Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.

The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 µg/g and maximal expression at approximately 400 µg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 µg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Myofiber Damage Evaluation by Evans Blue Dye Injection.

Evans blue dye (EBD) can be used in live mice to study muscle pathology or injury, including exercise-induced muscle damage. EBD is excluded from intact cell membranes but leaks into cells, including muscle fibers, when the cell membrane is ruptured. EBD can be visualized by its autofluorescence under a fluorescence microscope. EBD-stained myofibers can be quantified from microscope images of muscle cross-sections. These myofibers are often in clusters that lend themselves to morphometric analysis. When the damaged myofibers are interspersed among intact myofibers, however, a more suitable approach is to count individual myofibers in the field of view. A much faster approach to measure EBD in muscles from different strains of mice or between treatment groups is to extract the EBD from muscle samples and quantitate it using a spectrophotometric microplate reader. The advantages and disadvantages of using each of these approaches are discussed here. Curr. Protoc. Mouse Biol. 1:463-488 © 2011 by John Wiley & Sons, Inc.

Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD(+) muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra- and interlimb amounts of EBD in 2-7-month-old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse.

Functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection.

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

K12-biotinylated histone H4 marks heterochromatin in human lymphoblastoma cells.

Covalent modifications of histones play crucial roles in chromatin structure and genomic stability. Recently, we reported a novel modification of histones: biotinylation of lysine residues. Here we provide evidence that K12-biotinylated histone H4 (K12Bio H4) maps specifically to both heterochromatin (alpha satellite repeats in pericentromeric regions) and transcriptionally repressed chromatin (gamma-G globin and interleukin-2) in human lymphoblastoma cells. The abundance of K12Bio H4 in these regions was similar to that of K9-dimethylated histone H3, a known marker for heterochromatin. Likewise, K8-biotinylated histone H4 (K8Bio H4) mapped to heterochromatin, but the relative enrichment was smaller compared with K12Bio H4. Stimulation of interleukin-2 transcriptional activity with phorbol-12-myristate-13-acetate and phytohemagglutinin caused a rapid depletion of K12Bio H4 in the gene promoter. These data are consistent with a novel role for biotin in chromatin structure and transcriptional activity of genes.

The expression of genes encoding ribosomal subunits and eukaryotic translation initiation factor 5A depends on biotin and bisnorbiotin in HepG2 cells.

Biotin affects gene expression at both the transcriptional and the posttranscriptional level; biotin metabolites might have biotin-like activities with regard to gene expression. Here, human hepatocarcinoma (HepG2) cells were used (i) to identify clusters of biotin-dependent genes, (ii) to determine whether the naturally occurring metabolite bisnorbiotin affects gene expression and (iii) to determine whether biotin and bisnorbiotin affect the expression of genes coding for ribosomal subunits and translation initiation factors. HepG2 cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L, control) and pharmacological (10 nmol/L) concentrations of biotin; a fourth treatment group consisted of cells cultured in biotin-deficient medium (0.025 nmol/L) supplemented with bisnorbiotin (0.225 nmol/L). Gene expression was quantified by using DNA microarrays and reverse transcriptase polymerase chain reaction. The expression of 1803 genes depended on biotin concentrations in culture media; the expression of 618 genes depended on bisnorbiotin. Biotin deficiency was associated with increased expression of a gene cluster encoding ribosomal subunits and eukaryotic translation initiation factor 5A; this effect was reversed by supplementation with biotin and bisnorbiotin. Additional prominent clusters of (bisnor)biotin-dependent genes included DNA-, RNA-, and nucleotide-binding proteins, consistent with a role for biotin in cell signaling and gene expression. Collectively, these data suggest that bisnorbiotin has biotin-like activities regarding gene expression, and that clusters of (bisnor)biotin-dependent genes include genes that play roles in translational activity.

Biotin supplementation decreases the expression of the SERCA3 gene (ATP2A3) in Jurkat cells, thus, triggering unfolded protein response.

Protein folding in the endoplasmic reticulum (ER) depends on Ca(2+); uptake of Ca(2+) into the ER is mediated by sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3). The 5'-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells were cultured in media containing 0.025 nmol/L biotin (denoted "deficient") or 10 nmol/L biotin ("supplemented"). The transcriptional activity of the full-length human SERCA3 promoter was 50% lower in biotin-supplemented cells compared to biotin-deficient cells. Biotin-dependent repressors bind to elements located 731-1312 bp upstream from the transcription start site in the SERCA3 gene. The following suggest that low expression of SERCA3 in biotin-supplemented cells impaired folding of secretory proteins in the ER, triggering unfolded protein response: (i) sequestration of Ca(2+) in the ER decreased by 14-24% in response to biotin supplementation; (ii) secretion of interleukin-2 into the extracellular space decreased by 75% in response to biotin supplementation; (iii) the nuclear abundance of stress-induced transcription factors increased in response to biotin supplementation; and (iv) the abundance of stress-related proteins such ubiquitin activating enzyme 1, growth arrest and DNA damage 153 gene, X-box binding protein 1 and phosphorylated eukaryotic translation initiation factor 2alpha increased in response to biotin supplementation. Collectively, this study suggests that supplements containing pharmacological doses of biotin may cause cell stress by impairing protein folding in the ER.

HepG2 cells develop signs of riboflavin deficiency within 4 days of culture in riboflavin-deficient medium.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential coenzymes in redox reactions. For example, FAD is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/l) medium for up to 6 days; controls were cultured in riboflavin-sufficient (532 nmol/l) medium. The activity of glutathione reductase decreased by 98% within 4 days of riboflavin-deficient culture. Transport rates of riboflavin increased in response to riboflavin depletion, whereas expression of enzymes mediating flavocoenzyme synthesis (flavokinase and FAD synthetase) decreased in response to depletion. The oxidative folding and synthesis of plasminogen and apolipoprotein B-100 was impaired within 4 days of culture in riboflavin-deficient medium; this is consistent with impaired processing of secretory proteins in riboflavin-deficient cells. Riboflavin depletion was associated with increased DNA-binding activities of transcription factors with affinity for endoplasmic reticulum stress elements and nuclear factor kappaB (NF-kappaB) consensus elements, suggesting cell stress. Moreover, the abundance of the stress-induced protein GADD153 was greater in riboflavin-deficient cells compared with controls. Riboflavin deficiency was associated with decreased rates of cell proliferation caused by arrest in G1 phase of the cell cycle. These studies are consistent with the hypothesis that HepG2 cells have a great demand for riboflavin and that cell stress develops rapidly if riboflavin supply is marginally low.

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase.

Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

High-throughput immunoblotting identifies biotin-dependent signaling proteins in HepG2 hepatocarcinoma cells.

Biotin affects the abundance of mRNA coding for approximately 10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.025 nmol/L (denoted "deficient"), 0.25 nmol/L ("physiological" = control), and 10 nmol/L ("pharmacological") for 10 d before harvesting. The abundance of 1009 proteins from whole-cell extracts was quantified by using high-throughput immunoblots. The abundance of 44 proteins changed by at least 25% in biotin-deficient and biotin-supplemented cells compared with physiological controls. One third of these proteins participate in cell signaling. Specifically, proteins associated with receptor tyrosine kinase-mediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells than in controls. This was associated with increased DNA-binding activities of the transcription factors Fos and Jun, and increased expression of a reporter gene driven by activator protein (AP)1-binding elements in biotin-deficient cells compared with physiological controls. The abundance of selected signaling proteins was not paralleled by the abundance of mRNA, suggesting that biotin affects expression of these genes at a post-transcriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. This study is consistent with the existence of clusters of biotin-responsive proteins in distinct biological processes, including signaling by Fos/Jun; the latter might mediate the proinflammatory and antiapoptotic effects of biotin deficiency.

Biological functions of biotinylated histones.

Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase. Here we describe a novel peptide-based technique, which was used to identify eight distinct biotinylation sites in histones H2A, H3 and H4. Biotinylation site-specific antibodies were generated to investigate biological functions of histone biotinylation. Evidence was provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage.

Roles for nutrients in epigenetic events.

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA damage. Third, tryptophan and niacin are converted to nicotinamide adenine dinucleotide, which is a substrate for poly(ADP-ribosylation) of histones and other DNA-binding proteins; poly(ADP-ribosylation) of these proteins participates in DNA repair and apoptosis. Here we present a novel procedure to map nutrient-dependent epigenetic marks in the entire genomes of any given species: the combined use of chromatin immunoprecipitation assays and DNA microarrays. This procedure is also an excellent tool to map the enzymes that mediate modifications of DNA and DNA-binding proteins in chromatin. Given the tremendous opportunities offered by the combined use of chromatin immunoprecipitation assays and DNA microarrays, the nutrition community can expect seeing a surge of information related to roles for nutrients in epigenetic events.

Biotin deficiency stimulates survival pathways in human lymphoma cells exposed to antineoplastic drugs.

Cells may respond to nutrient deficiency or death signals with nuclear translocation of the transcription factor nuclear factor kappaB (NF-kappaB), which activates transcription of anti-apoptotic genes. Here we tested the hypothesis that biotin deficiency stimulates NF-kappaB-dependent survival pathways in human lymphoma cells, enhancing resistance to antineoplastic agents. Lymphoma (Jurkat) cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. If cells were treated with antineoplastic agents (taxol, doxorubicin or vinblastine), nuclear translocation of two NF-kappaB proteins (p50 and p65) was >25% greater in biotin-deficient compared with biotin-supplemented cells. The transcriptional activities of the following NF-kappaB-dependent reporter genes were 16-59% greater in biotin-deficient compared with biotin-supplemented cells treated with various antineoplastic agents: (1) reporter expression driven by a TATA box and five NF-kappaB repeats and (2) reporter expression driven by the regulatory region of the anti-apoptotic Bfl-1 gene. Collectively, these findings are consistent with activation of survival pathways in biotin-deficient lymphoma cells. Finally, cells were treated with antineoplastic agents for 48 h and cell survival was monitored at timed intervals. Biotin deficiency was associated with enhanced survival of cells treated with doxorubicin and vinblastine, but did not affect survival of cells treated with taxol. Collectively, these observations suggest that biotin deficiency may enhance resistance of cancer cells to antineoplastic agents.

Biotin supplementation increases expression of the cytochrome P450 1B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks.

DNA microarray studies provided evidence that biotin supplementation increases the abundance of mRNA encoding cytochrome P(450) 1B1 (CYP1B1) in human lymphocytes. CYP1B1 hydroxylates procarcinogens, generating electrophilic mutagens. Here, we sought to identify the signaling pathways that increase the expression of CYP1B1 in biotin-supplemented human T (Jurkat) cells and to determine whether activation of the CYP1B1 gene is associated with increased occurrence of single-stranded DNA breaks. Jurkat cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media. The transcriptional activity of a CYP1B1 reporter gene construct was 24% greater in biotin-supplemented compared with biotin-deficient cells (P < 0.01). Similarly, the abundance of CYP1B1 mRNA was 72% greater in biotin-supplemented than in biotin-deficient cells (P < 0.05). Electrophoretic mobility shift assays suggested that Sp1 sites in the regulatory region of the CYP1B1 gene play important roles in transcriptional activation by biotin. The abundance of CYP1B1 protein and activity of CYP1B1 were 124 and 35% greater, respectively, in biotin-supplemented compared with biotin-deficient cells (P < 0.05). The increased expression of CYP1B1 in biotin-supplemented cells was associated with an increase in the occurrence of single-stranded DNA breaks compared with biotin-deficient cells; synthetic inhibitors of CYP1B1 prevented strand breaks, suggesting that the effects of biotin were specific for CYP1B1. These studies provide evidence that transcription factors with an affinity for Sp1 sites mediate transcriptional activation of the CYP1B1 gene in biotin-supplemented T cells, increasing the occurrence of single-stranded DNA breaks.

Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells.

BACKGROUND: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development. AIM OF THE STUDY: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells. METHODS: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified. RESULTS: Biotin concentrations in culture media correlated negatively with expression of the biotin transporter SMVT, as judged by cellular transport rates of biotin, abundance of SMVT protein, and transcriptional activity of SMVT reporter-gene constructs. Notwithstanding this homeostatic mechanism, biotin concentrations in media correlated positively with activities of biotin-dependent propionyl-CoA carboxylase, abundance of biotinylated carboxylases, and with biotinylation of histones. Biotin deficiency was associated with decreased rates of thymidine uptake into JAr cells [pmol thymidine/( 10(6) cells x 24 h)]: 1.6 +/- 0.1 (25 pmol/L biotin) versus 2.3 +/- 0.2 (250 pmol/L biotin) versus 3.7 +/- 0.4 (10,000 pmol/L biotin), suggesting that cell proliferation depends on biotin. Secretion of progesterone was reduced in biotin-deficient cells; this effect was caused by reduced generation of new cells in deficient media rather than by an immediate effect of biotin on progesterone secretion at the singlecell-level. CONCLUSIONS: This study provides evidence that choriocarcinoma cells cannot maintain normal activities of biotin-dependent metabolic pathways if biotin concentrations in culture media are low. It is uncertain whether activities of biotin-dependent pathways in placenta affect fetal development in vivo.

The nuclear abundance of transcription factors Sp1 and Sp3 depends on biotin in Jurkat cells.

Biotin affects gene expression in mammals; however, the signaling pathways leading to biotin-dependent transcriptional activation and inactivation of genes are largely unknown. Members of the Sp/Krüppel-like factor family of transcription factors (e.g., the ubiquitous Sp1 and Sp3) play important roles in the expression of numerous mammalian genes. We tested the hypothesis that the nuclear abundance of Sp1 and Sp3 depends on biotin in human T cells (Jurkat cells) mediating biotin-dependent gene expression. Jurkat cells were cultured in biotin-deficient (0.025 nmol/L) and biotin-supplemented (10 nmol/L) media for 5 wk prior to transcription factor analysis. The association of Sp1 and Sp3 with DNA-binding sites (GC box and CACCC box) was 76-149% greater in nuclear extracts from biotin-supplemented cells compared with biotin-deficient cells, as determined by electrophoretic mobility shift assays. The increased DNA-binding activity observed in biotin-supplemented cells was caused by increased transcription of genes encoding Sp1 and Sp3, as shown by mRNA levels and reporter-gene activities; increased transcription of Sp1 and Sp3 genes was associated with the increased abundance of Sp1 and Sp3 protein in nuclei. Notwithstanding the important role for phosphorylation of Sp1 and Sp3 in regulating DNA-binding activity, the present study suggests that the effects of biotin on phosphorylation of Sp1 and Sp3 are minor. The increased nuclear abundance of Sp1 and Sp3 in biotin-supplemented cells was associated with increased transcriptional activity of 5'-flanking regions in Sp1/Sp3-dependent genes in reporter-gene assays. This study provides evidence that some effects of biotin on gene expression might be mediated by the nuclear abundance of Sp1 and Sp3.

Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells.

Here the hypothesis was tested that monocarboxylate transporters (MCT) mediate biotin transport in human lymphoid cells. Uptake of [(3)H]biotin was measured in human lymphoid cells [peripheral blood mononuclear cells (PBMC) and Jurkat cells] under conditions known to affect MCT-mediated transport. When biotin uptake into PBMC was measured in the presence of excess concentrations of competitors (MCT substrates) and MCT inhibitors, transport rates decreased significantly to <75 and <67%, respectively, of controls. Biotin uptake correlated with the concentration of protons in culture media, consistent with cotransport of protons and the carboxylate biotin by MCT. Efflux of biotin from PBMC was stimulated by extracellular lactate (a known substrate for MCT), consistent with countertransport of the two substrates by the same transporter. PBMC responded to proliferation with parallel increases of transport rates for both biotin and lactate, providing circumstantial evidence that the same transporter mediates uptake of the two substrates in PBMC. Transfection of Jurkat cells with an expression vector encoding MCT1 caused a 50% increase in biotin uptake; in contrast, overexpression of MCT1 did not affect biotin uptake in various nonlymphoid cell lines. These findings are consistent with the hypothesis that MCT mediate biotin uptake in human lymphoid cells.

Interleukin-2 receptor-gamma -dependent endocytosis depends on biotin in Jurkat cells.

Biotin has been credited with having beneficial effects on immune function despite observations that biotin supplementation causes decreased secretion of interleukin-2. Here this paradox was addressed by determining whether receptor-dependent internalization of interleukin-2 by immune cells depends on biotin. Theoretically, this would be consistent with both decreased net secretion of interleukin-2 by biotin-supplemented cells (causing increased endocytosis) and beneficial effects of biotin on immune function (causing increased receptor signaling). Jurkat cells were cultured in biotin-defined media (25, 250, or 10,000 pM). Secretion of interleukin-2 correlated negatively with biotin supply, but transcriptional activity of the interleukin-2 gene correlated positively with biotin supply, suggesting that decreased secretion of interleukin-2 by biotin-supplemented cells was not caused by decreased gene expression. Expression of the interleukin-2 receptor-gamma gene was greater at 10,000 pM than 25 pM biotin, mediating increased endocytosis of interleukin-2 in biotin-supplemented medium. Inhibition of endocytosis by genistein and overexpression of interleukin-2 receptor-gamma abolished the effect of biotin. These findings suggest that endocytosis of interleukin-2 depends on biotin.