Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

"Super p53" mice exhibit enhanced DNA damage response,are tumor resistant and age normally

The tumor suppressor p53 is critical in preventing cancer due to its ability to trigger proliferation arrest and cell death upon the occurrence of a variety of stresses, most notably, DNA damage and oncogenic stress. Here, we report the generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes. Prior to this, we demonstrate that the p53 transgenic allele (p53-tg), when present in a p53-null genetic background, behaves as a functional replica of the endogenous gene. "Super p53" mice, carrying p53-tg alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage. Importantly, "super p53" mice are significantly protected from cancer when compared with normal mice. Finally, in contrast to previously reported mice with constitutively active p53, "super p53" mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control. Together, our results prove that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.     

Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans

Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant.     

Effect of Macrovipera lebetina and Cerastes cerastes venoms on adherence to integrins of cancerous cells (IGR39, HT29-D4 and IGROV1)

In this work, we provide experimental arguments in favor of the fact that components from Macrovipera lebetina and Cerastes cerastes venoms bind to IGR39 melanoma cells but not to HT29D4 cells that derive from carcinoma adenome. Furthermore, Macrovipera lebetina and Cerastes cerastes venoms inhibit the adherence of IGR39 and HT 29-D4 to various extracellular matrix proteins. Macrovipera lebetina and Cerastes cerastes venoms did not inhibit the non specific adherence of IGR 39 cells to polylysine. In addition, binding of components from Cerastes cerastes venom to IGR39 cells is inhibited by GRGDS peptide and by monoclonal antibidy anti-av, while these two components have no effect on the adherence of IGR39 to Macrovipera lebetina venom.     

Roles for the two 1-butanol dehydrogenases of Pseudomonas butanovora in butane and 1-butanol metabolism

Pseudomonas butanovora grown on butane or 1-butanol expresses two 1-butanol dehydrogenases, a quinoprotein (BOH) and a quinohemoprotein (BDH). BOH exhibited high affinity towards 1-butanol (K(m) = 1.7 +/- 0.2 microM). BOH also oxidized butyraldehyde and 2-butanol (K(m) = 369 +/- 85 microM and K(m) = 662 +/- 98 microM, respectively). The mRNA induction profiles of BOH and BDH at three different levels of 1-butanol, a nontoxic level (0.1 mM), a growth-supporting level (2 mM), and a toxic level (40 mM), were similar. When cells were grown in citrate-containing medium in the presence of different levels of 1-butanol, wild-type P. butanovora could tolerate higher levels of 1-butanol than the P. butanovora boh::tet strain and the P. butanovora bdh::kan strain. A model is proposed in which the electrons from 1-butanol oxidation follow a branched electron transport chain. BOH may be coupled to ubiquinone, with the electrons being transported to a cyanide-sensitive terminal oxidase. In contrast, electrons from BDH may be transferred to a terminal oxidase that is less sensitive to cyanide. The former pathway may function primarily in energy generation, while the latter may be more important in the detoxification of 1-butanol.     

Nitrite reductase of Nitrosomonas europaea is not essential for production of gaseous nitrogen oxides and confers tolerance to nitrite

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.     

Structural properties of a collagenous heterotrimer that mimics the collagenase cleavage site of collagen type I

Collagens contain sequence- and conformation-dependent epitopes responsible for their digestion by collagenases at specific loci. A synthetic heterotrimer construct containing the collagenase cleavage site of collagen type I was found to mimic perfectly native collagen in terms of selectivity and mode of enzymatic degradation. The NMR conformational analysis of this molecule clearly revealed the presence of two structural domains, i.e. a triple helix spanning the Gly-Pro-Hyp repeats and a less ordered portion corresponding to the collagenase cleavage site where the three chains are aligned in extended conformation with loose interchain contacts. These structural properties allow for additional insights into the very particular mechanism of collagen digestion by collagenases.