Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite.

High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancer cells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in the hepatocellular carcinoma cell line Huh7. Treatment with 20 µM of selenite reduced the viability of Huh7 cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold. Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxic effect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutral sphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 µM of SK1-II prior to treatment with 10 µM of selenite caused induction of apoptosis and gave rise to a 2.5-fold increase in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 µM of SK1-II combined with 10 µM of selenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides. The formation of reactive oxygen species (ROS) after treatment with 10 µM of selenite was maximally enhanced by 1 µM of SK1-II. Moreover, combined treatment with SK1-II and 10 µM of selenite synergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell line MIHA remained unaffected by the same treatment. These results raise the possibility that a combination of selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant, at doses that are non-toxic to normal liver cells.

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney.

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.

Thioredoxin reductase 1 is upregulated in atherosclerotic plaques: specific induction of the promoter in human macrophages by oxidized low-density lipoproteins.

Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.

The core promoter of human thioredoxin reductase 1: cloning, transcriptional activity, and Oct-1, Sp1, and Sp3 binding reveal a housekeeping-type promoter for the AU-rich element-regulated gene.

The selenoprotein thioredoxin reductase 1 (TrxR1) carries many vital antioxidant and redox regulatory functions. Its mRNA levels are known to be post-transcriptionally modulated via AUUUA motifs (AU-rich elements (AREs)), but the promoter yet remains unknown. Here we have cloned and determined the sequence of a 0.8-kilobase pair human genomic fragment containing the proximal promoter for TrxR1, which has transcriptional activity in several different cell types. The core promoter (-115 to +167) had an increased GC content and lacked TATA or CCAAT boxes. It contained a POU motif binding the Oct-1 transcription factor and two sites binding Sp1 and Sp3, which were identified with electrophoretic mobility shift assays using crude nuclear extracts of A549 cells. The TrxR1 promoter fulfills the typical criteria of a housekeeping gene. To our knowledge this is the first housekeeping-type promoter characterized for a gene with post-transcriptional regulation via ARE motifs generally possessed by transiently expressed proto-oncogenes, nuclear transcription factors, or cytokines and influencing mRNA stability in response to diverse exogenous factors. Expression of TrxR1 as an ARE-regulated housekeeping gene agrees with a role for the enzyme to maintain a balance between intracellular signaling via reactive oxygen species and protection of cells from excessive oxidative damage.

Prominent expression of the selenoprotein thioredoxin reductase in the medullary rays of the rat kidney and thioredoxin reductase mRNA variants differing at the 5' untranslated region.

The mammalian selenoprotein thioredoxin reductase is a central enzyme in protection against oxidative damage or the redox control of cell function. Previously a neuroblastoma-cell-derived 2193 bp cDNA for rat thioredoxin reductase 1 (TrxR1) was characterized [Zhong, Arnér, Ljung, Aslund and Holmgren (1998) J. Biol. Chem. 273, 8581-8591]. Here, the major rat TrxR1 mRNA was determined as 3.5 kb by Northern blotting. A corresponding full-length 3360 bp liver-derived cDNA was cloned and sequenced, being extended in the 3' untranslated region (3' UTR) compared with the previous clone. Among tissues examined, lowest TrxR1 mRNA levels were found in spleen and testis and highest in liver and kidney. High expression in kidney was unexpected and in situ hybridization of adult rat kidney was performed. This revealed a highly structured expression pattern with the mRNA being prominently synthesized in the proximal tubules of the medullary rays. Analysing rat kidney cDNA, a 5' UTR domain of TrxR1 was found that was different from that in liver- or neuroblastoma-derived cDNA clones. The kidney-derived 5' UTR mRNA domain was instead highly similar to kidney-derived cDNA variants of murine apolipoprotein E. By sequence determination of the rat genomic sequence upstream of the open reading frame for TrxR1, an exon was encountered that encoded a third alternative 5' UTR domain that could also be expressed, as confirmed by its presence in a mouse skin TrxR1 cDNA clone. It can therefore be concluded that TrxR1 mRNA is expressed in at least three different variants that differ at their 5' UTRs.

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability.

A method of lambda-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)6 tag, into the chromosome of a strain lacking tufB.