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Tumor Biology |
Institut National de la Santé et de la Recherche Médicale U539, Faculté de Médecine, 44035 Nantes, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, anti-Fas antibody plus Adriamycin was characterized by the expression of early markers of apoptosis, whereas the key nuclear features of apoptosis were absent. In contrast, exposure of confluent cells to this treatment led to a typical apoptotic nuclear fragmentation. Both forms of apoptosis, in exponentially growing and confluent cells, were sensitive to the broad spectrum inhibitor of caspases, z-Val-Ala-Asp-fluoromethyl ketone. Our findings support the concept that the expression of ICAD is essential to the execution of full-blown apoptosis in colonic cancer cells. Altogether, our results point to ICAD as a potential target for restoring a normal apoptotic signal transduction pathway in colonic cancer cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture for Immunoblot Analysis.
For immunoblot analysis, confluent cells were harvested at least 3 days after confluency. For cells in the exponential phase of growth, Caco-2 (20 x 103 cells/ml), HGT-1 (20 x 103 cells/ml), HT29 (40 x 103 cells/ml), HT29-Cl.19A (60 x 103 cells/ml), and HT29-Cl.16E (60 x 103 cells/ml) cells were seeded in 30 ml of DMEM/FCS in 10-cm diameter Petri dishes (Costar, Brumath, France). After 3 days of culture, the medium was discarded and replaced by 6 ml of DMEM/FCS, and cells were submitted or not to a cytotoxic treatment as described below.
Treatment for Cell Death Induction.
HT29-Cl.16E cells were seeded at 60 x 103 cells/ml in 5 ml of DMEM/10% FCS in six-well culture plates (Costar). Three days later, the medium was discarded and replaced by 1 ml of DMEM/10% FCS. Cells were treated with a human activating anti-Fas antibody (10 ng/ml, clone CH-11; Upstate Biotechnology, Euromedex, Mundolsheim, France) in the presence or in the absence of IFN- (10 ng/ml; R&D System, Oxon, United Kingdom) for 24 h. Adriamycin (Sigma Chemical Co., St. Quentin Fallavier, France) was added to the culture medium for the last 12 h of treatment at the concentrations indicated. This treatment with the three agents is referred to as IFA treatment throughout the text. After treatment, both viable adherent cells and floating dead cells were collected.
Jurkat cells were seeded at 1 x 106 cells/ml, and anti-Fas antibody (10 ng/ml) was added to the medium for 24 h. In some experiments, the broad-spectrum, cell-permeable inhibitor of caspases, ZVAD-fmk (Calbiochem, Meudon, France) was added to the culture medium at 50 µM, 90 min before IFA treatment for HT29-Cl.16E cells or anti-Fas antibody treatment for Jurkat cells.
Isolation of Human Normal Colonic Epithelial Cells.
Histologically normal colonic epithelial cells were isolated from surgical specimens using a nonenzymatic dissociation technique as described previously (19)
. Preparations of colonocytes were devoid of contamination by immune cells (19)
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Immunoblot Analysis.
Cells from the various cell lines as well as human normal colonocytes were lysed for 30 min on ice in RIPA buffer [150 mM NaCl, 0.5% sodium deoxycholate (Sigma), 50 mM Tris-HCl (pH 8.0), 0.1% SDS from Bio-Rad (Ivry/Seine, France)] supplemented with protease inhibitors 0.1 mM Pefabloc (Roche Diagnostics, Meylan, France), 2.5 µg/ml pepstatin, 10 µg/ml aprotinin, and 5 µg/ml leupeptin (Sigma). Protein concentrations were determined using the Folin assay (DC protein assay kit; Bio-Rad). Fifty µg of protein were mixed in 2x loading buffer [125 mM Tris-HCl (pH 6.8), 4.6% SDS (Bio-Rad), 10% ß-mercaptoethanol, 20% glycerol, and 0.003% bromphenol blue (Sigma)], boiled for 3 min, and run on a 12% SDS-polyacryamide gel. Proteins were electrotransferred onto nitrocellulose membranes (Bio-Rad) using a Trans-blot apparatus (Bio-Rad). After membranes were blocked overnight (1% blocking reagent; Roche Diagnostics), they were then probed with rabbit polyclonal antibodies directed either to human caspase-3 (1:2,500; PharMingen, BD Biosciences, Le Pont-de-Claix, France), to human ICAD (1:500; PharMingen), or to human CAD (1:500; Chemicon, Euromedex, France), followed by horseradish peroxidase-conjugated sheep antirabbit antibody (1:10,000; Roche Diagnostics). The immunoreactive proteins were detected on films using an enhanced chemiluminescence substrate according to the manufacturer’s instructions (Roche Diagnostics).
Kinetics of ICAD-L/DFF45 and CAD/CPAN/DFF40 Expression.
Cell lysates prepared at different time points (50 µg of total proteins) were electrophoresed as described above and then were electrotransferred onto a polyvinylidene difluoride membrane (Bio-Rad) and immunoblotted with anti-ICAD or CAD antibodies. Bands were visualized by enhanced chemifluorescence using a STORM 860 imaging system (Molecular Dynamics; Amersham Pharmacia Biotech, Orsay, France) and quantified with the ImageQuant software (Molecular Dynamics; Amersham Pharmacia Biotech). All levels of ICAD were expressed relative to the lowest expression of ICAD (day 5 of culture), which corresponds to 100%. All levels of CAD were also expressed relative to CAD expression at day 5 (100%).
Caspase-3 Activity Assay.
Caspase-3 activity was determined using an ApoAlert caspase-3 colorimetric assay kit (Clontech, Ozyme, St. Quentin en Yvelines, France) according to the manufacturer’s protocol. This assay is based on the spectrophotometric detection of the pNA after cleavage from the labeled caspase-3 substrate, DEVD-p-nitroanilide. Anti-Fas antibody-treated Jurkat cells (200 ng/ml for 15 h) were used as a positive control according to the manufacturer’s instructions. Results are expressed in pmol of cleaved substrate/106 cells/min.
LDH Activity-based Cytotoxicity Assays.
LDH activity was assessed using a standardized kinetic determination (Enzyline LDH kit; Biomerieux, Marcy-l’Etoile, France). LDH activity was measured in both floating dead cells and viable adherent cells. Floating cells were collected carefully from culture medium by centrifugation (240 x g for 10 min at 4°C), and the LDH content from the pellets, referred to as LDHp, was used as an index of apoptotic cell death (20)
. The LDH released in the culture medium (extracellular LDH, or LDHe) was used as an index of necrotic cell death. The LDH present in the adherent, viable cells was referred to as intracellular LDH (LDHi). The percentage of apoptotic and necrotic cell death was calculated as follows: % apoptosis = LDHp/(LDHp + LDHi) x 100; and % necrosis = LDHe/(LDHe + LDHi) x 100.
Immunocytochemistry.
Cytocentrifuged preparations of HT29-Cl.16E cells from six-well plates were fixed for 10 min in cold acetone and incubated with the M30 monoclonal antibody (1:50; Roche Diagnostics) for 1 h at room temperature. This antibody specifically recognizes a cytokeratin 18 neo-epitope that becomes available at an early caspase cleavage event during apoptosis and can be considered as an early marker of apoptosis in epithelial cells (21)
. The incubation with M30 antibody was followed by a streptavidin-biotin-horseradish peroxidase method using aminoethyl carbazole as a chromogen (Histostain Plus kit; Zymed, Clinisciences, Montrouge, France) according to the manufacturer’s instructions. Nuclei were counterstained with hematoxylin. The percentage of cells stained with the M30 antibody was assessed by counting at least 400 cells under the microscope. This technique was also applied to HT29-Cl.16E cells cultured on Labteck chambers to ensure that the staining pattern observed on cytocentrifuged preparations was not attributable to the trypsinization process itself.
Hoechst Staining.
The DNA-specific dye Hoechst 33258 (Calbiochem), 0.5 µg/ml in HBSS without phenol red (Life Technologies, Inc.) was used on Carnoy-fixed cytocentrifuged preparations of cells. The percentage of apoptotic nuclei was determined by counting under a fluorescence microscope (x400, IX70; Olympus, Rungis, France). At least 600 cells/determination were counted.
Ultrastructural Studies.
HT29-Cl.16E cells were harvested and centrifuged at 100 x g. The cell pellets were fixed in cacodylate-buffer 3% glutaraldehyde and prepared for electron microscopy by standard methods.
Analysis of DNA Fragmentation by Agarose Gel Electrophoresis.
HT29-Cl.16E cells were pelleted and suspended in 100 mM NaCl, 10 mM Tris (pH 8.0), 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K and incubated for 3 h at 37°C. Then DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), precipitated with isopropanol overnight at -20°C, and centrifuged. DNA was then washed with 70% ethanol and centrifuged, and pellets were dried and suspended in 10 mM Tris/1 mM EDTA. Samples were incubated 30 min at 37°C with 100 µg/ml RNase A (Sigma), and DNA concentrations were determined. Samples were incubated for 20 min at 68°C after addition of loading buffer (Sigma). Ten µg of DNA were analyzed in ethidium bromide-containing 2% agarose precast gel (Invitrogen, Groningen, the Netherlands).
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RESULTS |
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. ICAD expression was found to be restricted to the functional isoform of Mr 45,000 in both normal colonocytes (Fig. 1A)
and in six postconfluent colonic cancer cell lines (Fig. 1, B and C)
. ICAD-L/DFF45 was expressed independently of the differentiation state of the cells; the clonal derivatives of HT29 cells, HT29-Cl.19A and HT29-Cl.16E cells (13)
, which differentiate at confluency into ion-transporting polarized cells and typical monolayers of mucus-secreting cells, respectively, expressed ICAD-L/DFF45 at the same level. In addition, the parental undifferentiated HT29 cell line also expressed ICAD-L/DFF45. In contrast, Jurkat cells, used as a control, expressed both the Mr 45,000 and Mr 35,000 isoforms (Fig. 1B)
, as described previously (10
, 22)
. Finally, we examined whether ICAD-L/DFF45 expression was dependent on the growth phase in cultured colonic cancer cells. In preliminary experiments, ICAD-L/DFF45 expression was examined in five cell lines (HT29, HT29-Cl.16E, HT29-Cl.19A, Caco-2, and HGT-1) at two time points, i.e., during the exponential phase of growth (day 4 of culture) and in confluent cells (data not shown). In these conditions, HT29-Cl.16E was the only one to exhibit down-regulation of DFF45 expression during the exponential phase of growth (Fig. 1C)
. HT29-Cl.16E cells were therefore chosen for additional studies. Time course experiments showed a decreasing expression of ICAD-L/DFF45 from seeding (day 0) up to day 5 of culture (Fig. 1E)
. Then ICAD-L/DFF45 expression increased over time to reach its maximum at confluency; the fluorescence level of ICAD-L/DFF45 band was 6.4-fold higher at day 14 than at day 5. In contrast, CAD remained essentially stable during the same period of culture (Fig. 1, D and E)
. These results prompted us to study the consequences of ICAD-L/DFF45 down-regulation on the cellular response of growing HT29-Cl.16E cells after treatment with proapoptotic agents.
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To induce cell death, growing HT29-Cl.16E cells were preincubated for 12 h with anti-Fas antibody (10 ng/ml) plus IFN- (10 ng/ml), and then Adriamycin was added to the incubation medium for an additional 12-h incubation (referred to as IFA treatment). The extent of cell death was evaluated by measuring LDH released in the culture medium (as an index of necrotic cell death) and LDH content of floating cells (as an index of apoptotic cell death; see "Materials and Methods"). Adriamycin dose dependently induced both apoptotic (Fig. 2A)
and necrotic (Fig. 2B)
cell death from growing HT29-Cl.16E cells treated with IFN- and anti-Fas antibody. Adriamycin was maximally effective at a concentration of 10 µg/ml. Anti-Fas antibody alone, up to a concentration of 100 ng/ml (data not shown); Adriamycin alone, up to a concentration of 50 µg/ml (Fig. 2)
; or the combination of anti-Fas antibody with IFN- or Adriamycin (data not shown) were unable to trigger cell death. Several preliminary experiments were performed to verify that cells released in the supernatant upon IFA treatment were actually dead cells; these cells were unable to readhere and/or proliferate upon reseeding in fresh culture medium.
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, most cells released in the supernatant upon IFA treatment (floating cells) scored positive with the M30 antibody. We then performed an immunoblot analysis of caspase-3 cleavage during the IFA treatment of growing HT29-Cl.16E cells. As shown in Fig. 3A
, the IFA treatment led to a typical pattern of caspase-3 activation in floating cells, associated with a loss of procaspase-3. Interestingly, caspase-3 cleavage was also observed to a lesser degree in adherent IFA-treated cells (Fig. 3A)
, suggesting that the proteolytical activation of caspase-3 was an early event preceding cell detachment. Measurements of caspase-3 activity using a spectrophotometric assay showed that IFA treatment resulted in caspase-3 activation in both adherent and floating cells [29.7 pmol DEVD-pNA/min/106 cells (n = 2) and 35.3 pmol DEVD-pNA/min/106 cells (n = 2), respectively], whereas untreated cells did not display any caspase-3 activity [0 pmol DEVD-pNA/min/106 cells (n = 2)]. This level of activity was in the same order of magnitude as that of anti-Fas antibody-treated Jurkat cells [26.3 pmol DEVD-pNA/min/106 cells (n = 3) versus 0 pmol DEVD-pNA/min/106 cells (n = 2) in untreated Jurkat cells]. Immunoblot analysis of both adherent and floating cells with anti-ICAD antibodies did not disclose any immunoreactivity (Fig. 3B)
. When confluent HT29-Cl.16E cells, used as a control, were treated with IFA, ICAD-L/DFF45 was found to be processed proteolytically, as shown by the loss of the Mr 45,000 band (Fig. 3D)
, in parallel with caspase-3 cleavage (Fig. 3C)
. Altogether, these results show that growing, IFA-treated HT29-Cl.16E cells expressed early markers of apoptosis, i.e., retention of cytosolic proteins, cleavage of cytokeratin 18, and proteolytic activation of caspase-3.
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. Electron microscopic examination of these cells showed only a peripheral pattern of chromatin condensation with neither chromatin compaction nor nuclear fragmentation (Fig. 4B)
. Finally, IFA treatment was unable to provoke the typical DNA laddering (Fig. 4E
, Lane 2). To understand the mechanisms underlying the IFA-induced cell death of growing HT29-Cl.16E cells, we evaluated the cellular responses to the broad-spectrum caspase inhibitor, ZVAD-fmk. This inhibitor (50 µM) was able to inhibit the IFA-induced apoptotic cell death of growing HT29-Cl.16E by 65.7% (±2.4; n = 6) without impairment of necrosis (data not shown), based on LDH activity assays. At this concentration, ZVAD-fmk almost fully protected Jurkat cells from apoptosis induced by anti-Fas antibody (92.1% inhibition ± 0.8; n = 3), as demonstrated by Hoechst staining.
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and electron microscopy (Fig. 4D)
. In addition, these nuclear features of apoptosis were associated with a typical DNA ladder (Fig. 4E
, Lane 4). This apoptotic cell death was inhibited by ZVAD-fmk (50 µM; data not shown). |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When it came to studying the functional and morphological consequences of ICAD down-regulation, our strategy, based on the HT29-Cl.16E model in the exponential phase of growth, was to examine the effects of agents known to trigger the activation of caspase-3. In fact, caspase-3 is responsible for ICAD cleavage and subsequent CAD release, with this nuclease giving rise to the nuclear changes that are the signature of apoptosis. The best characterized apoptotic pathway involving caspase-3 activation and cleavage of its substrate ICAD is the one induced through the surface molecule Fas. The recent demonstration that human colonocytes undergo apoptosis upon Fas ligation (24
, 25)
, together with the present demonstration that human colonocytes express the functional isoform of ICAD, point to a role of ICAD in mediating the apoptotic process triggered by Fas in colonocytes. In contrast, several cancer cell lines are resistant to Fas-induced apoptosis (24
, 26)
. This resistance can be accounted for by down-regulation of Fas receptor or receptor-proximal molecules. In fact, some of these cancer cell lines can be sensitized to the Fas cytotoxic effect by agents, e.g., IFN- or Adriamycin, which are known to up-regulate Fas receptor or receptor-proximal molecules (24
, 27, 28, 29, 30, 31, 32, 33)
. HT29-Cl.16E cells were resistant in our experimental conditions to Fas, and a cytotoxic response could be elicited only upon cell exposure to the combination of IFN-, anti-Fas antibody, and Adriamycin (IFA treatment), a finding in line with the concept that these agents trigger synergistic pathways. Indeed, in these conditions, the cytotoxic effect on growing HT29-Cl.16E cells was associated with caspase-3 activation and expression of early markers of apoptosis, i.e., intracellular retention of cytosolic proteins (LDH) and cleavage of cytokeratin 18, which is a caspase-3 substrate (23)
. In contrast, the late events of apoptosis, i.e., internucleosomal DNA cleavage and nuclear fragmentation, were absent, whereas confluent HT29-Cl.16E cells, which express ICAD-L/DFF45, exhibited nuclear fragmentation upon IFA treatment. Interestingly, the morphological nuclear features of growing HT29-Cl.16E cells in response to a cytotoxic treatment are remarkably similar to those of murine ICAD-/- cells and caspase-3-/- oncogenically transformed murine embryonic fibroblasts in response to apoptotic stimuli (11
, 34)
. In addition, the MCF-7 cell line, devoid of caspase-3 because of a 47-bp deletion within exon 3 of the caspase-3 gene, does not undergo typical nuclear changes when treated with tumor necrosis factor or staurosporine (35)
. Taken together, these findings as well as the present study support the concept that the apoptotic nuclear changes are dependent on the integrity of the caspase-3/ICAD pathway. Because both incomplete apoptotic phenotype displayed by dying proliferating cells and complete apoptotic process displayed by confluent cells were caspase dependent, it is concluded that ICAD inactivation is a dispensable step in caspase-mediated cell death.
During malignant transformation, colonocytes acquire different mechanisms to escape apoptosis mediated by various agents including p53 mutations (36) , Fas down-regulation (24) , inhibition of Fas capping, or enhanced expression of intestinal trefoil factor (37 , 38) . DFF45/ICAD down-regulation in proliferative colonic cancer cells could be another mechanism to escape the late changes of apoptosis including nuclear fragmentation and cell disintegration. Here we describe a new phenotypic feature of cell death induced by caspase activation, the biological significance of which remains to be determined in vivo.
In conclusion, this is the first report showing changes associated with ICAD down-regulation in a human colonic cancer cell line. Additional studies are needed to extend this characterization to a large panel of cell lines. ICAD is a potentially important target involved in the control of the apoptotic process in colonic cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Ligue Départementale de Loire-Atlantique Contre le Cancer. L. C. is the recipient of a fellowship from the Ligue Départementale de Vendée Contre le Cancer. 
2 To whom requests for reprints should be addressed, at Institut National de la Santé et de la Recherche Médicale U539, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes cedex 01, France. Phone: 33-240-41-28-30; Fax: 33-240-41-28-37;
3 The abbreviations used are: CAD, caspase-3-activated DNase; CPAN, caspase-activated nuclease; DFF, DNA fragmentation factor; ICAD, inhibitor of caspase-3-activated DNase; IFA, IFN-/anti-Fas antibody/Adriamycin; LDH, lactate dehydrogenase; pNA, chromophore p-nitroanilide; ZVAD-fmk, z-Val-Ala-Asp-fluoromethyl ketone.
Received 7/17/01. Accepted 1/23/02.
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A. Jarry, L. Charrier, C. Bou-Hanna, M.-C. Devilder, V. Crussaire, M. G. Denis, G. Vallette, and C. L. Laboisse Position in Cell Cycle Controls the Sensitivity of Colon Cancer Cells to Nitric Oxide-Dependent Programmed Cell Death Cancer Res., June 15, 2004; 64(12): 4227 - 4234. [Abstract] [Full Text] [PDF]
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) and CAD (
) expression over time in HT29-Cl.16E cells was performed using enhanced chemifluorescence as described in "Materials and Methods" (E). All levels of ICAD were expressed relative to the lowest expression of ICAD (day 5 of culture), which corresponds to 100%. All levels of CAD were also expressed relative to CAD expression at day 5 (100%). Data points represent one experiment, and another experiment gave essentially the same results.
