Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

The prima donna of epigenetics: the regulation of gene expression by DNA methylation

This review focuses on the mechanisms of DNA methylation, DNA methylation pattern formation and their involvement in gene regulation. Association of DNA methylation with imprinting, embryonic development and human diseases is discussed. Furthermore, besides considering changes in DNA methylation as mechanisms of disease, the role of epigenetics in general and DNA methylation in particular in transgenerational carcinogenesis, in memory formation and behavior establishment are brought about as mechanisms based on the cellular memory of gene expression patterns.

Early effects of estrogen on the rat ventral prostate

Complex interactions between androgen and estrogen (E2) regulate prostatic development and physiology. We analyzed the early effects of a high single dose of E2 (25 mg/kg body weight) and castration (separately or combined) on the adult 90-day-old male Wistar rat ventral prostate. Androgen levels, prostate weight, and the variation in the relative and absolute volume of tissue compartments and apoptotic indices were determined for 7 days. Castration and exogenous E2 markedly reduced ventral prostate weight (about 50 percent of the control), with a significant reduction in the epithelial compartment and increased stroma. The final volume of the epithelium was identical at day 7 for all treatments (58.5 percent of the control). However, E2 had an immediate effect, causing a reduction in epithelial volume as early as day 1. An increase in smooth muscle cell volume resulted from the concentration of these cells around the regressing epithelium. The treatments resulted in differential kinetics in epithelial cell apoptosis. Castration led to a peak in apoptosis at day 3, with 5 percent of the epithelial cells presenting signs of apoptosis, whereas E2 caused an immediate increase (observed on day 1) and a sustained (up to day 7) effect. E2 administration to castrated rats significantly increased the level of apoptosis by day 3, reaching 9 percent of the epithelial cells. The divergent kinetics between treatments resulted in the same levels of epithelial regression after 7 days (about 30 percent of control). These results show that E2 has an immediate and possibly direct effect on the prostate, and anticipates epithelial cell death before reducing testosterone to levels as low as those of castrated rats. In addition, E2 and androgen deprivation apparently cause epithelial cell death by distinct and independent pathways.

Aggrecan structure in amphibian cartilage

The structure of the large proteoglycan present in the bullfrog epiphyseal cartilage was studied by immunochemical and biochemical methods. The isolated monomer showed a polydisperse behavior on Sepharose CL2B, with a peak at Kav = 0.14. Chondroitin sulfate chains were identified by HPLC analysis of the products formed by chondroitinase digestion and mercuric acetate treatment. These chains have approximately 38 disaccharides, a Di45:Di68 ratio of 1.6 and GalNAc4S + GalNAc4,6S are the main non-reducing terminals. Keratan sulfate was identified by the use of two monoclonal antibodies in Western blots after chondroitinase ABC treatment. A keratan sulfate-rich region (~110 kDa) was isolated by sequential treatment with chondroitinase ABC and proteases. We also employed antibodies in Western blotting experiments and showed that the full length deglycosylated core protein is about 300 kDa after SDS-PAGE. Domain-specific antibodies revealed the presence of immunoreactive sites corresponding to G1/G2 and G3 globular domains and the characterization of this large proteoglycan as aggrecan. The results indicate the high conservation of the aggrecan domain structure in this lower vertebrate