Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells

Norah Defamie, Baharia Mograbi, Cyril Roger, Laurent Cronier1, André Malassine1, Françoise Brucker-Davis, Patrick Fenichel, Dominique Segretain,2 and Georges Pointis,3

INSERM EMI 00-09, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 02 and
1 INSERM U427, Faculté SC Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris, France


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Lindane ({gamma}-hexachlorocyclohexane) is a lipid-soluble pesticide that exerts carcinogenic and reprotoxic properties. The mechanisms by which lindane alters testicular function are unclear. Sertoli cells control germ cell proliferation and differentiation through cell–cell communication, including gap junction intercellular communication. Using the 42GPA9 Sertoli cell line, we show that lindane, at a non-cytotoxic dose (50 µM), abolished gap junction intercellular communication (GJIC) between adjacent cells. This change was associated with a time-related diminution and redistribution of Cx43 from the membrane to the cytoplasmic perinuclear region. A similar alteration was observed for ZO-1, a tight junction component associated with Cx43, but not for occludin, an integral tight junction protein. After a 24 h lindane exposure, Cx43 and ZO-1 colocalized within the cytoplasm and no modification of non-phosphorylated and phosphorylated isoforms of Cx43 was observed. By double immunofluorescent labelling we demonstrate that the cytoplasmic Cx43 signal was not present in either the endoplasmic reticulum/Golgi apparatus or lysosomes. These results suggest that lindane inhibits GJIC between Sertoli cells and that aberrant Cx43/ZO-1 localization may be responsible for this effect. The alterations in gap junctions induced by lindane in 42GPA9 Sertoli cells are similar to those observed in tumour cells and may be involved in the pathogenesis of neoplastic seminomal proliferation.

Abbreviations: Cx, connexin; GJIC, gap junction intercellular communication; FRAP, fluorescence recovery after photobleaching; PBS, phosphate-buffered saline; ZO-1, zonula occludens-1.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gap junctions are intercellular plasma membrane channels, formed by connexins (Cx), which allow direct intercytoplasmic exchange between neighbouring cells. There is strong evidence that this cell–cell communication plays an important role in the control of cell growth and differentiation (reviewed in ref. 1). Several in vitro and in vivo findings indicate that aberrant control of gap junction intercellular communication (GJIC) is one mechanism involved in carcinogenesis and that agents which impair GJIC have the ability to enhance neoplastic transformation (reviewed in ref. 2).

Recent epidemiological observations have suggested a relationship between pesticide exposure and the incidence of human cancers (3,4). Testicular cancer is the most common malignancy of young men and its incidence has increased in recent years (5). Because of this consistant trend, a chronic exposure to environmental polluants such as pesticides has been suspected. Lindane ({gamma}-hexachlorocyclohexane), which has been a widely used pesticide in agriculture, is known to promote several non-mutagenic carcinogenic effects in rodents (6). It is also known to concentrate in the ovary and testis (79), but the mechanisms by which this agent might lead to testicular neoplasia remain unclear.

It was reported that this agent was able to inhibit folliculogenesis in the ovary by altering gap junction and Cx43 expression in granulosa cells (10), as also described in different cell lines (1114). Within the seminiferous epithelium Sertoli cells, the equivalent of granulosa cells, control proliferation and differentiation of germ cells and are potential targets for environmental polluants. They form junctional complexes composed of gap and tight junctions which structure the blood–testis barrier (15). Cx43 is distributed in both Sertoli and germ cells (16) and plays an essential role in the spermatogenic cycle, as recently demonstrated by a Cx43 gene knock-out (17,18). Since GJIC is important in normal cellular growth control, inhibition of intercellular communication of Sertoli cells by lindane may be an important mechanism by which germ cells escape growth regulation by Sertoli cells and progress to neoplasia. However, no study has so far addressed the effect of lindane on GJIC and Cx43 expression in Sertoli cells.

We have recently established a Sertoli cell line (42GPA9) (19). At confluency, 42GPA9 Sertoli cells have the ability to form gap junctions and to express Cx43 (20). The present study was performed to analyze the effect of lindane on GJIC and on Cx43 in 42GPA9 Sertoli cells. The effects on two tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were also studied. In this analysis we provide evidence that lindane induces aberrant localization of Cx43 and ZO-1 but does not alter occludin expression.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Cell culture and cytotoxicity assay
The 42GPA9 Sertoli cell line was obtained from sexually mature polyoma virus large T (PyLT) transgenic mice and was maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 32°C (19). Exponentially growing cells were plated in 96-well plates at 45 000 cells/well for 24 h. Increasing concentrations of lindane (Sigma, St Louis, MO) dissolved in dimethylsulphoxide were added to the culture medium. Solvent vehicle-treated cells were used as controls. After 24 h, cytotoxicity was evaluated by the neutral red uptake assay. Briefly, cells were loaded for 3 h with neutral red (50 µg/ml culture medium), fixed with a mixture of formaldehyde and CaCl2 (1:1 v/v) and the dye was extracted with a mixture of acetic acid and ethanol (1:50 v/v). Plates were left overnight at 4°C and transferred to a microplate reader. Absorbance was recorded at 540 nm. Experiments were performed three times, using 4–8 wells per concentration of lindane.

Fluorescence recovery after photobleaching (FRAP) analysis
The cell to cell diffusion of fluorescent dye was measured by the gap FRAP method as previously described (21). Briefly, the cells were washed three times with phosphate-buffered saline (PBS) and loaded for 15 min at 32°C with 5,6-carboxyfluorescein diacetate (7 µg/ml in Ca2+/Mg2+-PBS). Dye transfer was monitored at 32°C using an interactive laser cytometer (ACAS 570; Meridian Instruments, Okemos, MI). Individual cells were bleached with a 488 nm laser and recovery of fluorescence intensity was monitored at 4 min intervals.

Reverse transcription–polymerase chain reaction (RT–PCR)
Total RNA was extracted from 42GPA9 Sertoli cells using RNeasy (Qiagen, Courtaboeuf, France). RNA was treated with RNase-free DNase I (Gibco BRL Life Technologies, Cergy Pontoise, France) at 22°C for 5 min. Briefly, RNA was reverse transcribed into cDNAs using oligo(dT)12–18 as primers (Roche Diagnostics, Meylan, France) and SuperScript II reverse transcriptase (Gibco BRL Life Technologies). This reaction product served as a template for PCR with a PTC-100 DNA thermal cycler as recommended by the supplier (MJ Research). Primers (Eurogentec, Seraing, Belgium) were designed from published sequences: rat Cx43, sense 5'-CGCTGGCTTGCTTGTTGTAA-3', antisense 5'-TGGTGTCTCTCGCTCTGAAT-3' (22); mouse ß-actin, sense 5'-ATGAGGTAGTCTGTCAGGTC-3', antisense 5'-CCGGTCGAGTCGCGTCCA-3' (23). To check for the absence of genomic DNA contamination, RT–PCR reactions were performed under identical conditions, except that SuperScript II was omitted from the cDNA synthesis step.

Gel electrophoresis
Cells were solubilized in NP40/Brij lysis buffer (50 mM Tris–HCl, pH 7.5, 1% NP40, 1% Brij 96, 1 mM Na3VO4, 10 mM ß-glycerophosphate, 10 mM sodium fluoride, 2 mM EDTA, 1 mM aprotinin, 25 mM leupeptin, 1 mM pepstatin, 2 mM phenylmethylsulfonyl fluoride). Whole cell lysates (75 µg) were separated by 10% SDS–PAGE and electroblotted onto a polyvinylidene fluoride membrane (PVDF Immobilon-P; Millipore) for 3 h at 4°C in 20 mM Tris, 150 mM glycine, 20% methanol using the TE 62X Transphor II Unit (Hoefer). The blot was saturated in TNB buffer (10 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.1% Tween 20, 3% BSA, 0.5% gelatin) and then incubated overnight at 4°C with either anti-Cx43 (1:2000) (Transduction Laboratories, Lexington, KY), anti-ZO-1 (1:1500) (Zymed Laboratories, CA) or anti-occludin (1:2500) (Zymed Laboratories) antibodies. After three washes with TNN buffer (10 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 1% NP 40), the presence of primary antibody was revealed with horseradish peroxidase-conjugated anti-mouse (1:5000) (Dako, Trappes, France) or anti-rabbit IgG (1:10 000) (Dako) and visualized with an enhanced chemiluminescence detection system (ECL; Amersham, little Chalfont, UK) as previously reported (20). The state of phosphorylation of Cx43 was analysed by pre-incubating the protein extracts with alkaline phosphatase.

Immunocytochemical procedures
Cells were fixed in cold methanol at –20°C for 5 min and washed with 0.1% Tween 20-PBS for 5 min. Fixed cells were incubated with the monoclonal Cx43 antibody (1:100) in PBS containing 3% non-fat dry milk overnight at 4°C. Cells were rinsed with 0.1% Tween 20-PBS and then incubated for 45 min with a FITC-conjugated goat anti-mouse IgG (1:50) (Jackson Immunoresearch Laboratories, Baltimore, MD) in PBS containing 3% non-fat dry milk. After washing five times with PBS, the slides were then mounted in Mowiol medium (Calbiochem, La Jolla, CA) for immunofluorescence. Controls omitted the primary antibody. Dual-label analysis was performed as recently reported (24). Briefly, slides were incubated with a mixture containing the anti-Cx43 antibody and for each co-localization one of the antibodies directed against specific markers of endoplasmic reticulum (25) at a dilution of 1:300, of Golgi apparatus (1:500) (CTR433, a gift from M. Bornens, Institut Curie, Paris, France), of lysosomes (CD107b/lamp-2; Pharmingen, Becton Dickinson, Le Pont de Claix, France; 1:100) or of tight junctions (ZO-1 at 1:100 and occludin at 1:250). Subsequently, the slides were incubated in a mixture containing FITC-conjugated goat anti-mouse IgG (1:50) (Dako) and rhodamine TRITC-conjugated anti-rat or anti-rabbit IgG (1:200) (Amersham) in PBS containing 3% bovine serum albumin. Sections were examined with a confocal laser scanning microscope (Leica TCS SP) fitted with a 488 or 543 nm krypton/argon laser allowing simultaneous analysis of the fluorescein and rhodamine chromophores.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Effects of lindane on Cx43 protein and mRNA levels
We first evaluated the lindane toxicity threshold by monitoring the viability of 42GPA9 Sertoli cells exposed for 24 h to different concentrations of lindane (0–150 µM). Concentrations of lindane between 10 and 50 µM did not exert a significant effect on 42GPA9 Sertoli cell viability (data not shown). In contrast, cell exposure to higher concentrations of lindane (100 and 150 µM) significantly reduced the number of cells (P < 0.05).

GJIC between 42GPA9 Sertoli cells was analysed by cell loading with a fluorescent dye and by the gap FRAP method (Figure 1AGo). At confluency, 42GPA9 Sertoli cells exhibited functional GJIC when cultured under control conditions as previously reported (20). A progressive decrease in GJIC was observed in the presence of increasing doses of lindane (10–50 µM). Complete inhibition of GJIC was obtained in the presence of 50 µM lindane, as indicated by the dramatic fall in the percentage of coupled cells (Figure 1AGo). For this reason this dose was choosen for further experiments. Western blots were performed to examine the biochemical changes in Cx43 protein levels associated with lindane-induced GJIC uncoupling. A representative blot from one of three separate experiments is shown in Figure 1BGo. Blot analysis revealed the presence of three bands at ~43 kDa in untreated 42GPA9 Sertoli cells. We have identified these bands as the non-phosphorylated (NP) and phosphorylated (P1 and P2) isoforms of Cx43 by alkaline phosphatase treatment (data not shown), as previously described (26). Cell treatment with 50 µM lindane resulted in a marked reduction in the relative levels of the three Cx43 isoforms. To determine whether lindane altered the steady-state levels of Cx43 transcripts, Cx43 mRNA was analysed by RT–PCR (Figure 1CGo). In control 42GPA9 Sertoli cells a single band for Cx43 PCR product was observed of the expected size (267 bp). Little if any change in Cx43 mRNA was detected after cell treatment with 50 µM lindane for 24 h. A slight decrease in Cx43 mRNA level was, however, observed for a longer time of exposure (48 h) to lindane (data not shown).



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Fig. 1. Effects of lindane on GJIC and on Cx43 expression in 42GPA9 Sertoli cells. Sertoli cells were cultured for 24 h in the presence or absence of 50 µM lindane. (A) Analysis of the percentage of coupled cells by the gap FRAP method in control (n = 62) and lindane-treated cells (n = 37). (B) Western blot analysis of Cx43 levels in control and lindane-treated Sertoli cells. Numbers on the left refer to molecular mass (kDa) based on protein standards. Non-phosphorylated (NP) and phosphorylated (P1 and P2) isoforms of Cx43 are indicated. (C) Cx43 and ß-actin mRNA identified by RT–PCR in control and lindane-treated Sertoli cells. The lane on the left indicates molecular DNA size markers. Representative of three separate experiments.

 
Time course effect of lindane on Cx43 expression and localization
Treatment of 42GPA9 Sertoli cells with 50 µM lindane resulted in a time-dependent decrease in expression of Cx43 protein, as observed in Figure 2AGo. This inhibitory effect of lindane on Cx43 expression was reversible after cell washing and re-feeding with lindane-free culture medium for 24 h (Figure 2AGo, lane 6). We next analysed the distribution of Cx43 in 42GPA9 Sertoli cells by indirect immunofluorescence. As shown in Figure 2BGo, a punctuate linear specific staining for Cx43 was observed at appositional plasma membranes between adjacent cells in control cultures. In cells treated with 50 µM lindane for 1 h the membranous immunoreactive signal for Cx43 was more diffuse. Thereafter the signal disappeared from cell membranes and appeared in intracytoplasmic spots. Delocalization of Cx43 immunoreactivity was time dependent and clearly appeared 4–6 h after treatment. After 24 h most of the Cx43 signal was observed within the cytoplasm. After cell washing and culturing in lindane-free medium for 24 h all the immunoreactive signal was relocated to the plasma membranes.



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Fig. 2. Time-dependent effects of lindane on Cx43 expression in 42GPA9 Sertoli cells. (A) Western blot analysis of Cx43 in Sertoli cells cultured without lindane (lane 1) or in the presence of 50 µM lindane for 1 (lane 2), 4 (lane 3), 6 (lane 4) and 24 h (lane 5). Cx43 level was also measured after washing the treated cells and re-feeding with lindane-free culture medium for a further 24 h (lane 6). Numbers on the right refer to molecular mass (kDa) based on proteins standards. Non-phosphorylated (NP) and phosphorylated (P1 and P2) isoforms of Cx43 are indicated. (B) Cx43 localization in Sertoli cells cultured without lindane (a) or in the presence of 50 µM lindane for 1 (b), 4 (c), 6 (d) and 24 h (e). (f) Immunolocalization of Cx43 after washing the treated cells and re-feeding with lindane-free culture medium for a further 24 h. All magnifications x400. Representative of three separate experiments.

 
Analysis of Cx43 delocalization after lindane treatment
To identify the vesicular compartment where Cx43 accumulated following lindane treatment, double immunofluorescent labeling of Sertoli cells with anti-Cx43 antibody and antibodies directed against specific markers of various cytoplasmic components were performed. Confocal images of the same field show that the immunoreactive Cx43 signal did not co-localize with either the endoplasmic reticulum (Figure 3a–cGo), the Golgi apparatus (Figure 3d–fGo) or a lysosomal marker (Figure 3g–iGo) after cell treatment with 50 µM lindane for 24 h.



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Fig. 3. Confocal images of double immunolabelling of lindane-treated 42GPA9 Sertoli cells for Cx43 and specific markers of endoplasmic reticulum, Golgi apparatus and lysosomes. Sertoli cells were cultured for 24 h in the presence of 50 µM lindane and double immunolabelled for Cx43 (a) and endoplasmic reticulum (b), for Cx43 (d) and Golgi apparatus (e) or for Cx43 (g) and lysosome (h). No co-localization of Cx43 in endoplasmic reticulum (c), in Golgi apparatus (f) and in lysosomes (i) was observed. All magnifications x600. Representative of three separate experiments.

 
Effect of lindane on ZO-1 and occludin expression
To determine whether Cx43 was the sole component of junctional complexes affected by lindane, we analysed the effect of lindane on the expression of ZO-1 and occludin, two tight junction proteins. As shown for Cx43, anti-ZO-1 western blots indicated that the pattern of ZO-1 expression was time-dependently reduced by exposure to 50 µM lindane (Figure 4AGo). The immunoreactive signal for ZO-1 was specifically detected at the cell membrane between adjacent 42GPA9 Sertoli cells and co-localized with Cx43 at this location in control cells (Figure 4BGo). After lindane treatment cells exhibited faint ZO-1 membrane staining and concomitantly a large increase in cytoplasmic staining. In this case, double immunolabelling experiments revealed that ZO-1 and Cx43 had the same cytoplasmic distribution. In contrast to Cx43 and ZO-1, western blot analyses showed that expression of occludin was not altered by exposure of 42GPA9 Sertoli cells to lindane (Figure 5AGo). Immunofluorescence staining of occludin indicated a normal peripheral distribution pattern at the cell–cell contact points under control conditions, which was maintained in lindane-treated cells (Figure 5BGo).



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Fig. 4. Time-dependent effects of lindane on ZO-1 expression in 42GPA9 Sertoli cells. (A) Western blot analysis of ZO-1 in Sertoli cells cultured without lindane (lane 1) or in the presence of 50 µM lindane for 1 (lane 2), 4 (lane 3), 6 (lane 4) and 24 h (lane 5). ZO-1 level was also measured after washing the treated cells and re-feeding with lindane-free culture medium for a further 24 h (lane 6). (B) Double immunolabelling for Cx43 and ZO-1 in control and lindane-treated Sertoli cells. Sertoli cells were cultured for 24 h in the absence (a–c) or presence of 50 µM lindane (d–f) and double immunolabelled for ZO-1 (a and d, red labelling) and Cx43 (b and e, green labelling). Note that ZO-1 and Cx43 immunosignals co-localized (yellow labelling) in the membraneous compartment under control conditions (c) and have a similar cytoplasmic localization after lindane treatment (f). All magnifications x350. Representative of three separate experiments.

 


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Fig. 5. Time-dependent effects of lindane on occludin expression in 42GPA9 Sertoli cells. (A) Western blot analysis in Sertoli cells cultured without lindane (lane 1) or in the presence of 50 µM lindane for 1 (lane 2), 4 (lane 3), 6 (lane 4) and 24 h (lane 5). Occludin level was also measured after washing the treated cells and re-feeding with lindane-free culture medium for a further 24 h (lane 6). (B) Double immunolabelling for Cx43 and occludin in control and 50 µM lindane-treated Sertoli cells. Sertoli cells were cultured for 24 h in the absence (a–c) or presence of 50 µM lindane (d–f) and double immunolabelled for occludin (a and d) and Cx43 (b and e). Note that occludin and Cx43 immunosignals were localized in the membraneous compartment under control conditions (c) and that lindane altered Cx43 but not occludin localization (f). All magnifications x400. Representative of three separate experiments.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
There is direct and indirect evidence that GJIC is involved in the regulation of normal cell growth and differentiation and that abnormal control of this communication may play a crucial role in neoplastic transformation (reviewed in ref. 2). Deregulation or loss of function of gap junctions has been shown in different cell types exposed to lindane (1114). In the testis, several deleterious effects of lindane on testicular function have been reported (27) and Sertoli cells may be a potential target for this toxicant (28). The present data show for the first time that lindane impairs GJIC in 42GPA9 Sertoli cells. This inhibition of GJIC did not appear to be due either to decreased Cx43 gene expression or to changes in the relative levels of non-phosphorylated and phosphorylated isoforms of Cx43. Our data clearly show that this alteration is mainly due to aberrant localization of Cx43 and of the tight junction-associated component ZO-1, without any change in expression of the integral membrane protein of tight junctions, occludin.

These findings were substantiated by our immunofluorescence studies showing that Cx43 and ZO-1 immunoreactive signals, primarily detected at the plasma membrane in control 42GPA9 Sertoli cells, relocated to the cytoplasm after lindane treatment. The current observation that localization of both Cx43 and ZO-1 were similarly affected by lindane treatment and co-localized in the perinuclear region is consistent with the recent demonstration of a physical association between the C-terminal domain of Cx43 and the N-terminal domain of ZO-1 (29,30). This linkage has been shown to be involved in Cx43 targeting to the plasma membrane in cardiac myocytes (30). There is also evidence that ZO-1 can bind directly to the cytoplasmic C-terminal tail of the integral tight junction protein occludin (31,32). In addition, several lines of evidence suggest that ZO-1 recruits and targets occludin to tight junctions (33). In the present study, although the major part of the immunoreactive ZO-1 was delocalized, a persistant signal was detected at the periphery of lindane-treated cells. This residual and normally localized ZO-1 could be responsible for normal expression of occludin.

The mechanism(s) by which lindane affects GJIC in 42GPA9 Sertoli cells remains unclear. Several effects of lindane have been ascribed to the oestrogenic activity of one of its isomers, ß-hexachlorocyclohexane (34,35). In the present study, however, it is unlikely that all the effects of lindane on Cx43 and ZO-1 delocalization could be dependent of this oestrogen- like activity, since the lindane used was of high grade, without ß-hexachlorocyclohexane contamination. Lindane is a lipid-soluble toxicant which has been described as accumulating in the membrane lipid layer and it is able to alter phospholipid levels (36) and the dynamic properties of plasma membranes in different cell types (3739). Thus, aberrant localization of the Cx43/ZO-1 complex may result from such membranous effects of lindane.

The current co-immunolocalization experiments with antibodies directed against specific markers of endoplasmic reticulum and Golgi apparatus were unable to localize Cx43 protein within these cellular compartments. Thus, our data suggest that lindane probably does not prevent transport of newly synthetized Cx43 to the plasma membrane. Short-term treatment of WB-F344 liver epithelial cells with lindane has been reported to promote endocytosis of gap junction plaques and degradation of phosphorylated forms of Cx43 in lysosomes (40). Our data that Cx43 did not co-localize with LAMP2, a specific marker of lysosomes, rules out the involvement of lysosomes in lindane-induced Cx43 effects in Sertoli cells. The delocalization of Cx43 signal rather might be consecutive to a block in Cx43 gap junction endocytosis before their degradation in the lysosomal compartment. The precise mechanisms responsible for Cx43 delocalization are under investigation in our laboratory.

Abnormal GJIC due to aberrant localization of Cx in the cytoplasm has been observed in different tumour cells (4143) and in carcinogenic tissues (4446). Such deleterious effects on Cx have been also reported in several cell types and in 42GPA9 Sertoli cells exposed to tumour promoters such as 12-O-tetradecanoyphorbol-13-acetate (20,47,48). Impaired expression or aberrant localization of ZO-1 has also been frequently observed in human maligant tumours (49,50) and in Ras-transformed MDCK cells (51). So far, there is no data addressing a direct effect of lindane on both Cx43 and ZO-1 expression. In the testis Cx43 and ZO-1 co-localize within the seminiferous epithelium (24). In addition, there is compelling evidence that testicular expression of Cx43 and ZO-1 is essential for normal germ cell proliferation and differentiation (1618,52). Altered Cx43 expression has been reported in different cases of spermatogenesis failure in rodents (16,24) and in man (53). Testicular cancer is the most common malignancy of young men and its incidence has increased constantly over recent years (5). However, the aetiology of human testicular tumours is poorly defined (reviewed in ref. 54). Exposure to chemical agents, such as pesticides, has been suggested as a possible carcinogenic factor (3,4). The present results suggest that lindane may disrupt the control of germ cell proliferation by Sertoli cells and promote testicular seminoma cell transformation by inducing aberrant localization of Cx43 and ZO-1.


    Notes
 
2 Present address: Histologie-Embryologie, CHU-Paris Ouest, Faculté de Médecine, 45 Rue des St Pères, 75006 Paris 2, France Back

3 To whom correspondence should be addressed

Email: pointis{at}unice.fr Back


    Acknowledgments
 
This work was supported in part by the European Chemical Industry Council (CEFIC), the Ligue Nationale contre le Cancer and the Association pour la Recherche sur le Cancer (no. 98-80). N.D. was supported by a grant from Agence de l'Environnement et de la Maîtrise de l'Energie. B.M. was the recipient of a post-doctoral fellowship from Fondation Fertilité Sterilité.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 25, 2001; revised June 11, 2001; accepted June 15, 2001.