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.
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Abstract |
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Key words: 1,2fucosyltransferase/apoptosis/tumorigenicity/rat/carcinoma
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Introduction |
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Expression of type 1 [Lewis b (Leb)] and type 2 [H type 2 (H-2) and Lewis Y (Ley)] structures, with the common motif Fuc12Galß-R, is a characteristic feature of tumor progression in the distal colon and rectum (Itzkowitz, 1992
) and it has been associated with poor prognosis (Naitoh et al., 1994
). In accordance with these observations,
2FT activity is clearly increased in colon cancer (Orntoft et al., 1991
; Yazawa et al., 1993
; Sun et al., 1995
). 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
1,2fucosylated antigens in colon carcinomas (Yazawa et al., 1993
). Nevertheless, it was unambiguously shown by Sun et al. (1995)
that FUT1 is transcribed in colon adenocarcinoma and more recently, by Nishihara et al. (1999)
, 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, 1991
; Garrigues et al., 1994
; Goupille et al., 1997
). We observed previously, in a rat model of colon carcinoma, that the presence of
1,2fucosylated structures at the cell surface increased tumorigenicity. In this model, described by Martin et al. (1983)
, 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., 1985
). This rejection is immunologically mediated since REG tumors can grow in cyclosporin-treated rats (Shimizu et al., 1989
) or in rats depleted of
ß TCR bearing lymphocytes (Ménoret et al., 1995
). Unlike REG cells, PRO cells present
1,2fucosylated antigens at their surface, possess
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., 1994
). 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., 1997
). 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
2FT transfection is able to modulate the cells sensitivity to apoptosis as well as their in vivo behavior.
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Results |
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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 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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Discussion |
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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 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
1,2fucosylation. The sensitivity of the various REG transfectants to UV-induced apoptosis was not different, suggesting that the cell death pathway with which
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, 1998
). 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)
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 2FT exist, and that they possess distinct catalytic properties (Masutani and Kimura, 1995
) 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
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., 1999
). 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
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., 1995
; Yu et al., 1997
). 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., 1996
). 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 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., 1997
; Albert et al., 1998
). 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., 1996
). The increased resistance to cell death conferred by
1,2fucosyltransferases could explain the previously observed associations between expression of carbohydrate antigens and poor prognosis in colorectal and lung carcinomas (Miyake et al., 1992
; Naitoh et al., 1994
).
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Materials and methods |
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FTA and FTB transfection of REG cells
Two partial cDNAs encoding for 2 distinct rat 1,2fucosyltransferases have previously been cloned in our laboratory (Piau et al., 1994
). 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 manufacturers instructions. Selection of stable transfectants was achieved by addition of 0.6 mg/ml G418 for 2 weeks. Transfected cells expressing
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.
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--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 (LAbresle, 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 manufacturers 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.
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Acknowledgments |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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