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Transcription profiling by array of human SK-MEL-2 cells infected with Ad-E2F-1 after treatment with doxorubicin

ABSTRACT: SK-MEL-2 cells treated with Ad-E2F-1 (MOI 2), Ad-E2F-1 (MOI 2)plus doxorubicin 0.1uM, or Ad-LacZ (MOI 2) plus doxorubicin 0.1uM. The combination of E2F-1 gene therapy and chemotherapy produces a synergistic effect on melanoma cell apoptosis. However, the molecular mechanisms have not been fully elucidated. The purpose of this study was to identify novel genes or pathways that may play key roles in apoptosis when E2F-1 gene therapy is combined with doxorubicin chemotherapy. MATERIALS AND METHODS: SK-MEL-2 melanoma cells were infected with Ad-E2F-1 alone, Ad-E2F-1 plus doxorubicin, or Ad-LacZ plus doxorubicin. After 16 hours of treatment, the total RNA was extracted from these cells and subjected to microarray analysis. Quantitative real-time PCR was performed to confirm the microarray data. RESULTS: Our results showed that the combination treatment of Ad-E2F-1 and doxorubicin affected the expression of cytokines, transcription factors, as well as genes involved in signal transduction, cell cycle regulation and apoptosis. CONCLUSION: Our findings have identified, for the first time, novel molecular targets and pathways that led to apoptosis in melanoma cells when Ad-E2F-1 was combined with doxorubicin. The molecular information provided here will enhance further mechanistic studies.

ORGANISM(S): Homo sapiens

SUBMITTER: Hao H

PROVIDER: S-ECPF-GEOD-2715 | biostudies-other | 2006 May-Jun

REPOSITORIES: biostudies-other

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Project description:Melanoma tumor antigen p97 or melanotransferrin (MTf) is an iron (Fe)-binding protein with high homology to serum transferrin. MTf is expressed at very low levels in normal tissues and in high amounts in melanoma cells. The over-expression of MTf in tumor cells was hypothesized to assist rapidly proliferating neoplastic cells with their increased Fe requirements. However, our recent characterization of the MTf knockout (MTf -/-) mouse demonstrated that MTf did not have an essential role in Fe metabolism. To understand the function of MTf, we utilized whole-genome microarray analysis to examine the gene expression profile of five models after modulating MTf expression. These models included two new stably transfected MTf hyper-expression models (SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type (SK-Mel-28 melanoma) where MTf was down-regulated by post-transcriptional gene silencing. These findings were compared to alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the gene array data were also assessed in a new model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased cellular proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and up-regulation, we identified three genes modulated by MTf expression. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and transcription factor 4 (Tcf4) were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and cell proliferation/survival, respectively. This study identifies novel molecular targets directly or indirectly regulated by MTf and potential pathways involved in its function. These molecular targets could be involved, at least in part, to the role of MTf in modulating proliferation. Experiment Overall Design: To prepare a construct capable of generating hairpin siRNA specific for human MTf mRNA, the expression vector, pSilencerâ¢ 3.1-H1 neo (Ambion, Texas, USA) was used. The vector was used to clone transgene of 66-bp that transcribe 19-mer double-stranded hairpin RNAs of the target gene. The transgene were specifically targeted to positions 2031-2049-bp in the MTf gene (Genbank Accession: NM_005929). Transfections of pS-MTf transgenes or pS-scrambled vectors into human SK-Mel-28 melanoma cells were performed with LipofectamineTM 2000 reagent (Invitrogen, Melbourne, Australia). Total RNA was isolated from the cells using TRIzol ReagentÂ® (Sigma-Aldrich) according to the manufacturerâs protocol. Total RNA from the stably-transfected SK-Mel-28 melanoma cell lines were prepared and hybridized onto Human Genome U133 Plus 2.0 Array. The human GeneChipÂ® U133 Plus 2.0 consists of greater than 47,000 transcripts and variants from over 38,500 well characterized human genes (Affymetrix, Santa Clara, CA).

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Transcription profiling by array of human SK-MEL-2 cells infected with Ad-E2F-1 after treatment with doxorubicin

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