title: Low concentrations of chlorpromazine and related phenothiazines stimulate gene transfer in HeLa cells via receptor-mediated endocytosis.
abstract: Chlorpromazine and related phenothiazine antipsychotic compounds at the low concentration of 10(-5) M stimulated luciferase pRSVL DNA uptake and expression in HeLa cells. On the other hand, chloroquine at a 10(-5) M was without effect at this low concentration. However, at the higher normally used concentration of 10(-4) M (100 microM), chloroquine strongly stimulated luciferase expression and activity. Unfortunately, at 10(-4) M, the phenothiazines were toxic to the cells and could not be tested at this concentration. Further experimental work was carried out to elucidate the mechanism of action of phenothiazines and chloroquine on DNA uptake and expression. Interaction of [3H] pBR 322 DNA with chlorpromazine, perphenazine, and chloroquine was studied using these compounds as their free bases dissolved in chloroform, followed by their impregnation onto Whatman No. 1 filter paper discs. Both phenothiazines on filter paper discs bound [3H] pBR 322 DNA to a far greater extent than chloroquine. The method of assay (free base) suggests that the major contribution to binding is through intercalation. A further possible assay for studying the interaction of phenothiazines and chloroquine made use of the ethidium bromide/calf thymus DNA intercalation method. Intercalated calf thymus (CT) DNA complexes with ethidium bromide (EB) were examined for possible dissociation into free DNA and EB on the addition of either chloroquine. SO4 or chlorpromazine.HCl (soluble salts). Partial dissociation was observed with both compounds. Further experiments on the stability of pBR 322 DNA-polylysine complexes were also carried out using an alternative method of assay. Chloroquine (10(-2)-10(-4) M) and chlorpromazine (10(-4) M) did not bring about a dissociation of [3H] pBR 322 DNA-polylysine(200) complexes when reactions were studied by nitrocellulose filter assays to measure released double-stranded DNA. The results indicate that chlorpromazine and related phenothiazines stimulate luciferase DNA uptake expression at 10(-5) M. Chloroquine at this concentration had practically no effect on expression of luciferase activity. Further studies of chloroquine and chlorpromazine on their interaction with plasmid DNA as well as DNA-polylysine complexes are reported.
authors.affiliation: Department of Pharmacology, University of Stellenbosch Medical School, Tygerberg, South Africa. jmvzyl@gerga.sun.ac.za
authors.forename: Arthur
authors.initials: A
authors.lastname: Hawtrey
authors.forename: Daniel
authors.initials: D
authors.lastname: Joubert
authors.forename: Pieter
authors.initials: P
authors.lastname: van Jaarsveld
authors.forename: Anton
authors.initials: A
authors.lastname: Pieterse
authors.forename: Johann
authors.initials: J
authors.lastname: van Zyl
authors.forename: Mario
authors.initials: M
authors.lastname: Ariatti
mesh_terms: D014150:Antipsychotic Agents / Q000494:pharmacology*; D002746:Chlorpromazine / Q000494:pharmacology*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000378:metabolism*; D014162:Transfection
chemical_list: D014150:Antipsychotic Agents; D011990:Receptors, Transferrin; D011107:Polylysine; D004247:DNA; D008156:Luciferases; D002746:Chlorpromazine