The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

Phosphotriester formation by the haloethylnitrosoureas and repair of these lesions by E. coli BS21 extracts.

The alkylation of phosphates in DNA by therapeutically active haloethylnitrosoureas was studied by reacting N-chloroethyl-N-nitrosourea (CNU) with dTpdT, separating the products by HPLC, and identifying them by co-chromatography with authentic markers. Both hydroxyethyl and chloroethyl phosphotriesters of dTpdT were identified; a similar reaction between CNU and dTR yielded 3-hydroxyethyl and 3-chloroethyl dTR as the major products of ring alkylation. A DNA-like substrate for repair studies was synthesized by reacting 14C-labelled N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (14C-CCNU) with poly dT and annealing the product to poly dA. An extract of E. coli strain BS21 selectively transferred a chloroethyl group from one of the chloroethyl phosphotriester isomers in this substrate to the bacterial protein; chemical instability of the hydroxyethyl phosphotriesters precluded definite conclusions about the repair of this product.     

Inhibition of influenza virus RNA (PB2 mRNA) expression by a modified DNA enzyme.

DNA enzymes are RNA-cleaving single stranded DNA molecules. The structure and the catalytic domain of a DNA enzyme were determined by Santro et al. in 1997. In this study, we have designed several types of DNA enzymes (PB2Dz) targeted to the PB2 mRNA translation initiation region of influenza A virus, and examined their cleavage kinetics, nuclease resistance, and a luciferase gene reporter assay. Using a synthetic substrate, these DNA enzymes were shown to have cleavage activity that is dependent on the length of the substrate recognition domain. To confer serum nuclease resistance to the DNA enzymes, we designed a new type of DNA enzyme that has the N3'-P5' phosphoramidate modification (PB2Dz-N) at each terminal. We examined the activity of this DNA enzyme in vivo. The DNA enzymes used in this study inhibited the expression of the PB2-luciferase gene in COS cells. These results suggest that DNA enzymes are potentially useful as gene inactivating agents of influenza A virus.     

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.     

Prevalence, development, and molecular mechanisms of bacteriocin resistance in Campylobacter

Bacteriocins (BCNs) are antimicrobial peptides produced by bacteria with narrow or broad spectra of antimicrobial activity. Recently, several unique anti-Campylobacter BCNs have been identified from commensal bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacter. However, no information concerning prevalence, development, and mechanisms of BCN resistance in Campylobacter exists. In this study, susceptibilities of 137 C. jejuni isolates and 20 C. coli isolates to the anti-Campylobacter BCNs OR-7 and E-760 were examined. Only one C. coli strain displayed resistance to the BCNs (MIC, 64 μg/ml), while others were susceptible, with MICs ranging from 0.25 to 4 μg/ml. The C. coli mutants resistant to BCN OR-7 also were obtained by in vitro selection, but all displayed only low-level resistance to OR-7 (MIC, 8 to 16 μg/ml). The acquired BCN resistance in C. coli could be transferred at intra- and interspecies levels among Campylobacter strains by biphasic natural transformation. Genomic examination of the OR-7-resistant mutants by using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic resistance and acquired resistance to the BCNs. Altogether, this study represents the first report of and a major step forward in understanding BCN resistance in Campylobacter, which will facilitate the development of effective BCN-based strategies to reduce the Campylobacter loads in poultry.     

Identification and mapping of loci involved in motility, adsorption to wheat roots, colony morphology, and growth in minimal medium on the Azospirillum brasilense Sp7 90-MDa plasmid.

We have constructed a cosmid library of the Azospirillum brasilense Sp7 90-MDa plasmid (p90) and established the EcoRI restriction map of this plasmid. The central regions of cloned p90 DNA fragments from several recombinant cosmids were deleted by restriction endonuclease digestion and replaced by a DNA cassette encoding kanamycin resistance. Using these in vitro constructed deletions for marker exchange in Sp7, we made six different p90 deletion derivatives spanning all together 50% of the total length of p90. Comparison of the deletion derivatives with Sp7 for several properties revealed p90 loci involved in colony morphology, growth on minimal medium, motility, and adsorption to wheat roots. In analogy with the rhizobial symbiotic plasmids (pSym), we propose to denote the p90 plasmid as a rhizocoenotic plasmid (pRhico), carrying several genes involved in the A. brasilense-plant root interaction.     

Topoisomerase II in multiple drug resistance

Topoisomerase II is a target of alkaloid, anthracycline and related antitumor agents. Two types of multiple drug resistance are associated with these enzymes. In classical (typical) multidrug resistance, inhibitors are actively effluxed from cells by P-glycoprotein. In atypical multidrug resistance, topoisomerase II is either reduced in cellular content or mutated to a form that does not interact with inhibitors. Because cytotoxicity of most antineoplastic topoisomerase II inhibitors is directly related to the number of active topoisomerase II molecules, a reduction in this number leads to resistance. In the topoisomerase II mechanism, through which the DNA linking number is altered, DNA double strands are cleaved, and the termini transiently bound covalently (5) or noncovalently (3) to the enzyme while a second double strand is passed through the break in the first. This transition state complex then decays to enzyme and DNA of altered linking number. Most cytotoxic topoisomerase II inhibitors stabilize these reaction intermediates as ternary complexes, which are converted to lethal lesions when cells attempt to utilize the damaged DNA as templates. Toxicity is related to topoisomerase II content as well as to drug concentration. Thus, multidrug resistance results from either 1) decreasing cellular content of the inhibitor by P-glycoprotein (typical) or 2) decreasing cellular content and/or activity of the target, topoisomerase II, as, for example, when its content or activity is modulated downward by decreased expression, deactivation, or by mutations to the TopII gene, producing an enzyme that reacts poorly with inhibitors (atypical). Mixed types,i.e., both typical and atypical, are known. Attempts to abrogate or prevent both typical and atypical multidrug resistance to topoisomerase II inhibitors have been described.Abbreviations atMDR atypical multidrug resistance - kDa kilodaltons - MDR multidrug resistance - Pgp P-glycoprotein - TOPO II topoisomerase II     

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.     

Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-alpha has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC(50) for TNF-alpha, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-alpha had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-alpha resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-alpha.     

Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities

In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA·Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA·Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA·Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA·Ub resistance to DNA damage, nor did PCNA·Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA·Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.     

Azide resistance in Rhizobium ciceri linked with superior symbiotic nitrogen fixation

Isolated azide resistant (AzR) native R. ciceri strain 18-7 was resistant to sodium azide at 10 microg/ml. To find if nif-reiteration is responsible for azide resistance and linked to superior symbiotic nitrogen fixation, transposon (Tn5) induced azide sensitive mutants were generated. Using 4 kb nif-reiterated Sinorhizobium meliloti DNA, a clone C4 that complemented azide sensitivity was isolated by DNA hybridization from genomic library of chickpea Rhizobium strain Rcd301. EcoRI restriction mapping revealed the presence of 7 recognition sites with a total insert size of 19.17 kb. Restriction analysis of C4 clone and nif-reiterated DNA (pRK 290.7) with EcoRI and XhoI revealed similar banding pattern. Wild type strain 18-7, mutant M126 and complemented mutant M126(C4) were characterized for symbiotic properties (viz., acetylene reduction assay, total nitrogen content, nodule number and fresh and dry weight of the infected plants) and explanta nitrogenase activity. Our results suggested that azide resistance, nif-reiteration, and superior symbiotic effectiveness were interlinked with no correlation between ex-planta nitrogenase activity and azide resistance in R. ciceri.     

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7.

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.     

Nucleotide excision repair and anti-cancer chemotherapy

DNA repair is an important effector of anti-cancer drug resistance. In recent years, it has become apparent that DNA repair is an extremely complex process. Processes within DNA repair that may contribute to one or more drug resistance phenotypes include; O-6-alkyltransferase activity, base excision repair, mismatch repair, nucleotide excision repair, and gene specific repair. Clearly, several of these processes may show increased activity within any single cell, or tumor, at any one time. This review attempts to touch briefly upon the question of the distinctions between each of these specific pathways; and then seeks to expand on nucleotide excision repair as a possible effector of cellular and clinical resistance to platinum-based anticancer therapy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. Previously, we identified a human Cdc7 homolog, HsCdc7. In this study, we report the identification of a human Dbf4 homolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity. They are low in G(1), increase during G(1)-S, and remain high during S and G(2)-M. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo (32)P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate. Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells.     

Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.