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We report a case of spherocytosis associated with erythrocyte band 3 deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of erythrocyte membrane proteins showed that the patient’s band 3 was reduced to about 80% of the control level. Molecular analysis revealed that this quantitative alteration was accompanied by a novel base change at codon 837 (ACG→GCG) of the AE1 gene, resulting in substitution of alanine for threonine. In bone marrow mononuclear cells, both mutant and wild-type mRNA were comparably detected, suggesting that this mutation interfered with band 3 processing or assembly, leading to impaired accumulation of mutant band 3 in the plasma membrane. [ABSTRACT FROM AUTHOR]

Background: Binding of integrins to the extracellular matrix elicits various responses. We have previously reported a megakaryocytic-erythroid cell line (JAS-R) that showed phenotypic changes after adhesion to plastic dishes. However, the matrix protein and the mechanism responsible for megakaryocytic differentiation still remain unknown., Materials and Methods: JAS-REN (erythroid) cells were cultured on dishes coated with various proteins. The cells were treated with RGDS, a tetrapeptide ligand to integrins, or phorbol ester (12-o-tetradecanoylphorbol-13-acetate, TPA) for 48 hours and then were harvested. Subsequently, the cell surface markers were analyzed using flow cytometry and gene expression was studied by RT-PCR., Results: The JAS-REN cells adhered to fibronectin-coated dishes, but showed poor adhesion to dishes coated with collagen, laminin or poly-D-lysine. The TPA-stimulated JAS-REN cells showed an increase in the expression of integrin alphaIIbbeta3 complex (CD41a) and integrin beta3 (CD61), while glycophorin A (CD235a) expression was decreased. JAS-REN cells that were adherent to fibronectin-coated dishes also showed a similar pattern of phenotype to TPA-treated cells, but the changes were not so prominent. RT-PCR revealed that TPA treatment altered the gene expression profile of JAS-REN cells, making it similar to that of JAS-RAD (megakaryocytic) cells. The RGDS-treated and fibronectin adherent JAS-REN cells also showed a mostly similar expression profile to JAS-RAD cells, but these two stimuli did not alter the gene expression profile as TPA stimulation did. Transcription factors, FLI1 and GFI1, were induced by all stimuli., Conclusion: Signals triggered by adhesion to fibronectin result in the induction of FLI1 that may play a pivotal role in the lineage shift of JAS-REN cells from erythroid to megakaryocytic.

Background: The myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1LP, also called KLIP1 and CENP-50) is reported to localize in both the nucleus and the cytoplasm. To investigate the functions of MLF1IP, its subnuclear localization was studied., Materials and Methods: MLF1IP was tagged with green fluorescent protein (EGFP). Fibrillarin was tagged with red fluorescent protein (DsRed). EGFP-tagged MLF1IP deletion vectors were also constructed. Plasmid-constructs were transfected into human cervical adenocarcinoma HeLa cells or monkey kidney fibroblast COS-7 cells, and the localization was studied by either confocal fluorescence microscopy or fluorescence microscopy., Results: Ectopically expressed MLF1IP was localized mainly in the nucleolus. In some cells, small dot-like particles of MLF1IP fluorescence were observed in the nucleoplasm. Co-staining of fibrillarin disclosed that MLF1IP was co-localized with fibrillarin in the nucleolus. Deletion mutants of MLF1IP revealed that the N-terminal bipartite nuclear localization signal (NLS) was responsible for nucleolar targeting., Conclusion: MLF1IP was localized mainly in the nucleolus through the N-terminal bipartite NLS and partly in the nucleoplasm featuring small dot-like particles. These findings suggest that MLF1IP may have multi-functions and its different localizations may contribute to carcinogenesis.

Background: The processes of leukemogenesis and differentiation of the megakaryo-erythroid lineage remain poorly understood. Leukemic cell lines derived from megakaryocytic leukemia are valuable reagents for studies on these events., Materials and Methods: A new cell line, JAS-R, was established from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Its characteristics were studied by morphological, immunophenotypic and molecular biological analysis., Results: Immunophenotyping showed that the JAS-R cells were positive for CD33, CD41 and CD61, as well as moderately to weakly positive for CD4, CD7, CD13 and glycophorin A. Chromosomal analysis revealed a composite karyotype, but no major translocation abnormalities were observed. Electron microscopy disclosed that the JAS-R cells had numerous surface blebs and some cells also had alpha-granules and demarcation membranes. The mRNAs of 4 major proteins (platelet factor 4, beta-thromboglobulin, selectin-P and thrombospondin 1) found in alpha-granules were all expressed by the JAS-R cells. In particular, expression of platelet factor 4 was high. To further characterize JAS-R cells, comparison with 4 other megakaryo-etythroid cell lines (CMK, MEG-01, K562 and KU812) was done by gene expression profiling using an oligo-DNA microarray. The results showed that JAS-R was a distinctive cell line. It was noteworthy that the JAS-R cells secreted erythropoietin and expressed erythropoietin receptor. A neutralizing antibody for erythropoietin partly inhibited the proliferation of the cells., Conclusion: JAS-R may be a useful cell line for investigating the differentiation and leukemogenesis of megakaryo-erythroid cells and for studying the influence of erythropoietin on these cells.

Objective: To elucidate the molecular pathogenesis behind increased levels of laminin in cardiac muscle cells in cardiomyopathy by using a yeast hybrid screen. The present study reports the cloning of a newly identified heart-specific troponin I isoform, which is putatively linked to laminin. Future studies will explore the functional significance of this connection., Methods: Yeast two-hybrid screen analysis was performed using MLF1-interacting protein (amino acids 1 to 318) as bait. The human heart complementary DNA library was screened by using the yeast-mating method for overnight culture., Results: Two final positive clones from the heart library were isolated. These two clones encoded the same protein, a short isoform of human cardiac troponin I (TnI) that lacked TnI exons 5 and 6. The TnI isoform has a heart-specific expression pattern and it shares several sequence features with human cardiac TnI; however, it lacks the troponin T binding portion., Conclusion: The heart-specific segment of the human cardiac TnI isoform shares several sequence features with human cardiac TnI, but it lacks the troponin T binding portion. These results suggest that the heart-specific TnI isoform may be involved in cardiac development and disease.

Background: Interferon (IFN) potentiates cytotoxicity by X-ray irradiation. To elucidate the mechanism of this potentiation, the biological markers related to DNA damage and cell survival were studied., Materials and Methods: IFN-alpha-sensitive Daudi and its resistant cells were used. Survival after treatment was assessed by clonogenic assays. DNA breaks were studied by pulse-field gel electrophoresis (PFGE). Production of reactive oxygen metabolites was measured using flow cytometry. Messenger RNA and protein were examined by RT-PCR and immunoblot, respectively., Results: IFN-alpha treatment for 24 h before irradiation potentiated the sensitivity of Daudi cells to X-rays. This combination induced 50 kb DNA fragmentation and activated caspase-3 in Daudi cells. Pretreatment with IFN-alpha inhibited the production of reactive oxygen species by irradiation. IFN-alpha pretreatment down-regulated most of the double-strand break (DSB) repair-related mRNAs, but did not affect the repair of DSBs studied by PFGE. The induction and phosphorylation of p21(Cip1/WAF1) (p21) was prominently suppressed in cells pretreated with IFN-a., Conclusion: Pretreatment with IFN-alpha potentiates the cytotoxic effects of X-rays. Inhibition of X-ray-induced p21 may cause the augmented sensitivity by IFN-alpha pretreatment.

Background: Imatinib mesylate (ST1571) is the first-line drugfor chronic myeloid leukemia (CML), but development of resistance to this drug is a clinical problem. To explore the effective use of ST1571, we studied the combination treatment with histone deacetylase inhibitor (depsipeptide, FK228)., Materials and Methods: FK228 and trichostatin A (TSA) were studied with respect to apoptosis of two Bcr-Abl-positive cell lines, K562 and TCC-S. Genetically-modified K562 cells by any of cyclin D1, c-Myc and active MEK genes were also studied. Apoptosis was examined by nuclear-morphology under a fluorescent microscope and by the expression of annexin V Changes of apoptosis-regulating genes and acetylated histone H4 were studied by immunoblot., Results: FK228 showed cytotoxicity at the nano-molar level. Combination treatment with STI571 and FK228 enhanced the induction of apoptosis significantly compared with each single treatment, although the histone acetylation level was not changed by the co-treatment. The combination treatment activated caspase-3 and cleaved PARP, but it did not induce any notable change in the expression of Bcl-XL, Bcl-2 and Bax compared with each single treatment. Enhanced apoptosis by the co-treatment was abrogated by ectopic expression of cyclin D1, c-Myc or active MEK CONCLUSION: The combination of FK228 with STI571 is a promising treatment for Bcr-Abl-positive CML, but the activation of the MEK/ERK pathway and its downstream target genes may bring resistance to the co-treatment in leukemic cells.

Background: Anticancer agents modulate gene expression and these changes are essential for tumor cell killing. To investigate the mechanism by which etoposide acts as an anticancer agent, the relationship between p21WAF1/CIP1 (p21) and c-Myc was studied., Materials and Methods: K562 cells with and without ectopic c-Myc expression were studied. Apoptosis was detected using propidium iodide and Hoechst 33342 double staining. The c-Myc and p21 levels were studied by RT-PCR and immunoblot. The p21 promoter (from -205 to +67) was investigated by the luciferase reporter gene assay., Results: Ectopic c-Myc-expressing K562 (K562/c-Myc) cells showed more extensive apoptosis than K562 cells after continuous exposure to 200 microM etoposide for 24 hours. During this treatment, p21 expression was not observed in K562/c-Myc cells, and the expression of c-Myc and p21 was mutually exclusive. Etoposide activated the p21 promoter in a concentration-dependent manner, and etoposide-induced luciferase activity was suppressed by co-transfection of c-Myc., Conclusion: p21 promoter activity was repressed by c-Myc in proliferating K562 cells, and detoposide-induced down-regulation of c-Myc released this suppression, resulting in the induction of p21.

Background: BCL10, a gene involved in the chromosomal translocation t(1;14)(p22;q32) found in mucosa-associated lymphoid tissue lymphoma (MALT lymphoma), has shown mutation in not only MALT lymphomas but also other lymphold tumors. However, the mutation rate remains controversial. One possible reason is variation in the source material (DNA or RNA), with most studies having been done using tumor DNA. Accordingly, we studied BCL10 mutations using tumor RNA., Materials and Methods: Fifty lymphoid malignancies (26 malignant lymphomas, 10 chronic lymphocytic leukemias and 14 acute lymphoblastic leukemias) were examined. Total RNA was extracted from the tumor cells and first-strand cDNA was subjected to single-strand conformation polymorphism and fragment length analysis. Then the results were confirmed by direct sequencing., Results: No mutations of the BCL10 gene were found in the 50 samples. There were four polymorphisms (3G to T, 24G to C, 485C to T and 638G to A). All samples showed at least one 24C allele., Conclusion: The BCL10 mutations studied are rare events at the RNA level and may not be associated with the mechanism of tumorigenesis in most lymphoid tumors.