ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation

Abstract

Bloom's syndrome (BS), a rare genetic disease, arises through mutations in both alleles of the BLM gene which encodes a 3' ± 5' DNA helicase identi®ed as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer a ecting the general population and BLM-de®cient cells display a strong genetic instability. We recently showed that BLM protein expression is regulated during the cell cycle, accumulating to high levels in S phase, persisting in G2/ M and sharply declining in G1, suggesting a possible implication of BLM in a replication (S phase) and/or post-replication (G2 phase) process. Here we show that, in response to ionizing radiation, BLM-de®cient cells exhibit a normal p53 response as well as an intact G1/S cell cycle checkpoint, which indicates that ATM and p53 pathways are functional in BS cells. We also show that the BLM defect is associated with a partial escape of cells from the g-irradiation-induced G2/M cell cycle checkpoint. Finally, we present data demonstrating that, in response to ionizing radiation, BLM protein is phosphorylated and accumulates through an ATMdependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream e ector.

The Role of the BRCA1 Tumor Suppressor in DNA Double-Strand Break Repair

“…Whether BRCA1 inhibits BLM function, perhaps predicted by this model, is not clear. Similarly to BRCA1, BLM is phosphorylated by ATM (48,49) and forms foci with Rad51 after ionizing radiation exposure (50). The functional consequence of the ATM-dependent phosphorylation of BLM is not known, although a BLM phosphorylation mutant does not complement the sister chromatid exchange defect in Bloom's syndrome cells (51).…”

Cleavage of BLM and Sensitivity of Bloom’s Syndrome Cells to Hydroxurea

“…The EBV-transformed lymphoblastoid B cell lines GM03403D, IPM1 and D1, Ramos Burkitt Lymphoma cells and K562 erythroleukemia cells were used as previously described. 7 Asynchronous cells were split 24 h prior to the experiments and seeded at a density of 4 x 105 cells/ml. The cells were exposed to UVC light (254 nm, 50J/m 2 ) with a germicidal UV lamp (Fisher Bioblock Scientific) or treated with 2 mM hydroxyurea (Sigma), and then returned to 37˚C for the indicated times.…”

“…13 These data, together with our results showing that BLM protein is modified by phosphorylation through an ATM-dependent pathway in response to ionizing radiation and through an ATM-independent pathway in response to UVC irradiation and HU treatment, prompted us to analyze BLM expression and BS cell sensitivity in response to apoptosis induction by UVC or HU. 7,14 …”

“…3 However, several lines of evidence strongly support an essential role for BLM during DNA replication and in the cellular response to DNA damage. [4][5][6][7][8][9] Recent data have shown that BLM protein is cleaved to 40-47 kDa N-terminal and 110-120 kDa C-terminal major fragments by caspase-3 in response to several apoptosis-inducing agents such as anti-Fas antibody and etoposide. 10,11 It has also been shown that p53-mediated apoptosis is defective in fibroblasts from BS patients and that lymphoblastoid cells derived from BS patients are resistant to both γ-radiation and doxorubicin-induced cell killing.…”

Syndrome de Bloom : hétérozygotie et prédisposition au cancer

“…Ces résultats ont été reproduits par l'équipe de Joanna Groden dans un modèle murin [2]. En effet, cette équipe a développé des souris chez lesquelles une copie de l'homologue murin du gène BLM a été invalidée, produisant ainsi un allèle mutant Blm Cin [7] dans des mécanismes de surveillance du génome [8], de réso-lution de structures anormales de l'ADN [9,10], et/ou de réparation de lésions de l'ADN, notamment en réponse à des stress génotoxiques [11,12]. Si la protéine BLM est impliquée dans de tels processus au sein de complexes protéiques, et dans le cadre d'interactions stoechiométriques bien déterminées, alors une réduction de la quantité de protéine BLM pourrait être délétère pour la cellule.…”