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Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

ABSTRACT: Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either the Arf tumor suppressor or its downstream target, the p53 transcription factor. Mouse p19(Arf) is encoded by the Ink4a-Arf locus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16(Ink4a). We surveyed bone marrow-derived cells from wild-type, Ink4a-Arf-null, or Arf-null mice for their ability to bypass senescence during continuous passage in culture. Unlike preB cells from wild-type mice, those from mice lacking Arf alone could be propagated indefinitely when placed onto stromal feeder layers engineered to produce IL-7. The preB cell lines remained diploid and IL-7-dependent and continued to express elevated levels of p16(Ink4a). By contrast, Arf-null bone marrow-derived macrophages that depend on colony-stimulating factor-1 for proliferation and survival in culture initially grew at a slow rate but gave rise to rapidly and continuously growing, but still growth factor-dependent, variants that ceased to express p16(Ink4a). Wild-type bone marrow-derived macrophages initially expressed both p16(Ink4a) and p19(Arf) but exhibited an extended life span when p16(Ink4a) expression was extinguished. In all cases, gene silencing was accompanied by methylation of the Ink4a promoter. Therefore, whereas Arf loss alone appears to be the major determinant of establishment of murine fibroblast and preB cell lines in culture, p16(Ink4a) provides an effective barrier to immortalization of bone marrow-derived macrophages.

SUBMITTER: Randle DH

PROVIDER: S-EPMC55507 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

Randle D H DH Zindy F F Sherr C J CJ Roussel M F MF

Proceedings of the National Academy of Sciences of the United States of America 20010731 17

Establishment of cell lines from primary mouse embryo fibroblasts depends on loss of either the Arf tumor suppressor or its downstream target, the p53 transcription factor. Mouse p19(Arf) is encoded by the Ink4a-Arf locus, which also specifies a second tumor suppressor protein, the cyclin D-dependent kinase inhibitor p16(Ink4a). We surveyed bone marrow-derived cells from wild-type, Ink4a-Arf-null, or Arf-null mice for their ability to bypass senescence during continuous passage in culture. Unlik ...[more]

PMID: 11481442

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Project description:We defined the time course of ionizing radiation-induced senescence in lung compared to bone marrow of p16+/LUC mice in which the senescence-induced biomarker (p16) is linked to a luciferase reporter gene. Periodic in situ imaging revealed increased luciferase activity in the lungs of 20 Gy thoracic irradiated, but not 8 Gy total-body irradiated (TBI) mice beginning at day 75 and increasing to day 170. In serial sections of explanted lungs, senescent cells appeared in the same areas as did fibrosis in the 20 Gy thoracic irradiated, but not the 8 Gy TBI group. Lungs from 8 Gy TBI mice at one year did show increased RNA levels for p16, p21, p19 and TGF-β. Individual senescent cells in 20 Gy irradiated mouse lung included those with epithelial, endothelial, fibroblast and hematopoietic cell biomarkers. Rare senescent cells in the lungs of 8 Gy TBI mice at one year were of endothelial phenotype. Long-term bone marrow cultures (LTBMCs) were established at either day 60 or one year after 8 Gy TBI. In freshly removed marrow at both times after irradiation, there were increased senescent cells. In LTBMCs, there were increased senescent cells in both weekly harvested single cells and in colonies of multilineage hematopoietic progenitor cells producing CFU-GEMM (colony forming unit-granulocyte, erythrocyte, monocyte/macrophage, mega-karyocyte) that were formed in secondary cultures when these single cells were plated in semisolid media. LTBMCs from TBI mice produced fewer CFU-GEMM; however, the relative percentage of senescent cell-containing colonies was increased as measured by both p16-luciferase and β-galactosidase. Therefore, 20 Gy thoracic radiation, as well as 8 Gy TBI, induces senescent cells in the lungs. With bone marrow, 8 Gy TBI induced senescence in both hematopoietic cells and in colony-forming progenitors. The p16+/LUC mouse strain provides a valuable system in which to compare the kinetics of radiation-induced senescence between organs in vivo, and to evaluate the potential role of senescent cells in irradiation pulmonary fibrosis.

Dataset Information

Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

Publications

Differential effects of p19(Arf) and p16(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages.

Similar Datasets

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