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Carcinogenesis, Vol. 24, No. 11, 1723-1728, November 2003
© 2003 Oxford University Press

CANCER BIOLOGY

Gap junctional intercellular communication and cellular response to heat stress

Nobuyuki Hamada, Seiji Kodama, Keiji Suzuki and Masami Watanabe1

Division of Radiation Biology, Department of Radiology and Radiation Biology, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctional intercellular communication (GJIC) is essential in the maintenance of tissue homeostasis and has been implicated in tumor suppression. Recent studies have indicated that GJIC is also involved in cellular stress responses to low dose ionizing radiation, UV light and hydrogen peroxide. However, the contribution of GJIC to the heat stress response has not yet been elucidated. We here demonstrate a potential link between GJIC and the heat stress response. First, we investigated whether the abolition of GJIC by lindane affects heat sensitivity in normal human cells. Lindane potentiated cell killing by heat shock at 43°C, whereas little or no cytotoxicity was observed at 37°C. Nuclear translocation of heat shock protein 72 (HSP72) was interrupted by lindane, although its induction was not affected. These results indicate that lindane exacerbates hyperthermic lethality via disrupted nuclear translocation of HSP72 protein. Second, we assessed whether heat shock alters GJIC and phosphorylation of gap junction connexin (Cx) proteins in normal human cells. Persistent heat treatment augmented Cx43 phosphorylation in a heat- and time-dependent fashion and this phosphorylation was recovered after heat shock. GJIC was also disturbed by heat shock. These results indicate that heat shock augments Cx43 phosphorylation leading to GJIC abrogation. Our present results imply that GJIC contributes to protection against heat stress and that loss of GJIC function during carcinogenesis exacerbates hyperthermic lethality.

Abbreviations: Cx, connexin; GJIC, gap junctional intercellular communication; HSP, heat shock protein; MAPK, mitogen-activated protein kinase; PKC, protein kinase C


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
We are daily exposed to various stresses, such as ionizing radiation, UV light, oxidative stress and a non-permissive temperature. Normal human cells can respond to these stresses, whereas the stress response machinery is disturbed in human tumors. Concerning heat stress, malignant cells are more susceptible than their normal counterparts (1,2). Although this is one of the underlying causes of hyperthermia, very little is actually known about this mechanism. We previously found that confluent cells were more heat resistant than both sparse cells and a trypsinized suspension of normal confluent cells (1,2). However, little difference has been observed in heat sensitivity between confluent and sparse malignant counterparts (1,2). Therefore, it is highly likely that cell-to-cell contact is indispensable for an adequate cellular response to heat stress in normal human cells.

Among the systems that mediate cell-to-cell interaction, gap junctional intercellular communication (GJIC) is the unique route that allows direct exchange of small cytoplasmic molecules (<1 kDa) between contiguous cells (3) and plays essential roles in growth control, embryonic development and tissue differentiation (4). A gap junctional channel is composed of two juxtaposed transmembrane hemi-channels (connexons) provided by adjacent cells and each connexon is a hexamer of connexin (Cx) proteins. While Cx forms a multigene family consisting of at least 15 members in mammals (5), Cx43 is the most abundant and extensively studied form of Cx protein. All Cx proteins except for Cx26 are phosphoproteins (6) and phosphorylation of Cx is involved in various Cx behaviors, such as channel gating, trafficking, assembly and turnover (7,8). There is a great deal of evidence to indicate that an aberration in GJIC, via the abnormal localization and phosphorylation of Cx proteins and/or via the down-regulation of Cx genes, is involved in carcinogenesis and that Cx proteins are tumor suppressor proteins (9,10). It has recently been shown that GJIC is involved in the cellular response to various such stresses as hydrogen peroxide (11), UV light (12,13) and low dose ionizing radiation (14–16). However, the contribution of GJIC to the heat stress response has not yet been elucidated.

In this study, we postulate that GJIC is involved in the heat stress response in normal human cells. To test this hypothesis, the present investigation was designed to reveal whether GJIC abrogation affects heat sensitivity and to elucidate whether heat shock alters Cx phosphorylation and GJIC.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell cultures
Primary normal human diploid fibroblasts (HE49) derived from 7- to 8-week-old embryos were obtained as previously mentioned (17). Human cervical cancer-derived HeLa cells were provided by Dr K.Komatsu (Kyoto University, Japan). All cell cultures were routinely subcultured every 3–4 days in 75 cm2 tissue culture flasks in Eagle's minimum essential medium (Nissui Pharmaceutical Co. Ltd, Tokyo, Japan) supplemented with 10% fetal bovine serum (Trace Biosciences Pty Ltd, Melbourne, Australia) as previously described (17). For experiments, HE49 cells at passages 8–14 initially seeded at 5 x 105 cells/25 cm2 flask were cultured for 8 days to obtain confluent, density-inhibited cells, which were re-fed the day before heat shock treatment. Unless otherwise stated, all cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.

Treatment with lindane and heat shock
Cells were pretreated for 2 h with 0.1 mM lindane and were subsequently heat shocked at 43°C in a circulating water bath in the presence of lindane, because lindane is a reversible GJIC inhibitor (18,19). After heat shock, the cells were maintained in the absence of lindane. In parallel, control cells were treated with 0.1% dimethylsulfoxide as the solvent for lindane. Both reagents were purchased from Sigma (St Louis, MO).

Cell survival
Cell survival was tested by a colony formation assay as previously mentioned (20). Briefly, cells were re-plated into a 100 mm dish immediately after heat shock and were further kept for 10–12 days in the absence of lindane. Only colonies containing >50 cells were scored as survivors.

Dye transfer assay
The capacity of GJIC was assayed according to the scrape loading and dye transfer technique of El-Fouly et al. (21). Briefly, cells pretreated with lindane were rinsed twice with phosphate-buffered saline with Ca2+ and 0.05% Lucifer yellow CH (Molecular Probes, Eugene, OR) in PBS+ was added. The cell monolayer was scraped with a needle and kept in the dark for 2 min. Then, the cell monolayer was rinsed, fixed and observed with an Olympus AX70 fluorescence microscope.

Antibodies
The following antibodies against human proteins were used: anti-p53 (Ab-3, clone BP53-12) and anti-p21WAF1/CIP1 (Ab-3, DCS-60.2) were purchased from NeoMarkers; anti-phospho-p53 (Ser15) was purchased from Cell Signaling Technology; anti-heat shock protein (HSP)72 (Ab-1) was purchased from Oncogene Research Products; anti-Cx43 (clone 2, catalog no. 610061) was purchased from BD Transduction Laboratories.

Western blot analysis
Cell lysates were prepared as previously stated (22). Briefly, cells were lysed in radioimmunoprecipitation assay buffer and cleared by centrifugation. The supernatants were then used for western blotting after the protein concentration was determined. Whole cell extracts (8–16 µg) were electrophoresed on SDS–polyacrylamide gels, electroblotted onto Immobilon-P (Millipore) and the blots reacted with the above-mentioned antibodies as previously described (23). The blots were analyzed by scanning densitometry using the NIH Image 1.56 software.

Indirect immunofluorescent microscopy
Cells grown on coverslips were fixed in methanol. Then, HSP72 protein was detected using anti-HSP72 and fluorescein isothiocyanate-conjugated anti-mouse secondary antibodies. Nuclei were visualized by staining with propidium iodide.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
GJIC reduction by lindane exacerbated hyperthermic lethality of normal human cells
To explore the role of GJIC in the heat stress response, the effect of lindane on the heat stress response was examined. Lindane ({gamma}-hexachlorocyclohexane) is an effective GJIC inhibitor (24), which causes aberrant Cx43 localization in the cytoplasmic perinuclear compartments without affecting Cx43 phosphorylation (19). As depicted in Figure 1, lindane efficiently abolished GJIC of HE49 cells, to a level similar to that of HeLa cells lacking GJIC (25,26).



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  		Fig. 1. GJIC of untreated HE49 cells (A), lindane-treated HE49 cells (B) and untreated HeLa cells (C) were assessed by the dye transfer assay. Experiments were repeated twice with similar results, and representative data are shown.

 
We examined the effect of lindane on heat sensitivity. As shown in Figure 2, lindane exacerbated hyperthermic killing of HE49 cells 14.2-fold when heated for 6 h, whereas lindane was not cytotoxic at 37°C. On the other hand, heat sensitivity of HeLa cells was almost unaffected by lindane (data not shown).



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  		Fig. 2. (A) Cytotoxicity of lindane at 37°C. HE49 cells treated at 37°C with lindane for the time stated were subjected to colony formation assay. (B) Heat-sensitizing effect of lindane. Lindane-pretreated HE49 cells were heat shocked at 43°C for the indicated periods, followed by colony formation. Solid and open circles represent heat sensitivity of cells with and without lindane, respectively. These data are presented as means ± SD of four independent experiments with quadruplicate measurements.

 
Next, we investigated the influence of lindane on p53 accumulation after heat shock. We previously reported that heat shock causes p53 accumulation and its phosphorylation at Ser15, leading to a cell cycle arrest in G1 phase in a p53-dependent fashion (27,28). Lindane did not affect the levels of p53 accumulation, its phosphorylation at Ser15 or p21 induction (Figure 3). Thus, G1 arrest was not involved in the thermosensitizing effect of lindane.



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  		Fig. 3. Influence of lindane on the accumulation of p53 proteins. Lindane-pretreated HE49 cells were heated for 2 h and were further incubated at 37°C for various intervals, followed by western blot analyses. Hours after the 2 h heat treatment are indicated. Three independent analyses showed a consistent pattern of data, and representative blots are shown.

 
Subsequently, the effect of lindane on HSP72 was assessed. HSP72 protein, which is the major inducible member of the HSP70 family and presents mainly in the cytoplasm under normal conditions, translocates into nuclei and nucleoli during heat stress and protects cells against heat-induced damage (29–33). As shown in Figure 4, the induction of HSP72 protein after heat shock was not affected by lindane. On the other hand, lindane interrupted nuclear HSP72 translocation (Figure 5). In heated cells without lindane there were many fine dense nucleolar HSP72 foci, but these were fainter and much fewer in lindane-treated heated cells. Therefore, we further compared the difference in nucleolar HSP72 foci formation. Lindane decreased the fraction of cells with nucleolar HSP72 foci, as depicted in Figure 5B. Thus, lindane attenuated intranuclear HSP72 translocation after heat shock.



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  		Fig. 4. Effect of lindane on the induction of HSP72 proteins. (A) Lindane-pretreated HE49 cells were heated for 2 h and were subsequently kept at 37°C for various intervals, followed by western blot analysis. Hours after the 2 h heat shock are indicated. Three independent analyses showed a consistent pattern of data, and representative blots are shown. (B) Band intensity of the blot was densitometrically measured and the relative HSP72 expression was calculated by dividing the intensity of each band by that of an untreated control (left-most lane). These data represent means ± SD of three independent experiments.

 


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  		Fig. 5. Influence of lindane on the subcellular localization of HSP72 proteins. Lindane-pretreated HE49 cells were heated for 2 h and were then recovered at 37°C for the stated times. Subsequently, fixed cells underwent immunofluorescence microscopy. (A) Comparison of HSP72 localization between before heat shock (upper panels) and immediately after a 2 h heat shock (lower panels). (B) Cells with nucleolar HSP72 foci were enumerated where >1000 cells were analyzed. Closed and open circles represent the fraction of foci-positive cells treated with and without lindane, respectively.

 
Heat shock transiently augmented phosphorylation of Cx43 protein, concomitant with GJIC abrogation
We next assessed whether heat shock affects Cx43 phosphorylation and GJIC. The western blots probed with antibody specific to Cx43 protein displayed the typical three bands, which include one faster and two slower migrating phospho isoforms, designated P0, P1 and P2, respectively (5,34).

Intriguingly, persistent heat treatment decreased P0 and increased P2 (Figure 6A and B). Thus, heat shock augmented Cx43 phosphorylation in a heat- and time-dependent fashion. Incidentally, although it has been reported that a 30 min heat shock at 43.5°C rapidly degrades Cx43 proteins via augmented proteasomal and lysosomal pathways in normal rat cardiomyocytes (35), such Cx43 degradation by heat shock at 43°C even for 8 h was not observed in normal HE49 human fibroblast cells. As depicted in Figure 6C and D, Cx43 phosphorylation augmented by a 4 h heat shock recovered to the basal level 24 h after heat shock. Thus, heat-induced Cx43 phosphorylation was transient. We next tested whether heat shock alters GJIC. As shown in Figure 7, GJIC was abrogated by a 4 h heat shock and recovered to the basal level 24 h after heat treatment. Thus, heat shock transiently abolished GJIC.



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  		Fig. 6. Effect of heat shock on the phosphorylation of Cx43 proteins in HE49 cells. (A) Cells heated for the time stated were harvested immediately after heat shock, followed by western blot analysis probed with anti-Cx43 antibody. Four independent analyses showed a consistent pattern of data, and a representative blot is shown. (B) Cells heated for 4 h were further kept at 37°C and were subjected to western blot analysis. Experiments were repeated three times with similar results, and a representative blot is shown. Corresponding graphs in (C) and (D) show the alteration of Cx43 phospho isoform expression in blots (A) and (B), respectively. Solid circles, open triangles and open circles indicate Cx43 isoforms P0, P1 and P2, respectively. The fraction of Cx43 isoform was calculated from the intensity of each isoform band relative to that of total Cx43 bands. These data are presented as means and standard deviations of four (C) and three (D) independent experiments.

 


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  		Fig. 7. Effect of heat stress on GJIC of HE49 cells. (A) GJIC abrogation by continuous heat treatment. Cells heated for the time indicated were subjected to dye transfer assay. (B) GJIC recovery after heat shock. Cells heated for 4 h were recovered at 37°C for the stated periods, followed by dye transfer assay. Experiments were repeated twice with similar results, and representative data are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
First, we have demonstrated here that lindane exacerbates hyperthermic killing of normal cells whereas almost no cytotoxicity was observed at 37°C (Figure 2) and that HSP72 induction was not affected by lindane while lindane attenuated nuclear translocation of HSP72 (Figures 4 and 5). We previously showed a possible link between heat sensitivity and nuclear translocation of HSP72 (1). Before heat shock, HSP72 expression level was higher in malignant cells than in normal cells (1). In contrast, after heat shock, HSP72 in normal cells was quantitatively induced and qualitatively translocated into nuclei and nucleoli, although both induction and nuclear translocation of HSP72 were attenuated in malignant cells (1). Hence, our previous findings suggest that HSP72 function does not depend on total HSP72 amount and that nuclear HSP72 translocation is critical for the protection against heat stress. Therefore, we conclude that lindane exacerbated hyperthermic lethality via inefficient nuclear HSP72 translocation. Previously, we also reported a potential link between cell-to-cell contact and heat sensitivity (1,2). In normal cells, confluent cells became more heat-resistant than sparse cells, whereas there was almost no difference in heat sensitivity between confluent and sparse malignant cells (1,2). Therefore, it is highly likely that cell-to-cell interaction is a prerequisite to acquire heat resistance in normal cells. There are some reports indicating that tight junctions and desmosomes alleviate cell killing by heat shock (36) and that the components of tight and adherent junctions are associated with Cx proteins (37–39). These results indicate that not merely GJIC but a coordinated network of intercellular junctions contributes to the protection against heat stress.

Second, we have demonstrated here that heat shock transiently augments Cx43 phosphorylation and disturbs GJIC (Figures 6 and 7). Phosphorylation of Cx43 proteins has been thought to regulate channel gating, gap junction assembly and trafficking (7,8). Various protein kinases and protein phosphatases have been implicated in the regulation of phosphorylation status of Cx43 proteins (40,41). While mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) are known to phosphorylate Cx43 on serine residues leading to GJIC impairment (42,43), we found that neither kinase was activated by heat shock and heat-induced Cx43 phosphorylation was not suppressed by simultaneous treatment with either of the specific inhibitors PD98059 and GF109203X (data not shown), suggesting that MAPK and PKC are not involved in heat-induced Cx43 phosphorylation. It has been shown that heat shock greatly increases tyrosine phosphorylation (44), activates pp60 c-Src tyrosine kinase (45) and causes reversible inactivation of receptor protein-tyrosine phosphatase {alpha} (46). Activated c-Src has been shown to phosphorylate Cx43 proteins on residue Tyr265, leading to GJIC inhibition (47). Therefore, one possible mechanism of heat-induced Cx43 phosphorylation is that heat shock augments Cx43 phosphorylation on tyrosine residues via activated tyrosine kinases such as c-Src and/or inactivated protein tyrosine phosphatases.

Taking our present results into consideration, we propose a possible mechanism responsible for alleviation of hyperthermic killing by GJIC in normal cells. As an immediate response to heat stress, HSP72 is translocated into nuclei and nucleoli. Subsequent heat shock augments Cx43 phosphorylation, concomitant with GJIC attenuation. Severe heat shock then abolishes GJIC and further nuclear translocation of HSP72 protein induced by heat shock. Under these conditions, denaturated proteins are repaired by HSP72 protein already translocated into the nuclei and nucleoli. If repaired, HSP72 returns to the cytoplasm and GJIC is recovered. However, an excess of denaturated proteins over nuclear HSP72 induces cell death. In cells with defective GJIC, such as tumors, HSP72 protein fails to accumulate in the nuclei after heat stress and to protect cells against heat shock.

In conclusion, the present study is the first to demonstrate that the abolition of GJIC by lindane exacerbates hyperthermic lethality via interrupted nuclear HSP72 translocation and that heat shock augments Cx43 phosphorylation leading to GJIC disruption. Our present results indicate that GJIC participates in the heat stress response and is involved in protection against heat stress. Our results also imply the possibility that the aberration of GJIC during carcinogenesis disturbs the heat stress response machinery, which includes nuclear HSP72 translocation, leading to potentiated susceptibility to heat shock.


   Notes
 
1 To whom correspondence should be addressed Email: nabe{at}net.nagasaki-u.ac.jp Back


   Acknowledgments
 
We are grateful to Mr Mitsuaki Ojima (Nagasaki University, Japan) for technical assistance with the dye transfer assay. We would also like to acknowledge Dr Kevin M.Prise (Gray Cancer Institute, UK) for his critical reading of this manuscript. This work was supported by a Grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received ; revised June 25, 2003; accepted July 25, 2003.


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