Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells

Dietary phytochemicals, HDAC inhibition, and DNA damage/repair defects in cancer cells

Genomic instability is a common feature of cancer etiology. This provides an avenue for therapeutic intervention, since cancer cells are more susceptible than normal cells to DNA damaging agents. However, there is growing evidence that the epigenetic mechanisms that impact DNA methylation and histone status also contribute to genomic instability. The DNA damage response, for example, is modulated by the acetylation status of histone and non-histone proteins, and by the opposing activities of histone acetyltransferase and histone deacetylase (HDAC) enzymes. Many HDACs overexpressed in cancer cells have been implicated in protecting such cells from genotoxic insults. Thus, HDAC inhibitors, in addition to unsilencing tumor suppressor genes, also can silence DNA repair pathways, inactivate non-histone proteins that are required for DNA stability, and induce reactive oxygen species and DNA double-strand breaks. This review summarizes how dietary phytochemicals that affect the epigenome also can trigger DNA damage and repair mechanisms. Where such data is available, examples are cited from studies in vitro and in vivo of polyphenols, organosulfur/organoselenium compounds, indoles, sesquiterpene lactones, and miscellaneous agents such as anacardic acid. Finally, by virtue of their genetic and epigenetic mechanisms, cancer chemopreventive agents are being redefined as chemo- or radio-sensitizers. A sustained DNA damage response coupled with insufficient repair may be a pivotal mechanism for apoptosis induction in cancer cells exposed to dietary phytochemicals. Future research, including appropriate clinical investigation, should clarify these emerging concepts in the context of both genetic and epigenetic mechanisms dysregulated in cancer, and the pros and cons of specific dietary intervention strategies

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response

ABCG2 is a plasmamembrane multidrug transporter with an established role in the cancer drug resistance phenotype. This protein is expressed in various tissues, including several types of stem cells. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines while exhibiting a heterogeneous expression pattern. In the present study we examined the role and function of this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between the expression of pluripotency markers in ABCG2 positive and negative hESCs, however, ABCG2 expressing cells had a higher growth rate following cell separation. We found that certain harmful conditions (physical stress, drugs and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused a rapid internalization of ABCG2, indicating that certain environmental factors may induce the removal of this transporter from the plasmamembrane. In the light of these results we suggest that a dynamic balance of ABCG2 expression at the population level has an advantage to promptly respond to changes in the cellular environment. Such an actively maintained heterogeneity might be evolutionarily favorable to protect special cell types, including pluripotent stem cells

Stem cell therapy and the retina.

Retinal degeneration culminating in photoreceptor loss is the leading cause of untreatable blindness in the developed world. In this review, we consider how photoreceptors might be replaced by transplantation and how stem cells might be optimised for use as donor cells in future clinical strategies for retinal repair. We discuss the current advances in human and animal models of retinal cell transplantation, focussing on stem cell and reproductive cloning biology, in relation to the practical issues of retinal transplantation surgery. Stem and progenitor cells can be isolated from a number of sources including embryonic tissue, adult brain and even the retina, prompting many researchers to investigate the potential for using these cells to generate photoreceptors for transplantation. Nevertheless, several obstacles need to be overcome before these techniques can be applied in a clinical setting. Embryonic or stem cells have so far shown little ability to differentiate into retinal phenotypes when transplanted into the adult retina. We have recently noted, however, that donor cells harvested much later, at the photoreceptor precursor developmental stage, can be transplanted successfully and restore visual function. The current challenge is to understand the developmental processes that guide embryonic or adult stem cells towards photoreceptor differentiation, so that large numbers of these cells might be transplanted at the optimal stage. Future advances in reproductive cloning technology could lead to the successful generation of stem cells from adult somatic cells, thereby facilitating auto-transplantation of genetically identical cells in patients requiring photoreceptor replacement