Accumulation of senescent cells in mitotic tissue of aging primates

Abstract

Cellular senescence, a stress induced growth arrest of somatic cells, was first documented in cell cultures over 40 years ago, however its physiological significance has only recently been demonstrated. Using novel biomarkers of cellular senescence we examined whether senescent cells accumulate in tissues from baboons of ages encompassing the entire lifespan of this species. We show that dermal fibroblasts, displaying markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16, accumulate in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reaches a value of over 15% of total fibroblasts, whereas 80% of cells contain high levels of the heterochromatin protein HIRA. In skeletal muscle, a postmitotic tissue, only a small percentage of myonuclei containing damaged telomeres were detected regardless of animal age. The presence of senescent cells in mitotic tissues might therefore be a contributing factor to aging and age related pathology and provides further evidence that cellular senescence is a physiological event.

Fig. 1

Telomere damage induced senescence in baboon fibroblast cultures. (A) Passage history of baboon skin fibroblasts. PD: population doublings (B) p21 and p16 levels in senescent baboon skin fibroblasts visualized by immunostaining with antibodies against p21 (green, top left panel), p16 using the monoclonal JC8 antibody (red, center column), and p16 using the polyclonal antibody H156 (green, bottom left panel). Merged images are shown in the right column. DNA was counterstained with DAPI (blue). (C) Accumulation of γH2AX foci in senescent baboon skin fibroblasts. Senescent cells were immunostained with a γH2AX antibody and the number of cells displaying one or more γH2AX foci were quantified. The bar is divided into the fraction of cells displaying 1 (blue), 2 (red), 3 (yellow), 4–5 (Green), or over 5 (black) γH2AX foci. A total number of 113 cell nuclei were scored. (D) Activated G1 DNA damage checkpoint proteins in senescent baboon fibroblasts. Cultures of senescent fibroblasts were immunostained with antibodies against γH2AX (green, center column, 53BP1 (red, top left), ATM (S1981) (PATM, red, center left), and p53 (S15) (red, bottom left). Merged images are shown in the right column. DNA was counterstained with DAPI (blue). (E) Senescent cells were processed by immunoFISH to visualize γH2AX foci (green) and telomeres (red). DNA was counterstained with DAPI (blue). Arrows point to enlarged images of TIF.

Fig. 2

Accumulation of telomere dysfunction induced foci (TIF) in dermal fibroblasts of old baboons. (A) 8 µm skin (left column) or 8 µm skeletal muscle (right column) tissue sections from young (5 yr, top row) and old (30 yr, bottom row) baboons were immunostained with a 53BP1 antibody (red) to detect sites of double strand DNA breaks. 53BP1 foci are indicated by the arrows for better visualization. Nuclear DNA was counterstained with DAPI (blue). (B) Quantitation of 53BP1 positive cells in skeletal muscle and skin biopsies from young (5 animals between the ages of 5–6 years, grey bars) and old (5 animals between the ages of 26–30 years, black bars) baboons. 200–600 cell nuclei were scored for each animal (C) Fibroblasts in baboon skin biopsies (top panel, TIF positive) and myonuclei (bottom panel, TIF negative) were processed by immunoFISH to visualize 53BP1 foci (green) and telomeres (red). DNA was counterstained with DAPI (blue). Arrows point to enlarged images of the 53BP1/telomere signal. (D) Quantitation of TIF in skeletal muscle and skin biopsies from young (5 animals between the ages of 5–6 years, grey bars) and old (5 animals between the ages of 26–30 years, black bars) baboons. 200–600 cell nuclei were scored for each animal. A cell was considered TIF positive when 50% or more of its 53BP1 foci colocalized with telomeric sequences.

Fig. 3

Fibroblasts in skin biopsies primarily contain a single 53BP1 focus and display markers of cellular senescence. (A) Accumulation of 53BP1 foci in fibroblasts of skin biopsies from baboons at indicated ages. Bars indicate the total number of 53BP1 positive fibroblasts in the tissue. The bars are divided into the fraction of fibroblasts displaying 1 (blue), 2 (red), 3 (yellow), 4–5 (Green), or over 5 (black) γH2AX foci. 200–600 fibroblasts were scored for each animal. (B) The number of 53BP1 positive fibroblasts displaying a single 53BP1 focus remains constant as animals age. 200–600 fibroblasts in baboon skin biopsies were scored for each animal. (C) Immunofluorescence analysis of fibroblasts in skin biopsies of a 25 year old baboon showing colocalization between 53BP1 and indicated proteins. P-ATM: ATM(Ser1981). Nuclear DNA was counterstained with DAPI (blue).

Fig. 4

The number of fibroblasts in baboon skin biopsies displaying elevated levels of the heterochromatin protein HIRA increases with age of the animal. (A) Immunostaining of dermal fibroblasts of a young (5 year, top row) and an old (29 year, bottom row) animal with an antibody against HIRA (green). Nuclear DNA was counterstained with DAPI (blue). (B) Quantitation of HIRA positive fibroblasts in skin biopsies of baboons at indicated ages.