Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast.

A plasmid was constructed for the expression of human DNA topoisomerase II alpha in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase II alpha were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35 degrees C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase II alpha purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase II alpha provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase II beta can be eliminated in this system. Overexpression of human DNA topoisomerase II alpha in yeast also provides a convenient source of the enzyme for in vitro studies.

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues.

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.

Novel HeLa topoisomerase II is the II beta isoform: complete coding sequence and homology with other type II topoisomerases.

DNA topoisomerase (topo) II mediates DNA strand passage in an ATP-dependent reaction. Human cell lines express at least two genetically distinct forms of the enzyme, topo II alpha (p170) and II beta (p180). Previously, we isolated a novel HeLa cDNA clone (CAA5) that partially encodes a protein homologous to topo II alpha (Austin, C.A. and Fisher, L.M. (1990) FEBS Lett. 266, 115-117). In this paper we show that CAA5 encodes a C-terminal segment of human topo II beta. We report here for the first time cDNA clones spanning the entire coding sequence. Overlapping clones specifying the 3' end of the cDNA have been isolated, mapped and sequenced. The missing 5' coding sequence was obtained by an inverse PCR protocol and from a specifically primed cDNA library. Human topo II beta is a 1621 amino acid protein which is closely homologous to topo II alpha in the N-terminal three quarters of its sequence. In contrast, the C-terminal segments of the alpha and beta sequences show considerable divergence suggesting these regions may mediate different cellular functions of the two isoforms. Southern blot analysis of yeast and Drosophila DNA using human alpha and beta specific probes detected a single topo II homologue in these lower eukaryotes. Comparison of the protein sequence for human topo II beta with other type II topoisomerases revealed several conserved motifs and has allowed identification of the likely ATPase- and DNA breakage-reunion domains.

Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication.

Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance.

Regulation of DNA supercoiling in Escherichia coli: genetic basis of a compensatory mutation in DNA gyrase.

Bacterial DNA supercoiling is controlled by balancing the supercoiling activity of DNA gyrase and the relaxing activity of DNA topoisomerase I. We have characterized the gyrB gene from a top A deletion mutant of Escherichia coli (DM800) that has a compensatory mutation in gyrB, lowering the activity of gyrase 10-fold, and thereby redressing the intracellular level of supercoiling. The mutant gene differs from the wild type in carrying three rather than two direct tandem repeats of a 6 bp sequence encoding Ala-Arg. We suggest this novel mutation affects domain spacing and was generated by an unequal crossing over event, possibly involving gyrase.

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells.

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.

Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity.

Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling. These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.

DNA binding and intercalation by novel porphyrins: role of charge and substituents probed by DNase I footprinting and topoisomerase I unwinding.

Porphyrins carrying four charged sidechains, e.g., meso-tetrakis[4-N-methylpyridiniumyl]- and meso-tetrakis[4-N-(2-hydroxyethyl)pyridiniumyl]-porphyrin, bound and intercalated similarly into DNA as measured by helix stabilization and DNA unwinding studies in the presence of DNA topoisomerase I. Despite their different bulky sidechains, these complexes gave essentially identical DNase I footprinting patterns. In contrast, tetrasubstituted porphyrins carrying three phenyl rings and a single positively charged pyridiniumyl sidechain did not intercalate and exhibited little affinity for DNA. Thus, the presence of charged sidechains on the porphyrin rather than their identity appears to be critical for efficient DNA intercalation. The results are discussed in regard to current models for the porphyrin-DNA intercalation complex.

Batroxobin-induced clots exhibit delayed and reduced platelet contractile force in some patients with clotting factor deficiencies.

Thrombin causes platelet activation via multiple pathways, and deficient thrombin generation reduces platelet contractile force (PCF) during clot retraction. We hypothesized that PCF in blood samples from clotting factor-deficient patients would be diminished due to delayed or deficient thrombin generation. Blood samples from patients with fibrinogen, and factor V, VII, VIII, IX, X, XI and XIII deficiencies were compared to samples from normal controls. PCF in patient blood clotted with thrombin (1 NIH UmL(-1)) was compared to PCF in clots formed with batroxobin (0.25 micro g mL(-1)). PCF in the former should be normal, but PCF in the latter is dependent on thrombin generation within the sample and might be deficient. In factor VII-(n = 2, P < 0.05), factor VIII-(n = 6, P < 0.005) and factor XI-(n = 2, P < 0.05) deficient platelet-rich plasmas, PCF in batroxobin-induced clots was significantly lower than in thrombin-induced clots. In factor IX deficiency (n = 2), one patient had a dramatic reduction in PCF while a second patient had increased PCF. PCF was insignificantly (P = 0.346) reduced in two patients with factor X deficiency, and was normal in one patient with factor V deficiency. The factor X result is consistent with work in model systems, which indicates that as little as 1-3% factor X activity is sufficient to restore thrombin generation to normal. The factor V result probably indicates that the deficiency is incomplete. PCF in thrombin-induced clots was normal in all of these patients. Low fibrinogen and factor XIII deficiency reduced PCF in both thrombin- and batroxobin-induced clots. These results indicate that PCF is reduced, probably due to delayed thrombin generation, in some factor-deficient platelet-rich plasma samples.

Ciprofloxacin and the fluoroquinolones. New concepts on the mechanism of action and resistance.

Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent. It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling. Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli. An important mutational locus in the gyrase A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised. A quinolone resistant gyrA gene has been previously cloned and sequenced from an E. coli clinical isolate. Genetic analysis indicated that resistance resulted from a Ser-83----Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678----Glu and Ala-828----Ser, were neutral. GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay. The in vitro DNA supercoiling activity of the A (Ser-83----Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones. In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity. Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids. In addition, other quinolone-resistant strains of E. coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP). This analysis revealed that seven of eight resistant clinical isolates of E. coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence. Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin. The RFLP method described should prove useful in examining strains for such mutations. These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

Relationship of major histocompatibility complex class II genes to inhibitor antibody formation in hemophilia A.

Approximately 14% of transfused hemophiliacs develop an anti-factor VIII inhibitory antibody which specifically neutralizes factor VIII procoagulant activity. In this study an association of the major histocompatibility complex (MHC) with inhibitor antibody formation was evaluated by restriction fragment length polymorphism (RFLP) analysis using BamHI, EcoRI, HindIII, PstI, PvuII and TaqI digested genomic DNA probed with DP beta, DQ alpha, DQ beta and DR beta class II MHC gene probes. The RFLP patterns for 16 non-inhibitor and 11 inhibitor hemophiliac patients were analyzed. These 24 enzyme:probe combinations generated 231 fragments. Fifteen (15) fragments associated with the inhibitor phenotype; odds ratios ranged from 5.1 to 45 and lower bounds of 95% confidence intervals were greater than 1.000 for all 15 fragments. Five (5) fragments associated with non-inhibitors, with odds ratios ranging from 6.4 to 51.7. This report establishes a MHC related genetic basis for the inhibitor phenotype. No statistically significant differences in the distribution of serologically defined HLA-DR phenotypes were observed between the inhibitor and non-inhibitor groups.

Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta.

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines. Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.

Recovery of Chlamydia trachomatis from patients of southeastern venereal disease clinic.

The authors determined the incidences of Chlamydia trachomatis colonization obtained by use of two methods of isolation in 169 specimens from individuals attending a venereal disease clinic. Speciments were collected in phosphate sucrose buffer and planted simultaneously onto McCoy cells previously treated with 5-iodo-2-deoxyuridine (IUDR) and with cycloheximide. As controls, specimens were obtained from 76 hospital employees or medical students without clinical signs of infection and cultured in a similar manner. Qualitatively, recoveries of Chlamydia trachomatis inclusions from the differently treated cells were the same, but the number of inclusions per ml was greater for the cycloheximide-treated cells. Recoveries of Chlamydia trachomatis from the venereal disease clinic population were 33% for those patients who also had simultaneous culture positive for Neisseria gonorrhoeae and 42% for those with nongonococcal urethritis. The recovery of Chlamydia trachomatis from the control group was 5%.

Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 beta construct allows the study of topoisomerase II beta inhibitors in yeast.

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.

Characteristics of telephone applicants to a residential rehabilitation program for homeless veterans.

This study gathered descriptive data on 163 individuals who applied by telephone to a residential rehabilitation program for homeless veterans and compared these data with general veteran and homeless populations. Ss were a young (M = 40.82 years) and educated (M = 13.34 years of schooling) subgroup of homeless men (98.16%) with histories of relatively high, stable functioning (e.g., previous successful employment) and high rates of medical (47.47%), substance abuse (67.1%), psychiatric (41.93%; primarily nonpsychotic), and legal (71.15%) problems. These characteristics appear to be different from those of other subgroups of homeless (e.g., homeless chronically mentally ill, skid-row alcoholics), and they provide a basis for beginning to develop distinct remedial strategies that are specific to this subpopulation. The advantages of studying subgroups of homeless and the utility of the telephone interview data collection methodology are discussed.

DNA topoisomerases: enzymes that change the shape of DNA.

The topological state of DNA plays a crucial role in many biological processes including DNA replication and gene expression. DNA topoisomerases are enzymes, present in all cells, that modulate and regulate the topology of DNA, e.g. by introducing or removing DNA supercoils. New insight into the reaction mechanisms and cellular functions of these biologically essential proteins has come from novel enzymatic and genetic approaches. Medical interest in topoisomerases has developed from the discovery that they are the intracellular targets for drugs used in the treatment of infectious diseases and cancer. This review describes mechanistic, biological and medical advances in our understanding of these intriguing proteins.

Effect of adenovirus type 1 and influenza A virus on Streptococcus pneumoniae nasopharyngeal colonization and otitis media in the chinchilla.

Considerable evidence has implicated respiratory tract virus potentiation of bacterial adherence, colonization, and superinfection as a significant factor contributing to the pathogenesis of otitis media (OM). Influenza A and B viruses, adenovirus, and respiratory syncytial virus are the primary respiratory tract viruses associated with this disease. Investigations have established a dramatic increase in the development of experimental OM in chinchillas co-inoculated with influenza A virus and Streptococcus pneumoniae (Spn). The mechanism underlying this phenomenon was suggested to involve, in part, viral compromise of eustachian tube mucosal integrity and function. This study was designed to assess and compare the effect of adenovirus and influenza A virus infection on adherence, the kinetics of colonization, and invasion of the middle ear by Spn in the chinchilla model of OM. Cohorts were inoculated intranasally with adenovirus type 1 or influenza A virus, and then inoculated intranasally 7 days later with Spn 6A. All cohorts were observed over a 14-day period after challenge with Spn, and the incidence and severity of OM were assessed by several methods, including culture of the nasopharynx and middle ear effusions. The data indicated that influenza A virus promotes a significant increase in nasopharyngeal colonization by Spn, an increased incidence and severity of OM, and a sustained presence of Spn in the effusions. Adenovirus infection, however, did not enhance colonization by Spn or result in an increased incidence or severity of OM.

Effect of tumor necrosis factor alpha and interleukin 1-alpha on the adherence of Streptococcus pneumoniae to chinchilla tracheal epithelium.

The trachea whole organ perfusion technique was used to study the effect of tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) on the adherence of otitis media pathogen Streptococcus pneumoniae (Spn) type 6A. Tracheas were removed from chinchillas and divided equally. One-half trachea was activated by incubation with 1-10 ng/ml of either TNF alpha or IL-1 alpha prior to the addition of Spn 6A to the organ culture perfusion chamber. Colony forming units (cfu) of Spn/millimeter trachea were determined for activated tracheas and controls. Dose response and kinetics data were generated for each cytokine. The specificity of each reaction was determined by neutralization studies with specific anti-cytokine antibodies. The data indicate that both TNF alpha and IL-1 alpha increase the adherence of Spn to the respiratory epithelium of this tubal organ and suggest a mechanism which may facilitate enhanced adherence in vivo and thereby contribute to the pathogenesis of otitis media and other upper respiratory tract diseases.

High-fat diet and prostate cancer: the controversial connection.

High-fat diet has been associated with conditions such as heart disease and colon cancer. Is there a link between high-fat diet and prostate cancer? The summation of an abbreviated literature search strives to answer this question.