Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: relation to down-regulation of gap junctional intercellular communication

Abstract

Pentachlorophenol (PCP), a promoter of murine hepatocarcinogenesis, inhibits gap junctional intercellular communication (GJIC) in rat liver epithelial cells in vitro. To test the hypothesis that both inhibition of GJIC and apoptosis contribute to tumor promotion, we investigated the effect of PCP on both GJIC and serum deprivation-induced apoptosis in v-myc-transfected rat liver epithelial cells. The results showed that PCP inhibited apoptosis, as measured by the TUNEL assay and DNA ladder formation. Inhibition of apoptosis was associated with a decrease in GJIC. The study demonstrated that PCP has a potential for inhibiting apoptosis and GJIC, supporting the hypothesis.

Introduction

In multicellular organisms, apoptosis is associated with normal regulation of development [1] and with the immune defense system via the elimination of damaged or mutated cells [2]. Furthermore, conditions that either enhance or inhibit normal apoptotic rates have been associated with many disease states [3]. In the setting of carcinogenesis, inhibition of apoptosis has been correlated with tumor promotion [4].

Gap junctions have been linked to the apoptotic process [5] as well as to the tumor promotion process [6]. The transfer of ions and small molecular weight molecules are mediated through gap junctions, which are composed of two juxtaposed connexons consisting of a hexamer of proteins, connexins [7], [8]. Tumor-promoting chemicals, by disrupting gap junctional intercellular communication (GJIC), have been postulated to isolate transformed cells from the suppressing regulation of over cell-growth by surrounding normal cells [9] and permitting their clonal expansion. Activated oncogenes, growth factors and hormones which act as tumor promoters, as well as anti-sense connexin 43 (Cx43), can also down-regulate GJIC [10]. On the other hand, anti-tumor promoters such as the tumor suppressor gene of human chromosome 11, lovastatin, and connexin-transfected tumor cells, can restore normal GJIC in GJIC-deficient tumor cells. Recently, a connexin 32 (Cx32) knock-out mouse was demonstrated to develop spontaneous and chemically-induced hepatic tumors as compared to the wild-type mouse [11], supporting the hypothesis that GJIC contributes to suppress the tumor development. Several tumor promoting chemicals such as phenobarbital [12], phorbol esters [13] and peroxisome proliferators [14], which are known to inhibit apoptosis, have been shown to inhibit GJIC, and reversely, several anti-tumor promoting chemicals such as retinoids [15] and dexamethasone [16], which are known to facilitate apoptosis, can enhance GJIC. These observations suggest a possible mechanistic link between GJIC and apoptosis. For example, GJIC may facilitate signal transduction needed for the apoptotic process in cells contacting each other in the tissues. Alternatively, inhibition of GJIC may interfere with the apoptotic process [6].

Pentachlorophenol (PCP), an agent used as a wood preservative, is non-mutagenic by the Ames test, but has been demonstrated to induce hepatic tumors in mice [17]. Results of several experiments have suggested that tetrachlorohydroquinone (TCHQ), a metabolite of PCP, may be the carcinogenic initiator, since it is capable of inducing oxidative damage to cellular DNA [18], [19], [20], [21], [22], [23], [24] and mutations in Chinese hamster cells [25]. Active oxygen species generated during further oxidation have also been postulated as initiating agents [18], [19], [20], [21], [22], [23], [24], [25]. Administration of PCP to mice produces oxidative stress in the target organ [22]. Interestingly, administration of PCP facilitates hepatic cell proliferation during oxidative stress [23], and has tumor-promoting activity in diethylnitrosamine-initiated mouse hepatocarcinogenesis [24]. The mechanism by which PCP promotes liver tumors has been hypothesized to be inhibition of GJIC by the epigenetic activity of the parent molecule but not by any mutagenic activity of its metabolite TCHQ. This hypothesis is supported by our previously published in vitro study, showing that PCP inhibited GJIC in rat liver epithelial cells, but not TCHQ [26]. These results suggested that the GJIC-inhibitory activity of PCP may potentially mediate not only a mitogenic effects but also an inhibitory effect on apoptosis.

The present study was aimed at testing a potential relationship between inhibition of GJIC and inhibition of apoptosis by PCP during the tumor promotion process. To this end, we examined the effects of PCP on the apoptotic frequency and on GJIC levels in WB-MYC cells grown in serum-free medium [27]. WB-MYC is a v-myc-transfected rat liver epithelial cell line which expresses high levels of GJIC as does the parent cell line (WB cell), and moreover whose apoptotic frequency triggered after serum deprivation is greater than WB cells. For these properties, WB-MYC was used in this study as a suitable system for detecting the inhibitory potential of both apoptosis and GJIC by PCP.