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. 2007 Apr 14;145(4):1300-8.
doi: 10.1016/j.neuroscience.2006.08.074. Epub 2006 Oct 19.

Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair

J E Cleaver  1 , E Hefner, R R Laposa, D Karentz, T Marti
Affiliations

Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair

J E Cleaver et al. Neuroscience. .

Abstract

Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth.

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Figures

Figure 1
Figure 1
Induction of p53 in normal and CS cells by 10 or 20 J.m−2 UV light; western blot (antibody SC126) of cells harvested 16 hr after UV irradiation.
Figure 2
Figure 2
Western analysis of p21 (antibody: SX118 mouse IgG, epitope last 20 aa) as a function of salt concentration. Upper band a high molecular weight aggregate that moved slightly below the loading well; lower band normal size p21. Salt concentration left to right: 50, 150, 250, and 500 mM.
Figure 3
Figure 3
A. Western analysis of p21 (antibody: SX118 mouse IgG, epitope last 20 aa) in lymphoid cells: normal (PP034), CS-B (GM10902, GM1712 respectively) and XP-A (GM2345) grown in 15% serum. Right panel shows fibroblasts from normal and CSB (GM10903). B. Histogram showing the ratios of p21 expression levels in two CSB cell lines GM10902 and GM01712 as compared to the normal PP034 cell line. Error bars represent the standard errors of the means.
Figure 4
Figure 4
A. Complementation of CS-B lymphoblast with cDNA for CSB corrects the UV sensitivity of CSB cells. control; CS-B; CS-B corrected with cDNA clone 1; CS-B corrected with cDNA clone 2. B. Complementation of CS-B lymphoblast with cDNA for CSB corrects the over-abundance of p21. Upper bands p21, lower bands βactin loading controls. Wt lane PP034 cells, CS-B lane GM10903, cDNA correction of GM10903 plus cDNA, clone 1, transfected and selected for continuous expression.
Figure 5
Figure 5
Relative levels of p21 in normal and CSB lymphoblastoid cells (GM10902) as a function of time in cycloheximide (50microgram/ml). Primary mouse monoclonal anti-p21 (1:250 dilution), secondary horse-radish peroxidase anti-mouse (1/2000 dilution). Values normalized to the ambient level in each cell type without cycloheximide.
Figure 6
Figure 6
CS-A and CS-B cells show increased DNA breakage, as determined by γH2AX foci formation, when grown under normal ambient oxygen. Values represent the standard errors of the means.
Figure 7
Figure 7
Western blots from normal and CSB fibroblasts showing over-abundance of Hsp90 protein; normal (PPO34) and CSB (GM10903) lymphoblasts.
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