STAT3 is a transcription factor that regulates various cellular processes with oncogenic potential, thereby promoting tumorigenesis when activated uncontrolled. STAT3 activation is mediated by its tyrosine phosphorylation, triggering dimerization and nuclear translocation. STAT3 also contains a serine phosphorylation site, with a postulated regulatory role in STAT3 activation and G2/M transition. Interleukin-6, a major activator of STAT3, is present in elevated concentrations in uveal melanomas, suggesting contribution of dysregulated STAT3 activation to their pathogenesis. Here, we studied the impact of chelidonine on STAT3 signaling in human uveal melanoma cells. Chelidonine, an alkaloid isolated from Chelidonium majus, disrupts microtubules, causes mitotic arrest and provokes cell death in numerous tumor cells. According to our flow cytometry and confocal microscopy data, chelidonine abrogated IL-6-induced activation and nuclear translocation, but amplified constitutive serine phosphorylation of STAT3. Both effects were restricted to a fraction of cells only, in an all-or-none fashion. A partial overlap could be observed between the affected subpopulations; however, no direct connection could be proven. This study is the first proof on a cell-by-cell basis for the opposing effects of a microtubule-targeting agent on the two types of STAT3 phosphorylation.

The heterodimeric receptor complex of IL‐9 consists of the cytokine‐specific α‐subunit and the common γ_c‐chain shared with other cytokines, including IL‐2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL‐9 and IL‐2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL‐9 and IL‐2 receptors. Complementing Pearson correlation data with co‐occurrence analysis of confocal microscopic images revealed that a minimum degree of IL‐9R/IL‐2R co‐localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL‐9/IL‐2R system. Binding of IL‐9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL‐9R and IL‐2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines. © 2018 International Society for Advancement of Cytometry

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that regulates a broad spectrum of genes with oncogenic potential, thereby serving as a critical driver of tumorigenesis. Canonical STAT3 function is mediated through tyrosine phosphorylation, which enables dimerization and transcriptional activation, whereas serine phosphorylation of STAT3 has a postulated role in fine-tuning canonical functions and contributes to non-canonical roles as well. One of the transcriptional targets of STAT3 is the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, itself subject to phosphorylation-dependent regulation. In this study, we investigated the effect of chelidonine, an alkaloid component of Chelidonium majus L., on STAT3/Bcl-2 signaling in human T leukemia/lymphoma cells, reported to have numerous effects in common with microtubule-targeting agents (MTAs). Flow cytometry and confocal microscopy revealed that chelidonine transiently increased both serine-phosphorylated STAT3 (pSer-STAT3) and Bcl-2 levels in a distinct subpopulation of cells, with near-complete overlap between the affected cells. This effect appeared at least partially independent of interleukin-2 (IL-2) and was associated with the M-phase of the cell cycle, as indicated by enhanced phosphorylation of Bcl-2 at serine 70 and nuclear morphology characteristic of mitosis. Our study provides the first single-cell evidence that STAT3 and Bcl-2 undergo concurrent serine phosphorylation as a response to chelidonine treatment, with the effect tightly linked to the M-phase.

The bipartite spatial relationship of interleukin-9 receptor (IL-9R) with IL-2R and membrane areas containing the major histocompatibility complex is revealed by using confocal microscopy, as described by L. Mátyus et al. on p. 3969. This relationship may affect the signaling capacity of IL-9R in human T cells.