Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Smooth muscle-specific gene delivery in the vasculature based on restriction of DNA nuclear import

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.     

Gene expression of flap endonuclease-1 during cell proliferation and differentiation

It has been shown that flap endonuclease-1 (FEN-1), a structure-specific nuclease, acts on the removal of RNA primers during Okazaki fragment maturation in DNA synthesis. To study whether the gene expression of FEN-1 is inducible during cell proliferation, we analyzed the FEN-1 mRNA levels in actively growing cells and non-growing cells. The gene expression of FEN-1 was higher in mitotic cells than in resting cells, and was markedly decreased, especially, when terminal differentiation was induced in promyelocytic leukemia cells (HL-60 cells). The decline correlated substantially with the ceasing of DNA synthesis. In the examination of tissue-specific gene expression, the human testis, spleen, thymus and mucosal lining of colon tissues expressed this gene actively, whereas the prostate, ovary, small intestine and peripheral blood leukocyte hardly expressed it. In addition, FEN-1 was co-localized with the proliferating cell nuclear antigen (PCNA) in young rat kidney according to immunohistochemistry. These findings suggest that FEN-1 gene expression is inducible during cell proliferation for DNA synthesis, and is down-regulated during cell differentiation.     

Cell and animal imaging of electrically mediated gene transfer

Electropermeabilization is a nonviral method successfully used to transfer genes into cells in vitro as in vivo. Although it shows promise in field of gene therapy, very little is known on the basic processes supporting the DNA transfer. The aim of the present investigation is to visualize gene electrotransfer and expression both in vitro and in vivo. In vitro studies have been performed by using digitized fluorescence microscopy. Membrane permeabilization occurs at the sides of the cell membrane facing the two electrodes. A free diffusion of propidium iodide across the membrane to the cytoplasm is observed in the seconds following electric pulses. Fluorescently labeled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable over a few minutes. Changing the polarity and the orientation of the pulses lead to an increase in gene expression. In vivo experiments have been performed in Tibialis Cranialis mice muscle. Electric field application lead to the in vivo expression of plasmid DNA. We directly visualize gene expression of the Green Fluorescent Protein (GFP) on live animals. GFP expression is shown to be increased by applying electric field pulses with different polarities and orientations.     

Enhanced gene amplification in human cells knocked down for DNA-PKcs

Gene amplification, a key mechanism for oncogene activation and drug resistance in tumour cells, involves the generation and joining of DNA double-strand breaks. Amplified DNA can be carried either on intra-chromosomal arrays or on extra-chromosomal elements (double minutes). We previously showed that, in rodent cells deficient in DNA-PKcs, intra-chromosomal amplification is significantly enhanced. In the present work, we studied gene amplification in human HeLa cell lines in which the expression of the DNA-PKcs gene was constitutively inhibited by shRNAs. These cell lines showed an increased sensitivity to ionizing radiations, an enhanced frequency of chromosomal aberrations and an increased rate of occurrence of methotrexate resistant colonies compared to the control cell lines (6-18 times). The main mechanism of resistance to methotrexate was extra-chromosomal amplification of the dihydrofolate reductase gene. These results indicate that, in human cells, inhibition of DNA-PKcs gene expression favours gene amplification occurring via the production of double minutes. In addition, they show that cell lines constitutively expressing shRNAs are good model systems to study the role of specific functions in gene amplification.     

In vivo plasmid DNA electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers

In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers.     

Host genetic requirements for DNA release of lactococcal phage TP901-1

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.

In order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.