Il2 In Human Carcinomas

3.1 Discovery of endogenous IL-2 in human carcinomas

While performing immunoperoxidase (IP) staining on carcinoma cell lines and tissue sections of human tumours, we observed what appeared to be specific although weak staining for IL-2 in these cells. Upon more careful examination, all carcinoma cells grown in chamber slides were found to constitutively express IL-2 in the absence of any exogenous IL-2 in TCM (Figure 7). These cells showed a characteristic-staining pattern, with immunoreactive IL-2 localized to a circumscribed area of the cytoplasm, corresponding to the Golgi zone (Figure 7a-c). Immunostaining with FITC-labeled Abs to the Golgi complex and PE-and PE labeled Abs toIL-2 demonstrated co-localization of these two cellular targets (Figure 7c). PHA-stimulated Jurkat cells or Con-A-activated normal T lymphocytes used as controls also showed the same staining pattern (36, 38). Normal human keratinocytes were positive for IL-2, but stained weakly compared to tumour cells or activated T cells (36, 38).

While IL-2 protein was detectable by immunostaining in permeabilized tumour cells, it was not detectable in cellular supernatant of carcinoma cells tested by ELISA or in biologic assays, using an IL-2-dependent CTLL line. Minimal levels of IL-2 were shown to be present in these supernatants (39, 40). However, carcinoma cells disrupted by cycles of rapid freezing and thawing in the presence of protease inhibitors yielded cytosols containing 4060 pg/m)/ 5x 10G of IL-2, as determined in IL-2-specific ELISA (32). Western blots of tumour cell lysates confirmed the presence of IL-2 protein in carcinoma cells (Figure 8). These data indicated that tumour cells produced endogenous IL-2, which was either not secreted or was secreted only at levels not detectable by immunoassays. At the same time, the presence of IL-2 on the cell surface of variable proportions of tumour cells was confirmed by flow cytometry (32, 38). We interpret this finding as evidence for existence of an autocrine IL-2 pathway in carcinoma cells. Endogenous IL-2 is transported from the cytokine to the cell surface, where it occupies IL-2R and signals via the P component of the high affinity IL-2R.

To confirm the presence of mRNA for endogenous IL-2 in tumour cells, RT-PCR was performed, followed by Southern blots with IL-2 cDNA. In tumour cell lines and lymphoid cells used as positive controls, mRNA for IL-2 was consistently detected, although semiquantitative analyses showed that the number of copies of the transcript was considerably lower in tumour cells than that in Jurkat T cells (38). By in situ hybridization (ISH) for IL-2 mRNA, we have also shown that our cell lines were positive for its expression (Figure 9; ref 32).

Figure 1. A HNC cell line (PCI-13) was cultured in a monolayer, and the tumour cells were fixed, permeabilized and stained with PE-!abeied Ab to human IL-2 (a) and FITC-labeled Abs to the Golgi complex (b). Co-localization of IL-2 with the Golgi complcx is evident in c. Original magnification x 1000.

Figure 1. A HNC cell line (PCI-13) was cultured in a monolayer, and the tumour cells were fixed, permeabilized and stained with PE-!abeied Ab to human IL-2 (a) and FITC-labeled Abs to the Golgi complex (b). Co-localization of IL-2 with the Golgi complcx is evident in c. Original magnification x 1000.

Figure 8. Western blots of tumour cell lysates and of human recombinant IL-2 used as a positive control. Anti-human H,-2 Ab was used to visualize the 14.4 kDa band. Recombinant IL-2 and lysates of YT, an NK cell line, were used as positive controls.

IL-2 expression in tissue biopsies of OSCC tumour cells and normal oral mucosa was evaluated next by immunohistology (33). IL-2 Protein was found to be expressed throughout the tumour tissue, although in normal oral mucosa, IL-2 protein (or mRNA for IL-2 by ISH) was localized to the basal epithelial layer (33).

3.2 Transduction of carcinoma cells with the IL-2 gene

Carcinoma cells (PCI-50, PCI-1 and HR) were transduced with the IL-2 gene, using a retroviral vector, selected for neomycin resistance and evaluated for the ability to produce and secrete biologically-active IL-2 (39, 40). These transduced tumour cells were shown to secrete between 10 and 30 ng of IL-2 /5x!05 cells /48h (39, 40). Importantly, growth of these transduced tumour cells in culture was significantly improved over that of the parental cell lines or control tumour cells transduced with the LacZ gene (Figure 10).

The results suggested that these IL-2R+ carcinoma cells utilized the secreted IL-2 for growth. As expected, Abs to IL-2 used at the concentrations of 25 and 50 ug/ml inhibited in part proliferation of the IL-2- secreting, transduced tumour cells (36). In addition, these cells were found to be significantly less susceptible to apoptosis mediated in vitro by IL-2 activated human effector cells than the parental or LacZ control tumour cells (39). However, these seemingly advantageous properties of transduced tumour cells were not useful for the tumour growing in vivo: secreted IL-2 facilitated tumour cell destruction and/or regression of metastases by inducing accumulation in situ of antitumour effector cells (NK cells and macrophages) in the xenograft carcinoma models established in nude mice (39, 40).

3.3 The role of endogenous IL-2 in carcinoma cell cycle progression

We have observed that in monolayers of tumour cells and in human tumour tissues, IL-2 was especially strongly expressed in dividing cells (Figure 11).

To examine the possibility that endogenous IL-2 is involved in the regulation of tumour cell division, we examined its expression as well as expression of its receptors in various phases of the cell cycle in tumour cell lines. By flow cytometry and immunostaining, expression of these proteins was found to be induced in the S phase, and significantly up-regulated in the G2/M phase of the cell cycle (Figure 12).

The level of mRNA for IL-2 was 5 to 10 fold higher in the M-phase than in the (.i,, XS phase, as shown by quantitative competitive RT-PCR. When tumour cell lysates were examined in Western blots, we found that expression of the cyclin-dependent kinase (CDK) inhibitor p27klt>1 was high in the Go/G|, nearly absent in the S phase, and reappeared in the G2/M phase. In contrast, expression of p21 remained relatively unchanged during the cell cycle (38). Our results indicate that IL-2 and the 1L-2R complex behave like cell-cycle-related proteins in human carcinoma cells. Analogous to its function in T cells, endogenous IL-2 is important in regulating expression of the pi?*"11 CDK inhibitor and thus controlling cell cycle progression of tumour cells. Additional studies showed that growth of carcinoma cell lines was inhibited by the well known immunosuppressive agents, cyclosporin A, FK506 and rapamycin, similar to the effect of these drugs on the IL-2/IL-2R pathway in lymphoid cells (36).

Figure 9. In situ hybridization (ISH) for IL-2 mRNA in a human HNC cell line (PCI-1). Cytocentrifuge smears of tumour cells were used for ISH with a 35[S]-labeled IL-2 cDNA probe (32). The slides were examined by light (a and b) and dark-field (c_and d) microscopy. A negative control was obtained by pretreatment of tumour cells with RNA-se (b and d). Original magnification x 200.

Figure 9. In situ hybridization (ISH) for IL-2 mRNA in a human HNC cell line (PCI-1). Cytocentrifuge smears of tumour cells were used for ISH with a 35[S]-labeled IL-2 cDNA probe (32). The slides were examined by light (a and b) and dark-field (c_and d) microscopy. A negative control was obtained by pretreatment of tumour cells with RNA-se (b and d). Original magnification x 200.

Figure 10. Proliferation of tumor cells transduced with the IL-2 gene is significantly increased (asterisks; p<0.05) as compared to growth of parental or LacZ-transduced tumour cells. The transduced tumor cells secreted IL-2 into the supernatant (see text).

HR PCI-1 PCI-13

Figure 10. Proliferation of tumor cells transduced with the IL-2 gene is significantly increased (asterisks; p<0.05) as compared to growth of parental or LacZ-transduced tumour cells. The transduced tumor cells secreted IL-2 into the supernatant (see text).

Figure II. Expression of IL-2 in the cytoplasm of dividing tumour cells (PCI-50) is more intense than in non-dividing cells, Immuno-staining of tumour cell monolayers with anti-IL-2 Abs was performed as described (35). Original magnification x 1000

Figure 12. Flow cytometry histograms demonstrating the presence of intracellular IL-2, JL-2RP and 1L-2Ry in PCi-1 cells in different phases of the cell cycle. Tumour cells were synchronized as previously described (33), isotype control Abs were used to control for non-specific staining (white peaks).

Figure 12. Flow cytometry histograms demonstrating the presence of intracellular IL-2, JL-2RP and 1L-2Ry in PCi-1 cells in different phases of the cell cycle. Tumour cells were synchronized as previously described (33), isotype control Abs were used to control for non-specific staining (white peaks).

3.4 Correlation of IL-2 expression with cellular proliferation in SCCHN

To demonstrate that expression of IL-2 in carcinoma cells in tissue correlated with that of the proliferation-associated Ki-67 antigen, immunohistology for these proteins was performed in 34 tissue samples obtained from patients with oral carcinoma (33). Expression of IL-2 and Ki-67 in these tissues was correlated to the histological grade of the carcinomas (33). The strongest IL-2 expression was seen in tumour cells undergoing mitosis, identified by double staining with the antibody to Ki-67 protein, a marker of cellular proliferation. In the tumour tissue, the highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with the labeling index (LI) of 67.2% for IL-2 and 68.8% for Ki-67. Well-differentiated carcinomas showed a significantly lower expression of both proteins (LI = 35.0 % for IL-2 and 26.5% for Ki-67). The correlation between the labeling indices was statistically significant (R = 0.747, p < 0.001). These results demonstrate that iL-2 expression in SCCHN is strongly associated with cellular proliferation, and that endogenous IL-2 might function as a growth factor for these carcinomas (33).

3.5 Immunoprecipitation of endogenous IL-2 in carcinoma cells

In view of the data indicating that the IL-2/IL-2R pathway was active in human carcinomas and that endogenous IL-2 was involved in tumour cell proliferation, we next wished to compare endogenous IL-2 in carcinomas to that expressed in human lymphocytes. Therefore, we performed metabolic labeling with 35[S]-methionine followed by immunoprecipitation of IL-2 with specific Abs in cell lysates of SCC cell lines and Western blotting (36). Our data indicated that IL-2 produced by tumour cells and precipitated by the IL-2-specific Abs had the same electrophoretic mobility under reducing conditions as lymphoid cell-derived IL-2. The specificity of the precipitating Ab for IL-2 was confirmed by absorption with rIL-2, which almost completely eliminated the ability of this Ab to precipitate IL-2 in cellular extracts (36).

3.6 Endogenous IL-2 is essential for SCC growth

If endogenous IL-2 is required for carcinoma growth, then interference with its expression should inhibit growth. We generated IL-2 specific antisense and sense oligonucleotide phosphorothioates and, using DOTAP as a carrier, delivered them to tumour cells, attempting to disrupt the endogenous pathway. This strategy resulted in a transient loss of IL-2 protein and mRNA expression as well as significant inhibition of tumour cell growth (36). These results, together with the data indicating that endogenous IL-2 regulates expression of p27Ipl, provide compelling evidence that in tumour cells, similar to lymphocytes, endogenous IL-2 is involved in the regulation of cellular division and proliferation (37).

Was this article helpful?

0 0
Chinese Herbs

Chinese Herbs

Worried For Your Health More Than Ever? Want To PREVENT Rather Than CURE Your Illness? Then WE are the only ones who can answer YOUR concern of time by presenting the exclusive work piece, the explicit and special eBook on CHINESE HERBS- the call of time.

Get My Free Ebook


Post a comment