Small molecule inhibition of p38 MAP kinase extends the replicative life span of human ATR-Seckel syndrome fibroblasts

Abstract

Ataxia-telangiectasia and rad3 (ATR)-related Seckel syndrome is associated with growth retardation and premature aging features. ATR-Seckel fibroblasts have a reduced replicative capacity in vitro and an aged morphology that is associated with activation of stress-associated p38 mitogen-activated protein kinase and phosphorylated HSP27. These phenotypes are prevented using p38 inhibitors, with replicative capacity restored to the normal range. However, this stressed phenotype is retained in telomerase-immortalized ATR-Seckel fibroblasts, indicating that it is independent of telomere erosion. As with normal fibroblasts, senescence in ATR-Seckel is bypassed by p53 abrogation. Young ATR-Seckel fibroblasts show elevated levels of p21(WAF1), p16(INK4A), phosphorylated actin-binding protein cofilin, and phosphorylated caveolin-1, with small molecule drug inhibition of p38 reducing p16(INK4A) and caveolin-1 phosphorylation. In conclusion, ATR-Seckel fibroblasts undergo accelerated aging via stress-induced premature senescence and p38 activation that may underlie certain clinical features of Seckel syndrome, and our data suggest a novel target for pharmacological intervention in this human syndrome.

Keywords: ATR; Caveolin-1.; Chromosome instability; Fragile sites; Premature aging; Progeroid syndromes; Replication stress; Telomeres; Werner syndrome; p53.

Figure 1.

Growth characteristics of ataxia-telangiectasia and rad3 (ATR)-related Seckel and normal dermal fibroblast (NDF) strains with or without p38 inhibitor treatment. (A) GM18366 cells, (B) AG06234 cells, and (C) AG16409 cells. Fibroblasts were grown in Earle’s Modified Eagle medium (EMEM) supplemented with 0.1% (v/v) dimethyl sulfoxide (DMSO; ○) or the p38 inhibitors VX-745 (■), SB203580 (□), and BIRB 796 (●). Growth is measured as population doublings (PDs) vs days. Observation of F-actin stress fibers in (D–G) ATR-Seckel cells, (H) AG16409 cells, and (I) ATR-Seckel cells at M1. The conditions for growth are indicated on the panels. Bar = 100 μm.

Figure 2.

Immunoblot analysis of p38 and HSP27. Protein lysates were prepared from GM18366 (ataxia-telangiectasia and rad3-related Seckel [ATR-S]) and AG16409 (normal dermal fibroblast strains [NDFs]) cells. Expression levels were compared for phosphorylated p38 (p-p38), p38, phosphorylated HSP27 (p-HSP27), and HSP27. Loadings were normalized for p38. For each series A and B, a control lane has been added (with ATR-S used as control for NDFs and vice versa). Lanes are D, dimethyl sulfoxide (DMSO); VX, VX-745; SB, SB203580; B, BIRB 796. “An” are AG16409 cells treated with 30 μM anisomycin for 45min to activate p38 and phosphorylate HSP27. The bands marked with arrows in lanes VX and B of A are nonspecific bands that are also seen in B.

Figure 3.

Immunoblot analysis of cell cycle proteins and caveolin-1. Protein lysates were prepared from low population doubling (PD) GM18366 (ataxia-telangiectasia and rad3-related Seckel [ATR-S]), AG16409 (normal dermal fibroblast strains [NDFs]), and AG05229 (Werner syndrome [WS]) cells. Expression levels were compared for p16^INK4A, p21^WAF1, phospho-caveolin-1 (p-cav), caveolin-1 (cav), phospho-cofilin (p-cof), cofilin (cof), and MK2. Actin is used as loading control for each panel. Lanes are D, dimethyl sulfoxide (DMSO); VX, VX-745; SB, SB203580; and B, BIRB 796. The horizontal arrow indicates the 24kDa p-cav band and the diagonal arrow indicates activated MK2. The vertical line in C indicates that the single lanes have been cut and pasted from the same gels as the rest of the samples, however, the images have been handled in the same manner otherwise.

Figure 4.

Effects of p53 abrogation and ectopic hTert expression on the growth of ataxia-telangiectasia and rad3 (ATR)-related Seckel cells. Growth in population doublings (PDs) vs days for (A) GM18366^p53 cells, (B) GM18366^hTert cells, and (C) GM18366hTert cells treated with p38 inhibitors as indicated. In B and C, the arrow indicates the point at which inhibitor treatment of GM18366^hTert began. Observation of F-actin stress fibers in (D) GM18366^p53 cells at M^int and (E) GM18366hTert cells. Bar = 100 μm. (F) Immunoblot analysis for GM18366 cells (proteins analyzed as for Figures 2 and 3; the arrow on the p-p38 row shows activated p38). Cy = low PD cycling cells; M1 = cells at senescence; p53 = GM18366 abrogated for p53; hTert = GM18366 cells infected with hTert.