Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation.

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.

Telomeres and chromosomal instability.

Telomeres are distinctive structures, composed of a repetitive DNA sequence and associated proteins, which enable cells to distinguish chromosome ends from DNA double-strand breaks. Telomere alterations, caused by replication-mediated shortening, direct damage or defective telomere-associated proteins, usually generate chromosomal instability, which is observed in senescence and during the immortalization process. In cancer cells, this chromosome instability could be extended by their ability to 'repair' chromosomes and terminate in break-fusion-bridge cycles. Dysfunctional telomeres can be healed by activation of telomerase or by the 'alternative mechanism' of telomere lengthening. Activation of such telomere maintenance mechanisms may help to preserve the integrity of chromosomes even if they play a role in chromosomal instability. This review focuses on molecular processes involved in telomere maintenance and chromosomal instability associated with dysfunctional telomeres in mammalian cells.

Rapid method for mean telomere length measurement directly from cell lysates.

Telomere length is involved in cell survival, tumorigenesis, and early aging. We present here an innovative method to determine the mean telomere length without any DNA purification. Our strategy is to measure both the DNA concentration and the number of telomeric units (TTAGGG) directly from cell lysate produced by the combined action of NaOH (pH>13) and sonication directly on cell pellet. Telomere units are quantified using an enzyme hybridization assay on 96-well microtiter plates grafted with a captor sequence. A biotin-coupled-tracer oligonucleotide hybridizes with telomere fragments and the enzymatic reaction is performed with a streptavidin-acetylcholinesterase conjugate, using the colorimetric method of Ellman. OD measure is directly proportional to the number of telomere units in cell lysate. This scalable technique allows the determination of mean telomere length simultaneously in many samples. This assay will be highly efficient to screen new drugs involved in chemotherapy targeting telomerase or directly telomeres.

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF.

Lipopolysaccharide (LPS) was found to be associated with the major outer membrane protein OprF of the psychrotrophic bacterium Pseudomonas fluorescens MF0, using two OprF purification procedures. OprF, purified under mild conditions, presented two types of association with LPS: tight (tLPS) and slight (sLPS), both of type R. LPS protected OprF from heat modification and trypsin degradation and facilitated the reincorporation of purified OprF into an artificial lipid bilayer without affecting its pore-forming activity. The size of the OprF channel depended on cell growth temperature, as did the extent of LPS phosphorylation: we suggest that LPS may be involved in modifications of OprF pore formation.