Inhibition of leukaemia cell proliferation by folic acid–polylysine-mediated introduction of c-myb antisense oligodeoxynucleotides into HL-60 cells

Abstract

Summary The inhibitory effect of c-myb antisense oligodeoxynucleotides (ODNs) conjugated to folic acid (FA) on HL-60 cell proliferation was examined. Folic acid was covalently linked to a polylysine chain and purified by gel chromatography. Sterile FA-polylysine was complexed with c-myb sense and antisense. Exposure of HL-60 cells to the FA-polylysine-c-myb antisense ODN complex resulted in a down-regulation of c-myb expression and a greater inhibition of proliferation than that obtained using free ODNs. Moreover, FA-polylysine conjugate alone or complexed to c-myb sense ODN was not toxic to cells. The antigenic properties and uptake of the vitamin were not affected by the polylysine chain. These data suggest that this strategy is potentially useful for the selective delivery of anti-oncogene-targeted ODNs into cancer cells.Antisense oligodeoxynucleotides (ODNs) have proven useful for selective inhibition of gene expression (Holt et al., 1988; Szczylik et al, 1991). However, their rate of cellular uptake appears to be quite slow, and consequently attempts have been made to enhance their stability and their delivery into cells. For instance, receptor-mediated endocytosis has been used to increase the uptake of synthetic ODNs and other foreign molecules such as proteins complexed to specific ligands (Wu & Wu, 1987, 1988Cotten et al., 1990;Leamon & Low, 1991;Citro et al., 1992;Manfredini et al., 1993). Since the receptors for some growth factors, vitamins and hormones are overexpressed in rapidly dividing tumour cells (Rothemberg & Da Costa, 1971;Asok et al., 1981;Sclhub & Franklin, 1984; Lacey et al., 1989), the ligands of these receptors can be exploited to selectively introduce therapeutic compounds into the cells. The use of modified ligands for specific cell-surface receptors as carriers of oncogene-targeted antisense ODNs represents a potentially useful therapy to be used alone or in combination with antineoplastic drugs.We have previously reported that a c-myb antisensetransferrin-polylysine complex produces an enhanced uptake into HL-60 cells, resulting in an increased biological effect. Recently, we have also observed that a polylysine chain covalently linked to compounds such as insulin, folic acid, retinoic acid, oestrone and testosterone can be used for specific interactions with nucleic acids in physiological ionic conditions (G. Citro, unpublished observation). The presented study describes the efficacy of folic acid receptor-targeted c-myb antisense in the HL-60 cell line. The effect of the complexed phosphodiester (PO) ODNs was compared with that of phosphorothioate (PS) ODN antisense given alone.With doses of 20 and 30 fig mlVl, we found that PS c-myb antisense actively inhibited the rate of the cell proliferation while free PO c-myb antisense had no effect. However, when free PO c-myb antisense ODNs were complexed to FApolylysine, their inhibitory effect on the cell proliferation was even greater than that obtained using the free PS oligos. Furthermore, whereas recent research has indicated there...

The myb gene family in cell growth, differentiation and apoptosis

“…Similar eects have been observed with HL-60 and several other myeloid leukemia cell lines where antisense inhibition of c-myb blocked their entrance into S phase resulting in a block to their proliferation. These ®ndings suggested that c-myb proto-oncogene is required for maintenance of proliferation and plays a direct role in cell cycle control of hematopoietic cells without aecting their dierentiation status (Anofossi et al, 1989;Citro et al, 1992Citro et al, , 1994. However, in another study, c-myb-antisense-treated HL-60 cells differentiated only into monocytes but not into granulocytes indicating that granulocytic dierentiation, unlike monocytic dierentiation, requires c-myb-mediated proliferation (Ferrari et al, 1990).…”

Ligand Binding and Kinetics of Folate Receptor Recycling in Vivo: Impact on Receptor-Mediated Drug Delivery

“…Molecules that have been targeted to tumors by conjugation to folic acid include radiopharmaceuticals (Wang et al, , 1997Mathias et al, 1996Leamon et al, 2002). enzymes for pro-drug activation (Lu et al, 1999), nanoparticles (Oyewumi and Mumper;2003), peptides, toxic proteins , immunologically potent haptens 2002b), antisense oligonucleotides (Citro et al, 1994;Wang et al, 1995), chemotherapeutic agents (Ladino et al, 1997), gene therapy vectors (Lee and Huang, 1996;Reddy et al, 1999Reddy et al, , 2002Leamon and Low, 2001), viruses (Lee and Low, 1994;Reddy and Low, 2000), and polymeric drug carriers and liposomes Gabizon et al, 1999). It is noteworthy that both a folate-targeted therapeutic drug and a folate-linked imaging agent are currently under evaluation in human clinical trials.…”

Antisense Inhibition of Silica-induced Tumor Necrosis Factor in Alveolar Macrophages

“…This method has been used successfully to aid ON delivery in selected cell types (8 -11). For example, asialoorosomucoidpolylysine conjugates have been used to target ONs to HepG2 and chloramphenicol acetyltransferase cells (8,9), and transferrin and folic acid conjugates have been employed to aid ON delivery to rapidly growing cells such as HL-60 cells (10,11). In the lung, alveolar macrophages (AMs) represent a major target for dust-induced pulmonary fibrosis.…”