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The antitumor effects of IFN-α are abrogated in a STAT1-deficient mouse
Gregory B. Lesinski, … , Joan Durbin, William E. Carson III
Gregory B. Lesinski, … , Joan Durbin, William E. Carson III
Published July 15, 2003
Citation Information: J Clin Invest. 2003;112(2):170-180. https://doi.org/10.1172/JCI16603.
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Article Oncology Article has an altmetric score of 3

The antitumor effects of IFN-α are abrogated in a STAT1-deficient mouse

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Abstract

IFN-α activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-α exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-α–stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-α treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-α. In contrast, STAT1–/– mice could not utilize exogenous IFN-α to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1–/– mice was identical regardless of treatment (IFN-α or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-α. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-α in this experimental system.

Authors

Gregory B. Lesinski, Mirela Anghelina, Jason Zimmerer, Timothy Bakalakos, Brian Badgwell, Robin Parihar, Yan Hu, Brian Becknell, Gerard Abood, Abhik Ray Chaudhury, Cynthia Magro, Joan Durbin, William E. Carson III

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Figure 4

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Reconstitution of functional STAT1 in the AGS-1 cell line. (a) Immunoblo...
Reconstitution of functional STAT1 in the AGS-1 cell line. (a) Immunoblot analysis of lysates from AGS-1STAT1 cells using an anti-STAT1 antibody indicated that expression of STAT1 protein was restored. Ctrl, control. (b) The ability of STAT1 protein from AGS-1STAT1 cells to undergo phosphorylation at Tyr-701 was confirmed by immunoblotting with an anti-P-STAT1 monoclonal antibody. (c) Overnight stimulation with IFN-α (103 U/ml) resulted in enhanced H2k expression on AGS-1STAT1 but not AGS-1MSCV melanoma cells. Shaded histograms represent fluorescence intensity of H2k-PE staining in IFN-α–stimulated EGFP-positive cells. White histograms represent H2k-PE staining in PBS-stimulated EGFP-positive cells. Determination of H2k-positive cells was based on isotype control antibodies (M1), which fell within the first log of fluorescence. (d) IFN-α inhibits proliferation of the AGS-1STAT1 cell line in a dose-dependent manner. In contrast, the antiproliferative effects of IFN-α were not evident in AGS-1MSCV cells. Results represent the mean of triplicate wells and are expressed as optical density at 570 nm ± SE. (e) The ability of the AGS-1STAT1 cell line to regulate ISGF3 gene transcription in response to IFN-α was confirmed by PCR. Total cellular RNA was isolated from cell lines (B16F1, AGS-1, AGS-1STAT1, and AGS-1MSCV) after overnight treatment with 104 U/ml of murine IFN-α and converted to cDNA using standard methods. PCR primers specific for muISG-54 detected increased ISG-54 expression in only the B16F1 (positive control) and AGS-1STAT1 cell lines after IFN-α treatment. Primers specific for 28s rRNA (housekeeping gene) were used as controls.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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