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.
Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (⌬TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of ⌬TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for ⌬TK-or TK-expressing cells, both in vitro and in transgenic mice. However, ⌬TK behaved differently from TK in two ways. First, it did not cause sterility in ⌬TK transgenic males. Second, low-level ⌬TK RNA expression did not confer sensitivity to GCV. The uses of ⌬TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.The herpes simplex virus type 1 thymidine kinase (TK) gene has gained considerable importance in clinical medicine and as a research tool. TK can substitute for cellular thymidine kinase in the metabolic pathway of thymidine, and eukaryotic cells lacking functional TK can be rescued from culture in hypoxanthine-aminopterin-thymidine (HAT) selection medium by TK expression (44). Furthermore, TK can phosphorylate certain nucleoside analogs that are not metabolized by cellular enzymes (21). This property has allowed the discovery of nucleoside analogs such as acyclovir and ganciclovir (GCV) that possess strong activity against herpesvirus infections (19,21). This same property turned out to be useful for negative selection of TK-expressing cells. Phosphorylated-nucleoside analogs such as acyclovir triphosphate or GCV triphosphate are potent toxic metabolites for dividing cells. They are incorporated into elongating DNA by cellular DNA polymerase and induce chain termination and eventually cell death (20,36,42).This conditional toxicity of TK can be utilized in vivo. TKmediated destruction of undesirable cells is being developed for gene therapy of cancer or human immunodeficiency virus (HIV) infection (10,11,15,36). Effectiveness has been demonstrated in animal models, and clinical trials for cancer gene therapy have already been initiated (14). In addition, Heyman et al. developed the concept of TK obliteration, the ablation of a specific cell type in transgenic mice (26). This conditional cell knockout is obtained by expressing TK with a cell-specific promoter. This system offers the valuable advantag...
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