Abstract
A protein formed by fusion of transforming growth factor α withPseudomonas exotoxin (TGFα-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate thein vitro cytotoxic effects of TGFα-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGFα-PE40 to bind EGFR, and the cytotoxic effect of TGFα-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR=4.9×103–11.4×103/cell) had ED50 values (the concentration of TGFα-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; forc 1 (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGFα-PE40 concentrations of 104 pM, 104 pM and higher than 104 pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR=32×103–126×103/cell) had ED50 values of 20 pM, 66 pM, and 330 pM respectively and T24 showedc 1 values of 103 pM. RU-P1 (EGFR =92.6×103/cell) had the highest ED50 value of 8000 pM. These data indicate that the susceptibility to TGFα-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGFα-PE40, and that normal urothelial cells are more resistant to TGFα-PE40 than are cancer cells. The differential effect of TGFα-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.
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References
Ahlering TE, Dubeau L, Jones PA (1987) A newin vivo model to study invasion and metastasis of human bladder carcinoma. Cancer Res 47:6660–6665
Azuma M, Momose H, Oyasu R (1990)In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure toN-methyl-N-nitrosourea. Cancer Res 50:7062–7067
Bubenik J, Baresora M, Viklicky V, Jakoubkova J, Sainerova H, Donner J (1973) Established cell line of urinary bladder carcinoma (T-24) containing tumor specific antigen. Int J Cancer 11:765–773
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936–942
Chaudhary VK, FitzGerald DJ, Adhya S, Pastan I (1987) Activity of a recombinant fusion protein between transforming growth factor type α andPseudomonas toxin. Proc Natl Acad Sci USA 84:4538–4542
Elliott AY, Cleveland P, Cervenka J, Castro AE, Stein N, Hakala TR, Fraley EE (1974) Characterization of cell line from human transitional cell cancer of the urinary tract. J Natl Cancer Inst 53:1341–1349
Heimbrook DC, Stirdivant SM, Ahern JD, Balishin NL, Patrick DR, Edwards GM, Defeo-Jones D, FitzGerald DJ, Pastan I, Oliff A (1990) Transforming growth factor α-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts. Proc Natl Acad Sci USA 87:4697–4701
Hicks RM (1975) The mammalian urinary bladder: an accommodating organ. Biol Rev 50:215–246
Hirata Y, Orth DN (1979) Epidermal growth factor (urogastrone) in human fluids: size heterogeneity. J Clin Endocrinol Metab 48:673–679
Johnson MD, Bryan GT, Reznikoff CA (1985) Serial cultivation of normal rat bladder epithelial cellsin vitro. J Urol 133: 1076–1081
Kameyama S, Kawamata H, Kawai K, Oyasu R (1993) A novelin vivo model to study invasion and metastasis of rat and human bladder carcinomas. Carcinogenesis 14:1531–1535
Kawamata H, Azuma M, Kameyama S, Nan L, Oyasu R (1992) Effect of EGF/TGF-α and TGF-β1 on growth in vitro of rat urinary bladder carcinoma cells. Cell Growth Differ 3:819–825
Kawamata H, Kameyama S, Nan L, Yamamoto M, Oyasu R (1993) Effect of epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) on growth and invasive potentials of newly established rat bladder carcinoma cell lines. Int J Cancer 55:968–973
Masters JRW, Hepburn PJ, Walker L, Highman WJ, Trejdosiewicz LK, Povey S, Parker M, Hill BT, Riddle PR, Franks LM (1986) Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines. Cancer Res 46:3630–3636
Messing EM (1990) Clinical implications of the expression of epidermal growth factor receptors in human transitional cell carcinoma. Cancer Res 50:2530–2537
Messing EM, Hanson P, Ulrich P, Erturk E (1987) Epidermal growth factor-interactions with normal and malignant urothelium:in vivo and in situ studies. J Urol 138:1329–1335
Middlebrook JL, Dorland RB (1976) Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa andCorynebacterium diphtheriae: differential cytotoxity. Can J Microbiol 23:183–189
Momose H, Kakinuma H, Shariff SY, Mitchell GB, Rademaker A, Oyasu R (1991) Tumor-promoting effect of urinary epidermal growth factor in rat urinary bladder carcinogenesis. Cancer Res 51:5487–5490
Neal DE, Sharples L, Smith K, Fennelly J, Hall RR, Harris AL (1990) The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer 65:1619–1625
Pai LH, Gallo MG, FitzGerald DJ, Pastan I (1991) Antitumor activity of a transforming growth factor α-Pseudomonas exotoxin fusion protein (TGF-α-PE40). Cancer Res 51:2808–2812
Pastan I, FitzGerald D (1991) Recombinant toxins for cancer treatment. Science 254:1173–1177
Rigby CC, Franks LM (1970) A human tissue culture cell line from a transitional cell tumor of the urinary bladder: growth, chromosome pattern and ultrastructure. Br J Cancer 24:746–754
Sarosdy MF, Hutzler D, Von Hoff DD (1992) TP-40 in bladder cancer: a hybrid protein with selective targeting and cytotoxicity. J Urol 147:261A
Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660–664
Siegal CB, Xu Y, Chaudhary VK, Adhya S, FitzGerald D, Pastan I (1989) Cytotoxic activities of a fusion protein comprised of TGFα andPseudomonas exotoxin. FASEB J 3:2647–2652
Soloway MS, Masters S (1980) Urothelial susceptibility to tumor cell implantation: influence of cauterization. Cancer 46:1158–1163
Wood DP Jr, Fair WR, Chaganti RSK (1992) Evaluation of epidermal growth factor receptor DNA amplification and mRNA expression in bladder cancer. J Urol 147:274–277
Yura Y, Hayashi O, Kelly M, Oyasu R (1989) Identification of epidermal growth factor as a component of the rat urinary bladder tumor-enhancing urinary fractions. Cancer Res 49:1548–1553
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Supported by National Institutes of Health Grant CA14649
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Kameyama, S., Kawamata, H., Pastan, I. et al. Cytotoxic effect of a fusion protein from transforming growth factora andPseudomonas exotoxin on rat and human bladder carcinoma cells in vitro. J Cancer Res Clin Oncol 120, 507–512 (1994). https://doi.org/10.1007/BF01221026
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DOI: https://doi.org/10.1007/BF01221026