Thrombospondin-1 (TSP-1) and TSP-1-derived Heparin-binding Peptides Induce Promyelocytic Leukemia Cell Differentiation and Apoptosis

Abstract

Thrombospondin-1 (TSP-1) is a multifunctional adhesive glycoprotein that is synthesized by several cell types and modulates cell growth and differentiation. In this study, we showed that the amount of TSP-1 secreted by two human leukemia cell lines, HL-60 and NB4, increased markedly during differentiation of these cells by all-trans retinoic acid (ATRA) (10^-7 M), reaching about 100 ng/10⁶ cells after 3 days. Addition of purified TSP-1 alone (10^-9 - 5 x 10^-8 M) to HL-60 or NB4 cell cultures dose-dependently inhibited cell growth and differentiation. Differently to ATRA, TSP-1-induced differentiation of HL-60 and NB4 cells occurred independently of Bcl-2 regulation, as shown by immunofluorescence and Western immunoblotting. At day 5, TSP-1 also induced promyelocytic leukemia cell apoptosis. The percentage of apoptotic cells in NB4 cultures was higher with TSP-1 (5 x 10^-8 M) than with ATRA (10^-7 M) (46±3% versus 19±7%, p<0.001), whereas similar levels of apoptosis (37±7% and 38±6%) were reached with both agents in HL-60 cultures. Studies performed with synthetic peptides derived from the TSP-1 sequence indicated that two heparin-binding peptides, Hep-I and GGWSHW, located within the NH₂-terminal and type 1 repeats respectively, were strong inducers of apoptosis of HL-60 and NB4 cells, suggesting that cell surface heparan sulfate molecules might be involved in the apoptotic effect of TSP-1 on promyelocytic cells.