Expression of histo-blood group A antigen increases resistance to apoptosis and facilitates escape from immune control of rat colon carcinoma cells

Séverine Marionneau, Béatrice Le Moullac-Vaidye and Jacques Le Pendu1

INSERM U419, Institut de Biologie, 9 Quai Moncousu, 44093 Nantes, France

Received on June 21, 2002;; revised on August 14, 2002; accepted on August 15, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A and B histo-blood group antigens are present on carcinoma cells at the early stages of cancerogenesis and tend to disappear at later stages, but it is not yet clear whether they take part to the process of tumor progression. To gain some insight into this issue, we used a rat colon carcinoma experimental model. To obtain expression of the A antigen, REG cells were cotransfected with the rat A enzyme cDNA and a rat {alpha}1,2fucosyltransferase cDNA, either FTA or FTB, whereas PRO cells that spontaneously have {alpha}1,2fucosyltransferase activity were only transfected with the A enzyme cDNA. All A antigen–expressing transfected cells derived from either REG FTA, REG FTB, or PRO parental cells were more resistant to apoptosis induced by either serum deprivation or heat shock than were their respective controls. When injected to syngeneic immunocompetent rats, A enzyme–transfected PRO cells formed tumors that grew faster than those formed by mock-transfected PRO cells. However, in immunodeficient SCID mice, no difference in growth could be observed between the two types of tumors, indicating that the faster tumor growth of the A antigen–positive cells in immunocompetent animals was due to their higher ability to escape immune control and that this was associated with their higher degree of resistance to apoptosis. These results might explain the slightly augmented incidence of carcinomas observed in A and B blood group individuals compared to O individuals.

Key words: A enzyme/apoptosis/blood group/carcinoma/tumorigenicity


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Many modifications in glycosylation of cell surface glycoconjugates have been described in cancer (Hakomori, 1996Go; Kim and Varki, 1997Go). For the most part, the biological roles of these alterations are not well defined. Thus the expression of ABH antigens is subject to considerable variations on malignant transformation, and its significance is unclear (Hakomori, 1999Go). A loss of A and B antigens is observed in most types of carcinomas, such as carcinomas from the buccal epithelium, stomach, proximal colon, pancreas, larynx, lung, endometrium, ovary, prostate, urinary bladder, and breast. However, these antigens appear on carcinomas derived from some tissues where they are normally not present, such as the colorectal epithelium, liver parenchyme, and thyroid. The loss of A and B antigens is associated with a poor prognosis in carcinomas of the lung, urinary bladder, and head and neck. Inversely, in the case of colorectal carcinomas, it is their presence that is a sign of unfavorable outcome (Le Pendu et al., 2001Go). In addition, a higher incidence of various types of carcinomas is observed for blood group A and B individuals compared with blood group O individuals (Annese et al., 1990Go; Bjorkholm, 1984Go; Henderson et al., 1993Go; Kaur et al., 1992Go; Mourant et al., 1978Go; Slater et al., 1993Go; Su et al., 2001Go; Vioque and Walker, 1991Go; You et al., 2000Go). Transfection of the A or B enzymes’ cDNA or selection of A-positive subpopulations of human colorectal carcinoma cell lines showed that the presence of the A and B antigens was associated with a reduced motility on matrigel (Ichikawa et al., 1997Go, 1998). These observations may explain why the loss of A and B antigens is associated with a bad prognosis in some types of carcinomas because the A and B antigens would decrease the cells metastatic potential. However, they cannot account for the increased frequency of cancers in blood group A and B individuals or for the meaning of the appearance of A and B antigens from the early stages of colorectal carcinoma development.

Synthesis of these antigens requires several glycosyltransferases acting on precursor oligosaccharides. These precursors can be converted into H antigenic structures after addition of a fucose in {alpha}1,2 linkage by an {alpha}1,2fucosyltransferase. The A and B enzymes can then use the H structures as substrates to catalyze the synthesis of the A and B antigens by addition of an N-acetylgalactosamine or a galactose, respectively. In humans, two genes, FUT1 and FUT2, encode {alpha}1,2fucosyltransferases. In colon cancer, the {alpha}1,2fucosyltransferase activity of both FUT1 and FUT2 is clearly increased (Nishihara et al., 1999Go; Sun et al., 1995Go). Using a rat model of colon carcinoma, our group observed earlier a correlation between the level of expression of the histo-blood group H antigen and the cells’ degree of tumorigenicity (Zennadi et al., 1992Go). In this model, described by Caignard et al. (1985)Go), a cell line was obtained from a single chemically induced colon carcinoma in a BDIX rat. Cellular clones derived from this cell line present distinct behavior in vivo. The PRO clone forms progressive tumors in syngeneic animals, synthesizes H antigenic determinants, and expresses the mRNA of both FTA and FTB, the rat genes orthologous to the human FUT1 and FUT2 genes. In contrast, the REG clone, which is devoid of H antigen and of {alpha}1,2fucosyltransferase actitivity, forms only smalls tumors that are immunologically rejected within a few weeks.

We previously showed by transfection experiments that {alpha}1,2fucosylation, and hence expression of H antigen, increases resistance to apoptosis of REG cells induced by serum deprivation. It also enhanced their tumorigenicity in syngeneic rats. However, in immunodeficient SCID mice, both control and {alpha}1,2fucosyltransferase transfected REG cells were fully tumorigenic indicating that the increased tumorigenicity mediated by {alpha}1,2fucosylation was associated with increased resistance to apoptosis and with escape from immune control (Goupille et al., 2000Go). Similarly, heat resistance was clearly associated with the level of cell surface expression of blood group H and A antigens on REG and PRO cells (Ménoret et al., 1995Go). The BDIX rat A enzyme cDNA has been recently cloned in our laboratory (Cailleau-Thomas et al., 2002Go), allowing to test the influence of the A histo-blood group antigen expression on the cells behavior in this cancer experimental model. In this article, we show that the histo-blood group A antigen is able to modulate the cells’ sensitivity to apoptosis as well as their in vivo behavior.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Expression of histo-blood group A antigen enhances resistance to apoptosis induced by serum deprivation and heat shock
Stable FTA and FTB transfectants of REG cells were separately transfected with the pDR2 plasmid containing the histo-blood group A enzyme cDNA from BDIX rats. As previously described (Goupille et al., 2000Go), both the REG FTA and REG FTB transfectants express H antigenic determinants, but as shown in Figure 1, they do not present detectable A epitopes. After transfection with the pDR2 plamid containing the A enzyme cDNA, both cell types expressed cell surface A antigen. Two clones strongly expressing the A antigen derived from either REG FTA and REG FTB cells were selected. Flow cytometry analysis using monoclonal antibody (mAb) 3–3A reveals the presence of A epitopes on these clones, FTA A1, and FTA A9 derived from REG FTA cells, as well as FTB A1 and FTB A3 derived from REG FTB (Figure 1). PRO cells spontaneously express {alpha}1,2fucosylated structures, which are precursors of the A antigen. A small subpopulation of PRO cells spontaneously synthesizes A determinants. To avoid selecting cells from this subpopulation, a clone from PRO cells was first isolated that does not show any detectable A epitopes but expresses H epitopes. These cells were directly transfected with the rat A enzyme cDNA inserted into the pBK-CMV plasmid. This allowed expression of cell surface A antigen, but no B antigen could be detected. Clones PRO A1 and PRO A8 isolated from the stably transfected population were selected for their strong expression (Figure 1). Unlike the parental or mock-transfected PRO cells, these clones synthesize the A enzyme mRNA, as detected by reverse transcription polymerase chain reaction (RT PCR) (not shown).



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Fig. 1. Cytofluorimetric analysis of cell surface A histo-blood group antigen expression revealed by mAb 3–3A. The log of fluorescence intensities in arbitrary units is plotted against cell number. Fluorescence intensities from REG FTA and REG FTB cells, transfected with the rat {alpha}1,2fucosyltransferases FTA and FTB cDNAs, respectively, are superimposed on those from the cells cotransfected with the rat A enzyme cDNA (A enzyme/FTA clones A9 and A1; A enzyme/FTB clones A1 and A3). Fluorescence intensities from the mock-transfected PRO PBK cells are superimposed on those from the A enzyme–transfected clones PRO A1 and PRO A8.

 
To assay their sensitivity to apoptosis, cells were submitted to serum deprivation or heat shock. We first set up experimental conditions under which cell death by apoptosis occurs. We have previously observed that REG cells were more sensitive to heat shock and serum deprivation than were PRO cells (Ménoret et al., 1995Go). Twenty-four hours after being submitted to heat shock at 44.5°C for 20 min, a large fraction of REG cells are no longer adherent but in suspension. As shown on Figure 2A, nuclei staining with Hoescht 33258 of such cells in suspension revealed nucleolar condensation characteristic of apoptosis. In addition, DNA degradation with typical ladder figures was observed for all REG-derived cells following heat treatment (Figure 2B), indicating that in the conditions used, cell death occurred by apoptosis. The same results were obtained for PRO cells and derived transfectants submitted to a 30-min heat shock at 44.5°C. Likewise, after 4 days of culture in absence of fetal calf serum (FCS), many REG cells are in suspension, but 6 days are necessary to observe a significant proportion of nonadherent PRO cells. These cells also present characteristic figures of apoptosis (data not shown). Therefore, to compare their sensitivity to apoptosis, REG transfectants were submitted to either a heat shock at 44.5°C for 20 min or to 4 days of FCS deprivation, whereas PRO transfectants were submitted to a heat shock at 44.5°C for 30 min or to 6 days without FCS. Cell survival was determined by a colony formation assay.



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Fig. 2. Characterization of apoptotic cell death after heat shock. (A) Nuclei from floating cells in culture from DN mock-transfected REG cells were stained by Hoescht 33258 and visualized by fluorescent microscopy. The same figures of apoptosis were observed for nuclei from cells in suspension of all other transfectants described in this study. (B) After heat treatment, DNA was extracted from cells in suspension (sn) or adherent cells (ad) from DN mock-transfected REG cells, FTA- or FTB-transfected REG cells, and A enzyme–cotransfected FTA and FTB REG cells. DNA was visualized by ethidium bromide staining after electrophoresis on 1.8% agarose gel.

 
As previously observed (Goupille et al., 2000Go), REG FTA and FTB transfectants were more resistant to both heat shock and serum deprivation than mock transfected REG cells (Figure 3). Doubly transfected REG cells expressing the A antigen proved even more resistant than the H antigen expressing FTA and FTB transfectants because the percentage of surviving colonies increased from 10% to 20%. Similarly, the A enzyme–transfected PRO cells were significantly more resistant to both heat shock and serum deprivation than mock-transfected control cells with a 10% increase in surviving colonies (Figure 4A, B). Although the effect is modest, it is clearly visible with all A antigen–expressing clones compared with their respective control clones. These results indicate that the addition of an N-acetylgalactosamine residue on the H structures increases the cellular resistance to apoptosis.



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Fig. 3. Sensitivity of REG transfectants to apoptosis. (A, C) Comparison between DN mock-transfected cells, FTA-transfected cells (24 and A11), and A enzyme/FTA–cotransfected cells (A1 and A9) or (B, D) comparison between DN mock-transfected cells, FTB-transfected cells (3 and 13), and A enzyme/FTB–cotransfected cells (A1 and A3) submitted to a 20-min heat shock at 44.5°C (A, B) or to 4 days in absence of FCS (C, D). Cell survival was measured in a colony formation assay as described in Materials and methods. Percentages of surviving colonies were determined by comparison with the number of colonies from cultures with FCS in absence of heat shock. The number of colonies from these control cultures varied from 300 to 500 according to the experiment and did not differ for the various cell types. The values represent the mean ± SD of three experiments.

 


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Fig. 4. Sensitivity of PRO transfectants to apoptosis. Comparison between PBK mock-transfected PRO cells and A enzyme–transfected PRO cells (A1 and A8) submitted to a 30-min heat shock at 44.5°C (A) or to 6 days in absence of FCS (B). Cell survival was measured in a colony formation assay as described in Materials and methods. Percentages of surviving colonies were determined by comparison with the number of colonies from cultures with FCS in absence of heat shock. The number of colonies from these control cultures varied from 300 to 500 according to the experiment and did not differ for the various cell types. The values represent the mean ± SD of three experiments.

 
Expression of histo-blood group A antigen enhances tumorigenicity in immunocompetent animals
To define if A antigen expression modifies the cells behavior in vivo, PRO transfectants were injected subcutaneously (SC) to groups of syngeneic animals. As expected, mock-transfected cells (PBK) gave tumors in all animals. Likewise, the two A enzyme transfectants, A1 and A8, gave progressive tumors in all animals. However, these tumors grew significantly faster than those from control PBK cells (Figure 5A). Rats were sacrificed at day 51 when the first skin wounds became apparent in some animals. At that time, tumors from the A antigen–positive transfectants were between two to three times as large as tumors from mock-transfected cells (Figure 5B). To determine if the immune system was involved in this phenomenon, the same cells were injected SC to SCID mice. Like in rats, the three clones gave progressive tumors, but in these immunodeficient animals there was no difference between the growth of tumors from the A enzyme transfectants and that of tumors from the mock transfectants (Figure 5C, D). This indicates that the immune system was responsible for the difference observed in immunocompetent animals between the two types of tumors.



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Fig. 5. Growth of mock-transfected PRO PBK cells (open squares) or of A enzyme transfectants A1 (closed circles) and A8 (closed squares) in either syngeneic immunocompetent BDIX rats (A, B) or immunodeficient SCID mice (C, D). 1 x 106 cells were injected SC, and tumor volumes were measured at different time points with calipers. Results represent means ± SE from five animals in each group (A, C). Tumor volumes from individual animals at the time of sacrifice (51 days for syngeneic rats and 37 for SCID mice) are shown for each group of tumor (B, D). Student t-test indicates that the difference in tumor volumes at day 51 between PBK tumors and either A1 or A8 tumors in syngeneic rats is significant (p < 0.01).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We previously observed that {alpha}1,2fucosylation increased the tumorigenicity of rat colon carcinoma cells (Goupille et al., 1997Go; Labarrière et al., 1994Go). Furthermore, this increased tumorigenicity was dependent on the ability of H antigen–expressing cells to escape immune control and was associated with their enhanced resistance to apoptosis (Goupille et al., 2000Go). To determine if the histo-blood group A antigen could also take part in these phenomena, rat colon carcinoma cells expressing H antigen as precursor were transfected with an A enzyme cDNA. The A enzyme cDNA from BDIX rats, syngeneic to the PRO carcinoma cells, was used to avoid the generation of an irrelevant immune response to the A enzyme itself because the newly introduced gene product could be recognized as foreign if originating from either another strain of rats or another species. We have recently shown that this enzyme presents a strong A activity and only a small B activity, much like the human A1 gene product (Cailleau-Thomas et al., 2002Go). Accordingly, transfection into either PRO cells or cotransfection with the rat {alpha}1,2fucosyltransferases cDNAs FTA or FTB into REG cells allowed strong expression of A antigenic determinants.

Analysis of the cells’ sensitivity to apoptosis induced by either heat shock or serum deprivation indicated that all the A antigen–expressing cells originating from REG FTA, REG FTB, or PRO cells were slightly more resistant than their respective A-negative parental cells. No difference in proliferation between the A-positive and A-negative cells could be observed (data not shown), and the tumorigenicity of the two cell types in immunodeficient SCID mice was identical. Nevertheless, the tumorigenicity of A-positive PRO cells in immunocompetent syngeneic animals was significantly higher than that of mock-transfected PRO cells, indicating that A-positive cells present an enhanced ability to escape immune control. An alternative mechanism to explain the difference in tumor growth observed between syngeneic rats and immunocompromised SCID mice could involve the generation of facilitating anti-A antibodies in rats. The existence of antibodies facilitating tumor growth has indeed been documented long ago (Prehn, 1994Go). Yet we did not find anti-A antibodies in the sera of rats with an A-positive growing tumor (data not shown). This was not surprising because BDIX rats are A-positive and strongly express A antigenic determinants in various tissues (Cailleau-Thomas et al., 2002Go). Thus, although the mechanisms by which the immune system slows down the growth of PRO tumors have not been defined, these results suggest that the higher ability of A-positive cells to resist apoptotic stresses may facilitate their escape from immune control because effectors of the immune system are known to induce apoptosis of target cells.

ABH antigens can be present on key receptors controlling cell proliferation, adhesion, and motility, such as the epidermal growth factor receptor, integrins, cadherins, and CD44 (Greenwell, 1997Go; Hakomori, 1996Go, 1999). The expression pattern of these various receptors differs according to the type of cancer, and therefore the role of ABH antigens in the biology of human cancers may also vary. In this regard, it is not clear at present how representative the results described here using a rat experimental model might be. Whatever the case, cellular mechanisms promoting apoptotic cell death are connected with cell proliferation and prevent the accumulation of deleterious mutations and the development of cancer. It is therefore to be expected that elements counteracting apoptotis should favor the survival of cells harboring genetic anomalies that lead to cancer progression (Green and Evan, 2002Go).

In A and B blood group individuals, at the early stages of carcinoma development, the A and B antigens are expressed, either because they are already present on the healthy tissues or because they appear at the precancerous stage as in the distal colon where they can be observed on polyps (Le Pendu et al., 2001Go). If they increase the cellular basal resistance to apoptosis, as we have observed here with the PRO and REG cells, the histo-blood group A and B antigens could increase the probability of survival of cells that have accumulated genetic alterations and therefore the probability of occurrence of a cancer. The increased resistance to cell death of A antigen–positive cells could also facilitate the escape of carcinoma cells from immune surveillance at the early stages of tumor progression. This could explain why the frequency of various types of carcinomas is slightly higher among blood group A and B individuals than among blood group O individuals. At later stages of tumor progression, when the cancerous phenotype has been fully acquired and the immune system has become tolerant to the tumor cells, the loss of A and B antigens could facilitate metastatic spread by increasing cellular motility (Ichikawa et al., 1997Go, 1998). Similarly, at these later stages, the decreased expression of {alpha}1,2fucosyltransferase activity, and hence of ABH antigens, may participate to the metastatic progression by releasing the competition with an {alpha}2,3sialyltransferase, leading to increased expression of the sialyl-Lea and sialyl-Lex selectin ligand, as shown by others (Aubert et al., 2000Go). Thus, the presence of A and B antigens would have opposite effects at different stages of tumor progression. They would facilitate cancerogenesis at early stages, as exemplified by the experimental model described here, but would limit metastatic spreading at later stages.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cells lines
REG and PRO rat colon adenocarcinoma cells (obtained from F. Martin, Dijon, France) are clones derived from a dimethylhydrazine-induced cell line (DHDK12). REG has been previously transfected with cDNAs encoding for the FTA or FTB rat {alpha}1,2fucosyltransferases (Goupille et al., 2000Go). Briefly, two independent transfections were performed; following transfection, cells expressing {alpha}1,2linked fucose residues were sorted by flow cytometry. The resultant populations were called REG FTA and REG FTB, respectively, and then cloned by limiting dilutions. Control cell lines were obtained after transfection with the empty vector and were called REG DN, REG DN3, and PRO PBK. Cells were cultured in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin (Gibco BRL, Cergy-Pontoise, France). They were subcultured at confluency after dispersal with 0.025% trypsin in 0.02% ethylenediamine tetra-acetic acid (EDTA). Stable transfectants were cultured in the same medium supplemented with 0.25 mg/ml G418 (Promega, Madison, WI). Cells were routinely checked for mycoplasma contamination by Hoechst 33258 (Sigma, St. Louis, MO) labeling.

Enzyme A transfection of REG FTA, REG FTB, and PRO cells
The complete coding region for the rat histo-blood group A enzyme (N-acetylgalactosaminyltransferase, EC2.4.1.40) has been cloned in our laboratory (GenBank accession number AF2(4018). It was inserted in the pBK-CMV vector (Stratagene, Cambridge, UK), deleted of the lacZ promoter by digestion with Spe1 and Nhe1, through the Eco R1 site of the multiple cloning site. It was also inserted in the pDR2 vector (Clontech, Palo Alto, CA) deleted of the sequences lying between the EcoRV and Cla I sites. REG FTA and REG FTB cells were transfected with the pDR2 Enzyme A vector using lipofectamin (Gibco BRL) according to the manufacturer’s instructions. PRO cells were transfected with the pBK enzyme A vector by the same method. Selection of stable transfectants was achieved by addition of 0.6 mg/ml G418 (pBK transfectants) or of 0.6 mg/ml hygromycin (pDR2 transfectants) for 2 weeks. Transfected cells expressing the A antigen were sorted by flow cytometry using the anti-A specific mAb 3–3A (Bara et al., 1988Go). The resultant populations were then expanded and cloned by limiting dilution, and strongly expressing clones were selected for further study. The PRO stable transfectants were cultured in medium supplemented with 0.25 mg/ml G418, and the REG and stable double transfectants were cultured in medium supplemented with 0.25 mg/ml G418 and 0.25 mg/ml hygromycin B (Sigma).

Cytofluorimetric analysis
Viable cells (2 x 105 cells/well) were incubated with the anti-A mAb 3–3A at 10 µg/ml in phosphate buffered saline containing 0.1% gelatin for 1 h at 4°C. After washing in the same buffer, a second 45-min incubation was performed with an fluorescein isothiocyanate–labeled anti-mouse IgG (Sigma). Following three washes, fluorescence analysis was performed on a FACScan (Becton-Dickinson) using the CELLQUEST program.

Determination of in vitro cell sensitivity to apoptosis
The sensitivity of cells to apoptosis was quantified after serum deprivation or heat shock treatment by a colony formation assay. Cells were cultured in completed medium for 48 h, until confluency, before treatment started. Cells were washed with serum-free medium and kept in the same deprived medium for either 4 days (REG transfectants) or 6 days (PRO transfectants). The medium was changed twice during this incubation time. Adherent cells were then detached with EDTA-trypsin, and 1 x 103 cells seeded in six-well flat-bottom culture plates. After culture in complete medium for 96 h, colonies were stained with methylene blue and counted. Cell death was also induced by heat shock treatment. The cells were heated by submersion of the culture plate in a precision-controled water bath at 44.5°C for 20 min (REG transfectants) or 30 min (PRO transfectants). Adherent cells were then detached with EDTA-trypsin, and 1 x 103 cells seeded in six-well flat-bottom culture plates as described. Percentages of surviving colonies were determined relative to the number of colonies from control cultures of untreated cells.

Detection of nucleolar condensation and in vitro DNA fragmentation
After culture in absence of FCS or treatment with heat shock, cells in suspension or adherent cells from control and treated cultures were stained with 5 µg/ml Hoechst 33258 (Sigma) for 30 min at 37°C, rinsed, and then examined by fluorescence microscopy (Olympus, BH-2).

For analysis of DNA fragmentation, cells in suspension and adherent treated or untreated cells were incubated for 2 h with proteinase K (20 µg/ml). The DNA was extracted with phenol-chloroform and then precipitated overnight at –20°C following addition of ethanol. After incubation for 3 h at 37°C in Tris-EDTA containing 10 µg/ml RNase A, the DNA fragments were resolved by electrophoresis for 2 h at 40 V on 1.8% agarose gel and visualized under UV light after ethidium bromide staining.

Tumorigenicity assays
Inbred BDIX rats were purchased from Iffa-Credo (L’Abresle, France) and housed and bred under standard conditions in our laboratory. SCID mice were purchased from Charles River France (St. Aubain-Les-Elbeuf, France) and housed under sterile conditions. Ten-week-old rats and 8-week-old mice were used. Confluents cells were trypsinized, and 1 x 106 cells suspended in serum-free RPMI, 0.5 ml in the case of rats or 0.2 ml in the case of mice, were injected SC in the flank of animals. Tumors were measured with calipers, and animals were sacrificed at day 51 for rats and day 37 for mice, before appearance of skin ulceration to avoid pain. These experiments were performed in agreement with the rules from the French Ministry of Agriculture under supervision of the Vetenary Services (Agreement A44565).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The authors are grateful to J. Bara for his gift of antibody and to P. Fichet and S. Minaut for animal care. This work was supported by grants from the Association pour la Recherche sur le Cancer (ARC) and the Association for International Cancer Research (AICR).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
EDTA, ethylenediamine tetra-acetic acid; FCS, fetal calf serum; mAb, monoclonal antibody; RT PCR, reverse transcription polymerase chain reaction; SC, subcutaneously.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: jlependu@nantes.inserm.fr Back


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 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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