We investigated the interaction between GM-CSF and its receptor on human granulocytes and on several human tumor cell lines. Specific high-affinity binding for GM-CSF was characterized by Scatchard plot analysis. The specific radioactivity of the 125I-labeled derivative of rH. GM-CSF was determined by self-displacement analysis and calculated to be 30 μCi/μg. The maximum concentration of binding sites (B max) in granulocytes was 40 fmol/mg protein (2,200 molecules GM-CSF bound/cell) and the dissociation constant (KD) was 0.42 nM. No binding sites for GM-CSF were found in two lung cancer cell lines, SCLC-16HV and NCI-N417 or in the urinary bladder carcinoma cell line 5637, whereas the promyelocytic leukemia cell line HL60 was positive for GM-CSF binding. Time course experiments showed maximum binding of GM-CSF in granulocytes after an incubation period of 60 min and a decrease in binding after an incubation period of 2 h. In parallel, we found a maximum biological signal when granulocytes were preincubated for 90 min with GM-CSF, and a decrease after an incubation time of 120 min. Preincubation of the cells with rH. GM-CSF induced an enhancement of the production of activated oxygen species by the cells in response to PMA.

Addition of epidermal growth factor (EGF) to cultures of the urinary bladder carcinoma cell line 5637 regulated proliferation and production of colony stimulating activity (CSA). The optimal concentration range of EGF for stimulation of cell proliferation was 5–20 ng/ml EGF and for production of CSA 2–20 ng/ml EGF. High EGF concentrations (100–200 ng/ml) showed inhibitory effects on proliferation and to a greater extent on CSA production. Also, EGF binding sites of high affinity (kD: 3.25 nM) were demonstrated on the cell surface. In the optimal concentration range for stimulation (5–20 ng/ml EGF) EGF binding sites were occupied half-maximally. The loss in EGF binding after long incubation at 37°C was prevented by the lysosomal inhibitory agent, chloroquine. Nonspecific binding of EGF was very low, the amount of maximally bound EGF was 1430 fmol/mg protein (130,000 bound EGF molecules/cell). A strong band of approximately 170,000 daltons could be detected by means of an anti-erbB serum which recognizes the EGF receptor protein. The protein became phosphorylated upon addition of γ-32P ATP. The data suggest that EGF initiates its action by binding to specific high affinity receptors and plays a role in growth regulation and differentiation of the urinary bladder carcinoma cell line 5637.