BACKGROUNDSite A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.

METHODSFifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.

RESULTSThe IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate.

CONCLUSIONSMutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

Catalytic Domain ; genetics ; Glioblastoma ; genetics ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Isocitrates ; metabolism ; Mutagenesis ; Mutation ; Protein Binding ; Structure-Activity Relationship

OBJECTIVESTo analyze the long-term survivors of glioblastoma and to identify any prognostic factors that potentially contribute to survival.

METHODSFifteen glioblastomas patients underwent surgery from June 2007 to April 2009 who survived longer than 3 years were enrolled in. Clinical characteristics such as age, location of tumor, extent of resection, and radiotherapy or chemotherapy were analyzed. The expressions of epidermal growth factor receptor (EGFR), tumor protein 53 (P53), phosphatase and tensin homolog (PTEN), O6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase 1 gene (IDH1), and neurofibromatosis type 1 (NF-1) in tumor samples were measured by immunohistochemical method, and the status of P53 and IDH1 were detected by direct DNA sequencing as well. And the patients who survived less than 1 year were set as control. Kaplan-Meier analysis was used to evaluate the prognostic factors.

RESULTSThe average age of patients at diagnosis was 45.6 years. And the overall survival time was 3-6 years (median survival time 3.5 years). Thirteen patients underwent a total resection, and 14 patients took orally temozolomide. The occurrence frequency of these molecular markers in long-term survivors was PTEN (13/15), IDH1 (13/15), IDH1 mutation (12/15), P53 (8/15), P53 mutation (7/15), EGFR (6/15), MGMT (4/15) and NF-1 (3/15). There was a good correlation between IDH1 protein expression and IDHI mutation, and between P53 protein expression and P53 mutation. And the survival analysis showed that age above 50 years at diagnosis (OR = 0.262, 95%CI: 0.102 - 0.672), total resection (OR = 0.372, 95%CI: 0.149 - 0.931) and combined oral temozolomide (OR = 0.131, 95%CI: 0.044 - 0.390) were favorable clinical prognostic factors. PTEN (OR = 0.201, 95%CI: 0.074 - 0.549) and IDH1 (OR = 0.151, 95%CI: 0.050 - 0.454) expression, IDH1 mutation (OR = 0.276, 95%CI: 0.108 - 0.709) in tumor cells contributed to a favorable prognosis.

CONCLUSIONSThere is probably no single molecular marker that is responsible for long-term survival of patients with glioblastoma, may be a balance between all these molecular events result in a favorable outcome.

Adult ; Brain Neoplasms ; diagnosis ; metabolism ; Female ; Glioblastoma ; diagnosis ; metabolism ; Humans ; Isocitrate Dehydrogenase ; metabolism ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Mutation ; PTEN Phosphohydrolase ; metabolism ; Prognosis ; Survivors

Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.
Corynebacterium glutamicum ; genetics ; metabolism ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Industrial Microbiology ; Isocitrate Lyase ; biosynthesis ; genetics ; L-Lactate Dehydrogenase ; genetics ; Succinic Acid ; metabolism ; Transduction, Genetic

OBJECTIVETo evaluate the incidence rate of IDH1 in acute myeloid leukemia and analyze its effect on clinical characteristics and prognosis.

METHODSMononuclear cells in bone marrow samples were collected from 192 adult patients with newly diagnosed AML. Polymerase chain reaction (PCR) and direct sequencing were used to amplify exon 4 of IDH1 gene, the gene sequencing was used to analyze the gene mutations, at same time, the detection of NPM1, FLT3-TKD, FLT3-ITD, C-KIT, CEPBA, TET2 and JAK2V617F and MLL mutations were carried out, the follow-up was used to determine its therapeutic efficacy and outcomes of patients. The clinical and laboratory data of these cases were collected, and their clinical characteristics and prognosis were then analyzed.

RESULTSAmong the 192 AML patients, 13 cases were detected with IDH1 gene mutation, the mutation rate was 6.77% [95% CI (5.70%-13.38%)]. The sequencing chart of IDH1 gene showed double peaks, the mutations were heterozygous, out of them c.G395A (p.R132H) was found in 8 cases, c.C394T was found in 4 cases (p.R132C), c.C394A (p.R132S) was found in 1 cases, R132H and R132C are common, 13 cases showed missense mutation. The median age in mutation group was 52 years old, the median age in unnutration group was 40 years, there was significant difference between them (P = 0.010). Mutation rate of IDH1 gene in M1 and M2 was significantly higher than that in other FAB subtypes. There were no significant difference in sex, newly diagnosed peripheral white blood cell count, hemoglobin, platelet count, peripheral blood and bone marrow original cell proportion of primitive cells between them. Mutation of IDH1 gene had certain correlation with NPM1 gene mutation, but no correlation with FLT3-TKD, FLT3-ITD, C-KIT, TET2 and JAK2V617F and MLL natations was found. In addition, the IDH1 mutation easily occurred in patients with normal karyotype or in patients with middle prognostic risk karyotype, IDH1 mutation occurred in 11 cases with normal karyotype, the mutation rate was 10.28%, IDH1 mutation were observed in 2 cases with abnormal karyotype, the mutation rate was 3.50%, there was significant difference. In AML patients with middle prognostic risk karyotype. The complete remission (CR) and the 3 year survival (OS) rate of IDH1 mut patients were less than that in IDH1 wt, there was significant difference (P < 0.05).

CONCLUSIONSThe IDH1 mutation more easily occurr in older AML patients and mutations effect of IDH1 on clinical characteristics may represent a molecular marker for poor prognosis in AML.

Abnormal Karyotype ; Adult ; Exons ; Heterozygote ; Humans ; Isocitrate Dehydrogenase ; metabolism ; Leukemia, Myeloid, Acute ; enzymology ; Leukocyte Count ; Mutation ; Mutation, Missense ; Platelet Count ; Polymerase Chain Reaction ; Prognosis ; Remission Induction ; Survival Rate

OBJECTIVETo investigate the immunohistochemical expression of isocitrate dehydrogenase 1 gene (IDH1) R132H in glioma and its diagnostic utility.

METHODSImmunohistochemical study of IDH1R132H expression was performed on formalin-fixed paraffin-embedded tissue samples of 75 gliomas, including 33 cases of grade II, 20 cases of grade III and 22 cases of grade IV tumors. Six cases of pilocytic astrocytoma and 12 cases of gliosis were used as controls.

RESULTSNineteen in 33 cases of grade II (57.6%), 8 in 20 cases of grade III (40.0%), 6 in 22 cases of grade IV (27.3%) showed positive cytoplasmic staining of IDH1R132H. Scattered invasive glioma cells at the tumor periphery also expressed IDH1R132H. Gliomas involving the frontal lobe showed more strong IDH1R132H staining. In contrast, none of the pilocytic astrocytomas and gliosis showed IDH1R132H staining. Moreover, the rate of p53 immunopositivities were 42.4% (14/33) in grade II, 65.0% (13/20) in grade III and 77.3% (17/22) in grade IV gliomas. There were no statistic correlations between expression of IDH1R132H and p53.

CONCLUSIONIDH1R132H tends to express preferentially in low-grade gliomas, and it thus may serve as a valuable marker in distinguishing low grade gliomas from gliosis.

Adolescent ; Adult ; Aged ; Astrocytoma ; metabolism ; pathology ; Brain Neoplasms ; metabolism ; pathology ; Child ; Diagnosis, Differential ; Female ; Glioma ; metabolism ; pathology ; Gliosis ; metabolism ; pathology ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Male ; Middle Aged ; Mutation ; Tumor Suppressor Protein p53 ; metabolism ; Young Adult

OBJECTIVETo correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors.

METHODSThe study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion.

RESULTSDeletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05).

CONCLUSIONSA significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.

Aged ; Brain Neoplasms ; genetics ; metabolism ; Chromosome Deletion ; Chromosomes, Human, 19-20 ; genetics ; Chromosomes, Human, Pair 1 ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Middle Aged ; Mutant Proteins ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Oligodendroglioma ; genetics ; metabolism

OBJECTIVETo highlight recent researches which may show promise for histomolecular classification and new treatments for gliomas.

DATA SOURCESAll articles cited in this review were mainly searched from PubMed, which were published in English from 1996 to 2010.

STUDY SELECTIONOriginal articles and critical reviews selected were relevant to the isocitrate dehydrogenase-1/2 mutation in gliomas and other tumors.

RESULTSExtraordinary high rates of somatic mutations in isocitrate dehydrogenase-1/2 occur in the majority of World Health Organization grade II and grade III gliomas as well as grade IV secondary glioblastomas. Isocitrate dehydrogenase-1/2 mutations are associated with younger age at diagnosis and a better prognosis in patients with mutated tumors. The functional role of isocitrate dehydrogenase-1/2 mutations in the pathogenesis of gliomas is still unclear.

CONCLUSIONIsocitrate dehydrogenase-1/2 mutations define a specific subtype of gliomas and may have great significance in the diagnosis, prognosis, and treatment of patients with these tumors.

Adult ; Age Factors ; Brain Neoplasms ; genetics ; pathology ; Genes, p53 ; Glioma ; genetics ; pathology ; Glutarates ; metabolism ; Humans ; Isocitrate Dehydrogenase ; genetics ; physiology ; Ketoglutaric Acids ; metabolism ; Middle Aged ; Mutation ; NADP ; metabolism ; Neoplasm Grading ; Prognosis

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of myeloid precursors arrested in their maturation, creating a diverse disease entity with a wide range of responses to historically standard treatment approaches. While significant progress has been made in characterizing and individualizing the disease at diagnosis to optimally inform those affected, progress in treatment to reduce relapse and induce remission has been limited thus far. In addition to a brief summary of the factors that shape prognostication at diagnosis, this review attempts to expand on the current therapies under investigation that have shown promise in treating AML, including hypomethylating agents, gemtuzumab ozogamicin, FLT3 tyrosine kinase inhibitors, antisense oligonucleotides, and other novel therapies, including aurora kinases, mTOR and PI3 kinase inhibitors, PIM kinase inhibitors, HDAC inhibitors, and IDH targeted therapies. With these, and undoubtedly many others in the future, it is the hope that by combining more accurate prognostication with more effective therapies, patients will begin to have a different, and more complete, outlook on their disease that allows for safer and more successful treatment strategies.
Aminoglycosides ; administration & dosage ; Antibodies, Monoclonal, Humanized ; administration & dosage ; Elafin ; genetics ; Histone Deacetylase Inhibitors ; therapeutic use ; Humans ; Isocitrate Dehydrogenase ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; pathology ; Proto-Oncogene Proteins c-pim-1 ; metabolism ; TOR Serine-Threonine Kinases ; genetics

Glutamate leads to neuronal cell damage by generating neurotoxicity during brain development. The objective of this study is to identify proteins that differently expressed by glutamate treatment in neonatal cerebral cortex. Sprague-Dawley rat pups (post-natal day 7) were intraperitoneally injected with vehicle or glutamate (10 mg/kg). Brain tissues were isolated 4 h after drug treatment and fixed for morphological study. Moreover, cerebral cortices were collected for protein study. Two-dimensional gel electrophoresis and mass spectrometry were carried out to identify specific proteins. We observed severe histopathological changes in glutamate-exposed cerebral cortex. We identified various proteins that differentially expressed by glutamate exposure. Identified proteins were thioredoxin, peroxiredoxin 5, ubiquitin carboxy-terminal hydrolase L1, proteasome subunit alpha proteins, isocitrate dehydrogenase, and heat shock protein 60. Heat shock protein 60 was increased in glutamate exposed condition. However, other proteins were decreased in glutamate-treated animals. These proteins are related to anti-oxidant, protein degradation, metabolism, signal transduction, and anti-apoptotic function. Thus, our findings can suggest that glutamate leads to neonatal cerebral cortex damage by regulation of specific proteins that mediated with various functions.
Animals ; Brain ; Cerebral Cortex ; Chaperonin 60 ; Electrophoresis, Gel, Two-Dimensional ; Glutamic Acid ; Humans ; Infant, Newborn ; Isocitrate Dehydrogenase ; Mass Spectrometry ; Metabolism ; Neurons ; Peroxiredoxins ; Proteasome Endopeptidase Complex ; Proteolysis ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thioredoxins ; Ubiquitin Thiolesterase

OBJECTIVETo investigate mutation status of isocitrate dehydrogenase (IDH) 1 and IDH2 genes in Chinese patients with gliomas in correlation with clinicopathological characteristics.

METHODSFormalin-fixed and paraffin-embedded (FFPE) tissue samples of 234 gliomas were collected including the matched blood samples in 30 patients. DNA was extracted, followed by PCR-Sanger sequencing to detect IDH1 and IDH2 gene mutations. Immunohistochemistry was performed using mutation-specific antibody recognizing IDH1R132H mutation. Immunostains for p53 and epidermal growth factor receptor (EGFR) were also performed. Oligodendroglial tumors with IDH mutation were double stained with IDH1R132H and GFAP by immunofluorescence to investigate the location of IDH1R132H expression.

RESULTS(1) By IDH1 heterozygous somatic mutation analysis, Arg132His (c: G395A) was found in 31.6% (74 of 234) of the cases. IDH mutations were more frequent in oligoastrocytomas (9/13), anaplastic oligoastrocytomas (7/11), oligodendrogliomas(18/26, 69.2%), anaplastic oligodendrogliomas (8/10), and less frequent in diffuse astrocytomas (17/47, 36.2%), anaplastic astrocytomas (5/18), and glioblastomas (10/69, 14.5%). The mutation rate inversely correlated with the tumor grade in a linear fashion in astrocytic tumors (P = 0.007). Primary glioblastomas were characterized by a lower frequency of mutations than secondary glioblastomas (5/55 vs. 5/14, P = 0.036); IDH mutation was not detected in pilocytic astrocytoma and ependymoma. No IDH2 mutation was identified in this study cohort. (2) Immunohistochemistry of IDH1R132H demonstrated a strong cytoplasmic staining in 80 cases, which was highly correlated with IDH mutation status (P = 0.001). IDH1R132H was highly specific to tumor cells. (3) p53 immunostain was significantly correlated the IDH mutation in diffuse astrocytoma, anaplastic astrocytoma and secondary glioblastomas (P = 0.007, 0.026, 0.038 respectively). (4) No correlation between EGFR and IDH mutation was found.

CONCLUSIONSHigh prevalence of IDH heterozygous somatic mutation occurs in the earlier stage of gliomas, which can be detected by mutation-specific antibody IDH1R132H. Furthermore, evaluation of p53 and EGFR expression combined with IDH mutation analysis may significantly aid in the diagnosis and differential diagnoses of gliomas in Chinese patients.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Astrocytoma ; genetics ; metabolism ; Brain Neoplasms ; genetics ; metabolism ; Child ; Ependymoma ; genetics ; metabolism ; Female ; Glioblastoma ; genetics ; metabolism ; Glioma ; genetics ; metabolism ; Humans ; Isocitrate Dehydrogenase ; genetics ; metabolism ; Male ; Middle Aged ; Oligodendroglioma ; genetics ; metabolism ; Point Mutation ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Young Adult