Isolation and characterization of HC1: a novel human DNA repair gene.

Nucleotide excision repair (NER) acts on a broad spectrum of large lesions, while base excision repair removes individual modified bases. Although both processes have been well studied in human cells, novel genes involved in these DNA repair pathways have been described. Using a heterologous complementation approach, we identified a fetal human cDNA that complemented two Escherichia coli mutants that are defective in 3-methyl adenine glycosylase and in three endonucleases, all of which are enzymes with important roles in base excision repair. The central cDNA open reading frame complemented NER mutant strains and promoted an increase in survival rate of bacteria exposed to UV light. The corresponding protein was able to restore nucleotide-excision-repair activity when added to a cell extract from Chinese hamster ovary cells deficient in the ERCC1 protein, an enzyme known to promote incision at the 5' end of the lesion during NER. In contrast, that protein was not able to complement XPG Chinese hamster ovary cells deficient in the 3' incision step of NER. These data indicate a new human repair gene, which we named HC1; it is involved in the recognition of two kinds of DNA lesions and it contributes to the 5' DNA incision step in NER.

Characterization of promoter regulatory elements involved in downexpression of the DNA polymerase kappa in colorectal cancer.

The low-fidelity DNA polymerases thought to be specialized in DNA damage processing are frequently misregulated in cancers. We show here that DNA polymerase kappa (polkappa), prone to replicate across oxidative and aromatic adducts and known to function in nucleotide excision repair (NER), is downregulated in colorectal tumour biopsies. Contrary to the replicative poldelta and polalpha, for which only activating domains were described, we identified an upstream 465-bp-long repressor region in the promoter of POLK. We also found an activating 237-bp region that includes stimulating protein-1 (SP1) and cyclic AMP-responsive element (CRE)-binding sites. Mutations at one CRE-binding site led to a dramatic 80% decrease in promoter activity. Alterations of the SP1-binding site also affected, to a lesser extent, the transcription. Gel shift assays confirmed the role played by CRE/SP1 recognition sequences. Moreover, ectopic expression of SP1 or CRE-binding protein (CREB) protein favoured polkappa transcription. Finally, we found that polkappa downexpression in colorectal biopsies correlated with a decreased level of CREB and SP1 transcripts. This work shows that the promoter of POLK is cis-controlled and suggests that silencing of CREB and SP1 proteins could contribute to downregulation of this repair polymerase in colorectal tumours.

Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer.

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

RAD51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors.

The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.

Overexpression of DNA polymerase beta sensitizes mammalian cells to 2',3'-deoxycytidine and 3'-azido-3'-deoxythymidine.

Mammalian DNA polymerase beta is a DNA repair enzyme expressed constitutively at a low level. In vitro, purified DNA polymerase (Pol) beta incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphosphate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate into DNA, causing chain termination. We have tested the possibility of enhancing the cytotoxicity of these chain terminators against mammalian cells by increasing the level of Pol beta. Chinese hamster ovary AA8 and murine melanoma B16 cell lines were stably transfected with rat pol beta cDNA under the control of a viral enhancer/promoter. We found that overexpression of Pol beta sensitized the cells to ddC and AZT. To confirm the role of this polymerase in this process, we prepared cell extracts from the control and Pol beta overexpressing Chinese hamster ovary cell lines and tested in vitro their capacity to incorporate ddC-triphosphate and AZT-triphosphate into DNA. We found that inhibition of DNA replication by both chain terminators was more pronounced when extracts from pol beta-transfected cells were used, providing a direct evidence of the involvement of Pol beta in the sensitization process. In addition, we showed that cotransfection with bacterial or viral thymidine/thymidylate kinase genes enhanced the Pol beta-mediated cytotoxicity of AZT, suggesting that phosphorylation and polymerization activities might be combined to potentiate their respective effects. These observations may be useful for improving therapeutic efficiency of DNA chain terminators.

Excess Polθ functions in response to replicative stress in homologous recombination-proficient cancer cells.

DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes.

Enhanced expression and activity of DNA polymerase beta in human ovarian tumor cells: impact on sensitivity towards antitumor agents.

DNA polymerase beta, one of the most inaccurate DNA synthesizing enzymes, has been shown to confer genetic instability when up-regulated in cells, a situation found in several human cancers. Here, we demonstrated that enhanced activity and expression of this enzyme occur in the human ovarian tumor 2008/C13*5.25 cells, which are resistant to the antitumor agent cisplatin and hypersensitive to 6-thioguanine. We found that translesion synthesis across platinated DNA crosslinks as well as increased incorporation into DNA of 6-thioguanine took place in the 2008/C13*5.25 cells compared to the parental 2008 cells. Such features being molecular signatures of DNA polymerase beta, these findings suggest that deregulation of its expression in cancer cells may contribute to the modulation of the response to antitumor treatments and therefore to tumor progression.

Mutator phenotype of BCR--ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase beta.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR-ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR-ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3-5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR-ABL transfection induced an overexpression of DNA polymerase beta, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase beta in this process.

Adaptation to DNA damage and stimulation of genetic instability: the double-edged sword mammalian DNA polymerase kappa.

A major tolerance mechanism that functions to replicate damaged genomic DNA across lesions that have escaped elimination by repair mechanism is translesion DNA synthesis (TLS). DNA polymerase kappa (Pol kappa), a specialised low-fidelity DNA polymerase which is able to perform DNA synthesis across several damaged bases, is one of the enzymes involved in the process. The mutagenic nature of Pol kappa implies that its expression must be tightly regulated to prevent the formation of excessive genetic disorders along undamaged parts of the genome. Indeed, Pol kappa overexpression, which is notably observed in lung cancer, results not only in increased spontaneous mutagenesis, but also in pleiotropic alterations such as DNA breaks, genetic exchanges and aneuploidy. This review will discuss both aspects of DNA polymerase kappa, which can be considered as a genomic supervisor participating in genome maintenance and when misregulated as a genetic instability enhancer as well.

Genetical and biochemical evidence for the involvement of the coprotease domain of Escherichia coli RecA protein in recombination.

RecA amino acid residue 204 is involved in the coprotease domain of the protein responsible for the induction of mutagenic repair. Two mutations were created at this site leading to the addition of either a methyl or an isopropyl group on the original glycine. Analyses of both the in vivo and the in vitro properties of these mutated proteins demonstrated that this residue 204 is involved in many RecA activities, suggesting that this site could allosterically direct conformational changes in the protein or could be situated in a region interacting with many RecA cofactors.

Increased proliferative activity, loss of beta-adrenergic receptor function and class I major histocompatibility complex antigen surface expression in a modified lymphoma cell line.

The molecular interaction of class I major histocompatibility complex (MHC) antigens (Ag) and of beta-adrenergic receptors was previously demonstrated on lymphocytes. By long-term culturing with high concentration of foetal calf serum, the murine S49 lymphoma cell line was modified (S49m) giving phenotypic alterations in beta-adrenergic receptors and class I Ag expression. S49m cells displayed a reduced number of beta-adrenergic sites that were uncoupled to the adenylate cyclase system. These were unable to respond to beta agonist stimulation, despite the fact that direct activation of Gs could be achieved with aluminium tetrafluoride. Although S49m cells showed normal expression of the thy 1.2 Ag, they displayed no expression of class I Ag of the d haplotype. This was assessed by the evident lack of cytotoxic activity of specific monoclonal antibodies (Mo Ab) and of their binding. When performing IFI staining on permeabilized cells, we found positive staining with anti-class d Ab inside the cell. This loss of expression and activity of beta-adrenoceptors and the internalization of class I Ag were accompanied by a higher rate of proliferation in S49m cells. The possibility that the loss of both molecules would modify the biology of the cell is also discussed.

Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA.

RecB and RecA proteins play key roles in the process of DNA recombination in Escherichia coli and both possess DNA unwinding activities which can displace short regions of duplex DNA in an ATP-dependent manner in vitro. We have examined the effect of the most abundant DNA adduct caused by the chemotherapeutic agent cis-diamminedichloroplatinum(II) on those activities. For this purpose, we have constructed a partially duplex synthetic oligonucleotide containing the intrastrand d(GpG) crosslink positioned at a specific site. We report here that both the DNA strand separating and DNA-dependent ATPase activities of the RecB protein are inhibited by the d(GpG) cis-DDP adduct. In contrast, neither the unwinding nor the ATPase activities of RecA protein appear to be perturbed by this lesion.

Down-regulation of beta-adrenergic receptors induced by mitogen activation of intracellular signaling events in lymphocytes.

The expression of beta-adrenergic receptors on murine lymphocytes stimulated with concanavalin A was studied. A decrease in beta-adrenoceptor number on T lymphocytes and a diminished response to specific agonist stimulation at the peak of proliferation was found. The blockade of cell proliferation by tyrosine kinases or protein kinase C inhibitors reversed the decrease in beta-adrenoceptor number. PMA plus ionophore or interleukin-2 but not PMA alone were able to induce beta-adrenoceptor down-regulation accompanying cellular proliferation. These results showed that the intracellular signals triggered during lymphocyte activation are involved in beta-adrenoceptor down-regulation and it would represent the loss of a mechanism that exerts negative neuroimmune control of cellular proliferation.

DNA polymerase beta imbalance increases apoptosis and mutagenesis induced by oxidative stress.

Oxidative stress has been proposed to be one of the major causes leading to the accumulation of mutation that is associated with the initiation and progression of cancers. Elevated expression of DNA polymerase beta, an event found in many human tumors, has been shown to generate a mutator phenotype. Here, we demonstrated that overexpression of DNA polymerase beta strengthens the mutagenicity of oxidative damages, concomitantly with a higher cellular sensitivity and increased apoptosis. Deregulated expression of DNA polymerase beta could represent a predisposition factor for mutagenic effects of oxidative stress and thus have implication in the generation and/or evolution of cancer.

Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Site-directed mutagenesis in the Escherichia coli recA gene.

Escherichia coli RecA protein plays a fundamental role in genetic recombination and in regulation and expression of the SOS response. We have constructed 6 mutants in the recA gene by site-directed mutagenesis, 5 of which were located in the vicinity of the recA430 mutation responsible for a coprotease deficient phenotype and one which was at the Tyr 264 site. We have analysed the capacity of these mutants to accomplish recombination and to express SOS functions. Our results suggest that the region including amino acid 204 and at least 7 amino acids downstream interacts not only with LexA protein but also with ATP. In addition, the mutation at Tyr 264 shows that this amino acid is essential for RecA activities in vivo, probably because of its involvement in an ATP binding site, as previously shown in vitro.

Altered beta-adrenoceptor function associated to protein kinase C activation in hyperproliferative T lymphocytes.

beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.

DNA synthesis, mismatch repair and cancer.

The entire nucleotide sequence of the genome must be transmitted from one generation to the next with no or few errors. Preservation of this integrity requires multiple genes whose alteration can lead to an early event in tumorigenesis by increasing the mutation rate. This mutator phenotype would provide a continuing pool of mutants upon which selection could act to promote a tumor. Recent evidence consistant with this hypothesis is the mutator phenotype of tumor cells of patients with a hereditary form of colon cancer (HNPCC) which exhibit a several hundred-fold increase in spontaneous mutations in addition to a high degree of microsatellite instability. The multiple genomic alterations increasingly reported as associated with most cancers may therefore be linked to a variety of DNA metabolic processes guardians of the genome, including fidelity of the DNA synthesis and mismatch repair. The connection between cancer and deregulation of nucleotide synthesis, imbalance of the pools of nucleotides, deficiency of DNA polymerases, and mismatch repair is the subject of this review. We consider how perturbation in these DNA transactions results in instability of the genome and cancer.

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine.

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.

The Mars Sample Return Project.

The Mars Sample Return (MSR) Project is underway. A 2003 mission to be launched on a Delta III Class vehicle and a 2005 mission launched on an Ariane 5 will culminate in carefully selected Mars samples arriving on Earth in 2008. NASA is the lead agency and will provide the Mars landed elements, namely, landers, rovers, and Mars ascent vehicles (MAVs). The French Space Agency CNES is the largest international partner and will provide for the joint NASA/CNES 2005 Mission the Ariane 5 launch and the Earth Return Mars Orbiter that will capture the sample canisters from the Mars parking orbits the MAVs place them in. The sample canisters will be returned to Earth aboard the CNES Orbiter in the Earth Entry Vehicles provided by NASA. Other national space agencies are also expected to participate in substantial roles. Italy is planning to provide a drill that will operate from the Landers to provide subsurface samples. Other experiments in addition to the MSR payload will also be carried on the Landers. This paper will present the current status of the design of the MSR missions and flight articles.