ALBERT

Description: Glycolysis is an essential metabolic pathway for survival of red blood cells (RBC), and mutations in the RBC-type of pyruvate kinase (R-PK) cause hereditary non-spherocytic hemolytic anemia. We have shown that R-PK deficiency induced apoptosis in erythroid progenitor cells (Blood96:596a, 2000; Am J Hematol74: 68, 2003). In this study, we examined whether ex vivo treatment of erythroid cells with high concentration of glycolytic intermediates ameliorated apoptosis of R-PK deficiency. For this purpose, we used the R-PK deficient Friend erythroleukemic cell, SLC3, which has been established from a mice model of R-PK deficiency (Jpn J Cancer Res90: 171, 1999). SLC3 and a control Friend cell were cultured with 0, 1, 3, and 5mM phosphoenolpyruvate (PEP) or pyruvate (PA). Supplementations of either PEP or PA resulted in about 2-fold accumulation of intracellular PA; however, ATP concentration was not significantly changed. Flow cytometric analysis showed that 24-hours treatment with 5mM PEP significantly reduced annexinV (+)/PI (−) cells. These data suggested that exogenously supplemented glycolytic intermediates increased intracellular PA concentration, resulting in amelioration of erythroid apoptosis. We have shown that a glutathione precursor, N-acetyl cysteine, reduced apoptosis of SLC3. Several studies revealed that PA, which effectively maintained the redox state and also prevented the loss of intracellular antioxidants, antagonized the apoptotic cell death. Taken together, oxidative stress caused by decreased supply of PA might account for erythroid apoptosis due to R-PK deficiency.

Description: ErbB2, c-Myc and p53 gene expressions are widely reported for the poor prognostic markers for medulloblastoma (MB) patients. Recently, Wnt pathway and Sonic Hedgehog (SHH) pathways are known to related to the oncogenesis of MB cells, and the PDGF receptor gene and some adhesion molecule gene expressions are also known to the markers of the dissemination or the metastasis of MB cells. For adult malignant brain tumors, the molecular targeted therapy of the antibody (trastuzumab, cetuximab), the tyrosine kinase inhibitor (imatinib, gefinitib) or other signal transduction inhibitor (cyclopamin) are clinically researched. We examined several important marker gene expressions of the molecular therapy in various children’s brain tumors and searched the possible molecular targeted therapy. [Materials and methods] Fifteen children’s brain tumor (five MB, seven glioma, three ependymoma) and four adult brain tumor (two glioma, two glioblastoma) were examind. The three MB cases were primary metaststic ones. The mRNA expressions of the marker genes (ErbB2, PDGF receptor, PCNA, SPARC, β-catenin, SUFU, c-Myc, p53, TrkC and so on) were examined by quantity polymerase chain (qPCR) reaction with fresh frozen tumor cells. For the normal control, we used the normal cerebellum total RNA samples of the Becton, Dikinson and Company. CYBR green coloring system was used for the qPCR and their primers were designed in the region between two exons. GAPDH gene expression was also examined as an internal control and all of the gene expression were normalized by GAPDH expression. [Results] ErbB2 gene expression in MB cells were various, but their expression were similar to clinical futures, such as, the higher expressed cases had some high risk factors. In glioma cells, ErbB2 gene expression were almost equal by lower levels. PDGF receptor gene expression were more than five to ten times elevated in all of the glioma cells, even if they were low grade malignancy glioma, and were higher than that in MB cells. PCNA was specially elevated in primary metastatic MB cells. [Conclusion] Our data shows that the molecular characteristics of the children’s brain tumors were thought to be different by cases, even if they had same histopathological characters. Some special gene tageting therapy, such as anti-ErbB2, PDGFR and/or PCNA therapy can be used for the various children’s brain tumors which are resistant for conventional therapies and are cured by adequate molecular therapy.

Description: Approximately 500 patients are diagnosed as having congenital hemolytic anemia every year in Japan, and the etiology in 75% of these patients involves red cell membrane disorders. The remaining cases are categorized as congenital non-spherocytic hemolytic anemia (CNSHA) and only about 25 % of CNSHA patients are diagnosed as either unstable hemoglobinopathies or erythroenzymopathies. As a result, up to 100 subjects remained diagnosed as having idiopathic hemolytic anemia. Recently, α-hemoglobin stabilizing protein (AHSP) has been identified, and found to play an important role as molecular chaperones in the stabilization of unstable free α-globin. Intact α-globin monomer generates reactive oxygen species, resulting in damage to red cell protein and lipids. AHSP knockout mice showed compensated hemolysis with Heinz bodies. Genetic defects in protection against oxidative stress such as glucose-6-phosphate dehydrogenase deficiency may account for acute hemolysis only when subjects are exposed to either oxidative drugs or infection, but chronic hemolysis is not evident in those cases. These observations led us to hypothesize that AHSP gene defects might account for either Heinz body-positive or drug/infection-induced hemolytic anemia. We examined 25 CNSHA patients with unknown etiology: 6 cases showing Heinz body-positive red cells and 19 cases showing episodes of drug/infection-induced acute hemolysis. Erythroenzymopathies and unstable hemoglobinopathies were ruled out using red cell enzyme analysis, isopropanol stability tests and DNA sequencing of globin genes. Seven SNPs (single nucleotide polymorphisms) surrounding the AHSP gene (rs 4889672, rs 8046452, rs 8050390, rs 4296276, rs 17677, rs 10843, and a novel SNP at 92-bp upstream of rs 4889672) were examined, and compared with 100 control subjects. We found that a certain haplotype, 5′-GTCGA**-3′, was significantly prevalent in CNSHA cases (p=0.0086). Each SNP within the haplotype is located in the 5′-flanking region of AHSP gene, suggesting that the SNP(s) might account for decreased expression of AHSP gene. The reporter gene assay using K562 cells revealed that a construct containing 5′-GTCGA**-3′ had about 30% of transcriptional activity compared to a major haplotype 5′-ACCAG**-3′. Taken together, we conclude that the AHSP gene is correlated to disease-susceptibility in a group of hemolytic anemia with either Heinz body-positive or drug/infection-induced episodes.