Calcium ions as efficient cofactor of polycation-mediated gene transfer.

We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.

Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Molecular pathogenesis of secondary acute promyelocytic leukemia.

Balanced chromosomal translocations that generate chimeric oncoproteins are considered to be initiating lesions in the pathogenesis of acute myeloid leukemia. The most frequent is the t(15;17)(q22;q21), which fuses the PML and RARA genes, giving rise to acute promyelocytic leukemia (APL). An increasing proportion of APL cases are therapy-related (t-APL), which develop following exposure to radiotherapy and/or chemotherapeutic agents that target DNA topoisomerase II (topoII), particularly mitoxantrone and epirubicin. To gain insights into molecular mechanisms underlying the formation of the t(15;17) we mapped the translocation breakpoints in a series of t-APLs, which revealed significant clustering according to the nature of the drug exposure. Remarkably, in approximately half of t-APL cases arising following mitoxantrone treatment for breast cancer or multiple sclerosis, the chromosome 15 breakpoint fell within an 8-bp "hotspot" region in PML intron 6, which was confirmed to be a preferential site of topoII-mediated DNA cleavage induced by mitoxantrone. Chromosome 15 breakpoints falling outside the "hotspot", and the corresponding RARA breakpoints were also shown to be functional topoII cleavage sites. The observation that particular regions of the PML and RARA loci are susceptible to topoII-mediated DNA damage induced by epirubicin and mitoxantrone may underlie the propensity of these agents to cause APL.