[Cytotoxicity of lysomustine and its isomers, and their potential use for selection of cells].

N epsilon-Nitroso-N epsilon- [N'-(2-chloroethyl)carbamoyl]-L-lysine (I) and N epsilon- [N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-lysine (II), the isomers being the constituents of antitumor agent Lysomustine, were obtained by RFHPLC. The study of cytotoxicity of the above compounds against K562 cells showed that the lesions induced by isomer (II) produce a significant cytotoxic effect but can be efficiently repaired by the action of MGMT (O6-methylaguanine DNA methyltransferase). Under similar conditions, the lesions induced by isomer (I) produce substantially smaller effect but are weakly if at all repairable by MGMT. The effects of a clinically approved agent Lysomustine, which is the mixture of isomers (I) and (II), are similar to those of isomer (II). The results obtained point to a different chemical nature of DNA lesions induced by two Lysomustine isomers. Our data indicate that Lysomustine and its isomer (II) can be used for in vitro selection of cells expressing MGMT.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders.

Effect of expression of Jagged1 protein in stromal sublayer on the maintenance of hemopoietic precursors in in vitro coculturing system.

In order to verify the relationship between the expression of Notch ligand in NIH 3T3 fibroblast clones and maintenance of hemopoietic activity in vitro, monolayers from three clones were co-cultured with mouse bone marrow mononuclears for 1-4 weeks and the total production of hemopoietic cells and myeloid colony-forming activity in methylcellulose were evaluated. One of the clones exhibited significant hemopoiesis maintaining activity and after 2 weeks of culturing promoted a more than 100-fold increase in the number of colony-forming precursors in comparison with control cells. Hemopoiesis maintenance activity of the second clone was much lower, and that of the third clone virtually did not differ from that of control cells. The results indicate that expression of Notch ligand can radically increase cell strain capacity to maintenance of in vitro hemopoiesis in the co-culturing system.