Id-1 stimulates serum independent prostate cancer cell proliferation through inactivation of p16INK4a/pRB pathway
Xue Song Ouyang,
Xianghong Wang,
Ming-Tat Ling,
Hing Lok Wong,
Sai Wah Tsao and
Y.C. Wong,1
Department of Anatomy, Faculty of Medicine, 5/F, Li Shu Fan Building, 5 Sassoon Road, University of Hong Kong, Hong Kong, SAR, China
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Abstract
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It has been suggested that the helixloophelix protein Id-1 plays an important role in tumourigenesis in certain types of human cancer. Previously, we reported that Id-1 was up-regulated during sex hormone-induced prostate carcinogenesis in a Noble rat model (Ouyang et al. (2001) Carcinogenesis, 22, 965973). In the present study, we investigated the direct effect of Id-1 expression on human prostate cancer cell proliferation by transfecting an Id-1 expression vector into a prostate cancer cell line LNCaP. Ten stable transfectant clones were isolated and the ectopic Id-1 expression resulted in both increased DNA synthesis rate and the percentage of S phase cells. To study the possible mechanisms involved in the Id-1 induced prostate cancer cell growth, we examined the expression of several factors responsible for G1 to S phase progression. We found that Id-1 expression induced phosphorylation of RB and down-regulation of p16INK4a but not p21Waf1or p27Kip1. Our results indicate that the Id-1 induced inactivation of p16INK4a/pRB pathway may be responsible for the increased cell proliferation in prostate cancer cells. Given the fact that both Id-1 over-expression and inactivation of p16INK4a/pRB are common events in prostate cancer, our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.
Abbreviations: BrdU, 5'-bromo-2'-deoxyuridine; FCS, fetal calf serum; HLH, helixloophelix.
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Introduction
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Id proteins (inhibitor of differentiation or DNA binding) are a group of helixloophelix (HLH) transcription factors that lack the DNA-binding domain. Therefore, their function is mainly to act as dominant inhibitors of basic HLH proteins by forming non-functional Id-bHLH heterodimers. Since most of the bHLH proteins positively activate genes in cell differentiation, the Id proteins are considered to be the negative regulators of differentiation (1,2). The fact that Id proteins can stimulate DNA synthesis and immortalize mammalian cells, either alone or incorporated with additional oncogenes (35), indicates that they may function as potential oncogenes. Increased Id-1 expression has been found in several types of primary tumours including breast (6), pancreatic (7,8), prostate (9) and head and neck (10). Recently ectopic expression of Id-1 induced increased aggressiveness and metastasis in breast cancer cells (6), and up-regulation of Id-1 has also been correlated with increased tumour stage in several human cancers (8,10). In addition, in Id-1+/Id3/ knockout mice, a significantly reduced metastatic ability of tumour xenografts has been reported (11). These lines of evidence strongly suggest that Id proteins play important roles not only in tumourigenesis but also in tumour progression.
Previously, using cDNA array technique, we reported an up-regulation of Id-1 during sex hormone-induced prostate carcinogenesis in a Noble rat model (9) and increased Id-1 expression was also correlated with progression of human prostate cancer (12). Although it has been suggested that Id-2 and Id-4 promote cell proliferation through direct inactivation of pRB in human osteosarcoma and glioma cells (13), there is little evidence on the mechanisms involved in the function of Id-1. Recently, Id-1 has been shown to facilitate the bypass of replicative senescence by directly inhibiting p16INK4a expression in mouse and young human diploid fibroblasts (14,15). To study the direct effect of Id-1 on human prostate cancer cell growth and the possible mechanisms involved, in the present study we transfected an Id-1 expression vector into a prostate cancer cell line LNCaP, which showed undetectable levels of Id-1 in the absence of fetal calf serum (FCS), and isolated 10 stable transfectant clones. Here we report that ectopic Id-1 expression stimulated serum independent prostate cancer cell proliferation through inactivation of p16INK4a/pRB pathway.
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Materials and methods
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Cell lines and cell culture conditions
Human prostate cancer cell line LNCaP was obtained from American Tissue Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 medium supplemented with 10% FCS and penicillin (50 units/ml) and streptomycin (50 mg/ml) 37°C.
Generation of Id-1 transfectants
The retroviral vector containing full length Id-1 cDNA (pBabe-Id-1) (6) or pBabe-puro was transfected into the PG13 packaging cell line (obtained from ATCC) using the calcium phosphate method. After one-week's selection in 4 µg/ml puromycin, the culture medium containing infectious viruses was harvested for retroviral infection of LNCaP cells. Briefly, the virus-containing supernatant was mixed with an equal volume of fresh medium containing 8 µg/ml polybrene and then added to LNCaP cells. Puromycin (1 µg/ml), which killed all of the parental cells, was added 24 h later and ten Id-1 stable transfectant clones were isolated ~14 days after drug selection to generate LNCaP-pBabe-Id-1 C1 to C10 clones. Vector control was generated from a pool of >20 individual clones transfected with pBabe.
Measurement of cell growth
Two thousand cells were plated in each well in 24-well plates in medium containing 5% fetal calf serum (FCS). Serum free medium replaced the FCS containing medium 24 h after plating and the cells were counted every day using trypan blue assay. Each data point was tested on triplicate wells and each experiment was repeated at least three times. Cell growth curves were drawn using the means of each experiment and the error bars represent the standard error of the means.
Cell cycle analysis
Cells (5 x 105) were trypsinized and washed once in PBS. They were then fixed in cold 70% ethanol and stored at 4°C. Before testing, the ethanol was removed and the cells were resuspended in PBS. The fixed cells were then washed with PBS and treated with RNase (1 µg/ml) and stained with propidium iodide (50 µg/ml) for 30 min at 37°C. Cell cycle analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter Electronics, Hialeah, FL).
5'-Bromo-2'-deoxyuridine (BrdU) incorporation
Cells grown on 4 mm Chamber slides (ICN, Biomedicals, Aurora, OH) were treated with BrdU (10 µM) for 2 h and then washed once with PBS. The cells were then fixed in cold methanol/acetone (1:1) for 5 min at room temperature and washed in PBS. The cells were incubated with monoclonal antibody against BrdU (1:10, Roche) for 1 h at 37°C and detailed procedures were described in the protocols provided in Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Each experiment was repeated three times and at least 1000 cells were evaluated in each experiment. The error bars represent the standard deviation (SD) from three independent experiments.
Western blotting
Cell lysate was prepared by suspending the cells in a modified radioimmunoprecipitation (RIPA) buffer (50 mM TrisHCl [pH 8.0], 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS) including proteinase inhibitors (1mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM PMSF), and protein concentrations were measured using the protein assay kit (Bio-Rad). Equal amounts of proteins (50 µg) were separated by electrophoresis on a 12.5% SDSpolyacrylamide gel (SDSPAGE) and blotted onto the nitrocellulose membrane (Amersham). After blocking with 5% non-fat dry milk/2% BSA in TBS for 1 h, the blots were incubated with primary antibodies for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham) for another 1 h. The immunoreactive signals were detected by ECL Plus western blot detection reagents (Amersham) following the manufacturer's instructions. Antibodies against Id-1 (1:200, C20, Santa Cruz Biotechnology), p16INK4a (1:500, N20, Santa Cruz Biotechnology), CDK4 (1:250, Transduction Laboratories), p21Waf1 (1:1000, N20, Santa Cruz Biotechnology), p27Kip1 (1:1000, Santa Cruz Biotechnology), CDK2 (1:2000, Transduction Laboratories) and pRB (1:500, Ab-1, Oncogene) were used. The relative amounts of each protein were quantitated as ratios to Actin (1:500, Amersham).
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Results
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Introduction of ectopic Id-1 expression and its effect on prostate cancer growth
The effect of FCS on Id-1 expression in LNCaP cells was studied using western blotting analysis. As shown in Figure 1A
, Id-1 expression was high when cultured in 10% FCS and reduced with decreased FCS concentrations (10% to 0%). In the absence of FCS for 24 to 48 h, Id-1 was undetectable in LNCaP cells. To study the effect of ectopic Id-1 expression on prostate cancer cells, a retroviral vector containing full-length human Id-1 cDNA (pBabe-Id-1) (6) was transfected into LNCaP cells and ten stable transfectants clones were selected in puromycin (1 mg/ml). Vector control was generated using a pool of multiple clones transfected with the control vector pBabe. As shown in Figure 1B
, in the absence of FCS, seven out of the ten transfectant clones expressed Id-1 at different levels. The effect of Id-1 on prostate cancer cell growth was studied on these transfectant clones and additional controls, including the parental LNCaP cells and LNCaP-pBabe (vector control) under the same culture conditions.

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Fig. 1. (A) Serum-dependent Id-1 expression in LNCaP cells. LNCaP cells were cultured in medium (RPMI 1640, Sigma) containing different concentrations of FCS for 24 to 48 h before being analyzed by western blotting. Note that Id-1 expression decreases with decreased FCS concentrations. (B) Id-1 expression levels in stable transfectant clones (Id-1-C1-10), vector control (pBabe) and parental LNCaP cells cultured in serum free medium for 48 h. Note that seven out of 10 clones express different levels of Id-1 protein while Id-1 is undetectable in the controls. Expression of actin was tested as an internal control.
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It has been shown that when cultured in serum free medium and drug selective conditions, LNCaP cells sometimes show unstable morphology, however, we did not observe any significant morphological changes in LNCaP cells after introduction of Id-1 or cultured in serum free medium for up to 72 h (Figure 2A
). However, introduction of Id-1 resulted in an increase in cell growth which was also correlated with the expression levels of Id-1 (Figure 2B
).

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Fig. 2. Cellular morphology and cell growth rate in LNCaP cells and the Id-1 transfectants. (A) Morphological changes before and after introduction of Id-1 in LNCaP cells. (1): pBabe cultured in 5% FCS; (2): pBabe cultured in serum-free (SF) medium for 48 h; (3) and (4): Id-1-C2 and C7 cultured in SF medium for 48 h. Photos were taken under 200x magnification. Note that there are no significant morphological changes before and after introduction of Id-1. (B) Growth curves of the Id-1 transfectants and pBabe. Each time point was derived from three independent experiments and the error bars represent standard deviation. Note that increased cell growth rate is correlated with the increased levels of Id-1 expression.
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Effect of Id-1 expression on DNA synthesis and cell cycle distribution in LNCaP cells
Next we studied if the Id-1 induced cell growth was due to its ability to initiate DNA synthesis in prostate cancer cells in serum-free medium. Cell cycle analysis showed that in the absence of FCS, there was 5.89% of S phase cells in the control LNCaP-pBabe cells but the percentage of S phase cells was significantly increased (1119%) in the Id-1 expressing transfectants (Id-1-C2-7 and C10) (Figure 3A
). The number of S phase cells present in these transfectants was comparable with the vector control LNCaP-pBabe cultured in 10% FCS (14%). However, there was no significant increase in S phase cells in Id-1-C1, 8 and 9 (68%), which showed undetectable levels of Id-1 under the same culture conditions, compared with LNCaP-pBabe. The Id-1 induced DNA synthesis was also evident when measured by BrdU incorporation (Figure 3B
). After 48 h in serum-free medium, all the Id-1 expressing clones showed increased BrdU incorporation (2580% increase) compared with the vector control (LNCaP-pBabe) or the Id-1 negative clones (C1, C8, C9). The level of increment was correlated with the levels of Id-1 expression, as C4 and C5 showed both higher Id-1 expression and BrdU incorporation compared with Id-1-C2 (Figures 1B and 3B
). The DNA synthesis rate in the clones with higher Id-1 levels was similar to the control LNCaP-pBabe cultured in 10% FCS.


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Fig. 3. Induction of prostate cancer cell proliferation in Id-1 expressing transfectants. (A) Cell cycle distribution in the cells cultured in SF medium for 48 h, unless indicated. Flow cytometric analysis was performed on an EPICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter). Note that there is an increased number of S phase cells in the cells expressing Id-1. (B) BrdU incorporation in Id-1 transfectants and controls. At least 500 cells were counted in each experiment and the percentage of BrdU positive cells was calculated and compared with the controls. All cells were cultured in SF medium for 48 h before testing, unless otherwise indicated. Note that increased BrdU incorporation rate is found in Id-1 expressing clones.
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Effect of Id-1 expression on RB/p16INK4a pathway
To investigate the mechanisms involved in Id-1-induced cell proliferation in prostate cancer cells, we studied the expression levels of p16INK4a, CDK4, p21Waf1, p27Kip1, CDK2 and RB in the Id-1 expressing clones and compared with the controls. As shown in Figure 4B
, p16INK4a was much lower or undetectable in all of the Id-1 expressing clones (Id-1-C2-7 and C10), while ~2 to 3-fold increase in p16INK4a levels was observed in the controls and the Id-1 negative clones (C1, C8 and C9). In the presence of 10% FCS, LNCaP-pBabe also showed decreased p16INK4a levels compared with the controls cultured in serum free medium. These results clearly demonstrate that expression of Id-1 reduced p16INK4a protein levels in LNCaP cells. We also found that the phosphorylated form of CDK4 (upper band) (Figure 4B
) and CDK2 (lower band) (Figure 4C
) was apparent in all of the Id-1 expressing clones but not in the controls or the Id-1 negative clones (Figure 4B
). However, we did not observe any significant changes in p21Waf1 or p27Kip1 levels in the Id-1 expressing clones (Figure 4C
). As shown in Figure 4D
, in the Id-1 expressing transfectants, phosphorylated RB (upper band) was found in all of the clones while there was no evidence of RB phosphorylation in the controls or Id-1 negative transfectants.

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Fig. 4. Western blotting analysis of p16INK4a, CDK4, p21Waf1, p27Kip1, CDK2 and RB expression in Id-1 transfectants and controls. Cells were cultured in SF medium for 48 h before harvesting, unless otherwise indicated. Results represent three independent experiments. (A) Id-1 expression levels in the transfectants and the controls; (B) Decreased p16INK4a expression and the presence of CDK4 phosphorylation are found in the cells expressing Id-1; (C) No significant changes in p21Waf1 and p27Kip1 levels after Id-1 transfection but there is an increase in the phosphorylation of CDK2; (D) Increased RB phosphorylation is present in the cells expressing Id-1 but absent in the controls.
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Discussion
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In this study, we have demonstrated the significance of Id-1 expression in serum independent proliferation of prostate cancer cells. In addition, our results indicate that inactivation of RB pathway may be responsible for its action. Our evidence may provide a possible novel mechanism on the molecular basis of prostate carcinogenesis.
After transfection of Id-1, LNCaP cells showed an increase in serum independent growth (Figure 2B
) which was accompanied with increased percentage of cell cycle S phase cells (Figure 3A
) and BrdU incorporation rate (Figure 3B
). Previously, it was reported that ectopic Id-1 expression led to cell cycle G1 to S progression in mouse 3T3 cells and human fibroblasts (17,18) and inactivation of Id-1 by antisense oligonucleotides resulted in decreased cell proliferation (16,19). Our results are consistent with previous findings on mouse cells and human breast cancer cells that ectopic Id-1 expression stimulated DNA synthesis and induced cell cycle progression from G1 to S phase (16,18). Our evidence further confirms the function of Id-1 as a promoter of cell proliferation in human cancers including prostate cancer.
Ectopic Id-1 expression induced RB phosphorylation in human keratinocytes (4) and down-regulation of p16INK4a in mouse and human young primary fibroblasts (14,15). In the present study, ectopic Id-1 expression resulted in down-regulation of p16INK4a in LNCaP cells (Figure 4B
). One of the functions of p16INK4a is to inhibit the function of cyclin dependent kinases such as CDK4 and prevents phosphorylation of RB. We also found an increase in the expression of phosphorylated CDK4 (Figure 4B
, upper band). This indicates that activation of CDK4 by phosphorylation was associated with down-regulation of p16INK4a in the Id-1 transfectants. One of the pathways that regulates RB phosphorylation and controls cell cycle from G1 to S progression is through cyclinD and CDK4/6 complex. The activated cyclinD/CDK4 complex can phosphorylate RB and prevents its binding to E2F, resulting in the entry from G1 to S progression (20). In the Id-1 transfectants, phosphorylated RB (upper band) was evident in all of the Id-1 expressing clones but absent in the Id-1 negative clones or the controls (Figure 4D
). These lines of evidence indicate that Id-1 expression resulted in the phosphorylation of RB protein possibly through down-regulation of p16INK4a. The decreased p16INK4a and increased RB phosphorylation in Id-1 transfectants also correlated with the increased S phase fraction (Figure 3A
) and BrdU incorporation rate (Figure 3B
) in these cells. These results suggest that the effect of Id-1 on growth stimulation on prostate cancer cells may be due to the decreased p16INK4a, in turn the inactivation of RB. Previously, partial inhibition of p16INK4a by ectopic Id-1 expression was observed in human keratinocytes, but no significant changes were found in CDK4 and RB levels (5). Our evidence, however, agrees with a separate study showing that Id-1 expression induced RB phosphorylation through inactivation of p16INK4a (4).
Like p16INK4a, p21Waf1 and p27Kip1 are other kinase inhibitors that have been shown to promote de-phosphorylation of RB by inhibiting CDK2 (21). In mouse 3T3 cells, overexpression of Id-1 leads to inhibition of p21Waf1 at both mRNA and protein levels which correlate with the increased cell growth (22). However, there was no evidence of p21Waf1 involvement in a separate study on human keratinocytes transfected with Id-1, even though the Id-1 induced cell growth was also observed (5). This indicates that interaction between Id-1 and p21Waf1 may be cell type specific. In the present study, we did not observe any significant changes in p21Waf1 or p27Kip1 levels in the Id-1 expressing clones (Figure 4C
). However, phosphorylated CDK2 levels were found to be increased in these cells, indicating the involvement of additional factors in the activation of CDK2. It is possible that increased CDK2 phosphorylation or possible activation of CDK2 is independent of either p21Waf1 or p27Kip1 and the mechanisms involved in this process are currently under investigation. Nevertheless, activation of CDK2 may facilitate the phosphorylation of RB observed in the Id-1 transfectants.
In summary, we provide evidence for the first time on Id-1 induced cell proliferation in prostate cancer cells. The evidence that decreased p16INK4a expression and increased CDK and pRB phosphorylation were observed in Id-1 expressing transfectants indicates that Id-1 may stimulate prostate cancer growth through inactivation of p16INK4a/pRB pathway. Both Id-1 overexpression (12) and inactivation of p16INK4a and RB (23,24) are common events in prostate cancer, and our results provide a possible mechanism on the molecular basis of prostate carcinogenesis.
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Notes
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1 To whom correspondence should be addressed Email: ycwong{at}hkucc.hku.hk 
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Acknowledgments
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This work was supported by RGC grants to Y.C.Wong (Project no: HKU 490/96M, HKU 7186/99M and HKU7314/01M).
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Received July 19, 2001;