Tsukuru Umemura

Thiazolidinedione Induces Apoptosis and Monocytic Differentiation in the Promyelocytic Leukemia Cell Line HL60

Thiazolidinedione (TZD) is known to be a potent activator of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that constitutes a heterodimer with retinoid X receptor (RXR). Since a considerable amount of PPARγ is expressed in various hematopoietic cells, the present study was undertaken to examine the effect of TZD in the absence or presence of LG100268, an RXR-selective ligand, on a cultured promyelocytic leukemia cell line, HL60. Treatment with TZD (25–50 µM troglitazone or pioglitazone) markedly suppressed cell proliferation of HL60. A cell cycle analysis revealed that the suppressive effect of troglitazone on HL60 cell proliferations was caused by G0/G1 cell cycle arrest as well as by an apoptotic effect. Treatment with the same concentration of troglitazone also induced the monocytic differentiation of HL60 cells. The apoptotic or the differentiating effect of TZD on HL60 cells was synergistically enhanced by the combined treatment with 1 µM LG100268, while LG100268 alone neither had an apoptotic nor a differentiating effect on HL60 cells. These results suggest that these actions are mediated through the nuclear receptor system constituted by the PPARγ: RXR heterodimer.

Late‐appearing Philadelphia chromosome in a patient with acute nonlymphocytic leukaemia derived from myelodysplastic syndrome: detection of P210‐ and P190‐type bcr/abl fusion gene transcripts at the leukaemic stage

Summary. We describe a patient with acute nonlymphocytic leukaemia (ANLL) derived from myelodysplastic syndrome in whom the Philadelphia chromosome (Ph1) first emerged at the late stage of ANLL transformation. Cytogenetically, the Ph1 chromosome was not detected until the late stage of ANLL transformation, 14 months after the transformation following a 3‐month history of refractory anaemia with excess of blasts. The cells with and without the Ph1 chromosome had a common abnormal chromosome, t(3;3) (q21;q26). The reverse transcription‐polymerase chain reaction analysis showed no bcr/abl message at diagnosis. However, the mRNA encoding P210bcr/abl was detected in the early stage of ANLL transformation. Furthermore, the mRNAs encoding both P210bcr/abl and P190bcr/abl were detected in the late stage of ANLL transformation when the Ph1 chromosome was detected by cytogenetic analysis. These evidences support a multistep pathogenesis of leukaemias, and the product of bcr/abl fusion gene may influence the course of disease.

Thiazolidinedione suppresses the expression of erythroid phenotype in erythroleukemia cell line K562

The activation of PPARgamma:RXR nuclear system induces monocytic differentiation of some myelogeneous leukemia cell lines. The present study was undertaken to examine the effect of PPARgamma ligand, TZD (troglitazone or pioglitazone) and/or RXR selective ligand, LG100268 on the erythroleukaemia cell line K562 which has both an erythroid character and a potential for differentiation into megakaryocytes. TZD suppressed cell proliferation and the erythroid phenotype of K562 cells. The suppression of erythroid phenotype of K562 cells by TZD was synergistically enhanced by the combined treatment with LG100268. Moreover, the marked suppression of erythroid phenotype in K562 cells was also accompanied by the downregulation of the erythroid lineage-transcription factor, GATA-1. These novel actions of troglitazone may provide a biochemical basis for anemia occasionally which is observed after the in vivo administration of TZD.

Enhanced erythroid cell differentiation in hypoxic condition is in part contributed by miR-210.

Erythropoiesis, a process of erythroid production, is controlled by several factors including oxygen level. In this study, the effect of oxygen tension on erythropoiesis was investigated in K562 erythroleukemic cell line and erythroid progenitor cells derived from normal and β-thalassemia/hemoglobin (Hb) E individuals. The enhanced erythroid differentiation specific markers including increased levels of α-, β- and γ-globin gene expressions, numbers of HbF positive cells and the presence of glycophorin A surface marker were observed during cell culture under hypoxic atmosphere. The result also showed that miR-210, one of the hypoxia-induced miRNAs, was up-regulated in K562 and β-thalassemia/HbE progenitor cells cultured under hypoxic condition. Inhibition of miR-210 expression leads to reduction of the globin gene expression and delayed maturation in K562 and erythroid progenitor cells. This indicated that miR-210 contributes to hypoxia-induced erythroid differentiation in both K562 cells and β-thalassemia/HbE erythroid progenitor cells.

Imatinib induces demethylation of miR-203 gene : An epigenetic mechanism of anti-tumor effect of imatinib

MicroRNA (miRNA) is an important regulator of cellular proliferation, differentiation and death. Leukemia-specific signature of miRNAs suggests that epigenetic dysregulation of miRNAs is important for leukemogenesis. We focused on the role of DNA methylation of miR-203 which targets BCR-ABL1 mRNA. The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment. Imatinib induced the demethylation of the miR-203 promoter region, resulting in low expression of targeted BCR-ABL1 gene, and loss of proliferation of leukemic cells. In conclusion, demethylation of miR-203 is one of the molecular mechanisms of imatinib-induced inhibition of BCR-ABL1-positive leukemic cells.

Modulation of expression of multidrug resistance gene (mdr-1) by adriamycin

The acquired resistance to various drugs in cancer is mediated by P‐glycoprotein (P‐gp) which is encoded by the mdr‐1 gene. An increased level of mdr‐1/P‐gp was demonstrated after chemotherapy administered to treat cancer in humans. To clarify the direct effect of anticancer drugs on mdr‐1/P‐gp expression, we investigated the change in transport of adriamycin (ADR), and the expression of the mdr‐1 gene and P‐gp in an ADR‐treated, multidrug‐resistant leukemic cell line (K562/ADR500). The addition of ADR induced the over‐expression of mdr‐1/P‐gp, which led to a transient decrease in the intracellular accumulation of ADR although the difference was not statistically significant. A maximal effect was observed after 4 h incubation, returning to the baseline level after further incubation for 12–24 h. The phosphorylation of P‐gp was inversely correlated with the levels of P‐gp. These observations suggest that ADR itself modulates both the expression and function of P‐gp. Determination of the optimal schedule for administering adriamycin is essential to achieving the optimal effect in treating cancer.

Clinical significance of human bone marrow stromal cell colonies in acute leukemias.

Human bone marrow-derived fibroblastoid colony-forming cells (CFU-F) and adipocyte colonies in 36 patients with acute leukemia were studied, and were serially analysed at different clinical stages. At untreated stage, CFU-F number in acute lymphoblastic leukemia (ALL) was lower than that in acute non-lymphoblastic leukemia (ANL). In ANLs, CFU-F number in M1 was lower than that in M2. Adipocyte colonies were frequently developed at regenerating and relapsing stages, but rarely at untreated and remission stages. The adipocyte colony formation did not correlate with any of CFU-F number, marrow cellularity nor number of leukemic cells, but might be associated with hemopoietic regeneration. The favorable prognosis was associated with normal CFU-F number and with adipocyte colony formation at regenerating or bottom stage. As adipocytes in marrow samples were completely removed before cultures, adipocyte colony was probably originated from preadipocytes. Thus, our results suggest that adipocyte precursor cells increase in regenerating marrow and that they are essential in active hemopoiesis.

Telomerase activity in myeloma cells is closely related to cell cycle status, but not to apoptotic signals induced by interferon-α

Telomeres, G-rich structures at the ends of chromosomes are essential for maintaining chromosomal integrity. Most tumor cells contain telomerase, a ribonucleoprotein that elongates telomeric repeats, and it plays an essential role in indefinite proliferation. To better understand regulatory mechanisms of telomerase, in relationship with apoptosis and the cell cycle, we examined telomerase activity in PCM6, an interleukin-6 (IL-6)-responsive, interferon-alpha (IFN-alpha)-sensitive multiple myeloma cell line, using a PCR-based assay. When PCM6 cells were cultured in serum-free media, the addition of IFN-alpha resulted in apoptosis of the cells, but with no influence on telomerase activity. When IFN-alpha was added to the culture with serum plus rIL-6 after serum deprivation, G1-S transition was inhibited and telomerase activity was lower compare to findings in culture with no IFN-alpha. Dose response experiments of rIL-6 and IFN-alpha, and the measurement of telomerase activity of sorted cells in S-phase using CD71, demonstrated a higher activity of telomerase in the samples which contained a larger proportion of cells in S-phase. These data indicate that regulation of telomerase activity is closely related to cell cycle status, in particular cells in S-phase have an high telomerase activity. While telomeres play an important role in cellular senescence, the regulation of telomerase is independent from apoptotic signals induced by IFN-alpha in myeloma cells.

Hematopoietic growth factors (BPA and Epo) induce the expressions of c-myc and c-fos proto-oncogenes in normal human erythroid progenitors.

We investigated serial expressions of eight proto-oncogenes during in-vitro differentiation of normal human burst-forming unit, erythroid (BFU-E), and found that c-myc and c-fos are expressed in progenies of BFU-E. The expressions of the two proto-oncogenes correlated to the replating efficiency and adversely to erythroid differentiation. The absence of hematopoietic growth factors decreased the expressions, but the addition of erythropoietin together with burst promoting activity induced a re-expression of the c-myc and c-fos after 2 h of incubation. These observations suggest that the c-myc and c-fos proto-oncogenes have a physiological role in the proliferation of erythroid progenitors and that activations of the two proto-oncogenes are early cellular events after the stimulation by hematopoietic growth factors.

Regulation of erythropoietin and burst-promoting activity production in patients with aplastic anemia and iron deficiency anemia.

To clarify the control mechanism of production of erythropoietic growth factors in anemic states, we compared erythropoietin (Epo) and burst-promoting activity (BPA) in patients with aplastic anemia and iron deficiency anemia, using in vitro erythroid progenitor assays. Although serum levels of Epo activity increased in the presence of anemia, the rise was more marked in patients with aplastic anemia. BPA was high only in the sera of aplastic anemia patients. Serum levels of BPA of patients with aplastic anemia negatively correlated with hemoglobin concentrations, while those of patients with iron deficiency anemia did not correlate. In 2 patients with aplastic anemia who responded well to androgen therapy, serum levels of Epo activity and BPA decreased after the hemopoiesis had recovered. These results suggest that serum levels of BPA do not rise in response to anemia only. The elevated BPA levels in sera in cases of aplastic anemia are probably related to a reduction in the number of hemopoietic stem cells. Moreover, we observed that BPA in bone-marrow-conditioned medium (BMCM) from patients with severe aplastic anemia increased more than in the BMCM from patients with severe iron deficiency anemia. Therefore, our findings suggest that the enhanced BPA production depends on a decrease in hemopoietic precursors rather than the anemic state.