Abstract
The human acute promyelocytic leukemia cells line, HL-60, produces an autostimulatory activity (ASA) which allows it to grow in high density liquid and semi solid cultures in the absence of exogenous colony stimulating factor (CSF). Addition of highly purified preparations of CSF I, CSF II or medium from high density cultures of HL-60 will stimulate growth at lower densities. Addition of CSF or ASA to semi solid cultures increased the number and size of colonies, but had little effect on differentiation. CSF stimulation of HL-60 colony formation was inhibited by anti-CSF I or anti-CSF II. Furthermore, antibodies specific to CSF I or CSF II inhibited spontaneous HL-60 colony growth.ASA also had the capacity to stimulate colony formation in normal mouse and human marrows. Human colonies were seen at day 13 of incubation and were predominately macrophage morphology. ASA stimulation of human and mouse colony growth was inhibited by anti-CSF I and anti-CSF II, indicating that ASA shares immunological determinants with both CSF I and II.ASA was partially purified by preparative isoelectric focusing, phenyl sepharose affinity chromatography and gel filtration chromatography. Characterization of ASA indicates that it is a sialic acid containing glycoprotein with an apparent molecular weight of approximately 25,000 daltons and an iso-electric point range of 4.3 to 5.2. Treatment of ASA with neuraminidase will sharpen the isoelectric important role in stability or function of ASA. Dithiothreitol and 2-mercaptoethanol treatment also reduced biological activity by about 60%, indicating the importance of intact disulfide bonds.Although ASA has similar biological activity and shares immunological determinants with CSF I, the smaller molecular weight and cross reactivity with anti-CSF II distinguish it. Partial trypsin digestion of highly purified CSF I did not yield any biologically active fragments, further supporting that ASA is a unique molecule.Synthesis of ASA appears to be a property of HL-60 cells. When stimulated to differentiate into macrophage-like cells with phorbol esters, the cells continue to elaborate ASA although the cells no longer divide. Additionally, as the cells differentiate, they begin to produce new species of colony stimulating activity with similar biological and physical properties to human CSF I and CSF II.