Identification of the Human PHLDA1/TDAG51 Gene: Down-Regulation in Metastatic Melanoma Contributes to Apoptosis Resistance and Growth Deregulation1

[CANCER RESEARCH 62, 5920–5929, October 15, 2002] Identification of the Human PHLDA1/TDAG51 Gene: Down-Regulation in Metastatic Melanoma Contributes to Apoptosis Resistance and Growth Deregulation1

Ru¨diger Neef, Martina A. Kuske, Elma Pro¨ls, and Judith P. Johnson2 Institute for Immunology, Ludwig Maximilians University of Munich, Munich, Germany 80336

ABSTRACT are up-regulated or induced during melanoma development. Compar- ison of gene expression between human melanoma cell lines selected To identify molecules involved in the progression of human melanoma for high and low metastatic behavior in immune-deficient mice has to metastatic disease, autologous primary and metastatic melanoma cells were compared by differential mRNA display. One cDNA, expressed in also led to the identification of molecules up-regulated in metastatic primary but not in autologous metastatic cells in three different patients, cells in vivo, for example the cell adhesion molecule ALCAM (11), was cloned and characterized, and shown to be the human homologue of melanoma inhibitory activity (12), and the GTPase RhoC (13). The the inducible, immediate early TDAG51/PHLDA1 (pleckstrin-homology- comparison of gene expression between syngeneic benign and malig- like domain family A, member1) gene. Monoclonal antibodies produced nant murine melanocytic cells has led to the identification of genes against the PHLDA1 protein revealed homogeneous strong expression by such as annexin-VI that are also differentially expressed in human benign melanocytic nevi, and progressively reduced expression in primary melanocytic lesions in vivo (14). In the study reported here, gene and metastatic melanomas in vivo. Analysis of stable cDNA transfectants in two different cell lines revealed that constitutive PHLDA1 expression is expression in cells derived from primary and metastatic lesions of the associated with reduced cell growth, cloning efficiency, and colony for- same patient has been compared using mRNA differential display mation but not with alterations in cell cycle parameters. However, (15). This approach has led to the identification of the human homo- PHLDA1 expression was associated with increased basal apoptosis as logue of the TDAG51/PHLDA13 gene (16). The expression of this assessed by live cell annexin V binding, terminal deoxynucleotidyltrans- gene was down-regulated in metastatic as compared with primary ferase-dependent nucleotide incorporation, and with increased cleavage of melanoma cells in three different patients. PHLDA1/TDAG51, iden- poly(ADP-ribose) polymerase and caspase-9. Constitutive PHLDA1 expression greatly enhances the sensitivity of human melanoma cells to the tified previously in murine T lymphocytes where it is required for chemotherapeutic agents doxorubicin and camptothecin. These results activation induced cell death (17), is here shown to be expressed suggest that PHLDA1 is constitutively expressed by melanocytic nevi in human benign melanocytic nevi and to be progressively down- where it may contribute to their benign phenotype. The progressive loss of regulated in primary and metastatic melanomas. Analysis of cDNA PHLDA1 expression in melanomas may play a role in deregulated cell transfectants indicates that constitutive PHLDA1 expression in mela- growth and apoptosis resistance in these tumors. nocytic cells is associated with increased apoptosis sensitivity and with reduced growth. These results suggest that changes in the ex- INTRODUCTION pression of PHLDA1 in melanocytic cells may contribute to the progression of malignant melanomas. The progression of human tumors to systemic, metastatic disease is responsible for the majority of cancer-associated morbidity and mor- tality. The identification of molecules contributing to this process can MATERIALS AND METHODS lead to an understanding of critical pathways involved as well as to potential targets for therapeutic intervention (1). Malignant melanoma Cell Lines and Tissues is a rapidly metastasizing, therapy-resistant tumor, which is increasing Cell lines were obtained from the ATCC (Manassas, VA), established in our in incidence (2, 3). Because of their pigmented nature and epidermal laboratory or obtained through exchange. The autologous cell lines GT-BS and location, a variety of benign and malignant melanocytic lesions have GU-BSA were established from a primary nodular melanoma and a lymph been identified and ordered into a scheme of distinct stages, which is node metastasis, respectively (18), and were kindly provided by Monika proposed to reflect the development and progression of malignant Vetterlein, Institute for Tumor Biology and Cancer Research, University of melanoma (4, 5). Despite extensive histopathological and clinical Vienna, Vienna, Austria. WM-115 and WM-266–4 were obtained from analyses, the changes in gene expression and subsequent functional ATCC, and IgR37 and IgR39 from Stefan Carrell (deceased); Swiss Institute consequences that characterize these stages are only beginning to be for Experimental Cancer Research, ISREC, Epalinges, Switzerland. All of the defined. Different approaches have been used to identify changes in cells were maintained in RPMI 1640 with 5–10% FCS, 1 mM sodium pyruvate, gene expression during melanoma development. Comparison of mela- and antibiotics. Cells were stimulated with 10 ng/ml PMA (Sigma Chemical nocytic protein expression in different stages has led to the identifi- Co.) for 24 h, or exposed to 2 ␮g/ml camptothecin (Sigma) or 1 ␮g/ml cation of several molecules, which appear to play a role in the doxorubicin (Sigma) for the indicated times. progression of this tumor in in vivo models. These include ␤3 integrin Tissue specimens and histological diagnoses were obtained from the Der- (6, 7) and the cell adhesion molecule MCAM/MUC18 (8–10), which matology Departments of the University of Hamburg (Hamburg, Germany), the University of Munich, and the Technical University of Munich (Munich, Germany). Tissue specimens were frozen in liquid nitrogen shortly after Received 1/30/02; accepted 8/15/02. Ϫ The costs of publication of this article were defrayed in part by the payment of page removal and stored at 80°C. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Deutsche Krebshilfe, Mildred Scheel Stiftung, W137/ 3 The abbreviations used are: PHLDA1, pleckstrin-homology-like domain family A, 94/Jo1. This work was presented in part at the 92nd annual meeting of the AACR (2001) member 1; ATCC, American Type Culture Collection; PMA, phorbol 12-myristate and at the 32nd Annual Meeting of the German Society of Immunology (2001). 13-acetate; RT-PCR, reverse transcription-PCR; GST, glutathione S-transferase; TUNEL, 2 To whom requests for reprints should be addressed, at Institute for Immunology, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; MTT, 3-(4,5- Goethestrasse 31, 80336 Munich, Germany. Phone: 49-89-5996-660; Fax: 49-89-5160- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; neo, neomycin-resistant; PARP, 2236; E-mail: [email protected]. poly(ADP-ribose) polymerase; FACS, fluorescence-activated cell sorter. 5920

Differential Display and Isolation of Full-Length P7 cDNA Signaling), and peroxidase-coupled second antibodies (Dako), and bound enzyme activity detected with a chemiluminescence substrate (ECL; Amer- Total RNA was extracted from GT-BS and GU-BSA cells using RNAzol sham Bioscience). Goat antiserum against human TDAG51/PHLDA1 was (Wacker Chemie, Munich, Germany). Using the RNAmap kit (GenHunter, obtained from Santa Cruz Laboratories (L-19; Santa Cruz, CA), and mono- ␮ Nashville, TN), 0.2 g of total RNA was reverse transcribed with the oligode- clonal antibody RN-6E2 (IgG2a) directed against the human PHLDA1 mole- oxythymidylic acid primers T12MA, T12MC, or T12MG and subsequently cule was produced in our laboratory. To control for loading and transfer the amplified by PCR combining each of the three different T MM primers with 12 filters were stripped [0.1 M glycine (pH 2.9), 2 ϫ 20 min] and reprobed with Ј Ј Ј the five arbitrary primers AP-1 (5 -AGCCAGCGAA-3 ), AP-2 (5 -GAC- antiactin antibody (A5441, IgG1; Sigma). CGCTTGT-3Ј), AP-3 (5Ј-AGGTGACCGT-3Ј), AP-4 (5Ј-GGTACTCCAC- 3Ј), and AP-5 (5Ј-GTTGCGATCC-3Ј). Three independent reverse transcrip- Apoptosis Assays, Cell Cycle Analysis tion and PCR reactions were performed and the [33P]ATP-labeled amplification products separated on a 6% denaturing polyacrylamide gel. Apoptosis was assessed by annexin V binding in the presence of propidium Differentially amplified PCR products were cut out from the dried gel, ream- iodide to distinguish living and dead cells, and by TdT-dependent dUTP plified, and cloned into the pCRII vector (Invitrogen, Groningen, Netherlands). incorporation (TUNEL). Assessment of apoptosis was performed on cells that The 350-bp P7 differential display fragment was used to screen a melanoma were ϳ70% confluent. Binding of FITC-annexin V was performed according ␭ (Mel JuSo) cDNA library in ZAP (Ref. 8; Stratagene, La Jolla, CA), and two to the manufacturer’s instructions (Apoptosis Detection kit; BD PharMingen, overlapping clones of 3.0 and 2.7 kb were isolated. Neither clone contained an San Diego, CA). TUNEL assays were performed using the In Situ Cell Death open reading frame, although the 3.0 kb clone contained a polyadenylic acid Detection Kit (Roche Biochemicals). Cells were incubated with FITC-dUTP Ј Ј tail at the 3 end, suggesting that they were not full length in the 5 region. with and without TdT. FITC incorporation in the absence of TdT served as Ј Application of the 5 rapid amplification of cDNA ends System (Invitrogen background control for each cell line. Stained cells were analyzed by flow Ј Life Technologies, Inc.) using the P7-specific primer 5 -CCAAGATTAG- cytometry (FACscan; Becton Dickinson) and Cell Quest software. Ј GAATTACTACG-3 and anchor primers with reverse-transcribed (P7-specific Cells were harvested for cell cycle analysis at various times after plating of Ј Ј primer 5 - GCAGCCTGGACAGGAGTAC-3 ) RNA isolated from Mel JuSo 300,000 cells/6-mm tissue culture well. The cells were washed in 0.1% glucose melanoma cells generated a 700-bp fragment containing an open reading frame in PBS, adjusted to 1 ϫ 106/ml, and fixed in 70% ethanol for at least 48 h (19). that overlapped with the cDNA clone GenBank Z50194, coding for a proline, Fixed cells were resuspended in 0.1% glucose containing 50 ␮g/ml pro- histidine-rich protein from fetal liver. pidium iodide and 100 ␮g/ml RNase for 30 min, and analyzed by flow For RT-PCR analysis, total RNA was treated with DNaseI and reverse Ϫ cytometry (FACscalibur; Becton Dickinson). Cell cycle parameters were ob- transcribed with oligo(dT)12–18 and SuperScript RNase H reverse tran- tained using curve fitting analysis with the ModFit program (Verity Software, scriptase (Invitrogen and Life Technologies, Inc.). The following primers were Inc., Topsham, ME). used for the RT-PCR analysis of PHLDA1 expression: 5ЈCCAGGACAGAT- GCTACTTGG 3Ј and 5Ј-GACTACATAACCTAGCAGTGG 3Ј. For Northern Transfectants blot analysis, 10 ␮g of total RNA was separated in formaldehyde containing ϩ agarose gels, transferred to Nylon membranes (Hybond N ; Amersham Bio- The 293 kidney epithelial cell line and the melanoma cell line Mel Rif were 32 science, Uppsala, Sweden), and hybridized with a P random-labeled probe transfected with a PHLDA1 expression plasmid in pcDNA3 or with the corresponding to the first 1.1 kb of the PHLDA1 gene. neomycin resistance gene, using Fugene 6 (Roche Molecular Biochemicals; 1 ␮g plasmid DNA/3 ␮l Fugene 6) according to the manufacturer’s instructions. Immunohistochemistry, Immunofluorescence, and Antigen Competition The PHLDA1 expression plasmid was constructed from two cDNA clones isolated from a fetal liver cDNA library (5Ј region; clones Z50194–3 and Tissue sections were blocked with 100 ␮g/ml heat aggregated human IgG, Z50194–1, kindly provided by Willy A. Flegel, Department of Transfusion and stained with an indirect immunoperoxidase method using a biotinylated Medicine, University of Ulm, Ulm, Germany) and a 2.7 kb cDNA clone second antibody (Zymed, South San Francisco, CA) and streptavidin-peroxi- isolated from a melanoma cDNA library (3Ј region). The PHLDA1 insert is 4.5 dase complex (Roche Biochemicals, Mannheim, Germany) together with kb long and begins on the reverse strand of chromosome 12 with bp M 3-amino-9-ethyl-carbazole [0.25 mg/ml in 0.1 acetate buffer (pH 4 ϩ 74026201. It contains two potential ATG start codons, bp 74026036 and bp 4.9)] 0.003% H2O2 as substrate. Serial sections were stained with antimelanoma proteoglycan antibody (G7A5, IgG1; Immunotech, Marseille, France) to 74025613, although the second ATG appears to be used in the transfectants localize the melanocytic cells, anti-CD45 (GAP 8.3; ATCC; IgG2a) to localize and in all of the cell lines examined (data not shown). Transfectants were leukocytes, and appropriate isotype controls (11–4-1, IgG2a anti-H-2 obtained selected with G418 (1 mg/ml; Sigma) and cloned by limiting dilution. Two from Gunther Hammerling, German Cancer Center, Heidelberg, Germany; independent neo and four independent PHLDA1 transfectants were generated FR4H12 IgG1, directed against CD66c produced in our laboratory). Staining in each cell line. PHLDA1 expression was assessed by immunofluorescence was evaluated semiquantitatively, and the fraction of PHLDA1-positive mel- and by Western blotting. anoma/nevus cells as well as the intensity of staining was estimated by comparison of RN-6E2 with the antiproteoglycan staining. Cell Growth Assays Intracytoplasmic immunofluorescence was performed using the Fix and Growth Assays. Five ϫ 103 cells/well were seeded in quadruplicate in Perm Kit (Caltag Laboratories, Burlingame, CA), and FITC-antimouse immu- 96-well flat-bottomed tissue culture plates and cultured for 6 days. MTT noglobulin (Dako, Copenhagen, Denmark). (Sigma) live cell ELISAs (20) were performed each day (days 0–6). Briefly The specificity of RN-6E2 binding was determined by a 1-h preincubation MTT (1 mg/ml) was added to the wells and incubated for4hat37°C, the cells of sections or cells with 1.25–1.4 mg/ml PHLDA1-GST or cMCAM-GST were lysed with 7.5% SDS and 7.5 mM HCl overnight, and the plates read in recombinant protein before the addition of RN-6E2 antibody. Recombinant an ELISA reader (Viktor 1420, Turku, Finland) at 570 nm. GST proteins were produced using pGEX expression vectors (Amersham 4 Bioscience) in Escherichia coli BL21-Codon Plus-RP (Stratagene, La Jolla, Thymidine Incorporation. Cells (10 per well) were seeded in quadrupli- CA) and purified on glutathione Sepharose according to the manufacturer’s cate in 96-well flat-bottomed tissue culture plates and pulsed for 16 h with 1 ␮ 3 recommendations C[H]thymidine/well (Amersham Bioscience) on days 0 and 5 of culture. The cells were disrupted by freeze thawing and were harvested using a Skatron Micro96 Harvester, and the filters counted in a Beta Plate scintillation counter Western Blotting (1205; Wallac, Turku, Finland). Fifty to 100 ␮g protein from total cell lysates per lane were separated on Cloning Efficiency. One-hundred cells were plated in each of four 96-well 10% reducing SDS-PAGE and transferred to nitrocellulose membranes (BA85; plates, and growing colonies were scored at 14 days. Schleicher & Schu¨ll, Dassel, Germany). The filters were blocked in 5% dried milk in PBS, stained with anti PHLDA1 antibodies, anticleaved caspase 9 4 Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/maps.cgi?CHR ϭ 12 (1:500; Cell Signaling, Beverly, MA), or anticleaved PARP (1:1000; Cell &BEG ϭ 74021350&END ϭ 74026201. 5921

Colony-forming Assay. Cells (350) were plated in each of three wells of indicating that it represents the human homologue of TDAG51/ a six-well plate. At day 16 the plates were fixed in 70% methanol, stained with PHLDA1 (16) gene. 0.1% crystal violet, and the number of colonies counted. Western blot analysis using a polyclonal antiserum confirmed that expression of the PHLDA1 protein, which migrates with an apparent

RESULTS molecular weight of Mr 40,000, is reduced in the cell lines derived from the metastatic lesion as compared with those derived from the Identification of PHLDA1 as an mRNA Differentially Ex- autologous primary tumor (Fig. 1, C and D). pressed in Cells Derived from Autologous Primary and Metastatic In addition to these three autologous cell pairs, Western blot anal- Melanoma. mRNA differential display was used to compare gene ysis of 22 melanoma cell lines revealed a significant correlation expression in GT-BS, a cell line established from a primary nodular between PHLDA1 expression and cell origin. Cell lines derived from melanoma, and GU-BSA, established from a lymph node metastasis melanoma metastases expressed significantly less PHLDA1 protein of the same patient. Among the nine cDNA fragments showing than those derived from primary tumors (P Ͻ 0.0219; Fig. 1, E and F). differential expression in three independent experiments, the 340-bp Southern analysis of DNA derived from melanomas and normal cDNA P7 was preferentially expressed in the primary tumor cell line leukocytes provided no evidence for PHLDA1 gene amplification or (Fig. 1A). RT-PCR analysis with specific primers that amplified a 126-bp fragment within the P7 sequence generated a signal only in the translocation in human melanoma cells (data not shown), whereas primary tumor cell line (Fig. 1B, G). Examination of two additional Northern blot analysis of mRNA derived from normal human tissues pairs of cell lines derived from autologous primary and metastatic showed low ubiquitous PHLDA1 expression (data not shown), similar lesions of different patients (Fig. 1B, I and W), suggested that down- to what has been reported for the murine and rat homologues (17, 21). regulation of the P7 gene may be a common occurrence in melanoma PHLDA1 Protein Is Expressed in Melanocytic Lesions. The metastases. results obtained from the differential display suggest that PHLDA1,an To obtain the full-length cDNA, the P7 fragment was used to screen inducible early immediate gene, is constitutively expressed in malig- human melanoma cDNA libraries, and the clones obtained were nant melanomas and that it is down-regulated during their progression extended with 5Ј rapid amplification of cDNA ends. This resulted in to metastatic disease. To evaluate PHLDA1 protein expression on sequence information overlapping with the GenBank cDNA clone melanocytic lesions in vivo, we generated monoclonal antibodies Z50194, encoding a proline-rich protein. The 4.5-kb P7 cDNA (ac- directed to the human PHLDA1. The antibody RN-6E2, which rec- cession no. NM 007350) shows a 87% identity (at the amino acid ognizes the Mr 40,000 PHLDA1 protein in Western blots, reacts with level) to the murine TDAG51 cDNA (accession no. NM 009344) the primary melanoma cell line Mel JuSo (Fig. 2, A and B, solid line),

Fig. 1. Differential expression of PHLDA1 in cells derived from primary (P) and metastatic (M) melanoma cell lines. Autologous cell line pairs: I, IgR37 (M)/IgR39 (P); G, GU-BSA (M)/GT-BS (P); W, WM- 115 (P)/WM-266–4(M). A, RNA differential display with cell line pair G, in triplicate (1–3). Arrow indicates P7 fragment (PHLDA1). B, P7 (PHLDA1)- specific RT-PCR in cell line pairs compared with RT-PCR products of an irrelevant gene P9. C, PHLDA1 protein expression in autologous cell pairs. Western blot was probed with anti-PHLDA1 L19 antiserum and anti-␤-actin. D, quantification of protein expression shown in C. Densitometric analysis of the PHLDA1 protein bands was normalized to the ␤-actin bands. For each cell line pair, the value for the metastasis derived cell was set as 100. E, Western blot analysis of PHLDA1 expression in melanoma cell lines using the L19 anti-PHLDA1 antiserum and anti- ␤-actin. F, summary of PHLDA1 protein expression in melanoma cell lines derived from primary or metastatic lesions. Indicated are the number of cell lines expressing strong or weak PHLDA1. Data were analyzed using Fisher’s exact test.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. FUNCTION OF PHLDA1/TDAG51 IN HUMAN MELANOMA and this reactivity is completely inhibited in the presence of recom- localization signal, microscopic examination of the stained Mel JuSo binant PHLDA1 protein (Fig. 2A, filled histogram) but is not influ- melanoma cells indicates that at least in these cells, PHLDA1 is enced by the presence of an unrelated protein (Fig. 2B, filled histo- localized primarily to the cytoplasm (Fig. 2I). gram). The identical results are obtained when recombinant protein is The expression of PHLDA1 was examined on frozen tissue sections used in the staining of frozen tissue sections. Antibody RN-6E2 of 15 melanocytic nevi, 22 primary melanomas, and 18 metastatic reactivity with melanocytic cells (labeled by the antiproteoglycan lesions from skin and lymph node. In all of the cases the percentage antibody in Fig. 2C) is inhibited by preincubation with recombinant of reactive melanocytic cells (based on the staining of a serial section PHLDA1 protein (Fig. 2D) but not by recombinant MCAM protein with antimelanoma proteoglycan antibody) was estimated and the (Fig. 2E). Additional evidence for the specificity of this antibody is staining scored as strong (e.g., Fig. 3B) or weak (e.g., Fig. 3D). Strong shown by its reactivity with the cell line HL60. PHLDA1 mRNA (Fig. and uniform expression was observed with 80% of the nevi, which 2G) as well as RN-6E2 binding (Fig. 2H) can only be detected in included congenital as well as acquired lesions (Fig. 3, B and E). A HL60 cells after phorbol ester stimulation. similar expression pattern was observed in only 41% of the primary Although the derived cDNA sequence contains a putative nuclear tumors and 22% of the metastases. Among the metastatic lesions, no

Fig. 2. Specificity of the anti-PHLDA1 antibody RN-6E2. A and B, FACS analysis of RN-6E2 binding to melanoma cell line Mel JuSo. Reactivity of RN-6E2 (solid line) is inhibited by preincubation with recombinant PHLDA1 protein (A, filled histogram) but not by recombinant MCAM protein (B, filled histogram). Dashed lines, isotype control. Numbers in parentheses indicate the ␦ mean channels (mean channel anti-PHLDA1-mean channel isotype control) without and with inhibitor protein. C–F, immunoperoxidase staining of frozen tissue serial sections of melanocytic nevus, N3. Reactivity of RN-6E2 with melanocytic cells (identified by reactivity with antiproteoglycan antibody), C, is inhibited by preincubation with recombinant PHLDA1 protein (D) but not by preincubation with recombinant MCAM protein (E); Isotype control (F). G, Northern blot analysis of PHLDA1 mRNA expression in HL60 cells with (ϩ) and without (Ϫ) PMA stimulation using a PHLDA1-specific probe (“Material and Methods”). H, FACS analysis of RN-6E2 binding to HL60 cells with (filled histogram) and without (solid line) PMA stimulation. Dashed line, isotype control. I, cytoplasmic expression of PHLDA1 in Mel JuSo. Green, FITC anti-PHLDA1; red, 4Ј,6-diamidino-2-phenylin- dole staining of nucleus.

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Fig. 3. PHLDA1 expression in melanocytic lesions. Serial frozen tissue sections were stained with an indirect immunoperoxidase method. A, nevus N11, antiproteoglycan; 125ϫ; B, nevus N11, anti-PHLDA1 (RN-6E2); 125ϫ; C, lymph node metastasis (LNM) M7, antiproteoglycan; 250ϫ; D, LNM M7 anti-PHLDA1 (RN-6E2); 250ϫ; E, summary of PHLDA1 reactivity on melanocytic lesions. Each investigated lesion is represented by a circle and ordered by the percentage of PHLDA1- positive melanocytic cells. Filled circles, strong staining; open circles, weak staining. The green- filled circle is nevus N11, the red, unfilled circle is LNM M7.

apparent differences were seen between lymph node (in Fig. 3E metastasis: lesions M 1, 2, 7, and 9–17), lung (in Fig. 3E metastasis: lesion M 3) and skin metastases (in Fig. 3E metastasis: lesions M 4, 5, 6, 8, and 18). Compared with the nevi, PHLDA1 expression by primary and metastatic melanomas was heterogeneous with many tumors showing only weak staining in a fraction of the melanoma cells. Comparing the primary tumors to the metastases, PHLDA1 expression was most heterogeneous in the metastatic lesions. Thirty- three percent of metastatic lesions showed staining in less than half of the tumor cells, compared with 10% of the primary tumors. These data, presented schematically in Fig. 3E, suggest that PHLDA1 expression is down-regulated during the progression of malignant melanomas. Within the primary tumors, no correlation could be observed between the intensity or degree of PHLDA1 expression and the tumor stage. Among the primary tumors showing strong, uniform PHLDA1 expression were melanoma in situ (in Fig. 3E, primary tumors: lesion PT 6), superficial spreading melanomas (in Fig. 3E, lesions PT 2, 3, and 8), and nodular melanomas (in Fig. 3E, lesions PT 5 and 7). The vertical thickness or Breslow index remains the most widely used prognostic parameter in melanoma (5). Neither thin tumors (Ͻ0.75 mm, lesions PT 3, 15, 16, and 20) nor thick tumors (Ͼ3.5 mm, lesions Fig. 4. PHLDA1 expression in stable transfectants. Tr1-Tr4, PHLDA1 transfectants; PT 1 and 11) showed uniformity in PHLDA1 expression pattern. Neo1 and Neo2, neomycin-resistant transfectants. A, FACS histograms. Filled histograms, PHLDA1 expression of the melanomas was also not associated with PHLDA1 expression (antibody RN-6E2); unfilled histograms, isotype control; numbers in parentheses indicate the ␦ mean channel values (mean channel PHLDA1-mean channel the degree of leukocyte infiltration. The expression pattern observed isotype control). B, Western blot analysis. PHLDA1 was detected with monoclonal on the melanocytic tumors contrasts starkly with the surrounding antibody RN-6E2. Anti-␤-actin was used as loading control. 5924

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. FUNCTION OF PHLDA1/TDAG51 IN HUMAN MELANOMA normal tissues. No immunoreactivity was observed with leukocytes, more slowly than the neo controls (Fig. 5). This difference was more vessels, smooth muscle, keratinocytes, or any other cells in the lymph pronounced after day 2, a time at which essentially all of the cells had nodes or skin, including epidermal melanocytes (Fig. 3, B and D). established cell-cell contacts. Reduced growth of the PHLDA1 trans- Constitutive Expression of PHLDA1 Is Associated with Re- fectants was observed both in assays for living cells (MTT ELISA; duced Cell Growth. To directly examine the influence of PHLDA1 Fig. 5, A and B) and in DNA synthesis (Fig. 5C). However, the expression on the biological characteristics of cells, four independent PHLDA1 transfectants did not demonstrate obvious derangements in stable transfectants constitutively expressing various levels of the cell cycle parameters when examined at 24, 48, or 72 h after PHLDA1 protein were produced in two different human cell lines. plating. Cell cycle analyses at 48 h, a time point at which a reduced Both of these cell lines, 293, an adenovirus 5 transformed embryonic growth rate is evident, are shown in Fig. 5D. kidney epithelial cell, and melanoma cell line Mel Rif, expressed low The PHLDA1 transfectants also demonstrated a reduced ability to endogenous levels of PHLDA1 protein. The PHLDA1 expression in form colonies in liquid medium (Fig. 6, A and B) and a reduced the eight transfectants and in neo control transfectants is shown in Fig. cloning efficiency (Fig. 6C), suggesting that autonomous growth 4, A and B. Whereas the different transfectants express different levels potential is also decreased. The number of colonies was not increased of PHLDA1, these are comparable with the endogenous levels ob- when measurements were made at a later time indicating that this is served in some melanoma cell lines derived from primary tumors (for not solely a reflection of the reduced growth rate. However, in both example, Mel JuSo, Fig. 2, A and B). assays, the size of the individual colonies was notably smaller in the The PHLDA1-expressing transfectants did not differ morpho- PHLDA1 transfectants than in the neo controls. logically from the neo controls, and no differences were observed Constitutive PHLDA1 Expression Is Associated with Increased in the expression of a variety of cell surface molecules including Sensitivity to Apoptosis. PHLDA1 was originally identified as a the ␤1 integrins (CD29 and CD49a-f) and CD95 (Fas; data not molecule necessary for activation-induced cell death in T lympho- shown). cytes (17). To determine whether the observed reduction in growth The PHLDA1-expressing transfectants were observed to grow observed for the human transfectants might be associated with an

Fig. 5. Constitutive PHLDA1 expression sup- presses cell growth. A and B, live cells measured with an MTT ELISA. Dashed lines, neomycin transfectants; solid lines, PHLDA1 transfectants. Values are given as mean absorbance with SD of four wells. Y axis, A570 nm. C, thymidine incorporation in growing 293 PHLDA1 (Tr1-Tr3) and control transfectants (Neo1 and Neo2) at days 0 and day 5. Data are presented as the mean of quadruplicate cultures; bars, Ϯ SD. D, cell cycle analysis. PHLDA1 (Tr2-Tr4) and neomycin (Neo1 and Neo2) transfectants in 293 and Mel Rif, 48 h after plating. Histograms showing cell number (Y axis) and DNA content as measured with propidium iodide (X axis), and the percentage of cells in the cell cycle phases as determined with the ModFit software.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. FUNCTION OF PHLDA1/TDAG51 IN HUMAN MELANOMA increase in basal apoptosis, the PHLDA1 and neo transfectants were PHLDA1 also increases the sensitivity of the melanoma cells to examined for surface binding of annexin V (Fig. 7A) and for TdT- camptothecin. As shown in Fig. 8B in the analysis of propidium dependent incorporation of FITC-labeled dUTP (TUNEL; Fig. 7B). iodide-stained cells, the fraction of cells with a sub-G1 DNA Compared with the neo controls, the PHLDA1 transfectants all dem- component (Ap), which represents apoptotic cells, increases in the onstrate an increase in annexin V binding by live cells (i.e., propidium PHLDA1 transfectant much more rapidly than in the neo transfec- iodide negative), manifest as a shift in fluorescence intensity. In tant. Similar results are seen when the expression of cleaved PARP addition, as shown for the 293 transfectants, the TdT-treated cells is compared between PHLDA1 and neo transfectants (Fig. 8C). demonstrate a population of small, FITC- positive cells (Fig. 7B, red) These results indicate that constitutive PHLDA1 expression clearly not seen in control cells incubated with FITC-dUTP in the absence of enhances the sensitivity of the melanoma cells to chemotherapeutic enzyme (Fig. 7B, green). This population is increased in all of the agents. The time course study presented in Fig. 8B also shows a PHLDA1 transfectants as compared with the neo controls. In addition, difference between the PHLDA1 and neo transfectants at time ϭ 0, cleaved caspase 9, an important effector of apoptosis and cleaved providing additional evidence that basal apoptosis is enhanced in PARP, a commonly used marker of apoptosis (22) are also increased the PHLDA1-expressing cells. in PHLDA1 transfectants as compared with neo transfectants (Fig. 7C). Taken together the results of these independent assays indicate DISCUSSION that constitutive expression of PHLDA1 is associated with an increase in basal apoptosis. The development and progression of malignant tumors is a To examine whether expression of PHLDA1 enhances sensitiv- complex multistep process involving alterations in the expression ity of melanoma cells to chemotherapeutic agents, the Mel Rif and function of a variety of different genes (23). In the study transfectants were cultured in the presence of doxorubicin and then presented here, a comparison of autologous primary and metastatic assessed for annexin V binding. Twenty-four h after doxorubicin melanoma cells led to the identification of the human PHLDA1 exposure, a separate population of strongly annexin V-positive gene as an mRNA that is down-regulated in cells derived from cells is visible in all of the PHLDA1 transfectants as compared metastatic lesions. Immunohistochemical analysis of melanocytic with the neo controls (Fig. 8A). Constitutive expression of lesions revealed PHLDA1 expression in benign melanocytic nevi

Fig. 6. Influence of constitutive PHLDA1 expression on colony formation and cloning efficiency. Tr1-Tr4, PHLDA1 transfectants; Neo1 and Neo2, neomycin transfectants. A, photograph of representative colony formation plates 16 days after plating. Left plates, 293; right plates, Mel Rif. B, quantitation of colony formation. Shown are mean colony numbers per 350 cells (triplicates); bars, ϮSD. C, cloning efficiency. Shown are the mean number of colonies per 100 cells (triplicates); bars, ϮSD.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. FUNCTION OF PHLDA1/TDAG51 IN HUMAN MELANOMA and confirmed its progressive down-regulation in primary and metastatic lesions in vivo. The PHLDA1 protein has a predicted size of 259 amino acids and contains a series of motifs indicative of protein-protein interactions. These include 16 proline-glutamine and 15 proline-histidine pairs in the COOH-terminal region and a stretch of 14 glutamine residues in the middle of the protein (17). Polyglutamine tracts are found in a variety of transcription factors as well as in neurodegenerative disease associated proteins, and are thought to present protein binding regions (24). Although PHLDA1 expression in melanoma cells was observed in the cytoplasm under normal culture conditions, the protein contains a putative nuclear localization signal suggesting that it may under

Fig. 8. PHLDA1 expression enhances sensitivity of melanoma cells to chemotherapeutic agents. Tr1, Tr2, and Tr4, PHLDA1 transfectants; Neo1 and Neo2, neomycin transfectants. A, annexin-V binding assay of Mel Rif transfectants after exposure to doxorubicin. Cells were cultured with 1 ␮g/ml doxorubicin for 24 h. X axis, fluorescence intensity; Y axis, cell number. The bar in each panel denotes the median fluorescence intensity for the neomycin transfectants. m, mean channel values; p1 and p2, peak channels for the two populations. B, cell cycle analysis. PHLDA1 and neo transfectants in Mel Rif, 24 h after plating were treated for the indicated time with camptothecin and stained with propidium iodide. Histograms showing cell number (Y axis) and DNA content as measured with propidium iodide (X axis). The percentage of cells in the cell cycle phases and the fraction of cells with a sub-G1 DNA content (apoptotic cells, Ap) were determined with the ModFit software. C, Western blot analysis of cPARP expression in representative PHLDA1 and neomycin Mel Rif transfectants after exposure to stimulation with camptothecin for the indicated time. The band intensities were normalized to the actin expression, and PHLDA1 expression by Tr1 at each time point was set to 100. The numbers represent the relative amount between PHLDA1 and control transfectants.

Fig. 7. Constitutive PHLDA1 expression is associated with increased apoptosis. certain conditions translocate into the nucleus. PHLDA1 does not Tr1-Tr4, PHLDA1-transfectants; Neo1 and Neo2, neomycin transfectants. A, annexin-V contain a predicted transmembrane region but does contain a pleck- binding of transfectants under normal growth conditions. Shown are FACS histograms of FITC- annexin-V binding of living (propidium iodide-negative) cells of 293 and Mel Rif strin homology region, which may mediate interaction with cellular transfectants; X axis, fluorescence intensity; Y axis, cell number. The red bar in each panel membranes and which is the basis for its inclusion in the PHLDA denotes the median fluorescence intensity for the neomycin transfectants. m, mean channel B, FACS analysis of a TUNEL assay in 293 transfectants. Shown are dot blot gene family (25). PHLDA1 is an immediate early gene, and is induced diagrams; X axis, fluorescence intensity (incorporated FITC-labeled nucleotides) versus in cell lines by a variety of external stimuli (17, 21) and in normal forward scatter (Y axis; cell size). Green represents the background fluorescence observed lymphocytes by mitogen treatment.5 in the absence of TdT and red the FITC-positive cells (apoptotic cells) observed only in the presence of TdT. The percentage of apoptotic cells (red/red ϩ green) is shown for The PHLDA1 gene has been identified independently in two other each transfectant. C, Western blot analysis of cleaved caspase-9 (cCas-9) and cleaved systems. The murine PHLDA1 was isolated in a search for genes PARP (cPARP) in representative Mel Rif PHLDA1 and neo transfectants. The numbers indicate the relative protein amount (PHLDA1 transfectant set to 100) after normalization to the ␤-actin expression. 5 Unpublished observations. 5927

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. FUNCTION OF PHLDA1/TDAG51 IN HUMAN MELANOMA involved in T-cell receptor-induced cell death in a T-cell hybridoma tumors with a tightly regulated growth control (38, 39) and a high (17), and the rat homologue was identified as an immediate early gene level of apoptosis (40). In transfectants, constitutive PHLDA1 expres- induced by fibroblast growth factor in a hippocampal neuronal cell sion was associated with a reduction in growth rate, a decrease in line (21). In all three of the systems expression of PHLDA1 has been cloning efficiency, and an increased apoptosis sensitivity. The con- associated with enhanced apoptosis. In the studies reported here, stitutive expression of PHLDA1 protein by nevi in vivo raises the constitutive PHLDA1 expression in melanoma cells and in 293 cells possibility that it contributes to the benign nature of these tumors, was associated with a reduction in growth rate, and a decrease in maintaining growth regulation and apoptosis sensitivity to loss of cloning efficiency and colony formation. The observed derangement survival signals provided by keratinocyte neighbors. Therefore, the in growth was not associated with an obvious alteration in cell cycle progressive loss of PHLDA1 expression with malignant transforma- suggesting that it may be a reflection of an increased sensitivity to tion may contribute to the loss of these characteristics in melanoma. apoptosis. In fact under normal growth conditions, the PHLDA1 The mechanisms involved in the down-regulation of PHLDA1 ex- transfectants showed broad evidence of an increased basal apoptosis pression remain unknown. TSSC3, a member of the PHLDA1 gene rate. PHLDA1 transfectants demonstrated increased annexin V bind- family, is imprinted during normal development (41) and also in brain ing, increased TdT-dependent dUTP incorporation, a higher fraction tumors (42). Although PHLDA1 is localized to a chromosomal region of cells with a sub-G1 DNA content, as well as higher levels of not yet implicated in imprinting (16, 24), methylation, which has been cleaved caspase 9 and cleaved PARP when compared with the neo shown to contribute to loss of Apaf-1 expression (29), could also be transfectants. The increase in the fraction of apoptotic cells in the involved in PHLDA1 silencing in melanoma. PHLDA1-expressing cells, as assessed by TdT-dependent dUTP incorporation or sub-G1 DNA content, averaged 5.7%. Apoptotic rates ACKNOWLEDGMENTS between 0.25 and 3% have been shown, in other systems, to lead to cell losses of 25–50%/day (26, 27). We thank Drs. Eckhard Breitbart (Buxtehude Hospital Dermatology Center, The development of resistance to apoptosis is a hallmark of malig- Buxtehude, Germany) and Sigfried Borelli (Department of Dermatology, Uni- nant cells, enabling them to survive despite apoptosis inducing envi- versity of Zurich, Zurich, Switzerland) for tissue specimens, and Dr. Willy ronmental signals and the loss of normal survival signals (23, 28, 29). Flegel (Department of Transfusion Medicine, University of Ulm, Ulm, Ger- Whereas mutation of p53 is a major mechanism used by tumor cells many) for cDNA clones. to escape from apoptosis, in malignant melanoma p53 mutations are rare (30). Nevertheless, melanomas are highly resistant to apoptosis, REFERENCES and this is thought to be, at least in part, an explanation for their 1. Yoshida, B. A., Sokoloff, M. M., Welch, D. R., and Rinker-Schaeffer, C. 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Rüdiger Neef, Martina A. Kuske, Elma Pröls, et al.

Cancer Res 2002;62:5920-5929.

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