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GATA-1 binding to the αV promoter negatively regulates expression of the integrin αV subunit in human leukemic K562 cells.

100%

Czyż M., Stasiak M., Boncela J., Cierniewski C. S.

Recently we observed that the transcription factors Sp1 and Sp3 bind to the CTCCTCCTC sequence located between positions -194 and -172 of the αV promoter region and are directly involved in the regulation of transcriptional activity of the αV gene in human umbilical vascular endothelial cells (HUVECs) (Czyz & Cierniewski, 1999, Eur. J. Biochem. 265, 638). In this report we provide evidence that the GATA-1 factor regulates αV expression during differentiation of pluripotent K562 cells induced either by phorbol 12-myristate 13-acetate (PMA) or butyric acid (BA) through interaction with the GATA element in the αV gene promoter. DNase I footprinting analysis revealed that region -413 to -408, covering the GATA binding site, was protected by nuclear extract from K562 cells. There was no protection of this region by HUVEC nuclear extract. Electrophoretic mobility shift assay (EMSA) analysis of nuclear extract of K562 cells treated with BA revealed an increase in GATA binding activity, which was associated with reduced αV mRNA and αV protein on the cell surface. Stimulation of K562 cells with PMA resulted in opposite effects: lower expression of GATA-1 was associated with increased levels of αV. We conclude that the GATA-1 transcription factor specifically binds to the GATA element in the αV gene promoter and negatively regulates αV gene expression.

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562

88%

Jakubowska J., Stasiak M., Szulawska A., Bednarek A., Czyz M.

2007

vol. 54

issue 4

839-846

STI571 (imatinib mesylate; Gleevec®) is an inhibitor that targets the tyrosine kinase activity of Bcr-Abl present in chronic myelogenous leukemia (CML) cells. Some preclinical studies have demonstrated that the combination of STI571 with chemotherapeutic drugs results in enhanced toxicity in Bcr-Abl-positive leukemias. We investigated the potential benefit of using STI571 to down-regulate Bcr-Abl activity for the enhancement of doxorubicin anti-proliferative action in K562 cell line derived from blast crisis of CML. At low concentrations of both drugs (40 nM doxorubicin combined with STI571 in the range of 100-150 nM), the antiproliferative effects were mainly due to cellular differentiation as assessed by benzidine staining for hemoglobin synthesis level and real-time PCR for γ-globin expression. Higher concentrations of STI571 used in combinations with doxorubicin caused mainly apoptosis as shown by DNA degradation and nuclear fragmentation visualized by fluorescence microscopy after DAPI staining, changes in cell morphology observed after Giemza-May Grünwald staining and cellular membrane organization estimated by flow cytometry after Annexin V staining. As compared with either drug alone, cotreatment with STI571 and DOX induced stronger cellular responses. A low concentration of STI571 in combination with a low concentration of DOX might be tested as an alternative approach to increasing the efficacy of chemotherapy against CML.

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2 Acta Biochimica Polonica

1 2007

1 2002

2 Stasiak M.

1 Bednarek A.

1 Boncela J.

1 Cierniewski C. S.

1 Czyz M.

1 Czyż M.

1 Jakubowska J.

1 Szulawska A.

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GATA-1 binding to the αV promoter negatively regulates expression of the integrin αV subunit in human leukemic K562 cells.

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562