Development of Adenoviral Vectors for Detection of Proliferation and Differentiation Stage Dependent Telomerase Reverse Transcriptase Expression in Single Living Hematopoietic Cells.

Elevated telomerase activity is an important molecular signature for proliferating hematopoietic progenitors, lymphocytes and a variety of leukemic cells. The human telomerase activity is predominantly determined by the transcriptional control of the catalytic subunit telomerase reverse transcriptase (hTERT). For prospective identification of single living hematopoietic cells with telomerase activity, we have developed telomerase-reporting adenoviral vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 hours (d2EGFP) driven by the hTERT promoter. To shield the hTERT promoter from adenoviral backbone cis-acting DNA sequences, vectors encoding the chicken β-globin locus HS4 insulator sequence or bovine growth hormone transcription stop signal were also engineered. Moreover, these vectors were retargeted with the Ad35 fiber receptor specificity (Ad5F35 vectors) and utilize ubiquitously expressed CD46 as a cellular receptor. The Ad5F35 vectors allow efficient gene delivery into hematopoietic cells (J Gene Med6:631, 2004). The telomerase activity dependent d2EGFP expression was demonstrated in telomerase positive HeLa, A549 or negative fibroblasts and WI-38 cells by infection with the telomerase-reporting vectors at 2000 physical particles per cells. Successful gene delivery was achieved as about 90% of the cells were EGFP+ following infection with control Ad5F35-CMV-EGFP vector. Between 40 to 95% of the individual HeLa and A549 cells were d2EGFP+, and the percentages of the d2EGFP+ cells positively correlated with the extent of the telomerase activity as assessed in Trap assay. In contrast, a barely detectable fraction of the WI-38 cells and lower than 6% of the fibroblasts were expressing lower levels of d2EGFP. The vector carrying the HS4 insulator sequence generated the lowest background d2EGFP expression in fibroblasts and WI-38 cells (called cTERTdGFP vector). Therefore the cTERTdGFP vector was used for all the subsequent studies. Importantly, no measurable d2EGFP expression was observed in normal primary myeloid cells. The cTERTdGFP vector mediated d2EGFP expression in hematopoietic cells is dynamically regulated during cell proliferation and differentiation. Promyelocytic leukemic HL-60 cells were infected with the cTERTdGFP or the control Ad5F35-PGK-EGFP vector. Nearly 100% of the HL-60 cells were EGFP+ following the control vector infection, however, around 50% of the HL-60 cells were d2EGFP+ following cTERTdGFP vector infection. The d2EGFP expression was reduced 2-fold with lower d2EGFP intensity in retinoic acid induced differentiating HL-60 cells compared with non-treated HL-60 cells. HL-60 cells expanded from sorted d2EGFP+ or d2EGFP- cells generated identical frequencies of d2EGFP+ and d2EGFP- cells following re-infection with the cTERTdGFP vector. Cell cycle analysis assessing cellular DNA content in combination with Ki-67 antigen staining or BrdU pulsing showed that the sorted d2EGFP+ HL-60 cells contained significantly fewer cells in the G1, more cells in the S/G2/M phase of cell cycle compared with the d2EGFP- HL-60 cells. In conclusion, our studies can provide a powerful tool for isolation of living normal hematopoietic or leukemic cells with telomerase activity from heterogeneous cell populations.

Coupling of Enhancer and Insulator Properties Identified in Two Retrotransposons Modulates Their Mutagenic Impact on Nearby Genes

ABSTRACT We recently reported a novel transposition system in which two retroelements from Drosophila melanogaster, ZAM and Idefix, are highly mobilized and preferentially insert within intergenic regions. Among the loci where new copies are detected, a hot spot for their insertion was identified at the white locus, where up to three elements occurred within a 3-kb fragment upstream of the transcriptional start site of white. We have used these insertions as molecular entry points to throw light on the mutagenic effect exerted by multiple insertions of retrotransposons within intergenic regions of a genome. Analysis of the molecular mechanisms by which ZAM and Idefix elements interfere with the regulation of the white gene has shown that ZAM bears cis-acting regulatory sequences able to enhance transcription of the white gene in the eyes of the flies. This activation may be counteracted by Idefix, which acts as an insulator able to isolate the white gene from the upstream ZAM enhancer. In addition to revealing a novel insulator sequence with its own specific features, our data clearly illustrate how retroelements can act as epigenetic factors able to interfere with the transcriptional regulation of their host.

Magnetoresistance, temporal evolution, and relaxation of the electrical resistivity in the re-entrant semiconducting La0.80Ba0.20CoO3 perovskite

We report here a study on the electrical and magnetic properties of La1−xBaxCoO3 in the re-entrant semiconducting region (x = 0.20). We find that in this material: (i) the insulator-metal-insulator sequence is unstable and evolves toward a purely semiconducting behavior; the initial r versus T curve can be reinstated upon appropriate annealing treatments; (ii) there are relaxation effects that can be seen by changing the polarity of the electrodes; (iii) there is a negative magnetoresistance Δρ/ρ ∼ 2–3%, for a field as low as 9 kOe, especially at the metal-insulating transition temperatures; and (iv) there are important fluctuations in the electrical resistivity. Taking into account these experimental observations, we can interpret this material as an inhomogeneous system where two thermodynamic phases, one semiconducting and the other metallic and ferromagnetic, coexist, although they are crystallographically indistinguishable.