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.

Internalization routes of cell‐penetrating melanoma antigen peptides into human dendritic cells

“…Very low amounts of inhibitors showed non‐significant paradoxical effects with a trend for an enhanced uptake of peptide. This observation is in accordance with earlier reports, but so far lacks a definite explanation . After establishing the optimal doses of inhibitors, internalization experiments with DCs and the Melan‐A peptides or ovalbumin were performed.…”

“…The Melan‐A‐TAT fusion peptide [CELAGIGILTVRKKRRQRRR, Melan‐A sequence underlined] was synthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The N‐terminal Cys residue was added to enable coupling of Alexa Fluor 488 (Alexa488) C 5 maleimide (Invitrogen, Carlsbad, CA, USA).…”

Novel two‐stage screening procedure leads to the identification of a new class of transfection enhancers

“…Wagner et al [7] suggested that the ability of chloroquine to increase the pH results in inhibition of DNA degradation and is responsible for the enhanced transfection efficiency in the presence of this drug. Hawtrey et al [29] proposed that the feature of removing H + -ions in endosomal compartments may not be the most important physiological action both of quinolines and phenathiazines, as the concentration of 10 µM of basic chlorpromazine present in the endosomes is too low to significantly affect the pH in these vesicles. Supporting the latter finding is the recent observation that endosomal escape of polymeric gene delivery complexes is not necessarily enhanced by polymers buffering at low pH [30].…”

Bioactive Phenothiazines and Benzo[a]phenothiazines: Spectroscopic Studies, and Biological and Biomedical Properties and Applications

“…Hawtrey's research group demonstrated the stimulation of a luciferase pRSVL DNA uptake and expression in HeLa cells at low concentrations (10 -5 M) of CPZ and related antipsychotic phenothiazine compounds [148]. Higher concentrations of phenothiazines were toxic to the cells, and could not be tested.…”