Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


The Integrity of Double Strand Break Repair in Pediatric Systemic Lupus Erythematosus Cells.

Davies2,  Robert C., Pettijohn2,  Kelly, Fike2,  Francesca, Wang2,  Jiexi, Nahas2,  Shareef A., Gatti3,  Richard A., McCurdy1,  Deborah K.

Mattel Children's UCLA-Rheumatology, Los Angeles, CA
UCLA-Pathology and Laboratory Medicine
UCLA-Pathology and Laboratory Medicine; Human Genetics

Introduction:

Our laboratory previously demonstrated a delay in single strand break (SSB) processing in cells from pediatric patients with systemic lupus erythematosus (SLE). Because SSBs are often converted to double strand breaks (DSBs) at the replication fork and some DSB repair proteins are immnunogenic in SLE, i.e., DNA ligase IV, Ku 70/80, DNAPKcs, and XRCC4, we assessed the integrity of DSB recognition, signaling, and repair mechanisms in B-lymphoblastoid cell lines (LCLs) derived from pediatric and adolescent patients with SLE. This study assesses the integrity of DSB recognition, signaling, and repair mechanisms in LCLs derived from 16 pediatric and adolescent patients with SLE (pSLE).

Methods:

Lymphoblastoid cells lines (LCLs) were established from the whole blood of 16 pSLE patients. Eight assays were used to assess four major pathways of repair of DSBs in pSLE LCLs. The assays included: (i) g-H2AX and (ii) 53BP1 IR-induced nuclear foci (IRIF); (iii) immunoblot analysis of the kinetics of IR-induced phosphorylation of Structural Maintenance of Chromosomes 1 (SMC1); (iv) a DNA ligation assay to evaluate the NHEJ (v) neutral comet assay (vi) monoubiquitination of FANCD2 (vii) flow cytometry to assess the S-phase checkpoint; and (viii) colony survival assay as a measure of general radiosensitivity.

Results:

Three of eight assays showed abnormal patterns of response to IR-induced DNA damage in some patients: 1) pSMC1 kinetics were intermediate in four patients, suggests defective repair, 2) the neutral comet assay was abnormal in half of the cells, showing delayed DSB repair, 3) colony survival assay showed radiosensitivity in half of the cell lines tested. We also observed reduced DNAPKcs, Ku70 and Ku80 proteins in one pSLE patient.

Conclusion:

Our data suggest that DSB repair is compromised in pSLE LCLs as shown in the broad-based DSB repair assays (neutral comet assay, colony survival assay). This contrasts with results from our functional assays (g-H2AX, 53BP1, NHEJ, FANCD2 monoubiquitination, S-phase checkpoint), which all appear normal. Although the majority of SLE cell lines exhibited DSB repair defects, the etiology of this remains unclear.

To cite this abstract, please use the following information:
Davies, Robert C., Pettijohn, Kelly, Fike, Francesca, Wang, Jiexi, Nahas, Shareef A., Gatti, Richard A., et al; The Integrity of Double Strand Break Repair in Pediatric Systemic Lupus Erythematosus Cells. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :509
DOI: 10.1002/art.28278

Abstract Supplement

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