Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice.

Serine/threonine protein phosphatase 5 (PP5) appears to play an underappreciated role in the regulation of cellular proliferation. In estrogen-responsive cells, PP5 expression is stimulated by 17 beta-estradiol, and in a variety of p53 wild-type tumor cells the suppression of PP5 expression with ISIS 15534 inhibits growth. To further explore the relationship between PP5 and the development of human cancer, here we tested the effect of elevated PP5 expression on tumor growth using a mouse xenograph model and a stable MCF-7 cell line in which the expression of wild-type PP5 was placed under the control of tetracycline-off regulated transactivator and operator plasmids. In the xenograph model a modest two fold increase in PP5 protein levels significantly enhanced the growth rate of estrogen-dependent tumors, suggesting PP5 plays a positive role in tumor development.

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo.

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.

Identification of a functional link for the p53 tumor suppressor protein in dexamethasone-induced growth suppression.

Serine/threonine phosphatase 5 (PP5) can act as a suppresser of p53-dependent growth suppression and has been reported to associate with several proteins, including the glucocorticoid receptor/heat-shock protein-90 complex. Still, the physiological/pathological roles of PP5 are unclear. To characterize the relationship of PP5, glucocorticoid receptor activation and p53, here we describe the development of chimeric antisense oligonucleotides that potently inhibit human p53 expression. This allowed us to regulate the expression of either p53 (e.g. with ISIS 110332) or PP5 (e.g. with ISIS 15534) in genetically identical cells. Studies with ISIS 110332 revealed that the suppression of p53 expression is associated with a decrease in the basal expression of the cyclin-dependent kinase inhibitor protein, p21(WAF1/Cip1), and a concomitant increase in the rate of cell proliferation. Suppression of p53 also blocks dexamethasone-induced p21(WAF1/Cip1) expression and G(1)-growth arrest. Furthermore, treatment with ISIS 110332, but not the mismatched controls, ablates the suppression of growth produced by prior treatment with dexamethasone. Additional studies revealed that dexamethasone-dependent p21(WAF1/Cip1) expression occurs without an apparent change in p53 protein levels or the phosphorylation status of p53 at Ser-6, -37, or -392. However, dexamethasone treatment is associated with an increase in p53 phosphorylation at Ser-15. Suppression of PP5 expression with ISIS 15534 also results in the hyperphosphorylation of p53 at Ser-15. Together, these findings indicate that the basal expression of p53 plays a functional role in a glucocorticoid receptor-mediated response regulating the expression of p21(Waf1/Cip1) via a mechanism that is suppressed by PP5 and associated with the phosphorylation of p53 at Ser-15.