Interleukin-2 Functions in Anaplastic Large Cell Lymphoma Cells through Augmentation of Extracellular Signal-Regulated Kinases 1/2 Activation

Abstract

In addition to intrinsic genetic alterations, the effects of the extrinsic microenvironment also play a pathological role in cancer development. Altered chemokine/cytokine networks in the tumor microenvironment may contribute to the dysregulation of cellular functions in cancer cells. Anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma caused by abnormal expression of anaplastic lymphoma kinase due to a chromosomal translocation. Notably, ALCL cells are also characterized by high-level expression of the high-affinity IL-2 receptor subunit CD25 on the cell surface. However, whether the IL-2/IL-2 receptor functions in ALCL cells and how this signaling affects the tumor remain unclear. In this study, we treated cultured ALCL cells with exogenous IL-2 and examined changes in cellular function and signaling pathways. IL-2 stimulated cell growth and augmented activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Additionally, IL-2 enhanced lymphoma cell survival by overcoming kinase inhibitor U0126-induced growth arrest and apoptosis. Subsequently, to identify the potential source of IL-2 for lymphoma cells in vivo, we performed gene expression and immunochemical analyses. RT-PCR revealed no IL-2 gene expression in cultured ALCL cells and ruled out the possibility of an IL-2 autocrine loop. Interestingly, immunostaining of lymphoma tumor tissues showed IL-2 protein expression in background cells within tumor tissue, but not in ALCL cells. Our findings demonstrate that IL-2 signaling plays a functional role in ALCL cells, and enhances lymphoma cell survival by increasing activation of the ERK1/2 pathway.

Keywords: CD25/IL-2 receptor; IL-2 signaling; anaplastic large cell lymphoma (ALCL); extracellular signal-regulated kinases (ERK1/2); tumor microenvironment.

Figure 1

ALCL tumor cells express a high level of CD25, a subunit of high-affinity IL-2 receptors. The formalin-fixed and paraffin-embedded lymphoma tumor tissues from two individual patients, ALCL tumor-1 and ALCL tumor-2, were evaluated for CD25 expression by immunohistochemical studies. (a) and (c), H&E stain of ALCL tumor tissues. (b) and (d) High-level CD25 expression detected in ALCL tumor cells. (e-g) Flow cytometric analysis detected high levels of expression of surface CD25 in ALCL cell lines (Karpas 299 and SUDHL-1 cells) but not in Jurkat cells, a human T leukemia/lymphoma cell line.

Figure 2

The presence of exogenous IL-2 enhanced ALCL cell growth. To reduce potential effects of serum-derived factor(s), the cells were cultured in medium containing 0.5 and 5% FCS. The cultured ALCL cells, Karpas 299 (a) and SUDHL-1 cell lines (b), were treated with exogenous IL-2 at different concentration as indicated. After culture for 5 days, the relative cell growth rates were calculated by comparing viable cell numbers in each condition and using untreated cultures as a baseline control. As a control, the known CD25-negative Jurkat and RPMI 8226 cell lines (c) were treated under the same conditions and the relative cell growth rates were examined as described above. The experiments were repeated three times and results analyzed by Student’s t-test: **p<0.01 versus controls.

Figure 3

IL-2 augmented phosphorylation/activation of cellular ERK1/2. (a) Western blotting showed that the presence of IL-2 (40 units/ml) in cultures significantly augmented the activation of cellular p-ERK1/2 in Karpas 299 and SUDHL-1 cells but exerted no effect on the protein expression of NPM-ALK as an indicator of equal protein loading. (b) Exposure of Karpas 299 cells to the kinase inhibitor U0126 (5 μM) resulted in complete inhibition of the constitutive ERK1/2 phosphorylation (lanes 1 and 2 of upper panel). IL-2 treatment overcame this U0126-induced decrease of ERK1/2 phosphorylation (lanes 2 and 4 of upper panel). Total ERK1/2 protein levels showed no change in the presence of U0126 and/or IL-2 treatments (lower panel). (c) The similar results were also observed with SUDHL-1 cells under the same treatments. The results are representative of three separate experiments with similar findings.

Figure 4

IL-2 regulated ALCL cell growth and apoptosis. To downregulate the ERK1/2 pathway, cultured Karpas 299 (a) and SUDHL-1 cells (b) were exposed to U0126 (5 μM) in the presence or absence of IL-2 (40 units/ml) as indicated. After culture for 36 hours, the number of viable cells in each condition was counted. The presence of exogenous IL-2 significantly reversed the U0126-induced growth arrest of ALCL cells. The treated cells were also stained with FITC-conjugated annexin V, and the apoptotic rate (%) was determined by flow cytometry. The presence of exogenous IL-2 almost completely suppressed the U0126-induced apoptosis of Karpas 299 (c) and SUDHL-1 cells (d). *p<0.05 and **p<0.01 versus controls.

Figure 5

IL-2 protein expressed in background cells of lymphoma tumors, but not in the ALCL cells. (a) Cultured Karpas 299 and SUDHL-1 cells were stimulated with 50 ng/ml of ConA or 10 nM of PMA for 3 hours. As a positive control, Jurkat cells, a human T-cell leukemia/lymphoma cell line known to produce IL-2, were utilized. Cellular IL-2 gene expression was examined by RT-PCR and gel electrophoresis. An 89-bp DNA fragment of the IL-2 gene sequence was detected in Jurkat cells, and this amount was markedly increased by ConA or PMA stimulation (lanes 7-9). In contrast, no IL-2 gene expression was detected in the cultured Karpas 299 and SUDHL-1 cells with or without stimuli (lanes 1-6). As an internal control of equal RNA loading, amplification of GAPDH gene is shown in the lower panel. The results are representative of three separate experiments with similar findings. (b) Immunostaining for IL-2 protein in the cultured ALCL cells was performed on cell block sections as described in “Materials and Methods.” Neither Karpas 299 nor SUDHL-1 cells produced IL-2 protein (100× magnifications). (c) To investigate the potential tissue source of IL-2 within tumor sites, immunostaining for IL-2 protein was performed on formalin-fixed and paraffin-embedded ALCL tumor tissue. IL-2 protein expression was detected in small background cells (solid arrowheads) of lymphoma tissues but not in the large ALCL cells themselves (open arrows) viewed at 100× (left) and 1000× (right) magnifications. (d) To detect any T cells present, the ALCL tumor tissue was stained for CD30 and CD3 separately. The admixed T cells were strongly positive for CD3 (solid arrowheads). In contrast, ALCL tumor cells were not stained for CD3 (open arrows), but were strongly positive for CD30.