The roles of ERK1/2 and p38 MAP kinases in the preventive mechanisms of mushroom Phellinus linteus against the inhibition of gap junctional intercellular communication by hydrogen peroxide
Jong-Ho Cho1,
Sung-Dae Cho1,
Hongbo Hu1,
Sung-Hoon Kim2,
Song Koo Lee3,
Yong-Soon Lee1 and
Kyung-Sun Kang1,4
1 Laboratory of Stem Cell and Tumor Biology, Department of Veterinary Public Health, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea,
2 Graduate School of East-West Medical Science, Kyunghee University, Suwon, South Korea and
3 Iljo Biological Industrial Company Limited, South Korea
 |
Abstract
|
---|
Modulation of gap junctional intercellular communication (GJIC) is a known cellular event associated with tumor promotion. The present study was undertaken to test the potential preventive effect of mushroom Phellinus linteus extract (PL) on the inhibition of GJIC, induced by hydrogen peroxide (H2O2), in WB-F344 rat liver epithelial cells (WB cells). Cells were pre-incubated with PL (5 and 25 µg/ml) for 24 h and this was followed by co-treatment with PL and H2O2 (500 µM) for 1 h. PL (at 5 and 25 µg/ml) prevented the inhibition of GJIC and blocked the hyper-phosphorylation of connexin 43 by H2O2. Moreover, H2O2 activated p38 kinase, extracellular signal-regulated protein kinases (ERK)1/2 and c-Jun N-terminal kinase (JNK) in WB cells. The present study indicates that PL is able to inactivate both ERK1/2 and p38 MAP kinases. However, PL did not affect the JNK pathway. For this reason, to elucidate the relation between MAP kinases and GJIC, we treated cells with PD98059 (an MEK inhibitor) and SB202190 (a p38 kinase inhibitor). These inhibitors were also found to prevent the inhibition of GJIC induced by H2O2, which suggests that PL may act as a natural anticancer product by preventing the inhibition of GJIC through the inactivation of ERK1/2 and p38 MAP kinases. In addition, our results indicate that the p38 kinase signaling pathway may be closely related functionally to the gap junction in rat liver epithelial cells.
Abbreviations: Cx43, connexin 43; ERK, extracellular signal-regulated protein kinases; GJIC, gap junctional intercellular communication; H2O2, hydrogen peroxide; JNK, c-Jun N-terminal kinase; PL, Phellinus linteus extract; SL/DT, scrape loading/dye transfer technique; WB cells, WB-F344 rat liver epithelial cells
 |
Introduction
|
---|
Intercellular communication is necessary in multicellular organisms to maintain tissue homeostasis and to control cell growth and differentiation (1). Gap junction channels play an important role in intercellular communication by providing a direct pathway for the movement of molecular information, including ions and polarized and non-polarized molecules up to a molecular mass of 1 kDa between adjacent cells (24). A gap junction channel consists of two juxtaposed hemi-channels, one from each of the participating cells. Although the mechanisms of gap junction formation and channel permeability regulation are poorly understood, the inhibition of gap junction intercellular communication (GJIC) is suspected to be involved in the mechanism of tumor promotion (5). Most tumor promoters inhibit GJIC (69), and the transfection of GJIC deficient cells with connexins suppresses tumor formation (1016). It has been reported that a number of pesticides, pharmaceuticals, dietary additives, polyhalogenated hydrocarbons and peroxisome proliferators inhibit GJIC through several diverse mechanisms. Moreover, chemopreventive natural chemicals, which have anti-oxidizing activities, such as vitamin C (17), germanium dioxide (18), honeybee propolis (19), green tea and Korean ginseng components (20), could prevent or recover the inhibition of GJIC induced by cancer promoters. Therefore, recovery of the inhibition of GJIC by cancer promoters has been proved to be a useful tool for the screening and assay of chemopreventive natural products, as well as mechanistic studies.
Hydrogen peroxide (H2O2) is a well-known cancer promoter that inhibits GJIC in WB-F344 (WB) rat liver epithelial cells in a dose- and time-dependent manner (21). Moreover, this inhibition correlates with reduced gap junction numbers and size, as well as the up-regulation of hyper-phosphorylated connexin 43 (Cx43) (21,22). Oxidative stress, including H2O2, has been shown to activate MAPKs (23,24). MAPKs, which include the extracellular signal-regulated protein kinases (ERK), the c-Jun N-terminal kinase (JNK) and the p38 subfamilies, are important regulatory proteins that transduce various extracellular signals into intracellular event (25). In other words, MAPKs are regulated by separate signal transduction pathways that control many aspects of mammalian cellular physiology including cell growth, differentiation and cell death (2528). In particular, it has been well documented that phospho-ERK (activated form) can inhibit GJIC in several kinds of cell lines, including WB cells, by Cx43 phosphorylation (2931). The p38 group kinases have been found to be involved in inflammation, cell growth, cell differentiation, the cell cycle and cell death (32). Although it is clear that the p38 pathway shares many similarities with the other MAPK cascades, the relation between p38 pathway and GJIC is poorly understood.
Phellinus linteus (PL) has been used as a traditional medicine in Korea, China, Japan and other Asian countries for the treatment of various diseases, including gastroenteric disorder, lymphatic disease and various cancers. It was reported previously that PL has the effect of stimulating cell-mediated and humoral immunity, and inhibiting tumor growth and metastasis (2334). In the present study, the anticarcinogenic effects of PL extracts and PL mycelia were evaluated in rat liver epithelial cells treated with H2O2 to inhibit GJIC, which has been associated with tumor promotion. The working hypothesis followed was that PL might prevent the blockage of GJIC in tumor promoter-treated cells. We also investigated the possibility of a relationship between the p38 pathway and GJIC.
 |
Materials and methods
|
---|
Chemicals
H2O2 was purchased from Showa Chemical (Tokyo, Japan). Lucifer yellow and monoclonal ß-actin antibodies were from Sigma Chemical (St Louis, MO), mouse monoclonal anti-Cx43 antibody from Chemicon International, rabbit anti-JNK1 and mouse monoclonal IgG against p38 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit anti-Map kinase, rabbit polyclonal anti-Cx43, HRP-goat anti-mouse IgG conjugate, rhodamine-conjugated goat anti-rabbit IgG and HRP-goat anti-rabbit conjugate antibody were all from Zymed Laboratories (San Francisco, CA). Anti-active MAPK, anti-active JNK, anti-active p38 and donkey anti-rabbit IgG HRP antibody were purchased from Promega (Madison, CA) and the Mek inhibitor, PD98059, and the p38 kinase inhibitor, SB202190, from Calbiochem (San Diego, CA).
PL extraction
PL was extracted using 30% ethanol at 90°C for 4 h and filtered through a 270 mesh filter. The sludge from the primary extraction was then extracted twice using the same procedure. The extract was concentrated using an Eyela rotating vacuum evaporator (Rikakikai, Tokyo, Japan) at 60°C, at 70 cmHg condition and spray dried.
Cell culture
WB rat liver epithelial cells were kindly provided by Dr J.E.Trosko at Michigan State University (USA). The procedure used for characterizing these cell lines has been described previously (34). Cells (passage 812) were cultured in D-media (Formula No. 78-5470EF, Gibco BRL, Grand Island, NY) containing 50 µg/ml gentamicin (Gibco BRL) in the presence of 5% fetal bovine serum (Gibco BRL). Cells were incubated in a 37°C humidified incubator containing 5% CO2 and 95% air. Cells were grown in 75 mm tissue culture plates and the culture medium was changed every other day.
Bioassay of cytotoxicity
Cytotoxicity was determined by the neutral red uptake assay according to the method of Borenfreund and Puerner (36). Cells were treated with PD98059, SB202190 or PL for 24 h, and the other cells were treated with PD98059 or SB202190 for 1 h or PL for 24 h before being treated with 500 µM H2O2 for 1 h. Following chemical treatment, the cells were rinsed three times with phosphate-buffered saline (PBS) and then 2 ml of fresh growth medium containing 0.033% neutral red, which had been incubated with D-medium for 4 h was added to the cells for 2 h. The time required for adequate uptake of the neutral red into the WB cells was 2 h. Extracellular neutral red was rinsed off with PBS and the cells were lyzed with 2 ml of an aqueous solution containing 1% acetic acid and 50% ethanol. Lyzed cells were analyzed for neutral red uptake at a wavelength of 540 nm using an ELISA reader.
Bioassay of GJIC
The scrape loading/dye transfer (SL/DT) technique was adapted using the method of El-Fouly et al. (37). Cells were pre-treated with PLs, for 24 h prior to the addition of H2O2 for 1 h, or pre-treated with the MEK inhibitor, PD98059, or the p38 kinase, SB202190, for 1 h followed by treatment with H2O2 for 1 h. The GJIC assay was conducted at non-cytotoxic dose levels of the samples, as determined by the neutral red uptake assay.
Following incubation, the cells were washed twice with 2 ml of PBS. Lucifer yellow was added to the washed cells and three scrapes were made with a surgical steel-bladed scalpel at low light intensities. These three scrapes were performed to ensure that the scrape traversed a large group of confluent cells. After a 3 min incubation period the cells were washed with 10 ml of PBS and then fixed with 2 ml of a 4% formalin solution. The distance traveled by the dye in a direction perpendicular to the scrape was observed with an inverted fluorescent microscope (Olympus Ix70, Okaya, Japan).
Western blot analysis
Cells were grown in a 100 mm tissue culture dish (Nunc, Rochester, NY) to the same confluency as in the SL/DT assay. The cells were then treated with test compounds in the same way as described in the SL/DT assay. Western blot analysis of Cx43 was performed, as described previously (37,39). Proteins were extracted with 20% SDS solution containing 1 mM phenylmethylsulfonyl fluoride (a protease inhibitor), 10 mM iodoacetoamide, 1 mM leupeptin, 1 mM antipain, 0.1 mM sodium orthovanadate and 5 mM sodium fluoride. Protein content was determined using the DC assay kit (Bio-Rad, Hercules, CA), and separated on 12.5% SDSPAGE according to the method of Laemmli (40). They were then transferred to nitrocellulose membranes at 100 V, 350 mA for 1 h. All antibodies were used according to the manufacturer's instructions and protein bands were detected using an ECL detection kit (Amersham, Piscataway, NJ).
 |
Results
|
---|
Cytotoxicity of PL, PD98059 and SB202190
Hasler et al. (41) and Upham et al. (21) showed that a dose of 500 µM H2O2 maximally inhibited GJIC in WB rat liver epithelial cells, and is non-cytotoxic, as determined by the lactate dehydrogenase activity and neutral red uptake tests. Therefore, we also used a dose of 500 µM H2O2 for all experiments. To select the appropriate doses of PL, PD98059 and SB202190 for this study, their cytotoxicities to WB cells were assessed using the neutral red uptake test. No cytotoxic effects were observed on cells treated with 80 µM PD98059, 8 µM SB202190 for 1 h or 100 µM PL for 24 h before treatment with 500 µM H2O2 for 1 h (Figure 1A
). In addition, cell viability did not change during 24 h incubation, following the addition of 100 µM PL. Neither 40 µM PD98059 nor 8 µM SB202190 had any cytotoxic effect on the cells after 1 h (Figure 2B
). We performed the following experiments using dose levels of chemicals
100 µM PL,
40 µM PD98059 and
8 µM SB202190.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 1. Cytotoxic effect of PL, PD98059 and SB202190 on WB cells. (A) Cells were pre-treated with PD98059 and SB202190 for 1 h or PL for 24 h before being co-treated with H2O2 500 µM for 1 h. (B) Cells were treated with PD98059 and SB202190 for 1 h or PL for 24 h. Cell viability was measured by the amount of neutral red accumulated in lysosomes of viable cells. Each bar represents the mean ± SD.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2. Effects of (A) PL extract and (B) PL mycelium on the GJIC using by SL/DT assay. (A) Cells were treated with (a) control, (b) H2O2 500 µM, (c) H2O2 plus PL extract 5 µg/ml, (d) H2O2 plus PL extract 25 µg/ml. (B) Cells were treated with (a) control, (b) H2O2 500 µM, (c) H2O2 plus PL mycelium 25 µg/ml, (d) H2O2 plus PL mycelium 50 µg/ml as described in the Materials and methods. (C) Quantification of recovery rate.
|
|
Effect of PL on GJIC
The GJIC of WB cells were assessed using SL/DT assay after treatment with the test compounds. The GJIC of control cells did not decrease during the experimental incubation period. After exposing the cells to 500 µM H2O2 for 1 h, an obvious inhibition was detected. However, the cells pre-treated with PL extracts (5 and 25 µg/ml) showed preventive effects on the inhibition of GJIC induced by 500 µM H2O2 (Figure 2A and C
). PL mycelia had not shown any preventive effects on GJIC (Figure 2B and C
)
Western blot analysis for a linkage protein group of GJIC
Western blot analysis, with antibodies specific to Cx43, was used to assess the phosphorylation status of the gap junctional proteins. The typical three bands (P0, P1 and P2) of Cx43 were detected in the control cells, and these were separated according to their degree of phosphorylation. The band P0 represented non-phosphorylated Cx43 and P2 represented a hyper-phosphorylation state. H2O2 treatment caused the P0 band to disappear and induced the P2 band. However, treatment of PL with H2O2 decreased the phosphorylation ratio (P2:P0) of Cx43 as induced by H2O2 (Figure 3
).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3. Western blot analysis on the changes in the phosphorylation pattern of Cx43. Total cellular protein extracts were prepared and western blot analysis was performed with 20 µg protein using antibody specific for Cx43.
|
|
Western blot analysis of MAPKs activation
We examined the activation of several MAPKs (ERK1/2, JNK and p38 kinase) to identify the protective action of PL on the inhibition of GJIC. H2O2 activated p38 kinase, ERK1/2 and JNK. PL pre-treatment (25 µg/ml) can block this activation of p38 and ERK1/2 MAP kinases, even though the proteins were constitutively expressed (Figure 4
). However, PL pre-treatment did not inhibit H2O2-induced activation of the JNK pathway (Figure 4
).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 4. Effect of PL extracts on H2O2-induced ERK, p38 and JNK phosphorylation. Total cellular protein extracts were prepared and western blot analyses were performed with 20 µg protein using antibody specific for (A) total ERK or phospho-ERK, (B) total p38 or phospho-p38 and (C) total JNK or phospho-JNK.
|
|
Effect of ERK1/2 and p38 MAP kinases on GJIC using inhibitors
To know the roles of ERK1/2 and p38 MAP kinase on the preventive effects of PL, we hypothesized if the preventive effects of PL are related to inactivated p38 and ERK1/2 kinases, the known inhibitors also can block activation of both MAP kinases and prevent the inhibition of GJIC by H2O2. For examination of this possibility, we treated cells with PD98059 (an MEK inhibitor) and SB202190 (a p38 kinase inhibitor). In an SL/DT assay, the results showed that those inhibitors also prevented the inhibition of GJIC induced by H2O2 as PL did (Figures 5 and 6
). In western blot analyses, H2O2 induced hyper-phosphorylation of Cx43 protein with loss of the P0 band of Cx43, while both inhibitors recovered the P0 band and showed a normal phosphorylation pattern of Cx43 protein (Figure 7A
). To examine the relationship between this recovery of GJIC and p38 and ERK1/2 MAP kinases, we treated cells with ERK1/2 MAP kinase inhibitor. There is no change of ERK1/2 protein expression between untreated cells and treated cells (Figure 7B
). However, H2O2 dramatically increased phosphorylated ERK1/2 MAP kinase, and this increased phosphorylated ERK1/2 MAP kinase was significantly reduced in PD98059-treated cells (Figure 7B
). In the case of the p38 MAP kinase, the inhibitor SB20219 also induced the disappearance of the phosphorylated p38 induced by H2O2 (Figure 7C
). These observations were the same as in PL-treated cells. Therefore, these results indicate that the ERK1/2 and the p38 kinase pathways may be closely related to the function of the gap junction and preventive effects of PL.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5. Effect of the MEK inhibitor PD98059 on GJIC using the SL/DT assay. Cells were treated by (A) (a) control, (b) H2O2 500 µM, (c) H2O2 plus PD98059 5 µM, (d) H2O2 plus PD98059 10 µM, (e) H2O2 plus PD98059 20 µM and (f) H2O2 plus PD98059 40 µM as described in the Materials and methods. (B) Quantification of recovery rate.
|
|

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6. Effect of the p38 inhibitor SB202190 on GJIC using the SL/DT assay. Cells were treated by (A) (a) control, (b) H2O2 500 µM, (c) H2O2 plus SB202190 0.5 µM, (d) H2O2 plus SB202190 1 µM, (e) H2O2 plus SB202190 2 µM and (f) H2O2 plus SB202190 4 µM as described in the Materials and methods. (B) Quantification of recovery rate.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 7. Effects of PD98059 and SB202190 on H2O2-induced ERK, p38 phosphorylation and Cx43 hyper-phosphorylation. Total cellular protein extracts were prepared and western blot analyses were performed with 20 µg protein using antibodies specific for (A) Cx43, (B) total ERK or phospho-ERK and (C) total p38 or phospho-p38.
|
|
 |
Discussion
|
---|
It is recognized that the function of GJIC can be modulated at many stages during the turnover of connexin by transcriptional, translational and post-transcriptional mechanisms. Moreover, GJIC is a target of different carcinogenic modulators (tumor promoters and anti-promoters) (11,42). Most tumor-promoting agents inhibit intercellular communications, and reduced GJIC capacity has been observed frequently during carcinogenesis (6,7). Tumor promoters such as H2O2 and TPA almost immediately disrupt GJIC in in vitro systems (21,22). Reactive oxygen species, and in particular, H2O2 play important roles in the multistep process of carcinogenesis and directly promote transformation in many in vivo and in vitro model systems (4345). Therefore, in this study, we investigated the anticarcinogenic effect of PL on WB cells after treatment with H2O2 using the SL/DT assay, and western blotting.
Significant prevention against the H2O2-induced inhibition of GJIC was observed at PL extract concentrations between 5 and 25 µg/ml (Figure 2A
).
Although it has been reported that PL inhibits tumor growth by stimulating humoral and cellular immunity (33,34), the present results show for the first time that PL extracts prevent the tumor promoter-induced inhibition of GJIC. However, the PL mycelium did not show the same preventive effect as the PL extract (Figure 2B
).
Tumor promoters can affect intercellular communication by mechanisms that distinguish between immediate and long-term responses. The immediate response of H2O2 on GJIC is associated with the hyper-phosphorylation of Cx43 (21). In the present study, western blot analysis showed that the intensity of the Cx43 P2 band increased after H2O2 treatment with loss of the P0 band of Cx43, and that it decreased slightly or remarkably when cells were pre-incubated with 5 or 25 µg/ml PL, respectively (Figure 3
).
In the present study, the final extraction yield was 15% from the raw mushroom. So, the concentration factor was 6.7. Therefore, the effective concentrations of PL extract (525 µg/ml) used in this study were equivalent to 33.5167.5 µg of raw mushroom.
To know the preventive mechanism of PL extract on GJIC, we examined MAP kinases. MAPKs, which include the ERK, JNK/stress-activated protein kinase and p38 subfamilies, are activated in response to stimuli such as treatment with DNA-damaging agents, growth factors and cytokines (2528). MAPKs regulate gene expression through the phosphorylation of downstream transcription factors (2528). Activation of JNK and p38 kinase is related to the stress response, growth arrest and apoptosis (2628), whereas ERK is important in mitogenesis and differentiation (46). However, reports exist that JNK activation occurs independently of cell death (47), and further that JNK activation actually promotes proliferation and cellular transformation (48,49). In addition to physiological response and activation pattern, MAPK activation is dependent upon types of MAPKs and cell type. For example, insulin can stimulate p38 in 3T3-L1 adipocytes (50), but down-regulates p38 activity in chick forebrain neuron cells (51). Again, momentary activation of JNK and p38 by tumor necrosis factor
is a survival signal, while the continued activation of these MAPKs is a death signal (52), and momentary activation of ERK can induce proliferation, while continued induction leads to growth arrest (53). In this manner, significant progress has been made in the understanding of MAPK function. Nevertheless, many questions regarding the regulation and function of this group of kinases remain unsolved. In the present study, three major MAPKs were activated in H2O2-treated cells (Figure 4
). This suggests that H2O2 can induce these MAPKs without cell death in this cell line because 500 µM of H2O2 is a non-cytotoxic dose and cells fully recover 4 h after the H2O2-containing cell media is replaced with H2O2-free media (21). PL extracts were found to inhibit ERK and p38 kinase activation but not JNK. Therefore, we hypothesized that blockage of ERK and p38 kinase activation is the primary mechanism of PL on GJIC. To confirm this hypothesis, we treated cells with PD98059 (Mek inhibitor) and SB202190 (p38 kinase inhibitor) before H2O2 treatment. As shown in Figures 57

, Mek and p38 kinase inhibitor blocked the down-regulation of GJIC by H2O2. Therefore, we suggest that not only ERK, but also p38 kinase can regulate Cx43.
In conclusion, this study shows that PL extracts increase GJIC and prevents the inhibition of GJIC by H2O2 through inhibition of ERK and p38 kinase activation. Moreover, this may be an important mechanism whereby the PL extract protects against tumor promotion. In addition, we found that the p38 kinase signaling pathway may be closely related to the function of the gap junction during cancer promotion. Therefore, the mushroom may give great health benefits to humans.
 |
Notes
|
---|
4 To whom correspondence should be addressed Email: kangpub{at}snu.ac.kr 
 |
Acknowledgments
|
---|
This work was supported by Brain Korea 21 Project.
 |
References
|
---|
- Loewenstein,W.R. (1981) Junctional intercellular communication: the cell-to-cell membrane channel. Physiol. Rev., 61, 829913.[Free Full Text]
- Bruzzone,R., White,T.W. and Paul,D.L. (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem., 238, 127.[Abstract]
- Kumar,N.M. and Gilula,N.B. (1996) The gap junction communication channel. Cell, 84, 381388.[ISI][Medline]
- Simon,A.M. and Goodenough,D.A. (1998) Diverse functions of vertebrate gap junction. Trends Cell Biol., 8, 477483.[ISI][Medline]
- Kang,K-S., Sai,K., Hirose,A., Hasegawa,R., Trosko,J.E. and Tohru,I. (2001) Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: relation to down-regulation of gap junctional intercellular communication. Cancer Lett., 173, 163174.[ISI][Medline]
- Trosko,J.E. and Ruth,J.R.J. (1998) Cell to cell communication in carcinogenesis. Front. Biosci., 3, 208236.
- Trosko,J.E. (1987) Mechanisms of tumor promotion: possible role of inhibited intercellular communication. Eur. J. Cancer Clin. Oncol., 23, 1929.[ISI][Medline]
- Klaunig,J.E. and Ruch,R.J. (1990) Role of intercellular communication in nongenotoxic carcinogenesis, Lab. Invest., 62, 135146.[ISI][Medline]
- Kang,K.-S., Lee,Y.-S., Kim,H.S. and Kim,S.H. (2001) Di-(2-ethylhexyl) phthalate-induced cell proliferation is involved in the inhibition of gap junctional intercellular communication and blockage of apoptosis in mouse sertoli. J. Toxicol. Environ. Health, 65 (Part A), 523535.[ISI]
- Mehta,P.P., Hotz-Wagenblatt,A., Rose,B., Shalloway,D. and Loewenstein,W.R. (1991) Incorporation of the gene for a cellcell channel protein into transformed cells leads to normalization of growth. J. Memb. Biol., 124, 207225.[ISI][Medline]
- Trosko,J.E. and Chang,C.C. (1988) Banbury report 31: nongenotoxic mechanisms in carcinogenesis: role of inhibited intercellular communication. In Hart,R.W. and Hoerger,F.D. (eds) Carcinogen Risk Assessment: New Direction in the Qualitative and Quantitative Aspects. Cold Spring Harbor, NY, pp. 139170.
- Masuda,M., Yamazaki,K., Matsunaga,T., Kanzaki,J. and Hosoda,Y. (1995) Melanocytes in the dark cell area of human vestibular organs. Acta Otolaryn., 519, 152157.
- Mesnil,M., Krutovskikh,V., Piccoli,C., Elfgang,C., Traub,O., Willecke,K. and Yamasaki,H. (1995) Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res., 55, 629639.[ISI][Medline]
- Rose,B., Nehta,P.P. and Loewenstein,W.R. (1993) Gap-junction protein gene suppresses tumorigenicity. Carcinogenesis, 14, 10731075.[Abstract]
- Hirschi,K.K., Xu,C.E., Tsukamoto,T. and Sager,R. (1996) Gap junction genes Cx26 and Cx43 individually suppress the cancer phenotype of human mammary carcinoma cells and restore differentiation potential. Cell Growth Differ., 7, 861870.[Abstract]
- Omori,Y., Dagli,M.L.Z., Yamakage,K. and Yamasaki,H. (2001) Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice. Mut. Res., 477, 191196.[ISI][Medline]
- Lee,K.W., Lee,H.J., Kang,K.S. and Lee,C.Y. (2002) Preventive effects of vitamin C on carcinogenesis. Lancet, 359, 172.[Medline]
- Kang,K.-S., Yun,J.-W., Yoon,B.S., Lim,Y.K. and Lee,Y.-S. (2001) Preventive effect of germanium dioxide on the inhibition of gap junctional intercellular communication by TPA. Cancer Lett., 166, 147153.[ISI][Medline]
- Na,H.-K., Wilson,M.R., Kang,K.-S., Chang,C.-C., Grunberger D. and Trosko,J.E. (2000) Restoration of gap junctional intercellular communication by caffeic acid phenethyl ester (CAPE) in a ras-transformed rat liver epithelial cell line. Cancer Lett., 157, 3138.[ISI][Medline]
- Kang,K.-S., Yun,J.W. and Lee,Y.-S. (2002) Protective effect of L-carnosine against 12-O-tetradecanoylphorbol-13-acetate or hydrogen peroxide-induced apoptosis on v-myc transformed rat liver epithelial cells. Cancer Lett., 178, 5362.[ISI][Medline]
- Upham,B.L., Kang,K.S., Cho,H.Y. and Trosko,J.E. (1997) Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells. Carcinogenesis, 18, 3742.[Abstract]
- Kang,K.S., Kang,B.C., Lee,B.J., Che,J.H., Li,G.X., Trosko,J.E. and Lee,Y.S. (2000) Preventive effect of epicatechin and ginsenoside Rb2 on the inhibition of gap junctional intercellular communication by TPA and H2O2. Cancer Lett., 152, 97106.[ISI][Medline]
- Wang,X., Martindale,J.L., Liu,Y. and Holbrook,N.J. (1998) The cellular response to oxidative stress: influences of mitogen-activated protein kinase signaling pathways on cell survival. Biochem. J., 333, 291300.[ISI][Medline]
- Chuang,S.M., Liou,G.Y. and Yang,J.L. (2000) Activation of JNK, p38 and ERK mitogen-activated protein kinases by chromium (VI) is mediated through oxidative stress but does not affect cytotoxicity. Carcinogenesis, 21, 14911500.[Abstract/Free Full Text]
- Marshall,C.J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell, 80, 175185.
- Whitmarsh,A.J. and Davis,R.J. (1996) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med., 74, 589607.[ISI][Medline]
- Kyriakis,J.M. and Avruch,J (1996) Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays, 18, 567577.[ISI][Medline]
- Ip,Y.T. and Davis,R.J. (1998) Signal transduction by the c-Jun N-terminal kinase (JNK)from inflammation to development. Curr. Opin. Cell Biol., 10, 205219.[ISI][Medline]
- Warn-Cramer,B.J., Cottrell,G.T., Burt,J.M. and Lau,A.F. (1998) Regulation of connexin-43 gap junctional intercellular communication by mitogen-actived protein kinase. J. Biol Chem., 273, 91889196.[Abstract/Free Full Text]
- Rummel,A.M., Trosko,J.E., Wilson,M.R. and Upham,B.L. (1999) Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity. Toxicol. Sci., 49, 232240.[Abstract]
- Ruch,R.J., Trosko,J.E. and Madhukar,B.V. (2001) Inhibition of connexin 43 gap junctional intercellular communication by TPA requires ERK activation. J. Cell Biochem., 83, 163169.[ISI][Medline]
- Koh,O. and Han,J. (2000) The p38 signal transduction pathway activation and function. Cell Signal, 12, 113.[ISI][Medline]
- Kim,H.M., Han,S.B., Oh,G.T., Kim,Y.H., Hong,D.H., Hong,N.D. and Yoo,I.D. (1996) Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom phellinus linteus. Int. J. Immunopharmacol.18, 295303.[ISI][Medline]
- Han,S.B., Lee,C.W., Jeon,Y.J., Hong,N.D., Yoo,I.D., Yang,K.H. and Kim,H.M. (1999) The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharmacology, 41, 157164.[ISI][Medline]
- Tsao,M.S., Smith,J.D., Nelson,K.G. and Grisham,J.W. (1984) A diploid epithelial cell line from normal adult rat liver with phenotype properties of oval cells. Exp. Cell. Res., 154, 3852.[ISI][Medline]
- Borenfreund,E. and Puerner,J.A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology, 24, 119124.
- El-Fouly,M.H., Trosko,J.E. and Chang,C.C. (1987) Scrape-loading and dye transfer: a rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res., 168, 422430.[ISI][Medline]
- Hayashi,T., Matesic,D.F., Nomata,K., Kang,K.S., Chang,C.C. and Trosko,J.E. (1997) Stimulation of cell proliferation and inhibition of gap junctional intercellular communication by linoleic acid. Cancer Lett., 112, 103111.[ISI][Medline]
- Sai,K., Upham,B.L., Kang,K.S., Hasegawa,R., Inoue,T. and Trosko,J.E. (1998) Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro. Cancer Lett., 130, 917.[ISI][Medline]
- Laemmli,N.K. (1970) Cleavage of structural proteins during the assembly head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
- Hasler,C.M., Frick,M.A., Bennink,M.R. and Trosko,J.E. (1990) TPA-induced inhibition of gap junctional intercellular communication is not mediated through free radicals. Toxicol. Appl. Pharmacol., 103, 389398.[ISI][Medline]
- Budunova,I.V. (1994) Alteration of gap junctional intercellular communication during carcinogenesis. Cancer J., 7, 228237.[ISI]
- Okamoto,M., Kaswai,K., Reznikoff,C.A. and Oyasu,R. (1996) Transformation in vitro of a nontumorigenic rat urothelial cell line by hydrogen peroxide. Cancer Res., 56, 26292653.
- Ruch,R.J., Cheng,S.J. and Klaunig,J.E. (1989) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10, 10031008.[Abstract]
- Muehlematter,D., Ochi,T. and Cerutti,P. (1989) Effects of ter-butyl hydroperoxide on promotable and non-promotable JB6 mouse epidermal cells. Chem. Biol. Interact., 71, 339352.[ISI][Medline]
- Hill,C.S. and Treisman,R. (1995) Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell, 80, 199211.[ISI][Medline]
- Yang,X., Khosravi-Far,R., Chang,H.Y. and Baltimore,D. (1997) Daxx, a novel Fas-binding protein that activate JNK and apoptosis. Cell, 89, 10671076.[ISI][Medline]
- Xu,X., Heidenreich,O., Kitajima,I., Mcguire,K., Li,Q., Su,B. and Nerenberg,M. (1996) Constitutively activated JNK is associated with HTLV-1 mediated tumorigenesis. Oncogene, 13, 135142.[ISI][Medline]
- Rodrigues,G.A., Park,M. and Schlessinger,J. (1997) Activation of the JNK pathway is essential for transformation by the Met oncogene. EMBO J., 10, 26342645.
- Sweeney,G., Somewar,R., Ramlal,T., Volchuk,A., Ueyama,A. and Klip,A. (1999) An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes. J. Biol Chem., 274, 1007110078.[Abstract/Free Full Text]
- Heidenreich,K.A. and Kummer J.L. (1996) Inhibition of p38 mitogen-activated protein kinase by insulin in cultured fetal neurons. J. Biol Chem., 271, 98919894.[Abstract/Free Full Text]
- Roulston,A., Reinhard,C., Amiri,P. and Williams,L.T. (1998) Early activation of c-jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor
. J. Biol Chem., 273, 1023210239.[Abstract/Free Full Text]
- Bennett,A.M. and Tonks N.K. (1997) Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science, 278, 12881291.[Abstract/Free Full Text]
Received January 18, 2002;
revised March 25, 2002;
accepted April 3, 2002.