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Changes in the gap junctional intercellular communication in renal tubular epithelial cells in vitro treated with renal carcinogens
Cancer Letters 122 (1998) 77–84
Changes in the gap junctional intercellular communication in renal tubular epithelial cells in vitro treated with renal carcinogens Mitsuru Noguchi*, Koichiro Nomata, Jun-ichi Watanabe, Hiroshi Kanetake, Yutaka Saito Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan Received 27 May 1997; received in revised form 28 July 1997; accepted 30 July 1997
Abstract Gap junctional intercellular communications (GJIC) are known as the channels for the direct transfer of cytoplasmic molecules between neighboring cells and are lost during transformation of normal cells. To study the function and the molecular mechanism for the loss of GJIC, the effects of dimethylnitrosamine, KBrO3 and FeSO4⋅7H2O, which are known as chemical tumor promoters of the kidney on the GJIC function and the expression of connexin 43 of Madin–Darby canine kidney (MDCK) epithelial cells, were examined. These tumor promoters inhibited the GJIC in MDCK cells. The expression of connexin 43 mRNA and connexin 43 protein was not altered by these treatments, whereas immunocytochemical study revealed that the distribution of connexin 43 protein was changed from the cell surface to the cytoplasma. These data suggest that blockage of GJIC in MDCK cells treated with renal carcinogens support the hypothesis that loss of GJIC might be important in renal carcinogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Gap junctional intercellular communication; Renal cancer; Connexin 43; Chemical tumor promoter
1. Introduction Gap junctions are membrane channels that permit the transfer of ions and small molecules of less than 1 kDa (for review see Ref. [1]). They are the only route for the direct transfer of cytoplasmic compounds between cells. Once the gap junctions in each cell are connected between the neighboring cells, direct transfer of molecules is observed. Many normal biological processes, including tissue homeostasis, embryonic development, glandular secretion, cellular differentiation and growth control, are modulated by the gap junctional intercellular communication * Corresponding author. Tel.: +81 95 8497340; fax: +81 95 8497343.
(GJIC) [2–4]. Approximately a dozen connexin genes, designated according to the molecular masses of the proteins they encode, have been cloned and characterized [2,5–7]. In human kidney, the expression of gap junctional proteins, connexin (Cx) 26, 32 and 43, was detected [8–11]. When the GJIC becomes disrupted changes in homeostasis occurs, which result in abnormal cell development as well as tumorigenesis [12–14]. It has been shown that a number of tumor promoting agents inhibited GJIC during transformation of normal cells and the inhibition of GJIC and decreased connexin expression in malignant tissues [15–20], indicating that GJIC is one of the important players in the processes of carcinogenesis. Tumor cells transfected by Cx genes showed restored normal cell char-
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0372-8
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acteristics and delayed growth rate [21]. This suggests that Cx genes act as tumor suppressor genes. Renal cell carcinoma is a common malignant tumor in the kidney and it is known that the tumor cells arise from renal tubular epithelial cells. However, the molecular mechanisms of renal carcinogenesis are almost unknown. In this study, we studied the GJIC function and Cx 43 protein expression during renal carcinogenesis using non-transformed renal tubular epithelial cell line, Madin–Darby canine kidney (MDCK cells) treated with a panel of chemical tumor promoters.
2. Materials and methods 2.1. Cell culture Madin–Darby canine kidney (MDCK) cells were obtained from the Japanese Cancer Research Resource Bank. These cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS; Life Tech Oriental, Tokyo, Japan) at 37°C in an atmosphere of 95% air and 5% CO2. A WB-F344 cell line was a kind gift from Dr Tomonori Hayashi (Institute for Radiation Effect, Hiroshima, Japan). WB-F344 cells were grown in DMEM supplemented with 7% FCS. 2.2. Lactate dehydrogenase assays of MDCK with chemical tumor promoter
grown to confluence were scraped with the sharp edge of a surgical blade in the presence of Lucifer Yellow dye (0.5 mg/ml). Lucifer Yellow medium was discarded 3 min later and cells were washed with PBS three times. Transfer of the Lucifer Yellow dye from the scraped edge to the neighboring cells was investigated under a fluorescent microscope. 2.4. Northern blot analysis of connexin 43 Total RNA from each cultured cell was isolated by using guanidine thiocyanate [24]. Total RNA (20 mg of each) was electrophoresed in 1.2% agarose–formaldehyde gels, transferred onto a Hybond-N Nylon membrane (Amersham, UK) and hybridized with the 1.4 kb fragment of human connexin 43 (Cx 43) cDNA (a kind gift from Drs Chia-Cheng Chang and James E. Trosko, Department of Pediatrics and Human Development and Institute of Environmental Toxicology, Michigan State University, MI) labeled with digoxigenin-H-dUTP (Boehringer Mannheim, Germany). The hybridized RNA bands were visualized by the chemiluminescent reaction using anti-digoxigeninAP Fab (Boehringer Mannheim, Germany) fragments and chemiluminescent formation Lumi-Phos 530 (Boehringer Mannheim, Germany). The membranes were then sealed in hybridization bags and exposed to X-ray film. The relative intensities of mRNA bands were corrected for the difference in 28S RNA band intensities. 2.5. Immunofluorescent staining of Cx 43
Dimethylnitrosamine (DMN) (Wako, Osaka, Japan), KBrO3, FeSO4⋅7H2O and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used as inducers for the chemical transformation of MDCK cells. DMN, KBrO3 and FeSO4⋅7H2O were dissolved in PBS and TPA was dissolved in a final concentration of 1% ethanol. To analyze the toxicity of these chemicals, the content of lactate dehydrogenase (LDH) in the culture medium was measured by using the in vitro toxicology assay kit, lactate dehydrogenase based (Sigma, St. Louis, MO) [22]. 2.3. GJIC assay GJIC assay was performed by using the scrape loading dye transfer technique [23]. Briefly, cells
Each cell line was grown to subconfluence in the wells of Lab-Tex (Nunc, USA) chamber slides and then the medium was changed to serum-free medium and the culture was continued for 24 h. They were incubated with or without chemical tumor promoters for 24 h in a serum-free medium. Cells were washed with PBS three times followed by fixation with a 50:50 mixture of acetone and PBS. After washing with PBS, cells were incubated with 2% bovine serum albumin (Wako, Osaka, Japan) for 1 h. Cells were then treated with monoclonal mouse anti-Cx 43 antibody (Zymed Laboratories, San Francisco, CA) diluted 1:100 for 1 h. As a negative control, cells were incubated with non-immune mouse IgG (Zymed Laboratories). After washing, cells were
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incubated with a biotinylated-F(ab′)2 fragment of goat anti-mouse IgG (H + L) (Zymed Laboratories) diluted 1:500 for 1 h followed by incubation with fluorescein-conjugated Streptoavidin (DAKO Japan, Kyoto, Japan) for 30 min. After extensive washing, photographs were taken using a fluorescent microscope. All procedures were carried out at room temperature. 2.6. Western blot analysis Cells were grown in an ordinary growth medium which was replaced with serum-free medium after reaching confluence and the culture was continued for 24 h. Cells were incubated with fresh serum-free medium in the presence or absence of chemical tumor promoters for 15 min as indicated in Fig. 1. MDCK cells were treated with a non-toxic concentration (0.1% DMN, 0.5 mM/ml KBrO3, 100 mg/ml FeSO4⋅7H2O and 1 ng/ml TPA). Cell lysate was made by adding 20% sodium dodecyl sulfate (SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysate was sonicated for 30 s on ice, followed by centrifugation for 5 min at 4°C. Each protein (40 mg) was loaded on the SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore Japan, Tokyo, Japan). After blocking the membrane with PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween 20, the membrane was incubated with anti-Cx 43 polyclonal antibody (Zymed Laboratories) followed by incubation with peroxidase-conjugated anti-mouse IgG (Amersham Japan, Tokyo, Japan). After extensive washing, the proteins were visualized with an ECL-chemiluminescence detection kit (Amersham).
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these chemical tumor promoters were used for renal carcinogens in this study. TPA does not induce or promote renal carcinogenesis [29], however, it is reported that TPA induced a drastic reduction of GJIC in MDCK cells [30]. Therefore, we used TPA for positive control of the GJIC disruption in MDCK cells. To determine the non-cytotoxic concentration of chemical tumor promoters, the LDH activity of MDCK cells released into the culture media was determined. Fig. 1 shows that 0.1% DMN, 0.5 mM/ ml KBrO3, 100 mg/ml FeSO4⋅7H2O and 1 ng/ml TPA did not show the cytotoxic effect on MDCK cells (Fig. 1A–D). 3.2. The inhibition of the GJIC by tumor promoters Lucifer Yellow is known to be transferred by GJIC between neighboring cells. To examine the GJIC function, the GJIC was disrupted mechanically by a surgical blade and then Lucifer Yellow dye was loaded on the cell. The dye uptaken into the wounded row of the cells will be transferred into the adjacent row of the cells if the GJIC function is intact and the transfer of dye will spread far away from the wounded cells. Fig. 2A shows the typical result of the assay. Lucifer Yellow dye was observed in cells growing on more than five rows from the wounded cells (just beside the wound). MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h (Fig. 2B) and for 24 h (Fig. 2C) showed impaired GJIC function. The GJIC of MDCK cells after the treatment with tumor promoters was diminished with time course; about 50% of the GJIC were blocked until 6 h and finally completely disappeared after 24 h (Table 1). 3.3. Northern blot analysis of Cx 43
3. Results 3.1. Determination of a non-toxic concentration of a panel of tumor promoters KBrO3 and FeSO4⋅7H2O are known as chemical tumor promoters for renal cell carcinoma [25,26]. DMN is known to induce renal mesenchymal tumors. However, it is reported that two types of renal neoplasm, renal mesenchymal tumor and cortical epithelial tumor, were induced by DMN [27,28]. Therefore,
Connexin 43 (Cx 43) is one of the major components of gap junctions in renal epithelial cells. To determine the mechanism of the disruption of the GJIC function in MDCK cells treated with chemical tumor promoters, total RNAs from MDCK cells treated with tumor promoters for 24 h were electrophoresed and transferred and the membrane was hybridized with human Cx 43. There was no remarkable difference of the expression of Cx 43 mRNA in each cell (Fig. 3). These results indicate that the alteration of the mRNA expression of Cx 43 is not
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Fig. 1. Cytotoxicity of chemical tumor promoters in cultured MDCK cells. Cells were plated into wells of six-well plates at a density of 5 × 104 cells/well in serum containing DMEM. Twenty-four hours later, the medium was changed to serum-free medium and the culture was continued for an additional 24 h. Cells were incubated with or without chemical tumor promoters at indicated concentrations for 24 h and LDH activity in the culture medium was measured by using a commercial kit. (A) DMN; (B) KBrO3; (C) FeSO4⋅7H2O; (D) TPA.
involved in the loss of the GJIC function in MDCK cells treated with chemical tumor promoters.
was no remarkable difference in the expression of Cx 43 protein in MDCK cells treated with chemical tumor promoters (non-cytotoxic concentration).
3.4. Altered localization of Cx 43 The disruption of Cx 43 protein in MDCK cells treated with chemical tumor promoters was examined. In untreated MDCK cells, Cx 43 containing gap-junctional plaques localized at the plasma membrane (Fig. 4A). In MDCK cells treated with tumor promoters, the Cx 43 membranous plaques were less evident and some Cx 43 plaques were detected in the cytoplasm (Fig. 4B). 3.5. Western blot analysis of Cx 43 To investigate the alteration of Cx 43 protein in renal carcinogenesis, Western blot analysis was studied (Fig. 5). WB-F344 cells as the control displayed the characteristic P0, P1 and P2 phosphorylation pattern previously observed by Matesic et al. [31]. There
4. Discussion The loss of GJIC has long been postulated to be important in carcinogenesis and in maintaining the transformed phenotype of initiated cells. Many cancer cells have been associated with defective GJIC which can occur at the level of connexin expression and transport or gap junction formation [32,33]. In this study, we have shown that the loss of GJIC function associated with the changes in the expression and the localization of Cx 43 was involved in renal tubular cell carcinogenesis. In MDCK cells treated with a panel of chemical tumor promoters, such as DMN, KBrO3 and FeSO4⋅7H2O, the GJIC function was inhibited and the localization of Cx 43 (usually at the plasma membrane) was altered (at the cyto-
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Fig. 2. The GJIC was inhibited in MDCK cells treated with chemical tumor promoters. The GJIC function was determined by using the scraped-loading dye transfer technique as described in Section 3.2. (A) MDCK cells without treatment; (B) MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h; (C) MDCK cells treated with 100 mM FeSO4⋅7H2O for 24 h. The magnification in (A–C) is 400×.
plasma). These alterations were observed in transformed renal tubular cells and renal carcinoma cells (data not shown). It is known that the MDCK cells are functionally coupled and that gap junctions are regulated by cell density [34]. Berthoud et al. [30] reported that Cx 43 is a major molecular component responsible for dye coupling between MDCK cells and that TPA induced a drastic reduction of GJIC. Furthermore, it is well known that different types of tumor promoters inhibit GJIC in cell cultures [15–18]. This strongly supports the hypothesis that the lack of GJIC between initiated cells and surrounding normal cells is important for clonal expansion of initiated cells. Among a variety of tumor promoters for renal epithelial cells, trisodium nitrilotriacetate monohydrate and sodium barbital are strong inhibitors of GJIC in rat renal epithelial cells [35]. However, there was no obvious information about the mechanism of GJIC inhibition. DMN, KBrO3 and FeSO4⋅7H2O are well known as tumor promoters of the kidney [25–28]. Oxidative damage
Table 1 The changes of the GJIC after treatment with chemical tumor promoters Time 15 min 30 min 1h 2h 3h 6h 12 h 24 h
Control DMN (0.1%) + + + + + + + +
+ + ± ± ± − − −
KBrO3 FeSO4⋅7H2O TPA (1 ng/ml) (0.5 mM) (100 mM) + ± ± ± ± ± ± −
+ + + + ± − − −
+ + + + ± − − −
+, more than four rows of dye transfer; ±, two to three rows of dye transfer; −, no transfer of the loaded dye. The GJIC assay was performed by using the scrape loading dye transfer technique. The GJIC of MDCK cells was investigated extensively by the transfer of Lucifer Yellow to adjacent cells. However, the GJIC of MDCK cells treated with tumor promoters was diminished time-dependently and finally completely blocked after 24 h. The vehicle (PBS, 1% ethanol) alone was used as the control.
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Fig. 3. The expression of Cx 43 mRNA was not affected in MDCK cells treated with chemical tumor promoters. Total RNA from each cell was electrophoresed, transferred onto a membrane and the membrane was hybridized by human Cx 43 cDNA probe as described in Section 2.
by KBrO3 was implicated in its toxic and carcinogenic factor [36] and the activity of the cadherin adherens junction-specific cell adhesion molecule was diminished by TPA in animal models [37]. It is suggested that free radical damage or alteration in cadherin expression are involved in renal carcinogenesis. In this study, inhibition of GJIC in MDCK cells treated with chemical tumor promoters was recognized. Our results revealed that DMN, KBrO3 and FeSO4⋅7H2O induced drastic reductions of GJIC in MDCK cells. Furthermore, we have observed that the number of Cx 43 membrane plaques was less evident and Cx 43 plaques moved into the cytoplasma. Recently, Feijter et al. [38] explained that altered localization of gap junction protein Cx 43 was mediated in part by changes in the phosphorylation of this protein. However, the relationship between Cx 43 localization and its phosphorylation in MDCK cells treated with tumor promoters (non-cytotoxic concentration) was not made clear by our Western blot analysis. In conclusion, our data concerning the blockage of GJIC in MDCK cells treated with renal carcinogens support the hypothesis that a loss of GJIC might be important in renal carcinogenesis.
Fig. 4. Localization of Cx 43 in MDCK cells was altered by tumor promoter treatment. Cells grown in wells of Lab-Tex chamber slides were fixed and stained with monoclonal anti Cx 43 antibody using an indirect immunofluorescent method. (A) MDCK cells without treatment; (B) MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h. The magnification in (A,B) is 1000×.
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[5]
[6]
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[9] Fig. 5. Cx 43 protein expression was unchanged in the MDCK cells treated with chemical tumor promoters. Each protein of cell lysate was electrophoresed, transferred onto a membrane and the membrane was immunoblotted by using the anti-Cx 43 polyclonal antibody as described in Section 2. WB-F344 cells (as the control) displayed the phosphorylated form of the Cx 43 band (Cx 43-P1, Cx 43-P2) and the dephosphorylated form of the Cx 43 band (Cx 43-P0).
Acknowledgements We are grateful to Dr Chia-Cheng Chang and Dr James E. Trosko (Department of Pediatrics and Human Development and Institute of Environmental Toxicology, Michigan State University, USA) for the kind gift of Cx 43 cDNA and to Dr Tomonori Hayashi (Institute for Radiation Effect, Hiroshima, Japan) for the kind gift of the WB-F344 cell line. We also thank Dr Shigeru Kanda for his advice and Takumi Shimogama for his excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 06671598).
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Changes in the gap junctional intercellular communication in renal tubular epithelial cells in vitro treated with renal carcinogens Mitsuru Noguchi*, Koichiro Nomata, Jun-ichi Watanabe, Hiroshi Kanetake, Yutaka Saito Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan Received 27 May 1997; received in revised form 28 July 1997; accepted 30 July 1997
Abstract Gap junctional intercellular communications (GJIC) are known as the channels for the direct transfer of cytoplasmic molecules between neighboring cells and are lost during transformation of normal cells. To study the function and the molecular mechanism for the loss of GJIC, the effects of dimethylnitrosamine, KBrO3 and FeSO4⋅7H2O, which are known as chemical tumor promoters of the kidney on the GJIC function and the expression of connexin 43 of Madin–Darby canine kidney (MDCK) epithelial cells, were examined. These tumor promoters inhibited the GJIC in MDCK cells. The expression of connexin 43 mRNA and connexin 43 protein was not altered by these treatments, whereas immunocytochemical study revealed that the distribution of connexin 43 protein was changed from the cell surface to the cytoplasma. These data suggest that blockage of GJIC in MDCK cells treated with renal carcinogens support the hypothesis that loss of GJIC might be important in renal carcinogenesis. 1998 Elsevier Science Ireland Ltd. Keywords: Gap junctional intercellular communication; Renal cancer; Connexin 43; Chemical tumor promoter
1. Introduction Gap junctions are membrane channels that permit the transfer of ions and small molecules of less than 1 kDa (for review see Ref. [1]). They are the only route for the direct transfer of cytoplasmic compounds between cells. Once the gap junctions in each cell are connected between the neighboring cells, direct transfer of molecules is observed. Many normal biological processes, including tissue homeostasis, embryonic development, glandular secretion, cellular differentiation and growth control, are modulated by the gap junctional intercellular communication * Corresponding author. Tel.: +81 95 8497340; fax: +81 95 8497343.
(GJIC) [2–4]. Approximately a dozen connexin genes, designated according to the molecular masses of the proteins they encode, have been cloned and characterized [2,5–7]. In human kidney, the expression of gap junctional proteins, connexin (Cx) 26, 32 and 43, was detected [8–11]. When the GJIC becomes disrupted changes in homeostasis occurs, which result in abnormal cell development as well as tumorigenesis [12–14]. It has been shown that a number of tumor promoting agents inhibited GJIC during transformation of normal cells and the inhibition of GJIC and decreased connexin expression in malignant tissues [15–20], indicating that GJIC is one of the important players in the processes of carcinogenesis. Tumor cells transfected by Cx genes showed restored normal cell char-
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0372-8
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acteristics and delayed growth rate [21]. This suggests that Cx genes act as tumor suppressor genes. Renal cell carcinoma is a common malignant tumor in the kidney and it is known that the tumor cells arise from renal tubular epithelial cells. However, the molecular mechanisms of renal carcinogenesis are almost unknown. In this study, we studied the GJIC function and Cx 43 protein expression during renal carcinogenesis using non-transformed renal tubular epithelial cell line, Madin–Darby canine kidney (MDCK cells) treated with a panel of chemical tumor promoters.
2. Materials and methods 2.1. Cell culture Madin–Darby canine kidney (MDCK) cells were obtained from the Japanese Cancer Research Resource Bank. These cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS; Life Tech Oriental, Tokyo, Japan) at 37°C in an atmosphere of 95% air and 5% CO2. A WB-F344 cell line was a kind gift from Dr Tomonori Hayashi (Institute for Radiation Effect, Hiroshima, Japan). WB-F344 cells were grown in DMEM supplemented with 7% FCS. 2.2. Lactate dehydrogenase assays of MDCK with chemical tumor promoter
grown to confluence were scraped with the sharp edge of a surgical blade in the presence of Lucifer Yellow dye (0.5 mg/ml). Lucifer Yellow medium was discarded 3 min later and cells were washed with PBS three times. Transfer of the Lucifer Yellow dye from the scraped edge to the neighboring cells was investigated under a fluorescent microscope. 2.4. Northern blot analysis of connexin 43 Total RNA from each cultured cell was isolated by using guanidine thiocyanate [24]. Total RNA (20 mg of each) was electrophoresed in 1.2% agarose–formaldehyde gels, transferred onto a Hybond-N Nylon membrane (Amersham, UK) and hybridized with the 1.4 kb fragment of human connexin 43 (Cx 43) cDNA (a kind gift from Drs Chia-Cheng Chang and James E. Trosko, Department of Pediatrics and Human Development and Institute of Environmental Toxicology, Michigan State University, MI) labeled with digoxigenin-H-dUTP (Boehringer Mannheim, Germany). The hybridized RNA bands were visualized by the chemiluminescent reaction using anti-digoxigeninAP Fab (Boehringer Mannheim, Germany) fragments and chemiluminescent formation Lumi-Phos 530 (Boehringer Mannheim, Germany). The membranes were then sealed in hybridization bags and exposed to X-ray film. The relative intensities of mRNA bands were corrected for the difference in 28S RNA band intensities. 2.5. Immunofluorescent staining of Cx 43
Dimethylnitrosamine (DMN) (Wako, Osaka, Japan), KBrO3, FeSO4⋅7H2O and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used as inducers for the chemical transformation of MDCK cells. DMN, KBrO3 and FeSO4⋅7H2O were dissolved in PBS and TPA was dissolved in a final concentration of 1% ethanol. To analyze the toxicity of these chemicals, the content of lactate dehydrogenase (LDH) in the culture medium was measured by using the in vitro toxicology assay kit, lactate dehydrogenase based (Sigma, St. Louis, MO) [22]. 2.3. GJIC assay GJIC assay was performed by using the scrape loading dye transfer technique [23]. Briefly, cells
Each cell line was grown to subconfluence in the wells of Lab-Tex (Nunc, USA) chamber slides and then the medium was changed to serum-free medium and the culture was continued for 24 h. They were incubated with or without chemical tumor promoters for 24 h in a serum-free medium. Cells were washed with PBS three times followed by fixation with a 50:50 mixture of acetone and PBS. After washing with PBS, cells were incubated with 2% bovine serum albumin (Wako, Osaka, Japan) for 1 h. Cells were then treated with monoclonal mouse anti-Cx 43 antibody (Zymed Laboratories, San Francisco, CA) diluted 1:100 for 1 h. As a negative control, cells were incubated with non-immune mouse IgG (Zymed Laboratories). After washing, cells were
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incubated with a biotinylated-F(ab′)2 fragment of goat anti-mouse IgG (H + L) (Zymed Laboratories) diluted 1:500 for 1 h followed by incubation with fluorescein-conjugated Streptoavidin (DAKO Japan, Kyoto, Japan) for 30 min. After extensive washing, photographs were taken using a fluorescent microscope. All procedures were carried out at room temperature. 2.6. Western blot analysis Cells were grown in an ordinary growth medium which was replaced with serum-free medium after reaching confluence and the culture was continued for 24 h. Cells were incubated with fresh serum-free medium in the presence or absence of chemical tumor promoters for 15 min as indicated in Fig. 1. MDCK cells were treated with a non-toxic concentration (0.1% DMN, 0.5 mM/ml KBrO3, 100 mg/ml FeSO4⋅7H2O and 1 ng/ml TPA). Cell lysate was made by adding 20% sodium dodecyl sulfate (SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysate was sonicated for 30 s on ice, followed by centrifugation for 5 min at 4°C. Each protein (40 mg) was loaded on the SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore Japan, Tokyo, Japan). After blocking the membrane with PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween 20, the membrane was incubated with anti-Cx 43 polyclonal antibody (Zymed Laboratories) followed by incubation with peroxidase-conjugated anti-mouse IgG (Amersham Japan, Tokyo, Japan). After extensive washing, the proteins were visualized with an ECL-chemiluminescence detection kit (Amersham).
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these chemical tumor promoters were used for renal carcinogens in this study. TPA does not induce or promote renal carcinogenesis [29], however, it is reported that TPA induced a drastic reduction of GJIC in MDCK cells [30]. Therefore, we used TPA for positive control of the GJIC disruption in MDCK cells. To determine the non-cytotoxic concentration of chemical tumor promoters, the LDH activity of MDCK cells released into the culture media was determined. Fig. 1 shows that 0.1% DMN, 0.5 mM/ ml KBrO3, 100 mg/ml FeSO4⋅7H2O and 1 ng/ml TPA did not show the cytotoxic effect on MDCK cells (Fig. 1A–D). 3.2. The inhibition of the GJIC by tumor promoters Lucifer Yellow is known to be transferred by GJIC between neighboring cells. To examine the GJIC function, the GJIC was disrupted mechanically by a surgical blade and then Lucifer Yellow dye was loaded on the cell. The dye uptaken into the wounded row of the cells will be transferred into the adjacent row of the cells if the GJIC function is intact and the transfer of dye will spread far away from the wounded cells. Fig. 2A shows the typical result of the assay. Lucifer Yellow dye was observed in cells growing on more than five rows from the wounded cells (just beside the wound). MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h (Fig. 2B) and for 24 h (Fig. 2C) showed impaired GJIC function. The GJIC of MDCK cells after the treatment with tumor promoters was diminished with time course; about 50% of the GJIC were blocked until 6 h and finally completely disappeared after 24 h (Table 1). 3.3. Northern blot analysis of Cx 43
3. Results 3.1. Determination of a non-toxic concentration of a panel of tumor promoters KBrO3 and FeSO4⋅7H2O are known as chemical tumor promoters for renal cell carcinoma [25,26]. DMN is known to induce renal mesenchymal tumors. However, it is reported that two types of renal neoplasm, renal mesenchymal tumor and cortical epithelial tumor, were induced by DMN [27,28]. Therefore,
Connexin 43 (Cx 43) is one of the major components of gap junctions in renal epithelial cells. To determine the mechanism of the disruption of the GJIC function in MDCK cells treated with chemical tumor promoters, total RNAs from MDCK cells treated with tumor promoters for 24 h were electrophoresed and transferred and the membrane was hybridized with human Cx 43. There was no remarkable difference of the expression of Cx 43 mRNA in each cell (Fig. 3). These results indicate that the alteration of the mRNA expression of Cx 43 is not
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Fig. 1. Cytotoxicity of chemical tumor promoters in cultured MDCK cells. Cells were plated into wells of six-well plates at a density of 5 × 104 cells/well in serum containing DMEM. Twenty-four hours later, the medium was changed to serum-free medium and the culture was continued for an additional 24 h. Cells were incubated with or without chemical tumor promoters at indicated concentrations for 24 h and LDH activity in the culture medium was measured by using a commercial kit. (A) DMN; (B) KBrO3; (C) FeSO4⋅7H2O; (D) TPA.
involved in the loss of the GJIC function in MDCK cells treated with chemical tumor promoters.
was no remarkable difference in the expression of Cx 43 protein in MDCK cells treated with chemical tumor promoters (non-cytotoxic concentration).
3.4. Altered localization of Cx 43 The disruption of Cx 43 protein in MDCK cells treated with chemical tumor promoters was examined. In untreated MDCK cells, Cx 43 containing gap-junctional plaques localized at the plasma membrane (Fig. 4A). In MDCK cells treated with tumor promoters, the Cx 43 membranous plaques were less evident and some Cx 43 plaques were detected in the cytoplasm (Fig. 4B). 3.5. Western blot analysis of Cx 43 To investigate the alteration of Cx 43 protein in renal carcinogenesis, Western blot analysis was studied (Fig. 5). WB-F344 cells as the control displayed the characteristic P0, P1 and P2 phosphorylation pattern previously observed by Matesic et al. [31]. There
4. Discussion The loss of GJIC has long been postulated to be important in carcinogenesis and in maintaining the transformed phenotype of initiated cells. Many cancer cells have been associated with defective GJIC which can occur at the level of connexin expression and transport or gap junction formation [32,33]. In this study, we have shown that the loss of GJIC function associated with the changes in the expression and the localization of Cx 43 was involved in renal tubular cell carcinogenesis. In MDCK cells treated with a panel of chemical tumor promoters, such as DMN, KBrO3 and FeSO4⋅7H2O, the GJIC function was inhibited and the localization of Cx 43 (usually at the plasma membrane) was altered (at the cyto-
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Fig. 2. The GJIC was inhibited in MDCK cells treated with chemical tumor promoters. The GJIC function was determined by using the scraped-loading dye transfer technique as described in Section 3.2. (A) MDCK cells without treatment; (B) MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h; (C) MDCK cells treated with 100 mM FeSO4⋅7H2O for 24 h. The magnification in (A–C) is 400×.
plasma). These alterations were observed in transformed renal tubular cells and renal carcinoma cells (data not shown). It is known that the MDCK cells are functionally coupled and that gap junctions are regulated by cell density [34]. Berthoud et al. [30] reported that Cx 43 is a major molecular component responsible for dye coupling between MDCK cells and that TPA induced a drastic reduction of GJIC. Furthermore, it is well known that different types of tumor promoters inhibit GJIC in cell cultures [15–18]. This strongly supports the hypothesis that the lack of GJIC between initiated cells and surrounding normal cells is important for clonal expansion of initiated cells. Among a variety of tumor promoters for renal epithelial cells, trisodium nitrilotriacetate monohydrate and sodium barbital are strong inhibitors of GJIC in rat renal epithelial cells [35]. However, there was no obvious information about the mechanism of GJIC inhibition. DMN, KBrO3 and FeSO4⋅7H2O are well known as tumor promoters of the kidney [25–28]. Oxidative damage
Table 1 The changes of the GJIC after treatment with chemical tumor promoters Time 15 min 30 min 1h 2h 3h 6h 12 h 24 h
Control DMN (0.1%) + + + + + + + +
+ + ± ± ± − − −
KBrO3 FeSO4⋅7H2O TPA (1 ng/ml) (0.5 mM) (100 mM) + ± ± ± ± ± ± −
+ + + + ± − − −
+ + + + ± − − −
+, more than four rows of dye transfer; ±, two to three rows of dye transfer; −, no transfer of the loaded dye. The GJIC assay was performed by using the scrape loading dye transfer technique. The GJIC of MDCK cells was investigated extensively by the transfer of Lucifer Yellow to adjacent cells. However, the GJIC of MDCK cells treated with tumor promoters was diminished time-dependently and finally completely blocked after 24 h. The vehicle (PBS, 1% ethanol) alone was used as the control.
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Fig. 3. The expression of Cx 43 mRNA was not affected in MDCK cells treated with chemical tumor promoters. Total RNA from each cell was electrophoresed, transferred onto a membrane and the membrane was hybridized by human Cx 43 cDNA probe as described in Section 2.
by KBrO3 was implicated in its toxic and carcinogenic factor [36] and the activity of the cadherin adherens junction-specific cell adhesion molecule was diminished by TPA in animal models [37]. It is suggested that free radical damage or alteration in cadherin expression are involved in renal carcinogenesis. In this study, inhibition of GJIC in MDCK cells treated with chemical tumor promoters was recognized. Our results revealed that DMN, KBrO3 and FeSO4⋅7H2O induced drastic reductions of GJIC in MDCK cells. Furthermore, we have observed that the number of Cx 43 membrane plaques was less evident and Cx 43 plaques moved into the cytoplasma. Recently, Feijter et al. [38] explained that altered localization of gap junction protein Cx 43 was mediated in part by changes in the phosphorylation of this protein. However, the relationship between Cx 43 localization and its phosphorylation in MDCK cells treated with tumor promoters (non-cytotoxic concentration) was not made clear by our Western blot analysis. In conclusion, our data concerning the blockage of GJIC in MDCK cells treated with renal carcinogens support the hypothesis that a loss of GJIC might be important in renal carcinogenesis.
Fig. 4. Localization of Cx 43 in MDCK cells was altered by tumor promoter treatment. Cells grown in wells of Lab-Tex chamber slides were fixed and stained with monoclonal anti Cx 43 antibody using an indirect immunofluorescent method. (A) MDCK cells without treatment; (B) MDCK cells treated with 100 mM FeSO4⋅7H2O for 6 h. The magnification in (A,B) is 1000×.
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[5]
[6]
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[9] Fig. 5. Cx 43 protein expression was unchanged in the MDCK cells treated with chemical tumor promoters. Each protein of cell lysate was electrophoresed, transferred onto a membrane and the membrane was immunoblotted by using the anti-Cx 43 polyclonal antibody as described in Section 2. WB-F344 cells (as the control) displayed the phosphorylated form of the Cx 43 band (Cx 43-P1, Cx 43-P2) and the dephosphorylated form of the Cx 43 band (Cx 43-P0).
Acknowledgements We are grateful to Dr Chia-Cheng Chang and Dr James E. Trosko (Department of Pediatrics and Human Development and Institute of Environmental Toxicology, Michigan State University, USA) for the kind gift of Cx 43 cDNA and to Dr Tomonori Hayashi (Institute for Radiation Effect, Hiroshima, Japan) for the kind gift of the WB-F344 cell line. We also thank Dr Shigeru Kanda for his advice and Takumi Shimogama for his excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 06671598).
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