This project continues to define components within the normal immune system possessing the ability to prevent or modify the process of carcinogenesis. Current efforts are directed at cloning the biologically active gene sequences of the anticarcinogenic cytokine leukoregulin, a 32 kD glycoprotein with the unique ability to induce permeability of tumor cell plasma membranes, to facilitate uptake of chemotherapeutic drugs, to enhance sensitivity of tumor cells to lymphocyte killing, and to prevent radiation and chemical carcinogen transformation of normal cells. Emphasis during the past year has focused specifically on isolating, by direct expression cloning in Xenopus laevis oocytes, cDNA clones expressing biologically active leukoregulin membrane- permeabilizing activity for tumor cells. Biological activity was identified by flow cytometric analysis of oocytes microinjected with multifunctional PBKCMV phagemids containing cDNA inserts from a cDNA library constructed from leukoregulin membrane-permeabilizing activity positive mRNA fractions isolated from normal human lymphocytes. Two clones, clone 1A and 15B, have been isolated, recloned and sequenced. Clone 1A has an 800 base pair cDNA insert and clone 15B a non-homologous 3000 base pair insert. Xenopus-expressed Clone 1A recombinant protein also has been shown to possess leukoregulin drug uptake enhancing and tumor cell NK lymphocyte sensitizing activity. E. coli has been successfully transformed with clone 1A and 15B cDNA and express recombinant protein with membrane permeabilizing activity. Molecular size exclusion and ion-exchange chromatographic purification of clone 1A recombinant protein stabilizes the recombinant biological activity. Continuing investigations are aimed at further purification and at characterizing the biological activity of the E. coli expressed recombinant proteins and comparison of the activities with the anticancer activities of native human leukoregulin.
