The relative value for ASCT2 expression in cells grown in standard glutamine-containing moderate for a minimum of 7 days in a separate experiment was 4

The relative value for ASCT2 expression in cells grown in standard glutamine-containing moderate for a minimum of 7 days in a separate experiment was 4.5. was derived from the human genome database. A 907 bp fragment of this sequence was directionally ligated into a luciferase reporter vector and was shown to exhibit promoter activity when transfected into HepG2 cells. Promoter activity was greatly reduced when transfection was performed in glutamine-free medium and was restored when glutamine was added post-transfection. The absence of other essential amino acids did not affect promoter activity, and glutamine deprivation did not affect the MCT1 (monocarboxylate transporter 1) promoter. These results indicate that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability. Keywords:ASCT2, glutamine, hepatoma, promoter, regulation, transporter Abbreviations:AP1, activator protein 1; DMEM, Dulbecco’s modified Eagle’s medium; MCT1, monocarboxylate transporter 1 == INTRODUCTION == Hepatoma cells transport and utilize glutamine at a much higher rate than do normal liver cells [1]. Glutamine is also an essential requirement for tumour cell growth. Expression of the cell-membrane glutamine transporter has been suggested to be an integral event in hepatocellular transformation, and this transporter has been considered as a potential target in approaches aimed at attenuating the growth of tumour cells. In some more differentiated human hepatoma cell lines, competitive inhibition of glutamine transport has been shown to decrease cell proliferation under certain conditions (see [2]). Glutamine transport across mammalian cell membranes is usually mediated by a number of transport systems (see [3]). In human [4] and rat [5] hepatoma cell lines, the predominant Na+-dependent glutamine transporter has properties similar to, but not identical with, proteins of the classical amino acid transport System ASC (alanine/serine/cysteine-preferring). cDNAs encoding ASC-like transporters have been cloned and sequenced from a number of different sources, and these have been termed ASCT2 [57] or ATB0[810]. Although there are minor species differences in the amino acid specificity of these transporters, it is clear from sequence similarity that they are all closely related members of the same protein family now denoted as SLC1A5 (see [11] for a recent review of transporter nomenclature). The importance of glutamine transport in hepatoma cell proliferation has been demonstrated recently by Fuchs et al. [12], who showed that attenuation of ASCT2 expression by inducible antisense RNA in a number of human hepatoma cell lines led to cell death via apoptosis. In the present paper, it is shown that both the expression of the glutamine transporter ASCT2 and the activity of the ASCT2 promoter in the human hepatoma cell line HepG2 are dependent upon glutamine availability. == EXPERIMENTAL == == Cell culture == The human hepatoma cell line HepG2 and the monkey kidney cell line COS were maintained in DMEM (Dulbecco’s modified Eagle’s Rabbit Polyclonal to MMP-8 medium; Gibco BRL) supplemented with 10% FBS (foetal bovine serum; Gibco BRL), 100 units/ml penicillin G and 0.1 mg/ml streptomycin (Sigma). In some experiments, glutamine-free DMEM (Gibco BRL), leucine-free DMEM (ICN) or methionine-free DMEM (ICN) replaced the standard medium. In growth-rate assays, cells were grown in the appropriate medium for 72 h. Multiple cell counts using a haemocytometer were taken every 24 h for each condition. == Antibodies == In order to produce an ASCT2-specific antibody, the C-terminal peptide CPTGDSSATFEKESVM was synthesized and coupled to keyhole limpet haemocyanin via the terminal cysteine residue. The conjugate was injected into rabbits following a standard protocol, and the unfractionated serum was used in Western blotting experiments. The anti-calreticulin KRAS G12C inhibitor 16 antibody used in some experiments was produced to a GST (glutathione S-transferase)-fusion protein as described in [13]. The glutamate dehydrogenase antibody was produced by injecting the commercially available purified enzyme into rabbits. == Western blot analysis == Cells were trypsinized, sedimented and then solubilized in the detergentN-decanoylmethylglucamide (2% final concentration). Cell protein concentration was measured by the method of Bradford [14]. Each sample (30 g) was separated by SDS/PAGE and proteins were transferred on KRAS G12C inhibitor 16 to nitrocellulose, which was then blocked by incubation with 5% (w/v) dried skimmed milk. Blots were probed with the appropriate primary antibody, followed by a suitable anti-rabbit secondary antibody and developed by ECL (enhanced chemiluminescence; Amersham Biosciences). The intensity of bands were quantified using ImageQuant (Molecular Dynamics). == Verification of specificity of the anti-ASCT2 antibody == The KRAS G12C inhibitor 16 full-length H4-ASCT2 cDNA in the pGem vector [5] was excised byEcoRI digestion and subcloned into the mammalian expression vector pCI-neo (Promega). Clones were checked for the correct orientation by PCR using a sequence-specific forward primer and T3 as the reverse primer. COS cells were transfected with 2 g of the construct using FuGENE 6 (Roche) according to the manufacturer’s protocol. Cells were harvested at 24 h and 48 h time points for Western blot analysis. == Cloning of the HepG2 ASCT2 promoter region == Genomic DNA was isolated from HepG2 cells using the Nucleospin Tissue Kit (Clontech) according to the manufacturer’s instructions. The following.