The Canadian Institutes of Health Research response to antimicrobial resistance.

Antimicrobial resistance (AMR) has been a research priority for the Canadian Institutes of Health Research (CIHR), Institute of Infection and Immunity (III) since its inception, and a number of strategic research initiatives have been launched to address this global health problem by promoting and supporting research related to mechanisms and processes that impact the emergence and spread of resistance among individuals and within the environment. Here we will present research initiatives on AMR led by CIHR-III, which include national programs as well as international partnerships with the United Kingdom and the European Union, in addition to interesting outcomes of these initiatives.

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.

6-Aminonicotinamide sensitizes human tumor cell lines to cisplatin.

The nicotinamide analogue 6-aminonicotinamide (6AN) is presently undergoing evaluation as a potential modulator of the action of various antineoplastic treatments. Most previous studies of this agent have focused on a three-drug regimen of chemical modulators that includes 6AN. In the present study, the effect of single-agent 6AN on the efficacy of selected antineoplastic drugs was assessed in vitro. Colony-forming assays using human tumor cell lines demonstrated that pretreatment with 30-250 microM 6AN for 18 h resulted in increased sensitivity to the DNA cross-linking agent cisplatin, with 6-, 11-, and 17-fold decreases in the cisplatin dose that diminishes colony formation by 90% being observed in K562 leukemia cells, A549 non-small cell lung cancer cells, and T98G glioblastoma cells, respectively. Morphological examination revealed increased numbers of apoptotic cells after treatment with 6AN and cisplatin compared to cisplatin alone. 6AN also sensitized cells to melphalan and nitrogen mustard but not to chlorambucil, 4-hydroperoxycyclophosphamide, etoposide, or daunorubicin. In additional studies undertaken to elucidate the mechanism underlying the sensitization to cisplatin, atomic absorption spectroscopy revealed that 6AN had no effect on the rate of removal of platinum (Pt) adducts from DNA. Instead, 6AN treatment was accompanied by an increase in Pt-DNA adducts that paralleled the degree of sensitization. This effect was not attributable to 6AN-induced decreases in glutathione or NAD+, because other agents that depleted these detoxification cofactors (buthionine sulfoximine and 3-acetylpyridine, respectively) did not increase Pt-DNA adducts. On the contrary, 6AN treatment increased cellular accumulation of cisplatin. Further experiments revealed that 6AN was metabolized to 6-aminonicotinamide adenine dinucleotide (6ANAD+). Concurrent administration of nicotinamide and 6AN had minimal effect on cellular 6AN accumulation but abolished the formation of 6ANAD+, the increase in Pt-DNA adducts, and the sensitizing effect of 6AN in clonogenic assays. These observations identify 6AN as a potential modulator of cisplatin sensitivity and suggest that the 6AN metabolite 6ANAD+ exerts this effect by increasing cisplatin accumulation and subsequent formation of Pt-DNA adducts.

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Interaction between the C. elegans cell-death regulators CED-9 and CED-4.

Programmed cell death (apoptosis) is an evolutionarily conserved process used by multicellular organisms to eliminate cells that are not needed or are potentially detrimental to the organism. Members of the Bcl-2 family of mammalian proteins are intimately involved in the regulation of apoptosis, but, their precise mechanism of action remains unresolved. In Caenorhabditis elegans, the Bcl-2 homologue CED-9 prevents cell death by antagonizing the death-promoting activities of CED-3, a member of the Caspase family of death proteases, and of CED-4, a protein with no known mammalian homologue. Here we show that CED-9 interacts physically with CED-4. Mutations that reduce or eliminate CED-9 activity also disrupt its ability to bind CED-4, suggesting that this interaction is important for CED-9 function. Thus, CED-9 might control C. elegans cell death by binding to and regulating CED-4 activity. We propose that mammalian Bcl-2 family members might control apoptosis in a similar way through interaction and regulation of CED-4 homologues or analogues.

Alteration of the nucleolar localization of poly(ADP-ribose) polymerase upon treatment with transcription inhibitors.

The effect of RNA, DNA, and protein synthesis inhibitors on the subnuclear localization of poly(ADP-ribose) polymerase (PARP) was examined. Indirect immunofluorescence indicated that PARP was distributed throughout the nuclei but concentrated in nucleoli of MDBK, HeLa, and CHO cells. Treatment with the DNA synthesis inhibitor cytosine arabinoside or the protein synthesis inhibitor cycloheximide did not change the distribution of PARP. In contrast, incubation with the RNA-synthesis inhibitor 5,6-dichloro-1-beta-ribofuranosylbenzimidazole (DRB) caused PARP immunofluorescence to become evenly distributed throughout the nucleus. This phenomenon was observed after a 1-h incubation with a DRB concentration that inhibited [5,6-3H]uridine incorporation by 75%. Similar results were obtained with actinomycin D. Immunoblotting showed that the DRB treatment did not cause any changes in the integrity and content of PARP. Removal of DRB from the media allowed PARP to reaccumulate in nucleoli within 1 h, suggesting that the nucleolar localization of PARP is dependent upon active RNA synthesis.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress.

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro. We report here that 1,5-dihydroxyisoquinoline, which was earlier shown to be a strong inhibitor of this polymerase in vitro, is also its potent inhibitor in vivo. Using sensitive techniques for measuring low levels of cellular poly(ADP-ribose) polymer, we show that this inhibitor can completely abolish oxidant-induced activation of the polymerase in C3H10T1/2 cells. We show that only a minor fraction of the poly(ADP-ribose) polymerase activity is sufficient in cellular recovery after sublethal oxidant damage. We also demonstrate that cells are unable to recover from oxidant damage in the complete absence of polymerase activity.

Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.

Temporal changes in chromatin, intracellular calcium, and poly(ADP-ribose) polymerase during Sindbis virus-induced apoptosis of neuroblastoma cells.

Sindbis virus (SV) induces apoptosis in many vertebrate cells, but the mechanism is unknown. To gain insight into this mechanism, the nature and time course of intracellular changes related to programmed cell death were studied in SV-infected mouse neuroblastoma cells. New virus production began at 5 h after infection and reach a peak at 12 h. Hoechst 33342 staining of DNA analyzed by flow cytometry demonstrated changes in chromatin beginning 6 h after infection. These chromatin changes were cell cycle dependent, affecting cells in G0/G1 but not S phase. Apoptosis was not dependent on increases in intracellular Ca2+ and occurred more rapidly in the absence of extracellular Ca2+. Nuclear changes were accompanied by activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), resulting in increased consumption of NAD which was apparent by 10 h after infection. SV-induced apoptosis also involved the proteolytic cleavage of PARP. This cleavage was detectable at 16 h after infection approximately the same time that DNA fragmentation was apparent by agarose gel electrophoresis. We conclude that SV-induced apoptosis of neuroblastoma cells is dependent on viral replication, is not dependent on a rise in intracellular Ca2+, and is accompanied by activation of PARP and of a protease that cleaves PARP.

Identification of a 116 kDa protein able to bind 1,3-bis(2-chloroethyl)-1-nitrosourea-damaged DNA as poly(ADP-ribose) polymerase.

SKI-1 is a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell line and SK-MG-1 is a BCNU-sensitive glioma cell line. Both cell lines do not express O6-methylguanine-DNA methyl transferase (MGMT) and exhibit comparable levels of 3-methyladenine DNA glycosylase. In order to detect DNA binding proteins involved in alternative DNA repair mechanisms of BCNU damage, we performed Southwestern analysis using a DNA probe damaged with BCNU and nuclear protein extracts from SKI-1 and SK-MG-1 cell lines. Both cell lines express a protein of M(r) 116,000 that is able to bind to BCNU-damaged DNA with higher specificity than to undamaged DNA. This protein was identified as poly(ADP-ribose) polymerase (PARP). Using glioma extracts depleted of PARP or using antibody to block the DNA binding domain of PARP no other protein binding to BCNU-treated probe was observed. Addition of methoxyamine, an inhibitor of DNA strand breaks, led to a significant reduction of PARP binding to BCNU-treated DNA. BCNU treatment of both glioma cell lines led to reduced nicotinamide adenine dinucleotide levels, indicating activation of PARP. Thus, the recognition and binding of PARP to BCNU-induced DNA nicks with concomitant PARP activation may be important processes that are involved in the initial stage of DNA repair of BCNU lesions in glial cells.

Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase.

Although the mechanism of mammalian apoptosis has not been elucidated, a protease of the CED-3/ICE family is anticipated to be a component of the death machinery. Several lines of evidence predict that this protease cleaves the death substrate poly(ADP-ribose) polymerase (PARP) to a specific 85 kDa form observed during apoptosis, is inhibitable by the CrmA protein, and is distinct from ICE. We cloned a ced-3/ICE-related gene, designated Yama, that encodes a protein identical to CPP32 beta. Purified Yama was a zymogen that, when activated, cleaved PARP to generate the 85 kDa apoptotic fragment. Cleavage of PARP by Yama was inhibited by CrmA but not by an inactive point mutant of CrmA. Furthermore, CrmA blocked cleavage of PARP in cells undergoing apoptosis. We propose that Yama may represent an effector component of the mammalian cell death pathway and suggest that CrmA blocks apoptosis by inhibiting Yama.

Biochemical properties and function of poly(ADP-ribose) glycohydrolase.

We describe here the latest observations on poly(ADP-ribose) glycohydrolase. There is now extensive evidence that this nuclear enzyme is an endo-exoglycosidase which has a key role to perform in the removal of polymers which interact with proteins through covalent and non-covalent interactions. Also, we have developed a zymogram which will permit the isolation of the various isoforms of the glycohydrolase and the eventual cloning of this enzyme. Finally, we have evidence that very short oligomers and even monomers of ADP-ribose covalently bound to proteins can be removed by poly(ADP-ribose) glycohydrolase.

Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.

Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.

Poly(ADP-ribose) polymerase can bind melphalan damaged DNA.

As a means of identifying damage recognition proteins involved in repair of nitrogen mustard lesions in chronic lymphocytic leukemia, we performed Southwestern analysis using a probe damaged with melphalan and protein extracts from chronic lymphocytic leukemia patients. We detected proteins with molecular weights of 116,000, 66,000, and 64,000 which bound the damaged probe with a higher specificity than the undamaged probe. The M(r) 66,000 and 64,000 proteins were determined to be degradation products of the M(r) 116,000 protein. The M(r) 116,000 protein was identified as poly(ADP-ribose) polymerase. The use of methoxyamine, an inhibitor of DNA strand breakage following depurination, significantly reduced binding of the melphalan damaged probe to poly(ADP-ribose) polymerase. Following depletion of poly(ADP-ribose) polymerase from the cell extracts, no other binding activity was discovered. Thus, poly(ADP-ribose) polymerase is the only demonstrable protein in chronic lymphocytic leukemia cells which can bind to a DNA probe damaged with melphalan.