Adhesion to fibronectin induces megakaryocytic differentiation of JAS-REN cells.

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.

Segregation of megakaryocytic or erythroid cells from a megakaryocytic leukemia cell line (JAS-R) by adhesion during culture.

Adhesion is one of the important biologic characteristics of leukemic cells. We previously reported a new megakaryocytic-erythroid cell line, JAS-R. In this study, JAS-R cells were segregated into two types by the differences of attachment to culture dishes. One type (designated as JAS-RAD cells) adhered to the substratum of the culture dishes, while the other (JAS-REN cells) grew as a single-cell suspension. Adhesion of JAS-RAD was inhibited by treatment with RGDS oligopeptide. Flow cytometric analysis revealed that JAS-RAD cells had high expression of CD41a and CD61 versus low CD235a expression, and JAS-REN showed low expression of CD41a, and CD61, and high CD235a. The two phenotypes were reciprocally exchangeable by selecting adherent or suspended cells from each type of culture. Microarray analysis and RT-PCR revealed that JAS-RAD cells expressed four major alpha-granule genes and JAS-REN cells expressed beta-globin. Interestingly, erythropoietin was only secreted by JAS-RAD cells. With regard to transcription factors, it was shown that GFI1, FLI1 and RUNX1 were strongly expressed in JAS-RAD cells while GATA1, FOG1 and NFE2 were equally expressed by both types. These findings indicate that adhesion via integrins is related to the phenotypic shift of JAS-R cells between megakaryocytic and erythroid lineages.

MLF1-interacting protein is mainly localized in nucleolus through N-terminal bipartite nuclear localization signal.

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.

Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyper-acetylated histones, and modulated gene expression profile.

Depsipeptide (FK228), a histone deacetylase inhibitor, is a promising new anticancer agent. The mechanism of resistance to this agent was studied using KU812 cells. Depsipeptide-resistant KU812 cells expressed P-glycoprotein (P-gp) and their resistance was abolished by co-treatment with verapamil. P-gp expression returned to the parental cell level when resistant cells were cultured in depsipeptide-free medium, while resistant cells cultured in the medium containing 16 nM depsipeptide still showed hyper-acetylation of histones. Moreover, resistant cells showed erythroid differentiation. Microarray analysis revealed that 28 genes showed increased expression and three genes showed decreased expression in resistant cells compared with parental cells. These 31 genes had various functions relating to signal transduction, cell cycle, apoptosis, and control of cell morphology and differentiation. Among the 28 genes that were upregulated, 15 genes also showed an increased expression in parental cells treated with 4 nM depsipeptide for 48 h, while the other 13 genes including P-gp were different. Among the three genes with decreased expression, HEP27 was most dramatically downregulated. These findings suggest that continuous exposure to depsipeptide reversibly induces P-gp, which contributes to the onset of resistance, but the altered gene expression profile of resistant cells may also play a role.

JAS-R, a new megakaryo-erythroid leukemic cell line that secretes erythropoietin.

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.

Cloning of a newly identified heart-specific troponin I isoform, which lacks the troponin T binding portion, using the yeast hybrid system.

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.

Pretreatment with interferon-alpha radiosensitizes Daudi cells modulating gene expression and biomarkers.

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.

Ectopic cyclin D1 expression blocks STI571-induced erythroid differentiation of K562 cells.

Bcr-Abl tyrosine kinase inhibitor induces apoptosis and erythroid differentiation of K562 cells. During this erythroid differentiation, c-Myc and cyclin D1 transcripts are transiently downregulated. Accordingly, we studied the effect of cyclin D1 overexpression on erythroid differentiation. After treatment with 250 nM STI571, 90% of K562 and 25% of K562/D1 cells underwent erythroid differentiation. The basal expression of glycophorin A in K562/D1 cells was markedly diminished compared with that by parental cells. STI571 treatment failed to induce glycophorin A expression in K562/D1 cells. During STI571 treatment, ERK activity was downregulated in parental cells, while it was constantly activated in K562/D1 cells. These results suggest that ectopic expression of cyclin D1 causes the resistance of K562 cells to erythroid differentiation by modulating ERK regulation.

No V(H) somatic hypermutation was detected in B-cells of a patient with macroglobulinemia due to splenic marginal zone lymphoma.

B-cell diseases are classified on the basis of the normal differentiation stages. We report here a case of a patient with a long history of leukocytosis, splenomegaly without lymphadenopathy, and hyperviscosity symptoms. Clinically, the patient's diagnosis was leukemic Waldenstrom macroglobulinemia. Chromosomal analysis revealed translocation t(2;7)(p11;q22) along with disease progression. Death occurred from pulmonary infection at 46 months after the initial presentation. At autopsy, malignant lymphocytes were found in the marginal areas of the spleen with spreading to the bone marrow and the liver. The histologic findings were consistent with splenic marginal zone lymphoma. We examined the sequences of the immunoglobulin V(H) gene in cells from the initial peripheral blood and from the spleen at autopsy and found that the sequences were identical and had no somatic hypermutation. Macroglobulinemia can occur in various B-cell disorders, including splenic marginal zone lymphoma, even with the transformation of unmutated B-lymphocytes.

Depsipeptide enhances imatinib mesylate-induced apoptosis of Bcr-Abl-positive cells and ectopic expression of cyclin D1, c-Myc or active MEK abrogates this effect.

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.

Lack of BCL10 mRNA mutation in lymphold malignancies.

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.

DNA topoisomerase II inhibitor, etoposide, induces p21WAF1/CIP1 through down-regulation of c-Myc in K562 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.

Detection of MUC1 and keratin 19 mRNAs in the bone marrow by quantitative RT-PCR predicts the risk of distant metastasis in breast cancer patients.

BACKGROUND: Early detection of micrometastasis in bone marrow is critical for the prognosis of breast cancer patients. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) has been used to detect cancer cells in bone marrow, but its utility as a prognostic factor still remains obscure. MATERIALS AND METHODS: Bone marrow samples were aspirated from the anterosuperior iliac spine of 34 patients, immediately after their surgical procedures had been completed. Control samples were also obtained from 10 healthy adult volunteers. The total RNA was extracted from the mononuclear cells, and the expression levels ofbeta-actin, MUC1 and keratin 19 mRNAs were studied by quantitative RT-PCR. Each mRNA level was scored according to the expression level. The sum of these expression scores was defined as the composite expression score, which was employed as the basis of the evaluation. RESULTS: The mean follow-up period was 45 months. Nine patients developed distant metastases, and one developed local recurrence. The 4-year disease relapse rates were 75% (RR=19.38; 95% CI: 1.94-193.20), 28% (RR=3.64; 95% CI: 0.43-31.18), and 8.3% for patients with composite expression scores of 5/6, 3/4 and 2, respectively. The difference among the three groups was statistically significant (log-rank test: p=0.0029), and multivariate analysis also found the composite expression score to be an independent prognostic factor. CONCLUSIONS: Breast cancer patients who show a high composite expression score in bone marrow have a significantly higher risk of recurrence.

Diffuse large B-cell lymphoma arising independently to lymphoplasmacytic lymphoma: a case of two lymphomas.

Richter's syndrome occurs in 5-10% of patients with chronic lymphocytic leukemia, either by transformation of the primary neoplastic lymphocyte, or as a distinct B-cell neoplasm. We report a Japanese patient with lymphoplasmacytic lymphoma in whom a diffuse large B-cell lymphoma developed after treatment with rituximab. Molecular examination on immunoglobulin VH genes revealed that the lymphomas had arisen in two separate clones. We reviewed clinical case reports in literature, and found 30-40% of cases with Richter's syndrome and composite lymphoma had a second B-cell lymphoma of a different origin.

Inactivation of ERK accelerates erythroid differentiation of K562 cells induced by herbimycin A and STI571 while activation of MEK1 interferes with it.

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ ERK pathway.

Both NUP98/TOP1 and TOP1/NUP98 transcripts are detected in a de novo AML with t(11;20)(p15;q11).

The NUP98 gene is involved in several chromosomal abnormalities associated with acute leukemia. The recurrent t(11;20)(p15;q11) chromosomal translocation results in generation of the NUP98/TOP1 chimeric gene. This abnormality has been observed primarily in therapy-related leukemias, and TOP1/NUP98 transcripts have not been demonstrated. We describe a case of de novo acute myeloid leukemia with t(11;20)(p15;q11), with no known history of exposure to chemicals. The translocation occurred in intron 13 of NUP98 and intron 7 of TOP1, as in the three previously reported cases. The breakpoint in NUP98 was exactly the same as that found in a previously reported case. In addition, a reciprocal TOP1/NUP98 transcript was detected for the first time in our patient.