Glucose-6-phosphate dehydrogenase correlates with tumor immune activity and programmed death ligand-1 expression in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare and highly malignant skin cancer. Some cases have a good prognosis and spontaneous regression can occur. Reported prognostic markers, such as Merkel cell polyoma virus infection or programmed death ligand-1 (PD-L1) expression, remain insufficient for precisely estimating the vastly different patient outcomes. We performed RNA sequencing to evaluate the immune response and comprehensively estimate prognostic values of immunogenic factors in patients with MCC. METHODS: We collected 90 specimens from 71 patients and 53 blood serum samples from 21 patients with MCC at 10 facilities. The mRNA was extracted from formalin-fixed paraffin-embedded tissues. Next-generation sequencing, immunohistochemical staining and blood serum tests were performed. RESULTS: Next-generation sequencing results classified MCC samples into two types: the 'immune active type' was associated with better clinical outcomes than the 'cell division type'. Expression of the glucose-6-phosphate dehydrogenase (G6PD) gene was highly significantly upregulated in the 'cell division type'. Among 395 genes, G6PD expression correlated with the presence of lymph node or distant metastases during the disease course and significantly negatively correlated with PD-L1 expression. Immunohistochemical staining of G6PD also correlated with disease-specific survival and exhibited less heterogeneity compared with PD-L1 expression. G6PD activity could be measured by a blood serum test. The detection values significantly increased as the cancer stage progressed and significantly decreased after treatment. CONCLUSIONS: G6PD expression was an immunohistochemically and serum-detectable prognostic marker that negatively correlated with immune activity and PD-L1 levels, and could be used to predict the immunotherapy response.

A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway.

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.

Lambda phage nanoparticles displaying HER2-derived E75 peptide induce effective E75-CD8+ T response.

We have investigated the in vitro immunogenicity and in vivo prophylactic and therapeutic potential of lambda (λ) phage particles displaying the E75 peptide (derived from HER2 protein) in an implantable TUBO breast tumor model of BALB/c mice. The mice were immunized with the E75-displaying phage (λF7-gpD::E75) every 2-week intervals over a 6-week period, and the generated immune responses were studied. Results showed in vitro induction of immune responses by the λF7 (gpD::E75) construct compared to the control λF7 and buffer groups. In the in vivo prophylactic study, all the control and vaccinated mice groups developed tumors. However, in the therapeutic experiments, we observed a significant difference in tumor size at days 14-36 for mice immunized with λF7 (gpD::E75) compared to control groups (P < 0.05). Moreover, the survival time prolonged in mice immunized with λF7 (gpD::E75). The discrepancy between the results obtained from the in vitro and in vivo studies may have been a result of the induction of Foxp3 CD4+CD25+ which has been previously reported to hamper effective T cell functionality. In conclusion, we observed a significant immune stimulatory response in the in vitro study, while in vivo, the vaccine was not able to exert significant tumor inhibitory effects. We suggest that the presence of Foxp3+ CD4+CD25+ cells may have impaired the anti-tumor response in mice challenged in vivo with the TUBO xenograft tumor.

The role of glucose-6-phosphate dehydrogenase in adipose tissue inflammation in obesity.

Obesity is closely associated with metabolic diseases including type 2 diabetes. One hallmark characteristics of obesity is chronic inflammation that is coordinately controlled by complex signaling networks in adipose tissues. Compelling evidence indicates that reactive oxygen species (ROS) and its related signaling pathways play crucial roles in the progression of chronic inflammation in obesity. The pentose phosphate pathway (PPP) is an anabolic pathway that utilizes the glucoses to generate molecular building blocks and reducing equivalents in the form of NADPH. In particular, NADPH acts as one of the key modulators in the control of ROS through providing an electron for both ROS generation and scavenging. Recently, we have reported that glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the PPP, is implicated in adipose tissue inflammation and systemic insulin resistance in obesity. Mechanistically, G6PD potentiates generation of ROS that augments pro-inflammatory responses in adipose tissue macrophages, leading to systemic insulin resistance. Here, we provide an overview of cell type- specific roles of G6PD in the regulation of ROS balance as well as additional details on the significance of G6PD that contributes to pro-oxidant NADPH generation in obesity-related chronic inflammation and insulin resistance.

BP5 regulated B cell development promoting anti-oxidant defence.

Bursa of Fabricius is the humoral immune system for B cell differentiation and antibody production. Bursopentine (BP5) is a novel immunomodulatory peptide and significantly stimulated an antigen-specific immune response in mice. BP5 was also found to protect LPS-activated murine peritoneal macrophages from oxidative stress. In this study, the effects of BP5 on B cell development were examined. The results suggested that BP5 markedly promoted B cell development by increasing CFU-pre B, and affected the redox homeostasis regulation of B cells. To study the molecular mechanism of effect of bursal-derived BP5, this research utilized 2D-E and MALDI-TOF/TOF to analyze the differentially expressed proteins of BP5-treated WEHI-231 cells. The results showed that BP5 affected the redox homeostasis regulation of WEHI-231 cells and induced alterations in the protein expression profiles related to the oxidoreduction coenzyme metabolic process, precursor metabolites and energy, proteolysis, RNA splicing and translation and cellular process, respectively. BP5 also induced glucose-6-phosphate dehydrogenase (G6PD) activity, an essential anti-oxidant cofactor. We found that the redox homeostasis regulation effect of BP5 was reduced in G6PD-deficient cells. These data suggested that BP5 affected the redox balance toward reducing conditions by promoting the expression of G6PD, which in turn regulated the glutathione redox cycle and other processes.

Real-time RT-PCR: considerations for efficient and sensitive assay design.

Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taq-man probes with a deoxyguanosine base at the 5' end fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer-dimer formation associated with RT enzymes is described.

The role of lipogenesis in the development of uremic hyperlipidemia.

BACKGROUND: It is well documented that hypertriglyceridemia in renal failure mostly is a result of impaired plasma triglyceride (TG) removal. However, the role of TG production in its development is obscure. Therefore, our attention was given to the gene expression of lipogenic enzymes participating in TG biosynthesis. METHODS: We measured some lipogenic enzyme activities, protein abundance (Western blot analysis), and messenger RNA level (Northern blot analysis) in liver and epididymal white adipose tissue (WAT) of rats with surgically induced renal failure (two-stage subtotal nephrectomy). Simultaneously, plasma TG and very low-density lipoprotein (VLDL) concentrations in uremic animals were determined. RESULTS: An increase in plasma TG and VLDL concentrations in rats with renal failure was observed. It was associated with an increase in fatty acid synthase and adenosine triphosphate-citrate lyase (ACL) gene expression in liver and WAT. Moreover, increased activities of malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were found. CONCLUSION: Results of the present study provide some evidence that the accumulation of TG-rich lipoproteins in renal insufficiency could be related in part to increased lipogenic enzyme gene expression and, consequently, TG overproduction.

Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.

A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VHbinding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27bpgene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-beta-delta-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bpcomplex showed that when the proteasomes of cells expressing DH27bpwere inhibited (N -acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bpare signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells. Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal.

[The detection of the location of glucose-6-phosphate dehydrogenase in the fibroblasts of voles and mice and in rat myoblasts by using monoclonal antibodies against glucose-6-phosphate dehydrogenase]. / Vyiavlenie lokalizatsii gliukozo-6-fosfatdegidrogenazy v fibroblastakh polevok i myshi i v mioblastakh krysy s pomoshch'iu monoklonal'nykh antitel protiv gliukozo-6-fosfatdegidrogenazy.

Monoclonal antibodies (MAs) were produced against glucose-6-phosphate dehydrogenase (G6PD) of two vole species--Microtus arvalis and M. subarvalis. The binding level of the MAs to G6PD in both species were almost the same, which suggested that these MAs may be specific for the antigenic determinants common to G6PD of these species. The MAs produced against the vole G6PD were used for its intracellular localization. The patterns obtained after staining cells with the use of MAs against G6PD were the same as those obtained after staining with the use of antibodies against F-actin. There was a good conformity between the results of light and electron microscopic immunoenzyme analyses with regard to the binding of MAs produced to the actin microfilaments. It is concluded that G6PD is closely associated with actin microfilaments of the cell cytoskeleton.

Immunohistochemical demonstration of increased glucose-6-phosphate dehydrogenase in preneoplastic and neoplastic lesions induced by propylnitrosamines in F344 rats and Syrian hamsters.

Changes in the level of expression of glucose-6-phosphate dehydrogenase (G6PD) within propyl nitrosamine-induced preneoplastic and neoplastic lesions in F344 rats and Syrian golden hamsters were investigated using an immunohistochemical approach. Previously demonstrated increases in G6PD activity in rat liver and hamster pancreatic foci of altered cells were revealed as being due to elevation in the quantity of enzyme protein, suggesting an underlying change in gene expression. Furthermore, strong positive binding of G6PD antibody in thyroid, lung, urinary bladder and kidney lesions indicated that increase in this enzyme protein might be a common marker for neoplastic alteration, regardless of organ. While the function of elevated G6PD may be related to growth requirements, the finding that preneoplastic lesions in some cases bind more strongly than more malignant populations suggests additional involvement of the enzyme in other biochemical pathway(s) relevant to tumorigenesis.

Cross-reacting material to monoclonal anti-G6PD in the absence of catalytic activity.

Monoclonal antibody prepared against highly purified rat liver G6PD was used to probe the mode of regulation of this enzyme in a mammalian model system. Material cross-reacting with antibody against liver G6PD was found in similar amounts in extracts of two genetically related rat hepatoma cell lines, only one of which exhibits detectable enzymatic activity when both are cultured under identical conditions in vitro. The data suggest a post-translational event is necessary for the expression of catalytic activity for G6PD in this model system.

Purification of hapten-enzyme conjugates for enzymoimmunoassay.

Chromatography on hydroxyapatite, including a potassium phosphate linear-gradient elution, was applied to the purification of hapten-enzyme conjugates to be used as immunoenzymatic tracers (namely, progesterone, phenobarbital, diphenylhydantoin coupled to glucose-6-phosphate dehydrogenase). The method effectively removed unreacted enzyme and separated the conjugate classes according to the number of substitutions between enzyme linking groups and hapten derivatives. The adequacy of the chromatographic procedures in improving the analytical performance of the enzymatic tracer appears to depend on direct interdependence between the degree of substitution and the immunological properties of the conjugates.

Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency.

A solid-phase radioimmunoassay for human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) was developed that allowed the specific activity of this enzyme protein to be measured in lysates from whole erythrocyte populations, in lysates from erythrocytes of different ages, and in purified samples. The enzyme was highly purified from erythrocytes of single donors by a simple procedure of affinity chromatography with insolubilized adenosine 2',5'-bisphosphate. These techniques were used in an attempt to elucidate the molecular mechanisms leading to deficiency of glucose-6-phosphate dehydrogenase activity in two genetic variants of the enzyme, i.e., the Mediterranean and the Seattle-like variants. The results indicate that the lowered activity of erythrocytes containing the Mediterranean variant of glucose-6-phosphate dehydrogenase is related to an enhanced rate of degradation of a catalytically defective protein synthesized at a nearly normal rate. Synthesis of a normally functioning protein and an increased breakdown of it are involved in the Seattle-like variant of the enzyme.

Characterization of the fatty acid-sensitive glucose 6-phosphate dehydrogenase from Pseudomonas cepacia.

The adenosone 5'-triphosphate-insensitive glucose 6-phosphate dehydrogenase from Pseudomonas cepacia has been found to be strongly inhibited by long-chain fatty acids and their acyl coenzyme A esters, suggesting that an important role of this isoenzyme might be to provide reduced nicotinamide adenine dinucleotide phosphate for reductive steps in fatty acid synthesis. The enzyme, which has been redesignated the fatty acid-sensitive glucose 6-phosphate dehydrogenase, has been purified to homogeneity using affinity chromatography with nicotinamide adenine dinulceotide phosphate-substituted Sepharose as a key step in the purification. The purified preparations were used to study the immunological properties and subunit composition of the enzyme and its relationship to the adenosine 5'-triphosphate-sensitive glucose 6-phosphate dehydrogenase present in extracts of P. cepacia. Although both enzymes were found to be composed of similar size subunits of about 60,000 daltons, immunological studies failed to demonstrate any antigenic similarity between them. Studies of the sedimentation behavior of the fatty acid-sensitive enzyme in sucrose gradients indicated that its apparent molecular weight is increased in the presence of glucose 6-phosphate and suggest that it may exist in an aggregated state in vivo. Palmitoyl coenzyme A, which strongly inhibited the enzyme, failed to influence its sedimentation behavior.

Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells.

Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs. By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P; these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes.

Sero-genetic studies on the Dama of South West Africa.

The Dama of South West Africa are a Negroid people living as a reproductive isolate in the desert and semi-desert areas of the north-west of the country. Until recent times a large proportion of them were held in bondage by the Khoikhoi (Hottentot) Nama, while the rest lived as hunter-gatherers in the mountains. This study and the work of Knussmann and Knussmann indicate that they are a Negro people, which probably has been cut off over a period from contact with other Negroes. They have received very little genetic contribution from the Khoikhoi or the San (Bushmen). The results of this investigation of 24 blood genetic marker systems in a carefully selected random sample of Dama support these conclusions.

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.

Post translational modifications of glucose-6-phosphate dehydrogenase in human leukemias.

The modifications of the electrofocusing pattern, the immunological reactivity and the kinetic properties of glucose 6 phosphate dehydrogenase have been studied in malignant blood cells of various leukemias and myeloproliferative disorders. 1. Granulocytic G-6PD forms with decreased isoelectric points have been found in all the acute myeloid leukemias and erythroleukemias, and in most of the chronic granulocytic leukemias and myelofibrosis. In contrast, granulocytic G-6PD from patients with polycythemia vera always was normal. On the same way leukemic lymphocyte or lymphoblast G-6PD was identical to that from normal lymphocytes. 2. The ratio of enzymatic activity to immunological reactivity (=molecular specific activity) was markedly decreased in the myeloblasts of two patients with acute myeloid leukemia, and in the erythroblast-rich cellular fraction of a patient with erythroleukemia. In these cells the decrease of molecular specific activity was parallel to the alteration of the electrofocusing pattern of G-6PD. 3. The enzymatic forms with decreased isoelectric point also exhibited an altered affinity for glucose 6 phosphate. These modifications are post translational alterations of the neosynthesized G-6PD, since this enzyme is a single molecule, coded by the same gene in all tissues; they seem to correspond to an accelerated molecular aging due to an increased concentration of "G-6PD modifying factors". The significance of such an increased concentration of these G-6PD modifying factors in malignant cells is discussed.