José Mp Freije

Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53

Aging is a multifactorial process that affects most of the biological functions of the organism and increases susceptibility to disease and death. Recent studies with animal models of accelerated aging have unveiled some mechanisms that also operate in physiological aging. However, little is known about the role of microRNAs (miRNAs) in this process. To address this question, we have analysed miRNA levels in Zmpste24‐deficient mice, a model of Hutchinson–Gilford progeria syndrome. We have found that expression of the miR‐29 family of miRNAs is markedly upregulated in Zmpste24−/− progeroid mice as well as during normal aging in mouse. Functional analysis revealed that this transcriptional activation of miR‐29 is triggered in response to DNA damage and occurs in a p53‐dependent manner since p53−/− murine fibroblasts do not increase miR‐29 expression upon doxorubicin treatment. We have also found that miR‐29 represses Ppm1d phosphatase, which in turn enhances p53 activity. Based on these results, we propose the existence of a novel regulatory circuitry involving miR‐29, Ppm1d and p53, which is activated in aging and in response to DNA damage.

Collagenase-3 Binds to a Specific Receptor and Requires the Low Density Lipoprotein Receptor-related Protein for Internalization

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that 125I-collagenase-3 binds specifically to two proteins (∼170 kDa and ∼600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

Reprogramming aging and progeria

The aging rate of an organism depends on the ratio of tissue degeneration to tissue repair. As a consequence, molecular alterations that tip this balance toward degeneration cause accelerated aging. Conversely, interventions can be pursued to reduce tissue degeneration or to increase tissue repair with the aim of delaying the onset of age-associated manifestations. Recent studies on the biology of stem cells in aging have revealed the influence of systemic factors on their functionality and demonstrated the feasibility of reprogramming aged and progeroid cells. These results illustrate the reversibility of some aspects of the aging process and encourage the search for new anti-aging and anti-progeria interventions.

Mutational analysis of BRCA1 and BRCA2 in hereditary breast and ovarian cancer families from Asturias (Northern Spain)

BackgroundThe prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies according to geographical origin of studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian populations (Northern Spain).MethodsIn the present work, 256 unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analyzed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to May 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened both by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA).ResultsA total of 59 families (23%) were found to carry a pathogenic germ line mutation, 39 in BRCA1 and 20 in BRCA2. Twenty nine additional families (12%) carried an unknown significance variant. We detected 28 distinct pathogenic mutations (16 in BRCA1 and 12 in BRCA2), of which 3 mutations in BRCA1 (c.1674delA, c.1965C>A and c.2900_2901dupCT) and 5 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.8042_8043delCA) had not been previously described.The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 8 and 6 families respectively and clustered in two separated small geographically isolated areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.211A>G (9 families), and the common BRCA1 mutation c.3331_3334delCAAG (6 families), account for approximately 50% of all affected families. By contrast, very frequent mutations in other Spanish series such as the BRCA1 Ashkenazi founder mutation c.68_69delAG, was found in only one family.ConclusionsIn this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differ from other areas of Spain. Our findings may help design a first step recurrent mutation panel for screening high-risk breast and/or ovarian cancer families from this specific area.

Germ-line mutations in epidermal growth factor receptor (EGFR) are rare but may contribute to oncogenesis: A novel germ-line mutation in EGFR detected in a patient with lung adenocarcinoma

BackgroundA subset of lung cancer patients harbour EGFR somatic mutations in their tumours and are candidates for treatment with EGFR tyrosine kinase inhibitors. In a few cases EGFR mutations have also been found in the germ line, suggesting a role in lung carcinogenesis. Objetives of this study were: 1) To analyze the EGFR gene mutations in a population diagnosed with lung adenocarcinoma from Northern Spain. 2) To determine the frequency of a new germ-line mutation found in our laboratory as well as the frequency in our population of three other EGFR germ-line mutations detected by other authors. 3) To determine whether the novel mutation detected may have a functional effect on the EGFR protein.MethodsTumour DNA samples were obtained from frozen or paraffin embedded tumour tissues. Samples of DNA from peripheral blood cells were obtained from 912 individuals with lung cancer recruited from the CAPUA study [1, 2], 477 unrelated healthy donor individuals and 32 individuals with other types of cancer. EGFR gene exons 18 to 21 were studied by direct standard dideoxy sequencing. Specific mutations were determined either by direct sequencing or by specific RFLP analysis. Cell lines were transfected with EGFR-mutant plasmids and analysed by western blot with antibodies specific for total or phosphorylated-EGFR.ResultsWe found EGFR mutation in 12 of the 71 tumour samples (17%). One tumour contained two mutations. One mutation (p.R776G) was present as a germ line. Using an RFLP analysis, this mutation was not found in 954 alleles from healthy individuals studied, concluding that it is not a polymorphism. The mutation was not found either in genomic DNA from 912 lung cancer patients. Three additional EGFR germ-line mutations that were already described were not found in any of the studied samples. These observations show that EGFR mutated alleles are rare in the population. In vitro studies revealed that tyrosine autophosphorylation is enhanced in p.R776G-mutant EGFR when compared with wild-type EGFR. This enhanced autophosphorylation in the absence of ligand may be associated with a proliferative advantage.ConclusionsGerm-line mutations in EGFR are rare but may contribute to oncogenesis

NF-κB signaling as a driver of ageing.

NF-κB signaling exerts essential roles in immunity and cellular stress responses, regulating many functions related with organism innate defense. Besides, NF-κB altered signaling has been causally linked to ageing and diverse pathological conditions. We discuss herein the functional involvement of this signaling pathway in ageing, visiting recent experimental evidence about NF-κB activation in this complex process, its functional consequences and the novel biological functions raised from these works. Moreover, we discuss ageing intervention strategies based on NF-κB inhibition, which have demonstrated to be effective at delaying and even reverting different ageing manifestations in human and mouse models of both normal and accelerated ageing. Altogether, the current evidence supports that NF-κB activation constitutes a driving force of the ageing process and a preferential target for rejuvenation-aimed approaches.

Luminescence-based in vivo monitoring of NF-κB activity through a gene delivery approach

BackgroundMonitoring activity of specific signaling pathways in vivo is challenging and requires highly sensitive methods to detect dynamic perturbations in whole organisms.ResultsIn vivo gene delivery of a luciferase reporter followed by bioluminiscence imaging allows measuring NF-κB activity in mice liver and lungs.ConclusionsThis protocol allows a direct measure of NF-κB activity through quantification of bioluminescence signal, demonstrating its accuracy and sensitivity in different animal models and experimental conditions. Variants could be also applied for the analysis of NF-κB activity in different tissues or for studying other signaling pathways in vivo.