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Japanese Journal of Clinical Oncology Pages 384-388

Expression of Fas in Renal Cell Carcinoma
Introduction
Materials And Methods
   Tissue Samples and Cell Culture
   Flowcytometric Analysis of Fas Expression
   Assessment of Fas ab-mediated Cytotoxicity and Apoptosis
   Statistical Analysis
Results
   Expression of Fas in RCC
   Susceptibility of Human RCC Cells to Fas-mediated Cytotoxicity
Discussion
Acknowledgments
References

Expression of Fas in Renal Cell Carcinoma

Expression of Fas in Renal Cell Carcinoma Shigeo Horie1,2, Munehide Kano2, Eiji Higashihara3, Nobuo Moriyama2, Etsuko Tanaka2, Akiko Hirose2, Tadao Kakizoe1 and Kazuki Kawabe2

1Division of Urology, National Cancer Center Hospital, Tokyo, 2Department of Urology, Faculty of Medicine, University of Tokyo, Tokyo, 3Department of Urology, Kyorin University, Tokyo, Japan

We have investigated whether the Fas-mediated cell death pathway is functional in renal cell carcinoma. The expression of Fas in surgical specimens and cell lines of renal cell carcinoma was examined. Fas expression was positive in six out of 18 tumors measured by flow cytometry and was prominent in advanced tumors. Three out of the six Fas-positive tumors had already metastasized at the time of surgery. A significant correlation was found between the tumor volume and the percentage of Fas-positive cells in a tumor (r = 0.70, P = 0.0007). Fas-positive tumors were larger than Fas-negative tumors [mean tumor volume (ml) ± SD, Fas(+), 265.6 ± 136.8; Fas(-), 65.8 ± 80.9, P = 0.0012]. All human renal carcinoma cell lines tested (ACHN, Caki-1, SMKT-R-2, SMKT-R-3 and SMKT-R-4) expressed Fas abundantly, as Fas-positive cells accounted for >50% in all cell lines by flow cytometry. Treatment with anti-Fas antibody caused apoptosis in Fas-positive renal cell carcinoma cell lines. However, the effectiveness of apoptosis induction in individual cell lines was not correlated with the level of Fas expressed. These data suggest that Fas targeting may be a therapeutic option for treatment of advanced renal cell carcinoma which is refractory to either chemotherapy or irradiation.

Key words: Fas - renal cell carcinoma - apoptosis - anti-Fas antibody

INTRODUCTION

Apoptosis is a physiological mechanism of cell death that is responsible for deletion of cells in normal tissues. Apoptosis also occurs in tumors and can be initiated by treatments such as radiotherapy and chemotherapy (1 ). Fas, a member of the nerve growth factor/tumor necrosis factor receptor superfamilies, is a cell surface protein known to trigger apoptosis in a variety of cell types upon binding to a specific cytotoxic monoclonal antibody. Fas and its natural ligand, Fas ligand, are involved in T cell-mediated cytotoxicity as well as down-regulation of immune reactions (2 ). Fas expression is widely distributed among solid tumors of various histologic types including renal cell carcinoma (RCC) (3 ), although the function ot the Fas expressed in the cancer has not been elucidated. RCC is resistant to cytotoxic chemotherapy and radiation therapy and the prognosis of the advanced cancer is very poor (4 ,5 ). Investigation of the inherent mechanisms by which RCC can become immune to apoptosis induced by the treatments is thus essential in order to develop a new therapeutic approach. The purpose of this study was to determine whether the Fas-induced apoptotic pathway functions in RCC. We examined the expression of Fas in surgical specimens and cell lines of RCC by flow cytometry. Fas-induced apoptosis was studied in cell lines of RCC to see whether they possess the machinery for apoptosis induction upon Fas ligation.

MATERIALS AND METHODS

Tissue Samples and Cell Culture

Tissue samples were obtained from 18 patients undergoing nephrectomy at the University of Tokyo Hospital and its affiliated hospitals as part of their medical care for RCC. Tissue was harvested from typical clear-cell type cancer under visual inspection. Samples were taken from relatively peripheral and viable portions of RCC not including necrosis, hemorrhage or fibrosis. The clinicopathological features were noted, including the tumor volume calculated from the size of the surgical specimen and the presence of any necrotic area inside the tumor. Tumors were graded by WHO classification and staged according to the 1992 TNM staging classification of the International Union Against Cancer. The human RCC cell lines Caki-1, ACHN, SMKT-R-2, SMKT-R-3 and SMKT-R-4 were maintained in modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin. ACHN was purchased from the American Type Culture Collection (Rockville, Maryland). Caki-1 was kindly provided by Dr M. Hayakawa (Saitama, Japan). SMKT-R-2, SMKT-R-3 and SMKT-R-4 (6 ,7 ) were kindly provided by Dr T. Tsukamoto (Sapporo, Japan).


Figure 1. Fas expression on renal cell carcinoma measured by flow cytometry. Isolated tumor cells were stained with anti-Fas ab CH-11 as described in [rdquor]Materials and Methods". Examined cells (R1) were selected by the intensity of fluorescence (FL1, FL2; arbitrary units). Fas-negative cells (R2) were defined using non-specific mouse IgM instead of anti-Fas ab. The data are presented as percentages of positive cells (R3) calculated directly from the gated scan (R1-R2).


Figure 2. Correlation of the tumor size with Fas expression. The percentage of Fas-positive cells in a tumor specimen is plotted on the Y-axis against tumor volume (ml). Distributions in 18 tumors are shown. Correlations were analyzed by Pearson's correlation test (correlation coefficient r = 0.70, P = 0.0007).

Flowcytometric Analysis of Fas Expression

Flowcytometric analysis of Fas expression was performed at Otsuka Assay Laboratories (Tokushima, Japan). Neoplastic tissues of RCC were digested in collagenase for 20 min at 25°C. Isolated cells were incubated with 20% normal goat serum for 10 min at 4°C. Cells were then incubated with anti-Fas antibody CH-11 (BML, Nagoya, Japan) for 30 min at 4°C and subsequently labeled with FITC-conjugated anti-mouse IgM (Tago, CA) for 30 min at 4°C. Labeled cells were washed twice in flow-cytometry buffer and fixed with 70% ethanol for 10 min at 4°C. Cells were resuspended in medium containing 10 µg/ml propidium iodide and a total of 10 000 viable cells were analysed on a FACScan cytometer (Becton Dickinson, San Jose, CA). The total population of examined cells was gated with a window set to exclude debris and aggregated cells (Fig. 1 , right small inset). Gating was determined by the intensity of fluorescence of propidiumiodide (FL2) and the extent of forward scattering (FSC) of applied cells. Gated cells (R1) were fractioned into Fas-negative (R2) and Fas-positive (R3) cells according to fluorescence intensity (FL1, CH-11; FL2, propidium iodide, arbitrary units). The percentage of Fas-positive cells was calculated from R3/R1.


Figure 3. (a) Killing of cultured cells by anti-Fas antibody CH-11, for ACHN and Caki-1 cells. Cells were treated with CH-11 for 72 h as described in [rdquor]Materials and Methods". Concentration of CH-11 added to the medium: 10 ng/ml (clear bars), 100 ng/ml (dotted bars), and 1µg/ml (solid bars). Individual columns show the viable recovery of cultured cells treated with CH-11, which is expressed as a percentage of the control. Data shown are mean ± SD from triplicate experiments. (b) The effect of the anti-Ras mouse IgM monoclonal ab MRG on the proliferation of cultured cells. Cell viability was calculated as described in (a). Concentration of MRG added to the medium: 100 ng/ml (dotted bars) and 1 µg/ml (solid bars). Individual columns show the recovery of viable cultured cells treated with MRG. Data represent mean ± SD of triplicate experiments.


Figure 4. Killing of cultured cells by CH-11 for SMKT-R-2, SMKT-R-3 and SMKT-R-4 cells. Cultured cells were treated with CH-11 for 72 h. Concentration of CH-11 added to the medium: 10 ng/ml (clear bars), 100 ng/ml (dotted bars) and 1µg/ml (solid bars). Individual columns show recovery of viable cultured cells treated with CH-11. Cell viability was calculated as described in Fig. 3a.


Figure 5. Apoptosis of Caki-1 cells treated with 1 mg/ml anti-Fas antibody for 4 h. Left, transmission electron microscopy. Right, scanning electron microscopy. Note cytoplasmic condensation and membrane blebbing.

Assessment of Fas ab-mediated Cytotoxicity and Apoptosis

Cell proliferation was assayed by plating cells at a density of 3000 cells/ml in 96-well plates in culture media. Anti-Fas antibody (CH-11), which was mouse IgM, or the anti-Ras mouse IgM antibody MRG (BML, Nagoya, Japan), was added at the final concentration indicated. After 72 h of incubation, cell proliferation was scored by the MTT (3-[4,5-dimethylthgiazol-2-yl]-2,5-diphenyltetrazolium bromide) uptake method (8 ). Dye uptake was determined by an automatic spectrometer based on the absorption at 565 nm. The optical density reading was normalized by the protein content that was determined in a reagent assay (Bio-Rad Laboratories, Richmond, CA). Absorbence was directly proportional to the number of cells. For the observation of cultured cells by electron microscopy, RCC cell lines treated as indicated were fixed and processed in the same way as for standard electron microscopy procedures (9 ).

Statistical Analysis

Statistical analysis was performed using unpaired t test, Fisher's PLSD test and Pearson's correlation test. Differences or correlations were considered significant at P < 0.05. Data are given with standard deviations.

RESULTS

Expression of Fas in RCC

Previous immunohistochemical study has shown that Fas is expressed abundantly in RCC (3 ). A total of 10 000 viable isolated cells freshly obtained from a surgical specimen immediately after resection were analysed for Fas expression by flow cytometry. The percentage of Fas-positive cells was calculated directly from the gated histogram (Fig. 1 ). Pathological profiles and the percentage of Fas-positive cells in a tumor measured by flow cytometry of 18 RCCs are shown in Table 1 . Flow cytometry revealed that six out of 18 RCCs (30%) expressed Fas. Most of the Fas-positive tumors were at an advanced stage with high grades. Three of the six Fas-positive tumors had already metastasized by the time of surgery. A significant correlation was found between the tumor volume and the percentage of Fas-positive cells in the tumor (r = 0.70, P = 0.0007; Fig. 2 ). Fas-positive tumors were larger than Fas-negative tumors [mean tumor volume (ml) ± SD: Fas(+), 265.6 ± 136.8; Fas(-), 65.8 ± 80.9, P = 0.0012]. Neither the cell type nor the subtype of the tumor was correlated with Fas expression. Interestingly, however, five of the six Fas-positive tumors contained areas of necrosis although the tissue samples were obtained from viable portions (Table 1 ). The human renal carcinoma cell lines ACHN, Caki-1, SMKT-R-2, SMKT-R-3 and SMKT-R-4 expressed abundant Fas, Fas-positive cells exceeding 50% in all of them: ACHN, 65.3%; Caki-1, 64.2%; SMKT-R-2, 72.9%; SMKT-R-3, 59.2%; SMKT-R-4, 63.7%.

Table 1. Clinicopathological features of patients with RCC, and tumor expression of Fas
Case Volume (ml) Cell type Subtype Grade pT Necrosis Metastasis Fas expr. (%)
1 1.8 clear alveolar 1 1 - none 0
2 1.8 clear cystic 1 2 - none 0
3 1.8 clear tubular > papillary 2 2 - none 0
4 11.5 clear alveolar 2 2 - none 0
5 14.1 clear alveolar > cystic 1 2 - none 0
6 22.4 clear alveolar 2 2 - none 0
7 33.5 clear > granular alveolar 1 2 - none 0
8 36.1 clear alveolar 1 2 - none 0
9 47.7 clear alveolar 2 2 + none 7.4
10 87.1 clear alveolar 1 3 - none 0
11 180 clear alveolar 2 2 - none 0
12 180 clear alveolar 2 3 - none 0
13 221 clear alveolar 1 2 + none 20.1
14 221 clear alveolar 1 2 - none 0
15 268 clear > granular alveolar 2 2 - none 19.0
16 268 clear alveolar 2 3 + lung 31.2
17 321 clear alveolar 2 3 + lung 25.9
18 468 clear > spindle tubular > solid 2 2 + lung 13.5
For flow cytometric analysis of Fas expression, neoplastic tissues were digested by collagenase. Isolated cells were incubated with anti-Fas antibody and subsequently labeled with FITC-conjugated anti-mouse IgM. A total of ten thousand viable cells were analysed on a FACScan cytometer. The percentage of positive cells was calculated directly from the gated histogram. Existence of necrotic areas inside the tumors was established by visual inspection. pT; pathological tumor stage.

Susceptibility of Human RCC Cells to Fas-mediated Cytotoxicity

Fas-mediated cytotoxicity was evaluated in cultured RCC cells. Treatment with 100 ng/ml of the anti-Fas antibody CH-11 was cytotoxic to RCC cells and inhibited cellular proliferation by less than 83% in ACHN and 77% in Caki-1 (Fig. 3 ). To eliminate the possibility that the effect was due to IgM monoclonal antibody rather than to anti-Fas antibody per se, the same experiments were repeated using anti-Ras mouse monoclonal IgM antibody MRG. MRG had no cytotoxic effect on either of the lines (Fig. 3 ). Thus it was concluded that anti-Fas antibody specifically kills human RCC lines. The induction of cell death upon Fas ligation in SMKT-R-2, SMKT-R-3 and SMKT-R-4 cells was not as effective as that in ACHN and Caki-1 cells (Fig. 4 ), although the numbers of Fas-positive cells were comparable. Exposure of the cell lines to anti-Fas antibody resulted in rounding and detachment of cells from the monolayer culture becoming apparent within 6 h after exposure to anti-Fas antibody. The cytotoxicity mediated by anti-Fas antibody was characterized as apoptosis by observations with electron microscopy, which showed condensation of nuclear chromatin and budding of cells to form apoptotic bodies (Fig. 5 ) (1 ).

DISCUSSION

Fas is a constitutively expressed apoptosis-related molecule in a variety of epithelial and neoplastic cells, but the biological role of the expressed Fas molecule has not been well elucidated except for Fas-mediated apoptosis. In the present study, Fas was expressed predominantly in advanced tumors, and tumors that had metastasized expressed high levels of Fas. In flow cytometric studies,contaminating cells other than cancer cells, such as vascular endothelium and infiltrating lymphocytes in resected tissue can be responsible for the expression of Fas. However, since cultured RCC cells strongly expressed Fas and immunohistochemistry also detected Fas on the cell surface of RCC (3 ), it was reasonable to assume that the expression of Fas was elevated in advanced cancer cells themselves. We showed that the expression of Fas was strongly correlated with tumor volume. In RCC, tumor size has been implicated as an independent prognostic index and tumor volume itself has been considered to reflect the invasive nature of RCC (10 ,11 ). The expressed Fas might have been induced in the tumor by cytokines such as interferon [gamma] as a part of the host's defense mechanism rather than being an inherent biological property of an advanced tumor (12 ). We also demonstrated that RCC cell lines were sensitive to the cell-killing effect of anti-Fas antibody. These cell lines were thus confirmed as possessing the cellular machinery required for Fas-mediated apoptosis. Whether or not expressed Fas actually interacts with Fas ligand produced by lymphocytes infiltrating RCC, resulting in apoptosis, remains to be studied, although we noticed that most RCC which expressed Fas in the viable portion had necrotic areas inside the tumor. Thus the necrosis frequently observed in advanced RCC may be associated with host-cancer interaction through Fas-induced apoptosis. Enhanced apoptosis has been reported in various high-grade advanced cancers such as non-small cell lung, prostate, bladder and breast cancer (13 ,14 ,15 ,16 ). In these studies, the extent of apoptosis observed in neoplastic tissue was correlated with the rate of cell proliferation and patient survival. The enhanced occurrence of apoptosis in high-grade advanced cancer is presumably a balance in the interaction between the host's defense mechanism and the cancer progression. Along these lines of evidence, it is possible to speculate that enhanced Fas expression per se has some relevance to the invasive and metastatic features of advanced RCC. The role of expressed Fas in relation to the invasive and metastatic features of a tumor warrants further study.

Inhibition of apoptosis would be an important process for the development of neoplasms and may contribute to tumor growth, clonal evolution and inherent resistance to chemotherapeutic agents (17 ). The fact that RCC is resistant to chemotherapy and radiotherapy suggests the existence of some inherent mechanism whereby the tumor can avoid apoptosis. In this study, SMKT-R cells were not as sensitive as ACHN and Caki-1 cells to Fas-induced apoptosis, in spite of their comparable levels of Fas expression. A prior study has shown that inherent susceptibility to anti-Fas-induced apoptosis is not necessarily correlated with the expression of this protein (18 ). In this respect, SMKT-R cells might have a mechanism for inhibiting Fas-induced apoptosis.

Cytotoxic chemotherapy and irradiation have been shown to have little anti-tumor activity against RCC, resulting in objective response rates of only 10-15% (4 ,5 ). The poor prognosis of advanced or metastatic RCC reflects the lack of effective treatments for patients who develop metastatic disease. Further elucidation of the signalling pathway of Fas-mediated apoptosis would be useful for the development of novel immunotherapeutic strategies for RCC.

In summary, this report documents that: (a) the expression of Fas is prominent in advanced RCC and is positively correlated with tumor volume and (b) the fact that anti-Fas antibody can induce apoptosis in cultured RCC cell lines may have therapeutic implications in the clinical setting.

Acknowledgments

We thank the following doctors who kindly provided surgical specimens of RCC: H. Asakage, S. Imao, N. Ishida, H. Kishi, S. Minowada and K. Nutahara. We are also grateful to Drs M. Hayakawa and T. Tsukamoto for providing the RCC cell lines.

References

1 Kerr JFR, Winterford CM, Harmon BV. Apoptosis: its significance in cancer and cancer therapy. Cancer 1994;73:2013-26.

2 Nagata S, Goldstein P. The Fas death factor. Science 1995;267:1449-56. MEDLINE Abstract

3 Leithäiser F, Dhein J, Mechtersheimer G, Koretz K, Brüderlein S, Henne C, et al. Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab Invest 1993;69:415-29. MEDLINE Abstract

4 Harris DT. Hormonal therapy and chemotherapy of renal-cell carcinoma. Sem Oncol 1983;10:422-30.

5 Finney R. The value of radiotherapy in the treatment of hypernephroma - a clinical trial. Br J Urol 1973;45:258-69. MEDLINE Abstract

6 Miyao N, Tsukamoto T, Kumamoto Y. Establishment of three human renal cell carcinoma cell lines (SMKT-R-1, SMKT-R-2 and SMKT-R-3) and their characters. Urol Res 1989;17:317-24. MEDLINE Abstract

7 Tsukamoto T, Kumamoto Y, Miyao N, Masumori N, Takahashi A, Yanase M. Interleukin-6 in renal cell carcinoma. J Urol 1992;148:1778-81. MEDLINE Abstract

8 Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63. MEDLINE Abstract

9 Moriyama N, Akaza H, Suzuki T, Kawabe K, Niijima T. Inverted papilloma: observation with scanning and transmission electron microscopy. Virchows Arch [Pathol Anat] 1985;407:25-32.

10 Tsukamoto T, Kumamoto Y, Takahashi A, Yanase M, Yamazaki K, Miyao N, et al. Tumor size of renal cell carcinoma: its clinical implication. Urol Int 1992;48:378-83. MEDLINE Abstract

11 Targonski PV, Frank W, Stuhldreher D, Guinan PD. Value of tumor size in predicting survival from renal cell carcinoma among tumors, nodes and metastases stage 1 and stage 2 patients. J Urol 1994;152:1389-92. MEDLINE Abstract

12 Weller M, Frei K, Groscurth P, Krammer PH, Yonekawa Y, Fontana A. Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells: induction and modulation of sensitivity by cytokines. J Clin Invest 1994;94:954-64. MEDLINE Abstract

13 Törmänen U, Eerola AK, Rainio P, Vähäkangas K, Soini Y, Sormunen R, Bloigu R, et al. Enhanced apoptosis predicts shortened survival in non-small cell lung carcinoma. Cancer Res 1995;55:5595-602. MEDLINE Abstract

14 Vesalainen S, Lipponen P, Talja M, Syrjänen K. Histological grade, perineural infiltration, tumour-infiltrating lymphocytes and apoptosis as determinants of long-term prognosis in prostatic adenocarcinoma. Eur J Cancer 1994;30A:1797-1803. MEDLINE Abstract

15 Lipponen PK, Aaltomaa S, Kosma VM, Syrjänen K. Apoptosis in breast cancer as related to histopathological characteristics and prognosis. Eur J Cancer 1994;30A:2068-73.

16 Lipponen PK, Aaltomaa S. Apoptosis in bladder cancer as related to standard prognostic factors and prognosis. J Pathol 1994;173:333-9. MEDLINE Abstract

17 Bedi A, Pasricha PJ, Akhtar AJ, Barber JP, Bedi GC, Giardiello FM, et al. Inhibition of apoptosis during development of colorectal cancer. Cancer Res 1995;55:1811-16. MEDLINE Abstract

18 Owen-Schaub LB, Radinsky R, Kruzel E, Berry K, Yonehara S. Anti-Fas on nonhematopoietic tumors: level of Fas and bcl-2 are not predictive of biological responsiveness. Cancer Res 1994;54:1580-6. MEDLINE Abstract

Received March 13, 1997; accepted June 23, 1997
For reprints and all correspondence: Shigeo Horie, Division of Urology, National Cancer Center Hospital, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan. Email: shorie@gan2.ncc.go.jp
Abbreviations: RCC, renal cell carcinoma; PCR, polymerase chain reaction.

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