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Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis

1998; Springer Nature; Volume: 17; Issue: 15 Linguagem: Inglês

10.1093/emboj/17.15.4358

1460-2075

Shuang Huang,

Protein Degradation and Inhibitors

Article3 August 1998free access Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis Suyun Huang Suyun Huang Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Didier Jean Didier Jean Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Mario Luca Mario Luca Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Michael A. Tainsky Michael A. Tainsky Department of Tumor Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Menashe Bar-Eli Corresponding Author Menashe Bar-Eli Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Suyun Huang Suyun Huang Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Didier Jean Didier Jean Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Mario Luca Mario Luca Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Michael A. Tainsky Michael A. Tainsky Department of Tumor Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA Search for more papers by this author Menashe Bar-Eli Corresponding Author Menashe Bar-Eli Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA Search for more papers by this author Author Information Suyun Huang1, Didier Jean1, Mario Luca1, Michael A. Tainsky2 and Menashe Bar-Eli 1 1Department of Cell Biology, The University of Texas, Houston, TX, 77030 USA 2Department of Tumor Biology, The University of Texas M.D.Anderson Cancer Center, Houston, TX, 77030 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:4358-4369https://doi.org/10.1093/emboj/17.15.4358 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Expression of the tyrosine kinase receptor, c-KIT, progressively decreases during local tumor growth and invasion of human melanomas. We have previously shown that enforced c-KIT expression in highly metastatic cells inhibited tumor growth and metastasis in nude mice. Furthermore, the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. Here we show that loss of c-KIT expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The c-KIT promoter contains three binding sites for AP-2 and EMSA gels demonstrated that AP-2 protein binds directly to the c-KIT promoter. Transfection of wild-type AP-2 into c-KIT-negative A375SM melanoma cells activated a c-KIT promoter-driven luciferase reporter gene, while expression of a dominant-negative AP-2B in c-KIT-positive Mel-501 cells inhibited its activation. Endogenous c-KIT mRNA and expression of proteins were upregulated in AP-2-transfected cells, but not in control cells. In addition, re-expression of AP-2 in A375SM cells suppressed their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of c-KIT is highly regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly through c-KIT transactivation and SCF-induced apoptosis. Therefore, loss of AP-2 expression might be a crucial event in the development of malignant melanoma. Introduction The molecular basis of human malignant melanoma progression has remained largely unknown despite the fact that the worldwide incidence of melanoma is increasing more than any other neoplastic disease (Kopf et al., 1995). The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not well defined. Since the production of metastases depends on the completion of a multistep process involving the survival and growth of a unique subpopulation of cells with metastatic properties (Fidler, 1990), more information is clearly needed regarding genetic changes underlying melanoma tumorigenesis and progression to provide insights into the development of this cancer. There has, however, been some progress. Of particular relevance to this paper are recent results with the c-KIT receptor in melanoma. Expression of the tyrosine kinase receptor encoded by the c-KIT proto-oncogene progressively decreases during local tumor growth and invasion of human melanomas (Lassam and Bickford, 1992; Natali et al., 1992; Zakut et al., 1993). The proto-oncogene c-KIT encodes a transmembrane tyrosine-protein kinase receptor related to the PDGF/CSF-1 (c-fms) receptor subfamily (Yarden et al., 1987). c-KIT has been found to play a pivotal role in the normal growth and differentiation of embryonic melanoblasts. In mice, c-KIT has been mapped to the dominant white spotting (w) locus (Chabot et al., 1988; Geisler et al., 1988), whose ligand is the product of the sl locus (Steel) (Zsebo et al., 1990) which encodes the stem cell factor, SCF (also known as KIT-ligand, KL, steel factor or mast cell growth factor, MCF). Mutations in the (w) locus or (sl) locus (Nocka et al., 1989, 1990; Reith et al., 1990; Tan et al., 1990; Brannan et al., 1991; Flanagan et al., 1991), or injection of neutralizing anti-KIT antibodies into pregnant mice (Nishikawa et al., 1991) results in the piebald phenotype, characterized by white spotting of the fur and attributed to a local reduction in the number of cutaneous melanocytes. Mutations in the c-KIT receptor also have been identified in human piebald patients (Fleischman et al., 1991; Gibel and Spritz, 1991; Fleischman, 1992), suggesting that normal function of c-KIT is required for human melanocyte development. These observations raise the question as to whether malignant transformation of melanocytes may be associated with changes in the expression of the c-KIT receptor. Indeed, several recent studies have demonstrated that the progression of human cutaneous melanoma is associated with loss of expression of the c-KIT proto-oncogene. About 70% of metastatic lesions and human melanoma cell lines do not express detectable levels of the c-KIT receptor (Lassam and Bickford, 1992; Natali et al., 1992; Zakut et al., 1993). To provide direct evidence that c-KIT plays a role in metastasis of human melanoma, we transfected the c-KIT gene into c-KIT-negative, highly metastatic human melanoma cells and subsequently analyzed their tumorigenic and metastatic potential in nude mice (Huang et al., 1996). Enforced c-KIT expression significantly inhibited tumor growth and metastasis. Exposure of c-KIT-positive melanoma cells in vitro and in vivo to SCF, the ligand for c-KIT, triggered apoptosis of these cells but not of normal melanocytes. These results suggest that loss of c-KIT receptor may allow malignant melanoma cells to escape SCF/c-KIT-mediated apoptosis, thus contributing to tumor growth and eventually metastasis (Huang et al., 1996). The mechanism(s) for the loss of c-KIT gene expression during melanoma progression are unknown. The promoter of the c-KIT gene has been cloned and sequenced (Yamamoto et al., 1993). The human c-KIT promoter region lacks a typical ‘TATA box’ but has a relatively high G+C content and strikingly, three putative AP-2-binding elements. These observations, coupled with our previous finding that highly metastatic human melanoma cells do not express the AP-2 transcription factor (Bar-Eli, 1997), led us to hypothesize that AP-2 may regulate c-KIT gene expression in human melanoma cells. AP-2, a 52 kDa protein, was first purified from HeLa cells. Partial peptide sequences led to the isolation of the cDNA from a HeLa cell library (Williams et al., 1988), and the gene was mapped to a region on the short arm of chromosome 6 near the HLA locus (Gaynor et al., 1991; Mitchell et al., 1991). The AP-2 protein binds to a consensus palindromic core recognition element with the sequence 5′-GCCNNNGGC-3′ (Williams et al., 1988). Functional AP-2-binding sites have been identified in the enhancer regions of viral genes such as simian virus 40 (SV40) (Mitchell et al., 1987), human T-cell leukemia virus type I (Nyborg and Dynan, 1990), and cellular genes such as murine major histocompatibility complex (H-2Kb), human metallothionein-IIa (huMTIIa), human proenkephalin, human keratin K14 genes, c-erbB-2, plasminogen activator inhibitor type I (PAI-1) and insulin-like growth factor binding protein-5 (Lee et al., 1981; Haslinger and Karin, 1985; Hyman et al., 1989; Leask et al., 1991; Descheemaeker et al., 1992; Bosher et al., 1995; Duan and Clemmons, 1995). The DNA-binding domain is located within the C-terminal half of the 52 kDa protein and consists of two putative amphipathic α helices separated by an 82 amino acid intervening span that is both necessary and sufficient for homodimer formation (Williams and Tjian, 1991). An alternatively spliced AP-2 protein, AP-2B, that differs in its C-terminus and acts as dominant-negative to AP-2 has recently been cloned (Buettner et al., 1993). AP-2 activity is regulated through a number of different signal transduction pathways. Phorbol esters and signals that enhance c-AMP levels induce AP-2 activity independently of protein synthesis, whereas retinoic acid treatment of teratocarcinoma cell lines results in a transient induction of AP-2 mRNA levels on a transcriptional level (Lüscher et al., 1989; Buettner et al., 1993). AP-2 is involved in mediating programmed gene expression both during embryonic morphogenesis and adult cell differentiation. Using in situ hybridization, a restricted spatial and temporal expression pattern has been observed during murine embryogenesis. In particular, regulated AP-2 expression was observed in neural crest-derived cell lineages (from which melanocytes are derived) and in facial and limb bud mesenchyme (Mitchell et al., 1991). Two recent reports of AP-2 null mutant mice have demonstrated that AP-2 is important for development of the cranial region and for midline fusions. The AP-2 null mice died at birth (Schorle et al., 1996; Zhang et al., 1996). In this study, we provide the first evidence that (i) there is a direct correlation between AP-2 and c-KIT expression in human melanoma cells; (ii) transfection of highly metastatic cells (c-KIT negative, AP-2 negative) with the AP-2 gene resulted in induction of c-KIT mRNA and protein; and (iii) re-expression of AP-2 in highly metastatic melanoma cells inhibited their tumor growth and metastatic potential in nude mice, possibly through transactivation of c-KIT. Our results add weight to the hypothesis that loss of AP-2 expression is a crucial event in the development of malignant melanoma, especially since other genes involved in the progression of human melanoma such as MCAM/MUC18, E-cadherin, MMP-2 and p21/WAF-1 are also regulated by AP-2. Results Direct correlation between c-KIT and AP-2 expression in human melanoma cell lines The mechanism(s) for lack of expression of c-KIT in metastatic melanoma cells is unknown. In an effort to determine the molecular basis for c-KIT's lack of expression in highly metastatic melanoma cells, we found that the c-KIT gene and its promoter in c-KIT-negative melanoma cells had no abnormalities (deletions, rearrangements or mutations) that can account for the lack of c-KIT expression (Huang et al., 1996; Bar-Eli, 1997; data not shown). These observations suggest that c-KIT expression might be regulated at the transcriptional level. To test this hypothesis, we subcloned the promoter of the c-KIT gene (−1215 to +1) (Yamamoto et al., 1993) in front of the luciferase reporter gene. Using the Dual-Luciferase Reporter System, we analyzed the luciferase activity driven by the c-KIT promoter in c-KIT-positive and c-KIT-negative melanoma cell lines. As shown in Figure 1, c-KIT-luciferase activity was higher in the human melanoma cell lines Mel-888 and Mel-501 (both express high levels of c-KIT mRNA) (Zakut et al., 1993), as compared with the activity in c-KIT-negative A375SM cells which was given the reference value of 1. We observed low luciferase activity in the WM-2664 cell line which does not express c-KIT mRNA and protein (Gutman et al., 1994). These results suggest that c-KIT expression is regulated at the transcriptional level in these melanoma cells. Figure 1.Luciferase activity driven by the c-KIT promoter in human melanoma cell lines. 2 μg of c-KIT luciferase reporter gene construct pKluc and 0.05 μg of pB-Actin-RL (Renilla) plasmid were co-transfected into 2×105 melanoma cells, and incubated for 60 h at 37°C. Firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay System. Luciferase activity was compared with c-KIT-negative A375SM cells which were given the reference value of 1. Higher luciferase activity was observed in the two c-KIT-positive cell lines, Mel-501 and Mel-888, than in the c-KIT-negative cell line, WM2664. The standard deviation bars represent replicates within the assay. This experiment is a representative of three performed. Download figure Download PowerPoint The 1.2 kb c-KIT promoter lacks TATA or CCAAT boxes, is highly G+C rich, and contains binding sites for SP-1, myb, GATA-1 and three putative AP-2 sites (Yamamoto et al., 1993). Deletion of the proximal 185 bp of the promoter abolished the transcription of c-KIT in HEL cells, suggesting that only the proximal 185 bp of the promoter are necessary and sufficient for c-KIT expression (Yamamoto et al., 1993). As we closely examined the proximal 185 bp promoter region, two AP-2 binding sites were identified within this region with a total of three putative AP-2 binding motifs within the 1.2 kb c-KIT promoter (Figure 3C). The presence of two AP-2-binding sites within the essential region of c-KIT promoter suggested that AP-2 might regulate expression of the c-KIT gene. Indeed, during mouse development, AP-2 and c-KIT are co-expressed in several tissues including hindbrain, kidney and heart, and in neural crest-derived lineages from which melanocytes originate (Orr-Urtreger et al., 1990; Mitchell et al., 1991). These observations prompted us to analyze whether the progression of human melanoma is associated with changes in the expression of the AP-2 transcription factor via regulation of the c-KIT gene. To study whether AP-2 plays a role in the regulation of c-KIT expression in human melanoma cells, we first examined the status of AP-2 in a panel of human melanoma cell lines exhibiting different metastatic capabilities in nude mice (Luca et al., 1995; Bar-Eli, 1997; Xie et al., 1997). Figure 2 demonstrates that expression of AP-2, correlated with the expression of c-KIT and the metastatic potential of human melanoma cells i.e. the non/low metastatic melanoma cell lines in nude mice such as MeWo, Mel-501, Mel-888, SB-2 and SB-3, expressed high levels of c-KIT and the two AP-2 transcripts. In contrast, the cell lines derived from metastatic lesions and also metastatic in nude mice (Luca et al., 1995) A375P, A375SM and WM-2664 were c-KIT negative and were also either negative for AP-2 (A375SM), or expressed negligible amounts of AP-2 mRNA (A375P, WM-2664). Expression of AP-2 in the nonmetastatic SB-2 cells and lack of expression of AP-2 in the highly metastatic A375SM cells was confirmed by Western blot analysis (Bar-Eli, 1997; see Figures 6 and 7). Figure 2.Northern blot analysis for the expression of c-KIT and AP-2 in human melanoma cell lines. c-KIT-positive melanoma cell lines expressed high levels of the two AP-2 transcripts, while c-KIT-negative melanoma cell lines were either negative for AP-2 (A375SM), or expressed negligible amounts of AP-2 mRNA (A375P, WM-2664). The upper band at ∼5 kb which is expressed in all lanes most likely represents the AP-2B transcript (Buettner et al., 1993). The same filter was hybridized with GAPDH to verify the integrity and the amount of RNA loaded in each lane. Download figure Download PowerPoint Figure 3.(A) Effect of AP-2 expression on c-KIT promoter. 2 μg of pKLuc, 1–6 μg of pSG5-AP-2 (expression vector for wild-type AP-2) or pSG5 (naked vector) were co-transfected into A375SM cells with PB-Actin-RL to monitor transfection efficiency. Fold activation was calculated relative to naked vector transfected control cells. Luciferase activity driven by the c-KIT promoter was activated by wild-type AP-2 in a dose-dependent manner. The standard deviation bars represent replicates within the assay. This is one of two experiments performed. (B) Effect of dominant-negative AP-2B on c-KIT promoter. 2 μg of pKLuc, 1–5 μg of pSG5-AP-2B (or pSG5) and 0.05 μg of pB-Actin-RL were co-transfected into Mel-501 cells. c-KIT promoter activity was inhibited by dominant-negative AP-2B. A 48% inhibition in luciferase activity was observed with 2.5–5 μg of AP-2B expression vectors. Fold inhibition was calculated relative to control cells transfected with the naked vector. (C) Schematic presentation of wild-type (pKluc) and mutant AP-2 DNA-binding sites −80 to −89 (Mut.1-pKluc) and −139 to −148 (Mut.2-pKluc), within the minimal region required of c-KIT activation. (D) Mutations within the AP-2 site located between −139 and −148 inhibited luciferase activity 4-fold in A375SM cells co-transfected with 2 μg of AP-2A expression vector. Mutations of the downstream site (−80 to −89) had no effect on the activation of the reporter gene. (E) Site-directed mutagenesis of the AP-2 site located between −139 to −148 bp upstream of the c-KIT initiation site significantly inhibited reporter activity (11-fold) in Mel-501 c-KIT-positive cells, while mutations in the AP-2 site located between −80 and −89 had no effect. Fold inhibition in (D) and (E) was calculated relative to control cells transfected with the wild-type c-KIT promoter pKluc. Download figure Download PowerPoint Transactivation of c-KIT promoter by AP-2A and repression by the dominant-negative AP-2B gene To assess the effect of AP-2 on c-KIT transcription, the c-KIT promoter–luciferase construct, pKLuc, was co-transfected into A375SM cells with increasing concentrations of an expression vector encoding for wild-type AP-2 (AP-2A, pSG5-AP-2) or with the control vector lacking AP-2A (pSG5). Using the β-actin-Renilla luciferase plasmid (pB-Actin-RL) vector as a control to normalize for transfection efficiency, Figure 3A shows that the luciferase activity driven by the c-KIT promoter was activated by AP-2A in A375SM cells in a dose-dependent manner. A 9.5-fold stimulation was observed in cells co-transfected with 6 μg of the plasmid expressing the AP-2A protein, which was not detected in transfections with the parent vector. Conversely, when the pKLuc construct was co-transfected into Mel-501 cells (which express high levels of c-KIT) and increasing concentrations of AP-2B, the dominant-negative form of AP-2 (Buettner et al., 1993), the luciferase activity was inhibited in these cells (Figure 3B). A 48% decrease in luciferase activity was observed with 3.5 μg of the plasmid encoding AP-2B. No further decrease in activity was observed with higher concentrations of AP-2B. It has been shown that AP-2A and AP-2B do not interact directly. The lack of further repression of the c-KIT promoter in Mel-501 cells by AP-2B might be due to the limited amount of a putative adopter protein required for interaction between AP-2A and AP-2B (Buettner et al., 1993). However, we cannot rule out the possibility that AP-2 is not the sole factor regulating c-KIT expression in these cells. These experiments indicate the presence of functional AP-2 elements within the c-KIT promoter that regulate c-KIT expression in human melanoma cell lines. To provide direct evidence that the AP-2 sites within the 185 bp proximal c-KIT promoter actually contribute to its activity, we performed site-directed mutagenesis of the AP-2 sites located between −80 to −89 and −139 to −148 upstream of the c-KIT transcription initiation site (Figure 3C). Disruption of the upstream AP-2 site located between −139 and −148 significantly inhibited reporter activity in c-KIT-positive Mel-501 cells (Figure 3E) and in c-KIT-negative A375SM cells co-transfected with 2 μg of expression vector for AP-2A (Figure 3D). Mutations of the downstream AP-2 binding site (−80 to −89) did not alter the luciferase activity in these cells, indicating that the AP-2 site located between −139 and −148 is crucial for the c-KIT promoter activation. This could be explained by the difference in the sequence of these two AP-2 sites in which the site located between −139 to −148 has more homology with the core AP-2 recognition sequence (Williams et al., 1988). Direct interaction of AP-2 with the c-KIT promoter To determine if AP-2 transactivation of the c-KIT promoter was due to direct AP-2 binding to the c-KIT promoter, we tested a 108 bp fragment from the c-KIT promoter (−68 to −175, that contains two AP-2 binding sites) for reaction with recombinant human AP-2 (r-h-AP-2) on EMSA gel. Figure 4 shows that r-h-AP-2 bound directly to this fragment. This binding was abrogated by an excess of unlabeled double-stranded AP-2-binding DNA sequences, but not by the AP-1-binding DNA motif. Furthermore, the observed DNA–protein complex was super-shifted by anti-AP-2 antibody but not by anti-CREB antibody (Figure 4). In repeated EMSA gel experiments, we were able to demonstrate that the shifted bands were eliminated by an excess of unlabeled DNA fragments corresponding to the c-KIT promoter region that was used as a probe (Figure 5A). To further determine the specificity of the AP-2 reaction with the c-KIT promoter, formation of these complexes was competed out again with an excess of unlabeled oligonucleotides corresponding to the AP-2 consensus binding motif (Figure 5B, lanes 2–4) but not with an excess of cold double-stranded oligonucleotides that were designed to harbor mutations that disrupt AP-2 binding (Figure 5B, lanes 6–8). Collectively, these data show that AP-2 bound directly to a region of the c-KIT promoter required for its transcription. Figure 4.Interaction of AP-2 with the c-KIT promoter. r-h-AP-2 binds directly to a fragment of the c-KIT promoter (−68 to −175) that contains two binding sites for AP-2. AP-2/c-KIT binding could be abrogated by an excess of AP-2 DNA binding sequences but not by AP-1 binding motifs. The specific DNA–protein complex was super-shifted by anti-AP-2 antibody but not by anti-CREB antibody. Download figure Download PowerPoint Figure 5.Interaction of AP-2 with the c-KIT promoter. r-h-AP-2 bound directly to a fragment of the c-KIT promoter described in Figure 4. This binding could be competed out by an excess of unlabeled double-stranded DNA fragments corresponding to the c-KIT promoter region (A), or by oligonucleotides corresponding to the AP-2 consensus binding motif (B) (lanes 2–4), but not by an excess of oligonucleotides with mutations that disrupt AP-2 binding (B) (lanes 6–8). Download figure Download PowerPoint Figure 6.(A) Northern blot analysis for the expression of AP-2 in parental A375SM, neo-transfected, and two AP-2 transfectants. High levels of AP-2 transcripts were observed in the two transfectants as compared with residual levels in parental and neo-transfected cells. The same blot was hybridized with GAPDH to verify the integrity of RNA and the amount loaded in each lane. (B) Western blot analysis for the expression of the AP-2 protein in A375SM parental, neo-transfected (neo.a and neo.b), and in the AP-2 transfectants A375SM-AP.T1 and A375SM-AP-2.T2. Note AP-2 expression was observed in the AP-2 transfected cells, while the parental and neo-transfected cells express a very faint band. r-h-AP-2 served as a positive control. The AP-2 band in the parental cells migrated more slowly on this gel. Download figure Download PowerPoint Figure 7.EMSA gel to determine that the AP-2 in the transfected cells is functional. Nuclear extracts from control and AP-2-transfected cells were reacted with the AP-2 DNA binding motif. SB-2 cells were used as a positive control for the presence of AP-2 that yielded three shifted bands (lane 2). Band 1 was super-shifted by anti-AP-2 antibody (lane 4), but not by anti-CREB antibody (lane 3). Note AP-2 binding activity was observed in the two AP-2 transfectants A375SM-AP-2.T1 (lane 6) and A375SM-AP-2.T2 (lane 7), but not in parental A375SM cells (lane 5). Band 1 was super-shifted in the AP-2 transfected cells by anti-AP-2 antibody (lanes 8 and 9). In addition, bands 1 and 3 were competed out by an excess of unlabeled double-stranded oligo- nucleotide probe, while band 2 was slightly affected. Download figure Download PowerPoint Ectopic expression of AP-2 in A375SM human melanoma cells To assess the contribution of the AP-2 transcription factor to c-KIT expression and to the acquisition of the metastatic phenotype in human melanoma cells, we decided to re-express AP-2 in A375SM cells. A375SM cells are highly metastatic in nude mice (Gutman et al., 1994; Luca et al., 1995; Xie et al., 1997) and also c-KIT negative, and they express low or negligible levels of endogenous AP-2 (Figure 6A and B). Following gene transfection with either an expression vector carrying a full-length human AP-2A (wild-type) cDNA or an empty vector, neo-resistant colonies were pooled and established in culture. Two independent transfections were performed. Therefore, two transfectants were obtained and designated A375SM-AP2.T1 and A375SM-AP2.T2, respectively. Northern blot analysis using AP-2 cDNA as a probe detected high levels of AP-2 mRNA transcripts in the two transfectants (Figure 6A), but only residual levels in parental A375SM or control neo-transfected cells. Expression of AP-2 in the transfected cells was also verified by Western blot analysis using nuclear extracts and anti-AP-2 antibody (Figure 6B). The 52 kDa protein was observed in the two transfectants as compared with faint bands in the two neo-transfected clones. To determine whether the AP-2 in the transfected cells is functional, we next analyzed the ability of nuclear extracts from the transfected cells to bind to a consensus AP-2-binding DNA oligonucleotide on an EMSA gel. We used the SB-2 cells as a positive control for cells expressing the AP-2 transcription factor. As shown in Figure 7, three shifted bands were observed with nuclear extracts from SB-2 cells (lane 2), with the upper band (band 1) being super-shifted when reacted with anti-AP-2 antibody (lane 4) but not with anti-CREB antibody (lane 3). In contrast, nuclear extracts from parental A375SM cells yielded a strong band 2 and very faint bands 1 and 3 (Figure 7, lane 5), while nuclear extracts from the two AP-2 transfectants T1 and T2 yielded three strongly shifted bands similar to the pattern observed with nuclear extracts from the SB-2 cells (Figure 7, lanes 6 and 7). The upper bands (band 1) in the two transfectants were super-shifted when reacted with anti-AP-2 antibody (Figure 7, lanes 8 and 9), and bands 1 and 3 were competed out using excess unlabeled double-stranded oligonucleotide probe (Figure 7, lanes 10 and 11). Band 2 was slightly competed out with ×100 molar excess of unlabeled AP-2 (Figure 7, lane 11). To further confirm that the AP-2 in the transfected cells is transcriptionally active, we took advantage of the observation that AP-2 transactivates the metallothionein promoter (Bauer et al., 1994). To that end, we constructed a luciferase reporter gene expression vector driven by three AP-2 consensus response elements from the human metallothionein gene IIA ligated in front of a minimal TK promoter (Figure 8A). The reporter constructs were transfected into control or AP-2-transfected A375SM cells, together with the pB-actin-RL plasmid that served as an internal control for transfection efficiency. Luciferase activity was 5- to 8-fold higher in the AP-2 transfectants A375SM-AP-2.T1 and A375SM-AP-2.T2, respectively, as compared with the neo-control cells (Figure 8B). These data demonstrate that the two AP-2 stably transfected T1 and T2 clones expressed high levels of active AP-2. Figure 8.(A) Construction of the luciferase reporter gene (3XAP2-Luc) driven by a trimer of AP-2 binding motifs and minimal TK promoter. (B) Luciferase activity of 3XAP-Luc in A375SM neo and AP-2 transfectants. Melanoma cells (2×105) were co-transfected with 1 μg of 3XAP2-Luc and 0.05 μg of pB-Actin-RL plasmid. Firefly and Renilla luciferase activities were measured by the Dual-Luciferase Reporter Assay System. Note luciferase activity was upregulated in the two AP-2 transfected clones but not in neo-transfected cells, which demonstrates that the transfected AP-2 was functional. Download figure Download PowerPoint Upregulation of c-KIT gene expression in AP-2 stably transfected human melanoma cells Our promoter analyses (Figure 3A) indicated that the AP-2 transcription factor is an important regulator of c-KIT gene expression. We therefore examined the effect of AP-2 re-expression in A375SM cells on c-KIT gene expression. To that end, the expression of the 145 kDa c-KIT receptor was analyzed in the two AP-2 transfectants, T1 and T2. To determine c-KIT protein expression, whole-cell lysates w