Cyr61 suppresses the growth of non-small-cell lung cancer cells via the β-catenin–c-myc–p53 pathway

Abstract

Cysteine-rich protein 61 (Cyr61) is a growth factor-inducible, immediate-early gene that has multifaceted activities in various cancers. In a previous study, we found that Cyr61 inhibited the growth of the H520 and H460 non-small-cell lung cancer (NSCLC) cell lines. In further studies, we now report that p53 plays a pivotal role in Cyr61-dependent cellular growth arrest. Blocking Cyr61 with a Cyr61 antibody resulted in the downregulation of expression of p53 and p21, as well as partially reversing the growth suppression of H520-Cyr61 cells. Proliferation of NSCLC cell lines (NCI-H157, H125, H1299), having a mutant p53, were not suppressed by Cyr61. Inhibition of wild-type p53, by either human papilloma virus type 16 E6 or a dominant-negative p53, resulted in the rescue of the growth suppression mediated by Cyr61 in the H520-Cyr61 cells. The enhanced levels of p21WAF1 and p130/RB2, in the Cyr61-expressing H520-Cyr61 cells, were also inhibited by blocking p53 showing that p21 and p130 were induced by p53 in these cells. In addition, levels of both c-myc and β-catenin increased in Cyr61 stably transfected H520 cells. Moreover, β-catenin was translocated into the nucleus in these cells. Inhibition of c-myc expression in the H520-Cyr61 cells with antisense c-myc resulted in their decreased levels of p53. Transfecting cells with a dominant-negative T-cell factor (TCF4), the specific inhibitor of the β-catenin/TCF4 complex, downregulated the expression of c-myc. Taken together, the data suggest that Cyr61 suppressed the growth of NSCLC cells by triggering a signal transduction pathway through β-catenin. In this pathway, Cyr61 activated the β-catenin/TCF4 complex, which promoted the expression of c-myc and the latter induced expression of p53, and p53 upregulated p21WAF1 and p130/RB2, resulting in growth arrest.

Introduction

β-catenin plays an important role in both cell–cell adhesion and intracellular signal transduction (Kemler, 1993; Cadigan and Nusse, 1997; Ben-Ze’ev and Geiger, 1998; Cox and Peifer, 1998; Willert and Nusse, 1998). On the one hand, β-catenin together with α- and γ-catenin binds to members of the cadherin superfamily and mediates adherence junctions (Kemler, 1993; Gumbiner, 1996), on the other, it is a pivotal constituent of the wnt/wingless pathway.

Cytoplasmic levels and subcellular localization of β-catenin are tightly regulated by its interaction with adenomatous polyposis coli (APC) (Munemitsu et al., 1995; Bienz, 1999; Clevers, 2000); axin (Zeng et al., 1997; Behrens et al., 1998) and glycogen synthase kinase 3β (GSK-3β) (Rubinfeld et al., 1996; Yost et al., 1996). In the absence of wnt-1 signaling, β-catenin is phosphorylated by GSK-3β and degraded through a ubiquitination and proteosome pathway (Hart et al., 1999; Sadot et al., 2000). Upon activation of the wnt-1 pathway, kinase activity of GSK-3β is inhibited and β-catenin is stabilized and translocated into the nucleus. In the nucleus, β-catenin binds to members of the T-cell factor/lymphocyte-enhancing factor (TCF/LEF) family and serves as a transcriptional regulator (Behrens et al., 1996; Riese et al., 1997; van de Wetering et al., 1997; Hsu et al., 1998).

Several genes have been identified as transcription targets of the β-catenin/TCF complex. c-myc is one of them, whose expression is directly activated by β-catenin/TCF in colon cancers having a mutation of either APC or β-catenin (He et al., 1998; Damalas et al., 2001; Shih et al., 2000). As a proto-oncogene, c-myc stimulates the expression of target genes, which play important roles in cell proliferation, growth arrest and apoptosis, by binding to consensus 5′-CACGTG-3′ nucleotide sequences in the region of these genes and behaving as a transcriptional activator (Blackwood et al., 1992; Dang, 1999; Evan et al., 1992; Marcu et al., 1992; Shi et al., 1992).

The tumor suppressor protein p53 is a target gene of c-myc (Reisman et al., 1993; Tavtigian et al., 1994). Deregulated, high levels of c-myc induce the expression of endogenous p53 (Hermeking and Eick, 1994; Roy et al., 1994). As a cellular gatekeeper, p53 plays the pivotal role in causing cell cycle arrest and apoptosis (Levine, 1997; Woods and Vousden, 2001). The p53-dependent cell cycle arrest is, in part, a consequence of the induction of p21WAF1 (el-Deiry et al., 1993, 1994; Deng et al., 1995; Huang et al., 2001). The p21WAF1 is a transcriptional target of p53. The accumulation of wild-type p53 often results in the upregulation of p21WAF1, which inhibits G1 cyclin-dependent kinases (CDKs) and causes G0–G1 arrest of the cell cycle (Harper et al., 1993; Xiong et al., 1993; Morgan, 1995; Huang et al., 2001).

Cysteine-rich protein (Cyr61) is a product of a growth factor-inducible, immediate-early gene (Kireeva et al., 1996). It is involved in tumorigenesis, but plays different roles in different tumor types. On the one hand, Cyr61 stimulates tumor progression in breast and gastric adenocarcinoma cells (Babic et al., 1998; Tsai et al., 2000; Xie et al., 2001), on the other, Cyr61 levels are decreased in prostate cancer and leiomyoma, but the functional significance of these low levels has not been studied (Pilarsky et al., 1998; Sampath et al., 2001).

In a previous report, we showed that Cyr61 was downregulated in lung cancers, and induced expression of Cyr61 in Cyr61-negative lung cancer cells resulted in their growth arrest in the G0–G1 phase of the cell cycle (Tong et al., 2001). In the present study, we further explored the mechanism of Cyr61-mediated inhibition of growth in lung cancer cells. Forced expression of Cyr61 in NCI-H520 squamous lung cancer cells caused the upregulation and nuclear localization of β-catenin, causing prominent the expression of c-myc, which in turn induced the expression of p53, which mediated the accumulation of p21 and p130/RB2. Further studies showed that the p53 played a pivotal role in Cyr61-induced suppression of cell growth.

Results

p21WAF1 and p130/RB2 were upregulated by p53 in H520-Cyr61 cells

NCI-H520 (squamous) and H460 (large cell) non-small-cell lung cancer (NSCLC) cells were stably transfected with Cyr61 (Tong et al., 2001). We previously demonstrated that transfection of Cyr61 into these cell lines resulted in a markedly decreased proliferation rate compared to the pcDNA3.1-transfected control cells (Neo cells) (see Tong et al., 2001 and Figure 2b and c). In our earlier report, the observed decrease in proliferation was accompanied by increased expression of the tumor suppressor proteins p53, p21, pRb and pRb2/p130. We investigated in more detail the molecular mechanism that was responsible for this growth suppression.

Figure 2
figure2

Inhibition of p53 in H520-Cyr61 cells and measurement of their growth. Either pcDNA1-DNp53 (dominant-negative p53) or pcDNA3.1-E6 (HPV16 E6) was cotransfected with the pTK-Hyg selection vector into the H520-Cyr61-1 cells. After 48 h, cells were treated with the medium containing both 400 μg/ml G418 and 200 μg/ml hygromycin for 2 weeks. Single colonies were picked and amplified. (a) The H520-Cyr61 cells stably transfected with either HPV16 E6 or DNp53 were subjected to Western analysis with antibodies against p53 and p21. Protein (50 μg) of each sample was used for blotting. Expression levels for each protein relative to the Neo cells in each cell type are recorded beneath each band. (b) Cell growth studies using HPV16 E6. A total of 3 × 103 cells of H520-Neo, H520-Cyr61-1 and two sublines (D4 and D5) of H520-Cyr61-1 stably transfected with E6 were plated into 96-well plates. After culturing for different durations, growth rates were measured by MTT assays. (c) Cell growth studies using DNp53. Proliferation of H520-Neo (control), H520-Cyr61-1 cells and sublines of these cells stably transfected with DNp53 (two sublines, A2 and C4) were measured by MTT assay as described above. Data represent mean±s.d. of four culture wells. Each experiment was repeated at least three times and similar results were obtained on each occasion

To examine whether the upregulation of p21 and p130 in H520-Cyr61 cells was p53 dependent or independent, replication-incompetent adenoviral recombinants expressing human papilloma virus type 16 (HPV16) E6 (Ad-E6) were used to inhibit p53. The virus expressing GFP (Ad-GFP) was used as a control. E6 protein of HPV 16 binds to p53 and targets it for degradation through the ubiquitin pathway (Scheffner et al., 1990; Werness et al., 1990; Seewaldt et al., 2001).

Levels of p53 in both H520-Cyr61-1 and H520-Cyr61-D4 stable sublines were markedly decreased by Ad-E6 (Figure 1). In parallel, p21 and the pocket protein p130/RB2 were also decreased by the addition of Ad-E6 to these cells, suggesting that both p21 and p130 are induced by p53 in H520-Cyr61 cells.

Figure 1
figure1

Effect of HPV16 E6 on the expression of p21 and p130. H520-Neo cells and H520-Cyr61 cells were infected by either Ad-GFP (recombinant adenovirus-containing GFP) or Ad-E6 (recombinant adenovirus-containing HPV16 E6) at an MOI of about 50. After 48 h, cells were harvested and 40 μg of total protein of each sample was subjected to Western blot analysis with antibodies against p53, p21, p130 and GAPDH (control to insure even loading). Expression levels for each protein relative to the Neo cells in each cell type are recorded beneath each band

Inhibition of p53 rescued the growth suppression of H520-Cyr61 cells

As described above, p53 might play an important role in the growth inhibition of H520-Cyr61 cells. To confirm this hypothesis, dominant-negative p53 (DNp53) and HPV16 E6 were stably transfected into H520-Cyr61 cells. DNp53 inhibited the expression of p21, and E6 down regulated levels of both p53 and p21 (Figure 2a). When transfected with E6, these H520-Cyr61/E6 cells grew more quickly than the H520-Cyr61 cells in liquid culture as measured by MTT assay (Figure 2b). In control experiments, H520-Neo cells transfected with E6 expressed less p53 and p21 (Figure 2a) and grew more rapidly (Figure 2b) compared to untransfected H520-Neo cells, demonstrating the correlation between E6, p53 levels and cell growth. Furthermore, the proliferative rate of the H520-Cyr61 cells expressing DNp53 was nearly the same as the Neo control cells (Figure 2c). In summary, inactivation of p53 by two specific inhibitors (DNp53 and E6) permitted the Cyr61-expressing cells (H520-Cyr61) to escape from growth suppression.

Inhibition of c-myc blocked expression of p53

The above results indicated that p53 was essential for the growth retardation caused by the expression of Cyr61. Thus, identification of upstream factors that enhanced p53 are important. Many studies have shown that activation of p53 often occurs in response to genomic damage, as well as the presence of deregulated oncogenes, such as ras and c-myc (Roy et al., 1994; Serrano et al., 1997; Zindy et al., 1998). In H520-Cyr61 clones, expression of c-myc was much greater than in the control cells transfected with pcDNA3.1 (Figure 3a). To determine if c-myc was upstream of p53, antisense oligonucleotides against c-myc (AS c-myc) were transfected into H520-Cyr61 cells. The control oligonucleotides with the same composition, but having a nonsense sequence (NS c-myc), were also transfected into parallel cells in a similar manner (Figure 3b). The cells treated with AS c-myc expressed a lower level of c-myc and p53 than the cells transfected with the NS c-myc. Experiments were repeated several additional times with similar results (data not shown).

Figure 3
figure3

Inhibition of p53 by AS c-myc. (a) Expression of c-myc was examined by Western blot in H520-Neo cells (control) and two H520-Cyr61 sublines. Protein (50 μg) from each sample was used for blotting. (b) c-myc AS study. A total of 20 μg of AS oligonucleotides against c-myc or NS oligonucleotides with the same nucleotide composition but scrambled sequences were transfected into H520-Cyr61 cells. After either 2 or 12 h, cells were harvested and 50 μg protein from each sample was subjected to Western blot analysis with antibodies against p53 and c-myc. Antibody against GAPDH was used as control. Expression levels for each protein relative to the Neo cells in each cell type are recorded beneath each band

C-myc inhibited growth of H520 cells and induced their expression of p53 and p21

H520 cells, which have wild-type p53, were transiently transfected with either pMV-c-myc or pMV empty vector (control). The H520 cells transfected with c-myc formed significantly fewer colonies than those transfected with pMV (Figure 4a). The clonal growth of the NSCLC cell line H1299, which is p53 null (Bodner et al., 1992; Mitsudomi et al., 1992), was not affected by transfection with c-myc (data not shown).

Figure 4
figure4

c-myc suppressed the growth of H520 cells. (a), c-myc inhibited colony formation in H520 cells. H520 were evenly split from one plate onto two 100-mm plates. After 24 h, the cells were transfected with the same amount (4 μg) of either pMV (control) or pMV-c-myc. After 2 days, cells were trypsinized and replated equally into 12-well plates. G418 was added at 400 μg/ml. Cultures were stained with 0.25% crystal violet in 50% methanol after 10 days of incubation. Triplicate wells are shown. (b) Effect of c-myc on the growth of H520 cells. H520 cells were transfected as described in (a). After 2 weeks, the cells were trypsinized and replated onto new dishes. After 1 week, the proliferation of the cells was measured by MTT assay. H520-Neo cells were used as control. (c) Expression of p53 and p21 in H520-c-myc cells. Cells transfected with either pcDNA3.1 (H520-Neo) or pcDNA3.1-c-myc (H520-c-myc) were harvested and 40 μg of total protein of each sample was subjected to Western blot analysis with antibodies against c-myc, p53, p21 and GAPDH (control to assess loading). Expression levels for each protein relative to the Neo cells in each cell type are recorded beneath each band

H520 cells were stably transfected with c-myc. The rate of proliferation of these H520-c-myc cells was much slower than the growth rate of the control H520-Neo cells (Figure 4b). Compared to the control cells (H520 Neo), the H520-c-myc cells had high expression of both p53 and p21 (Figure 4c).

Expression of c-myc was induced by β-catenin/TCF4 in H520-cyr61 lung cancer cells

Following expression of Cyr61 in H520 cells, total cellular levels of β-catenin increased modestly (Figure 5a). However, quite dramatically, β-catenin markedly accumulated in the nucleus of these H520-Cyr61 cells but not in the control H520-Neo cells (Figure 5b). Furthermore, culturing H520 wild-type cells in the presence of purified Cyr61 protein resulted in their nuclear translocation of β-catenin (Figure 5b). Conversely, overnight treatment of H520-Cyr61 cells with purified IgG from antiserum to Cyr61 caused redistribution of β-catenin into the cytoplasm (Figure 5b).

Figure 5
figure5

Inhibition of c-myc expression by DN-TCF4. (a) Expression of β-catenin in either H520-Neo cells and two H520-Cyr61 sublines was detected by Western blot; 50 μg protein of each sample was used for blotting. (b) H520-Neo and H520-Cyr61 cells grown overnight on coverslips were fixed with 1% paraformaldehyde for 15 min. Where indicated, anti-Cyr61 IgG was added at a concentration of 5 μg/ml and purified Cyr61 protein was added at a concentration of 700 ng/ml. After treating with cold methanol for 5 min, cells were blocked by 1% BSA for 15 min at 37°C, labeled with antibody against β-catenin and then rhodamine-linked anti-rabbit IgG. The samples were examined using a fluorescent microscope. (c) H520-Cyr61 cells were transfected with 12 μg of either pcDNA3.1 (vector control) or pcDNA3.1-DN-TCF4 (containing dominant-negative TCF4). After 48 h, cells were harvested and 50 μg protein from each sample was subjected to Western blot analysis with antibodies against β-catenin, c-myc and GAPDH (control). Expression levels for each protein relative to the Neo cells in each cell type are recorded beneath each band. (d) H520 wild-type cells were grown overnight in the presence or absence of purified Cyr61 at a concentration of 700 ng/ml

β-catenin/TCF4 can promote c-myc transcription (He et al., 1998; Damalas et al., 2001; Shih et al., 2000). To identify whether c-myc was induced by β-catenin/TCF4 in the H520-Cyr61 cells, a dominant-negative TCF4 (DN-TCF4) was used to inhibit the activity of the β-catenin/TCF4 complex. After transfection with pcDNA3.1-DN-TCF4, expression of β-catenin remained unchanged, but levels of c-myc protein decreased compared to the those of control cells transfected with the same amount of pcDNA3.1 vector (Figure 5c). Culturing H520 wild-type cells with purified Cyr61 protein upregulated expression of both β-catenin and c-myc protein levels (Figure 5d).

Discussion

The CCN (CTGF (connective tissue growth factor), Cyr61, Nov (nephroblastoma overexpressed) proteins are a family of growth factor-responsive immediate-early genes. Cyr61 is a secreted protein that associates with the extracellular matrix and can behave as a ligand for integrins (Kireeva et al., 1996; Jedsadayanmata et al., 1999; Chen et al., 2000; Grzeszkiewicz et al., 2001) and promotes a variety of cellular activities. A number of studies have shown that Cyr61 plays a role in either the development or progression of several types of cancers (Babic et al., 1998; Pilarsky et al., 1998; Tsai et al., 2000; Sampath et al., 2001; Tong et al., 2001; Xie et al., 2001).

We have paradoxically reported that Cyr61 acts as a tumor suppressor in NSCLC. It inhibits the proliferation of H520 and H460 cells by causing G1 cell cycle arrest with an upregulation of the expression of p53, p21WAF1 and p130 (Tong et al., 2001). To examine further the biologic interaction between Cyr61 and p53, we inhibited cellular p53 with either HPV16E6 or DNp53. E6 protein associates with p53 and accelerates its degradation (Scheffner et al., 1990; Werness et al., 1990; Seewaldt et al., 2001). DNp53 changes the conformation of the wild-type p53, so that it can no longer function (Kern et al., 1992; Levine, 1992; Marutani et al., 1999; Blagosklonny, 2000). When either E6 or DNp53 is expressed in the slowly growing H520-Cyr61 cells, the cells proliferated much more quickly (see Figure 2b and c). For example, the proliferation rate of the DNp53-transfected Cyr61 cells was the same as the Neo-expressing control cells.

The p53 is a transcription factor, which acts as a tumor suppressor and p21WAF1, also a tumor suppressor gene, is one of the downstream targets of p53 (el-Deiry et al., 1993, 1994; Deng et al., 1995). Enhanced expression of p53 results either in G1 arrest of these cells (Smith et al., 1994; Kuerbitz et al., 1992; Wang et al., 1998; Allan and Fried, 1999) or drives the cells toward apoptosis (Debbas and White, 1993; Lowe et al., 1993; Allan and Fried, 1999; Huang et al., 2001). The p53-dependent cell cycle arrest is often a consequence of the upregulation of expression of p21WAF1 (el-Deiry et al., 1993, 1994; Deng et al., 1995). In our experiments, arrest of cellular growth caused by p53 was also accompanied by an increase of p21WAF1. Inhibition of p53 by either E6 or DNp53 resulted in the down-regulation of p21WAF1 and increased cellular proliferation. Therefore, p21WAF1 is induced by p53 and leads to cell growth arrest in H520-Cyr61 cells.

One of the pocket protein, p130/RB2, is also upregulated in H520-Cyr61 cells. As a member of the pRB (retinoblastoma-related) family, p130 is the dominant pocket protein in quiescent and differentiated cells (Steigler and Giordano, (1999); Carroll et al., 2000). p21WAF1 inhibits the CDK associated with the G1 phase of the cell cycle. These CDKs phosphorylate pocket proteins and inhibition of CDKs leads to a G0–G1 arrest of the cell cycle (Harper et al., 1993; Xiong et al., 1993; Morgan, 1995; Weinberg, 1995). p130 protein accumulates as a result of the loss of CDK activity (Carroll et al., 2000). In our study, inhibition of p53 in H520-Cyr61 cells caused the decrease of p21WAF1 and p130, and p21WAF1 was shown to be an inhibitor of CDK (Tong et al., 2001).

Expression of p53 was markedly induced in H520-Cyr61 cells. Numerous stress signals including DNA damage and chromosomal alterations (Giaccia and Kastan, 1998; Prives, 1998; Ashcroft et al., 2000), as well as aberrant expression of oncogenes, such ras and c-myc (Roy et al., 1994; Serrano et al., 1997; Zindy et al., 1998), are able to cause the activation and accumulation of wild-type p53. Recent studies have revealed that β-catenin, a pivotal component of the wnt/wingless signal pathway, promotes the accumulation of p53 (Damalas et al., 1999, 2001). Our studies showed that both c-myc and β-catenin are upregulated after enhanced expression of Cyr61. Abundant expression of c-myc is reported to cause increased levels of p53 mRNA and protein (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994; Tavtigian et al., 1994; Yu et al., 1997). On one hand, the p53 gene is transcriptionally upregulated by c-myc. Furthermore, the p53 protein is stabilized by c-myc via ARF (Zindy et al., 1998). The prominent expression of p53 in H520-Cyr61 cells is inhibited by AS c-myc, indicating that p53 is induced by c-myc in these cells. Moreover, H520 cells directly transfected with c-myc undergo growth arrest with increased levels of p53 and p21WAF1, showing that c-myc is upstream of p53.

β-catenin, when translocated into the nucleus, associates with TCF and acts as a transcription factor (Behrens et al., 1996; Riese et al., 1997; van de Wetering et al., 1997; Hsu et al., 1998). c-myc is one of the target genes of β-catenin/TCF4 (He et al., 1998; Shih et al., 2000; Damalas et al., 2001; Danilkovitch-Miagkova et al., 2001; Waikel et al., 2001). We find a dramatic accumulation of β-catenin in the nucleus after the expression of Cyr61 in H520 cells, or following their treatment with purified Cyr61 protein. Blocking β-catenin by DN-TCF4, a specific inhibitor of the β-catenin/TCF4 complex, results in decreased expression of c-myc in H520-Cyr61 cells, suggesting that enhanced levels of c-myc is mediated by β-catenin/TCF4 in these cells.

The results generated in this investigation provide the data for the model, suggesting how Cyr61 suppresses growth of NSCLC cells (Figure 6). First, the secreted Cyr61 binds to several integrins in an autocrine and paracrine manner (Kireeva et al., 1998; Jedsadayanmata et al., 1999; Chen et al., 2000; Grzeszkiewicz et al., 2001) and triggers the signal transduction pathway to cause nuclear accumulation of β-catenin. How signals are transduced from integrin to β-catenin is not clearly determined, but some studies show that integrin-linked kinase can induce the activity of β-catenin/TCF4 (Delcommenne et al., 1998; Novak et al., 1998; Tan et al., 2001). The Cyr61 probably indirectly mediated the upregulation and translocation into the nucleus of β-catenin. Nuclear β-catenin associates with TCF4 to form a transcriptional complex (Behrens et al., 1996; Riese et al., 1997; van de Wetering et al., 1997; Hsu et al., 1998). β-catenin/TCF4 promotes expression of c-myc, c-myc upregulates p53 and p53 transactivates p21WAF1. As a result, p21WAF1 inhibits CDK2 (Tong et al., 2001) and induces the accumulation of p130. p130 cooperates with p21WAF1 to suppress cellular growth. Cells harboring a mutant p53 would have this pathway blocked at the c-myc/p53 interface.

Figure 6
figure6

Model of the pathway used by Cyr61 to suppress the growth of NSCLC cells. Cyr61 in an autocrine or paracrine manner interacts with integrin to activate β-catenin, which translocates into the nucleus and associates with TCF4 resulting in the transcription of c-myc. In NSCLC cells with wild-type p53 (e.g. H520, H460), c-myc induces the expression of p53, and the latter promotes the accumulation of p21WAF1 and p130/RB2. p21WAF1 and p130 cause growth arrest. In cells with mutant p53 (e.g. H1299, SW900), the pathway is blocked at the c-myc/p53 interface, and therefore, Cyr61 cannot suppress the growth

Cyr61 probably has alternate roles in different cancers (Babic et al., 1998; Pilarsky et al., 1998; Tong et al., 2001; Xie et al., 2001). Although many studies have confirmed the importance of Cyr61 in the development of selected tumors, the mechanism(s) by which Cyr61 mediates these effects are still poorly understood. In contrast, the results reported here demonstrate that Cyr61 suppresses growth of NSCLC cells with Cyr61 triggering signal transduction through β-catenin, and the p53 protein playing a pivotal role in the control of cell growth.

Materials and methods

Cell culture and stable transfection

NCI-H460, NCI-H520 and NCI-H1299 lung cancer cells were purchased from ATCC and maintained in RPMI 1640 medium (Life Technology, Inc.) supplemented with 10% fetal calf serum. Lung cancer cell lines, H520 (squamous cell) and H460 (large cell) stably expressing Cyr61, were obtained as described previously (Tong et al., 2001). Neomycin-resistant cells (Neo cells) were obtained by transfecting them with empty pcDNA3.1 vector.

To make sublines stably transfected with DNp53 (R175H) (Kern et al., 1992; Levine, 1992; Marutani et al., 1999), DNp53 was cloned into pcDNA3.1 and cotransfected with pTK-Hyg (Clontech) into H520-Cyr61 cells. After 48 h, cells were replated in the medium containing 400 μg/ml G418 and 200 μg/ml hygromycin. After 2 weeks, single colonies were replated into 24-well plates. The cDNA of E6 of HPV 16 was cloned into pcDNA3.1, and H520 cells stably transfected with pcDNA3.1-E6 were obtained as described above.

Transient transfection of cells

Each of the two sublines of H520-Cyr61 cells was split evenly into two 100-mm plates. After growing to approximately 60% confluence, cells were transfected with either c-myc AS phosphorothioate oligonucleotides (Calbiochem, 475959) or c-myc NS oligonucleotides (Calbiochem, 475961) using Geneporter reagent as described above. Oligonucleotides 20 μg were used for each 100-mm plate. After 2 and 12 h, cells were harvested for Western blot. Also, either pcDNA3.1 or pcDNA3.1-DN TCF4 (generous gift from Dr KW Kinzler) was also transiently transfected into H520-Cyr61 cells in a similar manner. In this case, 10 μg of plasmid was used for the transfection of each plate. After 2 days, cells were harvested for Western blot.

Colony formation assay

The H520 or H1299 cells were split evenly into two 100-mm dishes. After growing to approximately 60% confluence, cells were transfected with 4 μg of either pMV or pMV-c-myc. After 48 h, cells were resuspended in the medium containing 400 μg/ml G418 and replated into 12-well plates. After treatment with G418 for 2 weeks, the cells were stained with 0.25% crystal violet in 50% methanol.

Cell proliferation assay

The cells, which had been stably transfected with either Cyr61 (H520-Cyr61, H460-Cyr61) or pcDNA3.1 vector, which contains the neomycin-resistant gene, were plated into 96-well plates at 3.0 × 103 cells per well. After culturing for various durations, cell numbers were measured by MTT assay according to the protocol provided by Boehringer Mannheim. The proliferation rate of H520-Cyr61 cells transfected with either DNp53 or HPV16 E6, and H520 cells transfected with c-myc, were measured in a similar manner.

Viral infection

The recombinant adenovirus, Ad-GFP (containing GFP) and Ad-E6 (containing HPV 16 E6) were kindly provided by Dr KW Kinzler and Dr WS el-Deiry. Cells (H520-Neo, H520-Cyr61) were infected with either Ad-GFP or Ad-E6 at an MOI of about 50. After 24 h of incubation at 37°C, cells were harvested for Western blot.

Western blot

Cells were harvested for total cell lysates with RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5) containing protease inhibitor cocktail (Boehringer Mannheim) as well as 1 mM NaF and 1 mM NaVO4. Cell lysates were centrifuged at 13,000 r.p.m. for 10 min at 4°C. The supernatant was collected and the protein concentration was measured. The same amount of protein was added to each lane, resolved on 4–15% SDS–PAGE and transferred to PVDF membranes. Antibodies against p21WAF1 (Oncogene, OP64), p53 (Santa Cruz, sc-126), pRB2/P130 (Santa Cruz, sc-317), β-catenin (Santa Cruz, sc7199), c-myc (Santa Cruz, sc-764) and FLAG (M5, Sigma, F4042) were used to detect these proteins.

Immunofluorescence

H520-Neo and H520-Cyr61 cells were cultured on cover slips, rinsed twice with phosphate-buffered saline (PBS) and fixed with 1% paraformaldehyde for 15 min. After being permeabilized with −20°C methanol for 5 min, cells were blocked by 1% bovine serum albumin (BSA) for 15 min at 37°C and labeled with antibody against β-catenin for 1 hr at 37°C. Cells were washed with PBS three times, 5 min each time and incubated with rhodamine-conjugated goat anti-rabbit IgG for 30 min at 37°C. After washing, the samples were mounted and examined under the fluorescent microscope.

Production and purification of Cyr61 protein in Sf9 insect cells

Full-length Cyr61 was cloned into the transfer plasmid pVL1392 (BD Biosciences). Sf9 insect cells were maintained in TNM-FH Insect Medium (BD Biosciences). His-tagged Cyr61 protein was produced in Sf9 cells by infecting these cells with Cyr61-baculovirus according to the Baculovirus Expression Vector System (BD Biosciences). The conditional media containing Cyr61 were purified by a two-step-purification method; the conditional media were initially purified using Hitrap Heparin HP columns (Amersham Bioscience), and the eluted fraction was further purified using His Bind Resin (Novagen). Finally, the eluted fraction from the His Bind Resin column was applied to a PD-10 desalting column (Amersham Bioscience) to exchange the imidazole-containing elution buffer for PBS. Purification was confirmed by running an aliquot of the material on an SDS–PAGE gel, and either staining with coomassie blue or transferring the gel to a PVDF membrane and Western blotting using a Cyr61 antibody.

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Acknowledgements

This work was supported in part by the NIH Lung Spore grant CA90388 as well as support from the Parker Hughes Trust. HPK is the recipient of the Goodson Endowed Chair in Oncology Research and is a member of the Jonsson Cancer Center and the Molecular Biology Institute of UCLA.

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Correspondence to James O'Kelly.

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Tong, X., O'Kelly, J., Xie, D. et al. Cyr61 suppresses the growth of non-small-cell lung cancer cells via the β-catenin–c-myc–p53 pathway. Oncogene 23, 4847–4855 (2004). https://doi.org/10.1038/sj.onc.1207628

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Keywords

  • Cyr61
  • β-catenin
  • c-myc
  • p53, p21
  • p130

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