Mol Cancer Ther. 2005;4:743-750
© 2005 American Association for Cancer Research
Overexpression of hRFI (human ring finger homologous to inhibitor of apoptosis protein type) inhibits death receptor–mediated apoptosis in colorectal cancer cells
Tsuyoshi Konishi,
Shin Sasaki,
Toshiaki Watanabe,
Joji Kitayama and
Hirokazu Nagawa
Department of Surgical Oncology, University of Tokyo, Tokyo, Japan
Requests for reprints: Shin Sasaki or Tsuyoshi Konishi, Department of Surgical Oncology, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-35800-8653; Fax: 81-33811-6822. E-mail: SASAKI-1SU{at}h.u-tokyo.ac.jp or KONISHIT-SUR{at}h.u-tokyo.ac.jp
 |
Abstract
|
|---|
The acquisition of antiapoptotic properties is one of the essential mechanistic steps in colorectal carcinogenesis and is closely correlated with a loss of chemosensitivity and radiosensitivity. Human ring finger homologous to inhibitor of apoptosis protein type (hRFI) is a newly discovered gene encoding a ring finger domain highly homologous to that of X chromosome–linked inhibitor of apoptosis protein. Immunohistochemistry has revealed that the expression of hRFI increased in transition from normal colorectal mucosas to adenomas and from adenomas to carcinomas, suggesting an essential role in the early stage of colorectal carcinogenesis. However, the function role of hRFI in colorectal carcinoma has not been elucidated. To determine whether hRFI possesses an antiapoptotic function in colorectal cancer cells, HCT116 colorectal cancer cells stably overexpressing hRFI were established. The hRFI transfectant exhibited significant resistance to apoptosis induced by tumor necrosis factor-
or tumor necrosis factor–related apoptosis-inducing ligand compared with control. This antiapoptotic response was associated with decreased activity of caspase-3, -8, and -9. We also established an antisense down-regulation of hRFI, which effectively reversed the antiapoptotic activity of the hRFI transfectant. This confirmed that the antiapoptotic property of the hRFI transfectant was not due to the clonal effect but in fact dependent on hRFI function. In conclusion, hRFI possesses an antiapoptotic function in HCT116 colorectal cancer cells. Considering the progressive increase of hRFI expression in the advance of the colorectal adenoma-carcinoma sequence, hRFI is one of the important players in colorectal carcinogenesis through its effect on apoptosis regulation.
Key Words: hRFI • apoptosis • TNF- • TRAIL • colorectal cancer
|
Introduction
|
|---|
The acquisition of antiapoptotic properties is one of the essential steps in carcinogenesis and correlates with resistance to chemotherapy or radiotherapy, leading to further malignant transformation and poor prognosis (1). Inhibitor of apoptosis proteins (IAP) are key regulators of apoptosis and have been suggested as important molecules in colorectal carcinogenesis (2–5). For example, immunohistochemical analysis has revealed that survivin expression increases progressively in sequence from colorectal adenomas with low dysplasia to those with high dysplasia/carcinoma (6). Survivin expression also correlates with poor prognosis in colorectal carcinomas (7). Another immunohistochemical analysis has revealed that nuclear cIAP1 expression correlates with the relative risk of death in sporadic colorectal adenocarcinomas (8).
Human ring finger homologous to IAP type (hRFI; accession no. AB084914) is a newly discovered gene, which we isolated by means of a two-hybrid yeast screening method using hTID-1, an apoptosis regulator protein, as bait (9–11). hRFI encodes a ring finger domain highly homologous to that of X chromosome–linked IAP protein (XIAP), which is the most potent IAP among the IAPs (12, 13). hRFI also encodes a specific cleavage site targeted by caspase-3 at residues 230 to 233 (11). These structural evidence support a functional relationship of hRFI to the cell death pathway.
We have shown that hRFI is preferentially expressed in esophageal, gastric, and colorectal cancers, suggesting a possible association between hRFI and the development of digestive tract cancers (14, 15). Especially in the colorectal adenoma-carcinoma sequence, the expression of hRFI increased in the transition from normal colorectal mucosas to adenomas and from adenomas to carcinomas (15). This sequential increase of hRFI expression enables us to speculate that hRFI might play an essential role in the early stage of colorectal carcinogenesis, probably related to its effects on antiapoptotic transformation. Indeed, transient transfection of hRFI into HeLa cells resulted in a slight increase of surviving cells after treatment with tumor necrosis factor- (TNF-; ref. 11). However, the molecular mechanism of the increased survival in hRFI transfectant has not been fully clarified yet, and the effect of hRFI on cellular apoptosis or intracellular caspase activity has not yet been examined.
In this study, therefore, we examined whether hRFI molecule really possessed an antiapoptotic function in colorectal cancer cells and showed that stable overexpression of hRFI significantly inhibited death receptor–mediated apoptosis with decreased activities of caspase-3, -8, and -9 in HCT116 human colorectal cancer cells.
|
Materials and Methods
|
|---|
Cells and Culture Conditions
All cell lines were obtained from the American Type Culture Collection (Rockville, MD). HCT116 was maintained in McCoy's 5A modified medium (Invitrogen, Carlsbad, CA), and other cell lines were in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 100 units/mL penicillin, and 100 µg/mL streptomycin (Life Technologies, Inc., Grand Island, NY) at 37°C in a humidified 5% CO2 atmosphere.
Establishment of a Stable HCT116 Cancer Cell Line Consistently Expressing the hRFI Protein
The full-length cDNA of hRFI was subcloned into a pcDNA3.1/V5-His vector (Invitrogen) as described previously (11). Cells were seeded at a density of 1 x 105/mL in 35 mm dishes 24 hours before transfection. Cultures were then transfected with 2 µg/mL plasmids using 5 µg/mL Lipofectin (Invitrogen) in a serum-free Opti-MEM medium (Invitrogen) according to the manufacturer's protocol. Transfectants were incubated for 6 hours at 37°C and after replacement of the complete medium maintained for an additional 48 hours. For stable transfection, cells were trypsinized and incubated in the presence of 1,000 µg/mL G418 (BD Biosciences, Palo Alto, CA) for 2 to 3 weeks. Individual G418-resistant colonies were isolated and maintained in the presence of G418.
Western Blotting
Cells were scraped and lysed for 45 minutes at 4°C in a lysis buffer containing PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 0.01 mg/mL aprotinin, and 0.01 mg/mL leupeptin. Cell lysates were cleared by centrifugation. The concentration of protein was determined by the BCA protein assay reagent (Pierce Biomedical Co., Rockford, IL). Aliquots (20 µg) of whole cell lysates were electrophoresed in 15% Ready Gel J (Bio-Rad, Hercules, CA). Separated proteins were electrophoretically transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and incubated with the primary antibody overnight at 4°C followed by incubation with a rabbit anti-hRFI polyclonal antibody generated previously (11), an anti-V5-horseradish peroxidase antibody (Invitrogen), and an anti-ß-actin antibody (Sigma). Proteins were detected and visualized with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
Proliferation Assay
Cells were seeded in 96-well plates at a density of 5 x 103 per well and allowed to adhere for 24 hours. Then, the proliferative activity was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay (CellTiter 96 Non-Radioactive Cell Proliferation Assay; Promega, Madison, WI) to monitor the number of viable cells according to the manufacturer's instructions. Briefly, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt solution was added at 20 µL/well, and after 3 hours of incubation at 37°C in a humidified 5% CO2 atmosphere, the conversion of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt to formazan was measured in a plate reader at 490 nm. All experiments were done in triplicate, and the proliferation rate was calculated as the ratio of absorbance under each experimental condition to that of the control nontransfectant.
Flow Cytometry Analysis of Cell Cycle Distribution
Bromodeoxyuridine, at a final concentration of 5 µg/mL, was added to the subconfluent cells, which were incubated for a further 1 hour under the same conditions. Cells were then harvested by trypsinization and fixed with 70% ethanol. After staining with the propidium iodide (PI) and FITC-conjugated anti-bromodeoxyuridine antibody (PROGEN Biotechnik GmbH, Heidelberg, Germany), the percentage of cells in each cell cycle phase was measured using Becton Dickinson FACScan with CellQuest software (Franklin Lakes, NJ). The experiments were independently repeated thrice.
Nuclear Staining Assay
Cells were plated 24 hours before the induction of apoptosis. Apoptosis was induced by a combination of TNF- and cycloheximide. After apoptosis induction, cells were harvested by trypsinization and fixed with 70% ethanol. Fixed cells were treated with 0.5% Tween 20 and stained with 0.5 µg/mL PI for 15 minutes. Nuclear morphology was examined by fluorescence microscopy.
Flow Cytometry Analysis of Apoptosis
Cells were plated at a density of 1 x 105 per well in six-well plates 24 hours before the induction of apoptosis. Apoptosis was induced by a combination of TNF- and cycloheximide or TNF-related apoptosis-inducing ligand (TRAIL); then, cells were harvested by trypsinization and double stained with Annexin V-FITC and PI using an Annexin V-FITC Apoptosis Detection kit (BioVision Research Products, Mountain View, CA) according to the manufacturer's protocol. Samples were immediately analyzed with a FACScan flow cytometer with CellQuest software. This method allows a distinction to be made between early (Annexin V-FITC–positive/PI-negative) and late (Annexin V-FITC–positive/PI-positive) apoptosis cells, and the apoptosis rate was defined as the percentage of the Annexin V-FITC–positive rate in apoptosis-induced cells. Assays were done in triplicate.
Caspase Activity Assay
Cells were plated at a density of 1 x 106 in 10 cm dishes 24 hours before the induction of apoptosis. After TNF- and cycloheximide treatment, the activity of caspase-3, -8, and -9 was assayed using a caspase-3/CPP32, FLICE/caspase-8, and caspase-9/Mch6 Colorimetric Assay kit (BioVision Research Products) according to the manufacturer's protocol. Assays were done in triplicate.
Antisense Oligonucleotides
Antisense oligonucleotides and controls directed to hRFI have been made available by Biognostik (Gottingen, Germany). Two 18-mer phosphorothioate antisense oligonucleotides (AS1: TCATCTTCTGTGTTTGCT and AS2: GCTCCTGCAAATGCTGAC) and an 18-mer and a 16-mer phosphorothioate control oligonucleotides (C1: ACTACTACACTAGACTAC and C2: GCTCTATGACTCCCAG) were synthesized.
Delivery of Antisense Oligonucleotides
Cells were seeded at a density of 1 x 105 per mL in six-well plates 24 hours before transfection and were transfected with 1 µmol/L antisense oligonucleotides using Lipofectin according to the manufacturer's protocol. Transfectants were incubated for 6 hours at 37°C and, after replacement of the medium, maintained for an additional 24 hours before each assay.
|
Results
|
|---|
hRFI Was Expressed in All of Eight Human Colorectal Cancer Cell Lines
The expression levels of the hRFI protein in eight different human colorectal cancer cell lines were examined by Western blot analysis using an anti-hRFI antibody. A 46-kDa hRFI protein, whose molecular weight was expected from hRFI amino acid sequence, was expressed in all cell lines at variable levels (Fig. 1). Another immunoreactive band was also detected of 
60 kDa in all cell lines. The proportion of the two expression levels was variable among the cell lines examined.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 1. Western blot analysis of hRFI expression in colorectal cancer cells. Protein extracted from cells was analyzed for the hRFI protein in eight human colorectal cancer cell lines. Rabbit monoclonal anti-hRFI polyclonal antibody was used. Anti-ß-actin was used as a loading control. Bars, molecular weights of 68 kDa (top) and 41 kDa (bottom).
|
|
Selection of HCT116 Cells Stably Expressing hRFI
HCT116 cells exhibited a relatively low level of hRFI expression by Western blot analysis (Fig. 1), and HCT116 cells are relatively sensitive to TNF--induced apoptosis (16). Therefore, HCT116 cells were considered appropriate for examining the antiapoptotic function of hRFI by means of overexpression in human colorectal cancer cells.
To evaluate the functional role of hRFI in human colon cancer cells, we first established HCT116 cell lines stably overexpressing hRFI. HCT116 cells were transfected with an expression vector containing full-length cDNA of hRFI (HCT116/hRFI cells). After selection in G418, resistant cells were isolated and the relative level of hRFI was determined by Western blot. The Western blot analysis revealed that HCT116/hRFI cells exhibited an increased expression of hRFI compared with control cells transfected with LacZ (HCT116/LacZ cells; Fig. 2). The exogenously transfected hRFI with V5-His tag was detected at a larger molecular weight than the native hRFI.
View larger version (43K):
[in this window]
[in a new window]
|
Figure 2. Western blot analysis of hRFI expression in HCT116 cells stably transfected with hRFI or LacZ. HCT116 cells were transfected with an expression vector containing the full-length cDNA of hRFI or LacZ using Lipofectin. After 2–3 wks of selection in G418, resistant cells were isolated and analyzed for the hRFI protein by Western blot. An anti-hRFI polyclonal antibody and anti-V5-horseradish peroxidase antibody were used. Anti-ß-actin was used as a loading control.
|
|
Transfection of hRFI Had No Effect on Cell Proliferation and Cell Cycle Distribution
To determine whether hRFI expression affected cell proliferation, in vitro cell proliferation of HCT116/hRFI and HCT116/LacZ cells was assessed by the proliferation assay. No significant differences in proliferation were shown between these transfectants at 24, 36, 48, 60, or 72 hours after plating (data not shown). Next, we examined whether hRFI expression affected the cell cycle of HCT116/hRFI and HCT116/LacZ cells. As determined by flow cytometry, no differences were shown in the percentage of each cell cycle distribution between these transfectants (data not shown). These results indicate that hRFI expression does not affect cell proliferation and cell cycle distribution.
Overexpression of hRFI Inhibited Apoptosis Induced by TNF-
TNF- is a pleiotropic cytokine that can elicit a variety of biological responses, such as induction of differentiation and proliferation, but it causes apoptosis in the presence of cycloheximide (17–19). To investigate whether hRFI expression affected TNF--induced apoptosis, HCT116/hRFI and HCT116/LacZ cells were treated with 10 ng/mL TNF- and 1 µg/mL cycloheximide for 5 hours and subjected to a nuclear staining assay. Nuclear staining with PI revealed that apoptotic cells with typical apoptotic nuclei were fewer in HCT116/hRFI cells than in HCT116/LacZ cells (Fig. 3A). To quantify the apoptosis rates, HCT116/hRFI and HCT116/LacZ cells were exposed to TNF- and cycloheximide under various conditions and analyzed for the relative apoptotic rate after double staining with Annexin V-FITC and PI using flow cytometry. As shown in Fig. 3B, control cells exhibited no staining for either Annexin V-FITC or PI, and cells that underwent apoptosis stained positive for Annexin V. As shown in Fig. 3C and D, the apoptosis rate was seen in a dose- and time-dependent manner in both transfectants. However, the apoptosis rate in the HCT116/hRFI cells was significantly decreased compared with those of the HCT116/LacZ cells. The apoptosis rate was approximately three times less in the HCT116/hRFI cells than in HCT116/LacZ at 4 hours after exposure to 10 ng/mL TNF- and 1 µg/mL cycloheximide (Fig. 3D). The difference in apoptosis rate was less prominent, but still statistically significant, at 8 hours after apoptosis induction (P < 0.05, Student's t test).
View larger version (34K):
[in this window]
[in a new window]
|
Figure 3. Overexpression of hRFI inhibited apoptosis induced by TNF- and cycloheximide. A, representative fields of nuclear staining assay after 5 h of treatment with 10 ng/mL TNF- and 1 µg/mL cycloheximide, showing that the PI-stained apoptotic cells with typical apoptotic nuclei were fewer in HCT116/hRFI cells compared with HCT116/LacZ cells. B–D, flow cytometric analysis of apoptosis induced by TNF- and cycloheximide showing the effect of hRFI on HCT116 cells. As shown in B, cells without apoptosis exhibited no staining for both Annexin V-FITC and PI, and cells that underwent apoptosis stained positive for Annexin V. HCT116/hRFI and HCT116/LacZ cells were exposed to the indicated concentrations of TNF- and cycloheximide for 5 h (C) or to 10 ng/mL TNF- and 1 µg/mL cycloheximide for the indicated durations (D) and analyzed with a flow cytometer for the apoptosis rate after double staining with Annexin V-FITC and PI. Columns, mean of triplicate values; bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the control by Student's t test.
|
|
These results clearly indicate that the overexpression of hRFI inhibited the apoptosis induced by TNF- and cycloheximide in HCT116 cells.
Overexpression of hRFI Inhibited Apoptosis Induced by TRAIL
To determine whether the antiapoptotic effect of hRFI was specific to TNF--induced apoptosis or general to death receptor–mediated apoptosis, those transfectants were exposed to TRAIL under various conditions and assessed for apoptosis induction. As shown in Fig. 4A and B, the apoptosis rate by TRAIL was significantly decreased in the HCT116/hRFI cells. These results clearly indicate that the inhibition of apoptosis by hRFI overexpression is not specific to TNF--induced apoptosis but general to death receptor–mediated apoptosis.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 4. Overexpression of hRFI inhibited apoptosis induced by TRAIL. HCT116/hRFI and HCT116/LacZ cells were exposed to the indicated concentrations of TAIL for 4 h (A) or to 100 ng/mL TRAIL for the indicated durations (B) and analyzed with a flow cytometer for the apoptosis rate after double staining with Annexin V-FITC and PI. Columns, mean of triplicate values; bars, SD. *, P < 0.05; **, P < 0.01, compared with the control by Student's t test.
|
|
hRFI Overexpression Reduced Caspase-3, -8, and -9 Activation
To determine whether the antiapoptotic effect of hRFI was associated with the inhibition of the activation of caspases, we measured the activity of caspase-3, -8, and -9 in HCT116/hRFI and HCT116/LacZ cells after treatment with 10 ng/mL TNF- and 1 µg/mL cycloheximide. Caspase-8 activity in HCT116/hRFI cells was inhibited to the half of that in HCT116/LacZ cells at 2 hours after treatment (Fig. 5A). At this time point, caspase-9 activity did not exhibit any difference but exhibited similar inhibition at 4 hours after treatment (Fig. 5B). As shown in Fig. 5C, caspase-3 activation in HCT116/hRFI cells was similarly inhibited to the half of that in HCT116/LacZ cells at 2 hours after treatment, and this inhibition was more prominent at 4 hours.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 5. hRFI overexpression decreases caspase-3, -8, and -9 activation. Cells were plated at a density of 1 x 106 in 10 cm dishes 24 h before the induction of apoptosis. After treatment with 10 ng/mL TNF- and 1 µg/mL cycloheximide for the indicated durations, the activity of caspase-8 (A), caspase-9 (B), and caspase-3 (C) was assayed using a FLICE/caspase-8, caspase-9/Mch6, and caspase-3/CPP32 colorimetric assay kit, respectively. Columns, mean of triplicate values; bars, SD. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001, compared with the control by Student's t test.
|
|
Antisense-Mediated Down-Regulation of Overexpressed hRFI Reversed Its Antiapoptotic Effect
The difference in apoptosis sensitivity between HCT116/hRFI and HCT116/LacZ cells might be attributed to the clonal effect acquired through the selection in G418 and not to differences in the hRFI expression level. To exclude this possibility, antisense down-regulation of overexpressed hRFI was done in HCT116/hRFI cells using two antisense (A1 and A2) and two control (C1 and C2) oligonucleotides. Both antisense oligonucleotides effectively down-regulated exogenously overexpressed hRFI in HCT116/hRFI cells at 24 hours after transfection (Fig. 6A). The expression of hRFI partially recovered at 48 hours but was still down-regulated compared with the control. The efficiency of antisense down-regulation was higher in A2 oligonucleotides than in A1. Therefore, the following antisense experiments used A2 and the two control oligonucleotides.
Antisense Down-Regulation of Overexpressed hRFI Did Not Affect Cell Proliferation
To determine whether antisense down-regulation of hRFI affected cell proliferation, in vitro cell proliferation was compared by proliferation assay between antisense-treated HCT116/hRFI cells and control cells. No difference was seen at 24, 36, 48, 60, or 72 hours after transfection (data not shown). Thus, antisense down-regulation of hRFI did not influence the cell proliferation.
Antisense Down-Regulation of Overexpressed hRFI Reversed Its Antiapoptotic Effect
Finally, we examined whether the down-regulation of overexpressed hRFI reversed the inhibition of apoptosis. HCT116/hRFI cells transfected with AS2 or control oligonucleotides were treated with 30 ng/mL TNF- and 3 µg/mL cycloheximide for 6 hours, and the apoptosis rate was analyzed by flow cytometry after double staining with Annexin V-FITC and PI. The apoptosis rate of antisense-treated HCT116/hRFI cells showed a significant increase compared with that of the control (Fig. 6B). This indicates that the antisense down-regulation of overexpressed hRFI resulted in the recovery of apoptotic sensitivity, confirming the antiapoptotic effect of hRFI in HCT116 cells.
|
Discussion
|
|---|
In this study, we found that stable overexpression of hRFI in HCT116 human colorectal cancer cells inhibits TNF--induced apoptosis with an evidently decreased activity of caspase-3, -8, and -9. We also revealed that overexpression of hRFI inhibits TRAIL-induced apoptosis. These evidence indicate that hRFI overexpression can suppress apoptosis triggered by death receptor signals. Moreover, the antisense down-regulation of hRFI effectively reversed the antiapoptotic effect of hRFI overexpression. This indicates that the antiapoptotic property of the hRFI transfectants is not due to the clonal effect acquired via selection in G418 but is in fact dependent on hRFI function. This is the first report to show that hRFI molecule really elicits an antiapoptotic function in carcinoma cells.
Western blot analysis of eight colorectal cancer cell lines revealed that the hRFI protein was expressed in all of these lines, although the expression levels were variable. This result is also consistent with previous immunohistochemistry findings showing that hRFI is diffusely expressed in colorectal carcinomas (15). On the other hand, previous multitissue Northern blot analysis has shown hRFI mRNA to be more highly expressed in normal colonic mucosa than in SW480 colorectal carcinoma cells, and this is inconsistent with the immunohistochemical result that hRFI is not expressed in normal colonic mucosa (15). Interestingly, this discrepancy between mRNA and protein expression levels was likewise observed in other antiapoptotic genes, XIAP, cIAP1, and cIAP2, using various human tumor cell lines and human leukemia samples (20). These discrepancies suggest the existence of post-transcriptional regulation of the expression, such as different translational mechanisms or different protein turnover rates (20). Indeed, the existence of translational regulation of XIAP expression has been reported, with the identification of an internal ribosome entry site within the XIAP mRNA (21–23).
We found that the time course of caspase-3 and -8 activation preceded that of caspase-9 during the induction of apoptosis. In the extrinsic apoptotic pathway, stimulation of death receptors by extracellular death ligands, such as TNF- and TRAIL, results in the formation of death-inducing signaling complexes and the activation of caspase-8, initiating the sequentially subsequent cleavage of downstream effector caspases, such as caspase-3 (24). On the other hand, activation of caspase-8 also results in the cleavage of Bid, which acts as a bridge to a subsequent initiation of the mitochondrial apoptotic pathway, with the release of cytochrome c from mitochondria and the activation of the caspase-9-containing apoptosome complex (25, 26). The time course of caspase activation shown in our study is consistent with the time course of caspase activation in the extrinsic apoptotic pathway followed by interconnection to the mitochondrial apoptotic pathway.
The functional mechanism of caspase inhibition by hRFI is not yet clear. hRFI possesses a ring finger domain homologous to XIAP but lacks a BIR domain (11). The BIR domain has been shown to be essential for the caspase inhibition and subsequent suppression of apoptosis by the IAP family, whereas the ring finger domain does not seem to be essential for the inhibition of caspase activity by the IAPs (27), and the function of the ring finger domain is still not well defined (27). Recent studies have shown that the ring finger is associated with ubiquitin ligase activity, which has also been revealed in the ring finger of the IAPs (28). For example, the ring finger domain of cIAP2 has been shown to ubiquitinate caspase-3 and -7 in vitro, which are direct targets of binding and inhibition by the IAPs (29). Although the single ring finger domain of XIAP is unable to inhibit caspase activity by itself, the ring finger domain of XIAP does have the ubiquitin ligase activity, which is responsible for the proteosomal degradation of caspase-3, Smac, and XIAP itself, thus regulating the antiapoptotic effect of XIAP (27, 30–32). These previous findings have indicated that ubiquitination by the ring finger domain mediates, at least in part, the apoptosis regulation by IAP family. In the present study, overexpression of hRFI resulted in a marked inhibition of caspase-3, -8, and -9 activity during apoptosis in HCT116 cells. Considering that the ring finger domain of hRFI possesses a strong homology to that of XIAP, because 22 of the 53 XIAP ring finger residues are identical, this result suggests a possible relationship between hRFI and the ubiquitin ligase activity targeted to caspases, Smac, and XIAP (11).
In conclusion, hRFI overexpression in HCT116 certainly inhibits death receptor–mediated apoptosis with decreased activities of caspase-3, -8, and -9, and this inhibition is effectively reversed by antisense down-regulation. Considering the progressive increase of hRFI expression in the colorectal adenoma-carcinoma sequence, hRFI is evidently one of the important players in colorectal carcinogenesis through its effect on the regulation of apoptosis. Because apoptosis is closely associated with chemosensitivity and radiosensitivity, hRFI might be a new predictive marker for chemotherapy and a novel therapeutic target for gene therapy in colorectal carcinomas, as in the case of survivin (1, 33).
|
Acknowledgments
|
|---|
We thank all the members of the Tumor Biology Study Group for helpful discussions and C. Uchikawa for excellent technical assistance.
|
Footnotes
|
|---|
Grant support: Ministry of Education, Science, Sports, Culture, and Technology of Japan; Ministry of Health, Labour, and Welfare; and Public Trust of Surgery Research Fund.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1/17/05;
revised 2/22/05;
accepted 3/ 9/05.
|
References
|
|---|
Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456–62.[Abstract/Free Full Text]
Reed JC. Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol 1999;11:68–75.[CrossRef][Medline]
Miller LK. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol 1999;9:323–8.[CrossRef][Medline]
Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3:917–21.[CrossRef][Medline]
Reed JC. The survivin saga goes in vivo. J Clin Invest 2001;108:965–9.[Medline]
Kawasaki H, Toyoda M, Shinohara H, et al. Expression of survivin correlates with apoptosis, proliferation, and angiogenesis during human colorectal tumorigenesis. Cancer 2001;91:2026–32.[CrossRef][Medline]
Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998;58:5071–4.[Abstract/Free Full Text]
Ponnelle T, Chapusot C, Martin L, et al. Subcellular expression of c-IAP1 and c-IAP2 in colorectal cancers: relationships with clinicopathological features and prognosis. Pathol Res Pract 2003;199:723–31.[CrossRef][Medline]
Syken J, De-Medina T, Munger K. TID1, a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions. Proc Natl Acad Sci U S A 1999;96:8499–504.[Abstract/Free Full Text]
Syken J, Macian F, Agarwal S, Rao A, Munger K. TID1, a mammalian homologue of the Drosophila tumor suppressor lethal(2) tumorous imaginal discs, regulates activation-induced cell death in Th2 cells. Oncogene 2003;22:4636–41.[CrossRef][Medline]
Sasaki S, Nakamura T, Arakawa H, et al. Isolation and characterization of a novel gene, hRFI, preferentially expressed in esophageal cancer. Oncogene 2002;21:5024–30.[CrossRef][Medline]
Holcik M, Gibson H, Korneluk RG. XIAP: apoptotic brake and promising therapeutic target. Apoptosis 2001;6:253–61.[CrossRef][Medline]
Yang L, Cao Z, Yan H, Wood WC. Coexistence of high levels of apoptotic signaling and inhibitor of apoptosis proteins in human tumor cells: implication for cancer specific therapy. Cancer Res 2003;63:6815–24.[Abstract/Free Full Text]
Sasaki S, Kitayama J, Watanabe T, Konishi T, Nagawa H. Diffuse expression of hRFI is correlated with blood vessel invasion in gastric carcinoma. Jpn J Clin Oncol 2004;34:584–7.[Abstract/Free Full Text]
Sasaki S, Watanabe T, Konishi T, Kitayama J, Nagawa H. Effects of expression of hRFI on adenoma formation and tumor progression in colorectal adenoma-carcinoma sequence. J Exp Clin Cancer Res 2004;23:507–12.[Medline]
Zwacka RM, Stark L, Dunlop MG. NF-
B kinetics predetermine TNF- sensitivity of colorectal cancer cells. J Gene Med 2000;2:334–43.[CrossRef][Medline]
Kobayashi N, Takada Y, Hachiya M, Ando K, Nakajima N, Akashi M. TNF- induced p21(WAF1) but not Bax in colon cancer cells WiDr with mutated p53: important role of protein stabilization. Cytokine 2000;12:1745–54.[CrossRef][Medline]
Wang Z, Rao PJ, Castresana MR, Newman WH. TNF- induces proliferation or apoptosis in human saphenous vein smooth muscle cells depending on phenotype. Am J Physiol Heart Circ Physiol 2005;288:H293–301.[Abstract/Free Full Text]
Benoit V, Chariot A, Delacroix L, et al. Caspase-8-dependent HER-2 cleavage in response to tumor necrosis factor stimulation is counteracted by nuclear factor B through c-FLIP-L expression. Cancer Res 2004;64:2684–91.[Abstract/Free Full Text]
Tamm I, Kornblau SM, Segall H, et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res 2000;6:1796–803.[Abstract/Free Full Text]
Holcik M, Korneluk RG. Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation. Mol Cell Biol 2000;20:4648–57.[Abstract/Free Full Text]
Holcik M, Sonenberg N, Korneluk RG. Internal ribosome initiation of translation and the control of cell death. Trends Genet 2000;16:469–73.[CrossRef][Medline]
Holcik M, Yeh C, Korneluk RG, Chow T. Translational upregulation of X-linked inhibitor of apoptosis (XIAP) increases resistance to radiation induced cell death. Oncogene 2000;19:4174–7.[CrossRef][Medline]
Krammer PH. CD95's deadly mission in the immune system. Nature 2000;407:789–95.[CrossRef][Medline]
Debatin KM, Krammer PH. Death receptors in chemotherapy and cancer. Oncogene 2004;23:2950–66.[CrossRef][Medline]
Roy S, Nicholson DW. Cross-talk in cell death signaling. J Exp Med 2000;192:21–6.
Takahashi R, Deveraux Q, Tamm I, et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem 1998;273:7787–90.[Abstract/Free Full Text]
Deshaies RJ. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 1999;15:435–67.[CrossRef][Medline]
Huang H, Joazeiro CA, Bonfoco E, Kamada S, Leverson JD, Hunter T. The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7. J Biol Chem 2000;275:26661–4.[Abstract/Free Full Text]
Yang Y, Fang S, Jensen JP, Weissman AM, Ashwell JD. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 2000;288:874–7.[Abstract/Free Full Text]
Suzuki Y, Nakabayashi Y, Takahashi R. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proc Natl Acad Sci U S A 2001;98:8662–7.[Abstract/Free Full Text]
MacFarlane M, Merrison W, Bratton SB, Cohen GM. Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro. J Biol Chem 2002;277:36611–6.[Abstract/Free Full Text]
Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994;124:1–6.[Free Full Text]
Top
Abstract
Introduction
