Nuclear topography and expression of the BCR/ABL fusion gene and its protein level influenced by cell differentiation and RNA interference.
Nuclear topography, expression of the BCR/ABL fusion gene and its protein level/cellular pattern were studied in CML cell line K562 stimulated to differentiation, apoptosis and influenced by ABL-RNA interference (ABL-RNAi). Phorbol ester-induced maturation of K562 cells was accompanied by repositioning of down-regulated BCR/ABL genes closer to the nuclear membrane. This nuclear rearrangement could be connected with differentiation-related heterochromatinization of the amplified BCR-ABL locus, as demonstrated by increased histone H3(K9) dimethylation and decreased H3(K9) acetylation of B3A2 breakpoint. Topography of BCR/ABL in differentiated K562 cells was compared with other leukemic cell types: PMA-maturation of HL60 cells did not influence the nuclear positioning of individual BCR and ABL genes. Moreover, BCR and ABL genes in non-stimulated HL60 as well as in the bone marrow cells of CML patients, i.e. also BCR/ABL fusion genes, were positioned more interiorly in comparison with BCR/ABL multiple loci of K562 cells. Decreased expression of BCR/ABL gene was also found after cell stimulation by selectively pro-apoptotic agent etoposide and by ABL-RNAi leading to apoptosis. In order to compare the efficiency of selected experimental strategies, levels of Bcr/Abl and c-Abl proteins were determined and in all cases tested were reduced. In K562 cells the Bcr/Abl and c-Abl proteins were distributed homogeneously in both the cell nucleus and cytoplasm, while differentiation of K562 cells was characterized by a distinct pattern of Bcr/Abl and c-Abl proteins that were focally distributed rather in the cytoplasm while apoptotic population was completely absent of Bcr/Abl and c-Abl signals.
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.
Eva
E
Bártová
Andrea
A
Harnicarová
Jirí
J
Pacherník
Stanislav
S
Kozubek
D000954:Antigens, Surface / Q000187:drug effects / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D015536:Down-Regulation; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D034622:RNA Interference / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology
D000954:Antigens, Surface; C016658:phorbolol myristate acetate; D016044:Fusion Proteins, bcr-abl; D013755:Tetradecanoylphorbol Acetate