Cell differentiation and the multiple drug resistance phenotype in human erythroleukemic cells.

The gene expression of Oct-4, a transcription factor and hematopoietic stem cell marker, is higher in Lucena lines, which is MDR, and the gene Alox-5 has also been implicated in the differentiation of some cell lines. The aim of this study was to compare the response to PMA-induced differentiation in MDR and non-MDR cells. We observed the differentiation to megakaryocytes in the K562 cell line, which is non-MDR. The expression of Alox-5 and Nanog genes was downregulated and that of Mdr-1 was upregulated in K562 cells. The Lucena cell line contained a higher number of megakaryocytes than the non-MDR, but this number was not altered by PMA, as well as Mdr-1 gene expression. However, Alox-5 expression was downregulated. Alox-5, Mdr-1, Nanog, Oct-4 and Sox-2 basal expression was also evaluated in the K562, Lucena and FEPS (also MDR) cell lines. The transcription factors gene expression was similar in MDR cell lines. The expression of Alox-5 was higher in the non-MDR cell line, while FEPS had the lowest expression of this gene. The opposite pattern was observed for Mdr-1 gene expression. These results suggest that the Alox-5 gene might play a role in the differentiation of these cell lines.

Hemin induces mitophagy in a leukemic erythroblast cell line.

BACKGROUND INFORMATION: In eukaryotic cells, autophagy is considered a lysosomal catabolic process which participates in the degradation of intracellular components in a vacuolar structure termed autolysosome. This pathway plays a significant role in the erythropoiesis process, contributing to the clearance of some organelles (such as mitochondria) that are not necessary in the mature red blood cells. Nevertheless, the role of autophagy in erythrocyte maturation has not been fully established. RESULTS: Here, we have demonstrated that hemin (a physiological erythroid maturation stimulator) is able to induce the expression of critical autophagic genes (i.e., Map1a1b (LC3), Beclin-1 gen, Atg5) in an erythroleukemia cell type. We have also shown that hemin increased the size of autophagic vacuoles which were labelled with LC3 and the degradative lysosomal marker dye quenched-bovine serum albumin. In addition, we have determined by Western blot a rise in the lipidated form of the autophagic protein LC3 (i.e., LC3-II) upon hemin treatment. Moreover, we provide evidence that hemin induces mitochondrial membrane depolarisation and that mitochondria sequestration by autophagy requires the active form of the NIX protein. CONCLUSIONS: We have found that the physiological erythroid maturation stimulator hemin is able to induce mitophagy in K562 cells, and that the autophagy adaptor NIX is necessary for mitophagy progression. K562 cells have been used as a relevant model to determine the possible therapeutic role of new differentiating compounds. SIGNIFICANCE: It has been proposed that autophagy induction is a feasible new therapeutic key in fighting cancer. Our results suggest that hemin is favoring erythroid maturation by inducing an autophagic response in K562 cells, being a possible therapeutic candidate that may help in the chronic myelogenous leukemia (CML) treatment.

Stathmin 1 inhibition amplifies ruxolitinib-induced apoptosis in JAK2V617F cells.

The JAK/STAT pathway is constitutively activated in myeloproliferative neoplasms and can be inhibited by ruxolitinib, a selective JAK1/2 inhibitor. The JAK2(V617F) mutation leads to constitutive STAT3 phosphorylation and potentially leads to inhibition of Stathmin 1 activity via STAT3. In support of this hypothesis, we found that, in HEL JAK2(V617F) cells, ruxolitinib treatment decreased STAT3 and Stathmin 1 association, induced Stathmin 1 activation and microtubule instability. Silencing of Stathmin 1 significantly reduced cell proliferation and clonal growth, and increased apoptosis induced by ruxolitinib. Stathmin 1 silencing also prevented ruxolitinib-induced microtubule instability. To phenocopy the effect of Stathmin 1 inhibition, cells were treated with paclitaxel, a microtubule-stabilizing drug, in association or not with ruxolitinib; combined treatment significantly increased apoptosis, when compared to monotherapy. Notably, Stathmin 1 mRNA levels were highly expressed in CD34(+) cells from primary myelofibrosis patients. We then proposed that an undesired effect of ruxolitinib treatment may constitute Stathmin 1 activation and microtubule instability in JAK2(V617F) cells. Induction of microtubule stability, through Stathmin 1 silencing or paclitaxel treatment, combined with ruxolitinib could be an effective strategy for promoting apoptosis in JAK2(V617F) cells.

OCT4 mutations in human erythroleukemic cells: implications for multiple drug resistance (MDR) phenotype.

The OCT4 transcription factor is a crucial stem cells marker and it has been related to the cancer stem cells concept. Moreover, it has also been associated to the multiple drug resistance (MDR) phenotype. Our first results pointed out a straight relation between OCT4 and ABC transporters in K562-derivative MDR (Lucena) cells. Sequencing of ABC promoters did not reveal any mutation that could explain the differential expression of OCT4 in Lucena cells. Furthermore, sequencing of the homeobox domain region from the OCT4 gene isolated from both cell lines evinced, for the first time, that this transcription factor is a target of mutations and might be related to the MDR phenotype. The encountered mutations implied in several amino acids substitutions in both cell lines. K562 had seven amino acids substituted (three of them exclusive), while Lucena had 13 substitutions (nine of them exclusive). In addition, an in silico search for phosphorylation motifs within the amino acid stretch compared showed that human normal OCT4 has seven potential phosphorylation motifs. However, K562 has lost one phosphorylation motif and Lucena two of them. These findings bring OCT4 as an important target for cancer treatment, especially those resistant to chemotherapy.

Cytogenetic study of a case of childhood erythroleukemia.

We report a case of childhood erythroleukemia diagnosed by French-American-British Cooperative group (FAB) and by cytogenetic analysis of bone marrow cells. The following major chromosome anomalies were detected: hyperdiploidy with a modal number of 49, three markers consisting of translocations between chromosomes 3, 9, 20, and 15, deletion of the long arm of chromosome 16 (q22----qter), and karyotype instability. These changes were compared with others reported in the literature and discussed in terms of their importance for diagnostic confirmation.