Differential regulation of γ-glutamyltransferase mRNAs in four human tumour cell lines

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Abstract

Human γ-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-α (TNF-α), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-α and TPA did not alter these parameters. In LNCap cells, TNF-α, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-α, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.

Introduction

γ-Glutamyltransferase (GGT, EC 2.3.2.2), a glycosylated membrane enzyme, catalyses the transfer of γ-glutamyl groups from peptide donors such as glutathione to a variety of acceptors including dipeptides, free amino acids or water [1]. GGT activity is modulated in many physiological and pathological conditions, including differentiation and carcinogenesis. The GGT activity has been reported to be increased during kidney development and differentiation, but reduced in adult liver compared to fetal liver [2]. An increased GGT activity has also been detected in several neoplastic cells and notably in chemically induced tumours in rat liver [3], and in human ovary [4], prostate [5] and lung carcinomas [6]. GGT activity has been found increased in various tumour cell lines after anticancer drug treatment. This may be of some importance in drug resistance [7], [8], [9]. More recent data suggest that GGT could play an important role in maintaining cell proliferation [10]. This role was attributed to the oxidant stress generated from the GGT dependent glutathione metabolism. On the other hand, this oxidative stress can induce lipid peroxidation [11], protein oxidation [12], and could trigger the activation of transcription factors [13].

In humans, GGT is a multigene family of at least seven genes and pseudogenes [14]. Among these human GGT genes, one locus is of great importance since its transcription generates type I GGT mRNAs [14], [15], encoding an enzyme with catalytic properties that are identical to the enzyme isolated from human tissues [16]. Three subtypes of GGT mRNAs related to the type I and showing the same open reading frame (ORF) but differing in their 5′ untranslated region (UTR) have been isolated from HepG2 hepatoma [17], fetal liver [18], [19], and placenta [20]. For the sake of clarity we used the nomenclature proposed by Tsutsumi et al. [21]: subtype A corresponds to the mRNA firstly described in fetal liver, subtype B to the mRNA isolated from the human hepatoma cell line HepG2 and subtype C to the mRNA isolated from human placenta.

Diederich et al. [22] suggested that the 5′ UTRs of these GGT mRNAs play an important role in the regulation of translation. Analysis of the corresponding genomic sequences showed that these mRNAs are transcribed from the same gene. However, it is not clear if they are under the control of separate promoters as in rat and mouse [23], or if the observed polymorphism is due to differential splicing events.

Little information exists concerning the regulation of the expression of these GGT mRNAs. In their study, Tsutsumi et al. [21] analysed the distribution of GGT mRNAs in healthy and cancerous tissues and suggested that subtype B is closely related to the development of human hepatocellular carcinoma (HCC). The same authors demonstrated that analysis of GGT mRNA expression may provide a useful tool for the diagnosis of HCC at its early stages.

In this report we examined the modulation of the human GGT mRNAs type I by tumour necrosis factor-α (TNF-α), 12-O-tetradecanoylphorbol 13-acetate (TPA) and sodium butyrate in four human cell lines derived from different cancer tissues: HepG2, U937, LNCap and HeLa cells. We present evidence for the first time that these mRNAs are independently regulated and that their regulation is cell type specific.

Section snippets

Cell lines, culture conditions and treatments

HepG2, U937, LNCap and HeLa cells, obtained from ECCAC, were grown in RPMI 1640 medium (Sigma, France) supplemented with 10% (v/v) heat inactivated fetal calf serum (Boehringer), 1% (v/v) antibiotic–antimitotic solution (Sigma) and 2 mM glutamine (Sigma).

Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cells were treated with 200 U/ml TNF-α, 2.5 mM sodium butyrate, or 2 μM TPA (Sigma) in complete culture medium for 24 h before total RNA extraction, and for 48 h

Modulation of GGT activity after TNF-α, TPA and sodium butyrate treatment

The human cell lines HepG2, LNCaP, HeLa and U937 showed different basal activities, ranging from 16 mU/mg protein (HeLa cells) to 144 mU/mg (HepG2 cells). The regulation of GGT was studied in these cell lines after 48 h treatment with TNF-α, TPA or sodium butyrate. As shown in Table 1, GGT activity was largely decreased in HepG2 cells after sodium butyrate treatment (−50%), while TNF-α and TPA had no significant effects. In LnCap cells, the activity was dramatically decreased (∼70%) after TNF-α

Discussion

Most studies on the regulation of GGT mRNAs have been performed using rat and mouse models. For instance, it has been reported that rat GGT mRNA III is induced in rat hepatocytes in response to ethoxyquin [27], and in rat alveolar type II cells in response to quinone toxicity [28]. Rat mRNA II and mRNA IV have also been shown to respond to an oxidative stimulus in epididymis [29].

In humans, the more complex GGT gene organisation prevented rapid progress in the understanding of the regulation

Acknowledgements

The authors would like to thank for partial funding of this work the Ligue Nationale contre le Cancer, the Association Régionale pour l’Enseignement et la Recherche Scientifique (ARERS) and La Région Lorraine. S.D. was supported by a grant from the Ligue Nationale contre le Cancer, and M.J.A. was supported by a grant from ARERS and La Région Lorraine. I.P. was supported by a grant from the Norwegian Research Council.

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