Two Il2 Pathways In Carcinoma Cells

The opposite effects of endogenous and exogenous IL-2 in carcinoma cells deserve special attention. While the endogenous IL-2 at pM concentrations benefits the tumour cell, enhancing its proliferation, the exogenous ligand, working at nM or f*M concentrations, disrupts the cell cycle by increasing expression of p27, a CDK inhibitor, and arresting the cell cycle in the G1/S phase of growth. As suggested by the schema presented in Figure 13, exogenous IL-2 at nM or i.M concentrations binds to intermediate affinity IL-2R ( >,') and, since substantial numbers of these receptors are present on the tumour cell surface (4, 26), the ligand may be able to cross-link them. As a result, a negative signal is delivered to the cell, and CCA leads to growth inhibition. In contrast, endogenous IL-2 may interact with the IL-2R chains present in the cytoplasm, as it leaves the Golgi. The IL-2/IL-2R complexes are either transported to the cell surface and/or to the nucleus. Signaling associated with this intracrine IL-2 utilization leads to cellular proliferation (Figure 13).

Our data indicate that two IL-2 pathways might exist in carcinoma cells: an exocrine pathway resulting in the growth inhibition and an intracrine pathway promoting tumour growth. This implies that exogenous IL2 internalized by the tumour cell and endogenous IL2 synthesized by it would be found in distinct cellular compartments: in endosomal vesicles and in the Golgi, respectively. Indeed, as shown in Figure internalized exogenous (labeled) IL-2 is detected in the cytoplasm of tumour cells and is distributed similarly to IL-2R p or y chains (Figure 14 1> and ij. In contrast, endogenous IL-2 was localized in the Golgi by confocal microscopy (Figure 15a).

Furthermore, double staining for IL-2Rp and the endocytic compartment marker, rab7, followed by confocal microscopy showed that rab7 and IL chain co-localized to late endosomes in PCI-1 tumour cells (Fig 15 n). Together, these observations suggest that internalized IL-2 and the [5 and y chains of IL-2R are sorted toward the degradation pathway, similar to the intracellular fate of these chains in T lymphocytes, as described in the literature (41). The a chain of the IL-2R has been reported to recycle to the plasma membrane in T lymphocytes (41), and our preliminary evidence suggests that its fate is similar in carcinoma cells.

Figure 13. A schema depicting the hypothesized interactions between exogenous IL-2, endogenous IL-2 and their receptors in a carcinoma cell.

Figure 14. Localization of exogenous 1L-2 in tumour cells: immunostaining of permeabilized PCI-13 ceils following the uptake of labeled (red) exogenous IL-2 is shown in (a). Localization of IL-2R chains in tumour cells in (b), and (c). Immunostaining with specific Abs shows localization of IL-2Rp or y chains, respectively, to the cytoplasmic compartment in PCI-13 cells. Original magnification x 1000.

IL-2 R beta

Rab-7

Figure 15. Localization of endogenous IL-2 to the Golgi in PCI-13 cells. Following immunostaining of permeabilized tumor cells with labeled Abs to IL-2, confocal microscopy was performed (a). In (b), co-localization of of rab7 Ab(green) and anti-IL-2R[3 Ab (red) to late endosomal vesicles. Confocal microscopy was performed after PCI-13 cells were incubated in the presence of 200 pM of exogenous IL-2 and 50 jiM of cycloheximide, then fixed and permeabilized. IL-2R(3 was detected using TU27 mAB and localized to late endosomes visualized with FITC-labeled anti-rab7 Ab, which marks late endosomal vesicles. Original magnification xlOOO in a and x2000 in b.

IL-2 R beta

Rab-7

Figure 15. Localization of endogenous IL-2 to the Golgi in PCI-13 cells. Following immunostaining of permeabilized tumor cells with labeled Abs to IL-2, confocal microscopy was performed (a). In (b), co-localization of of rab7 Ab(green) and anti-IL-2R[3 Ab (red) to late endosomal vesicles. Confocal microscopy was performed after PCI-13 cells were incubated in the presence of 200 pM of exogenous IL-2 and 50 jiM of cycloheximide, then fixed and permeabilized. IL-2R(3 was detected using TU27 mAB and localized to late endosomes visualized with FITC-labeled anti-rab7 Ab, which marks late endosomal vesicles. Original magnification xlOOO in a and x2000 in b.

The presence of both the receptor chains and IL-2 in the cytoplasm of tumour cells, as demonstrated by immunostaining with specific Abs and confocal microscopy (Figures 14 and 15), and of receptor-bound IL-2 detectable by flow cytometry on the cell surface, as previously described (32, 36, 38), suggests that endogenous IL-2 might bind to the receptor inside the cell, and that the IL-2 receptor complex is then transported to the cell surface. As indicated earlier, no IL-2 is secreted by tumour cells, as none can be detected even in concentrated supernatants of these cells.

The existence of interactions between the endogenous and exogenous IL-2 pathways is possible and is under investigation in our laboratories. These interactions might be regulated at the level of the receptor (Figure 13) both at the cell surface and inside the cell. One possibility is that exogenous IL-2 interferes with the cell cycle progression and overrides the endogenous IL-2 pathway at the level of p27 CDK inhibitor. It is also possible that the competition for the available IL-2R exists between exogenous and endogenous IL-2. These and other mechanisms are currently under study in hope of elucidating the complexity of interactions between the two IL-2 pathways.

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