Remo Bottega

Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.

Cationic Liposomes for Direct Gene Transfer in Therapy of Cancer and Other Diseases

Cationic liposomes can mediate efficient delivery of DNA and DNA/protein complex to mammalian cells in vitro and in vivo. Cationic cholesterol derivatives mixed with phosphatidylethanolamine and sonicated to form small unilamellar vesicles can complex with DNA and mediate the entry into the cytosol from the endosome compartment. One of the liposome formulations, DC-Chol liposomes, is used in a gene therapy clinical trial for melanoma. Recently, we exploited these cationic liposomes for the delivery of trans-activating protein factors to regulate and control the expression of delivered transgenes in a protein dose-dependent manner. Bacteriophage T7 RNA polymerase was co-delivered with a reporter gene under the control of T7 promoter to allow cytoplasmic expression of the gene. Human immunodeficiency virus-1 transactivating protein was also codelivered with a reporter gene under the control of HIV-1 long terminal repeat. Finally, human tumor cells selected for cis-platin resistance or isolated from patients who have failed cis-platin therapy are highly transfectable with cationic liposomes. These results suggest a serial therapy protocol with cis-platin and gene therapy for malignancy.

Determination of the phase behaviour of phosphatidylethanolamine admixed with other lipids and the effects of calcium chloride: implications for protein kinase C regulation

The phase behaviour of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) was studied by differential scanning calorimetry and 31P-NMR spectroscopy. Modulation of the phase behaviour of POPE by 1-palmitoyl-2-oleoylphosphatidylserine (POPS). 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-di-olein (DOG), CaCl2, MgCl2, and combinations of these substances was studied. The bilayer-forming lipids, POPS and POPC, raise the bilayer-to-hexagonal phase-transition temperature of POPE. The POPC has a greater effect than POPS, probably because the former lipid is more miscible with POPE. Addition of 10 mM CaCl2 has little effect on the phase-transitions of POPE/POPC mixtures, but it greatly decreases the effectiveness of POPS in raising the bilayer-to-hexagonal phase-transition temperature of POPE. The effectiveness of DOG in lowering the phase-transition temperature of POPE is also greatly reduced in the presence of 10 mM CaCl2. This phenomenon may play a role in the negative feedback regulation of protein kinase C.

Inhibition of protein kinase C by sphingosine correlates with the presence of positive charge

The role of the 2-amino group of sphingosine on the in vitro inhibition of protein kinase C was investigated by comparing protein kinase C activity in the presence and absence of sphingosine at various pHs. Inhibition by sphingosine was found to be pH dependent. Above pH 7.75, sphingosine has little or no inhibitory effect. In fact, at pH 8.5, sphingosine slightly enhances enzyme activity above that which occurs when the enzyme is stimulated by diacylglycerol and phosphatidylserine. After correcting for electrostatic repulsion, we find that the intrinsic pK for sphingosine in Triton micelles is 8.5. Inhibition of protein kinase C by sphingosine at physiological pHs therefore correlates with the presence of a positive charge.

The Relationship Between the Bilayer to Hexagonal Phase Transition Temperature in Membranes and Protein Kinase C Activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3β-chloro-5-cholestene, 5β-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH2, Z-Gly-Leu-NH2, Z-Tyr-Leu-NH2, cyclosporin A and cholestan-3β, 5α, 6β-triol are protein kinase C inhibitors.

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca2+. It has a Ki of about 30 μm. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca2+-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.

Cholesteryl phosphate and cholesteryl pyrophosphate inhibit formation of the hexagonal phase

Abstract The effects of cholesteryl phosphate and cholesteryl sulfate on the LaHII phase transition temperature of dielaidoylphosphatidylethanolamine were compared. Both compounds raise the LaHII transition temperature. This effect is decreased with decreasing pH. Cholesteryl sulfate is a somewhat better bilayer stabilizer and the effect is observed to lower pH values. Cholesteryl pyrophosphate was synthesized. This compound raises the La