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Impaired alpha-interferon signaling in transitional cell carcinoma: lack of p48 expression in 5637 cells
S F Matin1, R R Rackley, P C Sadhukhan
1Department of Urology, Lerner Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Abstract:
The limited success of IFN-alpha therapy for clinical treatment of transitional cell carcinoma (TCC) has prompted us to investigate the responsiveness of TCC lines to IFN-alpha. The response to IFN-alpha in terms of 561 gene induction, an IFN-stimulated response element-containing IFN-alpha/beta-inducible gene, and IFN-stimulated gene factor 3 (ISGF3) formation was normal in primary human urothelial cells. We tested the antiproliferative effects of IFN-alpha in three TCC lines as a measure of IFN-alpha responsiveness, and variable patterns of growth inhibition were observed in three TCC lines. More than 90% growth inhibition was noted in TCCSUP cells, whereas only 40% and 10% inhibition by IFN-alpha was observed in 5637 and HT1197 cells, respectively. IFN-alpha treatment formed extremely low levels of ISGF3 in electrophoretic mobility shift assays in these later two relatively insensitive cells. In addition, expression of the 561 gene was significantly reduced in these two TCC lines by Northern blots. We have further identified a low expression level of Tyk2 in HT1197 cells compared with two other TCCs. This suggests that an extremely low ISGF3 level after IFN-alpha treatment may be due to low Tyk2 expression or other unidentified defects. In 5637 cells, p48 protein expression was undetectable. This undetectable p48 expression is not due to a deletion in the coding region because the correct size protein is detected following IFN-gamma treatment. Consequently, the ISGF3 complex formation and 561 gene induction were restored by IFN-gamma pretreatment plus IFN-alpha treatment. Introduction of p48 expressing plasmid into 5637 cells was sufficient to form the ISGF3 complex by IFN-alpha treatment, suggesting the defect lies in the expression of p48 protein in 5637 cells. Detailed mechanistic understanding of the action of IFNs in bladder cancer cell lines may explain the abrogated therapeutic response of IFN-alpha in the clinical treatment of TCCs.
Insights
Interferon-alpha (IFN-alpha) shows limited success in treating bladder cancer (transitional cell carcinoma, TCC). This study investigates TCC cell line responsiveness to IFN-alpha, identifying defects in signaling pathways that hinder treatment efficacy.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- Interferon-alpha (IFN-alpha) therapy has shown limited clinical success in treating transitional cell carcinoma (TCC).
- Understanding the molecular mechanisms of IFN-alpha responsiveness in TCC is crucial for improving therapeutic outcomes.
Purpose of the Study:
- To investigate the responsiveness of TCC cell lines to IFN-alpha.
- To identify potential molecular defects in IFN-alpha signaling pathways within TCC cells.
Main Methods:
- Assessed IFN-alpha responsiveness by measuring 561 gene induction and IFN-stimulated gene factor 3 (ISGF3) formation.
- Evaluated the antiproliferative effects of IFN-alpha on three TCC lines (TCCSUP, 5637, HT1197).
- Utilized electrophoretic mobility shift assays and Northern blots to analyze ISGF3 formation and gene expression.
- Investigated the role of Tyk2 and p48 protein expression in IFN-alpha signaling defects.
Main Results:
- Variable antiproliferative responses to IFN-alpha were observed across TCC lines, with TCCSUP showing >90% inhibition and HT1197 showing 10% inhibition.
- TCC lines with lower sensitivity exhibited significantly reduced ISGF3 formation and 561 gene expression upon IFN-alpha treatment.
- Defects identified include low Tyk2 expression in HT1197 cells and undetectable p48 protein expression in 5637 cells.
- Restoration of ISGF3 complex formation and 561 gene induction was achieved in 5637 cells by IFN-gamma pretreatment or p48 plasmid introduction.
Conclusions:
- Specific defects in IFN-alpha signaling pathways, such as low Tyk2 or p48 expression, contribute to the limited responsiveness of certain TCC cell lines.
- Understanding these molecular aberrations may provide insights into the abrogated therapeutic response of IFN-alpha in clinical TCC treatment.
- Targeting these specific signaling defects could potentially enhance the efficacy of IFN-alpha-based therapies for bladder cancer.