Arrest In K562 Cells Research Articles

Smenospongine, a Sesquiterpene Aminoquinone from a Marine Sponge, Induces G1 Arrest or Apoptosis in Different Leukemia Cells

Smenospongine, a sesquiterpene aminoquinone isolated from the marine sponge Dactylospongia elegans, was previously reported by us to induce erythroid differentiation and G1 phase arrest of K562 chronic myelogenous leukemia cells. In this study, we investigated the effect of smenospongine on the cell cycles of other leukemia cells, including HL60 human acute promyelocytic leukemia cells and U937 human histiocytic lymphoma cells by flow cytometric analysis. Smenospongine induced apoptosis dosedependently in HL60 and U937 cells. The smenospongine treatment increased expression of p21 and inhibited phosphorylation of Rb in K562 cells, suggesting the p21-Rb pathway play an important role in G1 arrest in K562 cells. However, the p21 promoter was not activated by the smenospongine treatment based on a luciferase assay using the transfected K562 cells. Smenospongine might induce p21 expression via another mechanism than transactivation of p21 promoter.

Smenospongine, a Sesquiterpene Aminoquinone from Marine Sponge, Induces G1 Arrest or Apoptosis in Different Leukemia Cells

Smenospongine, a sesquiterpene aminoquinone isolated from the marine sponge Dactylospongia elegans, was previously reported by us to induce erythroid differentiation and G1 phase arrest of K562 chronic myelogenous leukemia cells. In this study, we investigated the effect of smenospongine on the cell cycles of other leukemia cells, including HL60 human acute promyelocytic leukemia cells and U937 human histiocytic lymphoma cells by flow cytometric analysis. Smenospongine induced apoptosis dose-dependently in HL60 and U937 cells. The smenospongine treatment increased expression of p21 and inhibited phosphorylation of Rb in K562 cells, suggesting the p21-Rb pathway play an important role in G1 arrest in K562 cells. However, the p21 promoter was not activated by the smenospongine treatment based on a luciferase assay using the transfected K562 cells. Smenospongine might induce p21 expression via another mechanism than transactivation of p21 promoter.

Induction of G2/M phase arrest by squamocin in chronic myeloid leukemia (K562) cells

Squamocin is one of the annonaceous acetogenins and has been reported to have anticancer activity. Squamocin was found to inhibit the growth of K562 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in K562 cells following 24 h exposure to squamocin. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a dose-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that squamocin did not change the steady-state levels of Cdk2, Cdk4, cyclin A, cyclin B1, cyclin D3 and cyclin E, but decreased the protein levels of Cdk1 and Cdc25C. These results suggest that squamocin inhibits the proliferation of K562 cells via G2/M arrest in association with the induction of p21, p27 and the reduction of Cdk1 and Cdc25C kinase activities.

Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A

Purpose: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death.Materials and methods: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2 × 105 cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200 – 300 cGy/min. The cells were treated with 0.25 μM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins.Results: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation.Conclusions: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.

The modulation of radiation-induced cell death by genistein in K562 cells: Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.

The modulation of radiation-induced cell death by genistein in K562 cells: activation of thymidine kinase 1.

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression of TK1 mRNA and TK1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B1, provided the evidences of G1/S progression and G2-arrest, and their relationship with TK1 in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.

Smenospongine, a spongean sesquiterpene aminoquinone, induces erythroid differentiation in K562 cells.

The differentiation of K562 chronic myelogenous leukemia (CML) cells by smenospongine, which is a sesquiterpene aminoquinone isolated from a marine sponge, was examined. Smenospongine increased hemoglobin production in K562 cells at concentrations of 3-15 microM. In addition, flow cytometric analysis of smenospongine-treated K562 cells with FITC-labeled glycophorin A antibody showed an increase of glycophorin A expression, a marker for erythroid differentiation. Cell-cycle analysis showed G1 arrest in K562 cells after treatment with smenospongine for 24 h. The effect on expression of CIP/KIP family cyclin-dependent kinase inhibitors was investigated by Western blotting analysis and the result showed increased expression of p21, which is known to play an important role in differentiation. Furthermore, smenospongine was also found to inhibit the phosphorylation of Crkl, a substrate of Bcr-Abl tyrosine kinase, which is known as a causative protein of CML. In conclusion, our investigation indicated that smenospongine induced the differentiation of K562 cells into erythroblasts along with cell-cycle arrest at G1 phase and the mechanism might be attributed to the increased expression of p21.

Differentiation-inducing factor-1-induced growth arrest of K562 leukemia cells involves the reduction of ERK1/2 activity

The differentiation-inducing factor-1 (DIF-1) is a signal molecule that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 is a potent antileukemic agent that induces growth arrest in K562 cells. In this study, we investigated the mechanism of action of DIF-1 in K562 cells in the light of cell-cycle regulators such as cyclins, retinoblastoma protein (pRb), and the mitogen-activated protein kinase (MAPK) family. DIF-1 down-regulated cyclins D/E and a phosphorylated form of pRb (p-pRb), and thereby induced G 1 arrest of the cell cycle. DIF-1 inactivated the extracellular signal-regulated kinase (ERK) in a biphasic manner but did not affect the c-Jun N-terminal kinase (JNK) or p38 MAPK. The MEK (MAPK kinase) inhibitor, U0126, which has been shown to induce growth arrest, inactivated ERK and down-regulated cyclins D and E. Although DIF-1 activated the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway, neither wortmannin nor 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY294002; PI-3K inhibitors) cancelled DIF-1-induced growth arrest. The present results suggest that ERK inactivation may be involved in DIF-1-induced growth arrest and that PI-3K activity is not required for DIF-1-induced growth arrest in K562 cells.

Protein tyrosine kinase inhibitor, genistein, enhances apoptosis and cell cycle arrest in K562 cells treated with γ-irradiation

Genistein, is a natural isoflavone compound with a potent activity against protein tyrosine kinases. The leukemic cell line, K562, is a bcr/abl (Philadelphia chromosome) positive cell line that is resistant to DNA-damaging agents, including γ-irradiation. Treatment with genistein increased apoptosis and promoted G2-phase arrest in the non-apoptotic population of the γ-irradiated K562 cells. Irradiated cells that survived 72 h after the irradiation had a normal distribution in cell cycle, whilst genistein treatment kept cells arrested in the G2-phase, decreased the S-phase fraction and suppressed DNA-synthesis. Taken together, our results show that genistein augments apoptotic cell death after γ-irradiation in K562 cells and this result cannot be attributed to abrogation of the G2/M checkpoint.