Stabilization of β-catenin by genetic defects in melanoma cell lines

Science

Volumn 275, Issue 5307, 1997, Pages 1790-1792

  Rubinfeld, Bonnee ^a   Robbins, Paul ^b   El Gamil, Mona ^b   Albert, Iris ^a   Porfiri, Emilio ^a   Polakis, Paul ^a  

^a ONYX PHARMACEUTICALS   (United States)

^b NATIONAL CANCER INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BETA CATENIN; LYMPHOID ENHANCER FACTOR 1; TRANSCRIPTION FACTOR;

ARTICLE; HUMAN; HUMAN CELL; MELANOMA CELL; MISSENSE MUTATION; PRIORITY JOURNAL; PROTEIN STABILITY; RNA SPLICING; SIGNAL TRANSDUCTION; TUMOR SUPPRESSOR GENE;

ADENOMATOUS POLYPOSIS COLI PROTEIN; ANIMALS; BETA CATENIN; CELL LINE; CYTOSKELETAL PROTEINS; DNA-BINDING PROTEINS; GENE EXPRESSION REGULATION, NEOPLASTIC; GENES, APC; HUMANS; LYMPHOID ENHANCER-BINDING FACTOR 1; MELANOMA; MICE; MUTATION; POINT MUTATION; RNA SPLICING; RNA, MESSENGER; RNA, NEOPLASM; TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSFECTION; TUMOR CELLS, CULTURED; UP-REGULATION;

EID: 0030900696     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5307.1790     Document Type: Article

References (28)

1. B. M. Gumbiner, Curr. Opin. Cell Biol. 7, 634 (1995); M. Reifer, Trends Cell Biol. 5, 224 (1995); J. Klingensmith and R. Nusse, Dev. Biol. 166, 396 (1994).
2. B. M. Gumbiner, Curr. Opin. Cell Biol. 7, 634 (1995); M. Reifer, Trends Cell Biol. 5, 224 (1995); J. Klingensmith and R. Nusse, Dev. Biol. 166, 396 (1994).
3. B. M. Gumbiner, Curr. Opin. Cell Biol. 7, 634 (1995); M. Reifer, Trends Cell Biol. 5, 224 (1995); J. Klingensmith and R. Nusse, Dev. Biol. 166, 396 (1994).
4. J. Behrens et al., Nature 382, 638 (1996); M. Molenaar et al., Cell 86, 391 (1996); O. Huber et al., Mech. Dev. 59, 3 (1996).
5. J. Behrens et al., Nature 382, 638 (1996); M. Molenaar et al., Cell 86, 391 (1996); O. Huber et al., Mech. Dev. 59, 3 (1996).
6. J. Behrens et al., Nature 382, 638 (1996); M. Molenaar et al., Cell 86, 391 (1996); O. Huber et al., Mech. Dev. 59, 3 (1996).
7. R. S. Bradley, P. Cowin, A. M. C. Brown, J. Cell. Biol. 123, 1857 (1993); L. Hinck, W. J. Nelson, J. Papkoff, ibid. 124, 729 (1994); A. S. Tsukamoto, R. Grosschedl, R. C. Guzman, T. Parslow, H. E. Varmus, Cell 55, 619 (1988).
8. R. S. Bradley, P. Cowin, A. M. C. Brown, J. Cell. Biol. 123, 1857 (1993); L. Hinck, W. J. Nelson, J. Papkoff, ibid. 124, 729 (1994); A. S. Tsukamoto, R. Grosschedl, R. C. Guzman, T. Parslow, H. E. Varmus, Cell 55, 619 (1988).
9. R. S. Bradley, P. Cowin, A. M. C. Brown, J. Cell. Biol. 123, 1857 (1993); L. Hinck, W. J. Nelson, J. Papkoff, ibid. 124, 729 (1994); A. S. Tsukamoto, R. Grosschedl, R. C. Guzman, T. Parslow, H. E. Varmus, Cell 55, 619 (1988).
10. S. Munemitsu, I. Albert, B. Souza, B. Rubinfeld, P. Polakis, Proc. Natl. Acad. Sci. U.S.A. 92, 3046 (1995).
11. S. Munemitsu, I. Albert, B. Rubinfeld, P. Polakis, Mol. Cell. Biol. 16, 4088 (1996).
12. N. Funayama, F. Fagotto, P. McCrea, B. M. Gumbiner, J. Cell. Biol. 128, 959 (1995); S. Orsulic and M. Reifer, ibid. 134, 1283 (1996).
13. N. Funayama, F. Fagotto, P. McCrea, B. M. Gumbiner, J. Cell. Biol. 128, 959 (1995); S. Orsulic and M. Reifer, ibid. 134, 1283 (1996).
14. C. Yost et al., Genes Dev. 10, 1443 (1996).
15. P. F. Robbins et al., J. Exp. Med. 183, 1185 (1996).
16. J. Papkoff, B. Rubinfeld, B. Schryver, P. Polakis, Mol. Cell. Biol. 16, 2128 (1996).
17. This monomeric pool represents unbound β-catenin but does not reflect a lack of association of β-catenin with its binding proteins. For example, cells with this pool of excess β-catenin generally have much higher amounts of β-catenin associated with APC than do those without. There is a 100-to 1000-fold molar excess of β-catenin over APC in most cells and, therefore, saturation of APC with β-catenin would not significantly deplete the monomeric β-catenin pool.
18. D. E. Brash et al., Proc. Natl. Acad. Sci. U.S.A. 88, 10124 (1991).
19. B. Rubinfeld and P. Polakis, unpublished results.
20. 125I-labeled protein A at 0.5 μCi/ml (Amersham).
21. B. Rubinfeld et al., Science 262, 1731 (1993).
22. M. G. Prieve, K. L. Guttridge, J. E. Munguia, M. L. Waterman, J. Biol. Chem. 271, 7654 (1996).
23. The melanoma cell lines were generated from metastatic lesions (17) with the exception of the SK23 mel (21). The 888 and 1290 mel lines were derived from two independent metastases from the same patient; all others originated from separate patients. The SW480 cell line was obtained from the American Type Culture Collection (ATCC reference CCL228) and is a human colon cancer cell line. ATT20 (ATCC reference CCL89) is a murine pituitary tumor cell. Stable ATT20 clones expressing β-catenins were generated as previously described (5).
24. S. Topalian, D. Solomon, S. Rosenberg, J. Immunol. 142, 3714 (1989).
25. Transient transfection of 928 mel cells with plasmid encoding human WT APC was performed with lipofectamine (BRL) (4). Cells were fixed 48 hours later and stained for immunofluorescence microscopy (19). For detection of APC, cells were first incubated with COOH-terminal-specific APC3 antibody (14) and then with fluorescein isothiocyanate-conjugated goat antibody to rabbit immunoglobin G (IgG) (Sigma). We detected β-catenin by using mouse anti-β-catenin monoclonal antibody (Transduction Laboratories, Lexington, KY) and Texas red-conjugated donkey antibody to mouse IgG (Cappel, Durham, NC).
26. S. Munemitsu et al., Cancer Res. 54, 3676 (1994).
27. Cells were pulse-labeled (4) for 30 min and then incubated with media containing unlabeled methionine for the indicated times before lysis on the culture dish. After centrifugation of the lysates, β-catenin was immunoprecipitated from the melanoma cell supernatants with anti-β-catenin, and from the ATT20 or SW480 supematants by antibody to myc that had been covalently coupled to protein G-Sepharose. Immunoprecipitates were subjected to electrophoresis and fluorography on 8% SDS-polyacrylamide gels. In the transfection experiments (4), >50% of the SW480 cells expressed the ectopic cDNA. The APC25 construct encoded APC amino acids 1034 to 2130, and APC3 encoded amino acids 2130 to 2843. For isolation of β-catenin cDNAs, a cDNA pool was first obtained by reverse transcription of total mRNA (RNeasy kit, Qiagen), by use of a mixture of oligo(dT) and random primers. Polymerase chain reaction (PCR) was then performed on the cDNA pool, by use of six distinct primer sets specific for β-catenin cDNA, and the PCR products were cloned into pCR2.1 (Invitrogen) and propagated in Escherichia coli. Mutations of β-catenin were confirmed by sequencing analysis of PCR products obtained with the multiple primer sets.
28. We thank M. Waterman for antibody to Lef-1, T. Vuong and J. Heath for assistance with nucleotide sequencing, and T. Boon for SK23 mel cells. Supported in part by Small Business Innovation Research grant 1R43CA69931 from NIH.