Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase.

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 identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- 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 modifications 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. Oncogene (2000).

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

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 identified as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer affecting the general population and BLM-deficient 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-deficient 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 gamma-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 ATM-dependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream effector.

Prevention of graft-versus-host disease in mice using a suicide gene expressed in T lymphocytes.

Alloreactive T cells present in a bone marrow transplant are responsible for graft-versus-host disease (GVHD), but their depletion is associated with impaired engraftment, immunosuppression, and loss of the graft-versus-leukemia effect. We developed a therapeutic strategy against GVHD based on the selective destruction of these alloreactive T cells, while preserving a competent T-cell pool of donor origin. We generated transgenic mice expressing in their T lymphocytes the Herpes simplex type 1 thymidine kinase (TK) suicide gene that allows the destruction of dividing T cells by a ganciclovir treatment. T cells expressing the TK transgene were used to generate GVHD in irradiated bone marrow grafted mice. We show that a short 7-day ganciclovir treatment, initiated at the time of bone marrow transplantation, efficiently prevented GVHD in mice receiving TK-expressing T cells. These mice were healthy and had a normal survival. They maintained a T-cell pool of donor origin that responded normally to in vitro stimulation with mitogens or third party alloantigens, but were tolerant to recipient alloantigens. Our experimental system provides the proof of concept for a therapeutic strategy of GVHD prevention using genetically engineered T cells.

[Gender control and Olympics events from Albertville to Atlanta or good practices in gene biochemistry]. / Le contrôle de genre et les jeux olympiques d'Albertville à Atlanta ou du bon usage de la biochimie génique.

Gender verification in sport was introduced in 1967 based on the insufficient Barr test. For Albertville's Olympic Games we proposed the Medical Commission of IOC to carry out the control for the first time thanks to molecular biochemistry. PCR/SRY amplification is described giving more than 99.9% of good results. After Albertville, Barcelona and Lillehammer the method will be used in Atlanta and Nagano. Results and ethics are discussed. Easy and respectful of athletes, this reliable gender verification is indispensable for their protection. According to the good practices of CM/IOC, samples have to be discarded after the analysis is done.

A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice.

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 (delta TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of delta TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for delta TK- or TK-expressing cells, both in vitro and in transgenic mice. However, delta TK behaved differently from TK in two ways. First, it did not cause sterility in delta TK transgenic males. Second, low-level delta TK RNA expression did not confer sensitivity to GCV. The uses of delta TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved or bended helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy.

Pleiotropic derepression of developmentally regulated cellular and viral genes by c-myc protooncogene products in undifferentiated embryonal carcinoma cells.

We show here in mouse embryonal carcinoma (EC) cells that the endo A gene is negatively regulated and shares negative transacting factors with the Py and SV40 viruses. The products of the proto-oncogene c-myc derepress at the transcriptional level the appropriately initiated expression of the endo A gene and activate the Py early promoter in EC stem cells. C-myc products also activate the endo A and the Py early promoters in TDM epithelial cells, and the Py early promoter in 3T6 cells in which the two genes are already expressed or can be expressed. Furthermore we show that the myc exon 1 is essential for activation and that this activation might be mediated by AP1 family factors.

Exogenous c-myc gene overexpression interferes with early events in F9 cell differentiation.

The effect of c-myc gene expression on F9 teratocarcinoma stem cell differentiation was studied by transfecting mouse metallothionein I promoter-driven human c-myc genes into F9 cells. Human c-myc gene expression was triggered by cadmium treatment at different stages of differentiation. When this expression was induced before or just after addition of retinoic acid plus cyclic AMP (RA +cAMP), the death of 70-80% of the cells was observed. This cell mortality was not observed either when the parental F9 cells were induced to differentiate in the presence of cadmium or when the undifferentiated transfected clone was permanently grown in the presence of this inducer. The 30% surviving cells underwent terminal differentiation despite high c-myc mRNA levels and expressed differentiation-specific genes normally like their parental F9 cells. When cadmium was added 24 or 48 hr after RA +cAMP, no cell mortality or any other detectable effect on differentiation were observed, even though c-myc gene transcripts were induced. We therefore conclude that c-myc gene expression only inhibits F9 differentiation at an early stage in 70-80% of cells. The significance of these results will be discussed.

c-myc products trans-activate the adenovirus E4 promoter in EC stem cells by using the same target sequence as E1A products.

Using a short-term transfection assay, we show that the E4 early adenovirus promoter is expressed to a certain extent in undifferentiated F9 and PCC4 cells, which are known to possess cellular E1A-like activity. We have also observed that c-myc products trans-activate the E4 promoter in EC stem cells and HeLa cells. Using 5' deletion mutants of the E4 promoter, we show that the same target sequence is used by c-myc and E1A. This sequence is located between positions -179 and -158 upstream of the cap site and is known to contain an activating transcription factor (ATF)-binding site. Moreover, the basal of level of activity of the deletion mutants. is related to the number of ATF binding sites. We therefore suggest that c-myc is a functional cellular homolog of the viral E1A gene and that it might correspond to one of the cellular E1A-like activities previously described for EC stem cells. We have also observed that only a c-myc plasmid coding for both p67 and 64 proteins, in contrast to one coding for p64 only, is able to trans-activate the E4 and E2A adenovirus promoter, suggesting that the p67 protein plays an essential part in activation.