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Knocking-down cyclin A(2) by siRNA suppresses apoptosis and switches differentiation pathways in K562 cells upon administration with doxorubicin.

Wang X, Song Y, Ren J, Qu X - PLoS ONE (2009)

Bottom Line: Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways.The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin, China.

ABSTRACT
Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation. To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells.

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Knocking–down the expression of cyclin A2 in K562 cells significantly inhibited erythroid differentiation induced by low concentration DOX.Cells were transfected with cyclin A2 siRNA or not two hours prior to the addition of 0.4 µM DOX. Forty hours later, erythroid differentiation was scored by the benzidine staining method to determine the percentage of hemoglobin-positive K562 cells. Tests were done four times, counting a minimum of 300 total cells from at least three random microscope fields each. *p<0.05, **p<0.001 vs. control untreated cells by Student's t-test.
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pone-0006665-g007: Knocking–down the expression of cyclin A2 in K562 cells significantly inhibited erythroid differentiation induced by low concentration DOX.Cells were transfected with cyclin A2 siRNA or not two hours prior to the addition of 0.4 µM DOX. Forty hours later, erythroid differentiation was scored by the benzidine staining method to determine the percentage of hemoglobin-positive K562 cells. Tests were done four times, counting a minimum of 300 total cells from at least three random microscope fields each. *p<0.05, **p<0.001 vs. control untreated cells by Student's t-test.

Mentions: As mentioned above, cells treated with both cyclin A2 siRNA and DOX were much bigger than the control. Since enlarged phenotype may suggest cell differentiation, we performed the benzidine staining to assess erythroid differentiation, which was the differentiation pathway of K562 cells upon treatment with anthracycline antibiotics including DOX [42], [43]. Representative microscopy images of the benzidine staining in K562 cells after various treatments were shown in Fig. S5. For untreated cultures and cells administered with SWNTs, the percentages of benzidine positive cells were very low (less than 2%). Forty hours after incubation with DOX, around 14% benzidine positive cells were observed. However, down-regulation of cyclin A2 by siRNA in K562 cells substantially suppressed erythroid differentiation upon administration with DOX (less than 1% benzidine positive cells, as shown in Fig. 7).


Knocking-down cyclin A(2) by siRNA suppresses apoptosis and switches differentiation pathways in K562 cells upon administration with doxorubicin.

Wang X, Song Y, Ren J, Qu X - PLoS ONE (2009)

Knocking–down the expression of cyclin A2 in K562 cells significantly inhibited erythroid differentiation induced by low concentration DOX.Cells were transfected with cyclin A2 siRNA or not two hours prior to the addition of 0.4 µM DOX. Forty hours later, erythroid differentiation was scored by the benzidine staining method to determine the percentage of hemoglobin-positive K562 cells. Tests were done four times, counting a minimum of 300 total cells from at least three random microscope fields each. *p<0.05, **p<0.001 vs. control untreated cells by Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2721982&req=5

pone-0006665-g007: Knocking–down the expression of cyclin A2 in K562 cells significantly inhibited erythroid differentiation induced by low concentration DOX.Cells were transfected with cyclin A2 siRNA or not two hours prior to the addition of 0.4 µM DOX. Forty hours later, erythroid differentiation was scored by the benzidine staining method to determine the percentage of hemoglobin-positive K562 cells. Tests were done four times, counting a minimum of 300 total cells from at least three random microscope fields each. *p<0.05, **p<0.001 vs. control untreated cells by Student's t-test.
Mentions: As mentioned above, cells treated with both cyclin A2 siRNA and DOX were much bigger than the control. Since enlarged phenotype may suggest cell differentiation, we performed the benzidine staining to assess erythroid differentiation, which was the differentiation pathway of K562 cells upon treatment with anthracycline antibiotics including DOX [42], [43]. Representative microscopy images of the benzidine staining in K562 cells after various treatments were shown in Fig. S5. For untreated cultures and cells administered with SWNTs, the percentages of benzidine positive cells were very low (less than 2%). Forty hours after incubation with DOX, around 14% benzidine positive cells were observed. However, down-regulation of cyclin A2 by siRNA in K562 cells substantially suppressed erythroid differentiation upon administration with DOX (less than 1% benzidine positive cells, as shown in Fig. 7).

Bottom Line: Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways.The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin, China.

ABSTRACT
Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation. To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells.

Show MeSH
Related in: MedlinePlus