Heavy chain ferritin enhances serine hydroxymethyltransferase expression and de novo thymidine biosynthesis.

We have elucidated a biochemical mechanism whereby changes in iron metabolism cause changes in folate-dependent one-carbon metabolism. Although animal and clinical studies have demonstrated that perturbations in iron status and metabolism alter folate metabolism, the biochemical mechanisms underlying these associations have yet to be identified. The effect of altered ferritin expression on folate metabolism was determined in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells expressing rat heavy chain ferritin (HCF) exhibited markedly increased expression of the folate-dependent enzyme cytoplasmic serine hydroxymethyltransferase (cSHMT). These effects were not seen when rat light chain ferritin was expressed. Additionally, cSHMT expression was not altered when HCF expression was induced in MCF-7 cells cultured with supplemental ferric citrate. This indicates that cSHMT expression is increased by elevated HCF concentrations, independent of increased iron availability, suggesting that cSHMT expression may respond to HCF-induced chelation of the regulatory iron pool. Increased HCF expression did not alter cSHMT mRNA levels, but did increase translation rates of cSHMT mRNA. The increase in translation was mediated, at least in part, through the cSHMT 5'-untranslated region of the transcript. MCF-7 cells with increased expression of cSHMT displayed increased efficiency of de novo thymidylate biosynthesis, indicating that thymidylate synthesis is normally limited by cSHMT activity in MCF-7 cells. Our data suggest that the iron regulatory pool may play an important role in regulating folate metabolism and thereby thymidine biosynthesis.

Purification and properties of a folate-catabolizing enzyme.

We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivo and may be an important factor in regulating intracellular folate concentrations.

Mimosine is a cell-specific antagonist of folate metabolism.

Iron deficiency and iron chelators are known to alter folate metabolism in mammals, but the underlying biochemical mechanisms have not been established. Although many studies have demonstrated that the iron chelators mimosine and deferoxamine inhibit DNA replication in mammalian cells, their mechanism of action remains controversial. The effects of mimosine on folate metabolism were investigated in human MCF-7 cells and SH-SY5Y neuroblastoma. Our findings indicate that mimosine is a folate antagonist and that its effects are cell-specific. MCF-7 cells cultured in the presence of 350 microm mimosine were growth-arrested, whereas mimosine had no effect on SH-SY5Y cell proliferation. Mimosine altered the distribution of folate cofactor forms in MCF-7 cells, indicating that mimosine targets folate metabolism. However, mimosine does not influence folate metabolism in SH-SY5Y neuroblastoma. The effect of mimosine on folate metabolism is associated with decreased cytoplasmic serine hydroxymethyltransferase (cSHMT) expression in MCF-7 cells but not in SH-SY5Y cells. MCF-7 cells exposed to mimosine for 24 h have a 95% reduction in cSHMT protein, and cSHMT promoter activity is reduced over 95%. Transcription of the cSHMT gene is also inhibited by deferoxamine in MCF-7 cells, indicating that mimosine inhibits cSHMT transcription by chelating iron. Analyses of mimosine-resistant MCF-7 cell lines demonstrate that although the effect of mimosine on cell cycle is independent of its effects on cSHMT expression, it inhibits both processes through a common regulatory mechanism.

Primary biliary cirrhosis shows association with genetic polymorphism of tumour necrosis factor alpha promoter region.

BACKGROUND/AIMS: Primary biliary cirrhosis is an autoimmune disease in which increased prevalence in first-degree relatives and an association with HLA DR8 suggest a genetic background. TNFalpha is a mediator of inflammation and immunity, and is implicated in the pathogenesis of primary biliary cirrhosis, ex vivo studies having shown reduced production of TNFalpha by lymphocytes from patients. Our group has previously described a biallelic promoter-region polymorphism of the TNFA gene at position -308, and demonstrated that the rare allele, TNF*2, has increased promoter function compared with the common allele, TNF*1. A further biallelic base change has been described in the TNFA gene at -238. We conducted a case-control study to assess association of these gene polymorphisms with primary biliary cirrhosis. METHODS: Ninety-one patients and 213 controls were genotyped for both TNFA loci using restriction fragment length polymorphism analysis of PCR products. RESULTS: The high production TNFA-308*2 allele was significantly under-represented among subjects with primary biliary cirrhosis (27.5% PBC, 41.6% controls, p=0.02, pc=0.04, OR for carriage of TNF*1/*1 genotype=1.89, CI=1.10-3.32). No association was shown with the TNFA -238 polymorphism. CONCLUSION: Primary biliary cirrhosis is associated with reduced carriage of TNF*2. This is in keeping with a protective role of TNFalpha against the disease.

Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene.

The human cytoplasmic serine hydroxymethyltransferase (CSHMT) gene was isolated, sequenced and its expression characterized in human MCF-7 mammary carcinoma and SH_5Y5Y neuroblastoma cells. The 23-kb gene contains 12 introns and 13 exons; all splice junctions conform to the gt/ag rule. The open reading frame is interrupted by 10 introns, two of which are positionally conserved within the human mitochondrial SHMT gene. The gene is expressed with 330 nucleotides of 5' untranslated message within three exons. The 5' promoter region does not contain a consensus TATA, and primer extension and 5'-RACE studies suggest that transcription initiation occurs at multiple sites. Consensus motifs for several regulatory proteins, including SP1, mammary and neuronal-specific elements, NF1, a Y-box, and two steroid hormone response elements, are present within the first 408 nucleotides of the 5' promoter region. The human gene is expressed as multiple splice variants in both the 5' untranslated region and within the open reading frame, all due to exon excision. The splicing pattern is cell-specific. At least six CSHMT mRNA splice forms are present in MCF-7 cells; the gene is expressed as a full-length message as well as splice forms that lack exon(s) 2, 9 and 10. In 5Y cells, the predominant form of the message lacks exon 2, which encodes part of the 5' untranslated region, but does not contain deletions within the open reading frame. Western analysis suggests that the CSHMT gene is expressed as a single full-length protein in 5Y cells, but as multiple forms in MCF-7 cells. Multiple tissue Northern blots suggest that the CSHMT message levels and alternative splicing patterns display tissue-specific variations.

Immune status of mice tolerant of living cells. III. Presence and evolution of cells cytotoxic to the tolerated strain.

Spleen cells from CBA mice neonatally rendered highly tolerant to A/Jax (keeping a skin graft in perfect shape for more than 1 year and without detectable hemagglutinating or cytotoxic antibodies) contain cells cytotoxic for YAC 222 (A/Jax) in Cr release assay. The degree of the cytotoxicity depends on the age of the mouse, following a curve lower than, but parallel to, the one followed by the cytotoxicity of cells from CBA rendered immune by injecting them with A/Jax cells 1 week previously. The maximum of the cytotoxicity curve is reached during the 9th and 10th weeks. Normal CBA cells themselves are moderately cytotoxic to YAC 222. This "natural" cytotoxicity, significantly less intense and presumably directed against Moloney virus-related determinants, does not follow the same time pattern. The cytotoxic indices from both immune and tolerant cell populations are significantly reduced by CBA and anti-A/Jax immune serum. The tested sera of the tolerant mice did not contain hemagglutinating or in vitro-blocking antibodies in the Cr release assay (only the sera from unsuccessfully treated mice, having rejected their skin grafts, had some degree of blocking activity). However (and in agreement with previous experiments), these sera often contained synergistic hemagglutinins and in vivo enhancing properties of A/Jax tumors (Sal) grafted on CBA recipients.