Human Promyelotic HL-60 Cells Research Articles

Induction of Apoptosis by Alkaloids, Non-Protein Amino Acids, and Cardiac Glycosides in Human Promyelotic HL-60 Cells

The induction of apoptosis by 66 alkaloids of the quinoline, quinolizidine, pyrrolizidine, isoquinoline, indole, terpene, tropane, steroid, purine, and piperidine type, of 9 cardiac glycosides, 11 non-protein amino acids and 10 further secondary metabolites was assayed in HL-60 cell cultures and measured by quantification of the subdiploid DNA content by flow cytometry, detection of DNA fragmentation by gel electrophoresis, and cell morphology. Several alkaloids of the isoquinoline, quinoline, and indole type were active, whereas quinol-izidine, tropane, pyrrolizidine, terpene and piperdine alkaloids were mostly inactive. The proapoptotic alkaloids can be characterized by their property to inhibit protein biosynthesis and their intercalation into DNA at the same time, or by their inhibition of microtubule formation. All cardiac glycosides, which are both membrane detergents and Na+,K+-ATPase inhibitors, are potent apoptosis inducers. Also proapoptotic were a few non-protein amino acids, podophyllotoxin and the flavonoid quercetin.

Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8.

Human interleukin 8 (hIL-8), a neutrophil-activating and chemotactic cytokine, is known to play an important role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. This cytokine belongs to the family of CXC chemokines, mediating the response through binding to the seven-transmembrane helical G protein-coupled receptors CXCR1 and CXCR2. For the first time, we employed the expressed protein ligation (EPL) strategy to chemokine synthesis and subsequent modification. The ligation site was chosen with respect to the position of four cysteine residues within the hIL-8 sequence. Ligation with synthetic peptides that carry cysteine at their N-termini resulted in full-length hIL-8 and the specifically carboxyfluorescein-labelled analogue [K69(CF)]hIL-8(1-77). [K69(CF)]hIL-8(1-77) was fully active as shown by inhibition of cAMP production. Furthermore, this analogue was used to study receptor internalisation in human promyelotic HL60 cells that express CXCR1 and CXCR2 receptors. Binding and quenching studies were performed on HL60 membranes and suggest that the C-terminus of IL-8 is accessible to solvent in the receptor-bound state. Thus, we introduce here a powerful approach that allows the site-specific incorporation of chemical modifications into the sequence of chemokines, which opens new avenues for studying IL-8 function.