[show abstract][hide abstract] ABSTRACT: IFN-alpha 2b (IFN-alpha) has been used to treat patients with metastatic malignant melanoma and patients rendered disease-free via surgery but at high risk for recurrence. We hypothesized that IL-12 pretreatments would result in endogenous IFN-gamma production, and that this, in turn, would up-regulate levels of Janus kinase-STAT signaling intermediates and lead to increased expression of genes regulated by IFN-alpha. Treatment of PBMCs with IL-12 stimulated a significant and dose-dependent production of IFN-gamma. Pretreatment of PBMCs and tumor cells with IFN-gamma-containing supernatants from IL-12-stimulated PBMCs led to up-regulation of STAT1, STAT2, and IFN regulatory factor 9 (IRF9) and potentiated IFN-alpha-induced STAT signaling within PBMCs and tumor cells. These effects were abrogated by neutralization of IFN-gamma in the PBMC supernatants with an anti-IFN-gamma Ab. Pretreatment of HT144 melanoma cells and PBMCs with IFN-gamma or IFN-gamma-containing supernatants enhanced the actions of IFN-alpha at the transcriptional level, as measured by real-time RT PCR analysis of the IFN-stimulated gene 15. Experiments in wild-type C57BL/6 and IFN-gamma receptor knockout (B6.129S7-Ifngr(tm1Agt)) mice demonstrated that a regimen of IL-12 pretreatment, followed by IFN-alpha, could cure mice of i.p. B16F1 melanoma tumors (p < 0.007), whereas mice treated with either agent alone or PBS succumbed to fatal tumor burden. However, this treatment regimen did not significantly prolong the survival of IFN-gamma-deficient (B6.129S7-Ifng(tm1Ts)) mice compared with mice treated with IFN-alpha alone. These results suggest that the response to IFN-alpha immunotherapy can be significantly enhanced by IL-12 pretreatment, and this effect is dependent upon endogenous IFN-gamma production and its actions on melanoma cells.
The Journal of Immunology 06/2004; 172(12):7368-76. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: IntroductionInterferon-alpha (IFN-α) is currently administered to patients with metastatic malignant melanoma and those who are at risk for recurrence following surgery for high-risk lesions. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is activated by IFN-α and is thought to mediate the majority of its antitumor effects. Loss of STAT1 has been found in IFN-resistant melanoma cells. We developed a murine melanoma cell line in a STAT1-deficient mouse. We also transfected B16 melanoma cells with a wild-type form of STAT1 to induce its overexpression. Using the resulting cell lines and STAT1-deficient mice, we tested whether IFN-α could exert an antitumor effect on melanoma cells in the absence of STAT1-mediated signal transduction.
Journal of Surgical Research - J SURG RES. 01/2004; 116(1):129-136.
[show abstract][hide abstract] ABSTRACT: Interferon-alpha (IFN-alpha) is currently administered to patients with metastatic malignant melanoma and those who are at risk for recurrence following surgery for high-risk lesions. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is activated by IFN-alpha and is thought to mediate the majority of its antitumor effects. Loss of STAT1 has been found in IFN-resistant melanoma cells. We developed a murine melanoma cell line in a STAT1-deficient mouse. We also transfected B16 melanoma cells with a wild-type form of STAT1 to induce its overexpression. Using the resulting cell lines and STAT1-deficient mice, we tested whether IFN-alpha could exert an antitumor effect on melanoma cells in the absence of STAT1-mediated signal transduction.
A melanoma tumor was induced in STAT1-deficient mice via the application of DMBA (tumor initiator) followed by croton oil (tumor promoter). Immunohistochemical analysis confirmed that the resulting tumor was a malignant melanoma. Immunoblot analysis, intracellular flow cytometry, and gel-shift analysis were used to confirm the lack of STAT1 in the derivative cell line (AGS-1). In addition, the STAT1 protein was overexpressed in B16 melanoma cells by stable transfection with a plasmid construct encoding wild-type STAT1. The effects of IFN-alpha on these cell lines were studied in vitro and in vivo.
STAT1 was not expressed in the AGS-1 murine melanoma cell line. Treatment with IFN-alpha did not lead to activation of STAT1. Cell proliferation assays revealed that while IFN-alpha did not exert an antiproliferative effect on this cell line, it was capable of prolonging the survival of STAT1-competent C57BL/6 mice bearing 1 x 10(6) AGS-1 tumor cells in the intraperitoneal position (n = 20, P < 0.05), as compared to PBS-treated controls. Also, the survival of IFN-alpha-treated mice (as compared to PBS-treated controls) was not affected by the overexpression of STAT1 in B16 tumor cells.
This data suggests that IFN-alpha can enhance survival in an animal model where STAT1-mediated signal transduction and gene regulation is absent within the tumor but is present within the host. This data also indicates that the overexpression of STAT1 within the tumor does not significantly enhance the effects of exogenously administered IFN-alpha in this model. These findings indicate that the bulk of the antitumor actions of IFN-alpha may be derived from its effects on host tissues.
Journal of Surgical Research 01/2004; 116(1):129-36. · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: IFN-alpha activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-alpha 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-alpha-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-alpha treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-alpha. In contrast, STAT1-/- mice could not utilize exogenous IFN-alpha 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-alpha or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-alpha. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-alpha in this experimental system.
Journal of Clinical Investigation 08/2003; 112(2):170-80. · 12.81 Impact Factor