INSULIN BINDING TO THE HL-60 HUMAN PROMYELOCYTIC CELL LINE

Abstract

The HL-60 cell line derived from a patient with promyelocytic leukemia may be grown in liquid suspension cell culture. It binds [125I]-iodoinsulin in a highly specific, rapid, reversible, temperature and pH dependent manner. The pH maximum is approximately 7.8 and binding is maximal after 80 min at 22C with virtually 90% specific. Specific binding is linear with cell number over the range of 107 to 108 cells. The displacement of bound [125I]-iodoinsulin by unlabeled insulin yields curvilinear Scatchard plots at 22C. Using a 2 site model the insulin binding characteristics may best be defined by a “high” affinity site (n1=8.7±2.6(SD)·103 per cell; k1=2.7±0.55·108M-1) and a “low” affinity site (n2=1.12±0.32·105 per cell; k2=7.8±3.6·106M-1). To determine insulin release from its receptor, cells were exposed to [125I]-iodoinsulin for 90 min at 22C. Aliquots were diluted 100-fold in binding buffer or buffer plus 1.67·10-6M insulin. The excess unlabeled insulin accelerated the dissociation of labeled insulin at 22, 30 and 37C, but did not at 4 or 15C. Displacement of [125I]-iodoinsulin by vertebrate insulins and modified porcine insulins is in agreement with their known biological activities. Purified multiplication stimulating factor was less than 1 percent as effective as porcine insulin. These data indicate that the HL-60 cells maintain specific, high affinity insulin receptors. The release of insulin is accelerated by unlabeled hormone above 22C but not below 15C.