Arthur Hawtrey

Further Studies on Targeted DNA Transfer to Cells Using a Highly Efficient Delivery System of Biotinylated Transferrin and Biotinylated Polylysine Complexed to Streptavidin

Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to streptavidin have been prepared and found to transfer luciferase plasmid DNA very efficiently to HeLa cells in the presence of chloroquine. Transfection was dependent on (i) use of biotinylated short chain polylysine containing 70 lysine residues, (ii) biotinylated transferrin containing 1-2 biotin moieties, (iii) reaction of biotinylated transferrin with streptavidin followed by isolation of the resulting conjugate on Sephadex G-200 and (iv) interaction of streptavidin-biotinylated transferrin with biotinylated polylysine giving a complex suitable for DNA transfection. It was found that if the above sequence of steps resulting in the formation of streptavidin-biotinylated transferrin/biotinylated polylysine was followed without isolation of intermediate conjugates by Sephadex G-200 chromatography, pRSVL DNA transfer was still very efficient. Transfer of luciferase DNA by the streptavidin conjugates and subsequent expression of luciferase activity was almost completely inhibited by excess free transferrin, showing that gene transfer was through the transferrin receptor pathway via receptor-mediated endocytosis. The streptavidin (bio2-transferrin) bio10-pLys70 conjugate used in the present experiments was approximately one hundred times more efficient in pRSVL DNA transfection with the HeLa cells than the previously described avidin-pLys460 (bio-transferrin) complex.

Studies on the Transfer of DNA into Cells Through Use of Avidin-Polylysine Conjugates Complexed to Biotinylated Transferrin and DNA

A simple procedure has been developed for studying the transfer of DNA into cells using the process of receptor-mediated endocytosis. Poly-L-lysine 460 was covalently attached to the carbohydrate chains of avidin via periodate oxidation and NaBH4 reduction to give avidin-pLys460. Following purification through Sephacryl S-300, the conjugate was reacted with biotinylated transferrin. The resulting avidin-pLys460-[bio-transferrin] could be either fractionated by Superose 12 gel chromatography or used directly in experiments with DNA. Determination of the interaction between avidin-pLys460-]bio-transferrin] and DNA was carried out by nitro cellulose filter binding and agarose gel retardation assays. The conjugate was shown to bind DNA strongly, giving stable complexes soluble in 0.15-0.2 M salt solutions. Gene transfer using avidin-pLys460-[bio-transferrin] and the luciferase plasmid pRSVL was accomplished with Hela cells, alpha T3 pituitary cells and a human melanoma cell line used in the present study. Transfection was dependent on bio-transferrin and stimulated by the lysosomotropic agent chloroquine. The results are consistent with a receptor-mediated endocytosis mechanism of DNA delivery for Hela cells and a combination of receptor and adsorptive endocytosis for the alpha T3 pituitary and melanoma T-5 cell lines.

Low Concentrations of Chlorpromazine and Related Phenothiazines Stimulate Gene Transfer in HeLa Cells Via Receptor-Mediated Endocytosis

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 μM), 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 [3 H] 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 [3 H] 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. SO 4 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 [3 H] 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.

Development of a Reproducible Procedure for Plasmid DNA Encapsulation by Red Blood Cell Ghosts

AbstractObjective: The binding and encapsulation of [3H] pGL3 luciferase reporter plasmid DNA by red blood cell (RBC) ghosts, intended as a vehicle for transfection and ultimately for gene therapy, were studied using two methods for DNA compaction. Methods and Results: In the first approach, DNA was compacted through binding electrostatically to poly-L-lysine. Complexes were constructed to have a slight negative charge. Experimentally, it was found that a high percentage of binding was to the outside of the resealed RBC ghosts.An alternative approach using polyethylene glycol6000 at a final concentration of 15% (weight/volume) was used to collapse [3H] pGL3 DNA in the presence of 0.025M MgCl2. Addition of the reagents, premixed with DNA, to a pelleted suspension of RBC ghosts followed by a short incubation and then addition of 1.5M NaCl to restore tonicity, resulted in resealing of the ghosts. Uptake of [3H] pGL3 DNA by the ghosts was approximately 20% of the input amount of DNA. Further work showed that 60–70% of the DNA was inside the resealed ghosts and largely present in the supercoiled form. At no stage was any freezing and thawing used. Conclusion: Transfection studies have demonstrated that pGL3 DNA carrying the luciferase gene is successfully transferred from RBC ghosts to recipient HeLa cells in culture under mild fusion conditions.

A note on poly-L-lysine-mediated gene transfer in HeLa cells.

Poly-L-lysines of chain lengths varying from 70 to 300 residues are shown to bring about luciferase pRSVL DNA uptake and expression in HeLa cells. Transfection was approximately 50% that of the cationic liposome DOTAB. Expression was higher in the presence of chloroquine. Of interest was the fact that luciferase activity depended on the polysine/DNA charge ratio (+/−). Maximum activity occurred at a charge ratio (+/−) of 3, while at a charge ratio of 1 (conjugate electrically neutral) activity was much lower. At the higher charge ratios (+/−) of 4 and 5, luciferase activity decreased. The results obtained are discussed.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 ′-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5′-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)16-18 as template-primer with [3H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC50 values of approximately 60–80 μM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5′-phosphate, the inhibitory activity of this compound increased significantly (IC50 = 5 μM).

Are phosphorylated tyrosine residues of certain receptors involved in inducing transitory covalent protein cross-linking?

Following ligand binding a number of cell-surface receptors become phosphorylated at tyrosine residues of their cytosolic domains. These phosphorylations are associated with initiation of a signalling programme involving a sequence of tyrosine-phosphorylated protein-protein interactions. In the recognition process between phosphorylated proteins, electrostatic interactions between negatively charged phosphorylated tyrosines, serine and threonine residues and positively charged lysines play an important role as well as hydrophobic and H-bonding reactions. We suggest in this paper that the fairly high-energy phosphate bond of certain protein phosphorylated tyrosines are possibly involved in inducing transitory protein cross-linking reactions. Through a process involving transfer of an activated phosphate of phosphorylated tyrosine to a side-chain carboxyl group of the receptor or next protein of the signalling sequence, an acyl phosphate is formed. This then acylates a hydroxyl group on a serine, threonine or tyrosine residue of the protein not carrying the carboxyl phosphate to give an ester linkage, thus cross-linking the two proteins of the signalling pathway. The covalent ester linkage is labile to hydrolysis and depending on the protein-protein molecular environment it might have a finite half-life. On hydrolysis, the transitory covalent linkage is broken with separation of the proteins. It is suggested therefore that formation of a protein-protein ester linkage introduces a type of timing device into the system. Breakdown of the original protein-phosphorylated tyrosine in this case therefore does not involve a phosphatase enzyme.