Summary
Ligands for the epidermal growth factor receptor (EGFR) (e.g. EGF and transforming growth factor alpha, TGFα) may be included in membrane preparations from human breast tumors, either complexed or not to EGFR. Assessment of EGFR in human placental membranes (HPM) after preincubation with human EGF (hEGF) or human TGFα (hTGFα) indicated that the presence of these ligands in a membrane preparation did not affect the apparent number of binding sites, but only resulted in an increased apparent dissociation constant (Kd).
To obviate the effect of endogenously bound EGFR ligands in a radioligand binding assay, an acid treatment procedure has been used recently. In the present study we show that acid treatment of mammary tumor membranes does not result in increased EGFR levels but does result in an EGFR enriched membrane preparation by elimination of contaminating cytosol proteins. This however also occurred by washing the membranes with assay buffer at neutral pH.
From these experiments we conclude that endogenous ligands, if present in membrane fractions from human breast tumors and occupying EGFR, do not interfere in a multipoint radioligand binding assay.
This is a preview of subscription content, log in via an institution to check access.
Abbreviations
- EGF:
-
Epidermal Growth Factor
- TGFα:
-
Transforming Growth Factor alpha
- EGFR:
-
Epidermal Growth Factor Receptor
- HAP:
-
Hydroxylapatite
References
Carpenter G: Receptors for epidermal growth factor and other polypeptide mitogens. Ann Rev Biochem 56: 881–914, 1987
Klijn JGM, Berns PMJJ, Schmitz PIM, Foekens JA: The clinical significance of epidermal growth factor receptor (EGFR) in human breast cancer: a review on 5232 patients. Endocr Rev 13: 3–17, 1992
Dickson RB, Lippman ME: Estrogenic regulation of growth and polypeptide growth factor secretion in human breast carcinoma. Endocr Rev 8: 29–43, 1987
Macias A, Pérez R, Hagerstrom T, Skoog L: Transforming growth factor alpha in human mammary carcinomas and their metastases. Anticancer Res 9: 177–180, 1989
Foekens JA, Portengen H, Janssen M, Klijn JGM: Insulin-like growth factor-1 receptors and insulin-like growth factor-1 like activity in human primary breast cancer. Cancer 63: 2139–2147, 1989
Falette N, Lefebvre M-F, Meggouh F, Eynard M, Garin E, Saez S: Measurement of occupied and non-occupied epidermal growth factor receptor sites in 216 human breast cancer biopsies. Breast Cancer Res Treat 20: 177–183, 1991
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72: 248–253, 1976
Benraad ThJ, Foekens JA: Hydroxyapatite assay to measure epidermal growth factor receptors in human primary breast tumours. Ann Clin Biochem 27: 272–273, 1990
Kienhuis CBM: Measurement of the epidermal growth factor receptor. PhD Thesis, University of Nijmegen, The Netherlands, 1992
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kienhuis, C.B.M., Koenders, P.G., Klominska, H. et al. Endogenous ligands for the epidermal growth factor receptor in human breast tumors do not interfere in an epidermal growth factor receptor radioligand binding assay. Breast Cancer Res Tr 27, 271–275 (1993). https://doi.org/10.1007/BF00665697
Issue Date:
DOI: https://doi.org/10.1007/BF00665697