Local TAT-p27Kip1 fusion protein inhibits cell proliferation in rat carotid arteries.

INTRODUCTION: p27(Kip1) is a cyclin kinase inhibitor that induces cell cycle arrest. In this study, the efficacy of fusion protein TAT- p27(Kip1) to inhibit cell proliferation in rat perivascular injured carotid arteries was tested. METHODS: The cDNA of p27(Kip1) and GFP (green fluorescein protein) fused to the TAT epitope, which allows cell penetration, yielded TAT-p27 (Kip1) and TAT-GFP fusion proteins. In vitro biological activity on cell proliferation was evaluated by [(3)H] thymidine DNA incorporation in rabbit aortic endothelial cells (REC). An in vivo model used a silicone collar filled with saline positioned around the carotid vessel for 14 days to produce an increased adventitia cross-sectional area. RESULTS: TAT-p27(Kip1) inhibited REC proliferation in vitro using either 100, 200, and 500 nM compared to control (88.2 +/- 4.4, 81.3 +/- 7, 71.9 +/- 4.2 vs. 100 +/- 6.7%, N = 3, respectively, p < 0.05). This response was stable for purified proteins stored at -20*C for at least 23 days. In vivo , TAT-p27(Kip1) solution reduced adventitia cross-sectional area in a dose-dependent manner compared to TAT-GFP (area in mm(2) - TAT-p27(Kip1): 200 nM, 0.160 +/- 0.018; 500 nM, 0.050 +/- 0.005 vs. TAT-GFP: 500 nM, 0.595 +/- 0.066 vs. the contralateral: 0.047 +/- 0.005, N = 7, p < 0.01). CONCLUSION: Taken together, these results provide evidence that TAT-p27(Kip1) can inhibit vascular cells proliferation. It is the first successful demonstration that the cell permeable TAT-p27(Kip1) has potential as a vascular anti-proliferative agent.

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

BACKGROUND: Non-viral gene transfer efficiency is low as compared to viral vector systems. Here we describe the discovery of new drugs that are capable of enhancing non-viral gene transfer into mammalian cells using a novel two-stage screening procedure. METHODS: First, potential candidates are preselected from a molecular library at various concentrations by a semi-automated yeast transfection screen (YTS). The maximal transfection efficiency of every positive drug is subsequently determined in independent experiments at the optimal concentration and compared to the inhibitory effect of the drug on cell growth (IC50). In a subsequent mammalian cell transfection screen (MTS), the maximal transfection efficiency and the IC50 are determined for all preselected drugs using a human cell line and a luciferase reporter gene construct. RESULTS: Employing our novel system we have been able to identify a new class of transfection enhancers, the tricyclic antidepressants (i.e. doxepin, maprotiline, desipramine and amoxapine). All positive drugs enhanced gene transfer in both yeast and human cell lines, but lower concentrations were sufficient for mammalian cells. With a triple combination of doxepin, amoxapine and chloroquine we obtained a transfection efficiency that exceeded that of chloroquine, one of the best-known transfection enhancers of mammalian cells, by nearly one order of magnitude. CONCLUSIONS: Non-viral gene transfer efficiency can be increased significantly using new transfection enhancers that are identified by a novel, semi-automated two-stage screening system employing yeast cells in the first and specific human target cells in the second round.

Endocytosis is involved in DNA uptake in yeast.

The transfection of mammalian cells by endocytosis is frequently hampered by low efficiency. To identify the bottlenecks, a system that allows the analysis of the intracellular pathway of DNA along the endocytic compartments in the eukaryote Saccharomyces cerevisiae was developed. DNA uptake in yeast cells was achieved by endocytosis when the cells were incubated with episomal DNA in the presence of 34% sucrose and subsequently shifted to a hypotonic medium to induce osmotical lysis of accumulated endocytic intermediates. The compartments of the endocytic pathway after the intersection of the endocytic and the vacuolar sorting pathway are preferred sites of DNA degradation. Either a transport blockade before this point or, even better, the presence of chloroquine, which is known as an adjuvant for transfection in mammalian cells, are required for successful transfection. The transport blockade can be achieved by deleting a GTPase of the endocytic pathway, Ypt51p, or ethanol. Chloroquine affects the compartments of the late endocytic pathway, and no effect is seen on transfection in a strain that is defective for YPT51 and accumulates the DNA in the early endocytic intermediates. To our knowledge, this is the first report on endocytic DNA uptake in S. cerevisiae.