Senescence in Alveolar Epithelial Type II Cells Promotes Acute Lung Injury and Impairs Regeneration.

Mortality of acute lung injury (ALI) increases with age. Alveolar epithelial type 2 cells (AEII) are the progenitor cells of the alveolar epithelium and crucial for repair after injury. We hypothesize that telomere dysfunction-mediated AEII senescence impairs regeneration and promotes the development of ALI. To discriminate between the impact of old age and AEII senescence in ALI, young (3 months) and old (18 months) Sftpc-Ai9 mice and young Sftpc-Ai9-Trf1 mice with inducible Trf1 knockout-mediated senescence in AEII were treated with 1 µg lipopolysaccharide (LPS)/g BW (n=9-11). Control mice received saline (n=7). Mice were sacrificed 4 or 7 days later. Lung mechanics, pulmonary inflammation and proteomes were analyzed and parenchymal injury, AEII proliferation and AEI differentiation rate were quantified using stereology. Old mice showed 55% mortality by day 4, whereas all young mice survived. Pulmonary inflammation was most severe in old mice, followed by Sftpc-Ai9-Trf1 mice. Young Sftpc-Ai9 mice recovered almost completely by day 7, while Sftpc-Ai9-Trf1 mice still showed mild signs of injury. An expansion of AEII was only measured in young Sftpc-Ai9 mice at day 7. Aging and telomere dysfunction-mediated senescence had no impact on AEI differentiation rate in controls, but the reduced number of AEII in Sftpc-Ai9-Trf1 mice also affected de-novo differentiation after injury. In conclusion, telomere dysfunction-mediated AEII senescence promoted parenchymal inflammation in ALI, but did not enhance mortality like old age. While Differentiation rate remained functional with old age and AEII senescence, AEII proliferative capacity was impaired in ALI, affecting the regenerative ability.

Aberrant telomeric structures and serum markers of telomere dysfunction in healthy aging: a preliminary study.

Telomeres undergo a progressive shortening process as individuals age, and it has been proposed that severely shortened and dysfunctional telomeres play a role in the aging process and the onset of age-related diseases in human beings. An emerging body of evidence indicates that the shortening of telomeres in cultured human cells is also influenced by other replication defects occurring within telomeric repeats. These abnormalities can be detected on metaphase chromosomes. Recent studies have also identified a set of serological markers for telomere dysfunction and DNA damage (elongation factor 1α [EF-1α], stathmin, and N-acetyl-glucosaminidase). With this study, the correlation between telomere abnormalities (by FISH) and these biomarkers as measured in blood serum (by ELISA) from a cohort of 22 healthy subjects at different ages (range 26-101 years) was analyzed. A strong positive correlation between aging and the presence of aberrant telomere structures, sister telomere loss (STL), and sister telomere chromatid fusions (STCF) was detected. When serum markers of telomere dysfunction were correlated with telomere abnormalities, we found that stathmin correlated with total aberrant telomeres structures (r = 0.431, p = 0.0453) and STCF (r = 0.533, p = 0.0107). These findings suggest that serum stathmin can be considered an easy-to-get marker of telomere dysfunction and may serve as valuable indicators of aging.

Considerations on the scoring of telomere aberrations in vertebrate cells detected by telomere or telomere plus centromere PNA-FISH.

Given that telomeres play a fundamental role in maintaining genomic stability, the study of the chromosomal aberrations involving telomeric sequences is a topic of considerable research interest. In recent years, the scoring of these types of aberrations has been used in vertebrate cells, particularly human cells, to evaluate the effects of genotoxic agents on telomeres and the involvement of telomeric sequences on chromosomal aberrations. Currently, chromosomal aberrations involving telomeric sequences are evaluated in peripheral blood lymphocytes or immortalized cell lines, using telomere or telomere plus centromere fluorescence in situ hybridization (FISH) with Peptide Nucleic Acid (PNA) probes (PNA-FISH). The telomere PNA probe is more efficient in the detection of telomeric sequences than conventional FISH with a telomere DNA probe. In addition, the intensity of the telomeric PNA-FISH probe signal is directly correlated with the number of telomeric repeats. Therefore, use of this type of probe can identify chromosomal aberrations involving telomeres as well as determine the telomere length of the sample. There are several mistakes and inconsistencies in the literature regarding the identification of telomere aberrations, which prevent accurate scoring and data comparison between different publications concerning these types of aberrations. The aim of this review is to clarify these issues, and provide proper terminology and criteria for the identification, scoring, and analysis of telomere aberrations.

Silencing PinX1 enhances radiosensitivity and antitumor-immunity of radiotherapy in non-small cell lung cancer.

BACKGROUND: We aimed to investigate the effects of PinX1 on non-small cell lung cancer(NSCLC) radiosensitivity and radiotherapy-associated tumor immune microenvironment and its mechanisms. METHODS: The effect of PinX1 silencing on radiosensitivity in NSCLC was assessed by colony formation and CCK8 assay, immunofluorescence detection of γ- H2AX and micronucleus assay. Western blot was used to assess the effect of PinX1 silencing on DNA damage repair pathway and cGAS-STING pathway. The nude mouse and Lewis lung cancer mouse model were used to assess the combined efficacy of PinX1 silencing and radiotherapy in vivo. Changes in the tumor immune microenvironment were assessed by flow cytometry for different treatment modalities in the Lewis luuse model. The interaction protein RBM10 was screened by immunoprecipitation-mass spectrometry. RESULTS: Silencing PinX1 enhanced radiosensitivity and activation of the cGAS-STING pathway while attenuating the DNA damage repair pathway. Silencing PinX1 further increases radiotherapy-stimulated CD8+ T cell infiltration and activation, enhances tumor control and improves survival in vivo; Moreover, PinX1 downregulation improves the anti-tumor efficacy of radioimmunotherapy, increases radioimmune-stimulated CD8+ T cell infiltration, and reprograms M2-type macrophages into M1-type macrophages in tumor tissues. The interaction of PinX1 and RBM10 may promote telomere maintenance by assisting telomerase localization to telomeres, thereby inhibiting the immunostimulatory effects of IR. CONCLUSIONS: In NSCLC, silencing PinX1 significantly contributed to the radiosensitivity and promoted the efficacy of radioimmunotherapy. Mechanistically, PinX1 may regulate the transport of telomerase to telomeres through interacting with RBM10, which promotes telomere maintenance and DNA stabilization. Our findings reveal that PinX1 is a potential target to enhance the efficacy of radioimmunotherapy in NSCLC patients.

Review: Are moles senescent?

Melanocytic nevi (skin moles) have been regarded as a valuable example of cell senescence occurring in vivo. However, a study of induced nevi in a mouse model reported that the nevi were arrested by cell interactions rather than a cell-autonomous process like senescence, and that size distributions of cell nests within nevi could not be accounted for by a stochastic model of oncogene-induced senescence. Moreover, others reported that some molecular markers used to identify cell senescence in human nevi are also found in melanoma cells-not senescent. It has thus been questioned whether nevi really are senescent, with potential implications for melanoma diagnosis and therapy. Here I review these areas, along with the genetic, biological, and molecular evidence supporting senescence in nevi. In conclusion, there is strong evidence that cells of acquired human benign (banal) nevi are very largely senescent, though some must contain a minor non-senescent cell subpopulation. There is also persuasive evidence that this senescence is primarily induced by dysfunctional telomeres rather than directly oncogene-induced.

Telomere dysfunction in Tert knockout mice delays BrafV600E -induced melanoma development.

Telomerase activation is a crucial step in melanomagenesis, often occurring because of ultraviolet radiation (UVR)-induced mutations at the telomerase gene (TERT) promoter and rendering TERT transcription in response to the activated Raf-MAP kinase pathway by BRAFV600E mutation. Due to the excessively long telomeres in mice, this process does not occur during melanomagenesis in mouse models. To investigate the impact of telomere dysfunction on melanomagenesis, BrafV600E was induced in generations 1 and 4 (G1 and G4) of Tert-/- mice. Our findings revealed that, regardless of UVR exposure, melanoma development was delayed in G4 mice, which had shorter telomeres compared to G1 and wild-type C57BL/6J (G0) mice. Moreover, many G4 tumors displayed an accumulation of excessive DNA damage, as evidenced by increased γH2A.X staining. Tumors from UVR-exposed mice exhibited elevated p53 protein expression. Cultured tumor cells isolated from G4 mice displayed abundant chromosomal fusions and rearrangements, indicative of telomere dysfunction in these cells. Additionally, tumor cells derived from UVB-exposed mice exhibited constitutively elevated expression of mutant p53 proteins, suggesting that p53 was a target of UVB-induced mutagenesis. Taken together, our findings suggest that telomere dysfunction hampers melanomagenesis, and targeting telomere crisis-mediated genomic instability may hold promise for the prevention and treatment of melanoma.

TPP1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres / 第三军医大学学报

Objective To characterize the effects of TPP1 knockdown on Pot1a and Pot1b localization at telomeres and on the telomere end protection.Methods Knockdown of endogenous TPP1 in mouse embryonic fibroblasts(MEFs) with the retrovirus vector encoding shRNA targeting TPP1,IF/PNA-FISH was performed to determine the localization of Pot1a and Pot1b at telomeres,and TdT-FITC was applied to characterize the effects on the function of telomere end protection,cellular senescence was analyzed by SA-beta gal staining,and phosphorylated p53ser18 and p21 were examined by Western blotting.Results Pot1a and Pot1b were unable to localize at telomeres in about 65% of MEFs with TPP1 knockdown,while that was found in less than 5% of MEFs without TPP1 knockdown(t=10.96,P