Journal of Histochemistry and Cytochemistry, Vol. 46, 603-612, May 1998, Copyright © 1998, The Histochemical Society, Inc.


SYMPOSIUM PAPER

Endogenous IL-2 in Cancer Cells: A Marker of Cellular Proliferation

Torsten E. Reicherta, Simon Watkinsc, Joanna Stanson, Jonas T. Johnsonb, and Theresa L. Whitesidea,b
a Departments of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
b Otolaryngology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
c Cell Biology and Physiology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

Correspondence to: Theresa L. Whiteside, U. of Pittsburgh Cancer Institute, W 1041 Biomedical Science Tower, 211 Lothrop St., Pittsburgh, PA 15213-2582.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 -{gamma} 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 46:603–611, 1998)

Key Words: , human carcinomas, interleukin-2, cell cycle, Ki-67 antigen, mitosis, tumor grade


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

INTERLEUKIN-2 (IL-2) was first identified in 1975 as a growth-promoting factor for bone marrow-derived T-lymphocytes (Morgan et al. 1976 ). Since then, the spectrum of its known biological activities has expanded considerably. IL-2 regulates growth and differentiation of T- and B-lymphocytes, promotes growth of natural killer (NK) cells and enhances their cytolytic functions, and is also known to function in some non-lymphoid cells (reviewed in Smith 1984 ; Waldmann 1989 ; Minami et al. 1993 ; Whiteside and Herberman 1995 ). The biological effects of IL-2 are mediated through binding of IL-2 to the specific IL-2 receptor (IL-2R) complex, which is expressed on all classes of lymphoid cells (Smith 1984 ; Waldmann 1989 ; Minami et al. 1993 ; Taniguchi and Minami 1993 ; Whiteside and Herberman 1995 ) as well as in nonlymphoid cells (Plaisance et al. 1992 ; Arzt et al. 1992 ; Alileche et al. 1993 ; Ciacci et al. 1993 ). Human carcinomas, including squamous cell carcinomas of the head and neck (SCCHN), have also been shown to express IL-2 and IL-2R (Weidmann et al. 1992 ; Yasumura et al. 1994 ; Lin et al. 1995 ; McMillan et al. 1995 ). More recent results have shown that expression of endogenous IL-2 is required for cell proliferation (Reichert et al. submitted for publication) and that its absence, after anti-sense IL-2 treatment of tumor cells, results in growth inhibition (Reichert et al. submitted for publication). Therefore, it appears that in tumor cells, similar to lymphocytes, endogenous IL-2 is involved in regulation of cell division and proliferation (Reichert et al. submitted for publication).

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 Brown and Gatter 1990 ; Gerdes 1990 ). Detailed analysis of expression of this proliferation-associated antigen during cell division indicated that it is present in all active phases of the cell cycle (G1, S, G2, and mitosis) (Gerdes et al. 1984 ; Schrape et al. 1987 ; Bruno and Darzynkiewicz 1992 ). Furthermore, it has a characteristic topographical distribution during the cell cycle (Gerdes et al. 1983 ; Braun et al. 1988 ; Verheijen et al. 1989a, Verheijen et al. 1989b ).

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.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Heo et al. 1989 ; Shimizu et al. 1991 ). These lines were maintained in Dulbecco's modified minimal essential medium (DMEM) containing 10% (v/v) heat-inactivated (56C, 30 min) FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies; Grand Island, NY). The cells were passaged by trypsinization (0.25% trypsin solution; Life Technologies) and were routinely tested for mycoplasma (GEN-PROBE; San Diego, CA).

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 70–80% 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 streptavidin–biotin complex–immunoperoxidase (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 phosphatase–anti-alkaline phosphatase (APAAP) method, and then reacted with anti-IL-2 Ab, followed by an sABC-HRP technique (van Noorden 1986 ). Peroxidase staining was visualized with 3-amino-9-ethyl-carbazole (AEC; Biomeda, Foster City, CA) or 3,3-diaminobenzidine tetrahydrochloride (DAB; DAKO, Carpinteria, CA) and alkaline phosphatase staining with BCIP/NBT (Vector). The slides were counterstained with hematoxylin (Biomeda) and mounted, using a glycerol-based mounting medium.

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 fluorescein–phalloidin (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 (Pardee 1989 ) progressed through the remaining cell cycle, divided, and then stopped in G0/G1.

Using aphidicolin, an inhibitor of DNA polymerase-{alpha} (Huberman 1981 ), tumor cells were synchronized at the G1/S border. Tumor cell monolayers were incubated with 5 µg/ml aphidicolin for 16 hr, detached by trypsinization, released from the block by three washes in fresh medium without FCS, and resuspended in complete medium. The synchronized cells were placed in new T75 flasks and harvested after various incubation times (1–12hr) for flow cytometry and extraction of total RNA. PCI-13 cells reached the middle of the S-phase after 4 hr and were in the M-phase after 8.5 hr. PCI-1 cells needed 6 hr to reach the middle of the S-phase and 10 hr to reach the M-phase.

Synchronization of cells in the M-phase of the cell cycle was accomplished by mitotic "shake-off" of enriched cell monolayers, as described elsewhere (O'Connor and Jackman 1995 ). Enrichment of mitotic cells was achieved by addition of either colchicine (5 ng/ml; Sigma) or nocodazole (40 ng/ml; Sigma) for 16 hr to 70% confluent cell monolayers.

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 (Nagashima et al. 1997 )

Statistical Analysis
Results are given as means ± SD. For nonparametric analysis, the Kruskal–Wallis 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).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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).



View larger version (115K):
[in this window]
[in a new window]
 
Figure 1. IL-2 expression in PCI-13 (a) and PCI-1 (b) monolayers. Multicolor fluorescent images show immunoreactive IL-2 (red signal) in a juxtanuclear position in most of the tumor cells (compare to Figure 2). The tumor cell monolayers were counterstained with fluorescein–phalloidin (green signal), which binds to actin filaments, and with Hoechst dye 33342 (blue signal), which stains the nuclei. Bar = 12.5 µm.

Figure 2. Localization of the Golgi complex in PCI-1 tumor cells visualized by immunoperoxidase staining with an MAb specific for the Golgi complex. The staining reaction was developed with AEC and tumor cells were counterstained with hematoxylin. Bar = 10 µm.

Figure 3. Double immunostaining for IL-2 (red–brown signal) and Ki-67 (dark blue signal) in monolayers of PCI-1 tumor cells. The monolayers were first incubated with anti Ki-67, followed by an APAAP method and second with anti IL-2, followed by an sABC-HRP technique. The alkaline phosphatase staining was visualized with BCIP/NBT and the peroxidase staining with AEC. (a) A representative photomicrograph of the monolayer, showing IL-2-positive tumor cells in different phases of the cell cycle, identified by the characteristic distribution of Ki-67 antigen. (b) Ki-67-negative tumor cell in G0/early G1, showing juxtanuclear staining for IL-2. (c) IL-2-positive tumor cells in late G1 (arrow), as defined by the perinucleolar Ki-67 staining, and in the S-phase (arrowhead), as defined by finely dispersed Ki-67 staining in the nucleus, with still-prominent perinucleolar regions. (d) Increased perinuclear IL-2 expression in tumor cells in the G2-phase (arrow) and prophase (arrowhead). The Ki-67 distribution in G2 appears to be reticulum-like and, in prophase, chromosomes are coated with the antigen. (e) A tumor cell in metaphase of mitosis (center) with very intense, evenly distributed IL-2 staining throughout the cell. (f) Still strong but slightly decreased IL-2 staining in a tumor cell in telophase of mitosis (arrow). Ki-67 staining is still prominent around the chromosomes. Bar = 10 µm.

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).



View larger version (126K):
[in this window]
[in a new window]
 
Figure 4. Immunostaining for IL-2 in a frozen section of normal human oral mucosa (a), showing localization of the protein to the basal epithelial layer (see inset).(b) Immunostaining of moderately differentiated laryngeal carcinoma shows "dot-like" staining for IL-2 in most tumor cells. (c) Carcinoma cells (PCI-13) in culture stained for IL-2. Note the "dot-like" staining characteristic for cytokines. Bar = 25 µm.

Figure 5. Immunostaining for Ki-67 and IL-2 of well-differentiated (a,c), moderately differentiated (b,d), and poorly differentiated (c,f) SCCHN in situ. Staining for Ki-67 (upper row) reveals many positive nuclei in the tumor tissues. Staining for IL-2 (lower row) is localized to the cytoplasm of tumor cells, with a strongly stained juxtanuclear dot in many tumor cells. For both Ki-67 and IL-2, the numbers of positively stained cells are correlated with the histological grade of the tumor. Bar = 25 µm.

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 5a–c). 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 5d–f). 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).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 6. Correlation between Ki-67 and IL-2 expression in tumor samples from 34 patients with SCCHN. The correlation between labeling indices (LIs) was statistically significant (Pearson correlation coefficient 0.747; p<0.001).


 
View this table:
[in this window]
[in a new window]
 
Table 1. Labeling indices (LIs) for Ki-67 and IL-2 in SCCHN of different histological gradesa,b

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).



View larger version (15K):
[in this window]
[in a new window]
 
Figure 7. Quantitative competitive RT-PCR for IL-2 mRNA in SCCHN cells (PCI-13) and Jurkat cells used here as a positive control.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Sander et al. 1991 ). However, although IL-2 protein was detectable by immunostaining in permeabilized tumor cells, it was not detectable by ELISA in cell supernatants of carcinoma cells (Lin et al. 1995 ). With an IL-2-dependent CTLL line, very low levels of IL-2 were detected in these supernatants (Nagashima et al. 1997 ). Carcinoma cells disrupted by rapid freezing and thawing in the presence of protease inhibitors yielded cytosols containing 40–60 pg/ml/5 x 106 cells of IL-2, as determined by ELISA (Lin et al. 1995 ). Furthermore, mRNA for IL-2 was detectable by RT-PCR in tumor cells (Figure 7). These results indicated that tumor cells produced endogenous IL-2 , which was either not secreted or secreted only at levels not detectable by immunoassays. The present study confirms that normal epithelial and carcinoma (SCCHN) cells are capable of expressing IL-2 in vivo.

Because carcinoma cells produce endogenous IL-2 and express IL-2R (Weidmann et al. 1992 ; Yasumura et al. 1994 ; Lin et al. 1995 ), the question arises as to the biologic significance of this pathway in tumor cell growth and tumor progression. We have observed that, in monolayers of tumor cells and in human tumor tissues, IL-2 was especially strongly expressed in dividing cells. To examine the possibility that endogenous IL-2 is involved in the regulation of tumor cell growth, we examined its expression as well as its receptors in various phases of the cell cycle in tumor cell lines. To be able to follow IL-2 expression during the course of the cell cycle, we compared it to that of the Ki-67 antigen, which is cycle-dependent in its characteristic localization to various parts of the cell nucleus. Tumor cells in G0-, G1-, and S-phase showed the juxtanuclear distribution of IL-2. The expression level of IL-2 in various phases of the cell cycle was quite consistent, but a few IL-2-negative cells were also seen. The numbers of IL-2-negative tumor cells were clearly increased when monolayers were confluent, indicating a downregulation of IL-2 expression in resting cells (data not shown). Tumor cells in G2 and mitosis showed increased IL-2 expression, with the highest expression level in metaphase. In mitotic cells, the IL-2 protein was evenly distributed throughout the cell, probably reflecting fragmentation of the cell organelles into a set of smaller vesicles, which can be evenly distributed when the cell divides (Alberts et al. 1994 ). The observed modulation of IL-2 expression, with downregulation in resting cells and upregulation in dividing cells, leads to the hypothesis that IL-2 in tumor cells is involved in cell proliferation. This is highly plausible because of the observation that the tumor cells in mitosis also express increased levels of IL-2R chains (data not shown). We reported earlier that human SCCHN cell lines constitutively express mRNA and protein for IL-2R-{alpha}, -ß, and -{gamma} (Yasumura et al., 1984; Weidmann et al. 1992 ; Lin et al. 1995 ). In addition, when tumor cell lines were synchronized, expression of IL-2 and IL-2R was found to be induced in the S-phase and significantly upregulated in the G2/M-phase of the cell cycle (Reichert et al. submitted for publication). The level of the mRNA for IL-2 was five- to tenfold higher in the M-phase than in the G0/G1- phase, as shown by quantitative competitive RT-PCR (Reichert et al. submitted for publication). These results indicated that IL-2 and the IL-2R complex behaved like cell cycle-related proteins in human carcinoma cells (Reichert et al. submitted for publication). Additional in vitro studies with IL-2-specific anti-sense oligonucleotides showed that proliferation of carcinoma cells was significantly inhibited in the absence of endogenous IL-2 (Reichert et al. submitted for publication). In addition, growth of carcinoma cell lines was inhibited by the immunosuppresive agents cyclosporin A, FK 506, and rapamycin, similar to the effect of these drugs on the IL-2/IL-2R pathway in lymphoid cells (Reichert et al. submitted for publication).

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 (Brown and Gatter 1990 ; Gerdes 1990 ). In squamous cell carcinomas of the upper aerodigestive tract, expression of Ki-67 has been shown to correlate with proliferation, histological tumor grade, tumor invasion, lymph node status, and expression of the mutated tumor-suppressor gene p53 (Lorz and Meyer-Breiting 1988 ; Edstrom et al. 1991 ; Girod et al. 1993 ; Zoeller et al. 1994 ; Kosmehl et al. 1995 ; Youssef et al. 1995 ; Kawamura et al. 1996 ). In the present study, we confirmed the positive correlation of Il-2 expression with Ki-67 expression and the histological tumor grade. As expected, well-differentiated SCCHN had a low Ki-67 LI and poorly differentiated carcinomas showed a high level of expression of Ki-67, indicating that the latter have a higher proliferation rate and a poorer prognosis (Lorz and Meyer-Breiting 1988 ; Youssef et al. 1995 ). The highest level of co-expression of IL-2 and Ki-67 was observed in poorly differentiated carcinomas, with an LI of 67.2% for IL-2 and 68.8% for Ki-67 (significant correlation at p<0.001). Well-differentiated SCCHN had significantly lower expressions of both proteins (LI 35% for IL-2 and 26.5% for Ki-67) than poorly differentiated carcinomas. These results demonstrate that IL-2 expression in SCCHN is strongly associated with cell proliferation and, together with our previous data, indicate that endogenous IL-2 might be an important growth factor for these carcinomas.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
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, Muller–Hermelink 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[Abstract/Free Full Text]

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, Meyer–Breiting 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 Cycle—Materials 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, Augery–Bourget 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]