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Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human
1Department of Pathology, Columbia-Presbyterian Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Abstract:
Cultured human melanoma cells lose proliferative capacity and terminally differentiate after treatment with the combination of recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ). Subtraction hybridization of cDNA libraries prepared from actively proliferating human H0-1 melanoma cells from cDNA libraries produced from H0-1 cells treated with IFN-beta + MEZ identifies a novel melanoma differentiation-associated (mda) cDNA, mda-7, that displays elevated expression in differentiation inducer-treated H0-1 cells. mda-7 encodes a novel protein of 206 amino acids with a predicted size of 23.8 kDa. The level of mda-7 mRNA is elevated in actively proliferating normal human melanocytes versus primary and metastatic human melanomas. In the Matrigel-assisted melanoma progression model, mda-7 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. Treatment of human melanomas with IFN-beta + MEZ, and to a lesser extent with MEZ, results in growth suppression and induced or enhanced mda-7 expression. Immunoprecipitation analyses using peptide-derived rabbit polyclonal antibodies detect increases in mda-7 protein, and a higher molecular weight protein of approximately 90 to 100 kDa, in MEZ and IFN-beta + MEZ treated H0-1 cells. mda-7 is a highly conserved gene with an homologous sequence in the genome of yeast. Transfection of mda-7 expression constructs into H0-1 and C8161 human melanoma cells reduces growth and inhibits colony formation. These results confirm that mda-7 has antiproliferative properties in human melanoma cells and in this context may contribute to terminal cell differentiation. The mda-7 gene may also function as a negative regulator of melanoma progression.
Insights
A novel gene, melanoma differentiation-associated (mda)-7, shows reduced expression in melanoma. Its reintroduction suppresses melanoma cell growth, suggesting it acts as a tumor suppressor.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Cultured human melanoma cells exhibit reduced proliferation and terminal differentiation upon treatment with recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ).
- Identifying genes involved in melanoma differentiation is crucial for understanding and treating the disease.
Purpose of the Study:
- To identify and characterize novel genes associated with melanoma differentiation.
- To investigate the role of the identified gene, mda-7, in melanoma progression and its potential as a therapeutic target.
Main Methods:
- Subtraction hybridization was used to identify differentially expressed genes in melanoma cells treated with IFN-beta + MEZ.
- mda-7 gene expression was analyzed in normal melanocytes, primary, and metastatic melanomas.
- Melanoma cells were treated with IFN-beta + MEZ to assess growth suppression and mda-7 expression.
- mda-7 protein levels were detected using immunoprecipitation.
- mda-7 expression constructs were transfected into melanoma cells to evaluate its effect on cell growth and colony formation.
Main Results:
- A novel melanoma differentiation-associated (mda)-7 cDNA was identified, showing elevated expression in differentiation inducer-treated cells.
- mda-7 mRNA levels were higher in normal melanocytes compared to melanomas, and decreased during melanoma progression.
- Treatment with IFN-beta + MEZ suppressed melanoma cell growth and induced mda-7 expression.
- Transfection of mda-7 reduced melanoma cell growth and inhibited colony formation, confirming its antiproliferative properties.
Conclusions:
- The mda-7 gene has antiproliferative properties and may contribute to terminal cell differentiation in human melanoma.
- mda-7 may function as a negative regulator of melanoma progression, indicating its potential as a therapeutic target.