Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS 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. We developed polyclonal antibodies speci®c for the NH 2 -and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/ M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modi®cations in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process.
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.
Bloom's syndrome (BS) arises through mutations in both copies of the BLM gene that encodes a RecQ 3'-5' DNA helicase. BS patients are predisposed to developing all the cancers that aect the general population, and BS cells exhibit marked genetic instability. We showed recently that BLM protein contributes to the cellular response to ionizing radiation by acting as downstream ATM kinase eector. We now show that following UVC treatment, BLM-de®cient cells exhibit a reduction in the number of replicative cells, a partial escape from the G2/ M cell cycle checkpoint, and have an altered p21 response. Surprisingly, we found that hydroxyureatreated BLM-de®cient cells exhibit an intact S phase arrest, proper recovery from the S phase arrest, and intact p53 and p21 responses. We also show that the level of BLM falls sharply in response to UVC radiation. This UVC-induced reduction in BLM does not require a functional ATM gene and does not result from a subcellular compartment change. Finally, we demonstrate that exposure to UVC and hydroxyurea treatment both induce BLM phosphorylation via an ATM-independent pathway. These results are discussed in the light of their potential physiological signi®cance with regard to the role of BLM in the cellular pathways activated by UVC radiation or HU-mediated inhibition of DNA synthesis.
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