefeldin A-treated rat mammary tumor cells. J. Cell. Biol., 131: 1193-1203, 1995.
25. Kiang D. T., Kollander R., Lin H. H., LaVilla S., Atkinson M. M. Measurement of gap junctional communication by fluorescence activated cell sorting. In Vitro Cell. Dev. Biol. Anim., 30A: 796-802, 1994.
26. Plumb J. A., Miloy R., Kaye S. B. Effects of the pH dependence on diphenyl-tetrazolium bromide-formazam absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res., 49: 4435-4440, 1989.[Abstract/Free Full Text]
27. Yang L., Chiang Y., Lenz H. J., Danenberg K. D., Spears C. P., Gordon E. M., Anderson W. F., Parekh D. Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells. Hum. Gene Ther., 9: 719-728, 1998.[Medline]
28. Park J. Y., Elshami A. A., Amin K., Rizk N., Kaiser L. R., Albelda S. M. Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy. Gene Ther., 4: 909-917, 1997.[Medline]

This article has been cited by other articles:

				M. Ahtiainen, J. Toppari, M. Poutanen, and I. Huhtaniemi Indirect Sertoli Cell-Mediated Ablation of Germ Cells in Mice Expressing the Inhibin-{alpha} Promoter/Herpes Simplex Virus Thymidine Kinase Transgene Biol Reprod, November 1, 2004; 71(5): 1545 - 1550. [Abstract] [Full Text] [PDF]

				C. A Waldren Classical radiation biology dogma, bystander effects and paradigm shifts Human and Experimental Toxicology, February 1, 2004; 23(2): 95 - 100. [Abstract] [PDF]

				G. D. Carystinos, M. Kandouz, M. A. Alaoui-Jamali, and G. Batist Unexpected Induction of the Human Connexin 43 Promoter by the Ras Signaling Pathway Is Mediated by a Novel Putative Promoter Sequence Mol. Pharmacol., April 1, 2003; 63(4): 821 - 831. [Abstract] [Full Text] [PDF]

				J. H.-C. Lin, J. Yang, S. Liu, T. Takano, X. Wang, Q. Gao, K. Willecke, and M. Nedergaard Connexin Mediates Gap Junction-Independent Resistance to Cellular Injury J. Neurosci., January 15, 2003; 23(2): 430 - 441. [Abstract] [Full Text] [PDF]

				M. Mesnil and H. Yamasaki Bystander Effect in Herpes Simplex Virus-Thymidine Kinase/Ganciclovir Cancer Gene Therapy: Role of Gap-junctional Intercellular Communication1 Cancer Res., August 1, 2000; 60(15): 3989 - 3999. [Abstract] [Full Text]

				W. Harrouk, B. Robaire, and B. F. Hales Paternal Exposure to Cyclophosphamide Alters Cell-Cell Contacts and Activation of Embryonic Transcription in the Preimplantation Rat Embryo Biol Reprod, July 1, 2000; 63(1): 74 - 81. [Abstract] [Full Text]

				D. W. Laird, P. Fistouris, G. Batist, L. Alpert, H. T. Huynh, G. D. Carystinos, and M. A. Alaoui-Jamali Deficiency of Connexin43 Gap Junctions Is an Independent Marker for Breast Tumors Cancer Res., August 1, 1999; 59(16): 4104 - 4110. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carystinos, G. D. Articles by Batist, G. Search for Related Content PubMed PubMed Citation Articles by Carystinos, G. D. Articles by Batist, G.