Ikuya Sakai

FLJ10849, a septin family gene, fuses MLL in a novel leukemia cell line CNLBC1 derived from chronic neutrophilic leukemia in transformation with t(4;11)(q21;q23)

A t(4;11)(q21;q23) has been described in 50–70% of cases of infant acute lymphoblastic leukemia and, less frequently, in cases of pediatric and adult acute lymphoblastic leukemia and acute myeloid leukemia (AML). In t(4;11)(q21;q23) leukemias, the AF4 gene has been cloned as a fusion partner of the MLL gene. A human myeloid leukemia cell line, chronic neutrophilic leukemia (CNL)BC1, was established from a peripheral blood specimen of a patient with CNL in leukemic transformation. As with the original leukemia cells, the established line had a t(4;11)(q21;q23). We showed that the MLL gene on 11q23 was fused to the FLJ10849 gene on 4q21. The protein encoded by FLJ10849 belongs to the septin family, sharing highest homology with human SEPT6, which is one of the fusion partners of MLL in t(X;11)(q13;q23) AML. Our results suggest that FLJ10849 might define a new septin family particularly involved in the pathogenesis of 11q23-associated leukemia. The established cell line, CNLBC1, could provide a useful model for analyzing the pathogenesis of MLL-septin leukemias and chronic neutrophilic leukemia.

Novel RUNX1-PRDM16 fusion transcripts in a patient with acute myeloid leukemia showing t(1;21)(p36;q22).

The t(1;21)(p36;q22) is a recurrent chromosome abnormality associated with therapy‐related acute myeloid leukemia (AML). Although involvement of RUNX1 has been detected by fluorescence in situ hybridization analysis, the partner gene has not been reported previously. We identified a novel RUNX1 partner gene, MDS1/EVI1‐like‐gene 1 (PRDM16), in an AML patient with t(1;21). Alternative splicing of the fusion gene generates five different fusion transcripts. In two of them, the PRDM16 reading frame is maintained in the fusion with RUNX1, suggesting that the RUNX1–PRDM16 gene fusion results in the production of a protein that is highly homologous to the RUNX1–MDS1/EVI1 chimeric protein. It is suggested that PRDM16 and MDS1/EVI1 share a common molecular mechanism for the leukemogenesis of RUNX1‐associated leukemia. Characterization of the RUNX1–PRDM16 fusion protein and comparison with the RUNX1–MDS1/EVI1 protein will facilitate the understanding of the mechanisms underlying RUNX1‐associated leukemia.

Transcriptional Down-Regulation of CXC Chemokine Receptor 4 Induced by Impaired Association of Transcription Regulator YY1 with c-Myc in Human Herpesvirus 6-Infected Cells

We have recently reported that down-regulation of CXC chemokine receptor (CXCR) 4 in CD4+ T lymphocytes is induced by human herpesvirus (HHV) 6 infection. In this study, we further studied the mechanisms of HHV-6-induced CXCR4 down-regulation, focusing on the regulation of CXCR4 transcription. Down-regulation of CXCR4 transcription was detected in HHV-6A-infected JJHAN and HHV-6B-infected MT-4 cell lines, as we had previously reported for HHV-6-infected peripheral blood CD4+ T lymphocytes. Luciferase assays revealed that a YY1-binding site around −320 relative to the transcription start site is important for down-regulation of CXCR4 transcription in HHV-6-infected cells. The binding activity of YY1, which is a repressor of CXCR4 transcription, to the CXCR4 promoter appeared to significantly increase in HHV-6-infected cells compared with the binding activity in mock-infected cells. Immunoprecipitation assays showed that in HHV-6-infected cells association of c-Myc with YY1 was decreased and that of Max with c-Myc was increased, whereas association of Mad with Max appeared to be decreased. The amounts of each of YY1, c-Myc, Max, and Mad proteins synthesized in cells were not altered by HHV-6 infection. These data indicate that the decreased association of YY1 with c-Myc that is caused by impaired interaction in the c-Myc/Max/Mad network results in increased binding activity of YY1 to the CXCR4 promoter, mediating down-regulation of CXCR4 production in HHV-6-infected cells.

Absence of Chlamydial Infection in Japanese Patients with Ocular Adnexal Lymphoma of Mucosa-Associated Lymphoid Tissue

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immuno-globulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, orChlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies againstChlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Estimation of the Initial Dose Setting of Vancomycin Therapy With Use of Cystatin C as a New Marker of Renal Function

In recent years, it has been suggested that the glomerular filtration rate (GFR) can be predicted on the basis of serum cystatin C concentrations and that this measurement is more sensitive than serum creatinine concentration as a marker of renal function. In this study, to investigate the clinical utility of the initial dose setting of vancomycin by the population mean method with use of serum cystatin C as a marker of renal function, we compared the correlations between measured vancomycin concentrations and predicted vancomycin concentrations based on serum cystatin C or serum creatinine concentrations in elderly (≥65 years old) and nonelderly (<65 years old) patients. An analysis of prediction accuracy (bias) and precision was evaluated by calculating the mean prediction error (ME), the mean absolute error (MAE), and the root mean squared prediction error (RMSE). For nonelderly patients (n = 50), there was no significant difference in the MAE based on the use of serum creatinine or serum cystatin C concentration. However, for elderly patients (n = 105), the MAE based on serum cystatin C concentration was significantly better than that based on serum creatinine level. These results suggest that serum cystatin C is a good marker of renal function in comparison with serum creatinine for dose setting of vancomycin, especially in an elderly population.

Non-Hodgkin's lymphoma developing in a pacemaker pocket

A 29-year-old man developed diffuse large B-cell lymphoma in a subpectoral pacemaker pocket that 6 years previously had been created in the chest for a titanium-covered pulse generator. The patient had an 8-cm—diameter dark red tumor with necrotic tissue on a keloidal surgical scar in the left side of the chest. Left axillary lymphadenopathy also was present. Laboratory studies showed an increased level of soluble interleukin 2 receptor and a normal level of lactose dehydrogenase. A biopsy specimen showed a diffuse large B-cell phenotype and monoclonal immunoglobulin H gene rearrangement. A gallium scintigraphy study showed abnormal accumulation in the left chest and left axilla. On the basis of these findings, we diagnosed diffuse large B-cell lymphoma, stage II. The patient received THP-COP chemotherapy (pirarubicin, cyclophosphamide, vincristine, and prednisolone) and radiotherapy, achieved complete remission, and was free of disease for 16 months after treatment. This case suggests that there was a relationship between the development of non-Hodgkin’s lymphoma and the presence of chronic inflammation in the pulse generator pocket.

Cooperative Role of the Membrane-proximal and -distal Residues of the Integrin β3 Cytoplasmic Domain in Regulation of Talin-mediated αIIbβ3 Activation

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin β subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the β3 membrane-proximal and -distal residues in regulation of talin-mediated αIIbβ3 activation. Because a chimeric integrin, αIIbβ3/β1, in which the β3 tail was replaced with the β1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the β3 and β1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the β3 membrane-proximal and -distal regions, respectively, with the corresponding β1 residues or alanine rendered αIIbβ3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of Met-719 or Ser-749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation. These results suggest that the β3 membrane-proximal and -distal residues cooperatively regulate talin-mediated αIIbβ3 activation.

Existence of Leukemic Clones Resistant to Both Imatinib Mesylate and Rituximab before Drug Therapies in a Patient with Philadelphia Chromosome-Positive Acute Lymphocytic Leukemia

Imatinib mesylate and rituximab are molecularly targeted drugs against the BCR-ABL fusion protein and the CD20 antigen, respectively. Although these drugs have excellent anticancer effects, a major concern is drug resistance. We have investigated the case of a patient with Philadelphia chromosome-positive and CD20+ acute lymphocytic leukemia who acquired resistance to imatinib and rituximab. Imatinib therapy resulted in prompt cytogenetic remission, but resistance developed shortly thereafter. Sequencing of the kinase domain of the ABL gene and allele-specific polymerase chain reaction analysis revealed a point mutation resulting in an E255V substitution that was present before the therapy. After the patient received mild chemotherapy followed by rituximab administration, hematologic and cytogenetic remission was sustained for 5.5 months. The recurrent leukemic cells after the rituximab therapy showed not only the E255V mutation in the ABL gene but also loss of the CD20 antigen due to impaired transcription of the CD20 gene. The results of 2-color flow cytometry analysis showed that a small population of CD20- leukemic cells existed before the imatinib therapy. These results suggest that leukemic subclones carrying a genetic perturbation of the targeted molecules for both imatinib and rituximab were present before the therapies. The preexistence of primary resistant clones suggests the inability of combination therapy with 2 molecularly targeted drugs to overcome drug resistance in leukemia.

CD20-positive adult T-cell leukemia.

A 67‐year‐old woman was admitted to our hospital because of lymphadenopathy and lymphocytosis. Monoclonal integration of HTLV‐I provirus DNA was detected, and a diagnosis of adult T‐cell leukemia (ATL) was made. Flow cytometry revealed that the ATL cells expressed CD20 as well as T‐cell‐associated antigens, and expression of CD20 mRNA was also demonstrated. A novel T‐cell subpopulation expressing CD20 molecules has recently been identified. This is the first report of CD20‐positive ATL, suggesting that HTLV‐I can infect and transform CD20‐positive T cells. Am. J. Hematol. 66:39–41, 2001.