Nickel (II)-Induced Apoptosis and G2/M Enrichment
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EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 30, No 3, 171-176, September 1998 Nickel (II)-induced apoptosis and G2/M enrichment Sang-Han Lee,1,3 Do-Kwon Kim,1 Apoptosis, or programmed cell death, is an active, gene- Young-Rok Seo,2 Kee-Min Woo,1 tically controlled process which allows an organism to Chang-Se Kim1 and Man-Hee Cho1 eliminate unwanted cells through a safe, orderly process (Wyllie, 1980). Apoptosis is characterized morphologically 1 Department of Biochemistry, College of Medicine, by cell shrinkage, apoptotic body formation, chromatin Soonchunhyang University, Cheon-An 330-090, Korea condensation and fragmentation, and biochemically by 2 Toxicology Laboratory, Doping Control Center, KIST, Seoul 136-650, cleavage of DNA into oligonucleosomal DNA fragments. Korea The latter process results in DNA ladders on agarose et al 3 Corresponding author gel electrophoresis (Walker ., 1995). In many cultured cell lines, apoptosis can be readily et al Accepted 3 August 1998 triggered by DNA-damaging agents (Allday ., 1995), γ-irradiation (Sellins and Cohen, 1991), activation of Fas/ et al Abbreviations: PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; CHO, Apo1 receptor (Watanabe-Fukanaga ., 1992), or et al Chinese hamster ovary some anticancer agents (Lowe ., 1993). Activation of the cell death pathway is a safeguard to remove unrepairably damaged cells. Cells also can be restricted to a particular phase of cell cycle for the entry into apoptosis, although this cell cycle dependence does not appear to be a general process. Damaged cells stop et al Abstract DNA replication at G1 (Dulic ., 1994) or G2 phase (O’Connor et al., 1993). The arrest at the G1 and G2 Treatment with certain DNA-damaging agents induce checkpoints indicates the necessity of the cells to gain a complex cellular response comprising pertubation time to repair the damaged DNA before the next round of cell cycle progression and/or apoptosis on proli- of cell cycle. It seems that cell proliferation and ferating mammalian cells. Our studies were focused apoptosis are intrinsically linked, if not in all situations, on the cellular effects of nickel (II) acetate, DNA- at least in many. Recent studies have suggested an damaging agent, on Chinese hamster ovary (CHO) intricate relationship between apoptosis and cell cycle cells. Fragmented DNAs were examined by agarose (King and Cidlowski, 1995). However, it is yet unclear gel electrophoresis and cell cycle was determined whether cell cycle arrest is a prerequisite for the by DNA flow cytometry using propidium iodide activation of the apoptotic process. fluorescence. Apparent DNA laddering was observed In an effort to find the link between cell proliferation in cells treated with 240 M nickel (II) and increased and cell death, we first examined the induction of with a concentration-dependent manner. Tre a t m e n t apoptosis and the cell cycle distribution in Chinese of nickel (II) acetate resulted in apoptosis which hamster ovary (CHO) cells treated with diff e r e n t was accompanied by G2/M cell accumulation. quantities of nickel (II) acetate which induces genotoxic Proportion of CHO cells in G2/M phase was also e ffects such as DNA-protein crosslinking, DNA strand significantly increased in cells exposed to at least breaks, sister chromatid exchange, and oxidative DNA 480 M nickel (II) from 57.7% of cells in the G0/ G1 base damage (Kasprzak, 1995). phase, 34.7% in the S phase, and 7.6 % in the G2/M phase for 0 M nickel (II), to 58.6%, 14.5%, and 26.9% for 640 M nickel (II). These findings suggest Materials and Methods that nickel (II) can modulate cellular response through some common effectors involving in both Cell culture and treatment apoptotic and cell cycle regulatory pathways. Chinese hamster ovary cell variant, CHO-K1-BH4, grew in a monolayer culture in Ham’s F-12 medium (Biofluids, Keywords: DNA damage, apoptosis, cell cycle, flow Inc., Rockville, MD) supplemented with 5% fetal bovine cytometry, Sub-G1, DNA ladder serum, 1 mM glutamine, 100 unit of penicillin/ml, and 100 μg of streptomycin/ml. Cultures were maintained at Introduction 37˚C in a humidified air containing 5% CO2. To eliminate the possible interfering factor such as confluency- The critical balance between cell proliferation and death induced apoptosis (Brezden and Rauth et al., 1996), the is imperative for the survival of multicellular organisms. cells were differently seeded in 10-cm diameter culture 172 Exp. Mol. Med. plates, 5ለ1 05 or 1ለ1 06 cells/plate, 24 h prior to the iodide-stained cells were assayed at 488 nm on an EPICS addition of nickel (II) acetate (Sigma, St. Louis, MO), Profile flow cytometer (Coulter, Hialeah, FL) equipped with freshly dissolved in the above medium. Plates seeded an air-cooled 20 mW argon laser. A minimum of 10,000 with 5ለ1 05 cells were used for treatments with 0, 40, cells were collected in each run. The cells were selected 80, and 160 μM nickel (II) and plates seeded with 1ለ by pulse-height (doublet elimination) analysis and only 106 cells were used for treatments with 240, 360, 480, the integrated signals were collected to reject doublets. and 640 μM nickel (II). The cells were incubated with All histograms were evaluated by Multicycle software nickel (II) acetate for 24, 48, and 72 h. They were then (Advanced version, Phoenix Flow Systems, San Diego, harvested with 2 ml trypsin (0.25% w/v, without calcium CA) (Kallioniemi et al., 1994). The G0/G1 and G2/M peaks and magnesium, Biofluids, Inc.) for DNA flow cytometry were determined using Gaussian curves. Resolution of and DNA fragmentation assay. G0/ G1 peaks was evaluated by coefficient of variation which is calculated by dividing the half-peak width by Laser Scanning Microscopy the mean channel number. The S phase fraction was Cells were grown on coverslips for 3 days, and treated calculated by the area under the zero order polynomial / G p e a k with 240 μM nickel (II) acetate for 3 days. The media curve between mean channel number of G0 1 and that of G2/M peak. were removed, and then the cells were stained by direct applying with acridine orange (Sigma, St. Louis, MO; 40 μg/ml in PBS) and ethidium bromide (Sigma; 20 μg/ml Statistical analysis in PBS). Following washing once with PBS with special Statistical evaluation of the data was performed either care to avoid the excessive cell loss, the coverslip was by analysis of variance (ANOVA) or by Student’s t-test mounted on a slide and then observed with a LSM 410 for paired comparisons. Trends were examined by laser scanning microscope (Carl Zeiss, Jena, Germany). linear regression statistics using the SAS program. D i fferences with probability values ≤ 0.05 were Analysis of DNA ladder for apoptosis considered to be statistically significant. The analysis for DNA fragmentation was carried out by the method described by Gong et al. (1993). The floating and attached cells from each plate were pooled by Results and Discussion centrifugation and fixed in 70% ethanol at -20˚C for 72 h. The cells were then centrifuged at 800g for 5 min and CHO cell morphology the cell pellets (1-2ለ106) were resuspended in 40 μl of Morphological changes in response to the treatment of phosphate-citrate (PC) buffer, consisting of 192 parts of CHO cells with nickel (II) acetate were examined by 0.2 M Na2HPO4 and 8 parts of 0.1 M citric acid (pH 7.8) confocal microscopy after staining with acridine orange. for at least 30 min. After centrifugation at 1000g for 5 Nuclear fragmentation, indicative of apoptosis, was min, cell pellets were used in cell cycle analysis and the apparent in 240 μM treated adherent cells at day 3 after supernatant was transferred to new tube and concentrated treatment (Figure 1). Also at this time, 18% of the by vacuum in Speed Vac concentrator for 20 min. A 5 μl adherent cells exhibited pyknotic, fragmented nuclei. of a solution of DNase-free RNase (Sigma; 0.4 mg/ml) Phase-contrast microscopy of CHO cells treated with 480 was then added. After 1 h incubation at 37˚C, 5 μl of a μM nickel (II) acetate for 72 h showed an epithelial cell- solution of proteinase K (Sigma; 0.4 mg/ml) was added like morphology of untreated CHO cells to be elongated and extract was incubated for an additional 1 h at 37˚C. and resembled fibroblasts (Figure 2). The elongation After the incubation, 12 μl of loading buffer (0.25% brom- developed in the first 24 h of nickel (II) acetate and phenol blue, 0.25% xylene cyanol, 30% glycerol) was persisted throughout the remaining 72 h of exposure. added and the entire content of the tube was The change in morphology of CHO cells has been transferred to the gel. The DNA ladder was resolved in reported to be associated with increased intracellular a 1.8% agarose gel and visualized by ethidium bromide cAMP concentrations and activation of cAMP-dependent (0.5 μg/ml) staining. protein kinase (Costa, 1978). Flow cytometry DNA fragmentation assay Cell pellets removed from the PC buffer described above In initial assays of DNA fragmentation, the untreated were resuspended in 200 μl of Hanks’ buffered salt control cells and cells treated with ≤ 240 μM nickel (II) solution (HBSS) and counted with a hemocytometer. acetate proliferated rapidly and reached 90-95% confluency The cells (approximately 1ለ1 06 cells/ml) were then 72 h after treatment. The doubling time of untreated incubated with DNase-free RNase (0.1 mg/ml, Sigma) cells was determined to be 14 ±1 h. In contrast, cells and propidium iodide (50 μg/ml, Sigma) at 4˚C for at treated with 640 μM nickel (II) acetate proliferate more least 1 h prior to flow cytometric analysis.