Abstract
BackgroundKaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. There is consistent evidence of the role of Kaiso and its involvement in human tumorigenesis but there is no evidence about its role in hematopoietic differentiation or establishment of chronic myeloid leukemia (CML). We used, normal K562 cell line, established from a CML patient in blast crisis, and imatinib-resistant K562 cell line, to investigate the specific distribution of Kaiso and their contribution to the cell differentiation status of the blast crisis of CML (CML-BP).ResultsWe found cytoplasmic expression of Kaiso, in K562 cells and patients, confirmed by immunofluorescence, immunohistochemistry and western blot of cytoplasmic protein fraction. Kaiso was weakly expressed in the imatinib-resistant K562 cell line confirmed by immunofluorescence and western blot. The cytoplasmic expression of Kaiso was not modified when the K562 cells were treated for 16 h with imatinib 0.1 and 1 μM. In our study, small interfering RNA (siRNA) was introduced to down regulate the expression of Kaiso and p120ctn in K562 cell line. Kaiso and p120ctn were down regulated individually (siRNA-Kaiso or siRNA-p120ctn) or in combination using a simultaneous co-transfection (siRNA-Kaiso/p120ctn). We next investigated whether knockdown either Kaiso or p120ctn alone or in combination affects the cell differentiation status in K562 cells. After down regulation we analyzed the expression of hematopoietic cell differentiation and proliferation genes: SCF, PU-1, c-MyB, C/EBPα, Gata-2 and maturation markers of hematopoietic cells expressed in the plasma membrane: CD15, CD11b, CD33, CD117. The levels of SCF and c-MyB were increased by 1000% and 65% respectively and PU-1, Gata-2 and C/EBPα were decreased by 66%, 50% and 80% respectively, when Kaiso levels were down regulated by siRNA. The results were similar when both Kaiso and p120ctn were down regulated by siRNA. The increased expression of SCF and decreased expression of GATA-2 could be responsible by the higher cell viability detected in K562 cells double knock-down of both Kaiso and p120ctn. Finally, we studied the effect of knock-down either Kaiso or p120ctn, alone or in combination on CD15, CD11b, CD33 and Cd117 expression. Using siRNA approach a reduction of 35%, 8% and 13% in CD15, CD33 and CD117 levels respectively, were achieved in all transfections, when compared to scrambled knock-down cells.ConclusionThese results suggest that both Kaiso and p120ctn, contributes to maintaining the differentiated state of the K562 cells and similar to other cancers, cytoplasmic localization of Kaiso is related to a poor prognosis in CML-BP. By the broad and profound effects on the expression of genes and markers of hematopoietic differentiation produced by Kaiso knock-down, these findings reveal Kaiso as a potential target for selective therapy of CML.
Highlights
Kaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer
We started by characterizing its subcellular distribution in K562 cell line since it has been considered as a cellular model of chronic myeloid leukemia (CML)-BP
We investigated whether knock-down either Kaiso or p120ctn alone or in combination affects the global cell differentiation, evaluating the maturation markers of hematopoietic differentiation CD15, CD11b, CD33 and CD117 expressed in the plasma membrane of K562 cells by FACS analysis
Summary
Kaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. The disease follows a triphasic course: an initial chronic phase (CP) lasting 3–5 years, an accelerated phase (AP) lasting 6–18 months and the final phase called blast crisis (BC) or acute leukemia, defined hematologically by the increase of leukemic blasts (myeloid or lymphoid) in peripheral blood and/or bone marrow (more than 20%) At this stage of the disease, many patients died between three and six months, because they are refractory to most treatments, including resistance to imatinib [3]. Imatinib selectively induces growth arrest and apoptosis of bcr-abl-positive leukemia cells with minimal effect on normal hematopoietic progenitors [6,7,8] Of note, this agent has proven very effective in patients in chronic phase of CML [9] and to a lesser extent, in patients in accelerated phase and blast crisis [7]. Treatment with imatinib achieves complete hematologic remission in the great majority of patients with CML, total cytogenetic and molecular responses are relatively rare events [10]
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