Urinary Transforming Growth Factors in Neoplasia: Separation

Purified human epidermal growth factor (hEGF) from urine promotes anchorage-independent cell growth in soft agar medium. This growth is enhanced by transforming growth factor-/} (TGF-/8), and is specifically inhibited by hEGF antiserum. Transforming growth factors of the a type (TGF-a), potentially present in normal human urine or urine from tumorbearing patients, also promote anchorage-independent cell growth and compete with EGF for membrane receptor binding. Consequently, TGFa cannot be distinguished from urinary hEGF by these two functional assays. Therefore, a technique for separation of TGF-a and related peptides from urinary EGF based on biochemical characteristics would be useful. Radioiodination of characterized growth factors (mouse EGF (mEGF), hEGF, and rat TGF-a (rTGF-a)], which were then separately added to human urine, was used to evaluate a resolution scheme that separates TGF-a from the high level of background hEGF present in human urine. Commercially available methyl bonded microparticulate silica efficiently adsorbed the I2SI-labeledmEGF, '"I-labeled hEGF, and '"I-labeled rTGF-a that were added to 24-h human urine samples. Fractional elution with acetonitrile (MeCN) of the adsorbed silica re leased approximately 70 to 80% of the 125I-labeled mEGF and 125Ilabeled hEGF between 25 and 30% MeCN, and over 80% of the I25Ilabeled rTGF-a between IS and 25% MeCN, with retention after dialysis of less than 0.2 and 1.7% of the original urinary protein, respectively. Consequently, a single-step enrichment of about 400-fold for mEGF and hEGF, and 50-fold for rTGF-a were achieved rapidly. Subsequent BioGel P-10 chromatography indicated that '"I-labeled mEGF and I25Ilabeled hEGF eluted later than would be predicted on the basis of their reported molecular weight of approximately 6000, whereas <2sl-labeled rTGF-a eluted from Bio-Gel P-10 at an approximate molecular weight of 8000 to 9000. '-"I-laIn-led rTGF-a bound to carboxymethyl cellulose and eluted at a less acidic pH than did hEGF. On reverse phase high performance liquid chromatography using a linear gradient of 18 to 35% MeCN over 120 min, I25l-labeled rTGF-a was comparatively hydrophilic, eluting at 22% MeCN in contrast to the more Hydrophobie I2sl-labeled hEGF, which eluted at 27% MeCN. These observed biochemical differ ences between hEGF and TGF-a provide a rationale for resolution of these and perhaps other related putative transforming growth factors from bulk urine of tumor-bearing patients.