Cells and Culture Conditions - Regulatory Mechanisms of Mitotic Catastrophe Induced by Doxorubi

To explore the mode of cell death after induction of SLP by doxorubicin, we first determined the optimal conditions to induce SLP in most of the treated cells. Of the tested experimental protocols (including different doses of doxorubicin, pulse treatment with doxorubicin for different periods, intermittent or chronic treatment, etc.), we obtained the best results with chronic exposure of HCC cells to a low dose of doxorubicin (50 ng/ml for Huh-7 cells). Briefly, Huh-7 cells were first plated in 10 cm dishes with 6 ml of DMEM containing 10% fetal bovine serum (FBS). After overnight culture, this medium was replaced with 6 ml of fresh DMEM containing 10% FBS and 50 ng/ml doxorubicin. Three days later, 3 ml of fresh DMEM containing 10% FBS and 50 ng/ml doxorubicin was added into the preexisting medium (to avoid possible nutritional depletion in long-term culture and to maintain the long-term concentration of doxorubicin at 50 ng/ml). Thereafter, 3 ml of doxorubicin-containing fresh medium was added every 3 days in the same manner, until day 12. Well-defined human HCC cells (SNU-354, -398, -449, and -475) were obtained from the Korean Cell Line Bank (Seoul, Korea) (Park et al., 1995) and grown in RPMI 1640 (Gibco-BRL, Grand Island, NY) supplemented with 10% FBS.

In the respective HCC cell lines, the optimal concentration of doxorubicin to induce SLP was determined as follows: 60 ng/ml for SNU-354, 15 ng/ml for SNU-398, 120

19 penicillin/streptomycin and 10% fetal bovine serum.

B. SA-ββββ-gal Activity Assay

Cells were stained for β-galactosidase activity as described by Dimiri et al. (Dimiri

et al., 1995). Briefly, 1 X 10⁴ cells were seeded in 24-well plate. After appropriate exposure, the cells were washed twice with PBS, fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS, and washed twice in PBS. Cells were stained for 12 h in X-gal staining solution [1 mg/ml X-gal, 40 mmol/L citric acid/sodium phosphate (pH 6.0), 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, 150 mmol/L NaCl, 2 mmol/L MgCl₂]. Cells were then counterstained with 0.1%

haematoxylin solution or Giemsa stain solution.

C. Measurement of Cellular Viability

Huh-7 cells were treated with doxorubicin at the indicated concentrations for the fixed time points. Cellular viability was assessed by double labeling of cells with 2

µmol/L calcein-acetoxymethyl ester (calcein-AM) and 4 µmol/L ethidium

homodimer-1 (Etd-1). Calcein-positive live cells were counted under a fluorescence microscope (Nikon Diaphot 300, Japan), since Etd-1-positive dead cells were floated from the culture plate following treatment of doxorubicin. Alternatively, cellular viability was assessed by trypan blue exclusion assay. Following the treatment of Huh-7 cells with doxorubicin, cells were trypsinized. After addition of 0.4% trypan blue stain solution into the cells, total cell numbers and trypan blue-stained cells were counted using haemacytometer.

D. Transmission Electron Microscopic Examination

Huh-7 cells were treated with 50 ng/ml doxorubicin for the indicated time periods.

The cells were prefixed in Karnovsky’s solution (1% paraformaldehyde, 2%

glutaraldehyde, 2 mmol/L calcium chloride, 0.1 mol/L cacodylate buffer, pH 7.4) for 2 h and washed with cacodylate buffer. Post-fixing was carried out in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. After dehydration with 50 to 100% alcohol, the cells were embedded in Poly/Bed 812 resin (Pelco, Redding, CA) and polymerized, and observed under electron microscope (EM 902A, Zeiss, Oberkochen, Germany).

E. Annexin-V/Propidium Iodide Staining

Huh-7 cells grown on coverslips were treated with 50 ng/ml or 10 µg/ml doxorubicin

at different time periods. Adherent cells on coverslips were washed with PBS and externalized phosphatidyl serine and DNA was labeled with Annexin-V-fluorescein and Propidium iodide (1 µg/ml) in HEPES buffer (10 mmol/L HEPES/NaOH, ph 7.4,

140 mmol/L NaCl, 5 mmol/L CaCl2) for 15 min. The stained cells were observed under fluorescence microscope after washing and mounting of the coverslip on the slide glass. Floated dead cells were separately collected by centrifugation and washed with PBS. These cells were resuspended in HEPES buffer containing Annexin V-FITC and PI and incubated for 15 min. After centrifugation, the stained cells were observed under fluorescence microscopy by placing the resuspended cells in HEPES buffer onto a glass slide and covering with a coverslip. Percentages of the cells stained with Annexin V-FITC and/or PI relative to total cell numbers, the numbers of adherent cells on a coverslip plus the numbers of floated dead cells, were measured.

F. Immunoblotting

After treatments, cells were washed once with ice-cold PBS and lysed in sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mmol/L Tris [pH 6.8], 1% SDS, 10% glycerol, and 5% β-mercaptoethanol). The lysates were sonicated, boiled for 5 min, separated by SDS-PAGE, and transferred to an Immobilon membrane (Millipore, Bedford, MA). Immunoblotting was performed

using standard procedures. The membrane was incubated with antibodies against p53, p21, Cyc A, 14-3-3 σ (each diluted 1:500, purchased from Calbiochem, San Diego, CA), αtubulin, VDAC, Bid, phosphospecificp38, MKK3/6, SEK1, JNK, p38, -IkB-α, -Cdc2, -p53, -Chk1, -Chk2, -Histone H3 (each diluted 1:1000, purchased

from Cell Signaling Technology, Beverly, MA), caspase-9, FAK, Lamin B, Cdc2, Cdk2, Cyc B1, Cyc E, Cdc25A, Cdc25C, Cdc20, Cdc27, BubR1, Chk1, Chk2, Prc1 (each diluted 1:500, purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA), caspase-3, caspase-6, caspase-7, caspase-8 (1:500, Stressgen Biotechnologies Co., Victoria, BC), CENP-A, PARP, phosphor-specific H2AX (1:500, Upstate Biotechnology, Lake Placid, NY), securin (1:1000, Zymed Laboratories Inc., South San Francisco, CA), phosphor-specifin ATM (1:500, Abcam Inc., Cambridge, MA), actin, Mad2, Cdh1, Plk1, and cytochrome c (1:500, BD Transduction Lab., San Diego, CA) in blocking buffer. Bound primary antibodies were detected with HRP-conjugated secondary antibodies and enhanced chemiluminescence (Amersham, Arlington heights, IL).

G. Immunocytochemistry

Cells were washed twice with PBS and fixed in 4% formaldehyde for 10 min at room temperature, and then washed 3 times with PBS. To examine the expression of α-tubulin or cytochrome c, fixed cells were permeabilized in 0.1% Triton X-100/2%

BSA, and stained with mouse anti-α-tubulin antibody (1:200, Calbiochem, San

Diego, CA), mouse anti-cytochrome c antibody (1:50, BD Transduction Lab., San Diego, CA). Cells were further incubated with FITC-conjugated anti-mouse antibody (1:50, Molecular Probes, Inc., Eugene, OR). To examine the expression of lamin B, cells were stained with goat anti-lamin B antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) and further incubated with FITC-conjugated anti-goat antibody (1:50, Sigma, St. Louis, MI). Nuclei were further stained with Hoechst 33258, DAPI, or propium iodide (1 µg/ml, Sigma, St. Louis, MI). To examine the expression of actin, cells were incubated with FITC-conjugated phalloidin (1:50, Sigma, St. Louis, MI). Stained cells were examined by confocal or fluorescence microscopy (Olympus, Shinjuku-ku, Tokyo).

H. RT-PCR Analysis

Total RNA was isolated from cells treated with 50 ng/ml doxorubicin for the indicated time points using RNAZolB (Tel-Test, Friendswood, TX). Total RNA (2 µg) from each cell culture was reverse transcribed using oligo-dT primers and AMV

reverse transcriptase (TaKaRa, Otsu, Shiga). The cDNAs were amplified by PCR (94ôC for 30 sec, 60ôC for 30 sec and 72ôC for 1 min) with Taq DNA polymerase. To ensure exponential amplification, four aliquots were removed from each PCR assay at cycles 20, 25, 30, or 35 cycles (which were determined in preliminary experiments to produce the weakest detectable PCR product for each gene) and every 2-4 cycles thereafter. Amplified products were analyzed by agarose gel electrophoresis at cycles

within the linear range of mRNA amplification. The sequences of oligonucleotide primers used for RT-PCR and the expected transcript sizes are listed in Table 1.

I. Assessment of Nuclear Translocation of NF-κΒκΒκΒκΒ

After treatments, nuclear extracts of Huh-7 cells were prepared using an established protocol as described previously (Liu et al., 1998). The expression of the translocated NF-κB was assessed by Western blotting using ani-p65, c-rel, and p50 antibody (1:

500, Santa Cruz Biotechnology Inc., Santa Cruz, CA).

J. Cell Cycle Analysis

Trypsinized and floating cells were pooled, washed with PBS-EDTA, and fixed in 70% (v/v) ethanol. DNA content was assessed by staining cells with propidium iodide and monitoring by FACScan. Cell cycle distribution was determined with a ModFit LT program (Verity Software House, Inc).

K. Subcellular Fractionation for Analysis of the Release of Mitochondrial Cytochrome c

After treatments, Huh-7 cells were collected and washed twice in ice-cold PBS, resuspended in S-100 buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.9 mM MgCl2, 1 mM EGTA, 1 mM EDTA, mixture of protease inhibitors), and incubated on ice for 20 min. After 20 min incubation on ice, the cells were homogenized with a Dounce

glass homogenizer and a loose pestle (Wheaton, Millville, NJ) for 70 strokes. Cell homogenates were spun at 1,000 X g for 10 min to remove unbroken cells, nuclei, and heavy membranes. The supernatant was spun again at 14,000 X g for 30 min to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The mitochondria-rich fraction was washed once with extraction buffer, followed by a final resuspension in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% centrifugation at 13,000rpm for 15 min. Protein concentration was determined by Bio-Radassay,and 200 g of protein was used for each co-immunoprecipitation.

Immunoprecipitations were performed by incubating lysates with2 µg of the anti-Cyclin B1 or –Cdk2 antibody for Cdc2 or Cdk2 activity, respectively, for 3 h at 4 °C.

15µl of 50% protein A/G-agarose suspension (Santa Cruz Biotechnology Inc., Santa Cruz, CA)was added into the mixture, which was then further incubated for1 h.

Immune complexes were centrifuged at 2,500 rpm for 5 min and the precipitates were washed three times with buffer A andtwice with kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2,1 mM DTT). Cdk kinase assays on histone H1 were

performed by mixingthe respective immune complexes with 5 µg of histone H1 and 10µCi of [γ-³²P] ATP in 30 µl of kinase buffer. The kinase reactionwas performed at 30 °C for 30 min and then terminated with 2× SDS-PAGE sample buffer.

Radioactive ATP incorporation was measured by SDS-PAGE and autoradiography.

III. RESULTS

Part I. Two distinct modes of cell death induced by doxorubicin:

apoptosis and cell death by mitotic catastrophe accompanying senescence-like phenotype

1. LD doxorubicin induces SLP and cell death by mitotic catastrophe

While various cytotoxic agents have been reported to induce senescence in cancer cells at low doses (Wang et al., 1999; Chang et al., 1999a; Chang et al., 1999b), the biochemical changes associated with treatment-induced senescence are not clearly understood. Using doxorubicin, a widely used anti-cancer drug, we first determined the optimal condition to induce SA-β-gal activity, a biomarker for

cellular senescence (Dimiri et al., 1995) in Huh-7 human hepatoma cells. We found that chronic exposure to 50 ng/ml doxorubicin for 6 days could effectively induce SA-β-gal activity in over 82% of Huh-7 cells, but not in cells incubated in doxorubicin-free media for the same period, suggesting that doxorubicin is responsible for SA-β-gal expression (Fig. 1A and 1B). Moreover, Huh-7 cells treated

with 50 ng/ml doxorubicin for 6 days demonstrated a characteristic SLP, including enlargement of cell volume, flattened cell morphology, and the appearance of multinucleated and vacuolated cell forms (Fig. 1A). RT-PCR analysis of gene products commonly associated with cellular senescence showed that osteonectin,

29 doxorubicin were further assessed by Hoechst 33258 staining (Fig. 2A). After 3 days of LD doxorubicin treatment, the nuclei became significantly larger and some cells had several nuclei of unequal sizes. After 6 days, we observed an increased number of micronuclei, but did not observe the condensed or fragmented nuclei that were characteristic of apoptotic cells. More detailed electron microscopic observation demonstrated that cells became enlarged and the number of nuclei with intact nuclear membranes significantly increased after 3 days of treatment with LD doxorubicin (Fig. 2B). After 6 days, the number of micronuclei was further increased and multiple nuclei filled most of the cellular spaces. Furthermore, we observed considerable increases in the numbers of vacuoles, and electron-dense lysosomes, and we additionally observed the convolution or collapse of some nuclear membranes. After 9 days of LD doxorubicin treatment, most of the cells floated from the culture plate demonstrated necrosis-like characteristics such as diffuse chromatin,

Fig. 1. Induction of senescence-like phenotype by low-dose doxorubicin. (A) Expression of senescence-associated β-galactosidase in Huh-7 cells following chronic exposure to low dose (LD) doxorubicin. Huh-7 cells incubated with or without 50 ng/ml doxorubicin for 6 days and the control Huh-7 cells were fixed and then SA-β-gal assay was performed as described in Materials and Methods.

Representative pictures are shown (magnification, 200X). Note the substantial increase in cell volume and the blue staining of cells treated with doxorubicin. (B) Quantitation of β-galactosidase expression in Huh-7 cells treated with or without doxorubicin. Data are presented as the mean and SD (bars) of four independent experiments based on three random fields with 100 cell counts per field. (C) RT-PCR analysis of gene products associated with cellular senescence. RNA was prepared from Huh-7 cells treated with 50 ng/ml doxorubicin for the indicated time points and RT-PCR was performed as described in Materials and Methods. (d) Changes in cellular viability following treatment with LD doxorubicin, as analyzed by staining with trypan blue.

disruption of intact cellular boundaries, and losses of the distinctive nuclear membrane structures. We did not detect the DNA fragmentation characteristic of apoptosis, as assessed by agarose gel electrophoresis or TUNEL assay (data not shown). Next, we investigated whether abnormal nuclear morphologies are observed before or after the increase in the number of SA-β-gal positive cells. SA-β-gal assay and subsequent nucrear staining with Giemsa solution demonstrated that both the percentages of multinucleate cells and the percentages of SA-β-gal positive cells

were increased by 6 daays of 50 ng/ml doxorubicin treatment (Fig. 3A and 3B).

While the percentages of multinucleate cells were 8.16, 10.04, 22.15, 34.67, 40.24, 49.91, and 78.04% at 0, 1, 2, 3, 4, 5, and 6 day respectively, the percentages of

SA-β-gal positive cells were 3.87, 5.02, 4.95, 6.66, 21.41, 45.83, and 75.80% at the same incubation time points. Therefore, abnormal nuclear morphologies, which most likely arose as the consequence of abnormal mitosis, were observed earlier than the principal increase in the number of SA-β-gal positive cells. However, over 65% of total cells demonstrated both multinucleation and SA-β-gal positivity on 6-day

exposure, suggesting that multinucleation and SLP are induced in most cells treated with 50 ng/ml doxorubicin.

Next, to investigate whether multinucleation by doxorubicin was associated with abnormal mitosis, we examined the expression of α-tubulin, a major component of microtubules by staining with anti-α-tubulin antibody and nuclear morphologies by staining with DAPI. While the percentages of the cells with multiple nuclei were

Fig. 2. Changes in the cellular morphologies of Huh-7 cells treated with 50 ng/ml doxorubicin. (A) Changes in the nuclear morphologies. Representative pictures of cells stained with Hoechst 33258 and visualized under a fluorescence microscope (magnification, 200X). (B) Electron microscopic observation of Huh-7 cells treated with 50 ng/ml doxorubicin for the indicated time periods. Bar is 2.5 µm. Electron micrographs are same magnification. White arrowheads, black arrowheads, and white arrows denote electron-dense lysosomes, vacuoles, and the convolution of nuclear membranes, respectively. And higher magnification of the picture showing the collapse of the nuclear membrane at 6 day is demonstrated.

Fig. 3. Characterization of the time course of multinucleation and SA-ββββ-gal expression in Huh-7 cells treated with 50 ng/ml doxorubicin. (A) Microscopic analysis of multinucleation and SA-β-gal expression. Huh-7 cells were treated with 50 ng/ml doxorubicin for the indicated time points and SA-β-gal assay was

performed. Cells were countestained with Giemsa solution and photographed at bright field with 400-fold magnification. β+, β -, SN, or MN denotes SA-β-gal-positive, SA-β-gal-negative, single nucleus, multiple nuclei, respectively.

Representive pictures are shown. (B) Measurement of the percentages of cells with SA-β-gal negativity and single nucleus (β-gal (-) & SN), cells with SA-β-gal-positivity and multiple nuclei (β-gal (+) & MN), cells with SA-β-gal-SA-β-gal-positivity and single nucleus (β-gal (+) & SN) cells with SA-β-gal-negativity and multiple nuclei (β-gal (-) & MN

Fig. 4. Abnormal spindle formation in cells treated with 50 ng/ml doxorubicin. (A) Representative pictures of control Huh-7 cells and Huh-7 cells treated with 50 ng/ml doxorubicin for 3 days after immunostaining using

anti-α-tubulin antibody and DAPI. Images from confocal laser scanning microscopy are shown (magnification, 1200X). (B) Measurement of the percentages of cells with abnormal spindles or cells with multiple nuclei following treatment with 50 ng/ml doxorubicin for the indicated time periods.

gradually increased by treatment with 50 ng/ml doxorubicin, abnormal formation of spindles (tripolar, quadripolar or asymmetrical mircotubules) was observed with a peak at 3 days of doxorubicin treatment (Fig. 4A and 4B). These results suggest that abnormal spindle formation may contribute to the observed multinucleation by doxorubicin.

Next, we used FACS analysis to monitor the changes of DNA content in cells treated with LD doxorubicin (Fig. 5). At day 1.5 of doxorubicin treatment, the S phase cell population (from 23.7% to 50.2%) was significantly increased with a concomitant decrease in the G0/G1 phase cell population (from 52.5% to 25.3%). At day 3, about 37.7% of cells were detected at G₂/M phase and the fraction of hyperploid cell population (>4N DNA cells) increased (21.4%). Thereafter, the hyperploid cell population increased further and the DNA contents of the doxorubicin-treated cells became very heterogeneous, possibly due to the formation of multiple micronuclei of various sizes in the absence of cytokinesis. Interestingly, there was only a slight increase (7.5%) in the subG1 population after 9 days of LD doxorubicin treatment, whereas cellular viability, assessed by trypan blue exclusion assay, decreased to 54% at this time (Fig. 1D). Taken together, these results suggest that apoptosis might not be the major mode of the cell death in Huh-7 cells treated with LD doxorubicin, but that these cells likely died through mitotic catastrophe.

2. Several mitosis-controlling proteins are down-regulated during the induction of SLP and cell death through mitotic catastrophe by LD doxorubicin

Fig. 5. Changes in DNA contents following treatment with 50 ng/ml doxorubicin. Cells were fixed with ethanol and their DNA contents measured by FACS analysis. Representative histograms of three independent experiments are shown. Percentages of G₀/G₁, S, G₂/M and subG₁ phase cells were calculated by deconvolution of the DNA content histograms. Values are presented as the mean and SD of three independent experiments.

There is a broad consensus that p53-deficient cells are able to aberrantly reenter the cell cycle, where they undergo unchecked reduplication of their DNA, leading to increased nuclear content and subsequent chromosomal instability (Lanni and Jacks, 1998). In this context, p21 appears to be one of the major p53 target genes (Mantel et al., 1999). In Huh-7 cells harboring mutant p53 (Hsu et al., 1993), neither p53 nor p21 protein levels were altered following treatment with 50 ng/ml doxorubicin (Fig. 6), suggesting that induction of SLP by LD doxorubicin in Huh-7 is independent of p53 and p21. This is contrary to previous reports that p53 and p21 acted as positive regulators of doxorubicin-mediated senescence (Wang et al., 1999;

Chang et al., 1999a). We further analyzed the protein levels of mitosis-associated proteins following treatment with 50 ng/ml doxorubicin. We observed downregulation of the mitosis initiator Cdc2, the centromere protein CENP-A (Kalitis et al., 1998), spindle checkpoint control proteins Mad2 and BubR1 (Li and Benezra, 1996), and the DNA damage-induced checkpoint kinase Chk1 (Chan et al., 1999). In contrast, neither the DNA damage-induced checkpoint kinase Chk2 (Hirao et al., 2000) nor the mitotic checkpoint protein Plk1 (Seong et al., 2002) protein

levels were altered in response to 50 ng/ml doxorubicin. Our results suggest that doxorubicin may induce depletion of multiple proteins controlling mitosis, thus contributing to abnormal mitosis and subsequent cell death in Huh-7 cells.

3. Induction of SLP and cell death through mitotic catastrophe is observed in many human HCC cell lines treated with LD doxorubicin

Fig. 6. Changes in the expression of p53, p21 and some mitosis-controlling proteins following treatment with 50 ng/ml doxorubicin. Huh-7 cells were treated with 50 ng/ml doxorubicin for the indicated time periods and Western blotting was performed to detect the changes in the expression of the listed proteins. To confirm that anti-p21 antibody used in this experiment was immunologically active, Huh-7 cells were treated with 5 ng/ml TGF-β1 for 9 days and Western blotting of p21 was performed.

To analyze whether induction of SLP and cell death through mitotic catastrophe following to exposure to doxorubicin is restricted to a particular cell line, we examined the effect of LD doxorubicin on other human HCC cell lines including SNU-354, -398, -449, and -475. Although there were cell type-specific variations in the optimal concentrations of doxorubicin for induction of SLP and the incubation time to reach mitotic catastrophe, all tested cell lines demonstrated similar induction of SLP and cell death by mitotic catastrophe in response to doxorubicin treatment

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