Inhibiting homologous recombination by targeting RAD51 protein.

Homologous recombination (HR) is involved in repairing DNA double-strand breaks (DSB), the most harmful for the cell. Regulating HR is essential for maintaining genomic stability. In many forms of cancer, overactivation of HR increases tumor resistance to DNA-damaging treatments. RAD51, HR's core protein, is very often over-expressed in these cancers and plays a critical role in cancer cell development and survival. Targeting RAD51 directly to reduce its activity and its expression is therefore one strategy to sensitize and overcome resistance cancer cells to existing DNA-damaging therapies which remains the limiting factor for the success of targeted therapy. This review describes the structure and biological roles of RAD51, summarizes the different targeted sites of RAD51 and its inhibitory compounds discovered and described in the last decade.

Mariner transposons as genetic tools in vertebrate cells.

Transposable elements (TEs) are being investigated as potential molecular tools in genetic engineering, for use in procedures such as transgenesis and insertional mutagenesis. Naturally active and reconstructed active TEs are both being studied to develop non-viral delivery vehicles. To date, the active elements being used include three Mariner-Like Elements (MLEs). We review below the studies that have investigated the ability of these MLEs to insert a transgene in vertebrate cells.

Characterization and sequence analysis of manganese superoxide dismutases from Brachyura (Crustacea: Decapoda): hydrothermal Bythograeidae versus littoral crabs.

Hydrothermal vent conditions are particular and organisms living in these environments may have developed detoxification mechanisms and/or genetic adaptations. In particular, physico-chemical conditions are thought to generate reactive oxygen species, highly toxic for organisms. The enzyme superoxide dismutase constitutes the first line of defense against oxidative damage. To improve our understanding of the environmental impacts exerted on the vent organisms, we have characterized the two manganese superoxide dismutase cDNAs (mitochondrial: mMnSOD and cytoplasmic: cMnSOD) of three members of the Bythograeidae (Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica), the only endemic crab family living in hydrothermal vents. In comparison, the isolation of manganese superoxide dismutase cDNAs was also carried out in several littoral crab families. MnSOD signatures were found in both sequences from each species studied, as well as different residues involved in metal coordination and protein activity. The phylogenetic analysis performed confirms the probable ancient duplication that gave rise to the two MnSODs (cMnSOD and mMnSOD). This study describes two potential distinct mMnSOD isoforms presenting particular peptide signals. Nevertheless, no sequence particularity that could support the hypothesis of a genetic adaptation was found in Bythograeidae's MnSODs compared to the other sequences. The mRNA expression analysis performed by real-time PCR on B. thermydron and S. mesatlantica compared to Cancer pagurus and Necora puber revealed a higher cMnSOD and mMnSOD mRNA expression in hydrothermal crabs compared to littoral crabs.

Widespread occurence of mariner transposons in coastal crabs.

Mariner-like elements (MLEs) are ubiquitous DNA mobile elements found in almost all eukaryote genomes. Nevertheless most of the known copies are inactive and the question of the genome invasion by MLEs remains largely hypothetical. We have previously reported the presence of highly homologous copies of MLEs in the genome of phylogenetically distant crustacea living in the same hydrothermal environment suggesting the possibility of horizontal transfer. In order to further support the hypothesis that horizontal transmission of MLEs might occur between crustacean sympatric species, we described here 85 MLE sequences found in the genome of a large spectrum of coastal crab species. The number of the MLEs copies in genomes was variable. Half of these MLEs fit with the irritans subfamily of MLEs whereas the second half grouped in a new subfamily called marmoratus. In addition, a molecular phylogeny of crabs was established by using the 16S information. The comparison between 16S and MLEs based trees reveals their incongruence, and suggests either the existence of horizontal transfer events between phylogenetically distant species, or an ancestral MLE polymorphism followed by different evolution and stochastic loss.

Oxidative stress involvement in chemically induced differentiation of K562 cells.

The erythroid differentiation of K562 cells could be achieved by exposure to several pharmacologic agents, including hemin, butyric acid (BA), and anthracycline antitumor drugs such as aclarubicin (ACLA) and doxorubicin (DOX). When used at subtoxic concentrations, these drugs induce the overexpression of erythroid genes, leading to hemoglobinization of cells. Because anthracyclines are known to generate oxidative damage, we intended to demonstrate the involvement of an oxidative stress in the chemically induced differentiation process. The addition of antioxidants to anthracycline- and BA-induced cells decreased their growth and dramatically reduced the percentage of differentiated cells at day 3. Northern blot analysis showed that antioxidants also decrease the expression of erythroid genes and related transcription factors in induced cells. Moreover, analyses of oxidative stress markers showed that treatment with BA, ACLA, and DOX lead to a decrease in reduced glutathione and antioxidant enzymes (glutathione peroxidase [GPx], glutathione reductase [GRase], CuZn superoxide dismutase [SOD], and catalase [CAT]). In addition, DOX increased thiobarbituric acid reactants (TBARs), and MnSOD activity was decreased by BA and DOX. Finally, the production of reactive oxygen species (ROS) by differentiating agents was demonstrated using the dihydroethidium probe in a microspectrofluorometric assay. Altogether, these results strongly suggest the involvement of an oxidative stress generated by BA or anthracyclines as the first step in the irreversible differentiation process. Additionally, these results underline the differences between BA, ACLA, and DOX molecular mechanisms.

Inhibitory effect of nitric oxide on chemically induced differentiation of human leukemic K562 cells.

The effect of nitric oxide (NO) was investigated in the human K562 cell line during chemically induced erythroid differentiation. Butyric acid (BA) and the anthracycline antitumour drugs aclarubicin (ACLA) and doxorubicin (DOX) were used as differentiating agents. In all cases, cell hemoglobinization was dose dependently inhibited by NO donors such as sodium nitroprusside (SNP). A 50% inhibition of cell differentiation was obtained with 25 microM SNP, which generated less than 2 microM nitrite in 3-day culture media. Increasing SNP concentrations led to higher nitrite accumulation (up to 12 microM with 1 mM SNP) and total inhibition of cell hemoglobinization, but did not have a significant effect on cell proliferation. As shown by Northern blotting, high concentrations of SNP (1 mM) reduced the expression of gamma-globin and porphobilinogen deaminase, but did not change GATA-1 and NF-E2 mRNA levels in ACLA- and BA-treated cells. In contrast, hemin-induced erythroid differentiation was not affected by the presence of NO donors. Altogether, these results show that NO is able to inhibit cell differentiation induced by some (ACLA, DOX, BA), but not all (hemin), agents. The inhibitory effect of NO seems to take place downstream of the regulation of erythroid gene expression.

Requirement of GATA-1 and p45 NF-E2 expression in butyric acid-induced erythroid differentiation.

Butyric acid (BA) is known to induce overexpression of fetal hemoglobin and then erythroid differentiation. Therefore, BA is currently under clinical investigation as a potential therapy for the treatment of sickle cell disease and cancer. Nevertheless, the molecular mechanisms involved in BA-induced differentiation remain largely unknown. Previous reports have shown that BA-induced overexpression of erythroid genes occurred at the transcriptional level, suggesting the involvement of erythroid transcription factors. Here, we intend to demonstrate the requirement of GATA-1 and NF-E2 transcription factors in the BA-induced erythroid differentiation of human leukemic K562 cells. Time-course experiments showed that nuclear levels of GATA-1 and p45 NF-E2 proteins increased during BA treatment. Moreover, antisense oligodeoxynucleotides targeting either GATA-1 or p45 NF-E2 proteins inhibited both protein expression and BA-induced differentiation. In contrast, BA-induced cell growth inhibition was not affected. These results provide the first direct evidence for the requirement of GATA-1 and NF-E2 in BA-induced differentiation process.

Time-course of butyric acid-induced differentiation in human K562 leukemic cell line: rapid increase in gamma-globin, porphobilinogen deaminase and NF-E2 mRNA levels.

Butyric acid (BA) was shown to induce hemoglobinization of K562 cells in a dose- and time-dependent manner. The maximal differentiation (54% of hemoglobinized cells) was obtained with the 0.5 mM concentration, which induced a 60% inhibition of cell growth at day 3 without cytotoxicity. Parallel to the kinetics of hemoglobinization, a rapid increase in gamma-globin and porphobilinogen deaminase (PBGD) mRNAs was observed in BA-treated cells. This increase was time-dependent and higher for gamma-globin than for PBGD (six- and two-fold at day 3, respectively). In contrast, erythropoietin receptor mRNAs were not affected by BA treatment. Analysis of erythroid transcription factor mRNA levels during the time course of BA treatment showed, for the first time, an early and marked (up to three-fold) increase in p45 NF-E2 mRNA, contrasting with that of GATA-1 mRNA (<1.5-fold). Taken together, these results showed the rapid differentiating effect of BA and suggest the involvement of the NF-E2 transcription factor.

Anthracyclines as tumor cell differentiating agents: effects on the regulation of erythroid gene expression.

Tumor cells, and particularly leukemic cells, can be considered as maturation-arrested cells which have escaped some normal control and continue to proliferate. This maturation arrest can be reversed by differentiation agents such as antitumor drugs currently used in conventional cytotoxic chemotherapy. In this respect, anthracyclines have been shown to trigger the differentiation of leukemic and solid tumor cells, but the molecular mechanisms by which such drugs lead to the differentiating phenotype are still poorly understood. Using human leukemic multipotent K562 cells, we have demonstrated that subtoxic concentrations of aclacinomycin (ACLA) and doxorubicin (DOX) preferentially stimulate the hemoglobinic pathway (globins and heme synthesis) and the expression of mRNAs of globins and of porphobilinogen deaminase (PBGD). However, our results indicate that both drugs exert this differentiating effect along distinct regulatory pathways. Indeed, only ACLA and not DOX induces the expression of erythropoietin receptor (EpoR) mRNAs and of membrane EpoR, as well as an overexpression of the erythroid transcription factors GATA-1 and NF-E2 known to play a central role in erythroid gene regulation. Similarly, using transfection assays, ACLA but not DOX activates the regulatory regions (promoters and enhancers) of GATA-1, EpoR, PBGD, epsilon- and gamma-globin genes. Finally, results of run-on assays indicate that ACLA induces an enhancement of the transcription rate of these erythroid genes whereas DOX preferentially increases stability of GATA-1, NF-E2 and PBGD mRNAs. In conclusion, ACLA mainly acts at the transcriptional level via specific activation of erythroid regulatory regions whereas DOX rather acts at the posttranscriptional level by increasing the half-lives of erythroid mRNAs.

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome.

To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of L-NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFN gamma + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages, from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by L-NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.

Transcriptional and posttranscriptional regulation of erythroid gene expression in anthracycline-induced differentiation of human erythroleukemic cells.

Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX.

Involvement of nitric oxide synthase in antiproliferative activity of macrophages: induction of the enzyme requires two different kinds of signal acting synergistically.

Activated rodent macrophages inhibit micro-organism and tumour cell growth through a high output of nitric oxide; generated by an isoform of nitric oxide synthase which is induced, for example, in murine macrophages, by concomitant stimulation with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). We show here that LPS could be replaced as a co-stimulant by the mycobacterial derivative muramyl dipeptide (MDP) in macrophages, and by interleukin-1 (IL-1) in EMT-6 adenocarcinoma cells. Moreover, our results indicate that nitric oxide synthase RNA synthesis required either simultaneous or sequential exposure to IFN-gamma and MDP/IL-1; whereas exposure to MDP/IL-1 followed by exposure to IFN-gamma was ineffective. Thus, two kinds of signal could be distinguished: IFN-gamma on the one hand, acting first in an irreversible way, and LPS, MDP, IL-1 on the other hand, which seemed to be permanently required for continuous transcription of the nitric oxide synthase gene.

N omega-hydroxy-L-arginine, a reactional intermediate in nitric oxide biosynthesis, induces cytostasis in human and murine tumor cells.

Conversion of L-arginine to L-citrulline and nitric oxide (NO) by NO synthase induced in the murine EMT-6 cells resulted in the release of a large amount of the stable reactional intermediate N omega-hydroxy-L-arginine into the extracellular medium. We have prepared [3H]N omega-hydroxy-L-arginine biosynthetically, and shown that, after its uptake, this molecule can induce cytostasis in NO synthase-deficient P-815 and U-937 tumor cells. This long-lived intermediate could behave as a supplier of NO or other toxic molecules in cell-cell interactions.

High-performance liquid chromatographic analysis of the unusual pathway of oxidation of L-arginine to citrulline and nitric oxide in mammalian cells.

A very unusual pathway of the oxidation of L-arginine to citrulline and nitric oxide has been discovered recently in cytotoxic macrophages. In an attempt to detect molecules generated through this metabolic pathway, a fast radio high-performance liquid chromatographic method was developed to analyse the whole set of radiolabelled L-arginine-derived metabolites produced by mammalian cells after appropriate induction. A new intermediate which might be NG-hydroxy-L-arginine was found.

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells.

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.