Masato Shikami

Survival of human leukaemic B-cell precursors is supported by stromal cells and cytokines: association with the expression of bcl-2 protein.

We searched for cytokines with the potential to support the survival of human B‐cell precursor acute lymphoblastic leukaemia (pre‐B ALL) cells. 47 patients with pre‐B ALL were classified into four stages: stage I, CD19+CD10−CD20−; stage II, CD19+CD10+CD20−; stage III, CD19+CD10+CD20+cytoplasmic μ‐heavy chain (cμ)−; stage IV, CD19+CD10+CD20+cμ+. Interleukin (IL)‐3 receptor α chain (IL‐3Rα) was expressed in all stages, whereas the expressions of IL‐7Rα and IL‐2Rα were pronounced in stages IV and II, respectively. Neither IL‐3, IL‐7 nor IL‐2 supported the survival of pre‐B ALL cells. When pre‐B ALL cells were layered on stromal, MS‐10, cells, viability of the pre‐B ALL cells increased. Addition of IL‐3 to culture containing MS‐10 cells enhanced the survival of pre‐B ALL cells in all cases, whereas addition of IL‐7 augmented the survival of pre‐B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre‐B ALL cells was also supported by the conditioned media of MS‐10 cells. Stromal‐cell‐derived factor 1 (SDF‐1) supported the survival of pre‐B ALL cells. Effects of the conditioned media of MS‐10 cells were abrogated by an anti‐SDF‐1 neutralizing antibody. The extent of survival of pre‐B ALL cells supported by stromal cells and IL‐3 and IL‐7, correlated with the expression level of bcl‐2 protein. The effects of stromal cells may be in part related to SDF‐1.

Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin‐2 receptor α chain

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH‐1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase‐polymerase chain reaction (RT‐PCR). The immunophenotypes of the AML cells, and YNH‐1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA‐DR−/+. Cells from all samples strongly expressed c‐kit, granulocyte colony‐stimulating factor receptor (G‐CSFR), c‐fms (macrophage colony‐stimulating factor receptor), interleukin‐3 receptor α chain (IL‐3Rα), and granulocyte macrophage colony‐stimulating factor receptor α chain (GM‐CSFRα), and these data corresponded well to the growth responsiveness to the cytokines. IL‐2Rα expression was also found in all t(16;21) samples, but IL‐2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G‐CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c‐kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL‐2Rα expression) and FUS/ERG protein remains undetermined.

Biphasic expression of CD4 in acute myelocytic leukemia (AML) cells: AML of monocyte origin and hematopoietic precursor cell origin

In 227 of 495 (45.9%) Japanese adult patients with acute myelocytic leukemia (AML), leukemic cells expressed CD4. Incidence of CD4 expression in each FAB subtype was as follows: M1 37.4%, M2 33.7%, M3 35.4%, M4 65.0%, and M5 78.3%. The typical expression pattern of myelomonocytic differentiation antigens and cytokine receptors in CD4+ AML was CD34lowCD33high CD11bhighGM-CSFRhigh. AML cases with 11q23 abnormalities and with inv(16) were frequently CD4-positive. These data collectively indicate that CD4 expression in AML cells is associated with monocytic characteristics. However, CD4+CD34high AML cases appear to have unique immature characteristics including low expression of myelomonocytic differentiation antigens (ie CD33 and CD11b), and accumulation of chromosome abnormalities (ie t(8;21) in CD4lowCD34high AML and chromosome 7 abnormalities in CD4highCD34high AML). We speculate that these leukemia subsets originate from CD4+ hematopoietic precursor cells, therefore then should be considered separately from most of the CD4+ AML as represented by CD34lowCD33high CD11bhighGM-CSFRhigh. Overall survival of patients with CD4+ AML in our series was worse than that of those with CD4− AML (P = 0.0202).

Low BCL-2 expression in acute leukemia with t(8;21) chromosomal abnormality

In de novo t(8;21) AML which shows terminal neutrophilic differentiation, the BCL-2 expression was found to be significantly lower than that in types of other AML regardless of the phenotypic differentiation status. An inverse correlation between BCL-2 expression and the S/G2/M population cells was observed in AML. The S/G2/M population in t(8;21)AML was larger than in the other types of AML. In t(8;21)AML, spontaneous apoptosis after a 12-h liquid culture was prominent, and the autonomous DNA synthesis after a 72-h liquid culture was low. G-CSF and IL-5 promoted the colony formation of t(8;21)AML cells. The data suggest that, in vivo, the low BCL-2 in t(8;21)AML induced entry of cells from the G0/G1 phase to S phase, but the cells easily die by apoptosis, in vitro. The low BCL-2 expression and the supportive effects of G-CSF and IL-5 in t(8;21)AML is thought to be a key phenomenon which might be related to the formation of the in vivo blood picture, such as prominent neutrophilic differentiation and eosinophilia. Cellular extracts from t(8;21)AML cell line Kasumi-1 bound to both the AML1 and CRE binding sites in the bcl-2 promoter, but none of the cellular extracts from de novo t(8;21)AML bound to either of these sites. The DNA binding activity of transactivators in de novo t(8;21)AML is different from that in Kasumi-1 cells probably due to the phosphorylation status.

Overexpression of salivary-type amylase reduces the sensitivity to bortezomib in multiple myeloma cells

Amylase-producing myeloma exhibits refractoriness to chemotherapy and a dismal prognosis. In this study, we established a human myeloma cell line, 8226/AMY1, in which a lentivirally transfected AMY1 gene was stably expressed and explored its biological characteristics. 8226/AMY1 showed a survival advantage over mock control when treated with dexamethasone, bortezomib, and lenalidomide in vitro partly through inhibition of apoptosis induced by these reagents. In a xenograft murine model, 8226/AMY1 showed rapid tumor growth and reduced sensitivity to bortezomib compared with mock. A microarray gene expression analysis identified TCL1A, which functions as a coactivator of the cell survival kinase Akt, differentially up-regulated in 8226/AMY1. The expression of phosphorylated Akt was increased in the 8226/AMY1 cells following bortezomib treatment, but not in the mock cells. In addition, treatment with perifosine, an inhibitor of Akt phosphorylation, enhanced the anti-myeloma effect of bortezomib in the 8226/AMY1 cells. Our data suggest that amylase-producing myeloma reduced the sensitivity to bortezomib in vitro and in vivo, and the up-regulation of TCL1A may influence the drug susceptibility of 8226/AMY1 via the phosphorylation of Akt. These findings provide clues for developing treatment approaches for not only amylase-producing myeloma, but also relapsed and refractory myelomas.

Establishment and characterization of a novel vincristine-resistant diffuse large B-cell lymphoma cell line containing the 8q24 homogeneously staining region

Chromosome band 8q24 is the most frequently amplified locus in various types of cancers. MYC has been identified as the primary oncogene at the 8q24 locus, whereas a long noncoding gene, PVT1, which lies adjacent to MYC, has recently emerged as another potential oncogenic regulator at this position. In this study, we established and characterized a novel cell line, AMU‐ML2, from a patient with diffuse large B‐cell lymphoma (DLBCL), displaying homogeneously staining regions at the 8q24 locus. Fluorescence in situ hybridization clearly detected an elevation in MYC copy numbers corresponding to the homogenously staining region. In addition, a comparative genomic hybridization analysis using high‐resolution arrays revealed that the 8q24 amplicon size was 1.4 Mb, containing the entire MYC and PVT1 regions. We also demonstrated a loss of heterozygosity for TP53 at 17p13 in conjunction with a TP53 frameshift mutation. Notably, AMU‐ML2 cells exhibited resistance to vincristine, and cell proliferation was markedly inhibited by MYC‐shRNA‐mediated knockdown. Furthermore, genes involved in cyclin D, mTOR, and Ras signaling were downregulated following MYC knockdown, suggesting that MYC expression was closely associated with tumor cell growth. In conclusion, AMU‐ML2 cells are uniquely characterized by homogenously staining regions at the 8q24 locus, thus providing useful insights into the pathogenesis of DLBCL with 8q24 abnormalities.