SYMPOSIUM PAPER |
Correspondence to: Theresa L. Whiteside, U. of Pittsburgh Cancer Institute, W 1041 Biomedical Science Tower, 211 Lothrop St., Pittsburgh, PA 15213-2582.
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Summary |
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We have previously demonstrated that interleukin-2 (IL-2) receptors, IL-2 protein, and mRNA for IL-2 are present in human carcinomas in vitro and in vivo. Carcinoma cells synchronized in the G2/M-phase of the cell cycle express significantly more intracytoplasmic IL-2 as well as IL-2R-ß and - than tumor cells in the G0/G1-phase. Here we evaluated immunohistologically the cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen and expression of the cytokine IL-2 in four different carcinoma cell lines. In addition, 34 tissue samples from patients with squamous cell carcinomas of the head and neck were simultaneously analyzed for Ki-67 and IL-2 expression and the data were correlated to the histological grade of the tumors. All tumor cell lines were shown to express IL-2 in the Golgi complex. The strongest IL-2 expression was seen in tumor cells undergoing mitosis, identified by double staining with the antibody to Ki-67. In the tumor tissue, the highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with a 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 human carcinoma tissues is strongly associated with cell proliferation and significantly correlates with the histological tumor grade. (J Histochem Cytochem 46:603611, 1998)
Key Words: , human carcinomas, interleukin-2, cell cycle, Ki-67 antigen, mitosis, tumor grade
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Introduction |
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INTERLEUKIN-2 (IL-2) was first identified in 1975 as a growth-promoting factor for bone marrow-derived T-lymphocytes (
The antigen defined by MAb Ki-67 is a human nuclear protein expressed only in proliferating cells. Ki-67 is widely used in routine pathology as a proliferation marker to measure the growth fraction of cells in human tumors (reviewed in
In the present study we investigated expression of IL-2 protein and Ki-67 antigen in four different carcinoma cell lines and in 34 tissue samples of SCCHN. Expression of Ki-67 was used to identify proliferating tumor cells in tumor tissue and tumor cells in different phases of the cell cycle in the cell lines. To confirm that endogenous production and expression of IL-2 are associated with tumor cell proliferation in head and neck cancer, expression of IL-2 was correlated to that of Ki-67 antigen.
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Materials and Methods |
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Tumor Cell Lines and Tumor Tissues
Human SCCHN cell lines (PCI-1, PCI-2, PCI-4A, and PCI-13) have been established in our laboratory, as previously described (
Thirty-four biopsy tissues obtained from surgically resected SCCHN were used to study expression of IL-2 and Ki-67 in situ. Only tissue samples above those necessary for the pathological diagnosis were used for this study. Informed consent was obtained from all patients. The tissues were snap-frozen immediately after surgical removal, embedded in OCT compound (Miles Laboratories; Naperville, IL), and maintained at -70C until analyzed.
Immunohistochemistry
Tumor cell lines were cultured in Labtek chamber slides (CMS; Houston, TX). When the cultured cells became 7080% confluent, the chamber was removed and the slides were rinsed in cold PBS (twice for 3 min). The cells were pre-fixed for 5 min in 2% paraformaldehyde (PFA), permeabilized, and fixed with 0.1% Triton X-100/2% PFA for 10 min. Cryostat sections of tumor tissues were cut in an Ames cryostat (5 µm thick), air-dried, and fixed with cold acetone for 10 min.
Tumor cell monolayers and sections of tumor tissues were immunohistologically stained, using an anti-IL-2 polyclonal Ab (Becton Dickinson; Mountain View, CA), anti-Ki-67 (MAb) MM1 (Vector Laboratories; Burlingame, CA), and anti-Golgi complex MAb (BioGenex; San Ramon, CA). Optimal working dilutions of the primary Abs (anti-IL-2 20 µg/ml, anti-Ki-67 1:200, anti-Golgi complex 1:100) were determined in preliminary titration experiments. A standard streptavidinbiotin compleximmunoperoxidase (sABC-HRP) technique was used for staining. To perform double staining, tumor monolayers were first incubated with anti-Ki-67 MAb, stained by the alkaline phosphataseanti-alkaline phosphatase (APAAP) method, and then reacted with anti-IL-2 Ab, followed by an sABC-HRP technique (
For immunofluorescence, tumor cell monolayers were incubated with anti-IL-2 Ab followed by incubation with Cy3-labeled second Ab (Jackson Immunoresearch; West Grove, PA). The monolayers were counterstained with fluoresceinphalloidin (Molecular Probes; Eugene, OR) and Hoechst dye 33342 (2 µg/ml) (Sigma; St Louis, MO).
In all experiments, isotype-specific antibodies purchased from DAKO and Sigma were used as controls. Before staining, polyclonal anti-IL-2 Ab was preincubated with an excess of rIL-2 (Chiron; Emeryville, CA) to verify its specificity. This absorption was shown to completely eliminate cytokine-specific staining. In tissue sections, endogenous peroxidase was blocked with 0.3% H2O2.
Evaluation of Immunohistochemical Staining
Tumor cell monolayers stained by immunofluorescence were examined in an Oncor Multimode Microscope. The multicolor fluorescent images were collected with a cooled Photometrics CCD-camera using discrete bandpass filters configured for FITC (phalloidin), Cy3 (IL-2), and Dapi (Hoechst dye). The three filter sets had been previously aligned to ensure perfect image registration. The three images were collected in monochrome with a 12-bit grayscale reduced to 8-bit images and superimposed using the green, red, and blue channels to create a full-color RGB image.
Tissue sections and monolayers stained with immunoperoxidase and alkaline phosphatase were evaluated in an Olympus BH-2 light microscope to determine expression of IL-2 and Ki-67 proteins. A quantitative method was used to evaluate the proportion of Ki-67 and IL-2 positively stained tumor cells in tissue sections, as follows. The areas containing the largest number of positive cells were selected and the number of positive cells per 600 tumor cells was counted in each case at x 400 magnification. Tumor cells showing a definite nuclear staining with Ki-67 Ab and tumor cells with a clear cytoplasmic staining for IL-2, the latter often characterized by a strongly stained juxtanuclear "dot," were scored as positive. The percentages of positive cells were calculated and were expressed as Ki-67 and IL-2 labeling indices (LIs).
Synchronization of Tumor Cell Lines
Serum starvation was used to synchronize tumor cells in the G0/G1-phase of the cell cycle. Tumor cell monolayers were cultured in DMEM medium with 10% FCS until they became 80% confluent. The cells were next incubated in medium without FCS for 48 hr to arrest them in G0/G1. Those cells that had passed the restriction point (
Using aphidicolin, an inhibitor of DNA polymerase- (
Synchronization of cells in the M-phase of the cell cycle was accomplished by mitotic "shake-off" of enriched cell monolayers, as described elsewhere (
Quantitative Competitive RT-PCR for IL-2
Total RNA was extracted from tumor cells or PHA-activated Jurkat cells using the standard AGPC method, followed by DNase I (Promega; Madison, WI) treatment. For reverse transcription, aliquots of the internal standard RNA prepared from pQA-1 plasmid, a gift from Dr. David Shire (Sanolfi Elf Bio Recherches; Labege, France) were used. The following oligonucleotide primers were used for amplification of the IL-2 gene:
IL-2 sense 5'-GTCACAAACAGTGCACCTAC-3'
IL-2 anti-sense5'-CCCTGGGTCTTAAGTGAAAG-3'
To analyze the expression levels of IL-2m RNA in solid tumor or lymphoid cells, quantitative competitive (QC) RT-PCR established in our laboratory was performed as previously described (
Statistical Analysis
Results are given as means ± SD. For nonparametric analysis, the KruskalWallis test was used to test the variance of Ki-67 and IL-2 LIs in tumor groups with different histological grades. Pearson's correlation coefficient was used for comparison of the Ki-67 LI with the IL-2 LI. These analyses were performed using the statistical software system SPSS Version 7.0 (SPSS; Chicago, IL).
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Results |
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Expression of IL-2 and Ki-67 in Tumor Cell Lines
To study expression of IL-2 and Ki-67 in the four tumor cell lines, we performed immunohistochemical and immunofluorescence staining of cell monolayers grown on chamber slides in the absence of any exogenous IL-2. Almost all tumor cells in these monolayers constitutively expressed endogenous IL-2, with a characteristic accumulation of the protein in a juxtanuclear area of the cell (Figure 1). This IL-2-positive area co-localized with that stained for the Golgi complex (Figure 2). Furthermore, we consistently observed especially strong staining for IL-2 in dividing tumor cells. Our initial impression was that IL-2 expression was most prominent in cells undergoing mitosis (Figure 3a). To analyze expression of IL-2 more carefully, we performed double staining for IL-2 and Ki-67, which have a characteristic topographical distribution in the different phases of the cell cycle. Tumor cells in G0-, G1- and S-phase showed the characteristic distribution of IL-2 in the Golgi complex (Figure 3b and Figure 3c). Tumor cells in G2 and mitosis exhibited increased IL-2 expression, with the strongest staining in metaphase (Figure 3d and Figure 3e). Tumor cells in telophase still showed intensive IL-2 staining (Figure 3f), but after that IL-2 expression decreased to the starting level or almost disappeared. When the tumor cell lines were synchronized, as described in Materials and Methods, the staining patterns observed in various phases of the cell cycle were confirmed for both IL-2 and Ki-67 (data not shown).
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Expression of IL-2 in Tumor and Normal Tissues
IL-2 expression in tissue biopsies of SCCHN tumor cells and normal oral mucosa was next evaluated by immunohistology. As shown in Figure 4a, normal oral mucosa was weakly positive for IL-2, and the IL-2 protein (or mRNA for IL-2 by in situ hybridization; data not shown) was most strongly expressed in the basal epithelial layer. In the tumor tissue, most tumor cells showed "dot-like" staining for IL-2 (Figure 4b). Similar staining for IL-2 was seen in cultured carcinoma cells (Figure 4c).
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To test the relevance of IL-2 expression in vivo, we studied 34 tumor biopsy samples obtained from patients with SCCHN. On the basis of the results of the cell cycle-dependent expression of IL-2 in SCCHN cell lines, attempts were made to correlate IL-2 expression with that of the proliferation marker Ki-67, which is used to measure the growth fraction of human tumors in situ. In addition, the labeling indices (LIs = percentage of positive tumor cells per total number of tumor cells) were calculated for the different histological tumor grades.
Immunostaining with the Ki-67 Ab in tumor tissues showed a definite nuclear staining pattern (Figure 5ac). IL-2 staining was similar to that seen in the tumor cell lines and was located in the cytoplasm of the tumor cells. Many of the cells contained a strongly stained juxtanuclear "dot," a characteristic staining pattern for cytokines (Figure 5df). Expression of Ki-67 and IL-2 and SCCHN of different histological grades is shown in Table 1. The mean LIs for Ki-67 and IL-2 were positively correlated with the histological grade. Well-differentiated SCCHN had low levels of Ki-67 and IL-2 expression, whereas poorly differentiated carcinomas had a high level of expression of both these proteins (Figure 5). The differences in the mean LIS between the different histological grades were statistically significant (see Table 1). A positive correlation was found between the Ki-76 LI and IL-2 LI with a correlation coefficient of 0.747 and p<0.001 (Figure 6).
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mRNA for IL-2 in Solid Tumor and Lymphoid Cells
To confirm the presence of message for endogenous IL-2 in tumor cells, quantitative competitive RT-PCR was performed, followed by Southern blots with IL-2 cDNA (Figure 7). In tumor cell lines and lymphoid (Jurkat) cells used as a positive control, mRNA for IL-2 was consistently detectable, although the numbers of copies of the transcript were considerably lower in SCCHN cells than in PHA-activated Jurkat T-lymphocytes (i.e., 890 for PCI-13 vs 8400 for Jurkat cells).
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Discussion |
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While performing immunoperoxidase staining of carcinoma cell lines maintained in our laboratory and tissue sections of human solid tumors, we observed what appeared to be specific, although relatively weak, staining for IL-2 in these cells. On 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 tissue culture media. These cells showed a characteristic staining pattern, with immunoreactive IL-2 localized to a circumscribed area of the cytoplasm corresponding to the Golgi zone. PHA-stimulated Jurkat cells or concanavalin A-activated normal T-lymphocytes used as controls also showed the same staining pattern (Reichert et al. submitted for publication). Normal human fibroblasts and keratinocytes in primary cultures were likewise positive for IL-2 but stained weakly compared to tumor cells or activated T cells (Reichert et al. submitted for publication). The finding that IL-2 in SCCHN and other tumor cells was localized within the Golgi complex suggested that this cytokine was processed and secreted by tumor cells in the same way as in hematopoietic cells. IL-2, like most of the cytokines produced by hematopoietic cells, has hydrophobic amino acid-binding sequences, which lead to its accumulation and secretion through the Golgi complex (
Because carcinoma cells produce endogenous IL-2 and express IL-2R (, -ß, and -
(Yasumura et al., 1984;
To test the relevance of IL-2 expression in vivo, we studied tumor tissues obtained from patients with SCCHN. We compared IL-2 expression in tumors of different histological grades and correlated the LI for IL-2 to that for Ki-67. The proliferation marker Ki-67 is widely used to measure the growth fraction of human tumors in situ (
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Acknowledgments |
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Supported in part by R0-1 CA 63513 to TLW. TER was supported by the Deutsche Forschungsgemeinschaft (DFG), grant Re 1155/1-1.
Received for publication July 28, 1997; accepted August 22, 1997.
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Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) The mechanics of cell division. In Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Molecular Biology of the Cell. 3rd Ed. New York, Garland Publishing, 911-946
Alileche A, Plaisance S, Han DS, Rubinstein E, Mingari C, Bellomo R, Jasmin C, Azzarone B (1993) Human melanoma cell line M14 secretes a functional interleukin 2. Oncogene 8:1791-1796[Medline]
Arzt E, Stelzer G, Renner U, Lange M, Muller OA, Stalla GK (1992) Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures. J Clin Invest 90:1944-1951[Medline]
Braun N, Papadopoulos T, MullerHermelink HK (1988) Cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells. Virchows Arch [B] 56:25-33[Medline]
Brown DC, Gatter KC (1990) Monoclonal antibody Ki-67: its use in histopathology. Histopathology 17:489-503[Medline]
Bruno S, Darzynkiewicz Z (1992) Cell cycle-dependent expression and stablity of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif 25:31-40[Medline]
Ciacci C, Mahida YR, Dignass A, Koizumi M, Podolsky DK (1993) Functional interleukin-2 receptors on intestinal epithelial cells. J Clin Invest 92:527-532[Medline]
Edstrom SS, Gustafsson B, Stenman G, Lyden E, Stein H, Westin T (1991) Proliferative pattern of head and neck cancer. Am J Surg 162:412-416[Medline]
Gerdes J (1990) Ki-67 and other proliferation markers useful for immunohistological diagnostic and prognostic evaluations in human malignancies. Semin Cancer Biol 1:199-206[Medline]
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715
Gerdes J, Schwab U, Lemke H, Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 31:13-20[Medline]
Girod SC, Krueger G, Pape HD (1993) p53 and Ki-67 expression in preneoplastic and neoplastic lesions of the oral mucosa. Int J Oral Maxillofac Surg 22:285-288[Medline]
Heo DS, Snyderman C, Gollin SM, Pan S, Walker E, Deka R, Barnes EL, Johnson JT, Herbeman RB, Whiteside TL (1989) Biology, cytogenetics, and sensitivity to immunological effector cells of new head and neck squamous cell carcinoma lines. Cancer Res 49:5167-5175[Abstract]
Huberman JA (1981) New views of the biochemistry of eukaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha. Cell 23:647-648[Medline]
Kawamura T, Goseki N, Koike M, Takizawa T, Endo M (1996) Acceleration of proliferative activity of esophageal squamous cell carcinoma with invasion beyond the mucosa: immunohistochemical analysis of Ki-67 and p53 antigen in relation to histopathologic findings. Cancer 77:843-849[Medline]
Kosmehl H, Berndt A, Katenkamp D, Hyckel P, Stiller KJ, Gabler U, Langbein L, Reh T (1995) Integrin receptors and their relationship to cellular proliferation and differentiation of oral squamous cell carcinoma. A quantitative immunohistochemical study J Oral Pathol Med 24:343-348
Lin WC, Yasumura S, Suminami Y, Sung MW, Nagashima S, Stanson J, Whiteside TL (1995) Constitutive production of IL-2 by human carcinoma cells, expression of IL-2 receptor, and tumor cell growth. J Immunol 155:4805-4816[Abstract]
Lorz M, MeyerBreiting E (1988) Evaluation of proliferative activity in human head and neck tumors using the monoclonal antibody Ki-67. ORL J Otorhinolaryngol 50:183-187
McMillan DN, Kernohan NM, Flett ME, Heys SD, Deehan DJ, Sewell HF, Walker F, Eremin O (1995) Interleukin-2 receptor expression and interleukin-2 localization in human solid tumor cells in situ and in vitro: evidence for a direct role in the regulation of tumor cell proliferation. Int J Cancer 60:766-772[Medline]
Minami Y, Kono T, Miyazaki T, Taniguchi T (1993) The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol 11:245-267[Medline]
Morgan DA, Ruscetti FW, Gallo R (1976) Selective in vitro growth of T-lymphocytes from normal human bone marrow. Science 193:1007-1009[Medline]
Nagashima S, Reichert TE, Kashii Y, Suminami Y, Chikamatsu K, Whiteside TL (1997) In vitro characteristics of human squamous cell carcinoma of the head and neck cells engineered to secrete IL-2. Cancer Gene Ther in press
O'Connor PM, Jackman J (1995) Synchronization of mammalian cells. In Pagano M, ed. Cell CycleMaterials and Methods. Berlin, Springer-Verlag, 63-74
Pardee AB (1989) G1 events and regulation of cell proliferation. Science 246:603-608[Medline]
Plaisance S, Rubinstein E, Alileche A, Sahraoui Y, Krief P, AugeryBourget Y, Jasmin C, Suarez H, Azzarone B (1992) Expression of the interleukin-2 receptor on human fibroblasts and its biological significance. Int Immunol 4:739-746[Abstract]
Sander B, Andersson J, Andersson U (1991) Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119:65-93[Medline]
Schrape S, Jones DB, Wright DH (1987) A comparison of three methods for the determination of the growth fraction in non-Hodgkins lymphoma. Br J Cancer 55:283-286[Medline]
Shimizu Y, Weidmann E, Iwatsuki S, Herberman RB, Whiteside TL (1991) Characterization of human autotumor-reactive T-cell clones obtained from tumor-infiltrating lymphocytes in liver metastasis of gastric carcinoma. Cancer Res 51:6153-6162[Abstract]
Smith KA (1984) Interleukin 2. Annu Rev Immunol 2:319-333[Medline]
Taniguchi T, Minami Y (1993) The IL-2/IL-2 receptor system: a current overview. Cell 73:5-8[Medline]
van Noorden S (1986) Tissue preparation and immunological staining techniques for light microscopy. In Polak J, van Noorden S, eds. Immunocytochemistry: Modern Methods and Applications. 2nd ed, Bristol, Wright, 26-53
Verheijen R, Kuijpers HJ, Schlingemann RO, Boehmer AL, van Driel R, Brakenhoff GJ, Ramaekers FC (1989) Ki-67 detects a nuclear matrix-associated proliferation-related antigen I Intracellular localization during interphase. J Cell Sci 92:123-130[Abstract]
Verheijen R, Kuijpers HJ, van Driel R, Beck JL, van Dierendonck JH, Brakehoff GJ, Ramaekers FC (1989b) Ki-67 detects a nuclear matrix-associated proliferation-related antigen II. Localization in mitotic cells and association with chromosomes. J Cell Sci 92:531-540[Abstract]
Waldmann TA (1989) The multi-subunit interleukin-2 receptor. Annu Rev Biochem 58:575-611[Medline]
Weidmann E, Sacchi M, Plaisance S, Heo DS, Yasumura S, Lin WC, Johnson JT, Herberman RB, Azzarone B (1992) Whiteside TL Receptors for interleukin-2 on human squamous cell carcinoma cell lines and tumor in situ. Cancer Res 52:5963-5970[Abstract]
Whiteside TL, Herberman RB (1995) The role of natural killer cells in immune surveillance of cancer. Curr Opin Immunol 7:704-710[Medline]
Yasumura S, Lin WC, Weidmann E, Hebda P, Whiteside TL (1994) Expression of interleukin-2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin-2. Int J Cancer 59:225-234[Medline]
Youssef EM, Matsuda T, Takada N, Osugi H, Higashino M, Kinoshita H, Watanabe T, Katsura Y, Wanibuchi H, Fukushima S (1995) Prognostic significances of the MIB-1 proliferation index for patients with squamous cell carcinoma of the esophagus. Cancer 76:358-366[Medline]
Zoeller J, Flentje M, Sinn P, Born IA (1994) Evaluation of AgNOR and Ki-67 antigen as cell kinetic parameters in oral dysplasias and carcinomas. Anal Cell Pathol 7:77-88[Medline]