Marni Brisson

Diallyl trisulfide-induced G2-M phase cell cycle arrest in human prostate cancer cells is caused by reactive oxygen species-dependent destruction and hyperphosphorylation of Cdc25C

Molecular mechanism of cell cycle arrest caused by diallyl trisulfide (DATS), a garlic-derived cancer chemopreventive agent, has been investigated using PC-3 and DU145 human prostate cancer cells as a model. Treatment of PC-3 and DU145 cells, but not a normal prostate epithelial cell line (PrEC), with growth suppressive concentrations of DATS caused enrichment of the G2–M fraction. The DATS-induced cell cycle arrest in PC-3 cells was associated with increased Tyr15 phosphorylation of cyclin-dependent kinase 1 (Cdk1) and inhibition of Cdk1/cyclinB1 kinase activity. The DATS-treated PC-3 and DU145 cells also exhibited a decrease in the protein level of Cdc25C and an increase in its Ser216 phosphorylation. The DATS-mediated decrease in protein level and Ser216 phosphorylation of Cdc25C as well as G2–M phase cell cycle arrest were significantly attenuated in the presence of N-acetylcysteine implicating reactive oxygen species (ROS) in cell cycle arrest caused by DATS. ROS generation was observed in DATS-treated PC-3 and DU145 cells. DATS treatment also caused an increase in the protein level of Cdk inhibitor p21, but DATS-induced G2–M phase arrest was not affected by antisense-mediated suppression of p21 protein level. In conclusion, the results of the present study indicate that DATS-induced G2–M phase cell cycle arrest in human prostate cancer cells is caused by ROS-mediated destruction and hyperphosphorylation of Cdc25C.

Cell-active dual specificity phosphatase inhibitors identified by high-content screening.

Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institutes 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC(50) of <10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.

Subcellular trafficking of the cytoplasmic expression system

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in that it does not rely on nuclear DNA transport or mitosis for efficient expression.

A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes.

Inefficient nuclear transport of plasmid DNA continues to be a problem in nonviral vector-mediated gene transfer. This has made the cytoplasmic expression system an increasingly attractive idea. We have developed a new T7 RNA polymerase autogene for cytoplasmic expression containing both a CMV and a T7 promoter. The pCMV/T7-T7pol autogene does not encounter the problems associated with previously used autogenes. For instance, pCMV/T7-T7pol is easily amplified and purified from bacteria. Furthermore, the CMV promoter is used to drive the first round of synthesis of T7 RNA polymerase, thus negating the use of purified enzyme in the transfection complex. The endogenous T7 RNA polymerase produced from the CMV promoter could then act on the T7 promoter of pCMV/T7-T7pol in an autoregulatory mechanism. pCMV/T7-T7pol induces higher, more sustained levels (>7 days) of reporter gene expression than that observed with the previously used autogene pT7 AUTO 2C− or with the nuclear expression system pCMV-CAT. This seems to be due to the high levels of T7 RNA polymerase protein that are detected in cells transfected with pCMV/T7-T7pol. This vector also functions as an efficient autogene since at least 50 times more mRNA is transcribed from the cytoplasmic T7 promoter as compared with the nuclear CMV promoter in pCMV/T7-T7pol. Therefore, pCMV/T7-T7pol could replace existing autogenes for regeneration of T7 RNA polymerase and efficient target gene expression.

Delivery of a PCR amplified DNA fragment into cells: A model for using synthetic genes for gene therapy

Synthetic genes offer many potential advantages over conventional plasmid DNA, such as simplicity in purification, absence of endotoxin contamination, and more importantly, flexibility in chemical modifications to render them specific properties. We have used PCR amplified fragments as a model to test the feasibility of using synthetic genes for gene therapy. The CAT reporter gene driven by the CMV promoter (CMV-CAT), ie a nuclear expression system, or by the bacteriophage T7 promoter (T7-CAT), ie a cytoplasmic expression system, was used to evaluate this concept. The expression efficiency of both plasmids (pUCCMV-CAT and pT7-CAT) and their corresponding PCR fragments (fCMV-CAT and fT7-CAT) were compared on a molar basis. Limited expression of CAT was found with fCMV-CAT. However, fT7-CAT consistently gave a CAT activity comparable to that of pT7-CAT. When fT7-CAT was codelivered with pCMV/T7-T7pol (a self-amplifying T7 RNA polymerase autogene), high CAT activity could be detected up to 9 days. This expression was much longer than the duration of expression with a nuclear expression system. These encouraging results imply that gene therapy with synthetic genes could be both feasible and efficient.

Independent mechanistic inhibition of cdc25 phosphatases by a natural product caulibugulone.

Caulibugulones are novel but poorly characterized cytotoxic isoquinoline quinones and iminoquinones identified in extracts from the marine bryozoan Caulibugula intermis. We now report that the caulibugulones are selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases compared with either human vaccinia H1-related phosphatase (VHR) or tyrosine phosphatase 1B (PTP1B). The in vitro inhibition of Cdc25B by caulibugulone A was irreversible and attenuated by reducing agents or catalase, consistent with direct oxidation of the enzyme by reactive oxygen species. Mechanistically, caulibugulone A directly inhibited cellular Cdc25B activity, generated intracellular reactive oxygen species and arrested cells in both G1 and G2/M phases of the cell cycle. Caulibugulone A also caused the selective degradation of Cdc25A protein by a process that was independent of reactive oxygen species production, proteasome activity, and the Chk1 signaling pathway. Instead, caulibugulone A stimulated the phosphorylation and subsequent activation of p38 stress kinase, leading to Cdc25A degradation. Thus, caulibugulone inhibition of cellular Cdc25A and B phosphatases occurred through at least two different mechanisms, leading to pronounced cell cycle arrest.

IMPROVED GENE DELIVERY FORMULATIONS AND EXPRESSION SYSTEMS FOR ENHANCED TRANSFECTION EFFICIENCY

Effective gene therapy requires efficient delivery of DNA to transfected cells followed by high levels of gene expression. Our laboratory has developed two new delivery formulations LPDI containing the DNA condensing agent protamine sulfate and reconstitued chylomicrons containing a hydrophobic lipidDNA complex. LPDI can produce higher levels of gene expression than the corresponding liposome/DNA complexes alone both in vitro and in vivo. Reconstituted chylomicrons also induce high gene expression in the liver through intraportal injection. To increase overall gene expression of delivered DNA, an improved cytoplasmic expression system has been developed using a unique T7 RNA polymerase autogene pCMVIT7-T7pol. High levels of reporter gene expression were detected in the presence of this new autogene.