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Changes in expression of the antigen recognized by monoclonal antibody A7 in human pancreatic carcinoma cells following exposure to anticancer agents
Cancer Letters 126 (1998) 165–172
Changes in expression of the antigen recognized by monoclonal antibody A7 in human pancreatic carcinoma cells following exposure to anticancer agents Toshiharu Yamaguchi*, Nobuki Yamaoka, Kazuya Kitamura, Eigo Otsuji, Kazuma Okamoto, Hiroshi Tsuruta, Yoshihiro Yata, Toshio Takahashi First Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602, Japan Received 3 September 1997; received in revised form 25 November 1997; accepted 15 December 1997
Abstract Techniques which can increase the expression level of tumor-associated antigens may improve immunotargeting therapy. We studied the reactivity of MAb A7 toward an antigen expressed on the surface of the human pancreatic cancer cell line HPC-YS after treatment with various antitumoral agents. When we applied 1 mg/ml mitomycin C (MMC) or 0.1 mg/ml neocarzinostatin (NCS) for 1 h, A7 recognizing antigen expression was enhanced until 24 h after the treatments. At a dose that completely suppressed cell growth, increased antigen expression was maintained for 96 h. Therefore, this study suggests that the combined application of an anticancer drug and MAb A7 may be useful for immunotargeting chemotherapy. 1998 Elsevier Science Ireland Ltd. Keywords: Monoclonal antibody; Human pancreatic carcinoma; Anticancer agents; Flow cytometry
1. Introduction The prognosis of patients with pancreatic adenocarcinoma remains poor, with less than 2% of all patients surviving for 5 years [1]. Since there are currently no reliable diagnostic tests for detecting pancreatic carcinoma at an early treatable stage, monoclonal antibodies (MAbs) targeted against tumor-associated antigens may be useful in improving the diagnosis or therapy of this disease. In our laboratory, MAb A7 was produced against a human colon carcinoma. MAb A7 recognizes a 45 kDa glycoprotein on the cell * Corresponding author. Tel.: +81 75 2515527; fax: +81 75 2515522.
surface of colorectal cancer, stomach cancer, pancreatic cancer and breast cancer [3]. MAb A7 that bound to the surface of cancer cells was internalized into target cells. MAb A7 was conjugated covalently with the anticancer antibiotics, neocarzinostatin (NCS) and mitomycin C (MMC) [2,4–6]. The MAb–drug conjugate A7-NCS has already been administered to patients with colorectal carcinoma and has shown promising results [7,8]. In our recent histochemical study, MAb A7 reacted with 73% of the human pancreatic carcinoma cell lines tested [9]. Many other antibody–drug conjugates are less potent in in vitro cytotoxicity assays than the unconjugated drugs [10–12]. A7-NCS was approximately 2.7 times as effective as free NCS against the human pancreatic
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carcinoma cells which reacted with MAb A7 [13]. Moreover, the in vivo antitumor effect of intravenous A7-NCS conjugate was stronger than that of NCS alone. However, the A7-NCS conjugate could not suppress the tumor growth of pancreatic carcinoma cells as completely as that of colon carcinoma cells. This effect is hardly surprising since pancreatic carcinoma is resistant to most chemotherapy. As a method for increasing the efficacy of A7-NCS and A7-MMC conjugates, we propose to combine antibody therapy with other antitumoral agents. Before our attempt, we evaluated the influence of exposure to the antitumoral agents MMC, cis-platinum (CDDP) and NCS on the expression of the antigen recognized by MAb A7. In this study, we observed very interesting findings on the MAb A7-reactive antigen expression following cell exposure to the antitumor agents.
2. Materials and methods 2.1. Cell culture The human pancreatic ductal cell carcinoma cell line HPC-YS was used (a generous gift from Dr Nozomi Yamaguchi, Research Institute of Neurology and Geriatrics, Kyoto Prefectural University of Medicine, Japan) [14]. The cell line was kept in a standard tissue incubator at 37°C (95% air, 5% CO2, 100% relative humidity) and was propagated as a monolayer culture in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Flow Laboratories, Rockville, MD).
to the standard hybridoma technique by Kotanagi et al. [15]. The subclass of MAb A7 was IgG1 and MAb A7 could not mediate in the antibody-dependent cellmediated cytotoxicity (ADCC). MAb A7 has been reported to react with not only more than 50% of the fresh colorectal carcinoma tissues but also with 73% of the pancreatic carcinoma cell lines tested [9]. The pancreatic carcinoma cell line HPC-YS was one of the pancreatic carcinoma cell lines which reacted strongly with MAb A7. Purified mouse IgG was obtained from Zymed Laboratories (San Francisco, CA). FITC-conjugated rabbit anti-mouse immunoglobulin was purchased from DAKO (Glostrup, Denmark). 2.4. Incubation and cell counting HPC-YS cells (2 × 105/flask) were inoculated into 75-cm2 flasks. Forty-eight hours later, the cells received fresh culture medium with or without one of the following antitumor drugs: CDDP (1 and 10 mg/ml), MMC (0.1, 1 and 10 mg/ml) or NCS (0.01, 0.1 and 1 mg/ml). After 1 h of incubation in one of these antitumor drugs, the cells were washed twice with PBS, added to fresh media without any antitumor drugs and incubated at 37°C in a CO2 incubator. The media were changed every 48 h. The cells were harvested in 0.02% EDTA at 24, 48 and 96 h after the removal of the antitumor drugs and cell counts were performed with a hemacytometer. After a wash with PBS, the cells were resuspended in fresh culture medium to a concentration of 1 × 105 cells/ml. These cell suspensions were used as samples for the fluorescence staining procedure. 2.5. Fluorescence staining procedure
2.2. Antitumor drugs Mitomycin C (MMC) (Kyowa-Hakko, Tokyo, Japan), cis-platinum (CDDP) (Bristol-Myers, Tokyo, Japan) and neocarzinostatin (NCS) (Kayaku, Tokyo, Japan) were diluted to the indicated final concentrations in fresh medium and used immediately. 2.3. Antibodies The murine MAb A7 was developed against the human colonic adenocarcinoma, Colon-6, according
HPC-YS cells (1 × 105) were exposed to 30 mg/ml MAb A7 or normal mouse IgG in RPMI medium for 60 min at 37°C in 5% CO2. After three washes in PBS, the cells were fixed in 50% methanol at −20°C for 10 min, washed once with PBS and incubated with FITClabeled rabbit anti-mouse IgG diluted 1:40 in phosphate-buffered saline (PBS) for 30 min at 4°C. Cells were washed twice and stained for 30 min with 5 mg/ ml propiodium iodide (PI) (Sigma, St. Louis, MO) in PBS containing 1 mg/ml of RNase A (Sigma, St. Louis, MO).
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2.6. Expression of the antigen recognized by MAb A7 in each cell cycle phase of HPC-YS cells In order to evaluate the expression level of the antigen recognized by MAb A7 in each phase of the cell cycle, 30 000 cells each at the G0/G1, S or G2/M phases were obtained from the red pulse area versus pulse width dot plot. The mean green fluorescence intensity of each growth phase of the proliferating HPC-YS cells was calculated with the CellFit operating system (Becton Dickinson, San Jose, CA). 2.7. Flow cytometry Cells were analyzed on a FACScan flow cytometer equipped with a 15 mW air-cooled argon ion laser. The laser excitation wavelength was 488 nm and the fluorescence emission was collected after passing through band pass filters (530 nm for FITC and 585 nm for PI). Single and multiparameter histograms of FITC and PI were collected for 30 000 events. Flow cytometry list mode data were analyzed using the CellFit and Lysis operating systems (Becton Dick-
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inson, San Jose, CA). Doublets and debris were discriminated and gated out from the red pulse area versus the pulse width dot plot.
3. Results 3.1. Indirect fluorescence staining of the MAb A7reactive antigen on HPC-YS cells As shown in Fig. 1, the antigen detected by MAb A7 is present mainly on the surface of the cell membrane, while only weak green fluorescence of FITC was observed in the cytoplasm. The mouse control IgG did not bind to the HPC-YS cells. 3.2. Expression of the antigen recognized by MAb A7 in HPC-YS cells during the cell cycle Proliferating HPC-YS cells express the antigen recognized by MAb A7 recognizing antigen throughout the cell cycle, but the level of antigen expression is heterogeneous (Fig. 2). Cell progression through the
Fig. 1. Indirect fluorescence staining of the MAb A7-reactive antigen on HPC-YS cells. The cells were treated as described in the text and stained with propiodium iodide (to stain DNA) and FITC-labeled rabbit anti-mouse IgG (to stain any bound MAb A7 or normal mouse IgG). The antigen recognized by MAb A7 was detected mainly on the cell membrane (left). Positive staining was not observed with normal mouse IgG (right).
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0.1 mg/ml NCS (which transiently inhibit cell growth), the cells continued to accumulate at the G2/M phase until 24 h after the removal of the drugs.
Fig. 2. Expression of the antigen recognized by MAb A7 in each cell cycle phase of HPC-YS cells. The mean green fluorescence intensity indicating MAb A7 binding of 30 000 HPC-YS cells in the G0/G1, S and G2/M phases was calculated with the CellFit operating system. Error bars represent 1 SD.
cell cycle is paralleled by an increase in the per cell expression of antigen and cells in the G2/M phase express this antigen maximally. Antigen expression in S and G2/M phase cells was 1.20 and 1.46 times, respectively, as great as that in G0/G1 phase cells. 3.3. Changes in cell progression of HPC-YS cells and expression of the antigen recognized by MAb A7 after anticancer drug treatments Fig. 3 shows the growth curve of HPC-YS cells treated with various concentrations of the anticancer drugs MMC, CDDP and NCS. All these drugs inhibited cell growth in a dose-dependent manner. Cell division was suppressed completely by incubating the HPC-YS cells for 1 h with 10 mg/ml MMC, 10 mg/ml CDDP or 1 mg/ml NCS. With a concentration of 1 mg/ml MMC or 0.1 mg/ml NCS, cell growth was inhibited for 24 h following exposure but thereafter their rate of growth was the same as that of the untreated cells. To investigate the effects of these three anticancer agents on the cell cycle progression of HPC-YS cells, the DNA content in HPC-YS nuclei was measured by flow cytometric analysis. As shown in Fig. 4, the use of a dose which completely suppressed cell growth did not immediately arrest the progression of cells through the cell cycle but caused cells to accumulate at the G2/M phase and become over tetraploid for longer than 24 h. With doses of 1 mg/ml MMC or
Fig. 3. Inhibition of growth of HPC-YS cells by antitumoral agents. Cells (2 × 105/flask) were inoculated into 75-cm2 flasks. Fortyeight hours later, the cells received fresh culture medium with or without one of the following antitumor drugs: (A) MMC (0.1, 1 and 10 mg/ml); (B) CDDP (1 and 10 mg/ml); (C) NCS (0.01, 0.1 and 1 mg/ml). At various times later (0–96 h), aliquots from each flask were removed and the number of surviving cells was counted.
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Fig. 5 shows each three-dimensional isometric display of the DNA content (Y-axis), fluorescence intensity, i.e. cell surface expression of the A7 reactive antigen (X-axis), and the number of cells (Z-axis).
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When a concentration of antitumoral drugs was used which suppressed cell growth entirely, i.e. 10 mg/ml MMC, 10 mg/ml CDDP and 1 mg/ml NCS, upregulation of the antigen recognized by MAb A7 as well as a change in cell progression were observed and the expression of the antigen was enhanced out of scale. Moreover, for 24 h following the removal of 1 mg/ml MMC or 0.1 mg/ml NCS (doses that transiently suppressed cell division), A7 recognizing antigen expression increased out of scale in synchrony with the suppression of cell division.
4. Discussion
Fig. 4. Cell cycle analysis of HPC-YS cells after 1 h of anticancer drug treatment. After 1 h of incubation in anticancer drug, the cells were analyzed on a FACScan flow cytometer. (A) MMC-treated cells; (B) CDDP-treated cells; (C) NCS-treated cells. MMC, mitomycin C; CDDP, cis-platinum; NCS, neocarzinostatin.
Previous reports have indicated that the heterogeneity of target antigen expression, which is a characteristic of human carcinoma cell populations, and the existence of cells within the population which exhibit only weak expression of the target antigen can allow cancer cells to escape immunotargeting therapy because these cells do not bind enough of the appropriate antibody to kill the cells [16–18]. In this study we found that there was a difference in A7-reactive tumor-associated antigen expression during different phases of the cell cycle, with maximal expression at the G2/M phase. Therefore, we postulated that by increasing the percentage of cells at the G2/M phase, we could reduce antigenic heterogeneity and increase the efficacy of immunotargeting therapy. We used various types of chemotherapy which caused cells to accumulate at the G2/M phase. The first phase of this study was to examine the modulation of the expression of MAb A7-reactive antigen during the cell cycle. This required techniques which could simultaneously measure both the effect of the chemotherapy on cell cycle progression and the amount of the expression of the antigen recognized by MAb A7. We used flow cytometry to double stain the cells; we stained the DNA in the nucleus with propiodium iodide and the tumor-associated antigen on the cell membrane with FITC-labeled antibody [19]. Using this technique, we could measure the effects of the anticancer drugs against the cancer cells by recording the number of cells which had accumulated at the G2/ M phase. When we bathed the cells in 1 mg/ml MMC or 0.1 mg/ml NCS for only 1 h, the cell cycle distribution
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changed transiently as cells accumulated in the S and G2/M phases. This phenomenon may mimic the clinical situation when chemotherapeutic agents are administered intravenously but are not tumoricidal
Fig. 5. Expression of the A7 recognizing antigen in HPC-YS cells after the anticancer drug treatments. Each three-dimensional isometric display of DNA content (Y-axis), fluorescence intensity, i.e. cell surface expression of the A7 reactive antigen (X-axis) and the number of cells (Z-axis) is depicted in this figure. (A) MMC-treated cells; (B) CDDP-treated cells; (C) NCS-treated cells. MMC, mitomycin C; CDDP, cis-platinum; NCS, neocarzinostatin.
because they are present for only a relatively short time. However, the expression of the antigen recognized by MAb A7 may be increasing during this time, suggesting that this would be a better time for immunotargeting chemotherapy and tumor imaging. When we used a dose of chemotherapy that completely suppressed cell growth, the cells accumulated at the G2/M phase and their DNA content increased to over tetraploid while the increased reactivity of the MAb A7 was maintained. If such a condition can be achieved clinically, then using immunotargeting therapy during this time might selectively increase tumor cell death. This combined effect would be expected to be multiplicative. Monoclonal antibodies that localize to tumors are potentially valuable in the diagnosis and therapy of cancers. Several investigators have reported successful immunolocalization clinically, but more frequently such attempts have met with limited success, i.e. a low absolute amount of the injectate localized to the tumor sites. Such small amounts of antibody are hardly sufficient to allow the successful imaging of small lesions undiagnosed by conventional radiological techniques. They also are certainly not sufficient to deliver tumoricidal amounts of cytotoxic agents to the tumor cells without also damaging other normal organs such as the bone marrow, liver and kidney. The sensitivity of this approach is influenced by a number of variables, among which is the level of tumor-associated antigen expressed by tumor cell populations. Techniques that can increase the expression level of tumor-associated antigens have the ability to overcome this limitation. The modulation of tumor-specific antigen expression by drugs has been reported by several authors. Enhancing tumor-associated antigen expression by interferon treatment has been reported by many investigators [20–27], while other reports have focused on other agents. Muraoka et al. [28] reported that the a-fetoprotein (AFP)-secreting capacity per cell in human hepatocellular carcinoma and hepatoblastoma cell lines remained unchanged after in vitro chemotherapy with MMC, ADM, 5-FU or CDDP. Maas et al. [29] investigated with flow cytometric analysis the in vitro antiproliferative capacity of 5-FU and 5-fluoro-2′-deoxyuridine (FUdR) as well as their effects on the expression of carcinoembryonic antigen (CEA) in human colorectal cancer cell lines.
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They concluded that the expression of CEA was increased by 5-FU and FUdR but no relationship was found between the enhancement of CEA expression and changes in the cell cycle distribution upon exposure to these anti-metabolites. It has also been reported by different investigators [30,31] that the treatment of human cell lines with analogues of cyclic AMP selectively enhanced the expression of CEA or CA125. We revealed in this study that the antigen recognized by MAb A7 was expressed heterogeneously throughout the cell cycle of the pancreas carcinoma cell line and after treating the cells with anticancer drugs, which caused the cells to accumulate at the G2/M phase. However, the expression of this antigen was higher in the treated cells than in the untreated G2/M cells, indicating that this enhancing mechanism was not completely explained only by the changes in the cell cycle distribution of the cells. Nonetheless, this study suggests that the combined application of anticancer drugs and MAb A7 may be useful for the immunodetection and immunotargeting of chemotherapy against human pancreatic carcinoma.
Acknowledgements This work was supported by a grant-in-aid for the comprehensive ten-year strategy for cancer control from the Ministry of Health and Welfare, Japan.
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Changes in expression of the antigen recognized by monoclonal antibody A7 in human pancreatic carcinoma cells following exposure to anticancer agents Toshiharu Yamaguchi*, Nobuki Yamaoka, Kazuya Kitamura, Eigo Otsuji, Kazuma Okamoto, Hiroshi Tsuruta, Yoshihiro Yata, Toshio Takahashi First Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto 602, Japan Received 3 September 1997; received in revised form 25 November 1997; accepted 15 December 1997
Abstract Techniques which can increase the expression level of tumor-associated antigens may improve immunotargeting therapy. We studied the reactivity of MAb A7 toward an antigen expressed on the surface of the human pancreatic cancer cell line HPC-YS after treatment with various antitumoral agents. When we applied 1 mg/ml mitomycin C (MMC) or 0.1 mg/ml neocarzinostatin (NCS) for 1 h, A7 recognizing antigen expression was enhanced until 24 h after the treatments. At a dose that completely suppressed cell growth, increased antigen expression was maintained for 96 h. Therefore, this study suggests that the combined application of an anticancer drug and MAb A7 may be useful for immunotargeting chemotherapy. 1998 Elsevier Science Ireland Ltd. Keywords: Monoclonal antibody; Human pancreatic carcinoma; Anticancer agents; Flow cytometry
1. Introduction The prognosis of patients with pancreatic adenocarcinoma remains poor, with less than 2% of all patients surviving for 5 years [1]. Since there are currently no reliable diagnostic tests for detecting pancreatic carcinoma at an early treatable stage, monoclonal antibodies (MAbs) targeted against tumor-associated antigens may be useful in improving the diagnosis or therapy of this disease. In our laboratory, MAb A7 was produced against a human colon carcinoma. MAb A7 recognizes a 45 kDa glycoprotein on the cell * Corresponding author. Tel.: +81 75 2515527; fax: +81 75 2515522.
surface of colorectal cancer, stomach cancer, pancreatic cancer and breast cancer [3]. MAb A7 that bound to the surface of cancer cells was internalized into target cells. MAb A7 was conjugated covalently with the anticancer antibiotics, neocarzinostatin (NCS) and mitomycin C (MMC) [2,4–6]. The MAb–drug conjugate A7-NCS has already been administered to patients with colorectal carcinoma and has shown promising results [7,8]. In our recent histochemical study, MAb A7 reacted with 73% of the human pancreatic carcinoma cell lines tested [9]. Many other antibody–drug conjugates are less potent in in vitro cytotoxicity assays than the unconjugated drugs [10–12]. A7-NCS was approximately 2.7 times as effective as free NCS against the human pancreatic
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carcinoma cells which reacted with MAb A7 [13]. Moreover, the in vivo antitumor effect of intravenous A7-NCS conjugate was stronger than that of NCS alone. However, the A7-NCS conjugate could not suppress the tumor growth of pancreatic carcinoma cells as completely as that of colon carcinoma cells. This effect is hardly surprising since pancreatic carcinoma is resistant to most chemotherapy. As a method for increasing the efficacy of A7-NCS and A7-MMC conjugates, we propose to combine antibody therapy with other antitumoral agents. Before our attempt, we evaluated the influence of exposure to the antitumoral agents MMC, cis-platinum (CDDP) and NCS on the expression of the antigen recognized by MAb A7. In this study, we observed very interesting findings on the MAb A7-reactive antigen expression following cell exposure to the antitumor agents.
2. Materials and methods 2.1. Cell culture The human pancreatic ductal cell carcinoma cell line HPC-YS was used (a generous gift from Dr Nozomi Yamaguchi, Research Institute of Neurology and Geriatrics, Kyoto Prefectural University of Medicine, Japan) [14]. The cell line was kept in a standard tissue incubator at 37°C (95% air, 5% CO2, 100% relative humidity) and was propagated as a monolayer culture in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Flow Laboratories, Rockville, MD).
to the standard hybridoma technique by Kotanagi et al. [15]. The subclass of MAb A7 was IgG1 and MAb A7 could not mediate in the antibody-dependent cellmediated cytotoxicity (ADCC). MAb A7 has been reported to react with not only more than 50% of the fresh colorectal carcinoma tissues but also with 73% of the pancreatic carcinoma cell lines tested [9]. The pancreatic carcinoma cell line HPC-YS was one of the pancreatic carcinoma cell lines which reacted strongly with MAb A7. Purified mouse IgG was obtained from Zymed Laboratories (San Francisco, CA). FITC-conjugated rabbit anti-mouse immunoglobulin was purchased from DAKO (Glostrup, Denmark). 2.4. Incubation and cell counting HPC-YS cells (2 × 105/flask) were inoculated into 75-cm2 flasks. Forty-eight hours later, the cells received fresh culture medium with or without one of the following antitumor drugs: CDDP (1 and 10 mg/ml), MMC (0.1, 1 and 10 mg/ml) or NCS (0.01, 0.1 and 1 mg/ml). After 1 h of incubation in one of these antitumor drugs, the cells were washed twice with PBS, added to fresh media without any antitumor drugs and incubated at 37°C in a CO2 incubator. The media were changed every 48 h. The cells were harvested in 0.02% EDTA at 24, 48 and 96 h after the removal of the antitumor drugs and cell counts were performed with a hemacytometer. After a wash with PBS, the cells were resuspended in fresh culture medium to a concentration of 1 × 105 cells/ml. These cell suspensions were used as samples for the fluorescence staining procedure. 2.5. Fluorescence staining procedure
2.2. Antitumor drugs Mitomycin C (MMC) (Kyowa-Hakko, Tokyo, Japan), cis-platinum (CDDP) (Bristol-Myers, Tokyo, Japan) and neocarzinostatin (NCS) (Kayaku, Tokyo, Japan) were diluted to the indicated final concentrations in fresh medium and used immediately. 2.3. Antibodies The murine MAb A7 was developed against the human colonic adenocarcinoma, Colon-6, according
HPC-YS cells (1 × 105) were exposed to 30 mg/ml MAb A7 or normal mouse IgG in RPMI medium for 60 min at 37°C in 5% CO2. After three washes in PBS, the cells were fixed in 50% methanol at −20°C for 10 min, washed once with PBS and incubated with FITClabeled rabbit anti-mouse IgG diluted 1:40 in phosphate-buffered saline (PBS) for 30 min at 4°C. Cells were washed twice and stained for 30 min with 5 mg/ ml propiodium iodide (PI) (Sigma, St. Louis, MO) in PBS containing 1 mg/ml of RNase A (Sigma, St. Louis, MO).
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2.6. Expression of the antigen recognized by MAb A7 in each cell cycle phase of HPC-YS cells In order to evaluate the expression level of the antigen recognized by MAb A7 in each phase of the cell cycle, 30 000 cells each at the G0/G1, S or G2/M phases were obtained from the red pulse area versus pulse width dot plot. The mean green fluorescence intensity of each growth phase of the proliferating HPC-YS cells was calculated with the CellFit operating system (Becton Dickinson, San Jose, CA). 2.7. Flow cytometry Cells were analyzed on a FACScan flow cytometer equipped with a 15 mW air-cooled argon ion laser. The laser excitation wavelength was 488 nm and the fluorescence emission was collected after passing through band pass filters (530 nm for FITC and 585 nm for PI). Single and multiparameter histograms of FITC and PI were collected for 30 000 events. Flow cytometry list mode data were analyzed using the CellFit and Lysis operating systems (Becton Dick-
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inson, San Jose, CA). Doublets and debris were discriminated and gated out from the red pulse area versus the pulse width dot plot.
3. Results 3.1. Indirect fluorescence staining of the MAb A7reactive antigen on HPC-YS cells As shown in Fig. 1, the antigen detected by MAb A7 is present mainly on the surface of the cell membrane, while only weak green fluorescence of FITC was observed in the cytoplasm. The mouse control IgG did not bind to the HPC-YS cells. 3.2. Expression of the antigen recognized by MAb A7 in HPC-YS cells during the cell cycle Proliferating HPC-YS cells express the antigen recognized by MAb A7 recognizing antigen throughout the cell cycle, but the level of antigen expression is heterogeneous (Fig. 2). Cell progression through the
Fig. 1. Indirect fluorescence staining of the MAb A7-reactive antigen on HPC-YS cells. The cells were treated as described in the text and stained with propiodium iodide (to stain DNA) and FITC-labeled rabbit anti-mouse IgG (to stain any bound MAb A7 or normal mouse IgG). The antigen recognized by MAb A7 was detected mainly on the cell membrane (left). Positive staining was not observed with normal mouse IgG (right).
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0.1 mg/ml NCS (which transiently inhibit cell growth), the cells continued to accumulate at the G2/M phase until 24 h after the removal of the drugs.
Fig. 2. Expression of the antigen recognized by MAb A7 in each cell cycle phase of HPC-YS cells. The mean green fluorescence intensity indicating MAb A7 binding of 30 000 HPC-YS cells in the G0/G1, S and G2/M phases was calculated with the CellFit operating system. Error bars represent 1 SD.
cell cycle is paralleled by an increase in the per cell expression of antigen and cells in the G2/M phase express this antigen maximally. Antigen expression in S and G2/M phase cells was 1.20 and 1.46 times, respectively, as great as that in G0/G1 phase cells. 3.3. Changes in cell progression of HPC-YS cells and expression of the antigen recognized by MAb A7 after anticancer drug treatments Fig. 3 shows the growth curve of HPC-YS cells treated with various concentrations of the anticancer drugs MMC, CDDP and NCS. All these drugs inhibited cell growth in a dose-dependent manner. Cell division was suppressed completely by incubating the HPC-YS cells for 1 h with 10 mg/ml MMC, 10 mg/ml CDDP or 1 mg/ml NCS. With a concentration of 1 mg/ml MMC or 0.1 mg/ml NCS, cell growth was inhibited for 24 h following exposure but thereafter their rate of growth was the same as that of the untreated cells. To investigate the effects of these three anticancer agents on the cell cycle progression of HPC-YS cells, the DNA content in HPC-YS nuclei was measured by flow cytometric analysis. As shown in Fig. 4, the use of a dose which completely suppressed cell growth did not immediately arrest the progression of cells through the cell cycle but caused cells to accumulate at the G2/M phase and become over tetraploid for longer than 24 h. With doses of 1 mg/ml MMC or
Fig. 3. Inhibition of growth of HPC-YS cells by antitumoral agents. Cells (2 × 105/flask) were inoculated into 75-cm2 flasks. Fortyeight hours later, the cells received fresh culture medium with or without one of the following antitumor drugs: (A) MMC (0.1, 1 and 10 mg/ml); (B) CDDP (1 and 10 mg/ml); (C) NCS (0.01, 0.1 and 1 mg/ml). At various times later (0–96 h), aliquots from each flask were removed and the number of surviving cells was counted.
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Fig. 5 shows each three-dimensional isometric display of the DNA content (Y-axis), fluorescence intensity, i.e. cell surface expression of the A7 reactive antigen (X-axis), and the number of cells (Z-axis).
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When a concentration of antitumoral drugs was used which suppressed cell growth entirely, i.e. 10 mg/ml MMC, 10 mg/ml CDDP and 1 mg/ml NCS, upregulation of the antigen recognized by MAb A7 as well as a change in cell progression were observed and the expression of the antigen was enhanced out of scale. Moreover, for 24 h following the removal of 1 mg/ml MMC or 0.1 mg/ml NCS (doses that transiently suppressed cell division), A7 recognizing antigen expression increased out of scale in synchrony with the suppression of cell division.
4. Discussion
Fig. 4. Cell cycle analysis of HPC-YS cells after 1 h of anticancer drug treatment. After 1 h of incubation in anticancer drug, the cells were analyzed on a FACScan flow cytometer. (A) MMC-treated cells; (B) CDDP-treated cells; (C) NCS-treated cells. MMC, mitomycin C; CDDP, cis-platinum; NCS, neocarzinostatin.
Previous reports have indicated that the heterogeneity of target antigen expression, which is a characteristic of human carcinoma cell populations, and the existence of cells within the population which exhibit only weak expression of the target antigen can allow cancer cells to escape immunotargeting therapy because these cells do not bind enough of the appropriate antibody to kill the cells [16–18]. In this study we found that there was a difference in A7-reactive tumor-associated antigen expression during different phases of the cell cycle, with maximal expression at the G2/M phase. Therefore, we postulated that by increasing the percentage of cells at the G2/M phase, we could reduce antigenic heterogeneity and increase the efficacy of immunotargeting therapy. We used various types of chemotherapy which caused cells to accumulate at the G2/M phase. The first phase of this study was to examine the modulation of the expression of MAb A7-reactive antigen during the cell cycle. This required techniques which could simultaneously measure both the effect of the chemotherapy on cell cycle progression and the amount of the expression of the antigen recognized by MAb A7. We used flow cytometry to double stain the cells; we stained the DNA in the nucleus with propiodium iodide and the tumor-associated antigen on the cell membrane with FITC-labeled antibody [19]. Using this technique, we could measure the effects of the anticancer drugs against the cancer cells by recording the number of cells which had accumulated at the G2/ M phase. When we bathed the cells in 1 mg/ml MMC or 0.1 mg/ml NCS for only 1 h, the cell cycle distribution
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changed transiently as cells accumulated in the S and G2/M phases. This phenomenon may mimic the clinical situation when chemotherapeutic agents are administered intravenously but are not tumoricidal
Fig. 5. Expression of the A7 recognizing antigen in HPC-YS cells after the anticancer drug treatments. Each three-dimensional isometric display of DNA content (Y-axis), fluorescence intensity, i.e. cell surface expression of the A7 reactive antigen (X-axis) and the number of cells (Z-axis) is depicted in this figure. (A) MMC-treated cells; (B) CDDP-treated cells; (C) NCS-treated cells. MMC, mitomycin C; CDDP, cis-platinum; NCS, neocarzinostatin.
because they are present for only a relatively short time. However, the expression of the antigen recognized by MAb A7 may be increasing during this time, suggesting that this would be a better time for immunotargeting chemotherapy and tumor imaging. When we used a dose of chemotherapy that completely suppressed cell growth, the cells accumulated at the G2/M phase and their DNA content increased to over tetraploid while the increased reactivity of the MAb A7 was maintained. If such a condition can be achieved clinically, then using immunotargeting therapy during this time might selectively increase tumor cell death. This combined effect would be expected to be multiplicative. Monoclonal antibodies that localize to tumors are potentially valuable in the diagnosis and therapy of cancers. Several investigators have reported successful immunolocalization clinically, but more frequently such attempts have met with limited success, i.e. a low absolute amount of the injectate localized to the tumor sites. Such small amounts of antibody are hardly sufficient to allow the successful imaging of small lesions undiagnosed by conventional radiological techniques. They also are certainly not sufficient to deliver tumoricidal amounts of cytotoxic agents to the tumor cells without also damaging other normal organs such as the bone marrow, liver and kidney. The sensitivity of this approach is influenced by a number of variables, among which is the level of tumor-associated antigen expressed by tumor cell populations. Techniques that can increase the expression level of tumor-associated antigens have the ability to overcome this limitation. The modulation of tumor-specific antigen expression by drugs has been reported by several authors. Enhancing tumor-associated antigen expression by interferon treatment has been reported by many investigators [20–27], while other reports have focused on other agents. Muraoka et al. [28] reported that the a-fetoprotein (AFP)-secreting capacity per cell in human hepatocellular carcinoma and hepatoblastoma cell lines remained unchanged after in vitro chemotherapy with MMC, ADM, 5-FU or CDDP. Maas et al. [29] investigated with flow cytometric analysis the in vitro antiproliferative capacity of 5-FU and 5-fluoro-2′-deoxyuridine (FUdR) as well as their effects on the expression of carcinoembryonic antigen (CEA) in human colorectal cancer cell lines.
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They concluded that the expression of CEA was increased by 5-FU and FUdR but no relationship was found between the enhancement of CEA expression and changes in the cell cycle distribution upon exposure to these anti-metabolites. It has also been reported by different investigators [30,31] that the treatment of human cell lines with analogues of cyclic AMP selectively enhanced the expression of CEA or CA125. We revealed in this study that the antigen recognized by MAb A7 was expressed heterogeneously throughout the cell cycle of the pancreas carcinoma cell line and after treating the cells with anticancer drugs, which caused the cells to accumulate at the G2/M phase. However, the expression of this antigen was higher in the treated cells than in the untreated G2/M cells, indicating that this enhancing mechanism was not completely explained only by the changes in the cell cycle distribution of the cells. Nonetheless, this study suggests that the combined application of anticancer drugs and MAb A7 may be useful for the immunodetection and immunotargeting of chemotherapy against human pancreatic carcinoma.
Acknowledgements This work was supported by a grant-in-aid for the comprehensive ten-year strategy for cancer control from the Ministry of Health and Welfare, Japan.
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