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Carcinogenesis Advance Access originally published online on August 25, 2005
Carcinogenesis 2006 27(2):205-215; doi:10.1093/carcin/bgi217
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Carcinogenesis vol.27 no.2 © Oxford University Press 2005; all rights reserved.

Proteasome mediated degradation of Id-1 is associated with TNF{alpha}-induced apoptosis in prostate cancer cells

Ming-Tat Ling, Wai-Kei Kwok, Maggie K. Fung, Wang Xianghong * and Yong-Chuan Wong *

Cancer Biology Group, Department of Anatomy, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, SAR, China

* To whom correspondence should be addressed. Tel: +852 2819 9226; Fax: +852 2817 0857; Email: ycwong{at}hkucc.hku.hk; xhwang{at}hkucc.hku.hk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overexpression of the helix–loop–helix protein Id-1 has been reported in over 20 types of cancer. While a number of factors have been demonstrated to regulate Id-1 gene transcription, little is known about the mechanisms responsible for its degradation. In this study, we have demonstrated that Id-1 protein stability was regulated by TNF{alpha} in prostate cancer cells. We found that exposure of prostate cancer cell lines, DU145 and PC-3, to TNF{alpha} resulted in a rapid and significant downregulation of the Id-1 protein level. The fact that neither the Id-1 promoter activity nor the Id-1 mRNA level was affected by the TNF{alpha} treatment suggested that the decrease in Id-1 protein was not due to the suppression of gene transcription. In addition, the half-life of the Id-1 protein was decreased in both cell lines in the presence of TNF{alpha}, and the addition of an ubiquitin/proteasome inhibitor (MG-132) prior to the TNF{alpha} treatment completely blocked the effect of the TNF{alpha}-induced Id-1 protein degradation. Furthermore, introduction of a Flag-tag sequence into the N-terminus region of the Id-1 protein, which has been shown to stabilize the protein, was able to protect the Id-1 protein from TNF{alpha}-induced degradation. These results suggest that TNF{alpha} downregulated Id-1 through activation of the ubiquitin/proteasome degradation pathway in prostate cancer cells. Interestingly, in both DU145 and PC-3 cells, the decrease of Id-1 protein was associated with the activation of apoptotic pathway, as evidenced by the increased expression of cleaved PARP and caspase 3. In addition, TNF{alpha} failed to downregulate Id-1 in a sub-line of LNCaP cells that was resistant to TNF{alpha}-induced apoptosis. These results further suggest that the downregulation of Id-1 may facilitate TNF{alpha}-induced apoptosis in prostate cancer cells. In conclusion, our findings indicate that Id-1 protein may be regulated by TNF{alpha} through the ubiquitin/proteasome degradation pathway and the stability of the Id-1 protein appears to correlate with the sensitivity of TNF{alpha}-induced apoptosis.

Abbreviations: HLH, helix–loop–helix


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Id-1 is a helix–loop–helix (HLH) protein that belongs to the Id protein family (1), which binds with the basic-helix–loop–helix (bHLH) transcription factors. It lacks the basic domain for DNA binding, and thus functions as a dominant negative regulator of the bHLH transcription factor by inhibiting the bHLH from binding to target DNA sequences (1). Id-1 has a wide range of functions, including inhibition of cellular differentiation (2), activation of angiogenesis (3) as well as induction of cell proliferation (4). Recently, overexpression of Id-1 has been reported in over 20 types of cancers (5), including the breast (6), cervical (7) as well as prostate cancer (8). In addition, Id-1 has been shown to induce cancer cell proliferation through activation of MAPK (4) and inactivation of p16/RB (9) signaling pathways, suggesting that Id-1 may play an important role in cancer development. Meanwhile, Id-1 was also found to enhance cancer cell invasion in both cell lines and animal models, (3,10), and the downregulation of Id-1 has been shown to suppress metastasis of the mammary carcinoma (11). More importantly, in prostate cancer, Id-1 expression has been shown to correlate with Gleason grading (8) as well as the development of androgen independence (12), suggesting that Id-1 may play an essential role in cancer progression.

Recently, overexpression of Id-1 has been shown to protect cancer cells against apoptosis. For instance, in nasopharyngeal carcinoma cells, Id-1 has been shown to activate the MAPK signaling pathway, leading to reduction of sensitivity to Taxol-induced apoptosis (13 I). In addition, ectopic Id-1 expression has been shown to confer the androgen dependent prostate cancer cells (LNCaP), which are sensitive to TNF{alpha}-induced apoptosis, resistance to TNF{alpha} treatment through induction of the NF-Kappa B activities (14). Furthermore, suppression of Id-1 expression in androgen independent prostate cancer cell line DU145, which constitutively expresses high level of Id-1, was found to sensitize the cells to TNF{alpha}-induced apoptosis (14). Therefore, Id-1 may as well function as an inhibitor of apoptosis through the activation of multiple signaling pathways.

Although the oncogenic properties of Id-1 have been well demonstrated, the mechanism responsible for its upregulation cancer cell is still unclear. At transcriptional level, Id-1 expression can be induced by cytokines, such as BMP2 or BMP4, through direct activation of the Id-1 promoter (15,16). Meanwhile, growth factors such as IGF-1 or NGF have also been reported as potent inducers of Id-1 expression (17,18). Furthermore, a number of differentiation inducing agents, such as Vitamin D or TGF-ß1, have been demonstrated to suppress Id-1 expression in different types of cells (19,20). Since genetic or epigenetic changes of the Id-1 gene have not been reported to date, deregulation of the growth factors may be one of the possible mechanisms that contribute to the upregulation of Id-1 in human cancers.

Like other members of the Id family, Id-1 protein is degraded in a rapid manner by the ubiquitin/proteasome pathway, with a half-life of ~30–60 min (21,22). Through this pathway, the protein is first covalently attached with the ubiquitin, and is then subsequently degraded by the 26S proteasome complex. Therefore, the stability as well as the expression level of the Id-1 protein can be modulated by regulating the level of ubiquitinylation as well as the accessibility of the Id-1 protein to the 26S proteasome complex. For example, a recent study has shown that addition of a Myc-tag sequence at the N-terminus of the Id-1 protein can retard the ubiquitinylation as well as degradation of the Id-1 protein (22). In addition, dimerization of Id-1 with MyoD has also been demonstrated to protect the Id-1 protein from proteasome-mediated degradation (22). Meanwhile, binding of Id-1 to the COP9 signalosome, which is a homolog of the 26S proteasome lid (23), was found to enhance the ubiquitinylation as well as degradation of Id-1 (24). Nonetheless, the biological consequence of Id-1 degradation and the mechanisms controlling this process are still far from clear.

We report in this study that in androgen independent prostate cancer cell lines (DU145 and PC-3) TNF{alpha} treatment can drastically reduced the stability of the Id-1 protein through the ubiquitin/proteasome system. We found that TNF{alpha} treatment did not alter either the promoter activities or the mRNA level of the Id-1 gene, suggesting that the protein stability, but not the transcription of the Id-1 gene was affected by TNF{alpha}. Interestingly, we also found that downregulation of Id-1 by TNF{alpha} was associated with activation of the apoptotic pathway, which was correlated with increased sensitivity to TNF{alpha}-induced apoptosis. Our data suggest that downregulation of Id-1 through protein degradation may be essential for the induction of apoptosis by TNF{alpha}.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
Human prostate adenocarcinoma cell lines LNCaP, PC-3 and DU145 were obtained from American Type Culture Collection (Rockville, MD). All the cell lines were maintained in RPMI 1640 (Sigma, St Louis, MO) supplemented with 5% of fetal calf serum (FCS) (Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. TNF{alpha} (PeproTech EC, London, UK) was dissolved in H2O, Cycloheximide and MG-132 (Calbiochem, La Jolla, CA) were dissolved in DMSO and the synthetic androgen R1881 (Perkin-Elmer, Wellesley, MA) was dissolved in absolute ethanol.

Plasmids and transient transfection
To generate the vectors expressing the Flag-tagged full length or truncated form of Id-1, PCRs were performed using the full length Id-1 cDNA as the template, with the following primers: Flag-Id-1 (Forward-TAG GAT CCA TGG ATT ACA AGG ATG ACG ACG ATA AGA TGG GCA AGA CAG CGA, Reverse-ACG AAT TCT CAG CGA CAC AAG A), Id-1-Flag (Forward-TAG GAT CCT TAT GAA AGT CGC CA, Reverse-ACG AAT TCT CAC TTA TCG TCG TCA TCC TTG TAA TCG CGA CAC AAG ATG CGA) and Id-1-Flag-NT (Forward-TAG GAT CCG GCT GTT ACT CAC G). The PCR products were then cloned into the pcDNA3.1 vector. The Id-1 mutant [Id-1-Flag(101Y->D)] was generated using the site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the procedure described by the manufacturer. The Id-1 reporter construct contains the 2.1 kb of the Id-1 promoter and has been described previously (10). The resulting constructs were then transfected into the cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN).

Western blotting
Detailed experimental procedures were described previously (12). Briefly, whole cell lysates were prepared by resuspending the cell pellet in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS). Protein concentration was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA). Protein extract (20 µg for DU145 and PC-3 and 40 µg for LNCaP) was loaded onto a SDS-polyacrylamide gel for electrophoresis and then transferred to a PVDF membrane (Amersham, Piscataway, NJ). The membrane was then incubated with primary antibodies for 1 h at room temperature against Id-1, ß-actin, androgen receptor (AR) (Santa Cruz Biotechnology, Santa Cruz, CA), PARP, Caspase 3 (Cell signaling, Beverly, MA) or Flag (Sigma, St Louis, MO). After washing with TBS-T, the membrane was incubated with the secondary antibody against either mouse or rabbit IgG and the signals were visualized using ECL and the western blotting system (Amersham, Piscataway, NJ). The band intensities were then quantified by densitometry and were presented as the ratio to the control (assigned as 1), after normalization with actin.

Immunoprecipitation
Cells were co-transfected with vectors expressing the Flag-tagged and HA-tagged Id-1 protein using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN, USA) and were then lysed using RIPA buffer 48 h after transfection. The lysate was then precleared with 30 µl of protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C and then incubated with anti-Flag antibody (Sigma, St Louis, MO) for 1 h at 4°C. Protein A/G agarose (30 µl) was added into the mixture and incubated for another hour at 4°C. The agarose was then washed three times with 1 ml of RIPA buffer, boiled and then loaded onto SDS-polyacrylamide gel for electrophoresis and for western blot analysis, using the procedure described above.

Determination of the half-life of the Id-1 protein
The procedures for determination of the half-life of the Id-1 protein were as described by Trausch-Azar et al. (22). Briefly, cells were treated with cycloheximide (50 µM) for 2 h to inhibit protein synthesis and were then treated with TNF{alpha} (100 ng/ml). The cell pellet was collected at the indicated time point and lysed for western blot analysis using anti-Id-1 antibody. The band intensity of the western blot result was measured by the gel documentation system, with the reading normalized as the percentage of the initial Id-1 level (level at time = 0). The percentage was then plotted against time and the half-life of the Id-1 protein was calculated as the time required for degradation of 50% of the protein.

Semi-quatitative RT–PCR
Total RNA was isolated from treated and untreated cells using Trizol reagent, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). For RT–PCR, cDNAs were synthesized by using the SuperScriptTM First Strand Synthesis System (Invitrogen, Carlsbad, CA). The cDNAs were than amplified by PCR with Id-1 specific primers, with sequences and conditions as described previously (20). The cDNA of ß-actin was amplified as a control for the amount of cDNA present in each sample. PCR products were electrophoresed on 2% agarose gels and analyzed using the gel documentation system. The signals were quantified by densitometry, normalized with actin and were presented as the ratio to the control, which was assigned as 1.

Luciferase reporter assay
Luciferase reporter assay was performed by plating the cells (1 x 105 cells per well) into a 12-well culture plates, and the cells were allowed to grow for 24 h. pGL3-Id-1 (luciferase reporter containing the Id-1 promoter, a gift from Dr Desprez, USA) (10), was co-transfected with pRL-TK-Luc into the cells using Fugene 6 reagent (Roche Diagnostics, Indianapolis, IN, USA). Cells were lysed 48 h after transfection and were assayed for luciferase activity using the Dual-luciferase reporter assay system (Promega, WI). Luciferase activity in the presence of TNF{alpha} was compared with the untreated control, which was assigned the value of 100%. The standard deviation of the means was used as error bars and the results are presented for three independent experiments.

Cell cycle analysis
For the analysis, 5 x 105 cells were plated in 5% FCS culture medium. TNF{alpha} was added into the culture medium 24 hours later and the cells were harvested by trypsinization. The cells were then fixed in ice cold 70% ethanol and were then washed with PBS before incubation with propidium iodide (50 µg/ml) and RNase (1 µg/ml) for 30 min. Cell cycle analysis was performed on a flow cytometer EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter, Miami, FL).

3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay
Cell growth rate was measured using a MTT proliferation assay kit (Roche Diagnostics, Indianapolis, IN), and the detail procedures were as described previously (12). Briefly, 1000 cells were seeded in 96-well plates and then cultured in RPMI supplemented with 5% FBS for 24 h. Medium was then changed to RPMI supplemented with charcoal-stripped FBS for another 24 h. The synthetic androgen R1881 (0.01, 0.1 and 1.0 nM) or an equal volume of ethanol was then added into the medium. Cell viability was examined at 24 h and 48 h post-exposure. Before testing, 10 µl of MTT labeling reagent (5 mg/ml MTT in PBS) was added and the cells were incubated for a further 4 h at 37°C. Then 100 µl of dissolving reagent (10% SDS in 0.01 M HCl) was added and the plate was incubated overnight at 37°C to dissolve the formazan crystals. The optical density (OD) was measured at a wave length of 570nm on a Labsystem multiskan microplate reader (Merck Eurolab, Dietikon, Schweiz). Pilot experiments were conducted to determine the optimal cell concentration for comparison of the in vitro growth of cells. Each time point was performed in triplicate wells and each experiment was repeated at least three times. Results represented the OD ratio between the treated and untreated cells at indicated time points. Each data point represents the mean and standard deviation.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
TNF{alpha} downregulates Id-1 protein level in DU145 and PC-3 cells
Since overexpression of Id-1 has been shown to protect the cells from TNF{alpha}-induced apoptosis (14), we were interested in whether TNF{alpha} affects the expression of the Id-1 protein in prostate cancer cells. To examine the effect of TNF{alpha} on Id-1 expression, two androgen independent prostate cancer cell lines, DU145 and PC-3, were treated with TNF{alpha} (100 ng/ml). After the treatment, Id-1 protein level was analyzed by western blotting. As shown in Figure 1A, treatment of DU145 cells with TNF{alpha} for as short as 4 h resulted in significant downregulation of the Id-1 expression. In addition, the level of Id-1 remained low after 16 h of treatment, indicating that the effect of TNF{alpha} was not transient. Similarly, in PC-3 cells (Figure 1A) as well as in breast, colon and ovarian cancer cell lines (data not shown) exposure to TNF{alpha} also resulted in the downregulation of Id-1 expression, suggesting that the effect of TNF{alpha} on Id-1 may be common among different types of cancer cells.


Figure 1
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  		Fig. 1. TNF{alpha}-induced Id-1 downregulation in prostate cancer cells. (A) DU145 and PC-3 cells were treated with TNF{alpha} for an indicated period and then lysed for the analysis by SDS–PAGE and western blotting, with anti-Id-1 and anti-ß-actin (loading control) antibodies. (B) DU145 and PC-3 cells co-transfected with pGL3-Id-1-Luc and the internal control pRL-TK-Luc were treated with TNF{alpha} for 4 h, lysed and then measured for Luciferase activity. The result was compared with the control (–TNF{alpha}), which was assigned the value of 100%. Each experiment was performed at least three times in duplicate wells and each data point represented the mean and standard deviation. (C) RNA extracted from DU145 and PC-3 cells after 4 h of TNF{alpha} treatment was analyzed by semi-quantitative RT–PCR using Id-1 specific primers, using the detailed conditions described in Materials and methods. PCR products were then resolved in agarose gel. The expression of ß-actin was examined as an internal control.

 
To investigate whether the TNF{alpha}-induced Id-1 suppression occurred through regulation of Id-1 gene transcription, we then examined the effect of TNF{alpha} on the transactivation of Id-1 promoter. DU145 cells were first transfected with the Id-1 reporter construct and the Id-1 promoter activities in the presence or absence of TNF{alpha} was assayed using the Luciferase reporter assay. As shown in Figure 1B, Id-1 promoter activities in both DU145 and PC-3 cells were not affected by TNF{alpha} treatment. In addition, as reflected by the result of the semi-quantitative RT–PCR analysis (Figure 1C), Id-1 mRNA level was similar between the untreated control and the cells treated with TNF{alpha}. These results suggest that TNF{alpha}-induced downregulation of Id-1 protein is not regulated at the transcription level.

TNF{alpha} downregulates Id-1 through activation of proteasome pathway
Since TNF{alpha} does not affect the transcription of the Id-1 gene, we examined whether the decrease in the Id-1 protein level was due to the induction of protein degradation. To determine the stability of the Id-1 protein in these two cell lines, we first treated the cells with the protein synthesis inhibitor cycloheximide for 2 h, and the degradation profile of the Id-1 protein was then analyzed by western blotting. As shown in Figure 2A, Id-1 level decreased gradually in both cell lines due to protein degradation, with a half-life (t1/2) of ~250 min. In the presence of TNF{alpha}, however, the degradation of Id-1 protein became more rapid and the half-life decreased sharply in both the cell lines (t1/2 < 100 min in DU145 and t1/2 < 110 mins in PC-3, Figure 2A). These results suggest that TNF{alpha} suppresses the level of Id-1 by destabilization of the protein. To study if this process involved the ubiquitin/proteasome pathway, DU145 and PC-3 cells were pre-treated with 1.0 µM of MG-132, a commonly used proteasome inhibitor, prior to the TNF{alpha} treatment. Treatment of the cells with the proteasome inhibitor not only enhanced the stability of the Id-1 protein, but also successfully protected it from TNF{alpha}-induced degradation (Figure 2B), indicating that TNF{alpha} treatment leads to degradation of Id-1 through activation of the ubiquitin/proteasome pathway.


Figure 2
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  		Fig. 2. TNF{alpha}-induces degradation of the Id-1 protein. (A) Degradation profile of Id-1 protein was examined by blocking the protein synthesis, with 2 h of cycloheximide (50 µM) treatment, followed by the addition of TNF{alpha}. Id-1 level at indicated time points was then examined by the western blot analysis. (B) Effect of ubiquitin/proteasome inhibitor on Id-1 protein stability. Cells were treated with cycloheximide together with 1 µM of MG-132 for 2 h prior to the addition of TNF{alpha}. Protein was collected at the indicated time point for western blot analysis, with Id-1 antibody. (C) Signal intensity of the western blot result was measured by gel documentation system and the reading was normalized as percentage to that of the initial Id-1 level (level at time = 0). Log10 of the percentage was plotted against time and the half-life of the Id-1 protein was calculated as the time corresponding to the log10 of 50%.

 
Addition of Flag-tag sequence at the N-terminus protects Id-1 from TNF{alpha}-induced protein degradation
Most of the proteins targeted for ubiquitin-proteasome-mediated degradation are ubiquitinylated at the internal lysine residues, yet recent studies suggested that at least in some proteins, such as Id-1 or MyoD, ubiquitinylation occur at the N-terminal region instead of the internal lysine (22). This N-terminus-dependent ubiquitinylation can be interrupted by removal or masking of the N-terminal sequence, resulting in increase of the protein stability (22,25). For example, the addition of a Myc6 tag at the N-terminus of the Id-1 protein has recently been shown to increase the half-life of the protein (22). To examine whether TNF{alpha}-induced Id-1 degradation in prostate cancer cells was also through the N-terminus-dependent pathway, we introduced a single Flag-tag sequence at either end of the Id-1 protein. These Flag-tag-Id-1 constructs were then expressed in DU145 cells and confirmed by western blotting, using anti-Id-1 and anti-Flag antibodies (Figure 3A).


Figure 3
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  		Fig. 3. TNF{alpha}-induces Id-1 degradation through the N-terminus-dependent pathway. (A) DU145 cells were transiently transfected with the empty vector (pcDNA), the N-terminus Flag-tag-Id-1 (Flag-Id-1) or the C-terminus Flag-tag-Id-1 (Id-1-Flag). Cells were lysed 48 h after transfection and analyzed by western blotting with anti-Id-1 (upper panel) or anti-Flag antibodies (lower panel). (B) The half-life of the Flag-Id-1 or Id-1-Flag protein in DU145 cells was analyzed as described in the legend of Figure 2B. (C) DU145 cells expressing either the Flag-Id-1 or Id-1-Flag were treated with TNF{alpha} for the indicated period. Cells were then collected, lysed and examined by western blotting, with anti-Id-1 antibody. (D) PC-3 cells were transfected either with pcDNA3.1 or Id-1-Flag construct and then lysed for western blot analysis. (E) Analysis of Id-1 homodimerization by co-immunoprecipitation. PC-3 cells transfected with Flag-tagged (Id-1-Flag) and HA-tagged (Id-1-HA) Id-1 expression constructs were lysed and then subjected to immunoprecipitation and western blotting, as described in Materials and methods. (F) Effect of Id-1 mutants on endogeneous Id-1 expression in PC-3 cells. PC-3 cells were transfected with the full length Id-1, the N-terminus truncated form or the Id-1 mutant carrying a point mutation within the conserved helix–loop–helix domain. After transfection, cells were lysed for western blot analysis of Id-1 level.

 
Similar to the previous report (22), we found that Id-1 with masked N-terminus (Flag-Id-1) was highly stable, while the C-terminus-tag-Id-1 (Id-1-Flag) is still subjected to the proteasome-mediated degradation (Figure 3B). More importantly, we found that the N-terminus-tag-Id-1 was resistant to the TNF{alpha}-induced protein degradation (Figure 3C), and a slight decrease was observed only after 16 h of treatment. In contrast, TNF{alpha} was able to induce the degradation of the C-terminus-tag-Id-1 in DU145 cells (Figure 3C), although at a later time point than that of the endogenous Id-1 (8 h instead of 4 h). These results indicate that TNF{alpha} regulates the degradation of Id-1 protein through an N-terminus-dependent mechanism.

Interestingly, as shown in Figure 3A, ectopic Id-1 expression in DU145 cells resulted in upregulation of the level of endogenous Id-1 protein. This effect was even more signifcant when the Id-1-Flag construct was transfected into the PC-3 cells (Figure 3D). However, analysis of the promoter activity revealed that ectopic Id-1 expression did not lead to Id-1 promoter activation (data not shown). In addition, the co-immunoprecipitation assay revealed that Id-1 proteins are actively dimerized with each other (Figure 3E). Since dimerization of Id-1 with MyoD has been shown to stabilize the Id-1 protein (22), it is possible that the increase in endogenous Id-1 protein level is the consequence of dimerization of the ectopic Id-1 with the endogenous Id-1 protein. In fact, ectopic expression of the N-terminus truncated Id-1 (Id-1-Flag-NT) failed to induce Id-1 expression in the PC-3 cells (Figure 3F). Meanwhile, introduction of a single point mutation at the conserved helix–loop–helix domain of the Id-1 [Id-1-Flag (101Y->D)] also impaired its effect on the induction of endogeneous Id-1 (Figure 3F), suggesting that homodimerization of Id-1, which required an intact helix–loop–helix structure, is essential for the observed effect.

Downregulation of Id-1 is associated with TNF{alpha}-induced apoptosis
We have shown in our previous study that in DU145 cells downregulation of Id-1 leads to sensitization of the cells to TNF{alpha}-induced apoptosis (14). To investigate whether downregulation of Id-1 was associated with TNF{alpha}-induced apoptosis, DU145 and PC-3 cells were cultured with the TNF{alpha} containing medium for up to 5 days. While Id-1 was initially expressed at a high level in both cell lines, treatment with TNF{alpha} for 3 days resulted in a decrease of >50% of the Id-1 protein (Figure 4A). Continuation of TNF{alpha} treatment resulted in further downregulation of the Id-1 protein to a level that was almost undetectable by western blot analysis (Figure 4A). Interestingly, flow cytometry analysis (Figure 4C) revealed that, after 5 days of TNF{alpha} treatment, when Id-1 was almost absent, a significant proportion of the cells were undergoing apoptosis (20% in DU145 and 28% in PC-3, see the arrows). These results were also supported by the evidence that increased cleavage of the pro-apoptotic proteins PARP and Caspase 3 was observed in a time dependent manner, especially at day 5 of post-treatment time (Figure 4B). Taken together, our results indicate that downregulation of Id-1 is associated with TNF{alpha}-induced apoptosis in the androgen independent prostate cancer cells.


Figure 4
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  		Fig. 4. Downregulation of Id-1 correlates with induction of apoptosis by TNF{alpha}. (A) and (B) DU145 and PC-3 cells were treated with TNF{alpha} for the indicated period and cells were collected for western blot analysis, with anti-Id-1, anti-PARP and anti-caspase 3 antibodies, respectively. (C) The percentage of apoptotic cells after the TNF{alpha} treatment was examined by flow cytometry analysis and the arrow indicates the position of the sub-G1 peak.

 
Increased Id-1 protein stability correlates with resistance to TNF{alpha}-induced apoptosis in LNCaP cells
To substantiate our findings, we next investigated the effect of TNF{alpha} on Id-1 expression on the androgen dependent prostate cancer cell line, LNCaP. It has been reported that LNCaP cells, then progress to androgen independent phenotype can become resistant to TNF{alpha}-induced apoptosis (26). Since continuous passage of LNCaP cells for >80 passages has been shown to result in loss of androgen responsiveness of the cells (27), we examined whether there was difference in TNF{alpha} responsiveness between early and late passages of LNCaP cells. In agreement with the previous findings (27), the growth of the LNCaP cells at the late passage (p120) was much less sensitive to the stimulation of androgen when compared with the early passage (p60) (Figure 5A), despite the fact that they expressed similar levels of AR (Figure 5B). It was reported previously that expression of Id-1 in LNCaP cells was serum dependent (9). In the absence of serum, Id-1 was not detected in LNCaP cells, but under the normal culture condition (with serum supplementation) Id-1 protein expression was detectable in these cells (9). As shown in Figure 5B, when cultured in medium supplemented with serum, both the p60 and p120 of LNCaP cells expressed Id-1 at a similar level. However, when the cells were treated with TNF{alpha}, the response of Id-1 to the treatment was significantly different between the p60 and p120 of LNCaP cells (Figure 6A). For the cells at p60, TNF{alpha} treatment for 8 h resulted in drastic downregulation of the Id-1 protein, but under the same condition, there was no significant decrease in Id-1 level in the cells at p120. Consistent with this observation, we found that TNF{alpha} can only reduce the half-life of the Id-1 protein expressed in p60, but not that expressed in p120 (Figure 6B and C). These results suggested that Id-1 protein expressed in the late passage of the LNCaP cells is more stable in response to TNF{alpha} treatment.


Figure 5
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  		Fig. 5. Acquisition of androgen independent phenotype in late passage of LNCaP cells. (A) Androgen responsiveness of LNCaP cells at different passages was examined using MTT proliferation assay. Cells were first seeded in a 96-well plate for 24 h and were then incubated in RPMI supplemented with charcoal-stripped serum for another 24 h. The cells were then treated with different concentrations (0.01–10 nM) of the synthetic androgen (R1881) for 24 or 48 h. Cells were labeled with the MTT reagent, lysed and measured for the intensity at 570 nm, using procedures described in the Materials and methods. (B) Expression of Id-1 and AR in LNCaP cells (at p60 and p120) was examined by western blotting.

 

Figure 6
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  		Fig. 6. Id-1 expressed in late passage of LNCaP cells is less sensitive to TNF{alpha} treatment (A), LNCaP cells (at p60 and p120) were treated with TNF{alpha} for the indicated period and then lysed for western blot analysis of Id-1 expression (B). (C) Effect of TNF{alpha} on Id-1 protein degradation, as examined using the procedure described in the legend of Figure 2.

 
Importantly, as reflected by the results from the flow cytometry analysis (Figure 7A) as well as western blotting of PARP and Caspase3 proteins (Figure 7B), the LNCaP cells at p60 were more resistant to TNF{alpha}-induced apoptosis than at p120. As indicated by these results, the increase in Id-1 protein stability seems to correlate with the sensitivity of the cells to TNF{alpha}-induced apoptosis.


Figure 7
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  		Fig. 7. LNCaP cells at late passages were highly resistant to TNF{alpha}-induced apoptosis. LNCaP cells (at p60 and p120) were treated with TNF{alpha} for the indicated period and were collected either for flow cytometry analysis (A) or for western blot analysis, with anti-Id-1, anti-PARP and anti-caspase 3 antibodies (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The abundance of the Id-1 protein is maintained by the regulation of Id-1 gene transcription as well as by protein degradation. It is well documented that degradation of Id-1, like many other short-lived proteins, is through the ubiquitin/proteasome pathway (21). However, it is still not clear how this pathway is regulated with regard to Id-1 degradation, and more importantly, how this regulation may correlate with the function of Id-1. Several growth factors, such as TGF-ß1 or IGF-1, have been demonstrated to regulate the expression of Id-1 (17,20), but none have been reported to have any effect on the stability of the Id-1 protein.

In this study, we have demonstrated that Id-1 protein expression in DU145 and PC-3 cells was downregulated by the cytokine TNF{alpha}, through destabilization of the Id-1 protein (Figure 2). Although TNF{alpha} regulates the expression of its downstream targets mainly through the NF-KappaB pathway (28), recent studies suggest that TNF{alpha} also utilizes the ubiquitin/proteasome pathway as an alternative mechanism to regulate the abundance of the target proteins (29,30). For example, TNF{alpha} has been shown to inhibit myeloid leukemia cell proliferation by enhancing the ubiquitin-proteasome-mediated degradation of the D type cyclin (29). In addition, in muscle cells, TNF{alpha} treatment resulted in the degradation of MyoD protein, leading to inhibition of muscle regeneration (30). We therefore speculate that the effect of TNF{alpha} on Id-1 protein may act through the same mechanism. Indeed, we found in our study that addition of the proteasome specific inhibitor alone was able to completely abolish the effect of TNF{alpha} on Id-1 (Figure 2C), suggesting that the TNF{alpha}-induced degradation of Id-1 in prostate cancer cells is mediated through the ubiquitin/proteasome pathway. Interestingly, activation of the NF-Kappa signaling pathway was found to be essential for the TNF{alpha}-induced MyoD degradation (30). It is thus possible that the same pathway may be employed by TNF{alpha} in the regulation of Id-1 protein stability.

Although most of the proteins designated for proteasome degradation are ubiquitinylated at the internal lysine residues, in some proteins, ubiquitinylation may also occur at the N-terminal region (25,31). A typical example is the MyoD protein, where ubiquitinylation occurs at both N-terminal and internal lysine residues (25). This was proved by the finding that degradation of the MyoD protein is significantly interfered by either insertion of Myc-tag sequence at the N-terminus or removal of the internal lysine residue (25). Recently, it was found that degradation of Id-1 also utilized the N-terminus dependent-pathway, and the addition of the Myc-tag sequence at the N-terminus of the Id-1 protein greatly stabilized the protein (22). However, unlike MyoD, the removal of internal lysine residues did not affect the rate of Id-1 protein degradation (22). This suggests that N-terminal ubiquitinylation is the predominant mechanism for the degradation of Id-1 protein. In fact this is also true for the TNF{alpha}-induced Id-1 degradation, since we found in this study that Id-1 protein with its N-terminus fused with a Flag sequence was relatively resistant to the action of TNF{alpha} (Figure 3C).

Our finding that TNF{alpha} does not affect the transcription of Id-1 is somewhat contradictory to previous reports, which showed that TNF{alpha} can significantly affect Id-1 expression at transcription level (32,33). However, as demonstrated by the promoter assay as well as semi-quantitative RT–PCR, after TNF{alpha} treatment, where significant decrease of Id-1 protein level was observed, there was no detectable change in Id-1 transcription (Figure 1). Therefore, the decrease in Id-1 level in the prostate cancer cells by TNF{alpha} is not due to changes in Id-1 gene transcription. Since the cell types examined in our study (prostate epithelial cells) and previous studies [Epidermal (32) and neuronal cells (33)] are of different origin, it is possible that the response of Id-1 gene transcription to TNF{alpha} may be cell type specific. Another reason could be the differences in the dosage of TNF{alpha} used between these studies, which are worth further elucidation.

Although there are no reports suggesting that Id-1 can regulate its own expression, in this study, we observed that the endogenous Id-1 level was upregulated in the cells ectopically expressing the Flag-tagged Id-1 (Figure 3D). Since neither the N-terminus-tag nor the C-terminus-tag-Id-1 had any effect on Id-1 promoter activity (data not shown), the increase in Id-1 expression is not likely due to the upregulation of Id-1 transcription. Since dimerization of Id-1 with MyoD has been shown to protect the Id-1 protein from degradation (22), it is possible that the increase in Id-1 level may due to dimerization of the ectopic and endogenous Id-1. This was further supported by the finding that disuption of the helix–loop–helix structure significantly impaired the Flag-tagged Id-1 from upregulating the endogenous Id-1 (Figure 3F).

It is worth to note that Id-1 expressed in the prostate cancer cell lines was much more stable (t1/2 > 4 h) than that (t1/2 < 1 h) reported in previous studies (21,22). This is likely due to the use of difference cell lines, since examination of cell lines from other types of cancer revealed that the half-life of the Id-1 protein varied significantly among different cancers (data not shown). Whether this variation in Id-1 protein stability between these types of cancers correlates with their sensitivity to TNF{alpha} treatment is worth further investigation.

Previously, we have demonstrated that downregulation of Id-1 in DU145 cells by anti-sense oligonucleotide treatment sensitized the cells to TNF{alpha} treatment (14), suggesting that Id-1 plays an important role in TNF{alpha}-induced apoptosis. Since we found in this study that TNF{alpha} can downregulate Id-1 in both DU145 and PC-3 cells, we speculated that continuous treatment of TNF{alpha} may also lead to apoptosis induction. As expected, prolonged treatment of PC-3 and DU145 cells with TNF{alpha} resulted in drastic downregulation of Id-1, which was accompanied by significant increase of percentage of apoptotic cells (Figure 4C). In addition, in the late passage of LNCaP cells, which had acquired the androgen independent phenotype state, Id-1 expression was less sensitive to the TNF{alpha} treatment (Figure 5C). More importantly, these cells were also more resistant to TNF{alpha}-induced apoptosis than the cells of the early passage. These results suggest that degradation of Id-1 may be essential for the induction of apoptosis by TNF{alpha}. Since Id-1 has been shown to upregulate the anti-apoptotic protein bcl-XL in prostate cancer cells while to suppress the induction of the pro-apoptotic protein bax (14), we speculated that downregulation of Id-1 by TNF{alpha} may result in a shift of bcl-XL/bax ratio, which favor the induction of apoptosis.

In conclusion, we have shown in this study that TNF{alpha} regulates Id-1 protein degradation through the ubiquitin/proteasome pathway. Meanwhile, as demonstrated in prostate cancer cells, Id-1 degradation is associated with the induction of apoptosis by TNF{alpha}. Therefore, our results provide evidence that Id-1 may function as an inhibitor of apoptosis in prostate cancer cells and inactivation of Id-1 may therefore be a potential therapeutic strategy for prostate cancer treatment.


   Acknowledgments
 
This work was supported by HKU seed funding (N-HKU738/03) and RGC grants (HKU7470/04M) to Y.C.W. and to X.W. (HKU7478/03M).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 19, 2005; revised August 14, 2005; accepted August 19, 2005.


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X. Zhang, M.-T. Ling, Q. Wang, C.-K. Lau, S. C. L. Leung, T. K. Lee, A. L. M. Cheung, Y.-C. Wong, and X. Wang
Identification of a Novel Inhibitor of Differentiation-1 (ID-1) Binding Partner, Caveolin-1, and Its Role in Epithelial-Mesenchymal Transition and Resistance to Apoptosis in Prostate Cancer Cells
J. Biol. Chem., November 16, 2007; 282(46): 33284 - 33294.
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