Smad6 Gene and Suppression of Radiation-Induced Apoptosis by Genistein in K562 cells / 대한방사선종양학회지

PURPOSE: The genes involved on the suppression of radiation-induced apoptosis by genistein in K562 leukemia cell line was investigated. MATERIALS AND METHODS: K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. Forx-ray irradiation and drug treatment, cultures were prepared at 2x105 cells/mL. The cells were irradiated with 10 Gy (Clinac 1800C, Varian, USA). Stock solutions of herbimycin A (HMA, Calbiochem, UK) and genistein (Calbiochem, UK) were prepared in dimethylsulfoxide (DMSO, Sigma, UK). After incubation at 37degreesC for 24 h, PCR-select cDNA subtractive hybridization, dot hybridization, DNA sequencing and Northern hybridization were examined. RESULTS: Smad6 gene was identified from the differentially expressed genes in K562 cells incubated with genistein which had been selected by PCR-select cDNA subtractive hybridization. The mRNA expression of Smad6 in K562 cells incubated with genistein was also higher than control group by Northern hybridization analysis. CONCLUSION: We have shown that Smad6 involved on the suppression of radiation-induced apoptosis by genistein in K562 leukemia cell line. It is plausible that the relationship between Smad6 and the suppression of radiation-induced apoptosis is essential for treatment development based on molecular targeting designed to modify radiation-induced apoptosis.

Radiation-induced Apoptosis is Differentially Modulated by PTK Inhibitors K562 Cells / 대한방사선종양학회지

PURPOSE: The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induce d apoptosis in Ph-positive K562 leukemia cell line was investigated. MATERIALS AND METHODS: K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2x10' cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells throughth the cel l cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-X and bax protein levels. RESULTS: Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electro-phoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DN A fragmentation. Enhancement of apoptosis by H MA was not attributable to downregulation of the bcl-2 or bcl-X anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage cf cells in G2/M phase decreased to 30-40% at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. CONCLUSION: We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p2 10""'' failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bcl-2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation-induced apoptosis.