Introduction

NF-kB is a transcription factor present in the majority of cells as an inactive cytoplasmic precursor complexed with the inhibitory subunit IkB (Baeuerle and Baltimore, 1988). Activation of NF-kB in response to stimuli such as TNF- and IL-1 involves phosphorylation and degradation of IkB, accompanied by the release of activated NF-kB. Activated NF-kB then translocates to the nucleus, where it plays an important role in regulating apoptosis through activating transcription of NF-kB-dependent genes (Siebenlist et al., 1994; Baichwal and Baeuerle, 1997; Sonenshein, 1997). With the exception of mature B cells, virtually all normal cells have an inactive form of NF-kB in the cytoplasm sequestered by IkB (Doerre and Corley, 1999). However, the constitutive activation of NF-kB in certain neoplastic cells such as Hodgkin/Reed–Sternberg cells, lymphoma and acute leukemia cells has been reported (Bargou et al., 1996; Giri and Aggarwal, 1998; Krappmann et al., 1999; Kordes et al., 2000; Guzman et al., 2002).

Although the mechanism for constitutive activation of NF-kB is not clear, studies from several investigators have demonstrated that enhanced degradation of IkB and aberrant activation of IkB kinase (IKK) lead to constitutive nuclear NF-kB activity in some cases of neoplastic disease (Shattuck-Brandt and Richmond, 1997; Krappmann et al., 1999), as well as in normal B-lineage lymphoid cells (Miyamoto et al., 1994; Schauer et al., 1998). Whereas constitutive activation of NF-kB in normal B-lymphocytes is required for cellular growth and immune function (Gugasyan et al., 2000), constitutive NF-kB activation in neoplastic cells contributes to abnormal proliferation and resistance to apoptosis, resulting in disease progression (Bargou et al., 1997; Giri and Aggarwal, 1998).

Approaches to suppress NF-kB activation in malignant cells have been considered a potential treatment for neoplasia. For example, inhibition of constitutive NF-kB by protease inhibitors that stabilize the NF-kB-inhibitor IkB induces apoptosis in human Burkitt's lymphoma and acute leukemia cells (Chandra et al., 1998; Jeremias et al., 1998; Orlowski et al., 1998). Furthermore, inhibition of NF-kB activation by expression of a dominant-negative mutant form IkB (IkBm), which is resistant to phosphorylation and degradation, increased apoptosis following stimuli known to activate NF-kB such as TNF- (Van Antwerp et al., 1996; Wang et al., 1996).

The role of IkBm-mediated downregulation of NF-kB in sensitizing cells to chemotherapeutic drugs remains controversial. It has been reported that IkBm-mediated inhibition of NF-kB nuclear translocation enhanced apoptotic killing by certain chemotherapeutic drugs in human fibrosarcoma and pancreatic carcinoma cell lines (Wang et al., 1996; Arlt et al., 2001). However, several other studies have shown that IkBm-mediated inhibition of NF-kB in the human colon carcinoma, breast cancer and some pancreatic cell lines did not increase sensitivity to cytotoxic drugs (Bentires-Alj et al., 1999; Dong et al., 2002).

The function of IkB as an inhibitor of NF-kB in regulating activation of NF-kB has been well studied. Most recently, IkB has been found to inhibit p53 tumor suppressor protein by binding p53 to form a cytoplasmic p53.IkB complex (Chang, 2002), thus preventing p53 nuclear translocation. On the basis of this data, we hypothesized that certain apoptotic stimuli might induce the phosphorylation and degradation of IkB, releasing p53 from the complex and permitting nuclear translocation; activation of p53 would sensitize cells to p53-dependent drugs such as anthracyclines. Furthermore, transfection of IkBm might stably sequester p53 in the cytoplasm to block p53-induced cell death, resulting in increased resistance to p53-dependent drugs but increased sensitivity to p53-independent, NF-kB-dependent drugs. To test this hypothesis, we transfected IkBm into an ALL cell line with constitutively activated NF-kB, and evaluated the response of these cells to p53-dependent and independent chemotherapeutic agents.

Results

Inhibition of both constitutive and inducible activation of NF-kB by IkBM

To evaluate whether NF-kB can be inhibited in ALL cells expressing constitutively activated NF-kB, we transfected EU-1 cells with an IkBm cDNA. In EMSA analysis of nuclear extracts from both untransfected and transfected EU-1 cells, IkBm-transfected cells showed significant reduction of NF-kB activity compared with untransfected and control-transfected cells (Figure 1a). We next examined the expression of endogenous IkB and transfected IkBm in both untransfected and transfected EU-1 cells by Western blotting. As shown in Figure 1b, the expected 37-kDa endogenous IkB was detected with an anti-IkB antibody. However, an extra band (40 kDa) appeared only in extracts from IkBm-transfected cells (Figure 1b, lanes 3–5), but not in untransfected and control cells (Figure 1b, lanes 1 and 2), indicating that IkBm was stably expressed in EU-1/IkBm cells and inhibited the constitutive activation of NF-kB.

Figure 1.

Inhibition of NF-kB activation and NF-kB-mediated gene expression in EU-1 ALL cells by transfected IkBm. (a) EMSA analysis of constitutive NF-kB DNA-binding activity in parental EU-1 cells (lane 1), in EU-1 cells transfected with an empty vector (EU-1/neo, lane 2), and in three clones of EU-1 stably transfected with IkBm (EU-1/IkBm, lanes 3–5). (b) Western blot assay of endogenous IkB and transfected IkBm proteins in the same samples as in (a). (c) Gene reporter assay for the effect of IkBm on constitutive and inducible NF-kB-mediated E-selectin promoter activity. EU-1 cells were transiently cotransfected with 2 g E-selectin promoter-CAT reporter construct, and increasing amounts (1, 5 and 10 g) of IkBm (lanes 2–4). The total concentration of DNA was adjusted to 12 g per transfection with an empty neo vector. Transfection was performed by electroporation at 300 V and 950 F. In addition, cells transfected with either E-selectin promoter-CAT alone or the combination of E-selectin promoter-CAT and IkBm (10 g) were treated with 0.5 g/ml Dox (lanes 5 and 6) or 0.1 g/ml VCR (lanes 7 and 8) for 2 h, and then harvested. At 48 h after transfection, cell extracts were analysed for CAT protein expression. Data presented are representative of at least three independent transfections

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To further test whether IkBm was capable of inhibiting NF-kB-mediated gene expression, EU-1 cells were transiently cotransfected with IkBm and an NF-kB-dependent E-selectin promoter–reporter construct (Reed et al., 1997). As expected, IkBm repressed expression of the E-selectin promoter–reporter construct in a dose-dependent manner (Figure 1c, lanes 2–4). We also examined the effect of IkBm on NF-kB-mediated expression of E-selectin promoter–reporter plasmid in response to chemotherapeutic drugs Dox and vincristine (VCR). At 2 h post-treatment of EU-1 cells transfected with E-selectin promoter–reporter plasmid alone, Dox upregulated E-selectin promoter activity (Figure 1c, lane 5), whereas VCR downregulated E-selectin promoter activity (Figure 1c, lane 7), compared to untreated EU-1 cells transfected with E-selectin promoter–reporter plasmid alone (Figure 1c, lane 1). Similar treatment of Dox and VCR did not increase or decrease E-selectin promoter activity in EU-1 cells cotransfected with IkBm (10 g) and E-selectin promoter–reporter constructs (Figure 1c, lanes 6 and 8), compared to control (Figure 1c, lane 4).

Effect of IkBm on cell growth

Constitutive NF-kB activation is reportedly required for proliferation and growth of Hodgkin's lymphoma cells (Orlowski et al., 1998). In order to determine whether NF-kB is similarly required in ALL, we compared the growth rate of EU-1 cells with constitutive NF-kB activation (EU-1/neo and EU-1 parental) to cells stably transfected with IkBm plasmid (EU-1/IkBm, clone 1 as shown in Figure 1a). Neither line at low culture concentrations (10⁴/ml) showed a difference in total cell numbers during the first 3–4 days of culture. However, at higher cell concentrations (approximately 10⁵/ml), EU-1/IkBm showed significantly decreased cell growth compared with EU-1/neo and EU-1 parental cells. As shown in Figure 2a, the growth rate of EU-1/IkBm at the higher cell concentration was only half that for EU-1 parental and EU-1/neo cells after 5 or 6 days of culture. At this point, cell-doubling times were 39.3 and 20.5 h for EU-1/IkBm and EU-1/neo cells, respectively.

Figure 2.

Effect of IkBm transfection on cell growth and sensitivity to apoptosis induced by chemotherapeutic drugs. (a) EU-1 cells transfected with IkBm (EU-1/IkBm) and control cells (EU-1 parental and EU-1/neo) were cultured in 10 ml of RPMI 1640 medium supplemented with 10% FBS at an initial concentration of 10⁴/ml. Cells were counted at different time points. Data for the total number of cells (means.d. for triplicate cultures) are shown. (b, c), EU-1/IkBm and EU-1/neo cells were cultured with different concentrations of Dox and VCR for 48 h, and cell survival was determined by XTT assay. Cell survival was expressed as the percentage of control (i.e. cultures without drugs). Data represent means.d. detected in triplicate experiments. (d) Western blot analysis for activation of caspase 3 and cleavage of its substrate PARP in EU-1/neo and EU-1/IkBm cells following treatment with Dox (0.5 g/ml) and VCR (0.1 g/ml) for different time periods as indicated. Anti-caspase 3 recognizes both the unprocessed proprotease and the cleaved subunits. Anti-PARP detected a fragment cleaved from the PARP holoenzyme

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Effect of IkBm on cell death and apoptosis induced by Dox and VCR

It is well known that NF-kB not only promotes cell growth but also protects cancer cells from apoptosis induced by anticancer drugs. We evaluated the sensitivity of IkBm-transfected EU-1 ALL cells to chemotherapeutic agents Dox and VCR, which are commonly used for treatment of childhood ALL. XTT assay showed that inhibition of NF-kB by transfection of IkBm did not sensitize EU-1 cells to Dox (Figure 2b). However, IkBm sensitized EU-1 cells to VCR. As shown in Figure 2c, a significant difference was noted in mean cell survival after 48-h treatment between EU-1/IkBm and EU-1/neo cells at VCR concentrations of 1 ng/ml and greater (P<0.05, t-test). Consistent with these observations, a Western blot assay for detecting the activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP) showed that no difference in the activation of caspase 3 and cleavage of PARP was detected between EU-1/IkBm and EU-1/neo cells after treatment with Dox (Figure 2d, left). However, similar treatment with VCR for 48 h increased the activation of both caspase 3 and cleavage of PARP in EU-1/IkBm cells compared to EU-1/neo cells (Figure 2d, right).

Regulation of p53 and IkB by Dox and VCR

To further evaluate the possible mechanisms by which IkBm sensitizes EU-1 cells to VCR but not to Dox, we examined the expression and regulation of p53 and IkB in response to Dox and VCR treatment in both EU-1/neo and EU-1/IkBm cells. As shown in Figure 3a, Dox induced accumulation of p53 in both EU-1/neo and EU-1/IkBm cells, whereas VCR failed to activate p53. Contrastingly, Dox downregulated endogenous IkB in both EU-1/neo and EU-1/IkBm cells, whereas VCR slightly induced IkB expression in these cells; this is consistent with data (Figure 1c) showing that Dox induced NF-kB but VCR inhibited NF-kB-mediated gene expression in cells without IkBm transfection. The expression of transfected IkBm was not affected by treatment with either Dox or VCR. Moreover, we examined the cellular localization of p53 and IkB, including endogenous IkB, and transfected IkBm, in both EU-1/IkBm and EU-1/neo cells after treatment with Dox. At 2 h post-treatment, the expression of p53 in EU-1/neo cells was higher in the nucleus than in the cytoplasm. In contrast, p53 was predominantly expressed in the cytoplasm of EU-1/IkBm cells following similar treatment with Dox (Figure 3b). As also shown in Figure 3b, both IkB and IkBm were mainly located in the cytoplasm. Similar to data in Figure 3a, treatment of Dox downregulated cytoplasmic IkB, but did not affect expression of IkBm.

Figure 3.

Expression and regulation of p53 and IkB in response to chemotherapeutic agents. (a) EU-1/neo and EU-1/IkBm cells were treated with 0.5 g/ml Dox or 0.1 g/ml VCR for the indicated time, and assayed for expression of p53, IkB and IkBm by Western blot. (b) EU-1/neo and EU-1/IkBm cells were treated with 0.5 g/ml Dox for 2 h and assayed for expression of p53, IkB and IkBm in the cytosol and the nucleus by Western blot. (c) Binding of p53 to IkB and IkBm in vivo as examined by coimmunoprecipitation (IP) assay. Cytosolic proteins were extracted from EU-1/neo and EU-1/IkBm cells treated with 0.5 g/ml Dox and 0.1 g/ml VCR for different time periods as indicated, and then precipitated with anti-IkB antibody. The immunoprecipitated protein was analysed by Western blotting using anti-p53 antibody

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Binding of IkBm to p53

As an inhibitor of NF-kB activation, IkB binds the p50 and p65 subunits of NF-kB in the cytoplasm to prevent their nuclear translocation. IkB can also bind cytoplasmic p53 protein (Chang, 2002). Therefore, we wanted to know whether the predominantly cytoplasmic location of p53 in EU-1/IkBm cells after Dox treatment was due to binding of p53 to transfected IkBm. Immunoprecipitation of cytosolic IkB from EU-1/neo and EU-1/IkBm cells was performed using anti-IkB antibody. P53 was co-precipitated with IkB in the cytosol of untreated cells, as shown in Figure 3c. When cells were treated with Dox for 1–2 h, the co-precipitated p53 in EU-1/neo cells was significantly reduced, suggesting dissociation of the p53.IkB complex due to downregulation of IkB. In contrast, the amount of co-precipitated p53 was increased in similarly treated EU-1/IkBm cells, indicating that Dox treatment results in accumulation of p53, and that the p53.IkBm complex is stable. The co-precipitated p53 was neither decreased in EU-1/neo nor increased in EU-1/IkBm cells treated with VCR as compared to untreated cells.

Effect of IkBm on p53-mediated cell-cycle arrest and gene expression

Since IkBm can bind p53 as described above, we next examined the possible inhibition of p53 function by IkBm. Dox, a DNA damaging agent, typically induces accumulation of p53, which subsequently arrests the cells in G1 phase and upregulates target genes such as WAF1/p21, MDM2 and Bax. An experiment was conducted to examine the effect of IkBm on cell-cycle arrest in response to Dox. After treatment with Dox for 12 h, an increase in G0/G1 was observed with EU-1/neo but not EU-1/IkBm cells as shown in Figure 4a, in which 62.3% of EU-1/neo cells were in G0/G1 phase compared to 54.1% of EU-1/IkBm cells. Furthermore, we examined the effect of IkBm on p53-mediated gene expression. After treatment with Dox, the expression of p53 was induced equally in both EU-1/neo and EU-1/IkBm cells (Figures 3a, 4b). However, Dox significantly increased the expression of proteins for p21, MDM2 and Bax in EU-1/neo cells but not in EU-1/IkBm cells (Figure 4b). As also shown in Figure 4b, VCR treatment did not induce the expression of p53 and its target genes p21, MDM2 and Bax in either EU-1/neo or EU-1/IkBm cell lines. Consistent with these observations, a gene promoter–reporter assay showed that Dox induced a dose-dependent increase in p21 promoter activity in EU-1/neo cells (Figure 4c, lanes 2–4). P21 promoter activity induced by Dox in EU-1/neo cells was inhibited by cotransfection of MDM2 (Figure 4c lane 5). As also shown in Figure 4c, VCR had no effect on p21 promoter activity in EU-1/neo cells. Neither Dox nor VCR showed any effect on p21 promoter activity in EU-1/IkBm cells (Figure 4d).

Figure 4.

The effect of IkBm on p53-mediated cell-cycle arrest and gene expression. (a) EU-1/neo and EU-1/IkBm cells were treated with Dox (0.5 g/ml), and cell-cycle analysis performed 12 h post-treatment. (b) Western blot analysis for the effects of transfected IkBm on expression of p53, and its target genes p21, MDM2 and Bax following treatment with Dox and VCR. After treatment of EU-1/neo and EU-1/IkBm cells with Dox (0.5 g/ml) and VCR (0.1 g/ml) for 2 h, proteins were extracted and analysed for protein expression using antibodies as indicated. Actin served as control for equal protein loading and protein integrity. (c, d) Effect of stable IkBm expression on p53-mediated p21 promoter activity. EU-1/neo and EU-1/IkBm cells were transfected with either 5 g p21 promoter-luciferase (p21-L) alone, or in combination with 10 g MDM2 plasmid. Transfection was performed as described in Figure 1. At 46 h after transfection, cells were treated with increasing concentrations of Dox (0.05, 0.1 and 0.5 g/ml) or VCR (0.001, 0.01 and 0.1 g/ml) for another 2 h, and then cell extracts were prepared for luciferase activity assay as described in Materials and methods. Results were normalized to -galactosidase activity and plotted as the means of duplicates of a representative experiment out of at least three independent determinations

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Discussion

NF-kB is an apoptotic regulator that plays an important role in regulating the response of tumor cells to chemotherapy. Previous studies have shown that NF-kB in tumor cells is activated by treatment with TNF (Van Antwerp et al., 1996; Wang et al., 1996) and certain chemotherapeutic agents (Post et al., 1989; Hwang and Ding, 1995; Wang et al., 1996). Activation of NF-kB promotes cell proliferation, blocks apoptosis and induces resistance to various chemotherapeutic agents (Van Antwerp et al., 1996; Giri and Aggarwal, 1998; Wang et al., 1999a, 1999b). Similarly, constitutively activated NF-kB has been associated with increased cell proliferation and survival in cancer cells, and may be linked to drug resistance in these cells (Bargou et al., 1997; Nakshatri et al., 1997; Giri and Aggarwal, 1998). Inhibition of NF-kB activation by transfection of the dominant-negative inhibitor IkBm into malignant cells has been considered as a method to sensitize tumor cells to apoptosis induced by chemotherapeutic agents (Garg and Aggarwal, 2002). However, several studies have shown that stable inhibition of NF-kB activation in cancer cells by transfection of IkBm did not increase sensitivity to cytotoxic drugs. In particular, transfection of IkBm in the human colon carcinoma and breast cancer cell lines did not increase sensitivity to daunomycin or Taxol (Bentires-Alj et al., 1999; Dong et al., 2002).

In the present study, we have found that transfection of IkBm into the childhood ALL cell line EU-1 did not increase sensitivity to Dox, although IkBm significantly inhibited the growth rate of EU-1 cells. EU-1 cells express wild-type p53 phenotype and constitutively activated NF-kB. Treatment of EU-1 cells with Dox activated p53 and further induced NF-kB. Transfection of IkBm into EU-1 significantly inhibited both constitutive and inducible activation of NF-kB in these cells. Moreover, stable expression of IkBm in EU-1 cells inhibited p53 function, as demonstrated by the absence of G1 cell-cycle arrest and induction of p53 target genes, although expression of p53 was equally increased in both EU-1/IkBm cells and EU-1/neo control cells after treatment with Dox.

Structurally, IkB is composed of an N-terminus, a central domain containing five ankyrin repeats, and a highly acidic C-terminus (Turpin et al., 1999). The ankyrin repeats are responsible for the binding of IkB to the p65 and p50 subunits of NF-kB protein, whereas the C-terminus physically interacts with p53 (Chang, 2002). In response to apoptotic stimuli such as Dox, IKK kinase is activated, which in turn phosphorylates IkB at N-terminal serines 32 and 36. Phosphorylated IkB dissociates from the NF-kB/IkB complex, and is degraded by the proteasome/ubiquitin pathway, permitting activated NF-kB to translocate to the nucleus (Traenckner et al., 1995).

It has been shown that the cytosolic p53.IkB can dissociate without degradation of IkB (Chang, 2002). However, degradation of IkB following phosphorylation results in release of p53 from the IkB.p53 complex. IkBm is not susceptible to phosphorylation at N-terminal serines 32 and 36, which have been replaced by alanine. Transfection of IkBm stably sequesters both NF-kB and p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis. Therefore, transfection of IkBm into wild-type p53+EU-1 cells did not change their sensitivity to Dox.

Downregulation of NF-kB by transfection of IkBm may directly affect p53 function in EU-1 cells. Previous studies have demonstrated that the p53 promoter contains an NF-kB response element, and the p65 subunit of NF-kB could activate p53-mediated gene transfection (Wu and Lozano, 1994). Furthermore, downregulation of NF-kB in tumors that retain wild-type p53 may increase resistance to apoptosis in these cells (Ryan et al., 2000). However, other studies showed that NF-kB and p53 mutually repress each other's ability to activate transcription, and downregulation of NF-kB is required for p53-dependent apoptosis (Kawai et al., 1999; Wadgaonkar et al., 1999; Webster and Perkins, 1999). This discrepancy with regard to the relationship between NF-kB and p53 regulation may be attributed to different cell types and methods for activating/inactivating NF-kB and p53 used by different investigators. Consistent with the report by Ryan et al. (2000) that inhibition of NF-kB by IkB super-repressor inhibited p53-induced apoptosis in Saos cells, our results (i.e. decreased cell-cycle arrest and lack of p53-dependent gene activation in EU-1/IkBm cells treated with Dox) suggest that inhibition of NF-kB by means of IkBm in ALL cells seems to augment, rather than diminish, IkBm-mediated inhibition of p53.

In contrast to our data for Dox, transfection of IkBm into EU-1 cells significantly sensitized these cells to VCR. Unlike Dox, which induces cell death through a p53-dependent pathway (Lorenzo et al., 2002), VCR kills cells by inhibiting microtubule polymerization (Jordan et al., 1991). Recent studies have shown that VCR also induces cell death through an apoptotic pathway (Conway et al., 1998; Groninger et al., 2000). Our results confirmed that VCR induces apoptosis in EU-1 cells, as demonstrated by activation of caspase 3 and cleavage of PARP.

To date, the molecular events that link microtubule disruption to apoptotic signaling pathways are poorly understood. In the present study, treatment of both EU-1/IkBm and EU-1/neo cells with VCR did not induce p53 and its target genes p21, MDM2 and Bax. Furthermore, VCR appeared to reduce rather than induce NF-kB activation in EU-1 cells by increasing the expression of IkB, suggesting that downregulation of NF-kB is required for VCR-induced apoptosis. Therefore, we hypothesize that VCR induces apoptosis by a p53-independent mechanism that is negatively regulated by NF-kB.

NF-kB has been considered a target for cancer treatment (Garg and Aggarwal, 2002). NF-kB is activated by a variety of signals through mechanisms that result in phosphorylation and degradation of the inhibitory IkB protein. Methods to stabilize IkB, such as the use of protease inhibitors or transfection of the dominant-negative mutant IkB, have been employed to inhibit NF-kB activation in order to sensitize cancer cells to therapy-induced apoptosis. Findings from the present study suggest that only drugs that induce apoptosis by a p53-independent mechanism (e.g. VCR) will be enhanced by dominant-negative inhibitors of NF-kB. In contrast, drugs such as Dox that induce apoptosis by a p53-dependent mechanism may be inhibited by the use of dominant-negative IkBm constructs through inhibition of p53 function by these agents.

Materials and methods

Cell line and treatment

The EU-1 cell line was established in our laboratory from a child with BCP-ALL in relapse (Zhou et al., 1994). The cultured cells resembled the primary leukemic cells in expressing BCP-associated CD19 and CD10 antigens and wild-type p53 (Findley et al., 1997). EU-1 cells were grown in standard culture medium (RPMI 1640 containing 10% FBS, 2 mmol/l L-glutamine, 50 U penicillin and 50 g/ml streptomycin) at 37°C in 5% CO₂ – in air. Exponentially growing cells were treated with chemotherapeutic drugs Dox and VCR, commonly used for the treatment of childhood ALL patients. Cells were treated for different time periods at 37°C, and then harvested for studying gene expression and regulation, and for cell-cycle assay.

Gene transfection and reporter assay

For stable IkBm gene transfection, EU-1 cells in exponential growth were transfected with the dominant-negative mutant IkBm (IkB A32/36, provided by Dr M Lienhard Schmitz, Deutsches Krebsforschungszentrum, Germany); this IkBm is not susceptible to phosphorylation at N-terminal serines 32 and 36, which have been replaced by alanine. Cells transfected with empty vector served as a control. Transfection was performed by electroporation at 300 V, 950 F using a Gene Pulser II System (Bio-Rad, Hercules, CA, USA). The cells were seeded 48 h post-transfection into culture dishes for the selection of G418-resistant colonies. Colonies were grown in methylcellulose medium containing G-418 (500 g/ml) for 2–3 weeks, and clones were picked and grown in RPMI medium with or without G-418 for the duration of the experiments.

To examine the effect of IkBm on NF-kB-mediated E-selectin promoter activity in response to Dox and VCR treatment, EU-1 cells were cotransfected with -578 E-selectin promoter CAT provided by Dr T Collins (Harvard Medical School) and IkBm plasmid. Electroporation was performed as described above. Transiently transfected cells were treated with different doses of Dox or VCR for 2 h, and cell extracts were prepared. CAT activity was evaluated with the CAT ELISA Kit (Boehringer Mannheim, Germany). To examine the effect of IkBm on p53-regulated gene transcription in response to Dox and VCR treatment, EU-1 cells stably transfected with IkBm (EU-1/IkBm) or control-transfectant (EU-1/neo) were cotransfected with the p21-promoter-luciferase plasmid, provided by Dr M Oren (Weizmann Institute of Science, Israel) and MDM2 expression plasmid, provided by Dr B Vogelstein (Johns Hopkins University); Transfection with MDM2 served to inactivate p53. Transfections and drug treatments were performed as described above. For luciferase assay, cell extracts were prepared with 1 lysis buffer (Promega), and then 20 l aliquots of the supernatant were mixed with 100 l of luciferase assay reagent (Promega) and analysed on a Microplate Luminometer (Turner Designs). Luciferase activity was normalized to -galactosidase activity as an internal control.

Nuclear protein extraction and EMSA

Nuclear extracts were prepared using a Nonidet P-40 lysis method (Schreiber et al., 1989). EMSA for NF-kB DNA binding in EU-1 cells was performed using the annealed and [-³²P] ATP end-labeled kB consensus probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') in a 20 l reaction mixture for 15 min at 20°C, and the reactions were then terminated by the addition of EDTA, sodium dodecyl sulfate (SDS) and bromophenol blue. Samples were run on a nondenaturing 5% polyacrylamide gel and imaged by autoradiography.

Western blot analysis

Whole-cell protein samples were prepared by lysing cells in a buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin and 25 g/ml leupeptin for 30 min at 4°C. For detecting the cellular localization of p53 and IkB, nuclear and cytoplasmic fractions were isolated using the NE-PER kit (Pierce) according to the instructions of the manufacturer. After clarification, equal amounts of protein extracts were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to nitrocellulose paper. After blocking with buffer containing 20 mM Tris-HCl (pH 7.5) and 500 mM NaCl/5% non-fat milk for 1 h at room temperature, the filter was incubated with primary antibodies for 2 h at room temperature, followed by HRP-labeled secondary antibody. Blots were developed using a chemiluminescent detection system.

Preparation of cytosolic protein and immunoprecipitation

Cells pre- and post-treatment with chemotherapeutic drugs Dox and VCR were lysed in a hypotonic buffer composed of 10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, 0.5 g/ml leupeptin and 1 g/ml pepstatin, and incubated on ice for 10 min. After centrifugation to remove the nucleus, 30 g of the clarified cytosolic lysate was incubated with 1 g rabbit anti-IkB- antibody and 15 l Protein G plus/Protein A-agarose. After 24 h incubation, the protein–agarose conjugate was centrifuged, washed four times with ice-cold lysis buffer, suspended in electrophoresis sample buffer and boiled for 5 min. The immunoprecipitated protein was further analysed by Western blotting with mouse anti-p53 antibody as described above.

Determination of cell growth rate

IkBm-transfected EU-1 cells and control cells (EU-1 transfected with empty vector and EU-1 parental) were cultured in RPMI 1640 containing 10% FBS at an initial concentration of 10⁴/ml. Cells from triplicate cultures were then counted daily using a hemocytometer.

XTT assay

The effect of Dox and VCR on IkBm-transfected EU-1 cells was determined by the XTT cytotoxicity assay. Cleavage of XTT (a tetrazolium salt) by mitochondrial-associated dehydrogenase enzymes in metabolically active cells yields a colored formazan product that can be measured spectrophotometrically. The concentration of the products is directly proportional to the viability of the cells. Following treatment with different concentrations of adriamycin, cells were cultured in 96-well microtiter plates and incubated for 44 h. XTT (25 g/well) was then added and cells were incubated for an additional 4 h. The optical density (OD) of the wells was then read with a microplate reader at a test wavelength of 450 nm and a reference wavelength of 620 nm. Appropriate controls lacking cells were included to determine the background absorbance.

Flow cytometry

Flow cytometry was performed to analyse cell-cycle distribution. Following treatment with Dox, 5 10⁵ cells were collected, rinsed twice with PBS and fixed in 70% ethanol for 1 h at 4°C. Cells were then washed twice in PBS and resuspended in 30 l phosphate citrate buffer (0.1 M Na citrate/0.2 M Na₂HPO₄) at room temperature for 30 min. The samples were rewashed with PBS and finally suspended in 0.5 ml PBS containing 20 g/ml of propidium iodide (PI) and 20 g/ml of RNase A. After incubating at 4°C for at least 0.5 h, the samples were analysed using the FACScan (Becton Dickinson) and WinList software (Verity Software House Inc).

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Acknowledgements

This work was supported by grants from the NCI-NIH (R01 CA82323), CURE Childhood Cancer, Inc. and Children's Healthcare of Atlanta.