Pre-B-cell leukemia transcription factor 1 is a major target of promyelocytic leukemia zinc-finger-mediated melanoma cell growth suppression

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Abstract

Promyelocytic leukemia zinc-finger (PLZF) is a transcriptional repressor and tumor suppressor. PLZF is expressed in melanocytes but not in melanoma cells, and recovery of PLZF expression markedly suppresses melanoma cell growth. Several target genes regulated by PLZF have been identified, but the precise function of PLZF remains uncertain. Here, we searched for candidate target genes of PLZF by DNA microarray analysis. Pre-B-cell leukemia transcription factor 1 (Pbx1) was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB7. Reverse transcription–polymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB7 target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2 and matrix metalloprotease 9. These findings suggest that deregulation of Pbx1 expression owing to loss of PLZF expression contributes to the progression and/or pathogenesis of melanoma.

Introduction

Malignant melanoma is a highly aggressive neoplastic disease whose incidence is increasing rapidly. To develop new therapies, it is essential to elucidate the molecular mechanisms by which melanocytes are transformed into malignant melanoma cells. A recent report found that the promyelocytic leukemia zinc-finger (PLZF) protein is expressed in melanocytes but not in melanoma cells, and that melanoma growth is markedly suppressed by enforced expression of PLZF (Felicetti et al., 2004).

PLZF was first identified by its translocation with retinoic acid receptor alpha in t(11;17) acute promyelocytic leukemia (Chen et al., 1993). The PLZF protein is a sequence-specific transcription repressor characterized by a BTB/POZ domain, which is responsible for transcriptional repression, and nine zinc-finger domains that form the DNA-binding domain (Hong et al., 1997; Li et al., 1997; Wong and Privalsky, 1998). Some target genes regulated by PLZF have been identified. Studies of PLZF-knockout mice indicate that PLZF represses HoxD gene expression through chromatin remodeling, which in turn regulates limb and axial skeletal patterning (Barna et al., 2000, 2002). PLZF also binds to and represses the cyclin A2 promoter, and is involved in the regulation of cell cycle progression (Yeyati et al., 1999). Moreover, PLZF represses c-myc expression, which is central to the control of cellular proliferation, apoptosis and differentiation (McConnell et al., 2003). DNA microarray analyses have also revealed numerous candidate genes targeted by PLZF (McConnell et al., 2003; Costoya et al., 2004; Felicetti et al., 2004). These target genes may have a great influence on the progression of melanoma, but their role in the aggressiveness of this cancer remains unknown.

For this reason, we used DNA microarray analysis to search for additional PLZF target genes. One of the most strongly regulated genes identified was pre-B-cell leukemia transcription factor 1 (Pbx1). Pbx1 is also a transcriptional regulator and a binding partner for a number of Hox proteins (Mann and Chan, 1996; Shanmugam et al., 1997). We confirmed that Pbx1 is highly expressed in melanoma cells and that cell growth is markedly suppressed when its expression is knocked down. Furthermore, we identified HoxB7 as a binding partner of Pbx1 in melanoma cells. We show here that PLZF suppresses the expression of Pbx1-HoxB7 target genes as well as Pbx1 itself and that dysregulation of this network owing to loss of PLZF may play an important role in the progression of melanoma.

Results

PLZF suppresses Pbx1 gene expression

Felicetti et al. (2004) reported that primary melanomas and melanoma cell lines completely lack PLZF gene expression and that enforced expression of PLZF in melanoma cell lines suppressed their growth in vitro and in vivo. We confirmed these results using 15 melanoma cell lines, including A375, 397 cell lines and their PLZF transfectants (data not shown).

We then examined which genes are repressed by PLZF in the melanoma cells by DNA microarray analysis. For these experiments, we constructed adenovirus vectors carrying the genes encoding PLZF and LacZ and used them to infect A375 and 397 cells at a multiplicity of infection (MOI) of 200. Several genes exhibited different levels of expression in PLZF-negative and PLZF-positive cells. We found that Pbx1 was the most prominently downregulated transcription factor in PLZF-positive cells (Table 1). Reverse transcription–polymerase chain reaction (RT–PCR) confirmed that Pbx1 mRNA expression in A375 cells was clearly and substantially reduced in a time-dependent manner after infection with the PLZF adenovirus (Figure 1a). The expression of Pbx1 mRNA decreased to 25% of that in LacZ-infected cells. In addition, the Pbx1 protein levels in the PLZF-infected cells decreased by approximately 40% when compared with cells infected with the LacZ adenovirus or non-infected cells (Figure 1b). We also obtained similar results in 397 cells.

Table 1 Expression profile obtained by array hybridization
Figure 1
figure1

PLZF suppresses Pbx1 gene expression. (a) qRT–PCR of Pbx1 mRNA in A375 cells infected with LacZ or PLZF adenovirus. *P<0.05. (b) Upper panel: Western blot analysis of Pbx1 protein in A375 cells infected with LacZ or PLZF adenovirus and in uninfected cells. Lower panel: Relative intensity of Pbx1 proteins calculated as a ratio vs expression of β-actin. *P<0.05. AxLacZ, infection with LacZ adenovirus (MOI 200); AxPLZF, infection with PLZF adenovirus (MOI 200); no Ax, no infection. Each experiment was performed in duplicate.

Enforced expression of Pbx1 rescues growth suppression by PLZF

In order to determine the relative importance of PLZF-mediated downregulation of Pbx1 in the growth of melanoma cells, we examined the proliferation of stable PLZF-expressing melanoma cells transfected with the pME18S-Pbx1 or pME18S control vector. Pbx1 protein levels in the transfected cells were first estimated by Western blotting (Figure 2a). Pbx1 levels increased approximately 1.5- and 1.3-fold in A375P and A375 cells, respectively, in comparison with controls. We also examined transfection efficiency in these cells using the mixture of pME18S-Pbx1 and pME18S-ECFP (Enhanced Cyan Fluorescent Protein) vectors. We found that approximately 65% of the cells were CFP (Cyan Fluorescent Protein)-positive (Figure 2b, inset). We then investigated the growth of A375 and A375P cells after transient transfection with pME18S-Pbx1 or pME18S. Exogenous expression of Pbx1 did not cause further enhancement of A375 cell growth. This suggests that there was sufficient endogenous Pbx1 expression. A375P cells exhibited a marked downregulation of both endogenous Pbx1 expression and cell growth. This effect was partially (approximately 40%) rescued by the expression of exogenous Pbx1 (Figure 2b), thus suggesting the presence of a Pbx1-independent pathway in PLZF-transfected melanoma cells. We also obtained similar results in 397 and 397P cells (data not shown).

Figure 2
figure2

Enforced expression of Pbx1 rescues growth suppression of melanoma cells by PLZF. (a) Upper panel: Western blot analysis of PLZF and Pbx1 in A375 and A375P cells transfected with PME18S-Pbx1 or PME18S control vector. Lower panel: Relative intensity of Pbx1 protein, which was calculated as the ratio of Pbx1 vs β-actin. (b) Growth curves of A375 and A375P cells transfected with PME18S-Pbx1 and PME18S control vector. *P<0.05. Inset: Transfection efficiency of pME18S-Pbx1 was evaluated by counting CFP-positive cells transfected with both PME18S-Pbx1 and pME18S-ECFP.

PLZF physically interacts with the Pbx1 promoter

In order to demonstrate direct regulation of the Pbx1 gene by PLZF, we searched the Pbx1 promoter for a putative PLZF-binding site using the TFSEARCH program (Heinemeyer et al., 1998, http://mbs.cbrc.jp/research/db/TFSERCHJ.html), which suggested seven potential PLZF DNA recognition sequences located within a 3.0-kb region upstream of the transcription initiation site (Figure 3a). We then used a 3.0-kb fragment of the Pbx1 promoter in a reporter assay. The luciferase activity of Pbx1–3.0 was markedly suppressed in A375P, as compared to A375 cells (Figure 3b). Electrophoretic mobility shift assay (EMSA) was subsequently performed with nuclear extracts made from A375 cells infected with PLZF adenovirus and uninfected cells in order to identify the PLZF DNA recognition site in the Pbx1 promoter. Site 1 (2998 bp upstream) and Site 4 (2433 bp upstream) bound with factors in the A375 cell extracts without PLZF expression (stars in lane 1 and lane 7), and formed another complex in the A375 cell extracts expressing PLZF (arrow in lane 2, Figure 3c). On the other hand, Sites 2, 3, 4, 5, 6 and 7 did not form additional complexes in the A375 cell extracts expressing PLZF, as compared to A375 cell extracts without PLZF (lanes 3–14, see comments on lane 6 in figure legends). To confirm the specificity of the interaction between Site 1 and PLZF, we carried out dose–response analysis and competition analysis. The interaction between Site 1 and PLZF exhibited a dose-dependent relationship (Figure 3d), and was completely abolished by the addition of the competitor (unlabeled Site 1). The competitor with the mutation (mutated Site 1, see ‘Materials and methods’) failed to abolish the PLZF–Site 1 interaction (Figure 3e). On the other hand, deletion of Site 1 from Pbx1–3.0 (Pbx1–3.0/ΔSite 1) did not show significant recovery of Pbx1 promoter activity (data not shown), indicating that Site 1 is an interactive site for PLZF, and is not sufficient for complete repression of Pbx1 gene expression. These results suggest that repression of Pbx1 gene expression by PLZF is regulated by multiply direct and/or indirect mechanisms.

Figure 3
figure3

PLZF protein directly interacts with the Pbx1 promoter. (a) Schematic depiction of the Pbx1 promoter region and seven potential binding sites for PLZF (Sites 1–7). The +1 position corresponds to the initiation site of transcription. (b) Relative luciferase activity measured at 24 h after transfection of pGL Pbx1–3.0 into A375 cells and A375P cells. Each experiment was performed in triplicate and data represent the means±s.d. of three independent experiments. (c) EMSA carried out with nuclear extracts of A375 cells infected with PLZF adenovirus (MOI 100) and uninfected cells. White arrowhead in lane 6 shows stain. (d) Specificity of interaction between PLZF and Site 1 (an arrow) was confirmed by EMSA using nuclear extracts of A375 cells infected with PLZF (MOI 50, 100 and 200) or GFP adenovirus (MOI 200). (e) Interaction between PLZF and Site 1 (an arrow) was found to compete with unlabeled Site 1 but not with unlabeled mut. Site 1.

Melanoma cell growth is suppressed by Pbx1 small interfering RNAs

In order to further characterize the role of Pbx1 in melanoma cell growth, we examined the suppression of melanoma cell growth by Pbx1 knockdown using two types of small interfering RNA (siRNA) (siRNA1 and siRNA2). Mutated siRNAs (mut. siRNA1 and mut. siRNA2) were used as controls. We confirmed that transfection of A375 and 397 cells with Pbx1 siRNA1 significantly reduced Pbx1 mRNA (data not shown) and protein levels (Figure 4a). In addition, transfection of A375 and 397 cells with Pbx1 siRNA1, but not Pbx1 mut. siRNA1, caused marked and statistically significant suppression of cell growth (Figure 4b). These data suggest that Pbx1 is involved in cell growth of A375 and 397 cells. siRNA2 showed similar results as siRNA1 (data not shown).

Figure 4
figure4

Melanoma cell growth is suppressed by Pbx1 siRNAs. (a) Upper panel: Western blot analysis of Pbx1 in control siRNA-, mutated-Pbx1 siRNA- and Pbx1 siRNA-transduced melanoma cells. Lower panel: Relative intensity of Pbx1 protein expression calculated as the ratio of Pbx1 vs β-actin. *P<0.05. (b) Cell proliferation of melanoma cells transfected with each siRNA. *P<0.05. Each experiment was performed in triplicate.

Pbx1 binds HoxB7

Pbx1 forms complexes with a number of Hox proteins, and HoxB7 was expressed strongly in all melanoma cell lines tested (Care et al., 1996). We investigated whether Pbx1 interacts with HoxB7 in A375 and 397 cells. HoxB7 was immunoprecipitated from cell lysates and was then analysed by immunoblotting with anti-Pbx1 antibody. We detected a 52-kDa band corresponding to Pbx1 in both cell lines (Figure 5). We also found that an anti-Pbx1 antibody co-immunoprecipitated HoxB7 from the same cell lysates (data not shown).

Figure 5
figure5

Pbx1 binds HoxB7. Interaction between Pbx1 and HoxB7 as assessed by immunoprecipitation. A375 and 397 cell lysates were immunoprecipitated with anti-HoxB7 antibody and immunoblotted with anti-Pbx1 antibody. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was carried out under the non-reducing condition.

Expression of HoxB7 target genes is downregulated by reduction of Pbx1

We investigated the effects of Pbx1 knockdown on the expression of HoxB7 and its target genes. Knockdown of Pbx1 with the siRNAs slightly reduced the mRNA expression of HoxB7 (Figure 6a). It remains uncertain whether Pbx1 binds directly to the promoter region of the HoxB7 gene. HoxB7 is reported to regulate the expression of basic fibroblast growth factor (bFGF), angiopoietin-2 (Ang-2), matrix metalloprotease 9 (MMP9), growth-related oncogene-α (GROα), interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) (Care et al., 1996, 2001). We thus evaluated the effects of Pbx1 knockdown on the expression of the target genes. As shown in Figure 6b, knockdown of Pbx1 markedly reduced the level of bFGF mRNA, and mildly did those of Ang-2 and MMP mRNAs, whereas those of GROα, IL-8 and VEGF were unchanged. Pbx1 knockdown significantly reduced protein levels of Ang-2 and MMP9, but it did not seem to have any effect on the protein levels of bFGF. This may be owing to the short culture duration and/or long half-life of bFGF protein. We obtained similar results in 397 cells (data not shown).

Figure 6
figure6

Expression of HoxB7 and its target genes is modulated by knockdown of Pbx1. (a) Upper panel: RT–PCR analysis of HoxB7 expression in Pbx1-knockdown melanoma cells and control cells. Lower panel: Relative intensity of Pbx1 and HoxB7 mRNA expression in Pbx1-knockdown melanoma cells and control cells, which was calculated as a ratio vs the expression of GAPDH. *P<0.05. (b) RT–PCR analysis of HoxB7 target gene expression in each cell type. (c) Immunoblot analysis of Ang-2, MMP9 and bFGF in Pbx1-knockdown melanoma cells and control cells. Relative intensity of each protein was calculated as a ratio vs the expression of β-actin. *P<0.05.

We further examined whether enforced expression of PLZF in melanoma cells would reduce HoxB7, bFGF, Ang-2 and MMP9. Quantitative RT–PCR analysis revealed that transcripts of all of these were significantly reduced in both A375P and 397P cells (Figure 7). These data suggest that the PLZF–Pbx1 axis is important in regulating HoxB7 target genes via Pbx1/HoxB7 heterocomplex formation.

Figure 7
figure7

Expression of HoxB7, bFGF, Ang-2 and MMP9 is suppressed by the transduction of PLZF in melanoma cells. Upper panel: RT–PCR analysis for PLZF mRNA in A375, A375P, 397 and 397P cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. PLZF was evaluated at 35 cycles, whereas control GAPDH evaluated at 24 cycles. Lower panel: qRT–PCR for HoxB7, bFGF, Ang-2 and MMP9 mRNA in PLZF-expressing melanoma cells and parental cells. *P<0.05.

Discussion

In this study, we performed DNA microarray analysis and found that Pbx1 is a prominent transcription factor targeted by PLZF. Pbx1 was highly expressed in the A375 and 397 cell lines, and its expression was markedly reduced by the expression of PLZF. Moreover, we showed that the growth suppression caused by ectopic expression of PLZF is reversed by enforced expression of Pbx1. Knockdown of Pbx1 using siRNAs in A375 and 397 cells reduced its protein levels by approximately 80%. Pbx1 siRNAs significantly inhibited cell growth in culture, thus suggesting that Pbx1 is a downstream target of PLZF in melanoma cell growth regulation.

In order to determine whether PLZF suppresses Pbx1 gene expression directly or indirectly, we examined the ability of PLZF to repress the Pbx1 promoter. We found that the 3.0 kb upstream region of the Pbx1 gene (nucleotides −3.0 kb to 0) contains seven possible PLZF DNA-binding consensus sequences. We then performed reporter assay and EMSA to examine the direct binding of PLZF in the Pbx1 promoter region. EMSA suggested the presence of an interactive site (Site 1) for PLZF in the 3.0-kb of upstream Pbx1 gene sequences. Deletion of Site 1, however, did not recover Pbx1 gene expression. These data suggest multiple direct and/or indirect regulation mechanisms for Pbx1 gene expression by PLZF. Further analysis is required for elucidating these mechanisms.

Pbx1 was originally identified at t(1;19) chromosomal translocations in acute pre-B-cell leukemias (Kamps et al., 1990; Nourse et al., 1990), and it has also been described as a transcriptional activator. Pbx1 is known to interact with a number of Hox proteins through the YPWM motif located at the N-termini of the homeodomains in Hox proteins and to regulate the function of Hox proteins (Chang et al., 1995; Shanmugam et al., 1997). It has been reported that Pbx1 cooperates in the cellular proliferation and transformation induced by Hox proteins (Krosl et al., 1998). Krosl et al. (1998) showed that the ability of HoxB4 and HoxB3 to interact with Pbx1 is critical for transformation of Rat-1 cells, that Hox proteins have a notable effect on cell growth, and that Pbx1 further enhances this effect. Although HoxB7 is reportedly involved in melanoma growth (Care et al., 1996, 2001; Felicetti et al., 2004), there have been few reports on the relationship between Pbx1 and the progression of melanoma. We showed here that immunoprecipitation of HoxB7 from A375 and 397 melanoma cell lines co-precipitated a 52-kDa protein that was recognized by an anti-Pbx1 antibody, thus suggesting that Pbx1 physically interacts with HoxB7. Our data indicate that Pbx1 plays an important role in melanoma growth and that it forms heterocomplexes with HoxB7, which suggests that molecular events mediated by the Pbx1–HoxB7 heterocomplex are altered in melanoma cells when Pbx1 is suppressed by PLZF.

Previous studies have identified some HoxB7 target genes. bFGF was identified as the main target of HoxB7 in melanoma cell lines (Care et al., 1996). In addition, VEGF, GROα, IL-8, Ang-2 and MMP9 were found to be upregulated in HoxB7-transduced cells (Care et al., 2001). Based on these reports, we analysed the effect of Pbx1 knockdown on HoxB7 target genes. We found that knockdown of Pbx1 slightly downregulated HoxB7 itself and substantially downregulated bFGF, Ang-2 and MMP9, but not VEGF, GROα or IL-8. This suggests that HoxB7 target genes are differentially regulated by HoxB7–Pbx1 or by another HoxB7 complex. Interestingly, these factors are strongly associated with tumor angiogenesis and invasion (Becker et al., 1989; Ahmad et al., 2001; Etoh et al., 2001; Johnson et al., 2004). The Pbx1–HoxB7 complex may thus play a very important role in melanoma growth, and its suppression could be a major cause of the reduction in PLZF-mediated growth in melanoma. This is supported by previous reports that PLZF is expressed in melanocytes but not in melanoma cells, and that the pattern of PLZF expression inversely correlates with that of HoxB7 (Care et al., 1996, 1998).

In this study, we showed that the suppression of Pbx1 expression by PLZF downregulates some HoxB7 target genes, including bFGF, Ang-2 and MMP9. The data indicated that the changes in the molecular network caused by the loss of PLZF may play an important role in the progression of melanoma. Further analyses of the PLZF–Pbx1 network will assist in the discovery of target molecules for the development of novel antimelanoma drugs.

Materials and methods

Materials

Human melanoma cell lines A375 and 397 were generously provided by Dr Kawakami (Keio University, Tokyo, Japan) (Sumimoto et al., 2004). Cells were grown in Roswell Park Memorial Institute medium 1640 supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin sulfate (100 μg/ml) at 37°C in an atmosphere containing 5% CO2. Antibodies used were as follows: rabbit anti-Pbx1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-PLZF monoclonal antibody (Oncogene Research Products, San Diego, CA, USA), rabbit anti-HoxB7 polyclonal antibody (CeMines, Golden, CO, USA), rabbit anti-bFGF polyclonal antibody (Abcam, Cambridge, UK), mouse anti-Ang-2 monoclonal antibody, goat anti-MMP9 monoclonal antibody (R&D, Tokyo, Japan), goat horseradish peroxidase-conjugated anti-rabbit antibody, donkey horseradish peroxidase-conjugated anti-goat antibody and goat horseradish peroxidase-conjugated anti-mouse antibody (Promega, Madison, WI, USA).

Plasmid and adenovirus vector construction

A plasmid encoding PLZF was generated by subcloning the human PLZF cDNA into pcDNA3.1/Hygro. (Invitrogen, Carlsbad, CA, USA). A plasmid encoding Pbx1 was generated by subcloning Pbx1 cDNA into pME18S (Tanaka et al., 2004). The Pbx1 promoter 3.0 kb was subcloned into pGL3 (Promega). Transfection of plasmids was performed with LipofectAMINE (Invitrogen) according to the manufacturer's instructions. To assess transfection efficiency of pME18S-Pbx1 in 375P cells, both PME18S-Pbx1 and pME18S-ECFP plasmids were transfected into 375P cells and CFP-positive cells were counted. Adenovirus vector (Ax) carrying PLZF was prepared using an adenovirus expression vector kit (Takara Biomedicals, Kyoto, Japan). PLZF was subcloned into the cosmid cassette pAxCAw. Ax containing the CA promoter and PLZF (AxPLZF) was generated by the COS-TPC method according to the manufacturer's protocol. A375 and 397 cells were infected with Axs at an MOI of 50–200 for 1 h. Ax expressing LacZ (Ax-LacZ) and GFP (Ax-GFP) were used as controls to exclude the effects of Ax itself.

RT–PCR and quantitative real-time PCR

Primer sequences for RT–PCR are listed in Table 2. Total RNA was isolated from cultured melanoma cells using Trizol Reagent (Invitrogen), and was treated with DNase I (Clontech, Palo Alto, CA, USA) at 37°C for 30 min to remove contaminating genomic DNA. RT–PCR was performed using RT–PCR High Plus (Toyobo, Osaka, Japan) according to the manufacturer's instructions. cDNA was reverse transcribed from total RNA for 30 min at 60°C, and heated to 94°C for 2 min. Amplification was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) for 24 or 35 cycles. The cycle profile consisted of 1 min at 94°C for denaturation and 1.5 min at 60°C for annealing and primer extension. To evaluate amplification, 5 μl of the reaction mixture was electrophoresed on a 1.0% agarose gel containing ethidium bromide. We performed at least three independent studies and confirmed similar results. Quantitative real-time PCR (qRT–PCR) was performed using the ABI PRISM 7700 sequencer detection system (Perkin–Elmer Applied Biosystems, Foster City, CA, USA). Primers and probe were purchased from Applied Biosystems (Assays-on-Demand). RT–PCR mixtures were prepared according to the manufacturer's instructions for the TaqMan One-Step RT–PCR Master Mix Reagent kit (Perkin–Elmer Applied Biosystems). Probe was labeled with a reporter fluorescent dye (6-carboxyfluorescein) at the 5′ end. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection, Pre-Developed TaqMan Assay Reagent (Perkin–Elmer Applied Biosystems) was added. The thermal conditions were 48°C for 30 min for RT and 95°C for 10 min, followed by 45 amplification cycles of 95°C for 15 s for denaturing and 60°C for 1 min for annealing and extension. PCR products were sequenced to confirm proper amplification. To compare mRNA expression, results were determined as relative values against GAPDH as an internal reference. There were n=3 samples in each group.

Table 2 Primer sequences used in this study

siRNA experiments

siRNA sequences used in this study were as follows: Pbx1 siRNA sequences were 5′-IndexTermCCGCAGGGCATCAGTGCTA-3′ (siRNA1) and 5′-IndexTermCGACAGAAATCCTGAATGA-3′ (siRNA2), and the mutated Pbx1 siRNA sequences were 5′-IndexTermCCACAAGGCATTAGCGCTA-3′ (mut. siRNA1) and 5′-IndexTermCGACCGAGATCCTAAACGA-3′ (mut. siRNA2) (B-Bridge International, Sunnyvale, CA, USA). Scrambled siRNA directed against 5′-IndexTermGCGCGCTTTGTAGGATTCG-3′ was also used as a negative control. This sequence was not present in any mammalian mRNAs in the National Center for Biotechnology Information database. siRNA transfection was performed with CodeBreaker siRNA Transfection Reagent (Promega). Briefly, cells (5 × 105) were incubated in 35-mm plates with siRNA (final concentration, 40 nM)–CodeBreaker Reagent (15 μl) mixture in serum-free medium for 24 h. Cells were then grown in media with 10% FCS.

Microarray analysis

Total RNA was isolated from LacZ or PLZF Ax-infected melanoma cells at 48 h post-infection. The Acegene Human oligo chip 30K (Hitachi Software Engineering, Yokohama, Japan) containing 30 000 genes was used to compare gene expression in melanoma cells infected with PLZF or LacZ adenovirus. Arrays were screened according to the manufacturer's protocol. Fluorescent images of hybridized microarrays were obtained with a CRBIO IIe microarray scanner (Hitachi Software Engineering), and images were analysed with DNAsis Array software (Hitachi Software Engineering).

EMSA

Nuclear protein extracts were prepared using celLytic NuCLEAR Extraction Kit (Sigma, St Louis, MO, USA). Biotin-labeled oligomers containing PLZF-binding sequences (Sites 1, 2, 3, 4, 5, 6 and 7) were bound to nuclear extracts. Binding reactions were performed using the LightShift EMSA Optimization and Control kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Reaction mixtures were incubated on ice for 60 min. Biotin-unlabeled oligomers were used as competitors, and when present, were added to the mixtures 30 min before the other reagents. Binding reactions were subjected to EMSA on 4% non-denaturing polyacrylamide gels in 0.5 × Tris-Borate-EDTA buffer, and detection was performed using a LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer's instructions. Sequences of the probes used in this study were as follows: Site 1, 5′-IndexTermCCTCCAGATCCAGTTCATCC-3′; Site 2, 5′-IndexTermCGGGGTAAGACAGTTGCAAT-3′; Site 3, 5′-IndexTermTTTGAGTATATAGTTTTGTG-3′; Site 4, 5′-IndexTermTTTAAAAAAACAGTTTAAAA-3′; Site 5, 5′-IndexTermACTGTGTACACAGTCAGATT-3′; Site 6 5′-IndexTermAAAAATGACACAGTTTGGTA-3′; Site 7, 5′-IndexTermGCTGGAACTACAGTACCATT-3′ and mutated-Site 1, 5′-IndexTermCCTCCAGAGAAAGTTCATCC-3′.

Luciferase reporter assays

Melanoma cells were cultured in 24-well plates and grown to 70% confluence. Cells were transfected with 0.8 μg/well of pGL3-Pbx1 plasmid DNA using LipofectAMINE (Invitrogen). Transfected cells were harvested at 24 h post-transfection, and lysates were assayed for luciferase activity with a Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol.

Immunoprecipitation and Western blotting

A375 and 397 cells were transiently transfected with pME18S-Pbx1 plasmid. Cells were lysed 48 h after transfection and equivalent amounts of lysate protein were subjected into immunoprecipitation. Immunoprecipitation and Western blotting were performed as described previously (Goishi et al., 1995). After membranes were washed three times at intervals of 10 min with 0.05% Tween-20 in phosphate-buffered saline, horseradish peroxidase conjugate was detected by chemiluminescence with an ECL kit (Amersham, Buckinghamshire, UK) and autofluorography.

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Acknowledgements

We thank Drs T Tsuda, T Jyokou, E Tan, M Tohyama, H Iwabuki, T Kikugawa, Y Kinugasa and E Koya for technical assistance, helpful comments and discussion.

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Correspondence to S Higashiyama.

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Shiraishi, K., Yamasaki, K., Nanba, D. et al. Pre-B-cell leukemia transcription factor 1 is a major target of promyelocytic leukemia zinc-finger-mediated melanoma cell growth suppression. Oncogene 26, 339–348 (2007) doi:10.1038/sj.onc.1209800

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Keywords

  • Pbx1
  • PLZF
  • Hox
  • melanoma

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