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Soluble human MDA-7/IL-24: characterization of the molecular form(s) inhibiting tumor growth and stimulating
John B Mumm1, Suhendan Ekmekcioglu, Nancy J Poindexter
1Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Insights
Interleukin-24 (IL-24), a dual-function protein, was purified and characterized. This soluble, glycosylated IL-24 dimer shows immunomodulatory effects on monocytes and inhibits melanoma cell growth.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Interleukin-24 (IL-24), also known as melanoma differentiation-associated gene-7 (mda-7), is a member of the IL-10 family.
- IL-24 possesses both tumor suppressor and proinflammatory properties.
Purpose of the Study:
- To purify and characterize the biochemical and functional properties of soluble human IL-24.
- To investigate the immunomodulatory and anti-tumor effects of purified IL-24.
Main Methods:
- Purification of IL-24 from gene-transfected HEK 293 cell supernatant using size exclusion and cation exchange chromatography.
- Identification of IL-24 isoforms and associated serum components (albumin, C1q) via immunoblotting and SDS-PAGE.
- Assessment of IL-24 dimer function through cytokine secretion assays (TNF-alpha, IL-6) and melanoma cell line cytotoxicity assays.
Main Results:
- IL-24 forms a macromolecular complex (median 110 kDa) with various isoforms.
- Purified IL-24 dimers are N-linked glycosylated and covalently associated via disulfide bonds.
- These IL-24 dimers induced dose-dependent TNF-alpha and IL-6 secretion from human monocytes and exhibited cytotoxicity against human melanoma cell lines.
Conclusions:
- Secreted, glycosylated, dimeric human IL-24 is immunomodulatory to monocytes.
- IL-24 demonstrates tumor cell growth inhibition, supporting its dual role as a cytokine and tumor suppressor.
Abstract:
Interleukin-24 (IL-24), also known as melanoma differentiation-associated gene-7 (mda-7), is a member of the IL-10 family that exhibits both tumor suppressor and proinflammatory properties. We describe the purification of this novel dual-function tumor suppressor/cytokine from the supernatant of IL-24 gene-transfected HEK 293 cells and define the biochemical and functional properties of the soluble human IL-24 protein. Size exclusion chromatography demonstrates that an IL-24 macromolecular complex fractionates in a broad peak with a median of 110 kDa and comprises several IL-24 isoforms, identified by immunoblotting with anti-IL-24 polyclonal antibody after reducing SDS-PAGE analysis. IL-24 was found to associate with two serum components, albumin and C1q. Cation exchange purification results in the isolation of at least two N-linked glycosylated IL-24 dimers covalently associated via intermolecular disulfide bonds. These molecularly defined N-glycosylated IL-24 dimers elicited dose-dependent secretion of tumor necrosis factor-alpha (TNF-alpha) and IL-6 from human monocytes, as well as cytotoxicity to human melanoma cell lines. Thus, we demonstrated that the secreted, glycosylated, dimeric, human IL-24 is immunomodulatory to monocytes and exhibits tumor cell growth inhibition.
