Biotinidase knockout mice show cellular energy deficit and altered carbon metabolism gene expression similar to that of nutritional biotin deprivation: clues for the pathogenesis in the human inherited disorder.

Biotin is the prosthetic group of carboxylases that have important roles in the metabolism of glucose, fatty acids and amino acids. Biotinidase has a key role in the reutilization of the biotin, catalyzing the hydrolysis of biocytin (ε-N-biotinyl-l-lysine) and biocytin-containing peptides derived from carboxylase turnover, thus contributing substantially to the bioavailability of this vitamin. Deficient activity of biotinidase causes late-onset multiple carboxylase in humans, whose pathogenic mechanisms are poorly understood. Here we show that a knock-out biotinidase-deficient mouse from a C57BL/6 background that was fed a low biotin diet develops severe ATP deficit with activation of the energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK), inhibition of the signaling protein mTOR, driver of protein synthesis and growth, and affecting the expression of central-carbon metabolism genes. In addition, sensitivity to insulin is augmented. These changes are similar to those observed in nutritionally biotin-starved rats. These findings further our understanding of the pathogenesis of human biotinidase deficiency.

A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analyses, using BRCA1 as an example.

BACKGROUND: The detection of 2 recurrent mutations in the BRCA1 gene (Ashkenazi Jewish and Dutch populations) was studied with real-time polymerase chain reaction (PCR) and melting curve analyses. METHODS: PCR products were designed around the 185delAG in exon 2 and the single- base substitution 2841G>T in exon 11. Hybridization probe sets were designed for both PCR products, with each probe overlapping the specific mutation. The exon 11 probe set also covered another mutation, the 2814insA. The 39 end of the 59 probe was labeled with fluorescein isothiocyanate and the 59 end of the 39 probe with LightCycler Red 640 (Roche Diagnostics, Indianapolis, IN). RESULTS: The 185del and 2841G mutations were easily detected with the hybridization probes, resulting in dual peaks for heterozygotes in melting curve analyses. The differences in melting characteristics of the heteroduplexes in heterozygotes were not detectable with SYBR Green I. CONCLUSION: For known mutations, melting curve analyses using hybridization-specific probes provide a sensitive, rapid, and efficient approach to mutation detection.

TNF-alpha gene therapy with myeloid progenitor cells lacks the toxicities of systemic TNF-alpha therapy.

We examined the antileukemic activity and the toxicity of HPC transduced with human tumor necrosis factor (TNF) cDNA. Both clonal (32Dcl3) and BM-derived primary hematopoietic progenitors (BM-Prog) expressing hTNF-alpha gene (32DTNF-alpha and BMTNF-alpha cells, respectively) inhibited the development of leukemia in mice with a small dose of 32Dp210 cells, a myeloid leukemia cell line. Whether the trans-gene expressing 32DTNF-alpha cells produce toxicities commonly associated with systemic TNF-alpha therapy was determined by examining the effect of TNF-alpha-secreting progenitor cells on body weight, tissue histology, growth of HPC, and engraftment of BMT. Administration of a low or high dose of TNF-alpha-secreting 32DTNF-alpha cells to mice failed to produce loss in body weight, a measure of TNF-alpha-related cachexia. There was also no evidence of tissue necrosis or mononuclear cell (MNC) infiltration in lung, liver, kidney, or intestine of mice injected with transduced progenitor cells. Furthermore, 32DTNF-alpha cells showed no effect on the clonal growth of HPC in colony-forming assays or loss of cellularity in BM, spleen, or blood. Finally, TNF-alpha-secreting cells were found not to interfere with the engraftment of BM transplant and hematopoietic reconstitution thereafter. We conclude from these findings that unlike systemic administration of TNF-alpha, TNF-alpha gene therapy with transduced HPC is nontoxic and may have a role in eradicating residual leukemia after BMT.

Enhanced assessment of allogeneic bone marrow transplant engraftment using automated fluorescent-based typing.

Traditional qualitative gel electrophoresis approaches lack accurate' and quantitative assessment of mixed chimerism in BMT patients. The likelihood of informative markers is greatly increased using simultaneous amplification of 10 highly polymorphic loci with fluorescent-labeled primers in an automated DNA sequencer. This allows for more precise interpretation of mixed chimerism with a detection level approximating 1%. To evaluate this approach to quantitative assessment of chimeric populations we mixed varying proportions of samples from two unrelated donors, by either mixing aliquots of DNA isolated from whole blood, or by first counting the white blood cells and mixing varying proportions of cells together prior to DNA isolation. The allelic-peak area ratios were identical to allelic-peak height ratios and corresponded to the proportion of mixed DNA, regardless of the method used to create the mixture. Formulas to provide routine, consistent and quantitative interpretation of mixed chimerism are presented. We analyzed 14 allograft recipients and one autologous BMT patient with transfusion-induced GVHD. In all cases, at least four out of nine markers were informative. Inter-laboratory concordance of results was also obtained with an eight marker panel using an automated Alf-Express. In conclusion, the automated DNA fluorescent-labeled primer approach using an eight to 10 marker panel is quantitative and informative in assessing chimerism.

Nonselective inhibition of proliferation of transformed and nontransformed cells by the anticancer agent curcumin (diferuloylmethane).

We have investigated the antiproliferative effect of curcumin, an antitumor agent with antioxidant and anti-inflammatory properties, against a variety of transformed and nontransformed cell types. At equimolar concentrations ranging from 6.25 to 50 microM, curcumin inhibited DNA synthesis, as revealed by 3H-incorporation, in five leukemia lines, three nontransformed hematopoietic progenitor cell populations, and four nontransformed fibroblastic cell lines in a concentration-dependent manner. Curcumin also inhibited the cellular growth of both transformed and nontransformed cells in clonogenic assays. Without discriminating between transformed or nontransformed cells, the inhibition of cell proliferation by curcumin was not always associated with programmed cell death. These findings have implications for developing curcumin-based anticancer and anti-inflammation therapies.

Antileukemic activity of TNF-alpha gene therapy with myeloid progenitor cells against minimal leukemia.

Tumor necrosis factor-alpha (TNF-alpha) has exhibited antitumor activity against a variety of tumors in rodents and human tumor xenografts in nude mice, but it has been only marginally effective in cancer patients because of dose-limiting toxicity associated with systemic TNF-alpha therapy. To circumvent toxicity and to test the antileukemic activity against quantitated minimal leukemia, we have cloned human TNF-alpha (HuTNF-alpha) gene in an advanced myeloid progenitor cell line. 32Dcl3 myeloid progenitor cells transfected with HuTNF-alpha cDNA by the retroviral supernatant infection method stably express HuTNF-alpha gene and secrete substantial amounts of HuTNF-alpha. When injected i.v. into irradiated mice, transduced cells could be detected in the marrow but not in spleen or liver 10-12 days later. Injection of 5 x 10(6) transduced cells produced no obvious symptoms of TNF-alpha toxicity (i.e., weight loss, cachexia, or fever) suggesting that TNF-alpha producing cells are well tolerated by the recipient mice. Coinjection of 5 x 10(6) transduced cells and 10(2) or 10(3) 32Dp210 leukemia (BCR/ABL+) cells resulted in inhibition of leukemia development by 10(2) but not 10(3) 32Dp210 cells. An equal dose of nontransduced 32Dcl3 cells was ineffective in inhibiting leukemia progression by 10(2) 32Dp210 cells. Mice that rejected leukemia were BCR/ABL oncogene negative 8 weeks after leukemia cell injection. These data demonstrate the potential for TNF-alpha gene therapy for destroying residual leukemia, without the toxicity of systemic TNF-alpha therapy, following cytoreductive therapy and bone marrow transplant.

IL1α and TNFα Induction of Monocyte Chemoattractant Protein-1 (MCP-1/JE) in Bone Marrow Stromal Cells: The Role of Protein Kinase C (PKC) and Protein Tyrosine Kinase (PTK) Activity.

Chemotactic cytokines (chemokines) have been shown to influence myelopoiesis. Bone marrow stromal cell line +/+-1. LDA11 expresses MCP-1/JE chemokine upon stimulation with ILlα and TNFα. We have examined the role of PKC and PTK dependent protein phosphorylation in induction of MCP-1/JE by using PKC and PTK specific inhibitors. PKC inhibitors staurosporine and H-7, as well as PTK inhibitors herbimycin A and genistein suppressed MCP-1/JE expression (mRNA and protein) in a dose dependent manner. The suppression of MCP-1/JE by both classes of inhibitors was partially to completely reversible. While PKC only regulated gene expression posttranscriptionally (mRNA stability), transcription of MCP-l/JE gene by ILlα and TNFα depends both upon PKC and PTK activity, as demonstrated by nuclear run-on analyses. Furthermore, treatment of cells with IL1a and TNFα involved NF-kB mobilization. There was no effect of PKC inhibitors on NF-kB mobilization by either ILlα or TNFα. In contrast, mobilization of NF-kB was negatively affected by PTK inhibitors in a stimulus selective manner (e.g., herbimycin A and genistein inhibited IL1α and TNFα induced NF-kB mobilization, respectively). We conclude from these findings that while both PKC and PTK inhibitors suppress MCP-1/JE gene transcription, only PTK inhibitors do so by suppressing NF-kB activation.

Curcumin, a compound with anti-inflammatory and anti-oxidant properties, down-regulates chemokine expression in bone marrow stromal cells.

Chemotactic cytokines or chemokines play an important role in the regulation of myelopoiesis. Since the production of chemokines and colony stimulating factors (CSFs) by bone marrow stromal cells requires inflammatory conditions, we investigated the effect of curcumin, an agent with anti-inflammatory and anti-oxidant activities, on the expression of monocyte chemoattractant protein-1 (MCP-1 or MCP-1/JE) and interferon inducible protein-10kD (IP-10) in mouse bone marrow stromal cell line +/+-1.LDA11. Both chemokines are readily expressed in stromal cells after stimulation with pro-inflammatory interleukin-1alpha (IL-1alpha), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and endotoxin lipopolysaccharide (LPS). Curcumin attenuates the levels of MCP-1/JE and IP-10 mRNA expression by all of these stimulatory agents. A detailed analysis of the regulatory effects of curcumin on chemokine expression by IL-1alpha was performed. Curcumin inhibits both chemokine mRNAs in a dose- and time-dependent manner. The suppressive effect of curcumin on both mRNAs is reversible with complete recovery from suppression within 24 hours after removal of curcumin. The suppression of mRNA by curcumin is dependent on de novo synthesis of an intermediary protein(s), since suppression is abrogated by concomitant treatment with cycloheximide (CHX). Destabilization of mRNA transcripts is not the mechanism by which curcumin lowers the levels of mRNA; however, transcripts formed in the presence of curcumin are more stable, as indicated by their slower degradation kinetics. Run-on transcriptional assays demonstrate that curcumin inhibits the transcriptional activity of both genes. Finally, the attenuation of chemokine gene expression is associated with decreased production of chemotactic activity. Together, these findings indicate that while curcumin may post-transcriptionally stabilize mRNA transcripts formed in its presence, the overall reduction in mRNA levels by curcumin is mediated by inhibition of the transcription of chemokine genes.

Curcumin inhibits IL1 alpha and TNF-alpha induction of AP-1 and NF-kB DNA-binding activity in bone marrow stromal cells.

We have previously demonstrated that anti-inflammatory and antioxidant compound curcumin (diferuloyl-methane) inhibits the expression of monocyte chemoattractant protein-1 (MCP-1/JE) in bone marrow stromal cells by suppressing the transcriptional activity of the MCP-1/JE gene. Since both AP-1 (TRE) and NF-kB (kB) binding motifs are present in the promoter of MCP-1/JE gene, we examined the effect of curcumin on IL1 alpha- and TNF-alpha-induced activation of ubiquitous transcription factors AP-1 and NF-kB by electrophoretic mobility shift assay and Western blotting. IL1 alpha and TNF-alpha rapidly induced both AP-1 and NF-kB DNA binding activities in +/+(-)1.LDA11 stromal cells. However, treatment of these cells with curcumin blocked the activation of AP-1 and NF-kB by both cytokines. These data suggest that inhibition of MCP-1/JE transcription by curcumin involves blocking of AP-1 and NF-kB activation by IL1 alpha or TNF-alpha.

IL-4 upregulates IL-1-induced chemokine gene expression in bone marrow stromal cells by enhancing NF-kB activation.

Bone marrow stromal cells play a critical role in the proliferation and differentiation of hematopoietic stem and progenitor cells by secreting numerous hematopoietic growth factors and colony-stimulating factors (CSFs). We have previously reported that monocyte chemotactic protein-1 (MCP-1 or MCP-1/JE) and interferon-inducible protein 10 KD (IP-10) are both induced in murine bone marrow stromal cell line +/(+)-1.LDA11 upon stimulation with various inflammatory agents, including IL-1 alpha, IFN-gamma, TNF-alpha, or LPS. In addition, the expression of MCP-1/JE and IP-10 mRNA by these inducers is potentiated by IL-4 and TGF-beta 1. In the present study we have investigated the mechanism of IL-4-mediated upregulation of MCP-1/JE gene expression. Our results of nuclear run-on experiments show that IL-4 enhances the IL-1-induced transcription of MCP-1/JE gene. Because the transcription of genes is regulated by DNA binding nuclear factors and binding sites for transcription factors AP-1 and SP-1, and NF-kB in the enhancer region of MCP-1/JE have been demonstrated, we examined the effect of IL-4 on the levels of these factors in stromal cells stimulated with IL-1. Whereas AP-1 and SP-1 are constitutively expressed in stromal cells, NF-kB is detected only after stimulation with IL-1. Furthermore, while unable to induce the activation of NF-kB alone, IL-4 enhanced the activation of NF-kB by IL-1. Taken together, these data suggest that upregulation of NF-kB may be the mechanism by which IL-4 increases the transcription of MCP-1/JE gene resulting in overabundance of the chemokine mRNA.

Chemokine gene expression in bone marrow stromal cells: downregulation with sodium salicylate.

Chemotactic cytokines, chemokines, have been shown to influence the proliferation of hematopoietic progenitor cells. Thus, regulation of chemokine production by bone marrow accessory cells is a critical aspect of stromal cell regulation of hematopoiesis. We have previously reported that monocyte chemotactic protein-1 (MCP-1 or MCP-1/JE) and interferon inducible protein 10 kD (IP-10) are both induced in murine bone marrow stromal cells +/(+)-1.LDA11 after stimulation with the inflammatory agents interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), or lipopolysaccharide (LPS). In the present study, we have investigated the effect of sodium salicylate, an antiinflammatory agent, on the IL-1 alpha-induced expression of MCP-1/JE and IP-10 genes in stromal cells. Sodium salicylate attenuates the levels of MCP-1/JE and IP-10 mRNA in a concentration- and time-dependent manner. The suppression of MCP-1/JE mRNA is reversible, whereas IP-10 mRNA expression is more or less irreversibly affected as its recovery from the effect of sodium salicylate is slow and partial. Sodium salicylate-mediated suppression of mRNA expression is attributable neither to de novo synthesis of intermediary protein(s) nor to the destabilization of mature mRNA transcripts. On the other hand, sodium salicylate downregulates the transcriptional activity of both genes. Furthermore, IL-1 alpha induces activation of transcription factor nuclear factor (NF)-kB, and sodium salicylate suppresses it in a dose-dependent manner. We conclude that while posttranscriptional events remain unaffected, inhibition of NF-kB activation by sodium salicylate may account for the suppression of chemokine gene expression at the transcriptional level.

Induction of chemokine mRNA in bone marrow stromal cells: modulation by TGF-beta 1 and IL-4.

Bone marrow stromal cells produce cytokines that are essential for the proliferation and differentiation of hematopoietic stem and progenitor cells. Thus, regulation of cytokine production by bone marrow accessory cells is a critical aspect of stromal cell regulation of hematopoiesis. We have investigated the effect of two cytokines that have been demonstrated to modulate factor production by non-marrow accessory cells (i.e., transforming growth factor-beta 1 [TGF-beta 1] and interleukin-4 [IL-4]) on the induced expression of cytokine mRNA in a bone marrow-derived, cloned, murine stromal cell line +/+/-.LDA11. We showed that +/+/-.LDA11 cells can be induced with lipopolysaccharide (LPS), IL-1 alpha, or interferon-gamma (IFN-gamma) to express mRNA for monocyte chemoattractant protein-1 (MCP-1/JE), IFN-inducible protein-10 (IP-10), stem cell factor (SCF), and macrophage colony-stimulating factor (M-CSF) but not for IL-1 alpha, IL-3, or tumor necrosis factor-alpha (TNF-alpha). The expression of MCP-1/JE and IP-10 mRNA by these inducers was potentiated by TGF-beta 1 and IL-4. The augmentation by TGF-beta 1 of both mRNAs induced with IL-1 alpha was maximum when applied to the cells concurrently with the inducer; the IFN-gamma-induced expression of mRNAs was augmented even if the addition of TGF-beta 1 was delayed. Similarly, IL-4 potentiation of both mRNAs by either inducer progressively increased as the time between exposure to the inducer and exposure to IL-4 increased. Neither modulator altered the time course of mRNA expression by either inducer. TGF-beta 1- and IL-4-mediated augmentation of MCP-1/JE mRNA by IL-1 alpha or IFN-gamma was partially reversed by cycloheximide (CHX), whereas potentiation of IP-10 by either modulator remained unaffected. Increase in the stability of mRNA transcripts by TGF-beta 1 or IL-4 does not appear to play a role in the enhanced accumulation of mRNA in the presence of the modulators. These findings support a role for TGF-beta 1 and IL-4 as critical regulatory molecules in production of MCP-1 and IP-10 chemokines by stromal cells.