Conceived and designed the experiments: JR XQ. Performed the experiments: XW. Analyzed the data: XW YS JR. Contributed reagents/materials/analysis tools: YS. Wrote the paper: XW JR XQ.
The authors have declared that no competing interests exist.
Cyclin A2 is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A2 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 A2 and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A2 by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A2 in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A2 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 A2 and suggest that cyclin A2 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.
Tumor cells are characterized by deregulation of cell cycle checkpoints, leading to uncontrolled cell division and proliferation under conditions where non-transformed cells cannot enter and pass through the cell cycle. All these may come from over-expression of cyclins and the abnormal activation of cyclin-dependent kinases (CDKs)
Until recently it has been held that CDK2, presumed master of the known CDK isoforms, is a promising anticancer target for developing small molecule inhibitors. The first-generation CDK inhibitors, flavopiridol and CY-202, are in late-stage clinical trials, and have only modest activity
Apoptosis and differentiation are the predominant two mechanisms by which chemotherapeutic agents kill tumor cells. Low dose of doxorubicin (DOX) induces erythroid differentiation in K562 cells, while high concentration of DOX promotes apoptosis
Carbon nanotubes possess the unique features of being able to enter a living cell without causing its death or without inflicting other damage and can shuttle biological molecules into mammalian cells, indicating their potential application as a vector for the delivery of therapeutic molecules
DOX can inhibit growth of a variety of cancer cells
Apoptotic cells were determined by AO/EB staining. Tests were done in triplicate, counting a minimum of 300 total cells from at least three random microscope fields each.
RT-PCR (A) and western blotting (B) were performed 32 hours and 60 hours after drug administration, respectively.
We have previously demonstrated that functionalized single wall carbon nanotubes (SWNTs) can efficiently deliver siRNA targeting cyclin A2 into K562 cells, resulting in specific suppression of cyclin A2 expression
The viable cells were counted by trypan blue exclusion at indicated time points. The data shown here represent the average of two independent experiments.
Cells were transfected with cyclin A2 siRNA (○) or not (•) two hours prior to the addition of drug. The viability was assessed by MTT assay as specified in
To investigate whether cyclin A2 participates in cell apoptosis death and determine if reduction of cyclin A2 has an effect on apoptosis induced by DOX, siRNA specific for cyclin A2 was transfected into K562 cells by SWNTs, and low dose of DOX (0.4 µM) was administered. As shown in
Cells were stained with AO/EB dye mixture as described in
Early reports have demonstrated that there is a link between cyclin A2 subcellular localization and its cell function, and the level of cyclin A2 is correlated to cell apoptosis
Cells were administered with 0.4 µM DOX for 32 h. A significant fraction of cells underwent apoptosis. Immunofluorescence detection of cyclin A2 was performed as described in
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
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. *
To determine whether K562 cells with reduced cyclin A2 upon treatment with DOX underwent megakaryocytic pathway and monocyte-macrophage differentiation, which are the other two differentiation pathways of K562 cells, flow cytometric measurement of megakaryocytic specific surface antigen CD61 (GPIIIa) and nitro blue tetrazolium (NBT) reduction assay were carried out, respectively. As shown in
Cells were transfected with siRNA by SWNTs two hours prior to the administration of 0.4 µM DOX. Sixty hours later, expressions of CD61 (GPIIIa) were evaluated by using FITC-conjugated isotype control immunoglobulin and specific anti-CD61 (GPIIIa) FITC-conjugated monoclonal antibody. The marker placed to the right of histogram designates positive events.
Cells were transfected with siRNA by SWNTs two hours prior to the treatment of 0.4 µM DOX. Ninety six hours later, NBT dye reduction was used to qualitatively monitor monocyte-macrophage differentiation. Tests were done twice, counting a minimum of 300 total cells from at least three random microscope fields each. **
Taken together, these results indicate that knocking down the expression of cyclin A2 suppressed DOX-induced erythroid differentiation and a small fraction of K562 cells with reduced cyclin A2 were differentiated along megakaryocytic and monocyte-macrophage pathways upon treatment with DOX. These findings suggest that cyclin A2 is an important regulator of cell differentiation.
Cyclin A2 is particularly interesting among the cyclin family because it can activate two different CDKs and functions in both S phase and mitosis. In S phase, phosphorylated cyclin A2-CDK2 complexes are suggested to play an important role in the initiation of DNA replication. In mitosis, cyclin A2 may contribute to the control of cyclin B stability. Consistent with its role as a key cell cycle regulator, overexpression of cyclin A2 is associated with transformed cells
Single-walled carbon nanotubes (SWNTs) have been considered as the leading candidate for nanodevice applications ranging from gene therapy and novel drug delivery to membrane separations. We have previously showed that siRNA transfection efficiency of lipofectamine 2000 in K562 cells was low (28%) and some cells underwent apoptosis and necrosis during the process
DOX, a prominent member of anthracycline antibiotics, has been extensively used for treatment of solid tumors and leukemia. It exerts its cytotoxic activity against cancer cells mainly by intercalation into DNA, inhibition of topoisomerase II and helicase activity, leading to cell-cycle arrest at the G2/M phase and apoptosis
It has been reported that there is a link between cyclin A2 and apoptosis
We have demonstrated that SWNTs could effectively deliver cyclin A2 siRNA into K562 cells, significantly suppressing the expression of cyclin A2 with specificity and cell proliferation, and cells with reduced cyclin A2 showed a decrease in the percentage of cells in S phase
Most of chemotherapeutic agents show significant side effects and not all patients benefit from aggressive chemotherapy. Therefore, searching for tumor biological factors which can predict patient prognosis and chemotherapy response would be of most importance. Several studies have indicated that a high level of cyclin A2 expression may be a marker of poor prognosis in cancers
In several systems, it has been reported that down-regulation of cyclin A2 and its associated CDK 2 activity are important for successful differentiation
In conclusion, knocking down the expression of cyclin A2 by siRNA delivered by SWNTs suppresses apoptosis and erythroid differentiation, and promotes megakaryocytic and monocyte-macrophage differentiation in human chronic myelogenous leukaemia K562 cells upon administration with DOX. The results demonstrate the pro-apoptotic role of cyclin A2 and suggest that cyclin A2 is a key regulator of cell differentiation, supporting the notion that cyclin A2 is an important regulator for cell cycle as well as for cell apoptosis and differentiation.
The human erythroleukemic cell line K562
Doxorubicin (DOX), Acridine Orange (AO), Ethidium Bromide (EB), Methylthiazolyldiphenyl-tetrazolium bromide (MTT), DAPI, Nitro blue tetrazolium (NBT), benzidine and SWNTs (φ = 1.1 nm, purity>90%) were purchased from Sigma-Adrich (St. Louis, MO, USA). Annexin V apoptosis detection kit was obtained from Keygentec (Nanjing, China). The dye mix for the EB/AO staining was 100 µg/mL acridine orange and 100 µg/mL ethidium bromide in phosphate buffered saline (PBS).
The human erythroleukemic cell line K562
The siRNA oligonucleotides were synthesized by Genepharma Corporation (Shanghai, China). The sequence used for targeting silencing of cyclin A2 was
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The primers and conditions for cyclin A2 were
For Western blot analysis, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, pH 7.6) containing protease inhibitors for protein extraction. Protein concentrations were determined using the Bradford assay. 20 µg cell samples were denatured by addition of 2× reducing sample buffer (100 mmol/L Tris, 4% SDS, 25% glycerol, 10% β-mercaptoethanol, 0.01% bromphenol blue, pH 6.8), incubated for 10 minutes at 95°C, and separated on a 12% SDS-PAGE. The proteins were electroblotted to PVDF membrane. After blocking with Tris-buffered saline containing 0.05% Tween 20 (TTBS) and 2% BSA, the membranes were incubated overnight at 4°C with appropriate primary antibody diluted in TTBS. Working dilutions were: 1/500 rabbit polyclonal anti-cyclin A2 primary antibody (Lab Vision Corporation, CA, USA); 1/400 anti-GAPDH primary antibody (Santa Cruz, CA, USA). The membranes were washed three times in TTBS for 5 min each and then incubated for 1 h at room temperature with goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Immunoresearch, Stratech, UK). After extensive washing in TTBS, the protein–antibody complexes were visualized by CN/DAB substrate according to method described by Yong
K562 cells were plated in 96-well culture plate at a concentration of 0.5×104 cells/well, and were transfected with cyclin A2 siRNA or not by SWNTs. Two hours later, the cells were treated with various concentrations of DOX while the blank control wells were added medium without drug. Cells were then cultured for another 24 hours and 20 µL MTT (5 mg/mL) was added in each well, followed by additional four hour incubation. The supernatants were then discarded carefully and 150 µL dimethylsulphoxide was added and shaken vigorously to dissolve the purple precipitation formation. Optical density (OD) of each well was tested using Bio-Rad model-680 microplate reader with a wavelength of 490 nm.
Cells were collected by centrifugation at 200×
Cells were harvested, washed three times with PBS, fixed with 4% paraformaldehyde in PBS for 30 min at 4°C and permeabilized with 0.5% Triton X-100 in PBS for 5 min at 4°C. After blocking in 10% goat serum, primary cyclin A2 antibody (rabbit, polyclonal; Lab Vision Corporation, CA) was diluted to 1∶400. Goat anti-rabbit FITC-conjugated secondary antibody (Jackson Immunoresearch, Stratech, UK) was used at a 1∶200 dilution. If necessary, DAPI was used to visualize cell nuclei. Cells were observed and photographed by fluorescence microscopy with oil immersion objective and appropriate filters.
Erythroid differentiation was scored by the benzidine staining method for the determination of the percentage of hemoglobin-positive K562 cells induced by low concentration DOX. Briefly, cells were washed twice and then resuspended in 20 µL PBS. 10 µL of benzidine solution (0.2% benzidine, 0.6% H2O2, 0.5 M acetic acid) was added and incubated for 20 min at room temperature in the dark. Benzidine-positive cells (blue-black staining) were quantitated by light microscopy. At least 300 cells were counted in triplicate for each condition.
Expression of GPIIIa is considered the most selective marker of the megakaryocyte lineage since it is not expressed on cells of other hematopoietic lineages from normal human bone marrow. Hence, staining of cells for surface CD61 (GPIIIa) was used to evaluate megakaryocytic differentiation. It employed a mouse monoclonal antibody fluorescein isothiocyanate (FITC)-conjugated anti-CD61 (eBioscience) or isotype-matched immunoglobulin (IgG1-FITC, eBioscience) at a concentration of 0.6 µg/mL. Cells were harvested, washed, resuspended in PBS containing 10% fetus calf serum and 0.1% NaN3 and incubated for 30 min on ice with antibodies in the dark. After washing three times, cells were resuspended in PBS containing 0.5% formaldehyde and 0.1% NaN3, and then analyzed on a FACS Arial cytometer (Becton-Dickinson, San Diego, CA, USA). Viable cells were gated using forward and side scatter characteristics. Fluorescence intensity data were acquired using the BD FACSDIVA™ software.
NBT dye reduction was used to qualitatively monitor monocyte-macrophage differentiation. Briefly, cells were collected, washed with PBS and resuspended in IMDM medium without serum. Cells suspension were mixed with an equal volume of 0.1% NBT dissolved in PBS and incubated at 37°C for 40 min. NBT was reduced to insoluble formazan because of the intracellular oxygen radical release in the cells differentiated to monocytes-macrophages. The percentage of cells containing intracellular reduced blue-black formazan was determined by light microscopy. At least 300 cells per preparation were observed.
Data are expressed as mean±s.d. and analysis of variance was carried out using Student's t test with Origin 7.5 (OriginLab Corporation, Northampton, MA, USA), where
Knocking-down the expression of cyclin A2 in K562 cells significantly suppressed growth inhibition and apoptosis induced by DOX. Cells were plated in 6-well plate at a density of 0.8×105 cells/mL and transfected with cyclin A2 siRNA (A, B) or not (C, D) by SWNTs two hours prior to the administration of 0.4 µM DOX. 96 hours later, cells were viewed using an inverted microscope with objectives×10 (A, C) and ×40 (B, D). Pictures were taken with an Olympus digital camera. Shown here are the representative images of three independent experiments.
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Chemical interaction of DOX with MTT assay. The assay was performed as follows: 200 µL of medium containing DOX at different concentrations was placed in a 96-well plate; 20 µL of MTT solution (5 mg/mL) was added to each well; After incubation for 4 h at 37°C, 150 µL of DMSO was added to each well and absorbance at 490 nm was measured in a Bio-Rad model-680 microplate reader. DOX-free complete medium was used as control and was treated in the same way as the DOX-containing media. Variation (%) = (absorbance of DOX containing medium - absorbance of control)/absorbance of control×100.
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Annexin V-PI double staining of cells after various treatments. Upper left, untreated control; upper right, cells treated with transfection vector SWNTs; lower left, cells treated with 0.4 µM DOX for 32 h; lower right, two hours after cyclin A2 siRNA transfection, cells were then administered with 0.4 µM DOX for additional 32 h.
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Indirect immunofluorescence detection of cyclin A2 in control K562 cells. DAPI was used to visualize cell nuclei. Cells were viewed using an Olympus BX-51 optical system microscope (Tokyo, Japan) with oil lens and appropriate filters. Representative stained fields are shown: (A), DAPI staining (blue); (B), immunofluorescence detection of cyclin A2 (FITC, green); (C) merged image. As indicated, cyclin A2 was located at the nucleus of K562 cells without DOX treatment.
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Representative microscopy images of the benzidine staining of K562 cells after various treatments. 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 as described in
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Morphological changes of K562 cells after various treatments. Cells were transfected with cyclin A2 siRNA or not two hours prior to the administration of 0.4 µM DOX. Cells were then cultured for another 32 hours. Flow cytometry was used to show changes in size (forward scatter, on X-axis) and granularity (side scatter, on Y-axis). No significant morphological changes of K562 cells were observed upon RNAi mediated by SWNTs compared to the control. Cells administered with DOX underwent G2/M arrest and changes in granularity and cell size were clearly observed. Although knocking-down the expression of cyclin A2 by RNAi significantly inhibited growth suppression and apoptosis induced by DOX, cells co-administered with siRNA targeting for cyclin A2 and DOX showed similar increases in cell size and granularity compared to those treated with DOX alone.
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Flow cytometric measurement of megakaryocytic specific surface antigen CD61 (GPIIIa) in K562 cells. Cells were incubated with 0.4 µM DOX (A, B) or SWNTs (C, D) for 60 h. Expressions of CD61 (GPIIIa) were evaluated by using FITC-conjugated isotype control immunoglobulin (A, C) and specific anti-CD61 (GPIIIa) FITC-conjugated monoclonal antibody (B, D). The marker placed to the right of histogram designates positive events. K562 cells administrated with low concentration of DOX or SWNTs did not undergo apparent megakaryocytic differentiation.
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Representative microscopy images of the NBT reduction assay of K562 cells after various treatments. Cells were transfected with cyclin A2 siRNA or not two hours prior to the addition of 0.4 µM DOX. Ninety six hours later, NBT dye reduction was used to qualitatively monitor monocyte-macrophage differentiation. Cells were viewed and counted using an Olympus BX-51 optical system microscope (Tokyo, Japan) at 200× magnification. Two independent tests were performed. Pictures were taken with an Olympus digital camera.
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We thank Yong Chen (College of Life Science, Jilin University) and Professor Yongchen Zheng (Central Laboratory of the 2nd Hospital, Jilin University) for their technical assistance.