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Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype¹

MARTIN POOT², KATHERINE A. GOLLAHON, MARY J. EMOND^*, JOHN R. SILBER and PETER S. RABINOVITCH

Departments of Pathology,
^* Biostatistics and
Neurological Surgery, University of Washington, Seattle, Washington, USA

²Correspondence: Department of Pathology, University of Washington, Box 357 705, Seattle WA 98195-7705, USA. E-mail: mpoot{at}u.washington.edu

SPECIFIC AIMS

To investigate the role of Werner syndrome (WRN) helicase/exonuclease in the mesenchymal phenotype of Werner syndrome, we compared the response of primary cultures of diploid fibroblasts from WRN patients with cells from unrelated donors with WRN wild-type alleles to drugs that damage DNA. WRN-/- fibroblasts were compared to WRN+/+ fibroblasts and to WRN-/- and WRN+/+ lymphoblastoid cells with respect to decrease in proliferative survival and induction of apoptosis in response to DNA-damaging drugs.

PRINCIPAL FINDINGS

1. Differential sensitivity of WRN-/- vs. WRN+/+ fibroblasts to 4-nitroquinoline-N-oxide
WRN-/- lymphoblastoid cells (LCLs) respond to 4-nitroquinoline-N-oxide (4NQO) exposure with S phase-specific apoptosis and differential loss of proliferative survival. In pairwise comparisons, primary cultures of WRN-/- diploid fibroblasts showed greater decreases in proliferative survival than did WRN+/+ diploid fibroblasts after exposure to 4NQO (Fig. 1 ). This is the first demonstration of a drug sensitivity phenotype in a primary, mesenchymal cell type from patients with Werner syndrome.

View larger version (25K): [in this window] [in a new window]   Figure 1. Proliferative survival (A–C) and cell death (D–F) of diploid fibroblasts from three WRN patients (filled symbols) and three unaffected individuals (open symbols) previously matched according to proliferative potential. A, D) Strain AG 00780G (WRN) and strain 78–89 (unaffected); B, E) strain AOMOR 1010 (WRN) and strain 78–18 (unaffected); C, F) strain 73–26 (WRN) and strain 82–6 (unaffected). Error bars indicate the SD of triplicate determinations.

2. Differential sensitivity of WRN-/- vs. WRN+/+ LCLs and fibroblasts to induction of DNA interstrand cross-links
Exposure of cells to 4NQO may induce a variety of DNA lesions. To define the type of chromatin lesion(s) that leads to 4NQO sensitivity of WRN-deficient cells, we exposed LCLs to 8-methoxypsoralen (8MOP), followed by single and double UV irradiation. Exposure to 8MOP, followed by a single UV irradiation, leads to the formation of psoralen-DNA monoadducts that, after a second round of UV irradiation, are converted into DNA interstrand cross-links. WRN-/- LCLs from three different families exhibited stronger decreases in proliferative survival in response to DNA interstrand cross-links induced by 8MOP exposure followed by double UV irradiation than did WRN+/+ siblings from the same families (Fig. 2 A–C). Primary cultures of WRN-/- diploid fibroblasts vs. cultures of WRN+/+ fibroblasts from unrelated donors, but matched for initial proliferation rates, showed stronger decreases in proliferative survival than did WRN+/+ diploid fibroblasts after exposure to 8MOP, followed by double but not single UV irradiation (Fig. 2D-F ). This indicates that primary fibroblasts from WRN patients are sensitive to DNA interstrand cross-links.

View larger version (25K): [in this window] [in a new window]   Figure 2. : Proliferative survival of LCLs and diploid fibroblasts treated with 8MOP. A–C) The decrease in proliferative survival of LCLs from WRN patients (filled squares) and unaffected siblings (open squares) from SY, TUR, and SYR families, respectively, after 8MOP treatment. D–F) Proliferative survival of diploid fibroblasts from three WRN patients (filled symbols) and three unaffected individuals (open symbols). D) Strain AG 00780G (WRN) and strain 78–89 (unaffected); E) strain AOMOR 1010 (WRN) and strain 82–6 (unaffected); F) strain 73–26 (WRN) and strain 71–95 (unaffected). Error bars indicate the SD of triplicate determinations.

3. No induction of apoptosis in WRN-/- and WRN+/+ fibroblasts in response to 4-nitroquinoline-N-oxide
WRN-/- LCLs responded to 4NQO and 8MOP exposure with apoptosis from the S phase of the cell cycle. Cultures of WRN-/- diploid fibroblasts, in contrast, do not die in response to 4NQO (Fig. 1D-F ) or 8MOP (results not shown). This means that WRN-/- diploid fibroblasts are not eliminated and may persist after they have sustained DNA damage.

CONCLUSIONS

We found that WRN-deficient primary fibroblasts and LCLs show reduced proliferative survival in response to 4NQO and to 8MOP when compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand cross-links, but not 8MOP mono-adducts, produced S phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S phase apoptosis in WRN-deficient diploid fibroblasts in which drug hypersensitivity was due entirely to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S phase may promote genomic instability and help explain the increased risk of sarcoma in WRN patients. Since interstrand cross-links are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

View larger version (33K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0906fje; to cite this article, use FASEB J. (March 12, 2002) 10.1096/fj.01-0906fje

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