Multiparametric Flow Cytometric Analysis of Inter-Patient Variation in STAT1 Phosphorylation Following Interferon Alfa Immunotherapy

Lesinski, Gregory B.; Kondadasula, Sri Vidya; Crespin, Tim R.; Shen, Lei; Kendra, Kari; Walker, Michael J.; Carson, William E.

doi:10.1093/jnci/djh252

Cited by 71 publications

(75 citation statements)

References 30 publications

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“…5E). As had been noted in previous studies, each patient exhibited a unique dose-response curve for IFN-a -induced P-STAT1 in PBMCs (22). A paired sample t test revealed that mean levels of P-STAT1 were significantly higher in patient PBMCs following stimulation with 10 3 and 10 4 units/mL IFN-a (P = 0.046 and 0.038, respectively; n = 6) than in the corresponding tumor cells.…”

Section: Resultssupporting

confidence: 69%

“…Total levels of intracellular STAT1, STAT2, and Tyr 701 P-STAT1 were measured by flow cytometry as described previously (22). Data were expressed as specific fluorescence (Fsp = Ft -Fb), where Ft represents the median value of total staining and Fb represents the median value of background staining with an isotype control antibody (22).…”

Section: Methodsmentioning

confidence: 99%

“…Lysates were prepared from normal PBMCs, melanoma cell lines, or melanoma cells derived from patients and assayed for the expression of Jak-STAT proteins by immunoblot as described previously (22,24) with antibodies to STAT1, Jak1, IRF9, Tyk2, STAT5 (BD Biosciences), STAT2 (Biosource International), IFNAR (R&D Systems), STAT3, P-STAT3 (Cell Signaling Technology), or h-actin (Sigma).…”

Section: Methodsmentioning

confidence: 99%

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Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

Lesinski

Trefry

Brasdovich

et al. 2007

Clinical Cancer Research

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Purpose: IFN-a is administered to melanoma patients and its endogenous production is essential for immune-mediated tumor recognition. We hypothesized that a reduced capacity for signal transducer and activator of transcription (STAT) 1activation allows melanoma cells to evade the direct actions of IFN-a. Experimental Design: Tyr 701 -phosphorylated STAT1 (P-STAT1) was measured by flow cytometry in IFN-a^stimulated human melanoma cell lines, melanoma cells derived from patient tumors, and peripheral blood mononuclear cells (PBMC). Expression of other Janusactivated kinase (Jak)-STAT intermediates (STAT1, STAT2, Jak1, tyrosine kinase 2, IFN-a receptor, STAT3, and STAT5) was evaluated by flow cytometry, immunoblot, or immunohistochemistry. Results: Significant variability in P-STAT1was observed in human melanoma cell lines following IFN-a treatment (P < 0.05) and IFN-a^induced P-STAT1 correlated with the antiproliferative effects of IFN-a (P = 0.042). Reduced formation of P-STAT1 was not explained by loss of Jak-STAT proteins or enhanced STAT5 signaling as reported previously. Basal levels of P-STAT3 were inversely correlated with IFN-a^induced P-STAT1in cell lines (P = 0.013). IFN-a^induced formation of P-STAT1was also variable in melanoma cells derived from patient tumors; however, no relationship between P-STAT3 and IFN-a^induced P-STAT1 was evident. Because IFN-a acts on both tumor and immune cells, we examined the ability of IFN-a to induce P-STAT1 in patient-derived melanoma cells and PBMCs. IFN-a induced significantly lower levels of P-STAT1 in melanoma cells compared with matched PBMCs (P = 0.046). Melanoma cells and human melanocytes required 10-fold higher IFN-a doses to exert P-STAT1 levels comparable with PBMCs. Conclusions: Melanoma cells are variable in their IFN-a responsiveness, and cells of the melanocytic lineage exhibit a lower capacity for IFN-a^induced Jak-STAT signaling compared with immune cells.

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Section: Resultssupporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

Lesinski

Trefry

Brasdovich

et al. 2007

Clinical Cancer Research

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“…The commonly found dysfunction of NK cells in breast cancer patients [120][121][122] is probably the consequence of cytokine dysbalance due to the prevalence of immunosuppressive cytokines such as IL-10 and TGF [123], as well as tumor-produced inhibitory factors [124]. This finding is in concordance with the only previous study published for breast cancer patients [125] and also with several other investigations showing STAT dysregulation in PBL of melanoma and renal cell carcinoma patients [126,127]. Moreover, we showed that breast cancer patients' T and NK cell subsets have lower pSTAT1 level that could be a biomarker of decreased NK cell cytotoxicity and IFN production associated with progression of this disease [120,128,129].…”

Section: Stat Dysfunction Associated With Different Malignanciessupporting

confidence: 87%

STAT Transcription Factors in Tumor Development and Targeted Therapy of Malignancies

Konjević¹,

Radenković²,

Vuletić³

et al. 2013

Oncogene and Cancer - From Bench to Clinic

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“…In fact, recent data have shown that the occurrence of autoimmune sequelae and the presence of tumor-infiltrating lymphocytes correlate with clinical response in patients receiving IFN-a (23,24). Our previous analysis of IFN-a -induced JAK-STAT signal transduction in immune cells revealed reduced phosphorylation of these proteins at higher doses of IFN-a (25). We therefore hypothesized that intermediate and high doses of IFN-a might be equally effective in activating STAT proteins and inducing the expression of ISGs in patient peripheral blood mononuclear cells (PBMC).…”

mentioning

confidence: 93%

IFN-α-2b–Induced Signal Transduction and Gene Regulation in Patient Peripheral Blood Mononuclear Cells Is Not Enhanced by a Dose Increase from 5 to 10 Megaunits/m2

Zimmerer

Lehman²,

Ruppert³

et al. 2008

Clinical Cancer Research

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Multiparametric Flow Cytometric Analysis of Inter-Patient Variation in STAT1 Phosphorylation Following Interferon Alfa Immunotherapy

Abstract: This flow cytometry method can be used to monitor STAT1 activation within subsets of immune cells from patients undergoing IFN alpha immunotherapy.

Cited by 71 publications

References 30 publications

Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

Melanoma Cells Exhibit Variable Signal Transducer and Activator of Transcription 1 Phosphorylation and a Reduced Response to IFN-α Compared with Immune Effector Cells

STAT Transcription Factors in Tumor Development and Targeted Therapy of Malignancies

IFN-α-2b–Induced Signal Transduction and Gene Regulation in Patient Peripheral Blood Mononuclear Cells Is Not Enhanced by a Dose Increase from 5 to 10 Megaunits/m2

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