Abstract
AbstractThe biological function of germanium oxide (GeO2) was investigated in thisstudy. The function of germanium compounds at molecular and cellular levels arerarely investigated. In the first part, the effect of GeO2 on cell cycle progression wasinvestigated. GeO2 is not genotoxic to Chinese hamster ovary (CHO) cells and haslimited cytotoxicity. However, GeO2 arrests cells at G2/M phase. The proportion ofcells stopped at G2/M phase increased dose-dependently from 0 to 5 mM GeO2,for12 h but decreased at GeO2 concentration was greater than 5 mM. Analysis of5-bromodeoxyuridine-labeled cells indicated that GeO2 delayed S phase progressionin a dose-dependent manner, and blocked cells at G2/M phase. Confocal microscopicexamination confirmed that GeO2 treatment arrested cells at G2 phase. Similar toseveral other events that cause G2 block, the GeO2-induced G2 block can also beameliorated by caffeine in a dose- and time-dependent manner. To explore themechanism of G2 arrest by GeO2, cyclin content and cyclin-dependent kinase activitywere examined. Cyclin B1 level was not affected after GeO2 treatment in CHO cells.However, GeO2 decreased p34cdc2 kinase (Cdk1) activity. The kinase activityrecovered within 9 h after GeO2 removal and correlated with the transition of G2/Mto G1 phase of the cells. This result suggests that GeO2 treatment reduces Cdk1activity and causing the G2 arrest in CHO cells.In the second part, we investigated here the combined effect of GeO2 andradiation on cell viability. Cells were treated with 0 to 22 mM GeO2 for 12 hfollowed by 1 Gy X-irradiation. A synergistic cytotoxic effect was observed for thecombined treatment with a dose dependent reduction of cell viability. Completesurvival curves showed a 2.3- and 2.75-fold increase in radiosensitivity for 50% celldeath in the presence of 5 and 15 mM GeO2, respectively. The increasedradiosensitivity also occurred when GeO2 was given either 4 h prior to radiation orimmediately after radiation exposure. GeO2 did not affect total soluble thiol contentor the activities of catalase and glutathione S-transferase. Analysis of the productionof reactive oxygen species (ROS) revealed that the combined treatment dramaticallyincreased the synthesis of the ROS. Addition of N-acetyl cysteine (NAC, 20 mM)decreased the ROS production in cells. NAC, however, only slightly increased cellviability after GeO2 and radiation exposures. Thus, increased ROS production maypartly, if at all, attribute to cell death. The combination of GeO2 and X-irradiation,however, significantly increased DNA double-strand-break (dsb) frequency. Notably,the presence of GeO2 also reduced the efficiency of DNA repair. We conclude thattreatment with GeO2 followed by X-irradiation increases DNA dsb and cell death.In the third part, we investigated the apoptotic effect of GeO2 on hematogeniccells, HL-60. When cells were treated with 5 to 22 mM GeO2 for 24h, a G2/M arrestwas observed. Additionally, sub-G1 fraction (apoptotic cells) increased withincreasing concentrations of GeO2 as analyzed by flow cytometry, using agarose gelelectrophoresis and FITC-labeled annexine-V to analyze the GeO2-treated cells, asimilar result was found as that of flow cytometry. The apoptotic effect can bereduced by caspase inhibitor, z-VAD-fmk. For the GeO2-induced apoptosis, increaseof ROS, mitochondrial membrane potential, ratio of Bax/Bcl-XL were noted. Besides,the level of cytosolic cytochrome c and apoptotic-inducing factor also elevated.These increased were caspase-independent because z-VAD-fmk could not reverse theresults. However, caspase-3 was activated and caused the cleavage of PARP andresulted in apoptosis. Treatment of GeO2 activate the ERK, p38 and JNK. Theinhibitor of ERK, PD98059, stimulated the GeO2-induced apoptosis while the p38inhibitor, SB203580 had no effect. This result shows that JNK may be involved in theGeO2-induced apoptosis and ERK protects from cell death in the process.