ANTICANCER RESEARCH 26: 1947-1956 (2006)

Alteration of Expression in Melanoma Cells Following Combined Treatment with -1 and Doxorubicin

HONGYING HAO1, YAN BIN DONG1, MARIA T. BOWLING1, H. SAM ZHOU2,3,4 and KELLY M. McMASTERS1,5,4

Departments of 1Surgery, 2Medicine, 3Microbiology and Immunology and 4Pharmacology, University of Louisville School of Medicine, Louisville, KY; 5James Graham Brown Cancer Center, Louisville, KY, U.S.A.

Abstract. Background: The combination of E2F-1 gene therapy chemotherapy regimens have yielded disappointing results and chemotherapy produces a synergistic effect on melanoma cell (1, 2). The rapid increase in the incidence of melanoma and . However, the molecular mechanisms have not been the limited success of conventional therapy for advanced fully elucidated. The purpose of this study was to identify novel disease have driven the development of novel treatment or pathways that may play key roles in apoptosis when strategies, including the combination of gene therapy and E2F-1 gene therapy is combined with doxorubicin chemotherapy. chemotherapy (1-3). Materials and Methods: SK-MEL-2 melanoma cells were E2F-1 was first identified as a transcription factor that infected with Ad-E2F-1 alone, Ad-E2F-1 plus doxorubicin, or regulates a series of genes that promote transition Ad-LacZ plus doxorubicin. After 16 hours of treatment, the total from the G1- to S-phase (4, 5). Early studies with E2F-1 RNA was extracted from these cells and subjected to microarray suggested that this could function as an oncogene, analysis. Quantitative real-time PCR was performed to confirm based on its ability to stimulate cell proliferation. When the microarray data. Results: Our results showed that the overexpressed, it cooperates with the Ras oncogene, to combination treatment of Ad-E2F-1 and doxorubicin affected transform rat embryonic fibroblasts and promote the expression of cytokines, transcription factors, as well as genes tumorigenesis in mice (6, 7). Somewhat paradoxically, later involved in signal transduction, cell cycle regulation and studies showed that E2F-1 also has properties of a tumor apoptosis. Conclusion: Our findings have identified, for the first suppressor. Overexpression of E2F-1 has been shown to time, novel molecular targets and pathways that led to apoptosis induce apoptosis in many tumor cells (8, 9). Therefore, E2F-1 in melanoma cells when Ad-E2F-1 was combined with appears to be involved in both cell proliferation and the doxorubicin. The molecular information provided here will mediation of programmed cell death. enhance further mechanistic studies. Doxorubicin, also called adriamycin, is an anthracycline antibiotic produced by the fungus Streptomyces peucetius (10). Human malignant melanoma is characterized by both a Doxorubicin damages DNA by intercalation of the rapidly rising incidence and a growing lifetime risk. Because anthracycline portion, metal ion chelation, or by the of the limited success of surgery in treating metastatic generation of free radicals. Doxorubicin has also been shown melanoma, great interest has been shown in the search for to inhibit DNA topoisomerase II. Topoisomerase II is an effective systemic therapy. However, single and combination ATP-dependent enzyme that catalyzes topological changes in DNA. It also plays an important role in DNA metabolism (11). At present, a number of anticancer drugs used clinically act by targeting this enzyme (12, 13). Abbreviations: FBS, fetal bovine serum; MOI, multiplicity of Our previous work demonstrated that transduction of the infection. E2F-1 gene in melanoma cells markedly increased cell sensitivity to topoisomerase II inhibitors, thereby producing a Correspondence to: Kelly M. McMasters, MD, Ph.D., University of synergistic effect on apoptotic cell death. Topoisomerase II Louisville, Department of Surgery, Louisville, KY 40292, U.S.A. inhibitors also cooperated with Ad-E2F-1 to enhance Tel: 502-852-5447, Fax: 502-852-1704, e-mail: mcmasters@ louisville.edu antitumor activity in in vivo nude mouse models (14). However, the molecular mechanisms of E2F-1 gene therapy Key Words: E2F-1, doxorubicin, microarray, gene therapy, combined with chemotherapy have not been fully elucidated. chemotherapy. One of the genomic technologies, profiling,

0250-7005/2006 $2.00+.40 1947 ANTICANCER RESEARCH 26: 1947-1956 (2006) presents new and exciting opportunities to study complex containing doxorubicin at the concentration of 0.1 ÌM. At 16 hours mechanisms of gene therapy in combination with post-adenoviral infection or combined treatment, the cells were chemotherapy. In this study, a high-throughput gene chip was harvested for RNA isolation (some similarly treated plates were carried for 3 days to confirm that the cells underwent apoptosis as a used, containing 22,215 known genes, to determine the result of the combined treatment – data not shown). alteration of gene were expression profiles when SK-MEL-2 cells exposed to E2F-1 and doxorubicin. The combination Microarray analysis. Two control groups were chosen for the following treatment of Ad-E2F-1 and doxorubicin in melanoma cells microarray experiment. First, Ad-E2F-1 infection alone at an MOI affected the expression of a diverse range of genes, including of 2 focuses on identifying genes turned on or off by doxorubicin after cytokines, transcription factors, as well as genes involved in the combined treatment. Second, Ad-LacZ infection at an MOI of 2 signal transduction, cell cycle regulation and apoptosis. In this combined with doxorubicin at 0.1 ÌM focuses on identifying the study, several novel genes and potential pathways were different gene expression patterns after the combination treatment. identified, that may play key roles in apoptosis following Two completely independent experiments of the three samples were E2F-1 gene therapy in combination with doxorubicin performed. cRNA synthesis and labeling: At 16 hours after infection or infection chemotherapy. Our findings provided, for the first time, novel combined with doxorubicin treatment, total RNA was isolated with molecular targets and pathways that led to apoptosis in the RNeasy mini kit (QIAGEN, Valencia, CA, USA). This time-point melanoma cells when Ad-E2F-1 was combined with was chosen because it is a time of peak E2F-1 expression, prior to any doxorubicin. The molecular information provided here will evidence of apoptosis. First- and second-strand cDNA were enhance further mechanistic studies and provide potential synthesized from 5 Ìg to 15 Ìg of total RNA using the SuperScript clinical applications. Double-Stranded cDNA Synthesis Kit (Gibco Life Technologies, Bethesda, MD, USA) and oligo-dT24-T7 (5’-GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-3’) primer, Materials and Methods according to the manufacturer’s instructions. cRNA was synthesized and then labeled with biotinylated UTP and CTP by in vitro Cells and culture conditions. The human melanoma cell line, transcription using the T7 promoter coupled to the double-stranded SK-MEL-2, was purchased from the American Type Culture cDNA as a template and the T7 RNA Transcript Labeling Kit (ENZO Collection (Rockville, MD, USA). Cells were cultured in a 5% CO2 Diagnostics). Briefly, double-stranded cDNA synthesized from the incubator at 37ÆC and subcultured every 3 to 4 days (about 80% previous steps was washed twice with 70% ethanol and resuspended in confluent) in ·-MEM, supplemented with 10% heat-inactivated fetal 22 ÌL of RNase-free H2O. The cDNA was incubated with 4 ÌL of 10X bovine serum (FBS) and penicillin (100 U/mL)/streptomycin each reaction buffer, biotin-labeled ribonucleotides, DTT, RNAse (100 Ìg/mL) solution. All cell culture reagents were obtained from inhibitor mix and 2 ÌL of 20X T7 RNA polymerase for 5 hours at Gibco/BRL (Bethesda, MD, USA). For the experiments, the 37ÆC. The labeled cRNA was separated from unincorporated SK-MEL-2 cells were plated 24 hours prior to infection (1x106 cells ribonucleotides by passing it through a CHROMA SPIN-100 column in 100-mm dishes). The cells were then infected with Ad-E2F-1 or (Clontech) and then precipitating at –20ÆC for 1 hour to overnight. Ad-LacZ at a multiplicity of infection (MOI) of 2. Oligonucleotide array hybridization and analysis: The cRNA pellet was resuspended in 10 ÌL of RNase-free H2O, and 10.0 Ìg was Recombinant adenoviral constructs. Two replication-defective fragmented by heat and ion-mediated hydrolysis, at 95ÆC for 35 recombinant adenoviral vectors were used. The Ad5CMV-E2F-1 minutes in 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, and 150 vector has been deleted in the E1 subunit and contains the transgene mM MgOAc. The fragmented labeled cRNA was tested on the Test E2F-1 under the control of the CMV promoter (14). Ad5CMV-LacZ Chip (Affymetrix, Santa Clara, CA, USA) to ensure that the control (generously provided by Dr. Brent French, University of Virginia, transcript 3’/5’ ratio was ~1. Then, the fragmented cRNA was Charlottesville, VA, USA) was used as a control vector that expresses hybridized for 16 hours at 45ÆC to HG-U133A oligonucleotide arrays nuclear-localized ‚-galactosidase under the control of the same (Affymetrix) containing 22,215 full-length annotated genes together promoter (15). Both vectors were propagated in the 293 cell line, and with additional probe sets designed to represent EST sequences. titers were determined using standard plaque assays (14). Arrays were washed at 25ÆC with 6X SSPE (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA+0.01% Tween 20), followed by a stringent Chemotherapeutic agent. The chemotherapeutic agent, doxorubicin wash at 50ÆC with 100 mM MES, 0.1 M [Na+], 0.01% Tween 20. The (adriamycin) was purchased from Sigma Chemical Co. (St. Louis, MO, arrays were then stained with phycoerythrein-conjugated streptavidin USA). It was dissolved to 5 mM in H2O and stored at –20ÆC. It was (Molecular Probes), and the fluorescence intensities were determined then diluted in culture medium before being added to the cell cultures. using a laser confocal gene array scanner (Agilent). The scanned images were analyzed using Microarray MAS 5.0 or Adenovirus infections and combined treatment. For infections, 1x106 GCOS software (Affymetrix). Sample loading and variations in cells were plated in 100-mm plates and incubated overnight. The staining were standardized by scaling the average of the fluorescence following day, the media was removed and the adenoviral vectors were intensities of all genes on an array to a constant target intensity (250) added to 1 mL of ·-MEM at an MOI of 2 and incubated at 37ÆC. One for all arrays used. Data analysis was conducted using Microarray hour following infection, the medium was removed and replaced with Suite 5.0 (Affymetrix), following the user guidelines. The signal 10 mL of fresh ·-MEM with 5% FBS. For the combined treatment, intensity for each gene was calculated as the average intensity cells were infected with viruses as described above. One hour later, difference, represented by [™(PM-MM)/(number of probe pairs)], the medium was replaced with 10 mL of fresh ·-MEM-5% FBS where PM and MM denote perfect-match and mismatch probes.

1948 Hao et al: Altered Gene Expression after Combined Treatment with E2F-1 and Doxorubicin

Table I. Verification of microarray analysis by real-time PCR.

Gene symbol RT-PCR primers Accession # Microarray av. FC RT-PCR av. FC

‚-Actin F: CGATCCACACGGAGTACTTG NM_001101 NC NC R: GGATGCAGAAGGAGATCACTG NFKBIA F: CGTGGAGCTTTTGGTGTCCTT AI078167 3.2 3.0 R: CCAGGTCAGGATTTTGCAGGT TNFAIP3 F: TGTGTGCTTTGTGGTTGCTGTC AI738896 2.2 2.6 R:TACAGACCCCCAAAATGGCAG TRAF1 F: AACACCACGTCACTTCATGCAG NM_005658 2.7 4.3 R: GGAAGAGGAAAGTTTATGCCCC STAT-1 F: GGCAGTTTTCCATTGGTTTACC NM_007315 1.4 2.0 R: TCCCCCTACCAGATCCATGAT HRK F: CCCACTGTCACGTCAAGAGAAA NM_003806 4.0 2.0 R: CAGAATACCACAGCAGCACCAG MAP3K5 F: CCTGAAGCTTAAGTCCCAACCC NM_005923 2.2 2.5 R: GCTCCATTCACTCTCAGCCAGT SQSTM1 F: AAGAGACCTTGGCTGCTCACTG N30649 2.4 1.8 R: AGTCCCATCCAGCAGGTTACAG IL-8 F: GAAGGTGCAGTTTTGCCAAGGNM_000584 11.9 6.6 R: CGCAGTGTGGTCCACTCTCAAT CDKN2C F: AATGGATTTGGAAGGACTGCG NM_001262 3.5 3.4 R: CGAAACCAGTTCGGTCTTTCAA CXCL1 F: TTTCTGAGGAGCCTGCAACATG NM_001511 4.3 2.5 R: TGCACATACATTCCCCTGCCT E1 F: CACCAGCCACCTCCAGACA M74093 4.0 4.0 R: GTGGGAGTCCCTTAGGTCAAGTAG F: GAAGTAGCCGTTTACAAGCTAAGCA AF091433 3.7 4.3 R: GCCTGGATTATCTGGGCTTCT

In order to verify the alterations in gene expression at the mRNA level, which appeared on the microarray, the microarray results were confirmed with real-time PCR, using total RNA from 16 hours of infection or infection combined with doxorubicin. Primer express software from ABI was used to design the primers for real-time PCR. The GenBank ID numbers and gene symbols are as found at the NCBI website. The data are the average from two or three separate experiments, each in duplicate. The data are reported as the fold-change of gene expression level in Ad-E2F-1 plus doxorubicin-treated cells versus Ad-E2F-1 infection alone or Ad-LacZ plus doxorubicin treatment. FC=fold change.

Several criteria were used to identify genes whose expression was Real-time PCR. At 16 hours of infection or infection combined with affected by E2F-1 gene transfer. First, only genes that were up- or doxorubicin, RNA was extracted and prepared using an RNeasy mini down-regulated more than 2-fold, as determined by the Affymetrics kit along with an RNase Free DNase set to remove any traces of analysis, were selected. The second criteria employed was that the DNA contamination (both kits from QIAGEN, Valencia, CA, USA). difference call, as provided by the GeneChip software, was moderately cDNA was prepared from 500 ng of RNA (TaqMan Reverse induced (MI) or induced (I) for up-regulated genes and moderately Transcription Reagents, ABI/Roche, Branchburg, NJ, USA), based decreased (MD) or decreased (D) for down-regulated genes. In on the manufacturer’s instructions. The template cDNA addition, only genes whose absolute calls were both present, or one (12.5 ng/sample) from cells infected with Ad-LacZ plus doxorubicin present and one absent in Ad-E2F-1 plus doxorubicin versus Ad-E2F-1, 0.1 ÌM, Ad-E2F-1 plus doxorubicin 0.1 ÌM, and Ad-E2F-1 and water or Ad-E2F-1 plus doxorubicin versus Ad-LacZ plus doxorubicin, were (no template control) were mixed with the Syber Green master mix considered as E2F-1 plus doxorubicin-responsive genes. Two (ABI). The mixtures were aliquoted into a 96-well optical reaction completely independent experiments were performed. Only genes that plate (ABI) along with forward and reverse primers for each target had changed in the same direction in both experiments with the gene, and the samples were run in duplicates. The ABI Prism 7000 change p-value of <0.006 were considered for further analysis (16). sequence detection system was used, and the primers were designed These filtering criteria were used to reduce the false-positive responses using ABI primer express software. The amplicon sizes were all below and to ensure that only genes whose expression was reproducibly 200 bp, and the data were analyzed using the comparative CT altered were considered to be the result of E2F-1 gene transfer. The method. The fold-change of the target gene samples (Ad-E2F-1 plus final fold-changes were calculated based on the average signal log ratio doxorubicin) were normalized to the housekeeping gene (‚-actin), of the two experiments. The genes whose average expression differed which is relative to the calibrator (Ad-LacZ plus doxorubicin or by >2 fold when compared to the control-treated cells were grouped Ad-E2F-1). It is calculated by the equation: by functional category. Online data mining tools from three different 2–¢¢CT software programs were used: NetAffyx Analysis Center (Affymetrix), where ¢¢CT=¢CT (sample)– ¢CT (calibrator) and ¢CT is the CT Data Mining Tool 3.1 (Affymetrix) and Partek Pro 6.0 (Partek Inc., value of the target gene subtracted from the CT value of the St. Charles, MO, USA). housekeeping gene, all of which were determined in the

1949 ANTICANCER RESEARCH 26: 1947-1956 (2006)

Table II. Genes whose average expression differed by >2-fold are grouped by biological process or molecular function in SK-MEL-2 cells exposed to Ad- E2F-1 plus doxorubicin compared to Ad-E2F-1 infection alone.

Gene symbol/ GenBank Gene Other biological process or Fold- category No. name molecular function change

Cytokines IL8 NM_000584 Interleukin 8 Angiogenesis, cell motility, chemotaxis 11.9 CXCL1 NM_001511 Chemokine (C-X-C motif) ligand 1 Chemotaxis, inflammatory response 4.3 CXCL2 M57731 Chemokine (C-X-C motif) ligand 2 Chemotaxis, inflammatory response 2.2 CSF2 M11734 Colony stimulating factor 2 Cell surface receptor linked signal transduction 10.9 (granulocyte-macrophage) INHBA M13436 Inhibin, beta A (activin A) Induction of apoptosis, cell cycle arrest 10.4 Apoptosis NFKBIA AI078167 Nuclear factor of kappa light polypeptide Apoptosis, cytoplasmic sequestering of NF-kappaB 3.2 gene enhancer in B-cells inhibitor, alpha TNFAIP3 AI738896 Tumor necrosis factor, alpha-induced protein 3 Ubiquitin cycle, apoptosis, anti-apoptosis 2.2 SNCA NM_000345 Synuclein, alpha Anti-apoptosis –2.1 TRAF1 NM_005658 TNF receptor-associated factor 1 Protein complex assembly, apoptosis 2.7 PRLR NM_000949 Prolactin receptor Anti-apoptosis, cell surface receptor 3.6 linked signal transduction BIRC3 U37546 Baculoviral IAP repeat-containing 3 Regulation of apoptosis, cell surface receptor 2.5 linked signal transduction, protein ubiquitination SQSTM1 N30649 Sequestosome 1 Regulation of I-kappaB kinase/NF-kappaB 2.4 cascade, intracellular signaling cascade mda-5 NM_022168 Melanoma differentiation associated protein 5 Apoptosis 2.0 Cell-cell signaling EFNA1 NM_004428 Ephrin-A1 Cell-cell signaling 2.4 GCH1 NM_000161 GTP cyclohydrolase 1 L-phenylalanine catabolism, 2.0 tetrahydrobiopterin biosynthesis BMP2 AA583044 Bone morphogenetic protein 2 Cell-cell signaling, growth 2.0 NLGN1 NM_014932 Neuroligin 1 Protein targeting, cell adhesion –2.2 Protein kinase PLK1 NM_005030 Polo-like kinase 1 (Drosophila) Regulation of cell cycle, 3.0 protein amino acid phosphorylation, MAP3K12 NM_006301 Mitogen-activated Protein amino acid phosphorylation, protein –2.1 protein kinase kinase kinase 12 kinase cascade, JNK cascade JAK2 NM_004972 Janus kinase 2 (a protein tyrosine kinase) Protein amino acid phosphorylation, –3.4 JAK-STAT cascade TXK NM_003328 TXK tyrosine kinase Protein amino acid phosphorylation, –2.0 intracellular signaling cascade TNIK AF172268 TRAF2 and NCK interacting kinase Regulation of translation, protein amino –2.1 acid phosphorylation, protein kinase cascade Transcription factor NRF1 NM_005011 Nuclear respiratory factor 1 Regulation of transcription, regulation of –3.0 transcription from Pol II promoter ELK3 NM_005230 ELK3, ETS-domain protein Regulation of transcription, signal transduction –2.2 (SRF accessory protein 2) GATA2 AL563460 GATA binding protein 2 Regulation of transcription, transcription 2.3 from Pol II promoter GATA6 D87811 GATA binding protein 6 Regulation of transcription, positive 2.1 regulation of transcription Transporter activity TAP1 NM_000593 Transporter 1, ATP-binding cassette, Oligopeptide transport, immune response 2.0 sub-family B (MDR/TAP) KMO AI074145 Kynurenine 3-monooxygenase Electron transport, metabolism 3.4 (kynurenine 3-hydroxylase) ITPR1 L38019 Inositol 1,4,5-triphosphate receptor, type 1 Calcium ion transport, signal transduction –2.1 VDP AW973253 Vesicle docking protein p115 Intracellular protein transport 7.1

Table II. continued

1950 Hao et al: Altered Gene Expression after Combined Treatment with E2F-1 and Doxorubicin

Table II. continued

Gene Symbol/ Genµank Gene Other biological process or Fold- category No. name molecular function change

Cell adhesion CYR61 NM_001554 Cysteine-rich, angiogenic inducer, 61 Regulation of cell growth, cell adhesion, –2.2 cell proliferation ICAM1 AI608725 Intercellular adhesion molecule 1 (CD54) Cell-cell adhesion 2.7 VCAM1 NM_001078 Vascular cell adhesion molecule 1 Cell-cell adhesion 3.5 PCDH7 NM_002589 BH-protocadherin (brain-heart) Cell adhesion, homophilic cell adhesion –3.6 NLGN1 NM_014932 Neuroligin 1 Protein targeting, cell adhesion, –2.2 calcium-dependent cell-cell adhesion CTGF M92934 Connective tissue growth factor Regulation of cell growth, cell motility, –3.2 cell adhesion FLRT3 NM_013281 Fibronectin leucine rich transmembrane protein 3 Cell adhesion –2.0

Labeled cRNA prepared from SK-MEL-2 cells at 16 hours after combination treatment with Ad-E2F-1 (MOI 2) plus doxorubicin 0.1 ÌM or Ad-E2F-1 (MOI 2) infection alone were used for global gene profiling, employing the Affymetrix cDNA microarray chip (HG-U133A) which consists of about 22,215 genes. Data from the Ad-E2F-1 infection alone were used for comparative analyses. The housekeeping genes showed uniform hybridization intensity.

exponential phase of the reactions (17). (PCR primers and the doxorubicin versus Ad-LacZ plus doxorubicin. These genes accession numbers are listed in Table I.) are involved in cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 8 (IL-8), Results and NF-κB pathway-related genes, including the nuclear factor of the kappa light polypeptide gene enhancer in B-cell Our previous work (14) showed that SK-MEL-2 cells inhibitors, alpha (NFKBIA, also called I-κB·), TNF receptor- responded to doxorubicin in a dose-dependent manner: the associated factor 1 (TRAF-1) and sequestosome 1 (SQSTM1). viable SK-MEL-2 cells exposed to doxorubicin for 2 days at 0.1, 0.2, 0.4 and 0.8 ÌM were 97%, 65%, 58% and 20%, Gene expression profiling of Ad-E2F-1-doxorubicin combination respectively. In this study, it was observed that Ad-E2F-1 at compared to Ad-LacZ-doxorubicin combination. After an MOI of 2 caused a marked increase in E2F-1 protein comparing the combination treatment of Ad-E2F-1 plus expression in this cell line at 16 hours post-infection, but no doxorubicin to Ad-LacZ plus doxorubicin, it was found that the significant apoptosis or toxicity was detected until 3 days later gene expression pattern was different from that of Ad-E2F-1 (data not shown). The synergistic apoptotic effect was plus doxorubicin and Ad-E2F-1 only (Table III). Compared to observed when 0.1 ÌM of doxorubicin was combined with the gene expression results of Ad-LacZ infection plus Ad-E2F-1 at an MOI of 2, with more than 80% of dead cells doxorubicin, Ad-E2F-1 and doxorubicin in combination altered after 3 days of the combined treatment. Therefore, our the expression of some specific genes involved in the cell cycle microarray combination treatment was performed using (i.e., cyclin E1, cyclin E2, cyclin F) and some pro-apoptotic Ad-E2F-1 at an MOI of 2 combined with doxorubicin at 0.1 ÌM. genes in the Bcl-2 family (Bcl-2 and Bim).

Gene expression profiling following Ad-E2F-1 in combination Verification of microarray cDNA chip analysis by quantitative with doxorubicin compared to Ad-E2F-1 infection alone. In the real-time PCR. To verify the alterations in gene expression at microarray analysis, the combination treatment of Ad-E2F-1 the mRNA level, which appeared on the microarray, and doxorubicin (versus Ad-E2F-1 infection only) yielded 99 representative genes were chosen from several different genes that exhibited over a 2-fold (up to 13.6-fold) alteration categories for real-time PCR. RNA was prepared from two in expression. (Detailed information is available on the GEO separate experiments at the same time-point and with the repository website, attachment to account No. GSE2715.). Of same treatment as in the microarray. Real-time PCR was these 99 genes, 59 were found to be up-regulated and 40 were performed as explained in the Materials and Methods section, down-regulated. The above genes were classified based on using the gene-specific primers listed in Table I. For all of the their biological process or molecular functions (Table II). genes tested, the results of real-time PCR were in direct Compared to the gene expression results of Ad-E2F-1 agreement with the microarray data (Table I). The same infection only, Ad-E2F-1 and doxorubicin in combination alterations of gene expression were observed by real-time altered the expression of some specific genes that were not PCR analysis, although the fold-change in the expression level observed in the comparison between Ad-E2F-1 plus was not exactly the same between these two different

1951 ANTICANCER RESEARCH 26: 1947-1956 (2006)

Table III. Genes whose average expression differed by >2 fold are grouped by biological process or molecular function in SK-MEL-2 cells exposed to Ad-E2F-1 plus doxorubicin compared to Ad-LacZ plus doxorubicin.

Gene Symbol/ GenBank Gene Biological process or Fold category No. name molecular function change

Cell cycle SKP2 BG105365 S-phase kinase-associated protein 2 (p45) Regulation of cell cycle, G1/S transition 2.2 of mitotic cell cycle, ubiquitin cycle CDKN2C NM_001262 Cyclin-dependent kinase inhibitor 2C Cell cycle arrest, negative 3.5 (p18, inhibits CDK4) regulation of cell proliferation CCNF NM_001761 Cyclin F Regulation of cell cycle, , mitosis 2.3 CDK5R1 AL567411 Cyclin-dependent kinase 5, Regulation of cyclin-dependent protein 2.6 regulatory subunit 1 (p35) kinase activity CCNE2 NM_004702 Cyclin E2 Regulation of cell cycle, , 3.7 regulation of cyclin-dependent protein kinase activity UNG2 BC004877 Uracil-DNA glycosylase 2 Regulation of cell cycle, cytokinesis 3.3 CCNE1 AI671049 Cyclin E1 Regulation of cell cycle, G1/S transition 4.0 of mitotic cell cycle, cytokinesis Apoptosis HRK NM_003806 Harakiri, BCL2 interacting protein Induction of apoptosis, regulation of apoptosis 4.0 (contains only BH3 domain) BCL2L11 NM_006538 BCL2-like 11 (apoptosis facilitator) Apoptosis, induction of apoptosis, 2.0 positive regulation of apoptosis Protein kinase MAP3K5 NM_005923 Mitogen-activated protein MAPKKK cascade, protein amino acid 2.2 kinase kinase kinase 5 phosphorylation, apoptosis FGFR3 NM_000142 Fibroblast growth factor receptor 3 MAPKKK cascade, protein amino acid 4.0 phosphorylation, JAK-STAT cascade CDK5R1 AL567411 Cyclin-dependent kinase 5, Regulation of cyclin-dependent protein 2.6 regulatory subunit 1 (p35) kinase activity, cell proliferation Signal transduction CD83 NM_004233 CD83 antigen (activated B lymphocytes, Defense response, signal transduction 2.3 immunoglobulin superfamily) FZD1 NM_003505 Frizzled homolog 1 (Drosophila) G-protein coupled receptor protein 2.3 signaling pathway, development RIN2 AL136924 Ras and Rab interactor 2 Endocytosis, intracellular signaling cascade –2.0 PLCL2 AL117515 Phospholipase C-like 2 Lipid metabolism, intracellular signaling cascade 2.8 PDE3B AA888858 Phosphodiesterase 3B, cGMP-inhibited Signal transduction 3.0 RASSF4 AI890191 Ras association (RalGDS/AF-6) Signal transduction –3.5 domain family 4 Transcription FOXO1A NM_002015 Forkhead box O1A (rhabdomyosarcoma) Regulation of transcription from 2.5 Pol II promoter, anti-apoptosis EZH2 NM_004456 Enhancer of zeste homolog 2 (Drosophila) Regulation of transcription, DNA-dependent 2.3 CEBPD NM_005195 CCAAT/enhancer Regulation of transcription, transcription 2.0 binding protein (C/EBP), delta from Pol II promote TOX AI961231 Thymus high mobility group Regulation of transcription, DNA-dependent 2.8 box protein TOX MYB NM_005375 v-myb myeloblastosis viral Regulation of transcription, DNA-dependent 3.7 oncogene homolog (avian) GATA2 AL563460 GATA binding protein 2 Regulation of transcription, transcription 2.7 from Pol II promoter IRX5 U90304 Iroquois homeobox protein 5 Regulation of transcription, DNA-dependent 2.5 KLF11 AA149594 Kruppel-like factor 11 Regulation of transcription, negative 2.0 regulation of cell proliferation FLJ23311 NM_024680 FLJ23311 protein Regulation of cell cycle, regulation of 2.6 transcription, DNA-dependent

Labeled cRNA prepared from SK-MEL-2 cells at 16 hours after combination treatment with Ad-E2F-1 (MOI2) plus doxorubicin 0.1 ÌM or Ad-LacZ (MOI2) plus doxorubicin 0.1 ÌM were used for global gene profiling using Affymetrix cDNA microarray chip (HG-U133A) consisting of about 22,215 genes. Data from Ad-LacZ plus doxorubicin were used for comparative analyses. The housekeeping genes showed uniform hybridization intensity.

1952 Hao et al: Altered Gene Expression after Combined Treatment with E2F-1 and Doxorubicin methods. Both microarray and real-time PCR showed researchers found that metastatic melanoma cells considerably increased levels of IL-8, cyclin E1 and TRAF-1. constitutively secreted the cytokine IL-8, whereas non- The real-time PCR results supported the findings obtained metastatic cells produced low to negligible levels of IL-8, and from the microarray experiments. the expression levels of IL-8 correlated with disease progression in human melanomas in vivo (25, 26). IL-8 now Discussion appears to play contrasting roles in tumor suppression and tumor metastasis. How it cooperates with other cytokines or The outcome of single-agent chemotherapy or current anti-apoptosis genes and plays an anti-tumor function after chemotherapy combinations for malignant melanoma is the combined treatment needs further study. unsatisfactory, and durable responses are rarely achieved (2, NF-κB consists of a family of Rel-domain-containing 18). The combination of gene therapy and chemotherapy is (e.g., RelA (p65), RelB, c-Rel, p50, p52). Inactive one of the emerging treatment strategies with great scientific NF-κB consists of a heterotrimer of p50, p65 and I-κB·, and and application interest (3, 14). To date, there have been no is present in the cytoplasm of all cells. The phosphorylation, other reports that have studied the gene expression profile of ubiquitination and degradation of I-κB· released the p50-p65 E2F-1 and doxorubicin in combination in melanoma cells. heterodimer, which then activated and translocated to the Recent advances in microarray technology hold the promise nucleus (27). Following E2F-1 and doxorubicin combination of increasing our understanding of the mechanisms of the treatment, it was found that NFKBIA (I-κB·), TRAF-1 and combination treatments and providing new targets for treating SQSTM1 were all up-regulated. All of these genes are melanoma. involved in the regulation of the NF-κB pathway. This result From gene expression profiles, we found that following the suggested that the NF-κB pathway and related genes could be combination treatment of Ad-E2F-1 and doxorubicin, the an important molecular mechanism by which E2F-1 and molecular responses are complicated and are likely to be doxorubicin in combination inhibits the growth of melanoma mediated by a wide variety of regulatory pathways. When cells and induces apoptosis. Obviously, the mechanism by combined, E2F-1 and doxorubicin regulated the expression of which these genes cooperate together to render melanoma many important genes that control the cell cycle, apoptosis, cells sensitive to the combination treatment is very interesting. transcription and cell signaling. The role that NF-κB plays in regulating apoptosis following Among the up-regulated genes of Ad-E2F-1 and Ad-E2F-1 infection in combination with doxorubicin is an doxorubicin in combination treatment compared to ongoing investigation in our laboratory. Ad-E2F-1 infection only, the highest fold-change genes It should be noted that the above two pathways, cytokines were IL-8 (+11.9-fold) and the GM-CSF (+10.9-fold). and NF-κB pathways, were intertwined. NF-κB is activated by Other cytokines that were up-regulated following some cytokines and, at the same time, regulates the expression combination treatment with Ad-E2F-1 and doxorubicin of some of these cytokines. For example, TNF-· is the most were inhibin, beta A (INHBA) (up-regulated 10.4-fold), potent inducer of NF-κB and is also regulated by NF-κB (28, chemokine ligand 1 (up-regulated 4.3-fold), and chemokine 29). Like TNF, IL-8 might function through the recruitment ligand 2 (up-regulated 2.2-fold). Dranoff (19) identified of IRAK and TRAF6 followed by NIK and IKK. Finally, GM-CSF as the most potent molecule for augmenting IκB· is phosphorylated and degraded, which then activates tumor immunity for melanoma cells in a murine melanoma NF-κB. IL-8 mediates its action partially through induction of model. Vaccination with irradiated melanoma cells the GTPase pathway, which induces NF-κB (30). Cowburn et engineered to secrete GM-CSF enhanced host responses al. (31) reported that, in human neutrophils, TNF-· induced through improved tumor antigen presentation by recruited rapid IκB· degradation, NF-κB activation and IL-8 release, dendritic cells and macrophages (19, 20). Up-regulation of whereas GM-CSF stimulated NF-κB-dependent IL-8 synthesis GM-CSF suggests that GM-CSF could favor melanoma cell in the absence of IκB· degradation. Based on all of these apoptosis after Ad-E2F-1 is combined with doxorubicin. studies, it is apparent that the NF-κB and cytokines pathways IL-8 is a member of the CXC chemokine family and acts as are very complex, and probably no single gene will be found a chemoattractant and an activator of neutrophils (21, 22). solely responsible for mediating the apoptotic response High expression of IL-8 during the early stage of tumor following Ad-E2F-1 and doxorubicin in combination in development prevented tumor growth by inducing extensive melanoma cells. infiltration of neutrophils and tumor necrosis (23). Shibakura The human melanoma differentiation associated protein-5 et al. (24) demonstrated that doxorubicin markedly induced (mda-5), another apoptosis-inducing gene, was up-regulated IL-8 in small cell lung cancer (SCLC) cells and strengthened 2-fold after the combination treatment of Ad-E2F-1 and the antitumor effects by provoking the interaction between doxorubicin. The mda-5 was identified by subtraction inflammatory/immune cells and tumor cells, thus augmenting hybridization as a novel up-regulated gene in HO-1 human the cytotoxicity to kill cancer cells. However, other melanoma cells induced to terminally differentiate by

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Table IV. Common pathways that are involved in both comparisons of Ad-E2F-1 plus doxorubicin versus Ad-E2F-1 infection alone or Ad-LacZ plus doxorubicin.

Gene GenBank Gene Other biological process or FC in FC in symbol/ No. name molecular function Ad-E2F-1-dox Ad-E2F-1-dox category vs. Ad-E2F-1 vs. Ad-LacZ-dox

MAPK signaling pathway MAP3K5 NM_005923 Mitogen-activated protein MAPKKK cascade, protein amino NC 2.2 kinase kinase kinase 5 acid phosphorylation, apoptosis MAP3K12 NM_006301 Mitogen-activated protein Protein amino acid phosphorylation, –2.1 –2.3 kinase kinase kinase 12 protein kinase cascade, JNK cascade Ubiquitin-proteosome pathway BIRC3 U37546 Baculoviral IAP repeat-containing 3 Protein ubiquitination, 2.5 NC regulation of apoptosis E2-230K NM_022066 Likely ortholog of mouse Ubiquitin conjugating enzyme activity –4.1 –3.8 ubiquitin-conjugating enzyme E2-230K SENP7 NM_020654 SUMO1/sentrin specific protease 7 Ubiquitin cycle, protein sumoylation –2.3 –1.6 CYLD AA555096 Cylindromatosis Ubiquitin-dependent protein 2.4 1.2 (turban tumor syndrome) catabolism, ubiquitin cycle OTUB2 NM_023112 OTU domain, ubiquitin Ubiquitin cycle 2.1 NC aldehyde binding 2 SKP2 BG105365 S-phase kinase-associated Regulation of cell cycle, ubiquitin cycle NC 2.2 protein 2 (p45) TRIM36 NM_018700 Tripartite motif-containing 36 Protein ubiquitination, 1.3 2.4 ubiquitin-protein ligase activity TRIM49 NM_020358 Tripartite motif-containing 49 Protein ubiquitination 2.5 1.1

Cells were treated as in Table II and III. FC=fold change, Ad-E2F-1-Dox=Ad-E2F-1 plus doxorubicin, Ad-LacZ-Dox=Ad-LacZ plus doxorubicin.

treatment with IFN-‚+mezerein (MEZ) (32). Current data directly transactivates genes that are necessary for DNA document that overexpression of mda-5 induces apoptosis in synthesis and promotes cell cycle progression via the induction melanoma cells. The well-established JAK/STAT signaling of (CCND1) and (CCNE1) (34, 35). Here, pathway is directly involved in mda-5 expression (32). Our we showed that doxorubicin in combination with E2F-1 at an microarray data showed that two of the JAK/STAT pathway MOI of 2 altered several cell cycle genes. The possible genes were oppositely regulated during the combined interpretation of this result is that doxorubicin can inhibit cell treatment; JAK2 was down-regulated 3.4-fold, while PRLR growth by causing G2 arrest, while overexpression of E2F-1 was up-regulated 3.6-fold. These data suggested that induction has demonstrated induction of S-phase entry. When of the mda-5 gene through the JAK/STAT pathway might be doxorubicin and Ad-E2F-1 are combined, they can cooperate a novel target for the combination treatment induced together to regulate the cell cycle. Our results suggest that melanoma cell apoptosis. Ad-E2F-1 and doxorubicin in combination inhibited melanoma After comparing the combination treatment with Ad-E2F-1 cell growth through the regulation of the expression of some plus doxorubicin to Ad-LacZ plus doxorubicin, it was found important genes related to cell cycle. This finding is important that the gene expression pattern was different from that of for the development of therapeutic strategies. Ad-E2F-1 plus doxorubicin and Ad-E2F-1 only. Compared to BH3-only members of the Bcl-2 family proteins, including the gene expression results of Ad-LacZ infection plus PUMA, Bid, Bad, Bim and HRK, were proved to be able to doxorubicin, Ad-E2F-1 and doxorubicin in combination activate the apoptotic machinery in response to different altered the expression of some specific genes involved in cell conditions of stress. Unlike the combination of Ad-LacZ cycle and Bcl2 family members. The cell cycle-related genes plus doxorubicin, the combination treatment with Ad-E2F-1 include cyclin E1 (+4-fold), cyclin E2 (+3.7-fold), cyclin F plus doxorubicin up-regulated pro-apoptotic genes in the (+2.3-fold) and cyclin-dependent kinase inhibitor 2C Bcl-2 family, including HRK and Bcl2L11 (Bim). HRK was (+3.5-fold). Our previous microarray data (33) showed that up-regulated 4-fold and Bcl2L11 was up-regulated 2-fold. SK-MEL-2 melanoma cells treated with Ad-E2F-1 at an MOI Our previous data showed that another pro-apoptotic Bcl-2 of 100 caused changes in a set of cell cycle genes, including family member, PUMA, played an important role in the up-regulation of cyclin E1, cyclin E2, , and down- Ad-E2F-1-induced melanoma cell apoptosis (unpublished data). regulation of cyclin G1. Other reports have shown that E2F-1 Other studies (36) showed that in vitro MCF-7 breast cancer

1954 Hao et al: Altered Gene Expression after Combined Treatment with E2F-1 and Doxorubicin cells exposed to clinically achievable doxorubicin Conclusion concentrations for 6 hours revealed marked induction of PUMA mRNA as well. We expected that the PUMA gene Our findings provide, for the first time, novel molecular might also be involved in the apoptotic effect of this targets and pathways that lead to apoptosis in melanoma cells combination treatment. When we checked these microarray following combined treatment with Ad-E2F-1 and data, the PUMA gene signals were too low to be significant. doxorubicin. This treatment caused changes in the expression However, when we performed real-time PCR, the result was of many genes that are critically involved in the control of the a 2-fold up-regulation of this gene in the combination cell cycle, apoptosis, transcription and cell signaling. These treatment. Therefore, up-regulation of HRK, Bim and findings provide molecular information for further PUMA may play an important role in melanoma cell investigation of the mechanisms by which Ad-E2F-1 and apoptosis following the combination treatment. Taken doxorubicin in combination exert their therapeutic effects on together, these data suggest that the combination treatment melanoma cells. These results could also be important in caused melanoma cell apoptosis through the up-regulation revealing new mechanism-based and targeted therapeutic of some of the BH3-only family members. strategies for melanoma. However, further investigations are We noted that there were two groups of genes regulated needed to elucidate the mechanisms by which these altered in the Ad-E2F-1 plus doxorubicin treatment group genes communicate with one another in order to increase the compared to Ad-E2F-1 alone, or Ad-LacZ plus doxorubicin apoptotic effect in melanoma cells when Ad-E2F-1 is (Table IV). One group consisted of genes involved in the combined with doxorubicin. MAPK signaling pathway, such as MAP3K12 (down- regulated 2.1-fold) and MAP3K5 (up-regulated 2.2-fold). Acknowledgements Several recent studies have suggested that mitogen-activated kinases (MAPKs) can participate in the activation of NF-κB The authors would like to thank Ms. Sabine Waigel, from the in the cytoplasm, as well as in the modulation of its microarray facility laboratory at the University of Louisville (USA), transactivation potential in the nucleus (37, 38). MAPKs can for her help with cDNA gene chip analysis, the stem cell facility laboratory at the University of Louisville, for their help with real-time also activate cell death signals and induce cancer cell PCR, and Ms. Margaret Abby, in the Surgery Department at the apoptosis by modulating cell signaling pathways (39). University of Louisville, for her expert manuscript editing. This work MAPKs might also be involved in melanoma cell apoptosis was supported by NIH Grant R01 CA90784-01A1 to KMM. after combining Ad-E2F-1 and doxorubicin. The other group contained genes involved in the ubiquitin-proteasome References pathway. These genes included BIRC3 (up-regulated 2.5-fold, involved in the ubiquitin complex), E2-230K 1 Atkins MB: The treatment of metastatic melanoma with (down-regulated 4.1-fold, involved in ubiquitin conjugating chemotherapy and biologics. Curr Opin Oncol 9: 205-213, 1997. enzyme activity), and SENP7 (down-regulated 2.3-fold, 2 Serrone L and Hersey P: The chemoresistance of human involved in the ubiquitin cycle). Recent studies (40) found malignant melanoma: an update. Melanoma Res 9: 51-58, 1999. that dysregulation of this ubiquitin-proteasome enzymatic 3 Meng RD, Phillips P and El-Deiry WS: -independent increase in E2F-1 expression enhances the cytotoxic effects of etoposide system may also play a role in tumor progression, drug and of adriamycin. Int J Oncol 14: 5-14, 1999. resistance and altered immune surveillance, thereby making 4 Girling R, Partridge JF, Bandara LR, Burden N, Totty NF, the proteasome an appropriate and novel therapeutic target Hsuan JJ and La Thangue NB: A new component of in cancer. Our microarray data also suggested that the transcription factor DRTF1/E2F. Nature (Lond.) 362: 83-87, regulation of the ubiquitin-proteasome pathway might 1993. contribute to the induction of melanoma cell apoptosis. The 5 DeGregori J, Kowalik T and Nevins JR: Cellular targets for ubiquitin-proteasome pathway might be another potential activation by the E2F-1 transcription factor include DNA synthesis and G1/S-regulatory genes. Mol Cell Biol 15: 4215- therapeutic target in melanoma therapy. 4224, 1995. In this study, we found several novel genes not recognized 6 Xu G, Livingston DM and Krek W: Multiple members of the in previous studies. These genes included: the EHM2 gene E2F transcription factor family are the products of oncogenes. (cytoskeletal protein binding, up-regulated 6.0-fold), the Proc Natl Acad Sci USA 92: 1357-1361, 1995. FGFR3 gene (fibroblast growth factor receptor 3 gene, 7 Pierce AM, Fisher SM, Conti CJ and Johnson DG: Deregulated involved in the MAPKKK cascade, up-regulated 4.0-fold), expression of E2F-1 induces hyperplasia and cooperates with ras v-myb (DNA binding activity, involved in transcription in skin tumor development. Oncogene 16: 1267-1276, 1998. 8 Yang HL, Dong YB, Elliott MJ, Liu TJ, Atienza CJ, Stilwell A regulation, up-regulated 3.7-fold), NRF-1 (nuclear respiratory and McMasters KM: Adenovirus-mediated E2F-1 gene transfer factor 1, transcription factor, down-regulated 3.3-fold) and inhibits MDM2 expression and efficiently induces apoptosis in forkhead box O1A (transcription factor, also involved in mdm2-overexpressing tumor cells. Clin Cancer Res 5: 2242-2250, apoptosis, up-regulated 2.5-fold). 1999.

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