{alpha}1,2Fucosyltransferase increases resistance to apoptosis of rat colon carcinoma cells

Caroline Goupille1, Séverine Marionneau1, Valérie Bureau, Florence Hallouin, Marc Meichenin, Jézabel Rocher and Jacques Le Pendu2

INSERM U419, Institut de Biologie, 9 Quai Moncousu, 44035, Nantes, Cedex, France

Received on June 25, 1999; revised on October 15, 1999; accepted on October 23, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Accumulation of histo-blood group antigens such as Lewis b, Lewis Y and H in colon cancer is indicative of poor prognosis. It is accompanied by increase in {alpha}1,2fucosyl­transferase activity, a key enzyme for synthesis of these antigens. Using a model of colon carcinoma, we previously showed that {alpha}1,2fucosylation increases tumorigenicity. We now show that tumorigenicity inversely correlates with the cells’ sensitivity to apoptosis. In addition, poorly tumorigenic REG cells independently transfected with three different {alpha}1,2fucosyltransferase cDNAs, the human FUT1, the rat FTA and FTB were more resistant than control cells to apoptosis induced in vitro by serum deprivation. Inversely, PRO cells, spontaneously tumorigenic in immunocompetent syngeneic animals and able to synthesize {alpha}1,2fucosylated glycans, became more sensitive to apoptosis after transfection with a fragment of the FTA cDNA in the antisense orientation. Expression of {alpha}1,2fucosyl­transferase in poorly tumorigenic REG cells dramatically enhanced their tumorigenicity in syngeneic rats. However, in immunodeficient animals, both control and {alpha}1,2fuco­syltransferase transfected REG cells were fully tumorigenic and metastatic, indicating that the presence of {alpha}1,2fucosylated antigens allowed REG tumor cells to escape immune control. Taken together, the results show that increased tumorigenicity mediated by {alpha}1,2fucosyl­ation is associated to increased resistance to apoptosis and to escape from immune control.

Key words: {alpha}1,2fucosyltransferase/apoptosis/tumorigenicity/rat/carcinoma


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Among many phenotypic alterations of cancer cells, a set of changes in glycosylation of cell surface glycoproteins or glycolipids has been described (Hakomori, 1996Go; Kim and Varki, 1997Go). For a large part, the biological significance of these alterations is still unknown. Modifications of expression of ABH and Lewis histo-blood group-related antigens are a characteristic feature of carcinomas. Synthesis of these antigens requires several glycosyltransferases acting on precursor oligosaccharides. Four main types of precursor disaccharides are recognized which have in common a nonreducing galactose in ß linkage to either a N-acetylglucosamine (type 1: Galß1–3GlcNAcß1-R and type 2: Galß1–4GlcNAcß1-R) or a N-acetylgalactosamine (type 3: Galß1–3GalNAc{alpha}1-R and 4 Galß1–3GalNAcß1-R). These precursors can be converted into H antigenic structures after fucosylation in {alpha}1,2 linkage (Fuc{alpha}1–2Galß1-R) by GDPFuc:ß-D-galactoside {alpha}1,2-fucosyltransferases ({alpha}2FT), 2 genes for which have been cloned and designated FUT1 and FUT2 in humans (Rajan et al., 1989Go; Rouquier et al., 1995Go). These enzymes were previously termed H type and Secretor type, respectively.

Expression of type 1 [Lewis b (Leb)] and type 2 [H type 2 (H-2) and Lewis Y (Ley)] structures, with the common motif Fuc{alpha}1–2Galß-R, is a characteristic feature of tumor progression in the distal colon and rectum (Itzkowitz, 1992Go) and it has been associated with poor prognosis (Naitoh et al., 1994Go). In accordance with these observations, {alpha}2FT activity is clearly increased in colon cancer (Orntoft et al., 1991Go; Yazawa et al., 1993Go; Sun et al., 1995Go). Until recently it has been unclear whether both FUT1 and FUT2 enzymes or another as yet uncharacterized enzyme were involved in the synthesis of the {alpha}1,2fucosylated antigens in colon carcinomas (Yazawa et al., 1993Go). Nevertheless, it was unambiguously shown by Sun et al. (1995)Go that FUT1 is transcribed in colon adenocarcinoma and more recently, by Nishihara et al. (1999)Go, that both FUT1 and FUT2 participated in the synthesis of histo-blood group related antigens in this type of tumor. The mechanism by which such structures could play a role in tumor progression remains unclear. However, their participation to the phenomenon of cell motility has been noted in several experimental models (Miyake and Hakomori, 1991Go; Garrigues et al., 1994Go; Goupille et al., 1997Go). We observed previously, in a rat model of colon carcinoma, that the presence of {alpha}1,2fucosylated structures at the cell surface increased tumorigenicity. In this model, described by Martin et al. (1983)Go, a cell line was obtained from a single chemically induced colon carcinoma in BDIX rats. Cellular clones derived from this cell line present distinct behavior in vivo. The PRO clone forms progressive tumors and metastasis in syngeneic animals, whereas the REG clone forms only small tumors that spontaneously regress within a few weeks (Caignard et al., 1985Go). This rejection is immunologically mediated since REG tumors can grow in cyclosporin-treated rats (Shimizu et al., 1989Go) or in rats depleted of {alpha}ß TCR bearing lymphocytes (Ménoret et al., 1995Go). Unlike REG cells, PRO cells present {alpha}1,2fucosylated antigens at their surface, possess {alpha}2FT activity and synthesize mRNA for both the FTA and FTB enzymes which are homologous to the human FUT1 and FUT2 enzymes respectively (Piau et al., 1994Go). In addition, we have observed in situ expression of histo-blood group H antigens in pre-cancerous colonic mucosa as well as in chemically induced carcinomas in rats (Hallouin et al., 1997Go). The rat experimental model thus represents an excellent model to study the role of these blood group-related tumor associated antigens. Using this experimental model, we now report that the modulation of H antigen expression by sense and antisense {alpha}2FT transfection is able to modulate the cells sensitivity to apoptosis as well as their in vivo behavior.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Correlation between tumorigenicity and resistance to cell death
An interesting feature of the rat DHDK12 experimental model is that clones with distinct behavior in syngeneic animals can be derived from this parental cell line. As previously described, Figure 1 illustrates the high tumorigenicity of the PRO clone, the low tumorigenicity of the REG clone and the intermediate behavior of clone TS8F. When assayed for their sensitivity to cell death induced by serum deprivation, it appeared that the clones degree of sensitivity paralleled their in vivo behavior, with the PRO clone being largely resistant, REG being sensitive and TS8F standing between these 2 extremes.



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Fig. 1. Correlation between the tumorigenicity of clones derived from the DHDK12 cell line and their sensitivity to serum deprivation. (A) 1 x 106 cells were injected s.c. to the flanks of syngeneic BDIX rats and tumor volumes were measured weekly. Each line represents tumor growth in a single animal. (B) Cells were cultured for 96 h in absence of FCS and survival was measured by a colony formation assay as described in the Materials and methods section. Percentages of colonies were determined by comparison with number of colonies from cultures in the presence of serum.

 
H antigenic expression enhances resistance to apoptosis
We previously observed a correlation between the clones in vivo behavior and their ability to express cell surface {alpha}1,2fucosylated structures, PRO cells carrying such structures of which REG cells are almost completely devoid (Zennadi et al., 1992Go). In order to evaluate the importance of this phenotypic difference in the cells behavior, REG cells were transfected with cDNAs encoding for {alpha}2FT in the sense orientation and PRO with a cDNA in the antisense orientation. As previously described (Goupille et al., 1997Go), after transfection with the human FUT1 cDNA, REG cells express high levels of {alpha}1,2fucosylated structures as revealed by the UEA-I lectin (Figure 2). A similar result was obtained after transfection with the rat FTA and FTB {alpha}2FT cDNA. This cell surface expression of fucosylated structures correlated well with the enzymatic activities detected in the cell extracts (Figure 3). Inversely, transfection of PRO cells with a fragment of the rat FTA cDNA in the antisense orientation, significantly decreased the level of UEA-I reactivity (Figure 2) and of the fucosyltransferase activity, as previously reported (Hallouin et al., 1999Go). Binding of the lectin to these cell lines could be inhibited in the presence of 0.2 M fucose, confirming specificity (data not shown).



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Fig. 2. Cytofluorimetric analysis of cell surface {alpha}1,2fucosylation revealed by FITC-labeled UEA-I lectin. The log of fluorescence intensities in arbitrary units is plotted against cell number. Fluorescence intensities from REG mock transfected cells, DN1 and DN2 are superimposed on those from REG cells transfected with cDNAs from the {alpha}1,2fucosyltransferases FUT1, FTA, or FTB. Fluorescence intensity from control transfected PRO cells (R) are superimposed on fluorescence from the FTA antisense transfected clone A4. The fluorescence intensity of unlabeled R cells is shown as negative control (C).

 


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Fig. 3. {alpha}1,2fucosyltransferase activities of parental REG cells, DN1 and DN2 mock transfected REG cells and clones from REG cells transfected with the FTA or FTB cDNAs. Enzyme activities were tested as described in Materials and methods using Benzyl 2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-{alpha}-D-galactopyranoside (benzoylated type 3 precursor) as acceptor and GDP-L-[14C]-fucose as donor.

 
Since the cells tumorigenicity correlated with their sensitivity to cell death mediated by serum deprivation, the sensitivity to apoptosis of the transfectants was tested. In a first set of experiments, FUT1 transfectants of REG cells were cultured for either 72 h or 96 h in absence of FCS after what their ability to form colonies was compared to that of mock transfected cells treated similarly. As shown in Figure 4A, after 72 h, H1 and H2 cells proved to be more resistant than control cell lines DN1 and DN2 since their percentage of surviving colonies was almost twice higher. A similar difference was obtained after 96 h, although in this last case, the percentage of surviving colonies was lower for all cell types. The sensitivities to apoptosis of FTA and FTB transfected REG cells were next tested by the same method after 96 h of culture in absence of FCS. Cells transfected with the FTA cDNA were significantly more resistant than the control cells. However, clone 19 was more sensitive than clone 24 (Figure 4B). This could be related to the lower levels of {alpha}2FT activity and of H antigen synthesized by this clone (Figures 2 and 3). In the case of FTB transfectants (Figure 4C), the three clones gave 2–3 times higher percentages of surviving colonies than did control cell lines DN1 and DN2.



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Fig. 4. Sensitivity of transfectants to apoptosis induced by serum deprivation. (A) Comparison between DN1 and DN2 mock transfected REG cells, and FUT1 cDNA transfected REG cells; (B) comparison between mock transfected REG cells and FTA cDNA transfected REG cells; (C) comparison between mock transfected REG cells and FTB cDNA transfected REG cells; (D) comparison between R, a control transfected clone from PRO cells and A4, a clone from PRO cells transfected with a FTA cDNA fragment in the antisense orientation. Cells were cultured for 72 h (A) or 96 h (B–D) in the absence of FCS and survival was measured by a colony formation assay as described in Materials and methods. Percentages of surviving colonies were determined by comparison with number of colonies from cultures in the presence of serum. The number of colonies from these control cultures varied from 500 to 300 according to the experiment and did not differ for the various cell types.

 
After transfection of the PRO clone with an FTA antisense cDNA fragment, its expression of {alpha}1,2fucosylated structures decreased along with its enzymatic activity (Figure 2 and Hallouin et al., 1999Go). After 96 h in absence of FCS, the control clone R showed a 99% survival. In contrast, the percentage of surviving colonies was only 54% for the antisense transfectant clone A4 (Figure 4D).

Survival of the various transfected REG cells after UV treatment was also evaluated by the colony formation assay. Small differences in survival were visible among the different clones. However, they were not related to the presence of {alpha}1,2fucosylated structures at the cell surface (Figure 5). In order to confirm that serum deprivation induced cell death by apoptosis in the experimental cellular model, cell nuclei were stained with Hoechst 33258. Nuclei from floating cells from either H positive or H negative lines presented nucleolar condensation characteristic of apoptosis (Figure 6A). This was not the case for adherent cells. Similarly, characteristic DNA degradation was observed for floating cells but not for adherent cells, irrespective of the cell type (Figure 6B). In another set of experiments, floating cells were collected each day and pooled during the 96 h of culture in the absence of FCS and protein amounts were quantified using the Bradford reagent. DN1 control cells yielded twice more apoptotic cells than H1 cells measured as protein amounts (data not shown).



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Fig. 5. Comparison of sensitivity to apoptosis induced by UV irradiation between mock transfected REG cells, DN1 and DN2 and REG cells transfected either with the FUT1 cDNA, H2; the FTA cDNA, clones 19, 24; or the FTB cDNA, clones A3 and A8. Confluent cell cultures were submitted to UV irradiation for 1 min, and their survival was determined by a colony formation assay.

 


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Fig. 6. Characterization of apoptotic cell death after culture in absence of serum. (A) Nuclei from floating cells in cultures from DN1 mock transfected REG cells, were stained by Hoechst 33258 and visualized by fluorescence microscopy. The same figures of apoptosis were observed for nuclei from floating cells of all the other transfectants described in this study. (B) DNA was extracted from floating cells or adherent cells in cultures without serum from DN1 mock transfected REG cells or H1 REG cells transfected with the FUT1 cDNA and visualized by ethidium bromide staining after electrophoresis on 1.8% agarose gels. Lane 1, size markers; lane 2, DNA from adherent DN1 cells; lane 3, DNA from floating DN1 cells; lane 4, DNA from adherent H1 cells; lane 5, DNA from floating H1 cells. Similar results were obtained with adherent and floating cells respectively from the other transfectants.

 
{alpha}1,2Fucosylated REG cells escape immune rejection
We have shown earlier that REG cells expressing the H antigen after transfection with an {alpha}2FT cDNA increase their tumorigenicity in syngeneic rats (Goupille et al., 1997Go). In order to determine the importance of the immune system in this phenomenon, syngeneic rats and SCID mice were injected with mock transfected or {alpha}2FT transfected REG cells. As previously reported, H positive REG cells gave rise to progressive tumors in 4 out of 6 syngeneic rats in the experiment presented in Figure 7. At variance, in SCID mice, both cell types gave rise to progressive tumors in 5 out 5 animals, indicating that the immune system is responsible for the rejection of REGb cells in syngeneic rats and that {alpha}2FT transfected cells can overcome this immune rejection. In addition, two to three small lung metastatic foci were found in rats bearing large REGH1 tumors as well as in mice bearing either REGDN1 control tumors or REGH1 {alpha}1,2fucosylated tumors. No significant difference was observed between these two last groups. The absence of difference between REGDN1 and REGH1 tumor development in SCID mice is in keeping with the fact that {alpha}2FT transfection did not alter the cells proliferation or adhesion properties on various substrates such as fibronectin, collagen IV, hyaluronic acid, rat fibroblasts or vascular endothelial cell lines (data not shown). It should also be noted from comparison of Figures 1 and 7 that in syngeneic rats the growth of REGH1 tumors remained significantly slower than that of PRO tumors.



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Fig. 7. Growth in either syngeneic immunocompetent BDIX rats or in immunodeficient SCID mice of mock transfected REG cells (DN1) and FUT1 transfected REG cells (H1). 1 x 106 cells were injected s.c. and tumor volumes were measured weekly. Each line represents tumor growth in a single animal.

 
We next tested whether apoptosis could be observed in situ and whether a difference would exist between the control tumors from mock transfected REG cells and tumors from {alpha}2FT transfected cells. Three weeks after injection, massive apoptosis of cancer cells could be evidenced by the TUNEL assay in DN1 control tumors. In contrast, few apoptotic cells were detected in two out of three tumors originating from H1 {alpha}2FT transfected cells (Figure 8).



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Fig. 8. Comparison of apoptotic tumor cells in tumors from mock transfected REG cells (a) and from FUT1 transfected REG cells (b), 21 days after s.c. cell injection. Apoptotic cells are labeled in brown by the TUNEL assay and nonapoptotic cells are labeled in green by counterstaining with methyl green. Scale bar, 50 µm.

 
In order to get more insights into the mechanism of REG tumor rejection, syngeneic rats were depleted of either CD4 lymphocytes, CD8 lymphocytes or both CD4 and CD8 lymphocytes and REG tumor growth was determined in such depleted animals. A slightly increased tumor growth occurred in CD8 depleted animals. A more important effect of CD4 depletion was observed, while the fastest REG tumor growth occurred in rats depleted of both CD4 and CD8 lymphocytes subsets (Figure 9). These results indicate that the two cell types are involved in the rejection process, but that most of the effect is driven by CD4 lymphocytes.



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Fig. 9. Growth of REG tumors in groups of five rats depleted of either CD4 lymphocytes, CD8 lymphocytes, both CD4 and CD8 lymphocytes, or in a control group without lymphocytes depletion. Rats were depleted of the CD4 and CD8 lymphocytes subpopulations using antibodies OX-38 and OX-8, respectively. Twelve days after the first injection of antibody, they received 5 x 106 REG cells s.c. Tumor volumes were measured 14 days after injection of tumor cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We observed earlier that blocking {alpha}1,2fucosylation of the tumorigenic PRO cells by transfection of an {alpha}1,2fuco­syltransferase cDNA in the antisense orientation decreased tumorigenicity in syngeneic animals (Labarrière et al., 1994Go). Inversely, transfection of the spontaneously regressing REG cells by the FUT1 cDNA in the sense orientation allowed synthesis of H antigenic determinants and led to the acquisition of a progressive phenotype in syngeneic animals (Goupille et al., 1997Go). This result was reproduced here, and it was observed in addition that in SCID mice, {alpha}1,2fucosylated and control REG cells grew progressively at the same rate and formed the same number of metastatic foci. Thus, in this experimental model, the increased tumorigenicity conferred by {alpha}1,2fucosylation rests entirely on the modulation of the cells’ sensitivity to immune control.

Although the precise mechanism of REG tumor rejection is not defined, it required T lymphocytes, mainly of the CD4 subset, as evidenced by the depletion experiments. Rejection was characterized by massive apoptosis and correlated with in vitro sensitivity of cells to apoptosis induced by serum deprivation. Indeed, mock transfected REG cells, DN1 and DN2, were much more sensitive to serum deprivation than PRO control transfected cells (clone R), while {alpha}2FT transfected REG cells displayed an intermediate level of sensitivity together with an intermediate level of tumorigenicity in syngeneic animals. Thus, in immunocompetent animals, the difference in tumorigenicity between the PRO and REG cell types correlates with their degree of resistance to apoptosis, which itself depends, at least in part, on the level of {alpha}1,2fucosylation. The sensitivity of the various REG transfectants to UV-induced apoptosis was not different, suggesting that the cell death pathway with which {alpha}1,2fucosylation interferes is distinct from the one induced by UV irradiation. Proteins of the Bcl-2 family are well known modulators of the sensitivity or resistance to apoptosis (Hawkins and Vaux, 1998Go). It was therefore determined by ELISA that the levels of Bcl-2 and Bax proteins were not different between the various transfected clones (data not shown). This is in agreement with the results of Bonotte et al. (1998)Go who showed by Western blotting, that PRO and REG cells express similar amounts of Bcl-2 and Bax proteins. Therefore, the differences in sensitivity to apoptosis between the cell types studied here cannot be explained by differences in expression of these pro- or anti-apoptotic proteins.

Since various isoforms of {alpha}2FT exist, and that they possess distinct catalytic properties (Masutani and Kimura, 1995Go) they could mediate different biological effects. Yet, it was observed that FUT1, FTA, and FTB transfectants showed similarly increased resistance to apoptosis. We observed earlier that in PRO cells, which possess FTA and FTB mRNA at similar levels, 2 major glycoproteins carry {alpha}1,2fucosylated glycan chains. One of them, migrating at around 200 kDa, is a high molecular weight variant of CD44; the other at about 80 kDa has not been characterized as yet (Hallouin et al., 1999Go). In REG cells transfected with the FTA cDNA, the same observation was made. However, when transfected with the FTB cDNA, only the CD44 variant could be shown to carry {alpha}1,2fucosylated structures using either the UEA-I lectin or a panel of specific MAbs (Bureau et al., unpublished observations), indicating that these enzymes use different substrates in situ. Thus, the transfection experiments in REG cells revealed that the only detectable glycoprotein that the three enzymes fucosylate in common is the high molecular weight variant of CD44. Of particular interest is the observation that CD44 can inhibit apoptosis (Ayroldi et al., 1995Go; Yu et al., 1997Go). Furthermore, it was recently shown that CD44 molecules are shed by proteolytic cleavage in early steps of CD95-mediated apoptosis of colon carcinoma cells, suggesting that this shedding could contribute to the cell disintegration after detachment from the substrate (Günthert et al., 1996Go). Fucosylation of CD44 could possibly protect it from proteolytic cleavage. Alternatively, it could strengthen cell adhesion, slowing the process of detachment which precedes cell death. These hypotheses can now be tested.

In conclusion, we have shown that immune rejection of REG cells correlates with their sensitivity to apoptosis and that synthesis of H antigenic determinants by {alpha}2FT allows escape from immune rejection and increases resistance to cell death. Fucosylated cells could be more resistant to immune effector mechanisms. Alternatively, it has been recently shown that apoptotic bodies are potent immunogens (Boisteau et al., 1997Go; Albert et al., 1998Go). Therefore in absence of H antigen, a higher number of apoptotic bodies would be generated leading to an enhanced immune response. Resistance to apoptosis could be a factor of bad prognosis (Graeber et al., 1996Go). The increased resistance to cell death conferred by {alpha}1,2fucosyl­transferases could explain the previously observed associations between expression of carbohydrate antigens and poor prognosis in colorectal and lung carcinomas (Miyake et al., 1992Go; Naitoh et al., 1994Go).


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Cell lines
TS8F, REG, and PRO rat colon adenocarcinoma cells (obtained from Dr. F.Martin, Dijon, France) are clones derived from a dimethylhydrazine-induced cell line (DHDK12). REG has been previously transfected with a cDNA encoding for the FUT1 human {alpha}2FT (Goupille et al., 1997Go). Briefly, two independent transfections have been performed and following transfection, cells expressing {alpha}1,2-linked fucose residues were sorted by flow cytometry. The resultant populations were called REGH1 and REGH2. Control cell lines were obtained after two independent transfections with the empty vector. These mock transfected cells were called REGDN1 and REGDN2. PRO cells transfected with a fragment of the rat {alpha}2FT FTA cDNA in the antisense orientation have been previously described (Hallouin et al., 1999Go). A clone with stable expression of the antisense fragment, called A4 was selected. A subclone from A4, obtained after spontaneous loss of the antisense insert was used as control. This revertant clone was called R. Cells were cultured in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Gibco BRL, Cergy-Pontoise, France). They were subcultured at confluency after dispersal with 0.025% trypsin in 0.02% 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.

FTA and FTB transfection of REG cells
Two partial cDNAs encoding for 2 distinct rat {alpha}1,2fuco­syltransferases have previously been cloned in our laboratory (Piau et al., 1994Go). The complete cDNAs for the two enzymes homologous to human FUT1 and FUT2 have now been cloned and the two enzymes termed FTA and FTB, respectively (accession numbers: AF131237 and AF131238). They were inserted in the pBK-CMV vector (Stratagene, Cambridge, UK), deleted of the lacZ promoter by digestion with Spe1 and Nhe1, through the EcoRI site of the multiple cloning site. REG cells were transfected with the vector containing either the FTA or the FTB cDNA in the sense orientation using lipofectamin (GIBCO BRL) according to the manufacturer’s instructions. Selection of stable transfectants was achieved by addition of 0.6 mg/ml G418 for 2 weeks. Transfected cells expressing {alpha}1,2-linked fucose residues were selected by flow cytometry using FITC-labeled UEA-I lectin (Sigma, St. Louis, MO). The resultant populations were then expanded and cloned by limiting dilutions.

Cytofluorimetric analysis
Viable cells (2 x 105/well) were incubated with FITC-labeled UEA-I at 20 µg/ml in PBS containing 0.1% gelatin for 1 h at 4°C. After washings in the same buffer, fluorescence analysis was performed on a FACScan (Becton-Dickinson) using the CELLQuest software. Control of the specificity of the binding was performed by coincubation of the lectin with 0.2 M fucose.

{alpha}1,2Fucosyltransferase assay
Confluent cells were rinsed with ice-cold PBS pH 7.2 then recovered by scraping. After washing with ice-cold PBS, cells were solubilized in 50 mM potassium phosphate pH 6.0, containing 2% (v/v) Triton X-100 on ice for 30 min. Following a centrifugation at 13,000 x g for 10 min, the supernatant was collected and used as a crude enzyme preparation. Protein concentration was determined using bicinchoninic acid obtained from Pierce (Rockford, IL).

The reaction mixture contained: 20 µM GDP-L-[14C]-fucose (23 mCi/mmol, NEN Chemical Center, Dreieichenhain, Germany), 20 µM benzyl 2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-{alpha}-D-galactopyranoside (Sigma, St. Louis, MO), 10 mM L-fucose, 7.7 mM MgCl2, 1.9 mM ATP, and 50 µg protein extract in a final volume of 33 µl. After an incubation at 37°C for 3 h, the reaction mixture was quenched with 5 ml of distilled water and applied to a freshly conditioned C-18 Sep Pak cartridge (Waters-Millipore). The cartridge was washed with 20 ml water. The radiolabeled product was then eluted with 5 ml methanol and counted in 10 ml scintillation liquid (Ready Safe, Beckman, Palo Alto, CA). Background levels of radioactivity were obtained from controls without exogenous acceptor. Values obtained for the controls were then subtracted from those obtained for the assays.

Determination of in vitro cell sensitivity to apoptosis
The sensitivity of cells to apoptosis was quantified after serum withdrawal or UV treatment by a colony formation assay. Cells were cultured in complete medium for 48 h, until confluency, before treatment started. They were then washed with serum-free medium and kept in the same deprived medium for either 72 or 96 h. The medium was changed twice during this incubation time. Adherent cells were then detached with EDTA-trypsin and 1 x 103 cells seeded in triplicate wells of 6-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 UV treatment by exposing cell cultures flasks for 1 min under a UV light. Cells were then immediately detached with EDTA-trypsin and assayed for their ability to form colonies in 6-well flat bottom culture plates as above. 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 UV, floating 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, floating 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), housed and bred under standard conditions in our laboratory. SCID mice were purchased from Iffa-Credo and housed under sterile conditions. Ten-week-old rats and six-week-old mice were used. Confluent 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 subcutaneously in the flank of animals. Tumors were weekly measured with calipers and animals with large tumors were sacrificed before they became moribund. These experiments were performed in agreement with the rules from the French Ministry of Agriculture, under supervision of the Veterinary Services (Agreement A44565).

In situ analysis of apoptosis
Tumors from three rats with REGDN1 control tumors and from three rats with REGH1 control tumors were excised 3 weeks after cells’ injection, fixed in ethanol 95% for 48 h, and paraffin embedded. In situ detection of apoptotic cells was performed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method. Sections (5 µm) were deparaffinized and treated with terminal transferase and biotin-dUTP, followed by streptavidin-peroxidase using reagents from Oncogene Research Products (Cambridge, MA) and according to the manufacturer’s instructions. Sections were then counterstained with methyl green, which results in a green staining of non-apoptotic nuclei, whereas apoptotic nuclei are stained brown by the peroxidase substrate.

Depletion of CD4 and CD8 lymphocytes
Groups of six rats were used. One group was used as control without depletion. A second group received intraperitoneally 480 µg of purified anti-rat CD8 monoclonal antibody OX-8 and then twice 7 days apart to maintain depletion of CD8 lymphocytes. A third group was depleted of CD4 lymphocytes by three weekly i.p. injections of 640 µg of purified anti-rat CD4 antibody OX-38. A fourth group received both the anti-CD8 and the anti-CD4 with the same schedule as the groups that received a single antibody. Efficiency of the depletions were assessed by immunofluorescence on splenic lymphocytes and on circulating lymphocytes of one rat from each group, sacrificed 12 days after the first injection of antibodies. The same day, the five remaining rats of each group were injected with 5 x 106 REG cells s.c. and tumor growth was monitored.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We thank Drs. F.Valette, K.Meflah, and J.Sleeman for helpful discussions and Ms. P.Fichet and N.Ruellan for animal care. This work was supported by the Institut National de la Santé et de la Recherche Médicale and by grants from the Ligue Départementale des Pays de la Loire and the Association pour la Recherche sur le Cancer.


    Footnotes
 
1 The two first authors contributed equally to the work. Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Albert,M.L., Suater,B. and Bhardwaj,N. (1998) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumors. Nature, 392, 86–89.[ISI][Medline]

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