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
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Abstract |
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Key words: A enzyme/apoptosis/blood group/carcinoma/tumorigenicity
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Introduction |
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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 1,2 linkage by an
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
1,2fucosyltransferases. In colon cancer, the
1,2fucosyltransferase activity of both FUT1 and FUT2 is clearly increased (Nishihara et al., 1999
; Sun et al., 1995
). 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., 1992
). In this model, described by Caignard et al. (1985)
), 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
1,2fucosyltransferase actitivity, forms only smalls tumors that are immunologically rejected within a few weeks.
We previously showed by transfection experiments that 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
1,2fucosyltransferase transfected REG cells were fully tumorigenic indicating that the increased tumorigenicity mediated by
1,2fucosylation was associated with increased resistance to apoptosis and with escape from immune control (Goupille et al., 2000
). 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., 1995
). The BDIX rat A enzyme cDNA has been recently cloned in our laboratory (Cailleau-Thomas et al., 2002
), 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.
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Results |
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Discussion |
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Analysis of the cells sensitivity to apoptosis induced by either heat shock or serum deprivation indicated that all the A antigenexpressing 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, 1994). 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., 2002
). 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, 1997; Hakomori, 1996
, 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, 2002
).
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., 2001). 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 antigenpositive 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., 1997
, 1998). Similarly, at these later stages, the decreased expression of
1,2fucosyltransferase activity, and hence of ABH antigens, may participate to the metastatic progression by releasing the competition with an
2,3sialyltransferase, leading to increased expression of the sialyl-Lea and sialyl-Lex selectin ligand, as shown by others (Aubert et al., 2000
). 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.
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Materials and methods |
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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 manufacturers 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 33A (Bara et al., 1988). 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 33A 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 isothiocyanatelabeled 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 (LAbresle, 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).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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