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Endocrinology Vol. 138, No. 4 1697-1703
Copyright © 1997 by The Endocrine Society

ARTICLES

A Monoclonal Antibody against Rat Calcitonin Inhibits the Growth of a Rat Medullary Thyroid Carcinoma Cell Line in Vitro

Rusheng Zhang and Leslie J. DeGroot

Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Medullary thyroid carcinoma (MTC) cells synthesize large amounts of calcitonin (CT), which serves clinically as a useful tumor marker. To examine the possibility of CT serving as a target in immunotherapy for MTC, we raised and characterized more than 40 monoclonal antibodies (mAbs) against rat CT (rCT). The affinity constants for the mAbs were between 2.8 x 109 and 1.8 x 1011 M-1. Some mAbs react preferentially with solid phase rat CT, but not with liquid phase 125I-labeled rCT. Thirty-nine mAbs cross-react with human CT.

We evaluated the antitumor effect of the mAbs in vitro by analysis of [3H]thymidine incorporation into the rat MTC cell line CRL-1607. Some antibodies show an antiproliferative effect, but most are inactive. One mAb (2E5G5, IgG2b), which preferentially reacts with solid phase rCT, but not with liquid phase 125I-labeled rCT, exerts an antiproliferative activity on CRL-1607. At 6.25 x 10-7 M, 2E5G5 killed all of the tumor cells independently of complement in a cytotoxicity assay. We explored the cytotoxic mechanisms by assays for cell cycle arrest and DNA fragmentation. The antitumor effect was manifested by apoptosis and cell cycle arrest. Hence, a secreted peptide may serve as a target in tumor immunotherapy. Therapeutically antibodies may exert antitumor activity by a variety of mechanisms. The antitumor effect of this mAb in a rat animal tumor model is being tested.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MANY TUMORS express specific antigens that can be recognized by T cells, but these antigens rarely induce effective humoral immune reactions (1). Since few unique tumor-specific antigens have been identified, research in tumor immunology is often focused on tumor-associated target antigens. These antigens include oncofetal antigens such as {alpha}-fetoprotein, carcinoembryonic antigen, and other surface proteins, including epidermal growth factor (EGF) receptors and mucin. Some circulating tumor-associated antigens (TAA), such as {alpha}-fetoprotein and carcinoembryonic antigen, are present on the surface of tumor cells and serve as targets for tumor immunotherapy in animal models or clinical trials (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). A problem in using TAA for tumor immunotherapy is that the antigens are also produced and expressed on normal tissue cells, and in some tumors, the TAA are not suitable as an immunotherapeutic target because of their low expression level.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, always synthesizes and secretes large amounts of calcitonin (CT). Production of CT is mainly limited to C cells and MTC cells, and high levels of serum CT always indicate C cell neoplasm.

To investigate the possibility of CT serving as a target antigen for immunotherapy of MTC, we produced more than 40 monoclonal antibodies (mAbs) to rat CT (rCT). The effects of the mAbs were tested on a rat MTC (rMTC) cell line, CRL 1607. One of the mAbs effectively inhibits the growth of the cell line and has no effect on control cell lines.

The antitumor effector function of unconjugated mAbs in cancer therapy is complex. Mechanisms such as antibody-dependent cellular cytotoxicity, and complement-mediated cytolysis are very clear, and induction of apoptosis and cell cycle arrest also have been observed (15, 16). Our data show that the inhibitory effect was associated with apoptosis and cell cycle arrest.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of mAbs to rCT
mAbs were raised using an immunization protocol previously described, with modifications (17), and approved by the institutional animal care committee. Synthetic rCT conjugated to carrier protein keyhole limpet hemocyanin (Worthington Biochemical Corp., Freehold, NJ) was used to immunize 4-week-old female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) in the hind footpad. The spleen and popliteal lymphocytes were fused with the mouse myeloma cell line P3-NS-1–1Ag4–1 (P3, kindly provided by Dr. Jose Quintans, University of Chicago) according to a previously described procedure (18). Supernatants of growing hybridomas were screened using an enzyme-linked immunosorbent assay (ELISA) with solid phase rCT, and the positive cells were cloned by limiting dilution. Specific hybridomas were expanded for antibody production. mAbs were purified from ascites fluid by use of a protein A column (19). The affinity constants of the mAbs were calculated using Scatchard analysis (20). The determination of isotypes of mAbs was carried out using an ELISA method (21).

Tumor cell lines
A rMTC cell line (CRL-1607) and a human MTC (hMTC) cell line (CRL-1806) were purchased from American Type Culture Collection (Rockville, MD). CRL-1607 or CRL-1806 cell lines were maintained in culture by serial passage in DMEM or RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (complete medium). HepG2 (a human hepatoma cell line, kindly provided by Dr. Samuel Refetoff, University of Chicago) and GH3 (a rat GH tumor cell line, a gift from Dr. Herbert Samuels, New York University, New York, NY) cell lines were maintained in complete DMEM medium. Chinese hamster ovary (CHO) cells (a gift from Dr. Gilbert Vassart, Free University, Brussels, Belgium) were maintained in complete RPMI 1640. The cells were grown in a humidified atmosphere of 5% CO2 and air. Cell viability was determined by trypan blue exclusion.

Effect of anti-rCT mAbs in vitro
The activities of various mAbs on growth of rMTC were determined using a [3H]thymidine incorporation assay, as described previously (22). Cells (5 x 103/well) were plated in 180 µl culture medium into 96-well microtiter plates (Costar, Cambridge, MA). Twenty microliters of various mAb solutions were added. The plates were incubated for 72 h. [3H]Thymidine (ICN Pharmaceuticals, Irvine, CA) was added at 1.0 µCi/well in 20 µl solution for the last 18 h of this incubation. Cells were harvested onto filter paper using a cell harvester (Cambridge Technology, Watertown, MA). The incorporated radioactivity was determined by liquid scintillation counting. [3H]Thymidine incorporation was calculated and expressed as a percentage of that in untreated controls. All cultures were performed in triplicate.

Cytotoxic activity on CRL1607
The in vitro cytotoxicity of the mAbs on CRL 1607 was investigated by measuring cell survival using trypan blue exclusion. CRL 1607 cells (5 x 105) in the exponential growth stage were plated into 12-well plates in 1 ml culture medium containing various concentration of mAbs. After incubation at 37 C for 48 h, the cells were trypsinized, and the surviving cells were counted. Four other cell lines (GH3, HepG2, CHO, and CRL 1806) were studied under the same conditions to serve as controls.

Analysis of cell cycle progression
Cell cycle status was examined by flow cytometric analysis using the DNA-binding dye propidium iodide (PI; Sigma Chemical Co., St. Louis, MO). CRL 1607 cells (5 x 106) were incubated for 24 h at 37 C with either medium (control) or various mAbs. The concentration of mAbs was 200 µg/ml. Cells were harvested by trypsinization, washed once with complete medium, and then washed twice with sample buffer (0.1% glucose and 10 mM PBS without Mg2+ or Ca2+, pH 7.2). Cells were resuspended in sample buffer, and cell number was determined. The cell concentration was adjusted to 2 x 106 cells/ml in sample buffer. One milliliter of cell suspension was transferred into a 15 x 75-mm centrifuge tube and centrifuged for 10 min at 400 x g and 4 C. After carefully pouring off the supernatant, the cells were fixed with cold 70% ethanol for 24 h at 4 C. Cells were centrifuged, and the ethanol supernatant was removed. Freshly prepared PI staining solution (50 µg/ml PI and 100 U/ml ribonuclease in sample buffer) was added with gentle vortexing, and cells were incubated at room temperature for 30 min. After filtration through a 50-µm nylon mesh, samples were analyzed by flow cytometry (FACScan, Becton Dickinson Immunocytometry Systems, San Jose, CA) (23).

Examination of apoptosis by analysis of fragmented DNA
DNA fragmentation was determined as previously described (24, 25). CRL 1607 cells (1 x 106) were treated with various mAbs at 37 C for 24 h as described above, and untreated cells were used as a negative control or heated at 45 C for 1 h before incubation as a positive control (26). The cells were collected and washed by centrifugation at 200 x g for 10 min. The cell pellet was lysed with 0.5 ml hypotonic lysing buffer (TTE; 5 mM Tris, 5 mM EDTA, and 0.5% Triton X-100, pH 7.5). The lysates were immediately centrifuged at 13,000 x g for 15 min. Supernatants containing fragmented DNA were collected. Samples were extracted once with phenol plus chloroform (1:1, vol/vol) and then once with chloroform. The fragmented DNA was precipitated overnight at -20 C in 50% isopropanol and 130 mM NaCl. The precipitates were collected after centrifugation at 13,000 x g for 10 min. Pellets were air-dried and dissolved in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) at room temperature. The DNA extracts were analyzed on a 1.5% agarose gel that had been soaked overnight in distilled water containing 0.0001% ethidium bromide.

Quantitation of fragmented DNA
CRL-1607 cells were labeled by adding 1 µCi/ml [3H]thymidine to newly subcultured cells in complete medium in a 75-cm2 flask for 18 h. Cells were washed three times with 10 ml prewarmed medium. Cells were resuspended in complete medium, treated under different conditions (untreated cells as control), and incubated at 37 C for 24 h. Cells were transferred to 1.5-ml microcentrifuge tubes (labeled B) and centrifuged at 200 x g for 10 min at 4 C. Supernatants were transferred to another tube, labeled S, and set aside. Hypotonic solution (0.5 ml; TTE) was added to cell pellets in tube B and vortexed vigorously. Fragmented DNA was separated from intact chromatin as described above. Supernatants were transferred to a tube labeled T. Radioactivity in each tube was measured using a liquid scintillation counter. The percentage of fragmented DNA was calculated according to the following formula: % fragmented DNA = [(S + T)/(S + T + B)] x 100%.

Statistics
The statistical significance of the observations using various treatments or cell lines was determined by Student’s t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of mAbs to rCT
Table 1Go summarizes the characterization of mAbs raised to rCT. The mAbs detected have a high affinity constant (2.8 x 109 to 1.83 x 1011 M-1). We could not calculate the Ka values of some mAbs because they did not react with 125I-labeled rCT in liquid phase, but preferentially reacted with solid phase rCT. Cross-reaction with hCT was assayed using an ELISA method. Although there are only two amino acid differences in the sequence, several mAbs to rCT did not react or showed low reactivity to hCT. Cross-reactive mAbs to hCT were also used for immunohistochemical studies of hMTC samples. mAb 2E5G5 and other selected cross-reactive mAbs showed very strong positive cell staining of hMTC tissue (27).


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  		Table 1. Characterization of mAbs to rat calcitonin

 
Inhibitory effect of mAbs on growth of CRL 1607 in vitro
We examined the effects of various mAbs on proliferation of CRL 1607 in vitro. As shown in Fig. 1Go, 2EGo5G5 efficiently inhibited the incorporation of [3H]thymidine into this cell line, with an average IC50 of 2 x 10-8 M. mAbs 4H4E7, 11B7D4, 2H8C1, 2H10G8, and 1C10D8 had a slight inhibitory effect compared to 2E5G5.



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  		Figure 1. [3H]Thymidine incorporation in CRL 1607 cells pretreated with various mAbs. CRL 1607 cells (5 x 103/well·200 µl) were incubated with various concentrations of mAbs (1 x 10-13 to 1 x 10-6 M) for 54 h, then pulsed with [3H]thymidine for 18 h and harvested, and isotope incorporation was determined. Incorporation in cells treated with mAbs (percentage of that in control medium without mAb) was plotted against the concentration of mAb. Each point represents the mean percentage of triplicate determinations of [3H]thymidine incorporation. The SD never exceeded 10%. Data are representa-tive of four independent experiments. [3H]Thymidine incorporation in the presence of 2E5G5 is significantly different from that in the presence of other antibodies at concentrations of 10-6–10-8 M (P < 0.05).

 


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  		Figure 2. [3H]Thymidine incorporation in various cell lines pretreated with 2E5G5 mAb. Each point represents the mean percentage of triplicate determinations of [3H]thymidine incorporation. The SD was always less than 8% of the mean. Data are representative of three independent experiments. [3H]Thymidine incorporation in the CRL 1607 cell line is significantly different from that in other cell lines at antibody concentrations of 10-6–10-8 M (P < 0.05).

 
We next determined whether the effect of 2E5G5 on the CRL 1607 cell line was unique to these cells. Three cell lines (HepG2, GH3, and CHO), which do not produce CT, and CRL 1806, which produces a large amount of hCT, were tested. As shown in Fig. 2Go, the growth of hMTC cells was slightly inhibited by a high concentration of 2E5G5, whereas other cell lines were not affected by 2E5G5. Other mAbs were studied under the same conditions, and no effect was found at any concentration (data not shown).

When we performed our experiments, all mAbs used were purified by the protein A method and dialyzed at least three times in PBS (24 h, three times) to avoid any contamination. Different batches of purified mAbs were used for the study, and the same results were obtained.

Cytotoxicity of 2E5G5 in vitro
As shown in Fig. 3AGo, 2EGo5G5 was highly cytotoxic to CRL 1607. At a concentration of 10-6 M, 2E5G5 killed almost all of the cells after 48-h incubation, whereas other mAbs did not show visible cytotoxicity. We also checked the cytotoxic effect on four other cell lines (CRL 1806, HepG2, GH3, and CHO) under the same conditions. As shown in Fig. 3BGo, 2EGo5G5 has no cytotoxic effect on these four cell lines (Fig. 3BGo).



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  		Figure 3. Cytotoxic effect of various mAbs on CRL 1607 cells (A) and of mAb 2E5G5 on various cell lines (B). Each point represents the mean of triplicate assays of surviving cells (percentage of control). The SD was always less than 10%. Data are representative of three independent experiments. The numbers of CRL 1607 cells surviving after treatment with 2E5G5 were significantly reduced at concentrations of 10-6-10-8 M (P < 0.05).

 
As a logical action of 2E5G5 might be to bind CT present in the culture medium, we checked the possibility that added rCT inhibited the cytotoxic effect of 2E5G5 mAb on CRL 1607 cells. When a high concentration of rCT (100 µg/ml) was added to culture medium before treatment with 2E5G5, rCT did not inhibit the cytotoxicity on the cells induced by the specific mAb (data not shown).

To further confirm the cytotoxic effect of 2E5G5, CRL 1607 cells were treated with active or heat-inactivated 2E5G5 mAb. After 48-h incubation at 37 C, cells treated with active 2E5G5 died; cells treated with inactive 2E5G5 were still alive and showed no difference compared with control cells (data not show).

Mechanisms of cytoxic effect on CRL 1607 cells
We next determined whether the inhibitory effect of 2E5G5 was attributable to cell cycle arrest or apoptosis. Figure 4Go shows a representative FACS profile, and Table 2Go summarizes the data concerning cell cycle progression and the percentage of cells in different stages of the cell cycle after 24-h incubation either with medium (control) or various mAbs. 2E5G5 induced an increase in the number of cells in G2/M phase and a decrease in the number of cells in G0/G1 and S phases of the cell cycle. Other mAbs tested had no effect. This observation supports the occurrence of cell cycle arrest in the G2/M phase of the cell cycle.



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  		Figure 4. FACS analysis of the DNA in 5000 CRL 1607 cells. Cells were incubated for 24 h at 37 C with various mAbs (medium control, control mAb, and 2E5G5), stained with propidium iodide, and analyzed on a FACScan (Becton Dickinson). Representative examples of cell cycle progression analysis are shown. A, Medium control; B, mAb control; C, 2E5G5 mAb. M1, M2, and M3 indicate the cell numbers in G0/G1, S, and G2/M phases, respectively. The reduction and increase in the percentage of cells in S and G2/M phases after incubation with 2E5G5 mAb are indicated by arrows.

 

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  		Table 2. The effects of various mAbs on cell cycle progression

 
As shown in Fig. 5Go, in a qualitative analysis of fragmented DNA by gel electrophoresis, 2E5G5 caused detectable DNA fragmentation during a 24-h incubation, but other mAbs tested did not. We treated CRL 1607 cells with 2E5G5 for various intervals. We can detect apoptosis as early as after 12-h incubation with the specific mAb. At this time a clear smear band of fragmented DNA was observed in 1.5% agarose gel electrophoresis of cell DNA.



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  		Figure 5. Agarose gel (1.5%) analysis of DNA isolated from CRL 1607 cells. A: Lane 1, 2E5G5-treated, 24 h; lane 2, 4H4E7-treated, 24 h; lane 3, 2H8C1-treated, 24 h; lane 4, 2H10G8-treated, 24 h; lane 5, 11B7D4-treated, 24 h; lane 6, 1C10D8-treated, 24 h; lane 7, untreated; lane 8, DNA size markers. B: Lanes 1 and 14, DNA size markers; lanes 2 and 3, 2E5G5-treated, 12 and 24; lanes 4 and 5, 4H4E7-treated, 12 and 24 h; lanes 6 and 7, 2H8C1-treated, 12 and 24 h; lanes 8 and 9, 2H10G8-treated, 12 and 24 h; lanes 10 and 11, incubated at 45 C for 1 h then at 12 and 24 h at 37 C (mild hyperthermia as a positive control for apoptosis) (26). Lanes 12 and 13, Untreated.

 
Table 3Go summarizes the quantitative analysis of DNA fragmentation using the [3H]thymidine labeling method. After 24-h incubation with various mAbs (untreated cells as control), 2E5G5 induced more than 90% chromatin fragmentation.


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  		Table 3. Quantitation of DNA fragmentation induced by mAbs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many strategies have been applied to tumor immunotherapy. To improve tumor-specific T cell activation, investigators attempt to up-regulate the expression of MHC or costimulatory molecules by injecting recombinant cytokines systemically (28), by transfecting tumor cells with genes encoding cytokines (29, 30, 31, 32), or by injecting into tumors fibroblast cells transfected with cytokine genes (33). mAbs against tumors offer another approach to tumor immunotherapy. Conjugated and unconjugated specific mAbs have been directly infused into animals with tumors to kill the tumor cells, and there are impressive results from these trials. In a completed phase II trial, 21 patients with non-Hodgkin’s lymphoma receiving anti-CD20 mAb experienced a complete response. The response rate in patients whose tumors had become resistant to chemotherapy was about 70%, and the median duration of complete response exceeded 15 months (34). Positive results were obtained in animal and clinical trials using other mAbs (34, 35, 36, 37). A major problem for successful treatment using mAbs is the fact that few tumor-specific antigens have been found. Investigators, therefore, focus on TAA and related tumor targets. For example, antibodies may block growth factor receptors on the surface of a tumor cell and inhibit crucial growth signals to cancer cells. These targets include EGF receptor and vascular endothelial growth factor. Some preclinical data show that a combination of Ab to EGF and traditional chemotherapy has a synergistic effect to enhance cell killing (38, 39, 40, 41, 42, 43, 44, 45).

In the present study, we choose a unique peptide produced by MTC cells as an immunotherapeutic target and prepared more than 40 monoclonal antibodies. We performed a series of in vitro studies on the effect of the mAbs on MTC cells. One mAb (2E5G5), even at low concentration, efficiently inhibited [3H]thymidine incorporation into CRL 1607 cells. We examined the killing ability of this mAb in this rMTC cell line in vitro using a trypan blue exclusion method. At 200 µg/ml, this mAb killed all cells after 48 h of incubation. This effect is not caused by antibody-dependent cellular cytotoxicity or by complement-mediated cytolysis, because we used heat-inactivated FBS in all culture media. Also, when we treated this cell line with mAb and complement, no effect of complement was found (data not shown). Surprisingly, we could not detect surface binding of the antibody to rCT using whole cell ELISA or FACS methods (data not shown), although the mAb bound very strongly to surface-coated rCT in an ELISA and stained human MTC cells very efficiently by immunohistochemistry (27). When we examined cell cycle arrest and fragmentation of DNA after treatment with the mAb, we found that the mAb can induce both cell cycle arrest and apoptosis. We do not know how the mAb induces apoptosis. Although there are some reports of apoptosis induced by mAbs (46, 47, 48), all of these mAbs were against surface Ag and/or related to signal transduction, resulting in DNA fragmentation.

We considered that the rMTC cell line may be dependent upon stimulation by rCT for cell growth, implying that rCT is an autocrine hormone acting on surface rCT receptors on the cells. When we treated cells with mAbs to rCT, the cells might lose the stimulation from rCT and undergo apoptosis. Recently, Frendo and his colleague reported the presence of a truncated form of hCT receptor (hCTR) in TT cells (a cell line derived from MTC) (49). They found that hCTR2 is expressed in all MTC samples at an higher lever than normal C cells, and that the expression of hCTR2 messenger RNA is involved in TT cell proliferation, suggesting an autocrine role of CT in tumor cells. However, the antibody we found to kill rat MTC cells does not bind to 125I-labeled rCT in solution, and added rCT did not reduce cell death during in vitro culture.

Another hypothesis is that there is a transient phase in which rCT exists on the cell surface when it is secreted, that mAb can bind to the rCT on the cell surface, and that the binding of the mAb to the cell surface may transfer a negative signal, so that the cells undergo programmed cell death.

Our results suggest that rCT not only serves as a tumor marker in clinical diagnosis, but also may serve as a immunotherapeutic target for MTC. We are now examining the effect of the specific mAb on tumor growth in an animal model. Further studies of the mechanisms of mAbs underlying antibody-induced apoptosis and/or negative signal transfer should be helpful in designing future clinical trials for the immunotherapy of tumors.

Received October 21, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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