Identification of epidermal growth factor as a component of the rat urinary bladder tumor-enhancing urinary fractions

Using the heterotopically transplanted rat urinary bladder, we have shown that normal rat urine has a potent tumor-enhancing effect on bladder carcinogenesis. In an attempt to isolate tumor-enhancing factor(s), urine was fractionated by Bio-Gel P-100 column chromatography and each eluate fraction was examined for inducibility of ornithine decarboxylase (ODC) in a target rat bladder carcinoma cell line, 804G. We have identified two ODC-inducible peaks, one located in a high molecular weight region designated as Fraction I (Fr. I) and the second in a low molecular weight region designated as Fraction II (Fr. II). Fr. I consisted of two principal elements, transferrin and a component which induced ODC. The present investigation was conducted to characterize the ODC-inducible activity in Fr. I and II. Chromatographic analysis of Fr. I by Sephacryl S-200 and Fr. II by Bio-Gel P-10 chromatography separated several ODC-inducible peaks. However, the major ODC inducibility was due to a high concentration (460 ng/mg Fr. I residue, approximate Mr 54,000, and 580 ng/mg Fr. II residue, approximate Mr 6,100) of epidermal growth factor (EGF) as determined by radioimmunoassay. Aliquots obtained from these peaks competed with mouse EGF for EGF receptors in A431 cells. Preincubation of Fr. I and II with rabbit anti-rat EGF IgG significantly reduced ODC inducibility. Transforming growth factor alpha activity as determined by radioimmunoassay was also demonstrated in both Fr. I (34 ng/mg) and Fr. II (9 ng/mg). The results of the present study together with our previous data indicate that the majority of the ODC-inducing activity in the tumor-enhancing urinary components Fr. I and Fr. II is due to EGF itself and EGF-related growth factors of high molecular weight and that Fr. I also contains transferrin.