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[Cancer Research 63, 7128-7135, November 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Expression of CONNEXIN43 Is Highly Sensitive to Ionizing Radiation and Other Environmental Stresses1

Edouard I. Azzam, Sonia M. de Toledo and John B. Little2

Laboratory of Radiobiology, Harvard School of Public Health, Boston, Massachusetts 02115 [E. I. A., S. M. d. T., J. B. L.], and Department of Radiology, New Jersey Medical School, Newark, New Jersey 07103 [E. I. A., S. M. d. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
To gain a greater understanding of the mechanisms underlying the cellular responses to low-dose ionizing radiation, gene expression profiles were examined by microarray analysis of cDNA from confluent human diploid fibroblast cultures exposed to very low fluences of {alpha}-particles. The data, supported by Northern and Western analyses, indicate that radiation induces a significant up-regulation of CONNEXIN43 expression. This phenomenon was observed in a variety of irradiated cell types. These findings are consistent with our previous observations that connexin43 (cx43)-mediated gap-junction intercellular communication is involved in the bystander response observed in cell cultures exposed to fluences of {alpha}-particles by which only a very small fraction of the cell nuclei is traversed by a particle track (E. I. Azzam et al., Proc. Natl. Acad. Sci. USA, 98: 473–478, 2001). Increased mRNA levels in cells from irradiated cultures correlated with increased cx43 protein levels by ~4 h after irradiation. The induction of cx43 was observed by mean {alpha}-particle doses as low as 0.16 cGy, and also in cells exposed to {gamma}-rays, t-butyl hydroperoxide, and hyperthermia. Exposure to these stresses also resulted in post-translational modification of cx43; increased phosphorylation and hyperphosphorylation of the protein was observed. Up-regulation of cx43 expression in ionizing radiation exposed cells correlated with functional communication through gap junctions, as evidenced by dye transfer from irradiated to nonirradiated cells. In contrast, the response after UV radiation varied and was cell type-dependent. Overall, these data suggest a critical role for genes involved in intercellular communication in mediating the cellular responses to a variety of stresses.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
It is now well established that when mammalian cell populations are exposed to IR,3 biological effects in individual cells need not be a consequence of direct nuclear irradiation. For example, numerous reports have indicated that when confluent cultures are exposed to low fluences of {alpha}-particles, fluences by which only a very small fraction of the nuclei is traversed by a particle track, DNA damage, changes in gene expression, and genetic effects occur not only in the irradiated cells but also in the bystander, nonirradiated cells (1) . The occurrence of such bystander effects is considered to have important consequences to risk assessment, radiotherapy, and radiodiagnostics.

We have shown previously that cx43-associated GJIC and oxidative metabolism have a significant role in mediating radiation-induced bystander effects in human and rodent cell cultures exposed to very low mean doses of {alpha}-particles (2, 3, 4) . Gap-junctions are clusters of small aqueous channels that link the cytoplasms of contiguous cells and may mediate the bystander effect by allowing direct intercellular exchange of ions, small metabolites, and second messengers between irradiated and nonirradiated cells (5) . They may regulate the bystander effect independently or cooperatively with oxidative metabolism (4) . GJIC has been shown to contribute to tissue homeostasis, electrical coupling, tissue response to hormones, and regulation of embryonic development (6) . Gap-junctions are made of two hemi-channels called connexins, and each connexin is composed of six molecules of the membrane spanning connexin proteins. Connexins are an extensive family of proteins comprising several members (7) that are expressed in different tissues and have different selectivity related to the size and charge of the communicated molecules (8) .

To additionally understand the molecular events underlying the cellular response in cultures exposed to IR under bystander conditions, we carried out microarray cDNA analyses in confluent normal human cell cultures exposed to a mean dose of 1 cGy from {alpha}-particles under normal culture conditions or when GJIC had been down-regulated in the presence of lindane ({gamma}-isomer of hexachlorocyclohexane; Ref. 9 ). Our data, confirmed by Northern and Western analyses, indicate significant up-regulation of CONNEXIN43 expression in cultures irradiated in both the presence and the absence of lindane. They also show that IR regulates cx43 at the post-translational level by inducing its phosphorylation at mean {alpha}-particle doses by which <1% of the nuclei in the exposed population is traversed by a particle track. Furthermore, induction of cx43 was also observed in cultures exposed to {gamma}-rays, t-butyl-hydroperoxide, and hyperthermia indicating that modulation of its expression could serve as a sensitive marker of environmental stress.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Human Cells.
For experiments, AG1522 normal human skin fibroblasts were seeded at 104 cells/cm2, and subsequently fed on days 4 and 7 with Eagle’s Minimal Essential Medium supplemented with 10% heat-inactivated FCS and 0.025 mg/ml gentamicin sulfate (3) . Experiments were started 48 h after the last feeding when 95–98% of the cells were in G0/G1. As cellular radiation sensitivity changes at different phases of the cell cycle (10) , the cells were synchronized in G0/G1 by confluent, density inhibition of growth. Passage 10 or 11 cells maintained in a 37°C humidified incubator in an atmosphere of 5% CO2 in air were used. Control cells were sham-manipulated and handled in parallel with the test cells.

MEFs.
WT and cx43-/- primary MEFs were established from day 9 embryos, and cells in passages 6–9 were used (3) . They were grown in modified Eagle’s medium with Earle’s balanced salt solution supplemented with 50% more vitamins and essential amino acids except glutamine, 100% more nonessential amino acids, and 1 mM sodium pyruvate (Life Technologies, Inc.). The medium was supplemented with 15% heat-inactivated FCS. Cells were seeded at a density of ~5 x 104 cells/cm2; they were fed twice at 48-h intervals after seeding, and experiments were started 48 h after the last feeding when they were confluent.

Rat Liver Epithelial Cells.
The GJIC competent WB-F344 cell line was a gift from Dr. James Trosko (Michigan State University, East Lansing, MI). The GJIC-deficient WM-aB1 cells were derived from a mutant clone of WB-F344 cells (11) . The cells were seeded at a density of 104 cells/cm2 and cultured according to the protocol described for MEFs, except the growth medium was supplemented with 5% FCS.


   Irradiation, Hyperthermia, and Hydroperoxide Treatments
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
For {alpha}-particle irradiation, cells plated on a 1.5-µm thick mylar surface were exposed to a 238Pu-collimated source at a dose rate of 9.9 cGy/min as described previously (3) . Irradiation was carried out with {alpha}-particles of 3.65 MeV average energy at the cell layer. The fraction of cells of which the nucleus was traversed by an {alpha}-particle was derived from Poisson statistics, and estimates involving cell geometry, {alpha}-particle fluence, and energy loss (12) .

For {gamma}-irradiation, cell cultures were exposed in a 137Cs irradiator (J. L. Shepherd, Mark I, San Fernando, CA) at a dose rate of 0.9 Gy/min. For UV irradiation, the growth medium was removed, and the cells were rapidly rinsed with PBS. The salt solution was aspirated, and the cell monolayers were exposed to UVC radiation (254 nm light) at a dose rate of 10 J/m2/min. After irradiation, the conditioned growth medium was returned to the culture plates, which were incubated at 37°C. Control cultures were sham-treated and handled in parallel with the test cells.

For heat treatment, cell cultures flasks with 5 ml of growth medium were immersed in a 45°C water bath for 5 or 25 min, reimmersed in a 37°C bath for 5 min to ensure homogenous rapid cooling, and then transferred to the 37°C incubator.

For t-butyl hydroperoxide treatment, cell cultures were exposed to 10 or 100 µM in growth medium for 15 min, they were then rinsed in PBS, and 48 h-conditioned medium was added.

In confluent AG1522 fibroblasts, heating at 45°C for 5 min or exposure to 10 µM t-butyl hydroperoxide for 15 min resulted in minimal cell killing (~90% clonogenic survival). Exposure of these cells to either of the following treatments: 45°C for 25 min, 100 µM t-butyl hydroperoxide for 15 min, 4 Gy from {gamma}-rays, or 0.8 Gy from {alpha}-particles resulted in ~10% clonogenic survival. Whereas, doses of 12 Gy from {gamma}-rays and 1 Gy from {alpha}-particles reduced clonogenic survival to 1–10% in confluent WB-F344 cells, 16 Gy from {gamma}-rays and 1.5 Gy from {alpha}-particles were required to reduce survival to the same level in WM-aB1 cells.


   RNA Extraction, cDNA Microarray and Data Analysis, and Northern Analysis
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell monolayers were rinsed with PBS, and RNA was extracted by the guanidinium thiocyanate-phenol chloroform method (13) and resuspended in water to a final concentration of 1 µg/µl. For cDNA microarray analyses, total RNA (2 µg) was reverse transcribed, labeled, and hybridized to MICROMAX Human cDNA Microarray System 1.1 (2400 genes) slides according to instructions in the MICROMAX labeling and detection kit (Perkin-Elmer Inc.). Tyramide signal amplification was adopted in the analysis, and Perkin-Elmer, Inc. provided custom scanning and data processing services. In this system, mRNA for three different plant genes are added to the experimental mRNA samples as internal controls and corresponding cDNA spots are localized in the four quadrants of the microarray slide. In our experiment, equal signal from the two different tyramide dyes of the control cDNA spots was obtained indicating equal efficiency of the different reactions in the analysis of experimental mRNA samples.

For Northern analyses, 1–5 µg samples of total RNA were fractionated in 1% denaturing agarose gels, blotted and hybridized with a 32P-labeled connexin43 cDNA probe by standard methods. Level of 18S rRNA was used to verify equal loading of the samples.


   Western Analysis
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
RESULTS
 DISCUSSION
 REFERENCES
 
After experimental treatments, the cells were rinsed in PBS, proteins were extracted in chilled radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors, and analyzed as described previously (3) . Anti-cx43 and anti-{alpha}-tubulin (Ab-1), which were used to verify equal loading of the samples, were obtained from Chemicon International and Oncogene Research Products, respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
DISCUSSION
 REFERENCES
 
Monitoring of Gene Expression in Normal Human Fibroblast Cultures Exposed to Very Low Mean Dose of {alpha}-Particles: Up-Regulation of CONNEXIN43.
To gain insight into the mechanisms underlying the radiation-induced bystander effect, global changes in gene expression were monitored by cDNA microarray analyses in confluent AG1522 fibroblast cultures at 3 h after exposure to 1 cGy from {alpha}-particles, a dose by which 6% of the nuclei in the exposed culture would be traversed by an {alpha}-particle track. Of special interest was the up-regulation of connexin43 mRNA because of the role of cx43 GJIC in the response of cell cultures irradiated under bystander conditions (2 , 3) . Relative to respective controls, the results in Table 1Citation indicate a 1.9- and 5-fold increase in connexin43 expression in AG1522 cell cultures irradiated in either the absence or presence of the gap-junction inhibitor lindane. These results are consistent with the concept that cx43-gap junctions participate in the {alpha}-particle-induced bystander effect (1) and possibly other responses to IR. They suggest that regulation of GJIC is an important cellular response to IR. Consequently, we focused on additionally characterizing CONNEXIN43 regulation by Northern and Western analyses in various cell types exposed to IR and other stresses.4


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  		Table 1 Regulation of connexin43 mRNA expression, assayed by cDNA microarray analyses, in AG1522 confluent fibroblast cultures exposed to 1 cGy from {alpha} particles in the absence or presence of 50 µM lindane

Fold changes (irradiated/sham-irradiated control) are shown.

 
CONNEXIN43 Is Regulated by {alpha}-Particle as well as {gamma}-Radiation in Various Cell Types.
In the above microarray analyses in cultures exposed to 1 cGy under normal growth conditions, up-regulation of CONNEXIN43 was evident but <2-fold. However, examination by Northern and Western analyses of AG1522 cultures exposed to low fluences of {alpha}-particles under these conditions indicated that the gene is indeed activated. Relative to sham-irradiated controls, the data in Fig. 1, A and BCitation , indicate that CONNEXIN43 mRNA and protein levels are increased after exposure to mean doses extending from 1 to 24 cGy. The similar increases at all of the doses indicate that low fluences of {alpha}-particles induce molecular pathways that lead to maximal up-regulation of CONNEXIN43. Fig. 1BCitation indicates increased expression in three protein bands detected by the antibody used. These bands were described previously to represent the native, phosphorylated, and hyperphosphorylated isoforms of cx43 (14) . Our data show that low fluences of {alpha}-particles up-regulate both the native and the post-translationally modified isoforms in confluent normal human fibroblasts (AG1522).



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  		Fig. 1. Up-regulation of CONNEXIN43 in {alpha}-particle-irradiated cell cultures. A, Northern analyses of connexin43 expression in AG1522 fibroblast cultures exposed to {alpha}-particles doses ranging from 0 to 24 cGy and held at 37°C for 6 h. B, Western analyses of cx43 in AG1522 confluent cultures at 3 h after exposure to {alpha}-particles doses ranging from 0 to 10 cGy under normal growth conditions. C, Western analyses of cx43 expression in lysates from isogenic WT or cx43-/- MEF cultures exposed to {alpha}-particles (20 cGy) and harvested for analysis 2 or 4 h later. D, Western analyses of cx43 expression in confluent cultures of GJIC-proficient WB-F344 cells and GJIC-deficient WM-aB1 cells exposed to {alpha}-particles in a dose range from 0 to 10 cGy. Cells were harvested for analysis 4 h after exposure.

 
The Western analyses in Fig. 1CCitation in control and {alpha}-particle exposed MEFs that are WT or isogenic knockout for cx43 confirm that the protein detected with the antibody used in these studies is indeed cx43. Cx43 was detected in WT control cells, and its levels were increased in these cells at 2 and 4 h after exposure to 20 cGy. As expected, Fig. 1CCitation shows that cx43 was undetectable in the knockout cells.

The Western analyses in Fig. 1DCitation indicate that activation of CONNEXIN43 by {alpha}-particles is also observed in WB-F344 (GJIC proficient) and WM-aB1 (GJIC deficient) epithelial cells exposed to mean doses as low as 0.16 cGy. At these doses <1% of the nuclei in the exposed confluent culture would be traversed by a particle track. Interestingly, cx43 protein was up-regulated in both cell lines. The results with these epithelial cells confirm our observation with AG1522 fibroblasts (Fig. 1B)Citation and show that the effect was dose independent over the range extending from 0.16 cGy to 10 cGy. The up-regulation of cx43 in WM-aB1 cells is consistent with its up-regulation in lindane-treated cultures. Significantly, the magnitude of the induction at the very low mean doses and its monotony over a broad dose range suggests that cx43 may have been induced in nonirradiated bystander cells also.

The analyses in Fig. 2Citation extend the observations in the microarray analyses (Table 1)Citation and in Fig. 1Citation , and indicate that CONNEXIN43 is also regulated by {gamma}-rays. The data in Fig. 2ACitation indicate that connexin43 mRNA levels are increased in both lindane-treated and untreated cultures of WB-F344 cells exposed to {gamma}-rays. Relative to sham-irradiated/lindane-treated controls, a 2-fold increase was observed 4 h after exposure to 6 Gy (a dose that reduced survival to 50%). In addition, connexin43 mRNA levels were also increased in irradiated cultures that were not treated with lindane.



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  		Fig. 2. Up-regulation of CONNEXIN43 in {gamma}-irradiated cell cultures. A, Northern analyses of connexin43 expression in WB-F344 cultures exposed to 0 or 6 Gy from {gamma}-rays in the presence or absence of lindane (50 µM) and held at 37°C for 4 h. B, Western analyses of cx43 expression in WB-F344 or WM-aB1 cultures exposed to isosurvival doses of 12 or 16 Gy respectively from {gamma}-rays. Cells were harvested for analysis at times ranging from 1.5 to 9 h after exposure. C, Western analyses of AG1522 cells exposed to 0.5 or 4 Gy and harvested for analysis 3 h later.

 
The analyses in Fig. 2BCitation show that relative to sham-treated control cells, cx43 levels are increased in WB-F344 and WM-aB1 cells exposed to {gamma}-rays. The hyperphosphorylated, phosphorylated, and native forms of the protein were induced in WB-F344 cell lines by 3 h after exposure to 12 Gy. In WM-aB1 cells, at the iso-survival dose of 16 Gy, the native and phosphorylated forms were increased. As expected, the hyperphosphorylated form (top band) observed in WB-F344 cells and that is attributed to contribute to GJIC in these cells (11) is absent in sham-treated control and in irradiated WM-aB1 cells. Up-regulation of cx43 in both WB-F344 and WM-aB1 cells was also observed at lower doses (1–6 Gy; data not shown).

The data in Fig. 2CCitation indicate that a low dose of {gamma}-rays (0.5 Gy) up-regulates cx43 levels in AG1522 cells by 3 h after irradiation. Up-regulation of cx43 by IR appears to be a general response as it was observed in several other cell strains (e.g., GM5758 normal human fibroblasts, GBM-11T human glioblastoma cells, and MEFs; data not shown). Analyses (data not shown) with the various cell strains examined indicated that the kinetics of up-regulation and decay of CONNEXIN43 mRNA and protein expression levels may vary among experiments.

CONNEXIN43 Is Regulated in Cells Exposed to Hyperthermia, Hydroperoxide, and UV Light.
Next, we tested whether CONNEXIN43 is regulated by stresses other than IR. The data in Fig. 3, A and BCitation , indicate that CONNEXIN43 mRNA and protein are regulated by 6 h in WB-F344 cells heated at 45°C. A 5- or 25-min heat exposure increased connexin43 mRNA by 2-fold. It is of interest to note that whereas a 5 min exposure to 45°C resulted in increased levels of the phosphorylated isoforms with no apparent change in the native form, a 25-min heating at 45°C increased the hyperphosphorylated levels and resulted in the appearance of two additional, presumably super-hyperphosphorylated, isoforms; these changes were coupled with a decrease in the native form of the protein.



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  		Fig. 3. Regulation of CONNEXIN43 by hyperthermia, hydroperoxide, and UV radiation in WB-F344 cells. A, Northern analyses of connexin43 in WB-F344 cultures at 6 h after exposure to 45°C for 0, 5, or 25 min; B, parallel Western analyses to those in A; C, WB-F344 cultures exposed for 15 min to 10 or 100 µM t-butyl hydroperoxide and harvested for analysis 6 h later; D, WB-F344 cultures exposed to 10 J/m2 from UVC radiation and analyzed 6 h later.

 
Whereas a 15 min exposure to a mild dose (10 µM) of t-butyl hydroperoxide did not result in any change in cx43 expression, exposure to 100 µM significantly up-regulated the native and phosphorylated isoforms (Fig. 3C)Citation . Up-regulation of the hyperphosphorylated isoform in WB-F344 cells exposed to 500 µM hydrogen peroxide for 1 h was observed previously (15) .

In contrast to the above stresses, the data in Fig. 3DCitation show that exposure of WB-F344 cells to a UVC dose of 10 J/m2 resulted in significant reduction in levels of the cx43 native isoform by 2 h after exposure. No apparent changes in the expression levels of the phosphorylated isoforms were detected. These UVC effects appear to be cell type-specific, as they were not observed in AG1522 cells (data not shown).

Stability of the Induced Connexin43 Protein in Irradiated Cultures.
To examine the effect of radiation-induced post-translational modification of cx43 on its stability, cycloheximide (2 µg/ml) was added to WB-F344 confluent cultures 2 h before sham manipulation or exposure to {gamma}-rays (6 Gy). The data in Fig. 4Citation show that at 7 h after irradiation, the native form of cx43 was undetectable in either sham-manipulated control or irradiated cell cultures treated with cycloheximide. These data suggest that the increased level of native cx43 detected in control irradiated cells (Figs. 1Citation 2Citation 3)Citation is a result of de novo protein synthesis. In contrast to the native isoform, Fig. 4Citation indicates that the phosphorylated and hyperphosphorylated forms were detectable in both sham-manipulated control and irradiated cells treated with cycloheximide, with the hyperphosphorylated isoform levels being greater in irradiated than in control cells. Considering that the half-life of cx43 is short (1–3 h; Refs. 16, 17, 18 ), these data suggest that either the phosphorylated isoforms have a much greater half-life or that cycloheximide inhibited the synthesis of a protein that renders these isoforms substrate for degradation.



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  		Fig. 4. Stability of cx43 in IR exposed WB-F344 cells. Western analyses of cx43 expression in {gamma}-ray-irradiated (6 Gy) WB-F344 cultures in the presence or absence of cycloheximide (2 µg/ml). Cells were harvested for analysis 7 h after irradiation.

 
Expression and Post-Translational Modification of cx43 Correlate with the Ability of Stressed Cells to Functionally Communicate via Gap-Junctions with Nonstressed Cells.
Whereas increased expression of cx43 is associated with greater communication between contiguous cells (19, 20, 21) , phosphorylation of connexin proteins has been shown to both facilitate and restrict GJIC (22) . Importantly, activation of protein kinase C, which is radiation inducible (23 , 24) , has been shown to decrease junctional conductance and increase the turnover of cx43 (14) . To investigate the effect of cx43 regulation in stressed cells (Figs. 1Citation 2Citation 3)Citation on GJIC, a coculture experiment was performed. Confluent WB-F344 cultures were washed with PBS and dyed with CellTracker Orange CMTMR fluorescent dye (Molecular Probes, Eugene, OR) that remains tightly bound to cells. Additionally, the cells were dyed with Calcein AM (Molecular Probes). Once incorporated into the cell, this dye can migrate to adjacent cells through functional gap-junctions only. After labeling with the dyes, conditioned medium was returned to the cultures and 1 h later these cultures were {gamma}-irradiated (6 Gy), heated at 45°C for 5 min, or exposed to UVC (10 J/m2). Within 15 min after stress, the cultures were trypsinized and the cells were suspended with unstressed and undyed bystander cells originating from a confluent WB-F344 culture. Cell suspensions of stressed cells and bystander were plated at high densities at a respective ratio of 1:99 in conditioned growth medium. At different times thereafter, the cells were examined with a fluorescent microscope equipped with Texas Red (Fig. 5, A and C)Citation and FITC filters (Fig. 5, B and D)Citation . The data in Fig. 5ACitation represent a field of CellTracker Orange dyed/{gamma}-irradiated cells in the coculture. The data in Fig. 5BCitation are representative of Calcein AM dye transfer that typically occurs between {gamma}-irradiated cells and bystander cells. These data show that at about 1–3 h after plating, transfer of Calcein AM from {gamma}-irradiated cells to nonirradiated, undyed bystander cells was clearly observed. Similar Calcein AM dye transfer results were obtained between heat-stressed or sham-manipulated dyed cells and unstressed, undyed bystander cells (data not shown). These results indicate that irradiated or heated cells establish functional GJIC with bystander cells and suggest that the phosphorylation of cx43 in irradiated or heated cells does not inhibit GJIC in our studies. In contrast, relative to sham-manipulated control cocultures, Calcein AM transfer was significantly attenuated between UVC-irradiated and bystander cells (Fig. 5D)Citation . The decreased ability of UVC-irradiated cells to communicate via gap-junctions with bystander cells correlates with the down-regulation of cx43 in these cells (Fig. 3D)Citation . In UVC-irradiated AG1522 cells where no decrease in cx43 levels was observed, dye transfer between irradiated and bystander cells was clearly observed (data not shown).



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  		Fig. 5. Calcein AM transfer from stressed to bystander cells. Calcein AM (green fluorescence) and CellTracker Orange (red fluorescence) dyed WB-F344 cells were either sham-stressed or stressed and mixed with nondyed, nonstressed bystander cells at a respective ratio of 1:99. Cells were microscopically examined with either Texas Red (A and C) or FITC filters (B and D). A, representative field showing the {gamma}-irradiated/dyed cells present in the coculture. B, same field in A showing Calcein AM dye transfer that occurs between {gamma}-irradiated (6 Gy) and bystander cells at 1.5–3 h after coculture. Similar results were observed between sham-stressed or heated (5 min at 45°C) cells and bystander cells. C, representative field showing UVC-irradiated/dyed cells. D, same field in C showing minimal Calcein AM dye transfer between UVC irradiated (10 J/m2) and bystander cells up to 6 h after coculture.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
REFERENCES
 
Over the past decade, a number of distinct signal transduction pathways that couple environmental stimuli to specific changes in gene expression have been described. For example, the pathways that regulate cell cycle checkpoints in irradiated mammalian cells are presently well characterized (25) . This study indicates that cellular exposure to various environmental stresses results in highly sensitive regulation of CONNEXIN43 of which the product is thought to regulate the homeostatic response of tissues through its role in GJIC (26) . Of particular significance is the finding that exposure of confluent cell cultures to very low fluences of {alpha}-particles (mean doses as low as 0.16 cGy) that do not result in toxic effects significantly up-regulates cx43 expression. These results are consistent with our previous findings (3) that confluent cell populations exposed to low mean doses of {alpha}-particles respond to this type of radiation stress as an integrated unit rather than as separate individual cells that have been directly irradiated.

The equal sensitivity in the regulation of cx43 in low fluence {alpha}-particle irradiated GJIC competent (WB-F344) and deficient (WM-aB1) cell cultures (Fig. 1D)Citation highlights the complexity of the mechanisms underlying the bystander response. We have shown previously that, in addition to GJIC, oxidative metabolism is an important mediator of the {alpha}-particle-induced bystander effect (4) . Oxidative metabolism (27) or additional factors (28) may have contributed to up-regulation of cx43 in WM-aB1 GJIC-deficient cell cultures irradiated under bystander conditions. Significantly, the present study shows that the regulation of cx43 expression is sensitive to oxidative stress (Fig. 3C)Citation . Analysis of the promoter region of CONNEXIN43 has revealed binding sites for the redox-sensitive transcription factors nuclear factor {kappa}B (29) , activator protein 1 (30) , and ATF2 (31) ; our recent studies (4) showed that nuclear factor {kappa}B, ATF2, and activator protein 1 are significantly and rapidly activated in AG1522 cultures exposed to very low fluences of {alpha}-particles. Other mechanisms such as released factors from irradiated cells (32, 33, 34) that, in turn, have an effect on nonirradiated cells may also be involved.

This study has shown that post-translational modification of cx43 is a major cellular response to stress (Figs. 1Citation 2Citation 3)Citation . Phosphorylation of connexin proteins has been implicated in the regulation of multiple connexin processes among which are trafficking, assembly/disassembly, degradation, and gating of gap junction channels (14) . A rapid switch in phosphorylation is thought to represent a mechanism to restrict or facilitate transfer of selected signals between cells (35) , and the phosphorylation of cx43 has been reported to influence GJIC in both a positive and a negative manner that depends on cell type (14 , 36, 37, 38) . Generally, an increase in GJIC activity correlates with the phosphorylation of cx43 when cyclic AMP levels have been increased (39 , 40) . Our data showing functional communication between {gamma}-irradiated and nonirradiated cells (Fig. 5A)Citation is consistent with our previous studies that GJIC is an important mediator of the radiation-induced bystander effect, and correlates with increased native and phosphorylated cx43 levels in the exposed cultures. The fact that phosphorylation occurs soon after synthesis (17 , 18 , 41) supports the concept that the up-regulated phosphorylated cx43 isoforms we have observed in stressed cultures result from de novo synthesis. Cx43 is phosphorylated primarily on multiple serine residues, but also phosphorylation of threonine and tyrosine residues has been detected (14) , and the cx43 located in gap-junctions is primarily phosphorylated (42) . It has been suggested that the events leading to phosphorylation of cx43 may cause the formation or stabilization of gap-junctions (14) . Several kinases (e.g., mitogen-activated protein kinase, protein kinase C, protein kinase A, pp60src kinase, and p34cdc2) were reported to be involved in the phosphorylation events (14) . These kinases are known to be regulated by IR (43, 44, 45, 46) , and our previous studies had indicated that activation of mitogen-activated protein kinase and p34cdc2 in {alpha}-particle irradiated AG1522 cultures occurs in a bystander manner (2 , 4) .

Compared with other integral membrane proteins, the half-life of cx43 is quite short (1–3 h; Ref. 16 ). The data in Fig. 4Citation indicating increased expression of the hyperphosphorylated isoform in irradiated cells treated with cycloheximide suggests that IR regulates cx43 expression at the protein stability level also. A stable pool of cx43 in cells treated with cycloheximide was suggested previously to exist (47) . We show that this pool may consist of the phosphorylated isoforms (Fig. 4)Citation . Collectively, these results highlight the concept that the regulation of cx43 turnover is also an important factor in the control of GJIC in irradiated cultures. Considering that hyperphosphorylation of cx43 is closely correlated with incorporation of cx43 into gap-junction plaques (42 , 48) , these data emphasize the importance of cx43 GJIC in the cellular response to IR.

In contrast to studies with cx32 (49 , 50) , our data clearly show that IR, a net generator of superoxide and hydroxyl radicals, leads to up-regulation of cx43 expression in various cultured cell types. Ischemia/reperfusion studies in liver had suggested that oxygen radicals were responsible for the down-regulation of cx32 expression (49 , 50) . Liver perfusion with H2O2 resulted in a rapid degradation of cx32 (51) . Such differences suggest that the regulation of various connexin proteins by oxidative stress may vary.

Intercellular contact has long been considered of importance in mediating the cellular response to IR (52, 53, 54) , and gap-junctions have been postulated to play a role in radiation-induced biological effects (55) . Enhanced immunoreactivity of cx43 in mouse epidermal cells was observed 7 days after the start of daily irradiation with 3 Gy (56) and in rat lungs 1 week after irradiation (57) . In those studies, the enhanced expression was thought to be associated with accelerated repopulation in the irradiated tissue. The present study additionally develops these concepts, and shows that IR and other stresses result in rapid regulation of CONNEXIN43 mRNA and protein expression in mammalian cells. Regulation of cx43 expression by stress correlated with the ability of the stressed cells to functionally communicate through gap-junctions with neighboring nonstressed cells. Elucidation of the mechanisms underlying these effects will not only increase our understanding of the response to stress at the cellular and tissue level but will also increase our knowledge of intercellular communication.

Changes in the expression of genes other than CONNEXIN43 were also detected by cDNA microarray analyses in AG1522 confluent cultures exposed to 1 cGy from {alpha}-particles in the absence or presence of 50 µM lindane and are respectively described in Citation and Citation . Of 2400 genes examined, the data in Citation indicate that, relative to sham-treated controls, 28 genes involved in various cellular processes were up-regulated by at least 2-fold in cultures irradiated in the absence of lindane. Among these genes, BAL1 (58) was induced 4.5-fold. In addition, 26 genes had their mRNA expression levels decreased by at least 3-fold with the expression levels of {gamma}-ACTIN, COLLAGEN, and LAMININ B-2 decreased by 6–9-fold. The magnitude of regulation of the p53-inducible BAL1 gene in irradiated cultures where the majority of the cells were bystanders is consistent with our previous observations that p53 and its effector p21Waf1 are induced significantly in AG1522 bystander cells in {alpha}-particle irradiated cultures (Refs. 3 , 4 ; Note that WAF1 cDNA was not present on the microarray chip used in our experiments).


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  		4 Genes of which the transcripts are regulated by >2-fold (irradiated/sham-irradiated control) in AG1522 fibroblast cultures exposed to 1 cGy from {alpha}-particles

 

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  		4 Genes of which the transcripts are regulated by >2-fold (irradiated/sham-irradiated control) in AG1522 fibroblast cultures exposed to 1 cGy from {alpha}-particles in the presence of 50 µM lindane

 
The cDNA microarray analyses data in Citation show that relative to sham-treated cultures, 22 genes were up-regulated by at least 2-fold, and 27 genes were down-regulated by 3–7-fold when AG1522 cultures were exposed to 1 cGy under conditions by which GJIC was down-regulated by chemical inhibition with lindane (9) . Significantly, several genes were similarly regulated as in the nonlindane-treated, irradiated cultures Citation . Notably, another p53-regulated gene, 14-3-3-{varsigma} (59) , was also induced.


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  		4 Genes of which the transcripts were commonly regulated in AG1522 cultures exposed to a mean dose of 1 cGy from {alpha}-particles

Fold changes (irradiated/sham-irradiated control) in the presence or absence of 50 µM lindane are shown.

 
Interestingly, the results in Citation and Citation indicate that in addition to up-regulation of CONNEXIN43, {alpha}-particle irradiation of confluent cultures under bystander conditions significantly down-regulates the expression of cytoskeletal genes (e.g., {gamma}-ACTIN and ß-TROPOMYOSIN). A similar inverse relationship between cx43 and cytoskeletal proteins expression was reported previously in cultured smooth muscle cells (60) . In addition, our results indicate decreased expression of genes coding for adhesion molecules (e.g., COLLAGEN and LAMININ). The interaction between connexin proteins and adhesion molecules is under intense investigation (61) . It has been suggested that these two classes of cell surface proteins interact via a signal transduction system. Adhesion molecules have been proposed to participate in expression of the bystander effect through their role in the extracellular matrix (28) .


   ACKNOWLEDGMENTS
 
We thank Drs. Joseph Paulauskis, Aiman Al-Mutairi, Roger Howell, and Helene Hill for their input in this study, and Li Chen 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 FG02-98ER62685 and FG02-02ER63447 from the United States Department of Energy, 1RO1-CA92262-01A1 from the NIH, and 02-1081-CCR-S2 from the New Jersey Commission on Cancer Research. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Back

3 The abbreviations used are: IR, ionizing radiation; cx43, connexin43; GJIC, gap-junction intercellular communication; MEF, mouse embryo fibroblast; WT, wild-type. Back

4 Significant increases and decreases in the expression of genes other than CONNEXIN43 were also detected by cDNA microarray analyses in AG1522 fibroblast cultures exposed to 1 cGy in both the presence or the absence of 50 µM lindane. These results are presented and discussed in the Appendix: Citation , Citation , and Citation . Back

Received 4/24/03. Revised 8/11/03. Accepted 8/15/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation, Hyperthermia, and...
 RNA Extraction, cDNA Microarray...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 

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